i

EFFECT OF DIETARY INCLUSION OF SODIUM BICARBONATE ON PRODUCTION PERFORMANCE,

NUTRIENT DIGESTIBILITY AND BLOOD PROFILE OF CAGED LAYERS DURING SUMMER

By

Ghulam Abbas2000-ag-1523

M. Sc. (Hons.) Poultry Science

A thesis submitted in partial fulfillment of the requirement for the degree of

DOCTOROFPHILOSOPHY

IN

POULTRY SCIENCE

INSTITUTE OF ANIMAL AND DAIRY SCIENCESFACULTY OF ANIMAL HUSBANDRY,

UNIVERSITY OF AGRICULTURE,FAISALABAD,

PAKISTAN2017

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THIS HUMBLE EFFORT, FRUIT OF MY LIFE IS

DEDICATED TO MY RESPECTED BROTHER

MUHAMMAD AHMAD SHAHEEDThe Son of My Supervisor

DR. SULTAN MAHMOOD

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AcknowledgementsI offer my humblest thanks from the deepest core of my heart to “ALMIGHTY

ALLAH” who created the universe and bestowed the mankind with knowledge and wisdom

to research for its secrets. I bow before his compassionate endowments. I pay homage to

Holy Prophet Muhammad (Peace be upon Him and His Holy Descendants), the most

perfect and exalted among us who are forever a source of knowledge and guidance for

humanity as a whole.

It is my utmost pleasure to avail this opportunity to extend my heartiest gratitude to

my worthy supervisor Prof. Dr. Sultan Mahmood, professor, Institute of Animal Sciences,

University of Agriculture, Faisalabad (UAF) for providing and facilitating an encouraging

environment for competitive research. I feel highly privileged to pay cordial gratitude to my

reverend, zealot and distinguish supervisor for his keen personal interest, dynamic

supervision, immense cooperation, valuable suggestions, unfailing patience, constructive and

thoughtful criticism and, moral and economical support during my study.

I am also thankful to the members of my supervisory committee, Dr. AhsanulHaq,

Director of Institute of Animal Sciences at UAF and Prof.Dr. Haq Nawaz, Director

Graduate Studies, UAF for their constant support and help throughout the course of this

study.

I also extend my sincere words of admiration and appreciation to my family members

who supported me morally throughout my research. Special thanks are extended to my

brother Sajjad Hussain Hashmi and my wife Razia Abdual Majeed Qureshi for their

prayers and support. I further offer passionate thanks to especially Brother

SajjadHussainHashmifor his extended cooperation.

I extend my exorbitant and hearty thanks to my affectionate and generous parents

who taught me the first word to speak, the first alphabet to write and the first step to take.

They are up and above of all the blessings of ALMIGHTY ALLAH, I enjoyed during my

temporary stay in the world. I also pay sincere thanks to all of my sisters and brothers who

supported me during my study. May ALLAH give them a long, happy and healthy life

(Ameen)

Ghulam Abbas

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LIST OF CONTENTS

vii

2.14.8 Various egg quality parameters 35

viii

2.14.9 Body/rectal temperature and respiration rate 372.14.10 Mortality 382.14.11 Hematological profile 402.14.12 Serum metabolites and serum proteins 402.14.13 Plasma electrolytes and minerals 412.14.14 Serum lipids, hormones and enzymatic profile 442.14.15 Immune response 452.14.16 Digestibility of nutrients 46

CHAPTER 3 MATERIALS AND METHODS 483.1 Performance Trial 48

3.1.1 Experimental birds 483.1.2 Allocation of the birds to the cages 483.1.3 Management of the experimental birds 493.1.4 Experimental diets, groups and their feeding plans 49

3.2 Data Collection 493.2.1 Initial body weight of birds 493.2.2 Weight gain 493.2.3 Feed consumption 523.2.4 Egg production 523.2.5 Egg mass 523.2.6 Feed conversion ratio 523.2.7 Water consumption 523.2.8 Rectal temperature and respiration rate 523.2.9 Ambient temperature and Humidity index 52

3.3 Egg quality characteristics 533.3.1 Egg Weight 533.3.2 Shell thickness 533.3.3 Specific Gravity of Eggs 533.3.4 Albumen height 553.3.5 Yolk Height 553.3.6 Yolk Diameter 553.3.7 Blood and meat spots 553.3.8 Yolk index 553.3.9 Haugh unit score 553.3.10 Yolk pH and pH of albumen 56

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3.3.11 Egg yolk cholesterol 563.4 Mortality 563.5 Hematological profi le 57

3.5.1 Glucose 573.2.5 Hemoglobin 583.5.3 ESR 583.5.4 Packed Cell Volume (PCV) 583.5.5 Total leucocyte count 583.5.6 Red blood cells (RBCs) count 59

3.6 Serum proteins 603.6.1 Total serum protein 603.6.2 Serum albumen concentration 613.6.3 Serum globulin concentration 61

3.7 Serum lipids profile 613.7.1 Serum cholesterol concentration 613.7.2 Serum triglycerides 623.7.3 HDL cholesterol 64

3.8 Plasma electrolytes (i.e. Na+, K+, Cl-, HCO3-) and mineral (Ca

and P) profile66

3.8.1 Estimation of Blood pH 663.8.2 Estimation of Sodium (Na+) and potasium 663.8.3 Estimation of chloride 673.8.4 Calcium 673.8.5 Phosphorus 683.8.6 Estimation of HCO3

- 693.9 Hormono-enzymic performance 70

3.9.1 Assay procedure for Triiodothyronine (T3) 703.9.2 Assay procedure for Thyroxin (T4) 713.9.3 Assay procedure for cortisol 723.9.4 Assay procedure for estrogen 723.9.5 Assay procedure for progesterone 733.9.6 Serum Glutamic pyruvic transaminase (SGPT) 743.9.7 Serum Glutamic-Oxaloacetic Transaminase (SGOT)

Principle75

3.10 Serum metabolites 75

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3.10.1 Urea 753.10.2 Serum creatinine concentration 763.10.3 Uric acid 773.10.4 Alkaline Phosphatase (ALP) 78

3.11 Determination of Antibody titre against Newcastle disease virus in birds

79

3.12 Digestibility trial 813.13 Proximate composition 82

3.13.1 Dry matter 823.13.2 Crude protein 823.13.3 Ether extract 833.13.4 Crude fiber 833.13.5 Acid Insoluble Ash (AIA) 84

3.14 Mineral analysis 853.14.1 Wet digestion 853.14.2 Determination of calcium 853.14.3 Determination of Phosphorus 863.14.4 Determination of Sodium and potassium by flame

photometer87

CHAPTER 4 RESULTS AND DISCUSSION 88Results 88

4.1 Production performance 884.1.1 Live body weight 884.1.2 Feed consumption 884.1.3 Egg Production 904.1.4 Egg weight 904.1.5 Egg mass 904.1.6 Feed efficiency 92

4.2 Egg quality 934.2.1 Specific gravity 934.2.2 Shell thickness 934.2.3 Albumen height 954.2.4 Haugh unit 954.2.5 Yolk diameter 954.2.6 Yolk height 96

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4.2.7 Yolk Index 964.2.8 Egg yolk cholesterol 964.2.9 Yolk pH 974.2.10 Albumen pH 974.2.11 Meat and blood spots 98

4.3 Rectal temperature, respiration rate and water consumption 984.3.1 Rectal temperature 984.3.2 Respiration rate 984.3.3 Water intake 100

4.4 Mortality 1004.5 Hematological profile 100

4.5.1 Serum glucose 1024.5.2 Packed cell volume 1024.5.3 Blood hemoglobin 1024.5.4 Erythrocytes sedimentation rate 1034.5.5 Red blood cells count 1034.5.6 White blood cell count (WBCs) 103

4.6 Serum metabolites 1034.6.1 Serum urea 1044.6.2 Serum uric acid 1044.6.3 Serum creatinine 1064.6.4 Serum alkaline phosphatase 106

4.7 Serum proteins analysis 1064.7.1 Total proteins 1064.7.2 Albumen 1084.7.3 Globulin 108

4.8 Plasma electrolytes, minerals and serum pH 1084.8.1 Plasma sodium 1084.8.2 Plasma Potassium 1104.8.3 Plasma chloride 1104.8.4 Plasma bicarbonate 1104.8.5 Plasma calcium 1114.8.6 Plasma phosphorus 1114.8.7 Serum pH 111

4.9 Serum lipids profile 112

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4.9.1 Serum cholesterol 1124.9.2 Serum triglyceride 1124.9.3 Serum high density lipoprotein 1124.9.4 Serum low density lipoprotein 114

4.10 Hormones and enzymes 1144.10.1 Tri-iodothyronine (T3) and Thyroxin (T4) 1144.10.2 Oestrogen 1164.10.3 Progesterone 1164.10.4 Corticosterone 1174.10.5 Serum Glutamic-Oxaloacetic Transaminase (SGOT) and

Serum Glutamic-PyruvicTransaminase (SGPT)117

4.11 Immune response 1184.12 Economic Appraisal 120

Discussion 1234.13 Performance 123

4.13.1 Live body weight 1234.13.2 Feed consumption 1244.13.3 Egg Production 1254.13.4 Egg weight 1264.13.5 Egg mass 1274.13.6 Feed efficiency 129

4.14 Egg quality 1304.14.1 Specific gravity 1304.14.2 Shell thickness 1314.14.3 Albumen height 1324.14.4 Haugh unit 1334.14.5 Yolk diameter 1344.14.6 Yolk height 1354.14.7 Yolk Index 1354.14.8 Egg yolk cholesterol 1364.14.9 Yolk pH 1364.14.10 Albumen pH 1364.14.11 Meat and blood spots 137

4.15 Mortality 1374.16 Rectal temperature, respiration rate and water consumption 138

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4.16.1 Rectal temperature 1384.16.2 Respiration rate 1394.16.3 Water intake 140

4.17 Hematological profile 1414.17.1 Serum glucose 1414.17.2 Packed cell volume, ESR and Red blood cells count 1414.17.3 Blood hemoglobin 1424.17.4 White blood cell count (WBCs) 143

4.18 Serum metabolites 1444.18.1 Serum urea 1444.18.2 Serum uric acid 1444.18.3 Serum creatinine 1454.18.4 Serum alkaline phosphatase 145

4.19 Serum proteins analysis 1454.19.1 Total proteins 1454.19.2 Albumin 1454.19.3 Globulin 146

4.20 Plasma electrolytes, minerals and serum pH 1464.20.1 Plasma sodium 1464.20.2 Plasma Potassium 1474.20.3 Plasma chloride 1484.20.4 Plasma bicarbonate 1494.20.5 Plasma calcium and phosphorus 1494.20.6 Serum pH 150

4.21 Serum lipids profile 1514.22 Hormones and enzymes 151

4.22.1 Tri-iodothyronine (T3) and Thyroxin (T4) 1514.22.2 Oestrogen 1524.22.3 Progesterone 1534.22.4 Corticosterone 1534.22.5 Serum Glutamic-Oxaloacetic Transaminase (SGOT) and

Serum Glutamic Pyruvic Transaminase (SGPT)154

4.23 Immune response 155CHAPTER 5 DIGESTIBILITY TRIAL 157

5.1 Introduction 157

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5.2 Materials and methods 1575.2.1 Experimental diets 158

5.2.2 Chemical analysis of excreta 1585.2.3 Statistical analysis 158

5.3 Results 1585.3.1 Dry matter 1605.3.2 Crude protein 1605.3.3 Crude fiber 1605.3.4 Ether extract 161

5.4 Minerals 1615.4.1 Calcium 1615.4.2 Phosphorous 1635.4.3 Iron 1635.4.4 Sodium 1645.4.5 Potassium 164

5.5 Discussion 1655.5.1 Dry matter 1655.5.2 Protein 1665.5.3 Crude fiber 1675.5.4 Ether extract 169

5.6 Minerals 169CHAPTER 6 SUMMARY 173

LITERATURE CITED 177

TABLE OF CONTENTS

No. Title Page

xv

1 INTRODUCTION 1

2 LITERATURE REVIEW 04

3 MATERIALS AND METHODS 48

4 RESULTS AND DISCUSSION 88

5 DIGESTIBILITY TRAIL 157

7 SUMMARY 173

LITERATURE CITED 177

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LIST OF TABLES

Table # TITLE Page#

3.1 Proportion of ingredients used in experimental diets 50

3.2 Chemical composition of the experimental diets 51

4.1Effect of dietary inclusion of sodium bicarbonate on weight gain and

feed consumption of caged layers89

4.2Effect of dietary inclusion of sodium bicarbonate on production

performance of caged layers91

4.3Effect of dietary inclusion of sodium bicarbonate on egg quality

characteristics of caged layers94

4.4Effect of dietary inclusion of sodium bicarbonate on rectal

temperature, respiration rate and water consumption of caged layers99

4.5Effect of dietary inclusion of sodium bicarbonate on hematological

profile of caged layers101

4.6Effect of dietary inclusion of sodium bicarbonate on serum

metabolites of caged layers105

4.7Effect of dietary inclusion of sodium bicarbonate on serum proteins

concentration of caged layers107

4.8Effect of dietary inclusion of sodium bicarbonate supplementation

on plasma electrolytes and serum pH of caged layers109

4.9Effect of dietary inclusion of sodium bicarbonate on serum lipids

profile of caged layers113

4.10Effect of dietary inclusion of sodium bicarbonate on serum

hormones and liver enzymes of caged layers115

4.11Effect of dietary inclusion of sodium bicarbonate on immune

response of caged layers119

4.12

Economics of production of the layers fed different levels of sodium

bicarbonate, calculated for 12 weeks of production (27th -38th week

of age)

121

5.1 Effects of dietary inclusion of sodium bicarbonate on nutrient 159

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digestibility coefficient in layers

5.2Effect of dietary inclusion of sodium bicarbonate on absorbability of

minerals in layers162

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ABSTRACTThe intent of this study was to investigate the effect of dietary inclusion of NaHCO 3 on production performance, nutrient digestibility and blood profile of caged layers during summer. One hundred sixty commercial layers of 24 weeks old were bought from a commercial layer farm and were raised in a group for one week i.e. adaptation period. At the beginning of 26th week of age, these layers were further divided into 20 experimental units/replicates (8 layers/replicate). These 20 replicates/units were further allotted/distributed to five treatment groups (4 replicate/treatment). All the birds were offered diets containing 17% CP and 2700 Kcal/Kg ME with or without supplementation of NaHCO 3 for a period of twelve weeks. Group A served as control, which was provided layer ration without any supplementation, while group B, C, D, and E were offered ration supplemented with 0.5, 1, 1.5 and 2% sodium bicarbonate, respectively. All the diets were iso-nitrogenous (having same protein contents, CP, 17%) and iso-caloric (having same energy level, ME, 2700 Kcal/Kg). these diets were fed to the experimental birds ad libitum, for 12 weeks (26-37 weeks of age). Data on feed consumption, number of eggs produced, egg weight and egg mass laid by the birds were recorded. These data were used for the calculation of feed conversion ratios (FCR) on the basis of per dozen eggs and FCR on the basis of per kg egg mass produced. Five eggs from each replicate were checked weekly for their shell thickness (ST), yolk index (YI), albumen index (AI), Haugh unit (HU) score, yolk pH, albumen pH, specific gravity (SG) and yolk cholesterol. Results revealed that dietary inclusion of sodium bicarbonate significantly (P<0.05) increased feed consumption, weight gain, feed efficiency, egg production, egg weight, egg shell thickness, specific gravity, albumen height, Haugh unit, yolk height and yolk diameter of eggs produced by the birds. Yolk cholesterol was found to be minimum in the eggs laid by the birds fed rations containing 1% NaHCO 3

(group C). Whilst pH of yolk, egg albumen and Serum uric acid concentration were found to be higher in group E. Dietary inclusion of sodium bicarbonate significantly (P<0.05) decreased the rectal temperature and respiration rate of layers, whilst it increased the water intake of the birds significantly (P<0.05). Blood samples were collected from two birds selected randomly from each replicate 10 days post vaccination of 1st, 2nd, and 3rd vaccination to check antibody titer against ND virus. Blood samples were collected from two birds from each replicate at the last day of 37 th week for the analysis of blood profile. Serum glucose, white blood cells count, serum urea, plasma chlorides, serum cortisol and serum glutamic-oxaloacetic transaminase concentration were (SGOT) found to be significantly (P<0.05) higher in control group, whereas, blood hemoglobin concentration, red blood cells count, plasma sodium, potassium, bicarbonate, serum total protein and serum albumen concentration were found to be significantly (P<0.05) higher in birds of group C. However, yolk index, packed cell volume, erythrocyte sedimentation rate, serum creatinine, alkaline phosphatase, plasma calcium, plasma phosphorus, serum globulin and serum glutamic pyruvate transaminase concentration were not affected significantly (P>0.05) due to the dietary treatments. Serum cholesterol, triglycerides and low density lipo-proteincon centration were significantly (P<0.05) decreased, whereas, serum high density lipo-protein concentration was found to be significantly (P<0.05) increased by dietary inclusion of sodium bicarbonate. Birds of group C showed maximum concentration of estrogen, progesterone, T3, T4 and antibody titer against Newcastle disease. For digestibility trial thirty layers having similar body weight were obtained from the same batch which was used for the performance trial. These birds were maintained in individual metabolic cages and were randomly allotted to five experimental diets (same as in performance trial) in such a way that each ration was offered to 6 layer birds and each bird served as a replicate. Feces samples were collected at the end of 38th week of age for two days at the time interval of three hours. Results revealed that digestibility of dry matter , protein, ether extract and crude fiber as well as mineral absorption was found to be better in the birds fed diets containing 1% sodium bicarbonate.

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CHAPTER-1

INTRODUCTION

Pakistan is situated in the subtropical zone of Northern Hemisphere of the world

where temperature usually remains well above the higher side of thermo-neutral zone (25-37

°C) for the greater part of the year (Anjum, 2000). The environmental temperature of some

parts in the region reaches up to 52 °C (Vidal and Walsh, 2010). The optimum temperature

for efficient performance is 19-22°C for laying birds, however, ambient temperature

especially on the higher side is very disruptive and may reduce survival rate and production

(Charles, 2002). Heat stress during summer is a major problem in most parts of Pakistan, and

this has pronounced effects on production performance of layers (Mashaly et al., 2004). Egg

production declines drastically, thereby adversely affecting the economics of poultry

production, which may lead to increase in the number of culled birds.

High laying house temperature causes detrimental effects not only on egg production,

size of egg and egg quality (Farnell et al., 2001) but also adversely affects physiology of the

birds (Sahota et al., 1990) resulting in high mortality. The birds increase panting up to 10

times if ambient temperature is higher than thermo-neutral zone (Nillipour and Melog, 1999).

Ambient temperature and circadian rhythm influence liver glycogen and plasma carbohydrate

levels with distinct changes in blood glucose (Ahmad et al., 2005).

As ambient temperature shoots up, respiratory rate of birds increases resulting in

higher losses of CO2 that causes increase in blood pH and disturbs acid-base balance

(Toyomizu et al., 2005). Any change in acid-base balance does cause alkalosis or acidosis,

diverting the metabolic machinery used for homeostatic regulation rather than used for

production (Carlson, 1997). Alteration in levels of CO2 can cause disruption in blood pH and

deterioration in eggshell quality (Jones, 2006).

Different techniques are being used in poultry production to combat heat stress. These

techniques include nutritional manipulations such as dietary addition of oils (Ghazalah et al.,

2008), reduction in protein level of feed, supplementation of feed with limiting amino acids

(Daghir, 1995) and management practices like intermittent feeding, feeding the birds in

cool hours of the day, time limit feeding (Yalcin et al.,2001; Macleod et al., 1993),

sprinkling of water, evaporative cooling (Donald, 2000), improved ventilation (Nilipour,

1

2000) and supplementation of electrolytes (Ahmad et al., 2005). These techniques are

considered helpful in reducing heat stress. Time limit feeding during cool hours of the day is

a common practice for combating/manipulating heat stress. It has also been recommended

that birds should not be fed during hot hour periods (Mahmood et al., 2005) because it only

adds to body heat due to heat increment, which the birds has to dissipate. Whereas time limit

feeding during the cooler part of the day would increase the feed consumption at a time much

suited for its efficient utilization with minimum chances of heat stress. Although, such feed

practice is not likely to increase the overall daily feed intake, yet it is expected to improve the

feed efficiency and production performance.

The practice of feed withdrawal and intermittent feeding during the hottest hours of

the day is being practiced in many broiler producing areas for the prevention of heat stress

and to control mortality (Ahmad et al., 2006). Short term feed withdrawal has been shown to

lower the body temperature of birds and increase the ability to stay alive in acute stress

conditions (Siegel and Jordan, 1997). Practice of supplementation of ascorbic acid in

commercial feed has also been considered a useful and effective tool for heat stress

amelioration (Whitehead and Keller, Khattak et al., 2012).

Among the electrolytes, NaHCO3 may be used to maintain a correct plasma acid-base

balance to combat heat stress. Sodium bicarbonate is a cheap salt (electrolyte) and is also

used as a buffering agent, source of carbon dioxide, an antacid and for the production of

sodium carbonate (Whiting et al., 1991). It provides sodium and positively affects blood pH

supplying bicarbonate ions (Ahmad et al., 2006). Many studies have reported beneficial

effects of supplementing drinking water of broilers with sodium bicarbonate as a sodium

source (Hassan et al. , 2009). However, scientific research information regarding the use of

NaHCO3 in layer diet is still scarce.

Sodium bicarbonate is a good source of providing sodium and bicarbonate ions.

However, the potential benefits of including sodium bicarbonate as a source of sodium in

poultry diets are uncertain (Teeter et al . , 1985). Although it is adjusted with great care in

poultry diets, however, any imbalance of the sodium in poultry diet may lead to depress their

performance (Murakami et al., 2000). A fall in plasma sodium may cause an increase in

aldosterone secretion and increased re-absorption of sodium from the renal tubules (Rector et

al., 2004).

2

Supplementation of sodium bicarbonate resulted in better performance in broilers

subjected to high temperature and humidity stress (Khattaket al., 2012). During heat stress

(Gorman and Balnave, 1995) blood values for bicarbonate concentration are drastically

reduced due to excessive panting. Under such circumstances supplementing diets of birds

with bicarbonate may be useful. As feed additive, sodium bicarbonate helps to maintain

proper pH balance, eliminates acidosis and facilitates metabolic process, ensuring maximum

growth and productivity (Danny, 1995). Moreover, rise in blood bicarbonate concentration

favors increase in performance of chicken (Keskin and Durgan, 1997).

Keeping in view the information above, supplementation of NaHCO3 may be a useful

technique to combat heat stress, which still needs to be addressed in layers. Therefore, the

present study was planned to investigate the effect of dietary inclusion of NaHCO3 on

production performance, nutrient digestibility and blood profile of caged layers during

summer under the environmental conditions of Pakistan.

3

CHAPTER-2

REVIEW OF LITERATURE

Birds are able to maintain their body temperature within narrow limits (Khattak et al.

2012). An increase in body temperature due to higher ambient temperature or excessive

metabolic activities may cause irreversible thermoregulatory events that could be harmful for

the existence of birds (North and Bell, 1990). However, various techniques are being used in

birds such as addition/supplementation of various products in poultry rations to ameliorate

the effects of heat stress. Supplementation of sodium bicarbonate (NaHCO3) in water or feed

is one of these practices, which are used as an effort to combat the heat stress in broilers

(Ahmad et al., 1997; Mushtaq et al., 2005). However, scientific information regarding its use

in layers during summer is scanty, especially in the areas of Asiatic region.

The following review will address the effects of heat stress on poultry and

possibility of dietary use of NaHCO3 to reduce/mitigate the adverse effects of heat

stress in poultry.

2.1 What is stress?Any physical or physiological change in birds due to internal or external deviation in

birds caused by an unusual process is called stress (Reddy and Dinesh, 2004). It is an

additional burden on the birds, which possibly tends to produce a disharmony in various

physiological systems. Stress can arise from any of a number of internal or external factors

and can cause hormonal changes in the body though pituitary and adrenal glands. General

adaptation syndromes described by Selye (1973a) has become the basis of studies for many

scientists working on the subject of stress in animals. According to him, “stress describes an

animal's defense mechanisms, and thus stressor is any situation that elicits defensive

responses”.

The surrounding environment of the bird is amalgamated of interacting stress factors,

thus bird's success to compete with it depends on the intensity of stressor and bird's

physiological mechanism to react to the stressor (Chrrousos and Gold, 1992). Generally the

environment of the bird is of two types 1) external i.e. temperature, light, etc. and internal i.e.

disease organisms, parasites etc. Any changes in the environment of birds activate regulatory

mechanism in attempt to maintain homeostasis. There are 2 types of regulatory mechanisms:

4

1) specific, 2) nonspecific (Carrasco and Van de Kar, 2003). Any particular change in the

environment of the bird will educe/elicit a specific response. For example, when ambient

temperature is increased, it causes: body temperature of bird to rise, vasodilation for

rapid/quick heat dissipation, and feathers are rearranged to ease insulation. To regulate

adaptation process against stress, endocrine and nervous system work together.

2.1.1 Heat Stress mechanism

Stimulus of heat stress is received by the central nervous system, which excites

hypothalamus to activate pituitary glands (Selye, 1956) and in turn releases Adreno

corticotropic hormone (ACTH). Secretion of this hormone is regulated by the secretion of

corticotrophin releasing factor from hypothalamus. Adreno corticotropic hormone stimulates

adrenal gland to produce catecholamine (via adrenal medulla) and corticosteron (via adrenal

cortex). Catecholamine (epinephrine and nor-epinephrine) perform non-essential functions

i.e. increase glucose metabolism (Selye, 1973), blood flow through skeletal muscles (Sahin et

al., 2002), oxygen consumption, heart rate, elevates blood pressure and constricts arterioles

and venules (Grandin, 1998; Siegel, 1980). Adrenal cortex hormones (cortisol and

aldosteron) perform essential functions i.e. promote synthesis of glucose from fat and protein

(gluconeogenesis), maintain heat loss mechanism, blood flow rate and body temperature

(Maxwell, 1993). These hormones also maintain osmotic pressure, regulates sodium

retention and potassium loss through kidney, water balance and mediate response to stress

(Olanrewaju et al., 2006).

Stressors activate a compound array of responses i.e. endocrine system, nervous

system, immune system etc. and the process is called stress response (Carrasco and Van de

Kar, 2003). It causes a number of behavioral and physiological modifications that get better

animal existence when there are homeostatic challenges (Habib et al., 2001). Behavioral

adaptations for a stress response may involve increased awareness, euphoria, improved

cognition (Chrousos and Gold, 1992) and enhanced analgesia (Charmandari et al., 2005).

Physiological effects due to stress include: increase in cardiac activity, increased respiration,

increased intermediate metabolism and restraining of general vegetative functions such as

feeding, digestive, growth, reproductive and immune response (Sapolsky et al., 2000).

5

2.2 Thermoregulation mechanism in birdsThe birds being homoeothermic can regulate their deep body temperature within

certain limits of ambient temperature (Pickering, 2000). Birds are also endothermic and can

increase the body temperature by creating considerable quantity of heat within their cells and

tissue. Birds use plumage, fat insulation and salt glands to regulate their body temperature

(Sturkie, 1976). When ambient temperature of the birds goes beyond thermo-neutral zone,

chemical reaction speeds up in body, heat is generated and their body temperature rises

(North and Bell, 1990). In order to keep body temperature normal the birds make effort to

dissipate excessive heat via conduction, convection, radiation and evaporation. During humid

and hot weather evaporative cooling (panting) is a main source of heat loss from the body

which speeds up water to evaporate through respiratory tract resulting in removal of heat

from the body (Angiletta et al., 2010). This mechanism is also affected by ambient relative

humidity. Evaporative cooling, fogging and misting of birds are useful even in hot and humid

climates. As there are no sweat glands in birds hence they excrete heat through respiration

(Nillipour and Melog, 1999). Normally, when atmospheric humidity is increased, the humid

environment slows down the evaporation of water from the respiratory tract. In this situation

birds require more energy to lose heat from body which may cause exhaustion, resulting in

heat prostration (Remus, 2001).

Poultry birds physiologically perform most competently within a narrow comfort

zone of 25-37°C (Sturkie, 1976). This range of ambient temperature depends upon body

weight of birds, amount of plumage, shape of feathers along with their amount and

distribution in body, acclimatization and dehydration status of birds. The higher and lower

temperatures are known as upper and lower critical temperatures. A disturbance in ambient

temperature may cause a change in physiological and metabolic parameters such as rectal

temperature, diseases, metabolic disorders and production losses (Ahmad et al., 2007;

Anjum, 2000; Dibartola, 1992; Carlson, 1997).

2.3 Effect of heat stress on various physiological norms of birds2.3.1 Growth

Adverse effects of high ambient temperature (heat stress) on the production

performance of laying pullets and broiler is well known (Anjum, 2000; Yahav, 2000; Star

et al., 2008; Deng et al., 2012; Sohail et al., 2012). It is also well documented that growth

6

rate of birds varies with the fluctuations in environmental temperatures. Impact of different

environmental temperature on weight gain of birds may also vary depending on the age and

breed of birds (Hussan et al., 2011; Diarra and Tabuciri, 2014).

At high ambient temperature and humidity, efficiency of chickens to maintain their

normal body weight is adversely affected. High ambient temperature ranging 34 to 37.8 °C

has shown to reduce body weight gain in chickens (Geraert et al . , 1996) and quails

(Keskin and Durgun, 1997). Similarly adverse effect of heat stress on body weight gain in

birds has also been reported by Njoya and Picard (1994) and Njoya (1995). Macleod and

Hocking (1993) observed a significant loss in the weight of chicken with gradual increase

in ambient temperature. The study of Smith (1992) indicated that layer birds lost significant

body weight during higher ambient temperature. They also reported that the seasonal

variations affected the rate of daily protein requirements.

2.3.2 Feed consumption

Heat stress is known to cause noteworthy decrease in feed consumption in broilers

(Ahmad et al., 2006). At higher ambient temperatures body activities of birds are reduced

and the bird’s needs for energy to keep their body temperature normal are increased which

adversely affect the production performance and feed efficiency of the layers (Anjum, 2000).

Heat stress is reported to depress the feed intake of chickens (Tadtiyanant et al., 1991).

Balnave and Gormen (1993) observed a decrease in feed consumption of layers as the

ambient temperature increased. Roussan et al. (2007) studied the significant adverse effects

of thermal stress on feed consumption of turkeys. Balnave and Muheereza (1997) observed

the effects of high environment temperature (30 °C and 35 °C) on performance of laying

birds. They found significantly decreased feed consumption in birds exposed to higher

temperature.

Mack et al. (2013) observed that the birds kept under high environment temperature

spent more time in drinking, resting, wings elevating and panting whereas spent less time in

feed consuming, moving and walking activities. Feed consumption was reported to be

depressed at high ambient temperature (Geraet et al., 1996; Nakamura et al., 1992; Mikec et

al., 1992; Muiruri and Harrison, 1991a). Similarly, laying hens trained for two meals per day

showed decreased feed intake at higher temperature (Li et al . , 1992).

7

2.3.3 Feed efficiency

It is generally agreed that heat stress adversely affects feed efficiency of poultry birds

(Tanor et al., 1984; Anjum, 2000; Balnave and Gormen, 1993; 2005; Ahmad et al., 2007;

Star et al., 2008; Deng et al., 2012; Yahav, 2000; Sohail et al., 2012). Poor feed efficiency of

the poultry birds may perhaps be ascribed to the decrease in feed consumption of the birds

exposed to heat stress. Under these circumstances a major part of feed consumed is utilized

to fulfill the maintenance requirements of the birds. Moreover, a substantial part of energy is

utilized to dissipate heat from their body. Anjum (2000) reported poor feed conversion

efficiency in White Leghorn layers exposed to heat stress. Similarly, Li et al . (1986)

reported poor efficiency of feed utilization in birds exposed to heat stress. However, Muiruri

and Harrison (1991a) did not find any effect of environmental temperature on feed efficiency

in layers.

2.3.4 Egg production

Negative effects on egg production of layers due to high environmental temperature

conditions have been reported by many scientists (Muiruri and Harrison, 1991a; Bell and

Adams, 1992). Similarly, Melesse, (2011); Mashaly et al. (2004) and Peguri and Coon,

(1991) also observed a significant decrease in egg production in layer birds exposed to

heat stress.

Anjum (2000) reported that production performance of White Leghorn birds reduced

severely when they were exposed to an abrupt increase in their environmental temperature. It

was suggested that functioning of reproductive system might have been depressed under high

environmental temperatures. Findings of Odom et al . (1985) have also revealed a drastic

decrease in egg production of layers subjected to a high environmental temperature.

Peguri and Coon (1991) tested the effects of diverse ambient temperature on the

performance of layers. The birds were reared in rooms, which were constantly maintained at

six environmental temperatures i .e. 16.1, 18.9, 22.2, 25.0, 27.8 and 31°C. The results

revealed significant differences in egg production of the birds during 20 to 36 weeks of age,

which indicated specific effects of warm or cold environments. Deaton et al . (1982)

studied the effect of higher environment temperature i.e. heat stress, on egg production of

layer birds acclimated (adapted) to cyclic versus constant environmental temperature. The

birds were kept (acclimated) to twenty four hours of a linear thermal cycle of either 15.6 °C

8

to 3 °C to 15.6 °C and/or a continuous temperature of 25 °C. Heat stress temperature of 39

°C was maintained with a relative humidity (RH) of 26%. The results showed non-significant

variation in hen day egg production of layers kept (acclimated) to twenty four hours of linear

thermal cycles of either 15.6 °C to 3 °C to 15.6 °C and/or 25 °C. The decline in egg

production percentage was obviously higher for layer birds kept at even temperature of 25 °C

as compared to birds kept at 15.6 °C to 35 °C to 15.6 °C thermal cycle. Results revealed a

higher reduction in production at 39 °C.

In contrast to the findings discussed above, no effect due to heat stress has been

observed on egg production. Roland et al . (1996) checked the dietary manipulation of

calcium and environment temperature on first cycle egg production of commercial Leghorn

layers. Based upon the results of research experiment, warm and cool environment

temperatures did not exert any effect on egg production.

Heat stress has shown a noteworthy effect on the size of eggs in laying birds and has

been observed to decrease with increase in ambient temperature. Heat stress (Hyperthermia)

depressed egg weights in hens (Odom et al . , 1985). However, low ambient temperature has

been reported to increase egg weight of commercial Leghorn pullets housed at 15.6 °C to

23.3 °C (Ronald et al . , 1996).

2.3.5 Egg characteristics

It has been observed that birds exposed to high environmental temperature produced

eggs with poor shell thickness (El-Boushy and Raternick, 1993). Higher environmental

temperature has been shown to decrease shell thickness of eggs because of low feed intake

(Balnave and Muheereza, 1997), which probably caused reduction in calcium intake, an

important element required for shell formation (Karimian et al., 2004). Another probable

reason advocated regarding this aspect is decrease in serum calcium level due to high house

temperature (Hassan et al., 2003). Anjum (2000) has also reported adverse effects of high

environment temperature (heat stress) on egg shell thickness of the eggs produced by White

Leghorn layers and reported that birds exposed to heat stress laid thin shelled eggs.

However, Li et al . (1986) did not observe any effect on egg shell thickness as a

result of high environmental temperature. Slinger (1985) stated that egg shell thickness

decreased when body temperature rose above normal. Deaton et al . (1982) determined the

effects of high environmental temperature (heat stress) on layer birds acclimated (adapted) to

9

cyclic thermal environment versus constant ambient temperature. They conducted two trials

of eight weeks duration; each on 231 Dekalb layers acclimated (adapted) to twenty four

hours linear increase of ambient temperature of either, 15.6 to 35 °C and/or a constant

ambient temperature of 39 °C with a relative humidity of 26%. They observed insignificant

effects on shell thickness of the eggs of layer birds exposed to cyclic vs. (versus) constant

temperatures. Akram et al . (1999) reported that surface wetting of layers by water spraying

can improve thickness of the eggs produced by the birds.

Daniel and Balnave (1981) conducted a study to check the effect of high house

temperature on eggs quality characteristics of White Leghorn x Australorps layers of 32, 70

and 80 weeks old. Hens were exposed to high environmental temperature (35 °C) abruptly

and gradually, between the ranges of 60-80% relative humidity during summer months. No

detrimental effect of heat stress on the Haugh unit score was observed. However, North and

Bell (1990) stated that quality of albumen and yolk deteriorates with the increase in storage

time and temperature.

One of the serious problems in egg production is blood and meat spots in the eggs

which lowers quality as well as age of the eggs (shelf life). Higher protein percentage in diet

is positively correlated with the blood spot occurrence in laying hens (Bearse et al . , 1962).

Nair and Elizabeth (1983) reported that the percentage of eggs with blood spots was 4.5-9.5

in the various seasons and that of meat spots was 9.5-15.5; the differences between seasons

being significant.

Trail (1963) compared blood and meat spot problem in local chicken breeds of

Uganda with five imported ones. Indigenous breed was found to have less (0.7%) eggs

having blood and meat spots. Whereas, imported breeds were found to have more (2.1% to

9.1%) eggs experiencing blood and meat spots. This difference in the rate of eggs meat and

blood spots was attributed to less stress and good adaptation of hot environment rate

experienced by the birds. Akram et al . (1999) found less blood spots in restricted feeding of

hens under normal laying process.

2.4 PhysiologyAmong the factors affecting body temperature, important ones are humidity, heat

loss rate from the body, feed intake, nature of feed, water intake feeding schedules, vitamins,

minerals, acids-base balance, electrolytes, breed, strain, housing conditions and cooling

10

methods. Body temperature of adult chickens also differs with change in the environmental

temperature (Reddy and Dinesh, 2004). When the environmental temperature is equal to the

body temperature, heat cannot be lost from the bird by non-evaporative means. However,

excess heat losts through respiratory lining by evaporation of moisture (Mustaf et al., 2009).

Birds have air sacs for exchange of heat between the body and external environment. These

air sacs contribute to increase the surface area for exchange of gases and evaporative heat

loss (Fedde et al., 1998).

Environmental temperature causes a significant impact on physiological phenomena

in birds. Mack et al. (2013) studied the physiological responses of fowl reared at higher

environmental temperature and reported that birds exposed to higher ambient temperature

(heat stress) conditions spent more time for panting. Heat stress disrupts reproductive

hormones of layer birds secreted by the hypothalamus and ovary (Elnagar et al., 2010). A

decrease in volume of semen fluid, sperm cells concentration and live sperm cells count was

observed in males broiler breeders subjected to high temperature environment (McDaniel et

al., 2004).

Prieto et al. (2010) observed significant less circulating lymphocytes count but an

increase in the number of heterophils count of birds reared under heat stress. Moreover,

broilers subjected to high ambient temperature showed regressed lymphoid organs weights

(Niu et al., 2009), ultimately leading to a poor immune response in laying birds. Birds kept

under heat stress showed depressed systemic humoral immune response, less number of

intraepithelial lymphocytes and Immuno globulin A-secreting cells in their digestive tract

(Niu et al., 2009). The reduction in immune response of the birds was attributed to reduced

antibody response and phagocytic ability of macrophages in birds because of high ambient

temperature.

Teeter and Belay (1996) recommended fasting and starvation as a method of lowering

the rectal temperature in broilers reared under heat stress environment. Sahota et al . (1996)

observed effect of Vitamin C supplemented in diet during summer on rectal temperature of

LSB (Lyallpur Silver Black) and WLH (White Leghorn) layers at the age of 27 weeks when

exposed to heat stress for an experimental period of 12 week. The results of research

exhibited high rectal temperature (42.02 °C) in the control birds which were exposed to heat

stress than those kept under treated groups.

11

2.5 Hematological profile It is generally agreed that environmental temperature, age, season, feed, diurnal

effect, and fasting are related to hematological responses in poultry birds. Thermal

environment has shown a significant effect on white blood cells count (Maxwell et al., 1992).

Maxwell et al. (1992) examined the effects of hot weather environment on differential

leucocyte (DLC) responses in broilers, turkeys and ducks exposed to various degrees of feed

restriction. It was also reported that a mild to moderate heat stress may result in increase in

leucocyte count. An increased white blood cells count in laying birds at high environmental

temperature has also been reported by Anjum (2000).

Packed cell volume value of birds has shown an inverse relationship with high

ambient temperature (Parker et al . , 1982). Hypothermia (8 °C) caused an increase in the

hematocrits, whilst hyperthermia (30 °C) caused a decrease in hematocrit value.

Hyperthermia exhibited a reduction in packed cell volume in chicken and turkeys (Andrade

et al . , 1976; Parker and Boone, 1976). However, supplementation of anti-stress compound

like vitamin C and sodium bicarbonate have been reported to be effective in improving the

packed cell volume in layers exposed to heat stress. Sahota and Gilani (1995) determined the

effects of supplementation Vitamin C in the diet at level of 0, 50 and 100 ppm on the

hematocrit values of LSB and WLH layers kept under high ambient temperature of 31-45 °C

for 12 weeks. It was concluded that influence of ascorbic acid on packed cell volume might

be due to the rise in erythrocyte number. Similarly, Sahota et al .(1993) reported an

improvement in packed cell volume of LSB and WLH breeds of chicken with dietary

ascorbic acid supplementation during heat stress period.

Ekanayake et al. (2004) and Mubarak and Sharkawy, (1999) have reported an

increase in RBCs count in birds fed diets containing sodium bicarbonate. Whereas, Khattak

et al. (2012) reported an increase in WBCs count in the birds kept under heat stress condition

than those fed sodium bicarbonate containing diet at the same temperature. A significant

decrease in packed cell volume has been observed by Oladele et al. (2001) in birds exposed

to high environmental temperature. This increase was attributed to high ambient temperature

which might impaired the synthesis of blood cells in these birds. Heat stress increased blood

glucose and decreased liver glycogen levels in pigeons (Chakraborty and Sadhu, 1983).

Khmilyar (1983) concluded that an increase in ambient temperature above the optimum level

12

caused a considerable drop in serum glucose level in birds. Whereas, opposite to it, Yang et

al . (1992) observed highest blood sugar contents at 23 and 28 °C (223.6 and

221.7mg/100ml) at the exposure of broilers to 12, 18, 23, 28 and 32 °C temperatures.

Sahota and Gilani (1994) observed the effect of heat stress in Lyallpur Silver Black

and White Leghorn layer birds. The birds were exposed to 30 °C and 39 °C, respectively. An

increase in blood glucose in birds of control group was observed as ambient temperature was

raised. They observed a blood glucose level of 196 and 179 mg/dl in 5 weeks old Lyallpur

Silver Black and White Leghorn chicks at 17.5 °C, respectively, which increased to 206 and

195 mg/dl at 27.5 °C at 12 weeks of age.

Mubarak et al. (1999); Al-Hassani et al. (2001) and Ahmad et al. (2005) have

observed an increase in hematocrit values in birds treated with sodium bicarbonate. They

attributed this increase to high house temperature that might impair the synthesis of blood

cells in these birds. Similarly, results of a study executed by Oladele et al. (2001) has

reported a significant decrease in blood packed cell volume in birds exposed to high

environmental temperature when fed sodium bicarbonate supplemented diet.

2.6 Serum metabolites and serum proteinExposure of poultry birds to heat stress has been found to affect their serum

metabolites and the effect may be ameliorated by fortifying their diets with different levels of

sodium bicarbonate (Hassan et al., 2011). An increase in serum urea concentration in layers

kept at higher ambient temperature when compared to those reared under heat combating

systems has been observed by Anjum et al. (2000). Whereas, Yang et al . (1992) has

observed significantly higher serum urea concentration in birds kept at low temperature (12

°C) as compared to those kept at relatively higher ambient temperatures (23 and 28 °C). On

the other hand, Kurtoglu et al. (2007) investigated non-significant (P>0.05) effect due to

dietary inclusion of sodium bicarbonate on uric acid concentration in Brown-Nick layers.

Moreover, Koelkebeck and Odom (1995) did not observed significant effect of higher

ambient temperature on serum uric acid and creatinine concentration in laying birds.

Blood plasma proteins are of two types; albumen and globulin. A

marked decrease in plasma protein concentration has been observed in birds reared under

high ambient temperature (Anjum, 2000) and the decrease was ascribed to heat stress, which

hampered the synthesis of plasma proteins in liver. Higher ambient temperature causes a

13

decrease in serum protein contents in birds (Geraert et al . , 1996). Similarly, findings of

Yang et al . (1992) also revealed higher contents of serum total protein in broilers reared at

12 °C than those reared at 23 °C and 28 °C.

Three studies related to hemodynamic changes in blood protein of fowl kept under

high environmental temperature were conducted by Yahav et al . (1997) in which Cobb

broilers were raised for 4 weeks of age, in battery brooder at 26 °C. Thereafter, for the 1st

trial these birds were acclimated to constant environmental temperatures of 10, 20 and 30 °C

or to diurnal temperatures of low (10 °C) and high (30 °C) up to 8 weeks of age. In their 2nd

trial ambient temperatures were maintained at 15, 25 and 35 °C constantly, whereas in 3rd

trial, non-acclimatized 8 week (W) old birds were exposed to 35 °C. Results of these studies

revealed a decrease in plasma protein concentration at higher temperatures.

There is paucity of information about the response of birds with respect to the serum

uric acid fractions during hot summer conditions. However, Yang et al . (1992) reported

higher serum uric acid level in birds kept at 12 °C than those exposed to 23 °C and 28 °C.

Scientific information regarding the effects of inclusion of NaHCO3 in the diet on serum

alkaline phosphatase in the birds exposed to heat stress is scanty. However, findings of Bogin

et al. (1981) have depicted that broilers subjected to heat stress for two hours showed non-

significant effect on their blood serum alkaline phosphatase level. Findings of Koelkebeck

and Odom (1995) have also revealed non-significant effect of acute heat stress on alkaline

phosphatase enzyme in layers.

2.7 Plasma electrolyte and mineral profileAs ambient temperature exceeds 30ºC, it causes increase in respiration rate of birds,

which might go up to about 10 times more than normal rate (Nillipour and Melog, 1999). It

has been observed that birds started panting along with increase in blood pH during high

ambient temperature. A mild alkalosis (pH 7.55) develops at a temperature of 35 °C, with no

raise in body temperature. Further increase in temperature from 35 °C to 38 °C induces

moderate alkalosis, whereas, severe alkalosis (blood pH 7.65) occurs at higher ambient

temperature i.e. 41 °C (Leeson and Summer, 2001). Findings of Teeter et al. (1985) reported

that pH values higher than 7.25, lowered the performance of poultry birds. This increase in

blood pH can be prevented by reducing panting and rising water intake in birds (Belay and

Teeter, 1993). DEB and blood pH are directly related to each other; at 0 DEB blood pH is

14

always acidic, whilst at 350 DEB it shifts to basic (Ahmad et al., 2009). Acid base balance is

also considered to be important in controlling blood pH in fowls, which in turns improves

efficiency of enzymes and ultimately physiological functions (Patience, 1990).

An increase in blood pH has been shown to depress feed intake which ultimately

reduced the production performance of birds (Yahav et al., 2004). Probably bird performance

is directly associated to blood pH, which shows bird’s physiological and biochemical

condition. Production performance of broiler is higher when blood pH is optimum (7.28),

whereas a reduction in performance is exhibited when pH value is greater than 7.30 or lesser

than 7.20. In verity this narrow range of blood pH establishes the physiology of body enzymes

which is required in expressions of good general health and optimum production of animals

(Lehninger, 1970).

Scientific information regarding the consequence of dietary inclusion of sodium

bicarbonate (NaHCO3) on serum alkaline phosphatase in the birds exposed to heat stress is

scanty. However, findings of Bogin et al. (1981) have shown that broilers subjected to heat

stress for two hours showed non-significant effect on their blood serum alkaline phosphatase

level. Findings of Koelkebeck and Odom (1995) have also revealed acute heat stress had no

effect on alkaline phosphatase enzyme in laying hens. There is paucity of information about

the response of birds with respect to the serum uric acid fractions during hot summer

conditions. However, Yang et al . (1992) reported higher serum uric acid level in birds kept

at 12 °C as compared to those kept at 23 °C and 28 °C. Heat stress decreases the plasma

protein concentration in poultry birds with increase in the environmental temperature

(Anjum, 2000; Geraert et al . , 1996; Yang et al . , 1992). A decrease in blood Na+ level in

broilers kept under heat stress has been observed by Borges et al. (2004) and Takahashi and

Akiba (2002). Plasma potassium (K+) concentration/level was found to be decreased in birds

kept in heat stress (Harper et al., 1977). The decrease in K+ has been reported to be due to

either its increased excretion (Berne and Levy, 1993) or an increase in its uptake by the cells,

or both. Increases in excretion of K+ emerge to preponderate in chronic heat stress, whereas

its increase in uptake by the cells is manifested during acute high temperature environment.

The adverse effects of high environmental temperature/heat stress on blood K+ concentration

has also been found to be similar in different species of birds such as in broilers (Mushtaq et

al., 2005), layers (Ghorbani and Fayazi, 2009) and quails (Keskin and Durgan, 1997).

15

2.8 Serum lipids, hormones and enzymatic profileCholesterol is present in the cells of liver and aortic tissues as well as in the fluids of

animal body. Important factors which affect cholesterol level in the blood are: sex, age,

ration, hyperthermia and starvation. Hevia and Vinsek (1979) reported that fasting increased

blood cholesterol level by mobilizing fat through gluconeogenesis which ultimately increased

blood cholesterol level. High serum cholesterol concentration in birds kept at high

temperature has also been reported by Haazele et al . (1991); Takahashi et al . (1991);

Sahota et al . (1993) and Sahota and Gilani, (1994).

Heat stress affects performance as well as various biochemical processes including

hormone and enzymatic status of birds (Anjum, 2000). Adrenal gland has a central role in the

General Adaption Syndrome (Selye, 1973a) and hormones produced by this gland are

strongly correlated to the heat stress. Thyroid hormone is essential for the development and

normal growth of birds and its secretion rate accurately determines thyroid gland activity

(May et al . , 1974). Thyroid activity had been found to be adversely affected by high

ambient temperature and it was lowest in chickens reared under heat stress, which might

have been due to variations in photoperiod, seasonal reproduction behavior in species and

age of the birds (Bowen and Washburn, 1985).

Hypothalamus and pituitary respond to high environmental temperature by decreasing

the secretion of thyroid gland. Cogburn and Harrison (1980) observed low T3 values in birds

exposed to hot environment. Furthermore, El-Gendy et al . (1995) reported a lower plasma

T3 level in heat stressed broilers at 6 weeks of age. Similar responses on the secretion of T3,

due to heat stress have also been observed by Brigmon et al . (1992) in commercial layers.

Moreover, changes in environmental temperature and level of serum T3 have been found to

be negatively correlated with each other (Brigmon et al . , 1992).

Heat stress is known to influence reproduction performance in pullets (Tojo and

Huston, 1980) and estrogen is a key hormone for efficient reproductive performance in

layers. Kohne and Jones (1976), observed a continuous decrease in plasma levels of estrogen

during heat stress. Progesterone is also a vital hormone, which is related to ovulation process

of birds. Novero et al . (1991) reported that stoppage of progesterone because of malfunction

of ovary due to heat stress, may cause malfunction of positive feedback mechanism to the

hypothalamus resulting in a decrease in secretion of luteinizing hormone in birds.

16

2.9 Immune responseEnvironmental stressors affect immunity and innate resistance of the host directly or

indirectly. Layer birds kept under heat stress experienced a reduction in lymphocytes and a

rise in heterophil concentration (Anjum, 2000). Thaxton and Siegel (1972) reported that high

ambient temperature mediated immune depression. Heat stress caused decrease in total

leucocytes count (Ben Nathan et al., 1977) and thus affected immune response.

Environmental factors other than temperature have also shown to influence immunity in

birds, i.e microbial toxins (Michael et al., 1973), hypoxia (Tengerdy, 1970), non-ionic

radiation (McRee et al., 1977), social connections (Siegel and Latimer, 1975), heavy metals

(Morgan et al., 1975), pesticides (Glick, 1972), nest strain (Thaxton and Briggs, 1972) and

amount of nutrients intake (Tengerdy and Brown, 1977) etc.

The control of antibody mediated immunity at various environmental temperatures

had been studied by many investigators (El-Gendy et al., 1995). Birds exposed to a

temperature of 32.2 °C and higher than 32.2 °C reduced (P<0.05) the agglutinin level in their

blood. A petite exposure (2 or 4 times) to cold and subsequent antigen injection increased the

agglutinin and hemolytic response in birds. Whereas, 30 minutes cold contact for 2 or 4 times

considerably (P<0.05) augmented the IgM antibody concentration and markedly

abridged/decreased the IgG (Suba-Rao and Glick, 1977). El-Gendy et al. (1995) observed

that serum antibodies concentration against Newcastle disease vaccine (NDV) was lesser in

heat stressed broilers as compared to those kept under normal temperatures.

Anti-sheep erythrocyte values have been known to be affected due to high ambient

temperature. Savic et al. (1993) exposed broiler chicks to heat stress at different intervals of

time. The control (maintained at thermo-neutral zone) and heat stressed birds were

vaccinated at 12 days of age using Lasota strain vaccine against Newcastle disease virus. At

the time of vaccination, HI titer of both the groups was < 1:23 and was found to be 1:24 at 18

days post vaccination in control and 1:23 in heat stressed group. Environmental stressors have

been known to affect immunity and innate resistance of the host directly or indirectly

(Robertson, 1998). Bains et al. (1996) investigated a significant (P<0.05) lower immune

response in turkeys due to high ambient temperature Similarly, Tuekam et al. (1994)

observed a negative correlation between serum corticosterone concentration and antibody

titers in heat stressed birds.

17

2.10 DigestibilityHigh ambient temperature may exert a significant influence on digestion and

absorption of nutrients and their metabolism (Macleod, 2004). Increaese in ambient

teperature has been shown to reduce feed consumption in birds to prevent thermogenic effect

(heat increment) associated with nutrient utilization, absorption and assimilation (Koh and

Macleod, 1999). Ambient temperature above 30 °C has shown to cause a decrease in blood

flow towards digestive tract (Wolfenson, 1986). Consequently it may reduce hydrolytic

activities of the respective enzymes in upper part of digestive system (Haiet al.,2000) and

hence may lead to decrease in digestibility of protein.

Brake et al. (1998) reported that at high ambient temperatures (32 °C), arginine

uptake was reduced in birds. Puvadolpirod and Thaxton (2000) observed significantly lower

protein and carbohydrate digestibility in broilers in which ACTH was dispensed to induce

stress. Zuprizal et al. (1993) noted a reduction in the digestibility of rapeseed meal and soya

bean meal protein, at high ambient temperature. However, Virden et al. (2007) investigated

that physiological stress had no effect on amino acid digestibility.

Factors, which may influence digestibility of nutrients include, ambient temperature

(Macleod, 2004; Hai et al., 2000 ; Puvadolpirod and Thaxton, 2000), level of feed intake and

passage rate of digesta (Ravindran et al., 2008; Ahmad et al., 2007), anti-nutritional factors

present in feed ingredients (Hughes and Choct, 1999), age and physiological status of the

bird (Batal and Parsons, 2002; Huang et al., 2007; Garcia et al., 2007) and nutritional

composition of the diet (Leeson and Summer, 2001a).To improve digestibility of feed

ingredients different nutritional manipulations have been used such as adding enzymes in

feed (Selle et al., 2010; Bryden et al.,2009), heat treatment and processing of feed

ingredients and (Friedman, 1996; Amerah et al., 2007) addition of electrolytes in feed/water

during hot weather (Ravindran et al., 2008).

2.11 Significance of electrolytes in combating heat stress Several methods have been proposed to ameliorate high ambient temperature in the

poultry house and to decrease body temperature of birds for successful poultry production

(Daghir, 1995). Dietary electrolyte balance (DEB) in poultry birds plays a significant role

for the better performance. An optimum dietary electrolyte balance is thus required for

efficient performance, proper bone development and good litter quality (Oliveira et al.,

18

2010). However, if DEB is not maintained in normal limits, the performance of the birds is

adversely affected. Maiorka et al. (2004) recommended a dietary DEB of 174mEq/kg for

better feed intake and 163mEq/kg for the best weight gain as compared to 250mEq/kg of

DEB. It has also been observed that a DEB of 175mEq/kg may improve performance in

broilers until 21 days of age (Szabó et al., 2011), but DEB should be 250mEq/kg during the

grower and the finisher phases in broilers. However, role of DEB on performance of layer

birds has not been much studied. Ghasemi et al. (2014) have reported that, under tropical

conditions, using a DEB of 250mEq/Kg achieved a correction of the lay-induced metabolic

acidosis and results in a positive effect on performance of layers.

Electrolytes maintain ionic and water balance in living systems. It is important to note

that requirements of electrolytes cannot be considered individually because there must be an

overall balance among these to achieve homeostasis. Maintaining acid-base balance is a key

strategy to avoid harmful effects of heat stress. Acid base balance is mainly affected by

environmental and nutritional status of the birds. High anions (negative charged ions i.e. Cl-)

may cause acidemia in chickens, whilst high cation contents (positive charged ions i.e. Na+,

K+) in diet cause alkalemia. Both these adverse situations, therefore, may affect performance

of fowls. Dietary electrolyte balance may be calculated using equations developed by various

scientists. However, for the calculation of DEB, it be concerned that oncentration of sodium

(Na+), K+ and chloride (Cl-) should be within adequate range (Mongin, 1981). Physiological

stress, however, tends to cause deviation in electrolyte balance of poultry birds (Yalcin et al.,

2004; Sandercock et al., 2001; 2003; Borges et al., 2003, 2004).

In young birds, Cl− at high levels i.e. 160-240mEq/kg, significantly decreased blood

H+ concentration (Ruiz-Lopez and Austic, 1993). Sodium sulphate has been found to be

relatively more acidic as compared to calcium sulphate and potassium sulphate (Ahmad et

al., 2005). Gorman and Balnave (1994) investigated that heat stress can cause an increased

metabolic need for HCO3− ions. Patience (1990) observed that acid base and electrolytes

balance effect the growth, appetite, thermal stress response and the metabolism of different

nutrients in birds. Borges (2001) viewed that a complete electrolyte equation would be (Na+

+K++Ca+2+ Mg+2) - (Cl− + SO4−2 + 2PO4

−2) and also reported that maximum feed intake was

noted at DEB 264mEq/kg. Rondon et al. (2000) reported 250mEq/kg DEB when Na+ level

were different and 319mEq/kg when K+ level manipulate.

19

Murakami et al. (2001) recommended optimal DEB between 246 and 315mEq/kg for

broilers during starter phase and for the growers between 249 and 257mEq/kg to achieve

efficient performance. Borges et al. (2002) investigated that ideal DEB was found to be

between 246 and 277mEq/kg. Borges et al. (2003a) observed that dietary electrolyte balance

of 240mEq/ kg influenced beneficial effect on body weight and feed efficiency versus dietary

electrolyte balance of 0, 120, and 360mEq/kg, in chicken reared under heat stress. They

concluded that an optimum DEB range of 220 mEq/kg to 240mEq/kg be maintained for

adequate performance. Barbosa et al. (2014) revealed that electrolyte balance may affect

intestinal length, water intake and heart and liver relative weights. They concluded that

electrolyte balance of 120mEq/kg in feed and 30mEq/L in drinking water may cause an

increase in water intake of European quails reared under hot temperature.

Johnson and Karunajeewa (1985) investigated the dietary effect of mineral inclusion

i.e. calcium and available phosphorus and electrolytes i.e. sodium, potassium and chloride on

physiological response of chickens. They did not observe any change in plasma ions

concentration (Ca, inorganic P, Mg, Na, K and Cl) of birds due to treatments.

2.12 Buffering action of sodium bicarbonateFor normal metabolic events such as maintaining the normal structure and functions

of proteins etc., blood pH of poultry birds must be very near to narrow physiological range of

7.35 to 7.45 (Carlson, 1997). Moreover, blood pH is closely related to HCO3– buffering

system, which is the major buffering system for maintaining blood pH and can be described

with the following equation.

CO2 + H2O ⇔ H2CO3⇔ H+ + HCO3–

Blood bicarbonate concentration is primarily under the control of kidneys and to a

less extent, the lungs. Kidneys organize the concentration/level of HCO3– by adjusting its re-

absorption from the renal tubules. Increased breathing under heat stress decreases pCO2

which in turn causes an increase in pH that induces respiratory alkalosis (Belay and Teeter,

1993). The bicarbonate buffer system functions works with double regulatory control of the

lungs and kidneys. In HCO3– buffering system blood pH is represented by the expression

(Berney and Levy, 1993) called Henderson Hasselbalch equation as follows.

pH = 6.1 + log [HCO3–] / 0.03 pCO2

Where;

20

pCO2 = partial pressure of CO2

In normal physiological phenomenon the ratio of HCO3− to pCO2 is 20:1. In an

attempt to keep the body temperature normal, respiration rate of birds increases which lowers

the pCO2, hence increasing the Log term in the Henderson Hasselbalch equation. This causes

an increase in the pH (respiratory alkalosis). In such conditions sodium bicarbonate might be

used as a buffering agent to nullify the problem (Whiting et al., 1991).

2.13 Attempts to improve feed efficiency during hot weatherHigh ambient temperature has shown to cause significant adverse effect on efficiency

of feed utilization in birds. Anjum (2000) reported a poor feed conversion ratio in White

Leghorn layers exposed to heat stress whereas; Muiruri and Harrison (1991) found that

environmental temperature had no effect of on feed efficiency in layers. These types of

contradictory findings observed by various scientists are still causing confusion regarding the

effect of hot weather on feed efficiency of birds reared under different climatic temperatures,

which are direly needed to be addressed.

Several nutritional and managemental manipulations have been used to combat heat

stress. These practices include provision of maximum insulation and improving ventilation of

the poultry house/shed (Nilipour, 2000), use of evaporative cooling systems (Donald, 2000),

thermal conditioning (Yahav, 2000), use of ventilating fans, reducing bird density in the

house (Lott, 1991), provision of adequate cool drinking water, feed withdrawal for certain

periods of time or fasting prior to beginning of heat stress, feeding during cool hours of the

day and acclimation (Yamauchi et al., 1995; Yahav and Hurwitz, 1996).

Al-Zujajy et al . (1978) examined the effect of use of air coolers in poultry house of

birds/broilers kept under the subtropical conditions (Iraq). They reared chicks for 56 days in

two broiler houses in such a manner that one house was provided with two air coolers to

provide cool environment (21.2-29.5 °C), whilst environmental conditions in the other house

were hot and dry (30.1- 39.9 °C). They observed that efficiency of feed utilization was

significantly better in chicks kept under cool housing conditions.

Srivastava et al . (1980) observed the effect of cooling on feed utilization of broiler

chicks in two trials. In 1st trial (hot and dry atmosphere), feed conversion ratios were found

to be 2.34, 2.56, 2.45 and 2.31 in birds kept in house provided either the exhaust fan, a

fogging system plus ceiling fan, an evaporative cooling system or with no cooling system,

21

respectively, at 8 weeks of age. Whereas, corresponding results for trial 2 (hot and humid

atmosphere) were found to be 2.67, 2.78, 2.74 and 2.62, in the respective groups. Wang

(1995) reported that broilers subjected to time limited feeding during cooler hours showed

better feed efficiency than those exposed to heat stress. Moreover, use of air cooler improved

feed conversion efficiency of the birds by 4.3 to 9.7 percent. Therefore, time limit feeding

during cool hours can be a useful practice in poultry birds for combating heat stress. It has

also been recommended that birds should not be fed during hot hour periods (Mahmood et

al., 2005) because it only adds to body heat due to heat increment, which the birds has to

dissipate. Moreover, time limit feeding during the cooler part of the day would increase feed

consumption at a time much suited for its efficient utilization with minimum chances of heat

prostration. Although, this feed practice is not likely to increase the overall daily feed intake,

yet it is expected to improve the feed efficiency and production performance of birds.

Johnson and Karunajeewa (1985) investigated the effect supplementation of calcium

and available phosphorus (minerals) and the sodium (Na), potassium (K) and chloride

(electrolytes) in feed on growth and physiological response of broiler. Results exhibited that

dietary electrolyte balance (DEB) does not define the growth rate of chicken. Lower (<

180mEq/kg) or higher (> 300mEq/kg) electrolyte balance (DEB) in the feed depressed live

weight of the birds at the age of 42 days. Growth rate of chicken fed diets with DEB (dietary

electrolyte balance) higher than 300mEq/kg depends on the type of cat-ions (Na or K). The

range of Na: K ratio for proper growth was found to be 0.5-1.8. Barton (1998) found that

dissolved bicarbonate in drinking water improved feed conversion in turkeys.

2.14 Effect of dietary inclusion of NaHCO3 on various physiological

norms of birdsIn the following text, a brief review regarding the effects of including sodium

bicarbonate in the diets or/and in drinking water of different species of poultry on various

parameters related to their production performance have been presented.

2.14.1 Growth

High ambient temperature is generally known to reduce the average body weight in

layers as compared to those kept under a cool environment (Anjum, 2000). To improve

weight gain under different heat stress conditions, different feeding manipulations have been

practiced (see section 2.8), which have shown positive effects. However, use of feed

22

manipulation alone is not enough to mitigate adverse/devastating effect of heat stress on

body weight in birds completely (Spinu and Degen, 1993; Zakia et al . , 1995). Under such

conditions, dietary inclusion of sodium bicarbonate has shown significant (P<0.05) effect on

growth rate and efficiency of feed efficiency in birds exposed to heat stress (Ramezani et al.,

2011).

Inter-relationship between levels of NaCl, NaHCO3, phosphorus and calcium in the

diet of layers was studied by Junqueira et al. (1984). Three experiments were performed on

Hy-line layer birds kept in individual wire cages. The layers were given a diet based on

yellow maize and soybean meal with or without addition of sodium chloride, calcium

phosphate or sodium bicarbonate. Sodium chloride did not affect body weight gain of the

birds. However, addition of NaHCO3 in the diets of the birds produced a noteworthy increase

(P<0.05) on body weight of layer birds. Moreover, the diets high in sodium and low in

chloride caused increase in mortality of the hens. Supplementation of diet with 0.5% sodium

bicarbonate resulted in 9% increase in body weight gain in broilers suffering with chronic

hyperthermia (Teeter et al., 1985). Barton (1998) investigated the impact of water quality on

the performance of turkeys and found that dissolved bicarbonates in water were positively

correlated with the weight of broilers.

Harms (1982) replaced sodium chloride with sodium bicarbonate in the diet of

turkeys during starting phase. He observed maximum body weight and feed utilization when

NaHCO3 was added to the diet which already had 0.056% Na+ provided by NaCl. It was

concluded that mutual replacement of NaCl with NaHCO3 for the adjustment of sodium and

chloride might have resulted in better growth rate.

Bonsembiante et al . (1988) performed a trial to test the effect of supplementation of

sodium bicarbonate and ammonium chloride on the performance of chicken during hot

weather. Addition of sodium bicarbonate in broiler feed improved the growth rate and

enhanced efficiency of feed utilization of birds than birds of control group. However, chicks

receiving 0.5% sodium bicarbonate plus 1% ammonium chloride did not perform better than

that of controls. Effect of adding NaHCO3 (0.5%) and KCl (0.5%) in water was investigated

on the production performance and carcass parameters of chicken reared in thermo-neutral or

cyclic heat stress (Whiting et al . , 1991b). The results revealed no appreciable improvement

in weight gain of broilers due to the supplementations when compared to those offered water

23

without any supplementation.

Influence of dietary supplementation sodium bicarbonate, ascorbic acid and acetyl

salicylic acid was observed on broiler’s performance kept under the hot weather conditions

(Puron et al., 1994). For this 3 research trials were conducted, in experiment 1 and 2, sodium

bicarbonate was added at the level of 0.5% and 0.6% in the diets of broilers. Whereas, in

experiment 3, the experimental rations for broiler were prepared by dietary inclusion of 0.6%

bicarbonate, 200 ppm ascorbic acid and 250 ppm acetyl salicylic acid. However, control diet

was kept without any supplementation. These diets, when fed to the experimental groups did

not exhibit any significant difference on performance of the broilers, when compared

between control and treatment groups.

Teeter et al., (1985) observed meaningful (P<0.05) increase in body weight gain of

chicken fed diet containing NH4CI (1%) and sodium bicarbonate (0.5%) during hot weather

conditions. Fox et al . (1997) carried out a research experiment to explore the effects of

dietary addition of sodium bicarbonate (NaHCO3) on production performance and intestinal

pH of chicks infested with Eimeria acervulina (Coccidia) . Outcomes of the trial proved

that dietary supplementation of sodium bicarbonate significantly (P<0.05) increased the

growth and efficiency of feed utilization of the birds. Moreover, dietary inclusion of sodium

bicarbonate significantly increased body weight gain in birds infested with coccidiosis.

Hooge et al. (2000) evaluated the influence of dietary NaHCO3 (0 or 0.25%), monensin (0 or

99 ppm), or coccidial inoculation (0 or 2 Eimeria species), singly and in various

combinations on weight gain in broilers. Dietary sodium bicarbonate significantly (P<0.05)

improved the body weight and feed efficiency of the broiler than those fed diets without the

addition of sodium bicarbonate (control).

Kidd et al. (2003) evaluated the impact of dietary addition of NaHCO3 in high and

modest (moderate) temperature conditions on performance of broilers. Results revealed that

dietary treatments had minimum effect on body weight gain and breast meat synthesis of the

birds. Effects of supplementation of Vitamin C (62.5 mg/liter water), acetylsalicylic acid

(62.5 mg/liter water), NaHCO3 (75 mg/ liter water), and KCl (125 mg/L) in water was tested

on broilers exposed to heat stress (Roussan et al., 2007). However, better growth rate was

noted in the birds fed sodium bicarbonate.

Ramezani et al. (2011) conducted an experiment using 216 male Ross chicken to

24

assess the effect of organic selenium and supplementation of NaHCO3 on blood parameters,

body weight gain and carcass weight of broilers kept in heat stress condition. Results of the

study depicted that sodium bicarbonate significantly increased growth performance, along

with their breast and thigh weight. However, selenium supplementation significantly

(P<0.05) reduced the abdominal fat contents and liver weight of the birds.

Effect of dietary inclusion of betaine, vitamin C, vitamin E and sodium bicarbonate

was studied on the production performance of chicken during heat stress (Khattack et al.,

2012). Results showed a significant role of sodium bicarbonate in improving the performance

of broilers reared under heat stress. Supplementation of betaine and sodium bicarbonate were

also found to be useful in protection against heat stress related adverse effects. Balnave and

Gorman (1993) reported an improved weight gain in broilers fed diet added with sodium

bicarbonate. Genedi (2000) also reported that adding anti-stressors like NaHCO3 in to

drinking water (DL) of White Leghorn (WLH) and Matrouh layers increased their weight

gain under heat stressed condition.

In contrast to the findings of various studies mentioned above, Junqueira et al. (2003)

and Osman et al. (2015) found no effect (P>0.05) on growth rate of chicken due to dietary

supplementation of different levels of sodium bicarbonate. Similarly, findings of Hayat et al.

(1999) Wideman et al. (2003) and Saedi and KhajaliI (2010) did not find any effect (P>0.05)

of supplementation of NaHCO3 on body weight gain of birds. Whereas, Squires and Julian,

(2001) and Peng et al. (2013) reported a significant decrease in body weight gain in broilers

fed NaHCO3. Similarly, Wideman et al. (2003) observed 7% reduction in growth rate in

broilers fed diet containing 1% NaHCO3.

2.14.2 Feed consumption

Several nutritional manipulations have been recommended to minimize depression in

feed intake due to heat stress (Baghel and Pradhan, 1989; Fethiere et al., 1994; Anjum, 2000;

Khattak et al., 2012) in species of fowls. Different levels of electrolytes are reported to be

beneficial for broiler birds kept under heat stress conditions (Mushtaq et al., 2007).

Managing acid base balance by dietary supplementation of various electrolytes salts such as

sodium bicarbonate, KCl, ammonium chloride (NH4Cl) and calcium chloride is one of the

preeminent methods used to combat hot weather stress (Teeter et al., 1985; Borges, 1997;

Borges et al., 2003a, b; Ahmad et al., 2005).

25

Dietary addition of NaHCO3 in laying hens has shown a significant improvement in

their performance during heat stress (Ghorbani and Fayazi 2009). Teeter et al. (1986) and

Marinez et al. (1993) also found that dietary supplementation of sodium bicarbonate in

broilers reared under heat stress conditions has shown a significant improvement in their feed

consumption.

Bonsembiante and Chiericato (1990) offered feed to meat type turkeys without or

with 0.50% sodium bicarbonate and observed an average final weight of 8817 and 9046g

with an average daily gain of 63.4 and 65.9g, respectively. However, feed intake or feed

efficiency and levels of electrolytes among treatment groups were non-significantly affected

by the dietary treatments. Fethiere et al. (1994) executed an experiment to check efficacy of

dietary addition of sodium present in sodium zeoilte-A in broilers. Corn soybean meal basal

rations (iso-caloric and iso-nitrogenous) were formulated containing sodium from either

sodium zeolite-A (SZA) or sodium chloride. The addition of sodium in the diet resulted in an

improvement in feed consumption.

Junqueira et al. (1984) examined the effect of NaCl, sodium bicarbonate, calcium and

phosphorus supplemented in diet on performance of Hy-line layers reared in cages

maintained in open house. The layers when fed diet containing sodium bicarbonate at the

level of1 or 6 % showed significantly better performance as compared to those fed sodium

chloride free diets. However, performance of the hens remained unaffected due to the

addition of sodium chloride at the rate of 0.37 and 1.11 % in their diets.

Branton et al . (1986) conducted an experiment in which broilers were exposed to

acute heat stress and were provided water containing sodium bicarbonate and ammonium

chloride. The results indicated 20% increase in water intake by the birds using water

containing sodium chloride (6.25 g/L). However, both water and feed intakes were limited

when consumed sodium chloride at the rate of 31g/L of water. Cooke and Raine (1986) used

bicarbonate as sodium source rather than chloride in the diets of broilers. The results showed

that diets containing 0.13% and 0.19% sodium content decreased water intake (0.2L/bird)

and also exhibited improvement in the quality of litter by about 20%.

Darmon et al . (1986) investigated the effect of NaHCO3 and NaCl supplementation

in broilers on their water intake. They observed increased water consumption due to

increasing level of sodium chloride. Based upon the results it was concluded that sodium

26

from sodium bicarbonate might have been used in a way similar to that of from NaCl.

Balnave and Gorman (1993) studied the efficacy of supplementation of sodium bicarbonate

in broiler birds and observed increase in feed consumption and production performance

(growth rate) of the birds. The response was credited to the bicarbonate ions.

Effect of dietary NaHCO3 supplementation on feed consumption during late laying

period was studied by Yoruk et al. (2004). Hisex Brown layers were randomly divided in to

four groups to obtain one of the four diets (0, or 0.1, or 0.2, or 0.4% level of NaHCO 3) for a

period of 75 days. The results revealed that feed consumption was higher (P<0.05) in hens

fed diets supplemented with NaHCO3. It was also observed that there was a gradual raise in

feed intake of birds with raise in the levels of NaHCO3. Balnave and Gormen (1993) brought

it to fact that feed intake of chicken kept under heat stress can be increased by adding their

diet or water with NaHCO3. The significant beneficial effect might be due to the HCO3 ion or

raising the water intake. However, measuring dietary or retained values for dietary electrolyte

balance (DEB) were found to be inadequate for calculating the feed conversion (FCR) of

chickens.

Puron et al. (1997a) and McDowell (1992) have reported an increase in feed

consumption of birds due to more sodium ions concentration in rations containing sodium

bicarbonate. Ahmad et al. (2006) and Balnave and Gorman (1993) observed a significant

increase in feed consumption in broilers fed diet added with sodium bicarbonate during high

ambient temperature. Gongruttananun and Ratana et al. (2004) determined the response of

dietary addition of NaHCO3 on feed consumption of laying birds. Three experimental diets

were formulated as; control layer diet, diet with 1% inclusion of NaHCO3 and diet with 1.5%

inclusion of NaHCO3.The findings of the study did not reveal any significant effect on feed

consumption in the birds.

Mandal et al. (2010) investigated the effect of adding Vitamin C (ascorbic acid) and

sodium bicarbonate in layer diets, on egg production of the layers exposed to heat stress.

Daily feed consumption was lowered (P<0.02) in ascorbic acid supplemented group in

comparison to those of non-supplemented group (control). Khattak et al. (2012) have also

observed a reduction in feed intake in broilers fed sodium bicarbonate supplemented diets

during heat stress conditions.

However, in contrast to the results of various studies discussed above, findings of

27

research trial conducted by Whiting et al. (1991) did not (P>0.05) reveal any beneficial result

of dietary addition of NaHCO3 production performance of poultry birds reared under hot

weather conditions. Similarly, Puron et al. (1994) found that supplementation of NaHCO3 did

not affect the performance of chicken. They attributed these findings to the lack of response

of NaHCO3, which might be due to climatic conditions and environment of the experiment

which might produced only mild heat stress. Bonsembiante and Chiericato (1990) observed

no (P>0.05) distinction in feed consumption of birds using rations with or without

supplemented sodium bicarbonate. Similarly, Senkoylu et al. (2005) who tested the effects of

inclusion of various levels of NaCl, NaHCO3 and K2CO3 in poultry diets, on feed

consumption of layers during peak production did not observe any effect due to the dietary

inclusion of these compounds on feed intake of the layers. Findings of Balnave and

Muheereza (1997) and Waldroup et al. (2005) have also shown that feed intake of broilers

fed diets with or without sodium bicarbonate remained unaffected. Fuentes et al. (1997)

found no effect of adding different levels of sodium bicarbonate (0.6, 1.2, 1.8 and 2.4%) in

diets on feed consumption of guinea fowls reared at high ambient temperature.

2.14.3 Feed efficiency

Different levels of sodium bicarbonate have been proved beneficial for broilers reared

under different hot climatic conditions but with varying results. Differences in the levels of

sodium bicarbonate used, climatic conditions, species of birds and differences in precision

and accuracy in measurements among different experiments might be a cause of

disagreements among different studies reported. However, dietary supplementation of

sodium bicarbonate in broilers might improve their body weight gain and decrease losses

caused by heat stress (Mirsalimi and Julian, 1993).

It is generally agreed that beneficial effects of NaHCO3 can be only achieved when its

recommended/optimum levels are administered in the diet. Moreover, use of excessive levels

of this chemical compound in the diet has been reported as nephro-toxic and toxic in White

Leghorn layers (Davison and Wideman, 1992), which indicates that there is a dire need to

define the proper dietary level of sodium bicarbonate having proper dietary electrolyte

balance (DEB) for optimum performance of laying hens kept under environmental conditions

of Pakistan. In the following paragraphs a concise review regarding scientific findings has

been reported about the effects of different levels of NaHCO3 used in poultry birds.

28

Drinah et al . (1990) used sodium bicarbonate (0.25%, 0.5%, and 0.75%) to detoxify

the tannins present in the sorghum fed to starting broiler chicks. Findings of the study

revealed that a dietary addition of 0.25% sodium bicarbonate may overcome anti-nutrient

effects of tannins present in sorghums ultimately can cause better body weight and efficiency

of feed utilization. Findings of Keskin and Durgan (1997) have also reported an improved

FCR in quails fed diet supplemented with NaHCO3 (1%), KCl (1%), CaCl2 (1%), NH4Cl (1%)

and CaSO4 (1%).

Hooge et al. (1999) tested incorporation of sodium bicarbonate in the diet of broilers

at level of 0 to 0.4%. Dietary inclusion of NaHCO3 at level of 0.2 to 0.4% acquiesced

significant (P<0.05) increase in growth, feed efficiency/utilization and survivability of the

birds, whereas the level of 0.1% sodium bicarbonate did not exert such beneficial effects. A

range of 0.2 to 0.3% of sodium bicarbonate in the diet of broilers from day-old to market age,

was recommended.

Atlan et al. (2000) investigated the outcome of dietary addition of NaHCO3 on feed

consumption, feed efficiency and rectal temperatures in two strains of layers (brown and

white) reared during summer. Findings of the study indicated that dietary inclusion of sodium

bicarbonate at a level of 0.3% significantly improved feed conversion in both strains of the

layers. Rectal temperatures recorded during the hottest hours of the day were found to be

significantly higher in brown layers than in white layers. However, rectal temperature of the

birds remained unaffected due to NaHCO3 supplementation in their diets. Yoruk et al. (2004)

find out the dietetic effect of NaHCO3 on feed efficiency in layers during their late laying

period. Hisex Brown layers were indiscriminately allocated to obtain one of four treatments

diets having either, 0 or 0.1or 0.2, or 0.4% sodium bicarbonate for 75 days. The results of the

study depicted improved feed conversion ratios in the layers with increase in the level of

NaHCO3 in their diets.

Naseem et al. (2005) surveyed the outcomes of dietary supplementation of KCl and

NaHCO3 on body weight and feed conversion ratio (FCR) in the birds exposed to heat stress.

Hyperthermia in these birds led to a significant (P<0.05) reduce in their weight gain and poor

FCR. Balnave and Gormen (1993) reported that feed consumption and weight gain of

chicken reared under high temperature can be enhanced by adding their diet or drinking

water with NaHCO3. This might be due to an increase in HCO3 ions and may also be linked

29

with boost in water intake. Senkoylu et al. (2005) accounted no (P>0.05) effect of dietary

inclusion of varying levels of NaCl, NaHCO3 and K2CO3 on FCR (calculated on the basis of

gram of feed/grams of egg produced by layers) during peak production. Fuentes et al. (1998)

also observed a no effect (P>0.05) of sodium bicarbonate on FCR values calculated on the

basis of per kg egg mass produced in guinea fowls raised under high ambient temperatures.

2.14.4 Egg production

In order to improve egg production during summer, scientists have used diets

supplemented with varying levels of NaHCO3 and depicted different results. A percentage of

1.5% NaHCO3 was reported to be ineffective in laying hens (Grizzle et al., 1992), whereas

dietary addition of 0.3% to 2% increased the shell quality (Davison and Wideman, 1992) of

the eggs produced by the birds using these diets. Moreover, Davison and Wideman (1992)

noted that dietary supplementation of 3% NaHCO3 led to eggs without shells. In adding

together inclusion of macro minerals, micro minerals, salts and vitamin D, adjustment of

acid-base balance (DEB) by supplementation sodium bicarbonate (Grizzle et al., 1992;

Davison and Wideman, 1992) are existing emergent to get better layers performance.

Harms et al . (1995) conducted a study to access dietary sodium requirements of

Arbor Acres hens. In this study, NaCl was mixed to a corn-soybean diet to endow intakes of

either, 35, 65, 95, 125 or 150mg per hen daily. Egg production was markedly decreased

during 4th and 7th week of the experimental period, when the hens had an intake of 35mg

sodium/day and 65mg sodium/day, respectively. Daily requirements for maximum

production were found to be 113.8 and 96mg and the requisite for egg mass was 105mg and

100mg/day, during the respective production periods.

Dai et al. (2009) observed an improved egg production of the layers fed diet

containing sodium bicarbonate supplemented diet. Ghorbani and Fayazi (2009) studied the

consequence of dietary addition of NaHCO3 on egg production of layers kept under persistent

heat stress and found significant increase in egg production due to dietary inclusion of

sodium bicarbonate. Findings of Hassan et al. (2011) also discovered that addition of sodium

bicarbonate at a level of 0.25 and 0.50% in poultry diet may improve egg production of

layers exposed to heat stress. An increase in egg production has also been observed by

Balnave and Muheereza (1997) because of dietary addition of NaHCO3 (1%). Similarly,

Yoruk et al. (2004) scrutinized the effect of different levels of NaHCO3 (0.1%-0.4%) on

30

production performance of layers. Inclusion of different levels of sodium bicarbonate (0.1%-

0.4%) in laying hens diet improved their egg production significantly.

Makled and Charles (1987) compared the effects of adding calcium source, sodium

bicarbonate in the diets and photoperiod on 240 Hy-line Leghorn hens of 25 weeks age.

Photoperiod and dietary addition of these substances showed a noticeable effect on

production performance of the layers. Gongruttananun and Ratana (2005) also found non-

significant effect of adding varying level of sodium bicarbonate on production performance

in laying birds. They fed diets added with 1.0-1.5% sodium bicarbonate to Thai native hens

and observed that differences in egg production between treated and non-treated birds were

non-significant, even due to the different levels of sodium bicarbonate used. Similar results

regarding egg production are also observed by Grizzle et al. (1992); Gongruttananun et al.

(1999) and Waldroup et al. (2005). Egg production has also been found to remain unaffected

due to the inclusion of different levels of NaCl, NaHCO3 and K2CO3 in the diets of layers

(Senkoylu et al., 2005), during their peak production period. Mandal et al. (2010)

investigated the effect of adding ascorbic acid and sodium bicarbonate in layer diets on egg

production under heat stress. Two additives i.e. ascorbic acid (300mg/kg) and sodium

bicarbonate (1%) in diets were used but egg production was not affected by the dietary

treatments.

2.14.5 Egg weight/size

In order to improve egg weights during summer various scientists have used diets

supplemented with varying levels of sodium bicarbonate and have observed varying results.

Makled and Charles (1987) reported significantly (P<0.05) better weight of eggs in layers,

when fed diet containing 0.5% NaHCO3. Balnave and Muheereza (1997) fed either basal

diet or treated diet containing either 1% sodium bicarbonate or treated diet containing 0.05%

Zinc methionine or treated diet containing 0.04% vitamin C, to layers kept under high

ambient temperature and found significantly higher weight of eggs in the birds fed diet

supplemented with 1% sodium bicarbonate. Similar effects of sodium bicarbonate on egg

weight have also been reported by Ghorbani and Fayazi (2009) in layers. They studied the

significance of feeding various level of sodium bicarbonate (0.5%-1.5%) and rearing systems

on egg weight of layers kept under chronic heat stress and found that dietary levels of sodium

bicarbonate (0.5%-1.5%) in laying hens diet improved their egg weight whereas hens fed diet

31

supplemented with 1.5% sodium bicarbonate produced heavier eggs than its counterparts.

Yoruk et al. (2004) conduct a trial to find out the effects of dietary NaHCO3

supplementation on egg weight during late laying period of hens. Hisex Brown layers were

randomly distributed to get one of four diets having 0, or 0.1, or 0.2 or 0.4% NaHCO 3,for 75

days. Egg weight was more for hens fed diets having NaHCO3. Raising the level of NaHCO3

caused a linear raise in egg weight of the hens. Yin et al. (2001) observed that adding 0.3%

sodium bicarbonate as water supplement caused better egg laying rate and egg mass in laying

hens.

In contrast to the findings discussed above, no effect due to dietary addition of

NaHCO3 has been observed on egg weight of the poultry birds. Ernst et al. (1975) fed diets to

commercial Leghorn layers, with or without addition of sodium bicarbonate to improve their

egg weight. They adjusted Na+ level to 0.23% and Cl- level to 0.18% of the diets. However,

they did not discover any disparity in egg weight of layers due to these treatments. Senkoylu

et al. (2005) tested the effect of inclusion of diverse levels of NaCl, NaHCO3 and K2CO3 in

diet, on egg weight in layers during their peak production period and found no effect due to

dietary inclusion of these compounds on egg weight of the layers. Comparable results have

also been observed by Gongruttananun and Ratana (2005) who found no effect (P>0.05) on

egg weight of Thai native hens due to dietary addition of varying levels of NaHCO3 (1.0-

1.5%). Stevenson (2006) also observed that sodium bicarbonate containing diets did not exert

any effect on egg weight in layers. Similarly, egg weight of layers using diet containing 1%

sodium bicarbonate remained unaffected (Waldroup et al., 2005).

Higher egg mass production of the eggs produced by the birds using sodium

bicarbonate treated rations was reported by Dai et al. (2009). Yoruk et al. (2004) found

beneficial result of dietary addition of NaHCO3 on egg mass of turkeys. Balnave and

Muheereza (1997) fed either basal diet or diets containing 1% sodium bicarbonate, 0.05%

Zinc methionine or 0.04% Vitamin C to layers kept under high ambient temperature. Results

showed a significant improvement in egg mass produced by the bird fed diet supplemented

with 1% sodium bicarbonate. However, in contrast to the findings of various studies

mentioned above, Senkoylu et al. (2005) reported no effect (P>0.05) on egg mass production

due to inclusion of varying levels of NaHCO3 in the diet of layers during their peak

production period.

32

2.14.6 Egg shell thickness

Poor egg shell quality is the main trouble in birds during the summer/high ambient

temperature. Environmental temperature, seasonal changes, bicarbonates, calcium, ascorbic

acid, light intensity and duration, nutrition, genetics and age of hens are possibly the

important factors, which are related to egg shell quality of the birds. Wideman and Buss

(1985) studied the bicarbonate, CO2 and pH values in layers to observe egg shell quality.

Results of the study showed that the hens producing thin egg shell suffered with metabolic

acidosis during the first six hours of oviposition also had markedly lower blood pH and

bicarbonate contents than those producing eggs with thick shell. Ergun (1992) reported a

decline in blood pH and bicarbonate level at 22nd hour of laying cycle.

Austle and Keshavarz (1988) conducted two experiments to determine the results of

supplementation of Na and Cl on egg shell quality of Leghorn layers. In the first experiment,

they observed that presence of chloride in the diets reduced feed intake, egg shell thickness

and strength in the hens receiving 2 % calcium in their diet. In the second experiment,

influence of the dietary addition of sodium and Cl on egg shells of the hens was determined.

Increase in the concentration of sodium contents relative to those of chlorides, reduced feed

intake of the birds but significantly improved egg shell strength and thickness, bicarbonate

concentration, blood pH and base excess.

Balnave and Muheereza (1997) executed two trials to explore the effects of sodium

bicarbonate during heat stress. Layer birds were reared at higher house temperatures (30 and

35 °C) and fed sodium bicarbonate supplemented diet, at the end of lay. Shell thickness was

improved due to dietary addition of sodium bicarbonate. A significant improvement in egg

shell thickness in layers fed NaHCO3 added diet was observed by Hayat et al. (1999). Similar

beneficial effect of including NaHCO3 in poultry diets have also been reported on egg shell

thickness by Davison and Wideman (1992). An improvement in eggshell thickness was also

observed in hens fed diets added with 0.5% NaHCO3 (Makled and Charles, 1987).

Different levels of dietary inclusion of NaHCO3 have shown considerable effects on

egg shell quality of layers. Atlan et al. (2000) investigated the effect of dietary

supplementation of NaHCO3 on egg quality parameters during summer, in two strains of

layers (brown and white). Results of the trial showed that addition of NaHCO3 at the level of

0.3% significantly improved egg shell quality in both strains. Gongruttananun and Ratana et

33

al. (2004) observed the effect of different levels of dietary NaHCO3 (1 to 1.5%)

supplementation on shell thickness of eggs produced by Thai native hens. Three

experimental diets were formulated as; control layer diet, diet with 1% inclusion of NaHCO3

and diet with 1.5% inclusion of NaHCO3.Layers using diets added with NaHCO3 produced

eggs with higher shell thickness (P<0.05) than those of control group. It was concluded that

at moderate temperatures, eggshell quality of the hens could be improved by dietary

supplementation with 1.5% NaHCO3.

Kaya et al. (2004) tested the effect of dietary inclusion of NaHCO3 on blood gases,

blood pH and egg shell thickness in geese birds. Fourteen geese of two year age were

distributed into 2 groups as; 1) control, 2) 0.5% NaHCO3 supplemented group. Unlike the

results of various studies discussed above, in this dietary addition of NaHCO3 revealed no

significant improvement in egg shell thickness of the eggs produced by the birds. Yoruk et

al. (2004) have also observed non-significant effect on egg shell thickness of laying hens due

to dietary addition of different levels of NaHCO3 (0.1%, o.2%, 0.4%).

A significant decrease in shell thickness of eggs laid by the laying hens exposed to

heat stress and fed diets containing different levels of NaHCO3 (0, 0.5, 1 and 1.5%) was

observed by Ghorbani and Fayazi (2009). Findings of Mandal et al. (2010) who investigated

the effect of adding ascorbic acid and sodium bicarbonate in layer diets, on shell thickness of

eggs produced by laying birds kept under heat stress, have revealed that egg shell thickness

was higher in layers maintained at low energy level or ascorbic acid group. EL-Sheikh and

Salama (2010) investigated that adding 100 mg sodium bicarbonate in drinking water did not

significantly affect egg shell weight of layers but albumen weight and yolk weight were

significantly affected as compared to the control group during summer season. Whereas, Dai

and Bessei (2007) found that egg shell thickness was higher (P>0.05) and eggs having shell

deformities were lower in the birds treated with potassium chloride supplementation through

drinking water.

2.14.7 Specific gravity of intact egg

The effect of different dietary levels of sodium bicarbonate (0.1%-0.4%) on specific

gravity of eggs in layers was studied by Yoruk et al. (2004). The results revealed that

inclusion of different levels of sodium bicarbonate in laying hens diet markedly increased

specific gravity of the eggs produced by the hens. It was also reported that higher levels of

34

sodium bicarbonate (0.2%-0.4%) in the diet reduced the specific gravity of eggs laid by the

birds. However, Grizzle et al. (1992) research results do not support the opinion of such

effect on specific gravity of eggs due to dietary supplementation of 1% sodium bicarbonate

in layers. Similarly, Makled and Charles (1987) found no change in specific gravity of eggs

laid by hens fed diet supplemented with 0.5% sodium bicarbonate during their peak

production period. Connor and Arnold(2004) fed pullets (Australorp X White Leghorn) with

diets containing sodium bicarbonate and did not observe any effect due to dietary addition of

sodium bicarbonate on specific gravity of the eggs.

2.14.8 Various egg quality parameters

There are many factors, which may influence egg quality characteristics in birds but

heat stress is the most important factor which has shown pronounced effect on most of the

important egg quality parameters (Anjum, 2000). Although, many scientists have used

sodium bicarbonate in the diet of layers in order to improve egg quality characteristics during

hot weather, yet there is some difference of opinion among the investigators. In the following

paragraphs, scientific information concerning the effects of adding sodium bicarbonate in the

diets of layers, on variables like, Haugh unit values, albumen index and yolk index are

reviewed.

Change in Haugh unit score is affected by many factors and heat stress is one of them

(Anjum, 2000). As the laying birds turns old, Haugh unit score of their eggs decrease in

value (Coutts et al., 1990). In such conditions inclusion of sodium bicarbonate in diet may

prove beneficial. An improved absorption of mono-saccharides and amino acid (Johnson and

Karunajeewa, 1985; Ravindran et al., 2008) due to sodium bicarbonate may cause an

increase in protein content of eggs hence Haugh unit and albumen quality of eggs produced

by these birds.

Hussan et al. (2011) observed that sodium bicarbonate when added in layer diets at

the rate of 0.25%, 0.50% and KCl at 0.2%, 0.3%, alone or in amalgamation exhibited

beneficial effect on egg quality characteristics as compared to those provided diets without

supplementation. Additives (NaHCO3 and KCl) and their arrangement/combination

improved (P>0.05) the various traits of egg characteristics in Golden Montazah hens reared

in hot weather conditions. Yoruk et al. (2004) studied the effect of different levels of sodium

bicarbonate (0.1%-0.4%) on Haugh unit score of layers during late laying period. They found

35

an increase in albumen height of eggs produced by the birds fed sodium bicarbonate added diets.

In contrast to the findings of various studies mentioned above, Haugh unit score of

the eggs produced by the layers kept under chronic heat stress was found to be not affected

due to dietary inclusion of sodium bicarbonate and rearing system. Moreover, neither

addition of sodium bicarbonate nor its various levels (0.5%-1.5%) exhibited any effect on

Haugh unit score in layers (Ghorbani and Fayazi, 2009). Findings of Gongruttananum and

Ratana (2005) have also revealed that different dietary levels of sodium bicarbonate (1%-

1.5%) did not improve Haugh unit score of the egg laid by hens.

Information regarding the effect of sodium bicarbonate on yolk index of eggs is

scanty. Yolk index of eggs was found to be significantly improved by dietary inclusion of

sodium bicarbonate at 0.1% level (Yoruk et al., 2004) when different levels of this chemical

compound (0.1%-0.4%) were used in the ration of layers during late laying period. In

contrast to the findings of the previous study, Ghorbani and Fayazi (2009) observed that

neither dietary inclusion of sodium bicarbonate nor its levels (0.5%-1.5%) exhibited any

effect on yolk index of the eggs produced by the hens. Similarly, Gongruttananum and

Ratana (2005) have also reported that dietary levels of sodium bicarbonate (1%-1.5%) in

laying hens did not improve yolk quality of the eggs produced by the birds.

Presence of sodium is necessary for absorption/uptake of amino acids and

carbohydrates from gastrointestinal tract (Leeson and Summers, 2001a), thus deficiency of

this essential element may render the digested protein and carbohydrates unavailable to the

body. Therefore, addition of this beneficial chemical compound in the ration of poultry birds

may tend to affect Haugh unit, albumen index, albumen quality and other egg quality

parameters.

Ghorbani and Fayazi (2009) and Gongruttananum and Ratana (2005) studied the

effects of dietary addition of NaHCO3 on egg quality characteristics in layers kept under

chronic heat stress. They reported that dietary levels of sodium bicarbonate (0.5%-1.5%) in

the diets of laying hens did not show any improvement in the albumen quality of the eggs

produced by these birds. Similarly dietary addition of ascorbic acid and sodium bicarbonate

in layer diets did not exhibit any effect on albumen index of eggs produced by laying birds

kept under heat stress (Mandal et al., 2010).

Results of the various studies discussed in the paragraphs above, are in accord with

36

observations of EL-Sheikh and Salama (2010) who investigated that adding 100mg NaHCO3

or 75mg KCl/L in drinking water did not affect (P>0.05) albumin weight, yolk weight, yolk

index and Haugh units score, but on the other hand, albumin weight and yolk weight were

affected (P<0.05) by NaHCO3 or KCl supplementation.

The manner, in which different factors influence egg quality parameters have not

been fully determined yet. Although, many research workers have studied the effects of these

factors on some parameters of eggs quality, but still there is dearth of information regarding

the effect of sodium bicarbonate on important egg quality variables i.e., Haugh unit values,

albumen index and yolk index. Hence the expected changes in these factors under various

hyperthermic conditions still need to be addressed. Therefore, this project was planned to

check the effect of dietary inclusion of sodium bicarbonate on egg quality characteristics of

caged layers during summer.

2.14.9Body/rectal temperature and respiration rate It has been observed that birds experiencing heat stress have higher body temperature

than those reared under a comfortable zone of temperature and hence spend more time

panting (Mack et al., 2013). Birds utilize multiple ways to maintain body temperature and

homeostasis when subjected to heat stress, which include increased radiation, convection and

evaporation along with the heat loss by vasodilatation and perspiration (Mustaf et al., 2009).

Increase in respiration rate is correlated with high environment temperature along

with increase in moisture contents of the air. When ambient temperature is increased

chemical reactions speed up in the body, heat is generated and body temperature of birds

rises (North and Bell, 1990). Birds have no sweat glands; hence they dissipate their body heat

mainly through respiration (Nillipour and Melog, 1999). When environmental temperature

exceeds the thermo-neutral zone, respiration rate increases up to 10 times, from a normal rate

of 25 breaths/minute (Remus, 2001), which results in respiratory alkalosis. In such

conditions sodium bicarbonate can be used as a buffering agent to ameliorate the problem

(Whiting et al., 1991a). Angiletta et al. (2010) observed increased respiration rate in birds

exposed to hyper-thermal environment.

Ahmad et al. (2005) investigated the influence of addition of baking soda in the feed

on rectal temperature in broilers experiencing heat stress. They noted a reduction (P<0.05) in

body temperature of birds fed diets having sodium bicarbonate. The birds which were

37

supplied sodium bicarbonate containing diets also exhibited lower respiratory rate and thus

produced less heat for this physiological norm. However, Mushtaq et al. (2007) did not find

any correlation between rectal temperature of poultry birds and dietary sodium levels.

Similarly, findings of Junqueira et al. (2000) showed that layer birds exposed to heat stress at

33 °C receiving diets having varying levels of sodium bicarbonate (0.67 to 2.56%), were not

differed in their body temperature. Similar results are also observed by Dai and Bessei

(2007), that body temperature of birds was not affected due to dietary addition of KCl (0.2

and 0.4 %). In addition, the same trend i.e. non-significant effect due to the inclusion of

sodium bicarbonate (0.65 %) in drinking water of layers exposed to heat stress was observed

by Genedi (2000).

2.14.10Mortality Heat stress not merely reduces productive potential, but it also causes high mortality

leading to a reasonably harmful effect on economics of production in poultry birds. Mortality

of birds increases with increase in ambient temperature than that of thermo-neutral zone

(Anjum, 2000). Seley (1973) suggested that if an animal is unable to adjust it into a new set

of environmental conditions, it exhausts and ultimately dies. It was further reported that

response of the animals to higher temperature was different for a different set of

environment. High environmental temperatures have shown to increase mortality rate in

fowls as has been observed by Zakia et al . (1995) and Mandal et al. (2010)

There exists a significant difference in livability of heat resistant and vulnerable lines

of poultry birds. Mortality rate in heat resistant and susceptible lines of WLH, after twenty

hours of heat stress at 40.6 °C were found to be 44.4% in total (Bohren et al . , 1982).

However, the birds of heat resistant bird’s lines showed less (P<0.05) mortality than the birds

of heat susceptible lines. It was further reported that the birds kept in cold climatic conditions

exhibited higher (P<0.05) mortality under subsequent heat stress conditions.

Smith (1992) observed the effects of feed withdrawal and acclimation on livability in

heat stressed broilers. Chicks were exposed to ambient temperature of 38 °C for twenty four

hours from 1st to 9 days of age. In experiment I, chickens were kept under cycling

temperature of 24 °C to 35 °C for the next 44 days. In experiment 2, chicken were reared at

24 °C for 23 days followed by feed withdrawal for 0 to 24 hours and a rapid temperature

increase to 37 °C for the final 4 hours of feed withdrawal. They found that chickens on full

38

feed showed lower livability than those maintained on feed withdrawal program. Zakia et al .

(1995) studied the effect of feeding time and light on mortality in heat stressed birds and

observed that the birds subjected to heat stress with access to food and light showed higher

mortality (10%).

Dietary addition of anti-stress compounds, like sodium bicarbonate have proved to be

beneficial in controlling mortality of birds during heat stress (Khattak et al., 2012). Mushtaq

et al. (2005) observed no mortality in broilers fed diet supplemented with sodium bicarbonate

(0.025% Na+) under subtropical summer conditions. Similarly Owen et al. (1994) reported

that sodium bicarbonate when included into the poultry diet at the level of 1% reduce the

ascites related mortality because of its alkaline nature. Whereas, inclusion of ammonium

chloride at 1% level resulted in an increase (P<0.05) in ascites related disorders, which was

assumed to be due to its acidic nature (inducing acidosis). These results support the findings

of Genedi (2000) who reported that supplementing anti-stressors (NaHCO3, KCl and

NaHCO3+ KCl) decrease (P<0.05) the mortality level in layers kept under heat stress.

In a study, Merkley and Miller (1983) used non-conventional sodium salt sources

(sodium fluoride and sodium silicate) in broiler ration and found that efficiency of feed

utilization and mortality rate is not influenced by Na+ when provided in the form of these

chemical compounds. Lack of response of sodium supplied by non-conventional source can

be attributed to the absence of bicarbonate ions, which are necessary to combat heat stress.

Mandal et al. (2010) investigated the effect of adding ascorbic acid and sodium bicarbonate

in layer diets on mortality in layers kept under heat stress. Results revealed maximum

mortality in the birds fed sodium bicarbonate. Increase in mortality due to sodium

bicarbonate supplementation was attributed to higher levels of this compound. NaHCO3 at

1.45g/L has shown to increase ascites related mortality (Julian et al., 1986) whilst, a level of

7.50g/L resulted as toxic (Mirsalimi et al., 1993).

Puron et al. (1997a) reported slightly higher mortality in birds given NaHCO3 added

diets. They stated that such type of results might be due to higher stocking density rate,

which may probably be the cause of slightly higher mortality in treated group. Shane (1994)

observed that both humidity and temperature determine the level of heat stress in birds. He

pointed out that at temperature above 36 °C only production is adversely affected; whilst,

mortality losses occurs only when ambient temperature exceeds 47 °C.

39

2.14.11Hematological profile

It has been well documented that high environmental temperature depresses

hemoglobin level in birds (Anjum, 2000) and increase in environmental temperature and

blood hemoglobin concentration in birds, have been found to be negatively correlated.

Moreover, factors like age, sex, season, hormones and hypoxia may also influence quantity

of hemoglobin in blood (Sturkie, 1976a). Hemoglobin concentration was found to be

decreased in the male broiler chicks, as ambient temperature rose from 10 to 30 °C or 15 to

35 °C in two different trials conducted by Yahavet al . (1997). It was concluded that

hemodynamic variations are depicted only in the chicken acclimated to constant temperature

or to fast temperature changes e.g. those taking place during diurnal cycle but not happening

in birds reared under acute high or lower temperature. This might be responsible for the

inability of birds to regulate their body temperature.

Decrease in blood hemoglobin concentration in layers reared under high ambient

temperature is also reported by Vecerek et al. (2002). However, Ahmad et al. (2005)

observed increase in hemoglobin concentration in birds due to inclusion of sodium

bicarbonate in their diet. Similarly, findings of Genedi (2000) have also shown that addition

of anti-stressors like NaHCO3 in drinking water of Leghorn and Matrouh layers markedly

increased their hemoglobin concentration, even under heat stress conditions.

Elevation of blood glucose in experimental birds under heat stress is usually

attributed to hydrolysis of glucagon as a result of increased body temperature. Al-Hassani et

al. (2001) have reported a significant decrease in plasma glucose level in Hisex brown layers

subjected to heat stress, when fed diets containing sodium bicarbonate as compared to those

fed diets without any supplementation (control).

Ahmad et al. (2005) examined a decrease (P<0.05) in glucose level in broilers fed

diet containing sodium bicarbonate as compared to those fed diets without its addition

(controls). However, Koelkebeck and Odom (1995) did not observe any effect on glucose

concentration of the layers, due to high ambient temperature. Similarly, Zakia et al. (2009)

reported no effect of dietary addition of sodium bicarbonate on glucose level in chickens.

2.14.12 Serum metabolites and serum proteins

Heat stress has shown to decrease plasma protein concentration in poultry birds with

increase in the environmental temperature (Anjum, 2000; Geraert et al . , 1996; Yang et al . ,

40

1992). However, dietary inclusion of sodium bicarbonate has shown its potential beneficial

effect in improving serum protein concentration in laying birds during high ambient

temperature. Kurtoglu et al. (2007) reported an increase (P<0.05) in serum total protein due

to dietary inclusion of sodium bicarbonate in Brown-Nick layers. These results are in

agreement with Badran (2003) who resulted out that NaHCO3 supplementation at levels of 2,

3 and 4% increased (P<0.05) the blood protein concentration during heat stress. However,

Genedi (2000) reported that addition of NaHCO3 or KCl into drinking water of heat stressed

Leghorn and Matrouh layers did not cause any effects on the total protein and globulin

concentration in the birds. Heat stress has shown to increase serum uric acid level in poultry

(Yang et al . , 1992) and no effect on serum alkaline phosphatase (Bogin et al., 1981;

Koelkebeck and Odom, 1995). However, scientific information regarding the effect of

dietary inclusion of NaHCO3 on serum alkaline phosphatase and uric acid in the birds

exposed to heat stress is scanty.

Based upon scientific information available, it can be envisaged that high

environmental temperature can negatively influence serum protein concentration in poultry

birds. However, dietary addition of sodium bicarbonate during hot weather may improve

bird’s serum protein concentration. Therefore, it was required to initiate a project to check

response of dietary addition of sodium bicarbonate on serum protein concentration in layers

kept under hot weather conditions.

2.14.13Plasma electrolytes and minerals

Inter-relationship among NaCl, NaHCO3, calcium (Ca) and phosphorus (P) in the

diets of laying hens was studied by Junqueira et al. (1984) and for this purpose; 3

experiments were executed. For this Hy-line layers were kept in individual wire cages

installed in open sheds. The birds were given a diet based on yellow maize and soybean meal

with or without additional sodium chloride, calcium phosphate or sodium bicarbonate. The

results revealed a significant increase in blood pH, base excess and bicarbonate concentration

due to the dietary addition of sodium bicarbonate.

Incidence of respiratory alkalosis in broilers during heat stress period has also been

reported by Teeter et al . (1985). They observed a higher blood pH value of 7.39 in heat

stressed (32 °C) panting birds than those of non-panting (pH 7.28) birds, reared at 24 °C.

Induced heat stress from 32 °C to 41°C for 20 minutes, further elevated blood pH to 7.52 in

41

the same birds. Moreover, chronic hyperthermia caused an intermittent respiratory alkalosis

during panting, whereas an acute hyperthermia induced continuous panting and alkalosis in birds.

To investigate the effect of tap water, carbonated water, NaHCO3 and CaCl2 on blood

acid bases balance, an experiment was conducted on twenty Hubbard broilers (Bottje and

Harrison, 1985). A solution of 2 % Sodium chloride or 3.5 % Calcium chloride having pH

8.0 and pH 7.4, correspondingly, was introduced into the crop of birds and blood pH was

noted. Infusion of Sodium bicarbonate increased blood pH whereas, Calcium chloride

infusion reduced (P<0.05) the blood pH. However, high levels of sodium bicarbonate and

calcium chloride may cause a change in the DEB (dietary electrolyte balance). It is also well

documented that inclusion of NaHCO3 in diet may increase blood pH of poultry birds

(Squires and Julian, 2001; Glahn et al., 1988).

Branton et al. (1986) conducted an experiment in which they used NaHCO3 and

NH4Cl in broilers feed exposed to acute heat stress. The results revealed that sodium

bicarbonate did not exert any significant effect on blood pH of broilers. However, in a similar

study, Brenes et al . (1988) detected that sodium bicarbonate supplementation showed a

noteworthy (P<0.05) effect on distribution of calcium in bones of broilers. Turkeys suffering

from gout were observed for their biochemical profile when fed diets supplemented with

sodium bicarbonate (Mert, 1991). Concentration of bicarbonate was increased in the birds

and found to be 27.37and 35.73mEq/L in healthy and sick chickens, respectively.

Infusion of sodium chloride at10g/L into the crop of broilers, exposed to heat stress

for 90 minutes, resulted in metabolic acidosis due to reduction in blood bicarbonate

concentration. Its concentration was also found to be decreased in birds fed sodium chloride

treated diets (Bottje et al . ,1989). Harms (1991) conducted two experiments with Hyline

hens using corn soybean meal basal diet. Four diets; 1) control; 2) diet containing no sodium

chloride; 3) diet containing sodium chloride but sodium supplied as sodium bicarbonate; and

4) diet containing no added sodium and chloride supplied as calcium chloride, were

formulated and fed to the layers for 19 days period. Based upon the results, it

wasrecommended that provision of sodium from sodium bicarbonate may be used as

substitute of sodium chloride to avoid the presence of chloride in layer diets. Moreover, Kaya

et al. (2004) reported that the NaHCO3 supplementation in the diet proved helpful in

maintaining the venous blood pH, DEB, HCO3– and pCO2 levels at the physiological ranges.

42

Deyhim and Teeter (1991) conducted an experiment to assess the effect of iso-molar

KCl (0.5 %) and NaCl (0.39%) for drinking water supplementation on blood pH, HCO3- and

water intake of birds raised in heat stressed and thermo-neutral environments. At 35 °C,

supplementation of sodium chloride in drinking water of the birds decreased HCO3- contents

in their blood. Austle and Keshavarz (1988) launched 2 experiments to find out the effect of

addition of sodium and chloride contents in the diet, on bicarbonate ions concentration in

Leghorn layers. Blood bicarbonates and base excess were increased due to the presence of

chloride contents in the diet.

A decrease in blood sodium level in broilers kept under heat stress has been observed

by Borges et al. (2004) and Takahashi and Akiba (2002).Whereas, Ahmad et al. (2006)

observed an increase in plasma Na+ concentration in birds fed diets supplemented with

sodium bicarbonate. Similarly, Mushtaq et al. (2005) reported an increased serum sodium

concentration due to the addition of different dietary sodium levels. Bonsembiante and

Chiericato (1990), however, did not observe any effect of dietary inclusion of sodium

bicarbonate on sodium ion concentration in meat type turkeys.

Ruiz-Lopez et al. (1993) reported that elevated levels of chlorides in the diet may

lower the pH of the blood and blood HCO3- concentration. It may also lead to Tibial

dyschondroplasia (TD) and cartilage abnormalities (Nelson et al., 1981). Studies in hot

summer conditions have reported a raise in plasma sodium ions concentration along with

hem-dilution (Whithow et al., 1994; Ait-Boulahsen et al., 1989).

Gongruttananun and Ratana et al. (2004) determined the effect of supplementation of

NaHCO3 in the birds’s diet on plasma sodium and pH. Three experimental diets were

formulated as; control (diet without addition of NaHCO3), diet with 1% inclusion of NaHCO3

and diet with 1.5% inclusion of NaHCO3.Layers fed diet added NaHCO3 exhibited a higher

level of plasma sodium concentrations. An increase (P<0.05) in plasma pH was noted in

birds fed diet supplemented with 1.5%NaHCO3. Findings of Wideman and Buss(1985) have

revealed that the layers which produced thin egg shell had significantly lower blood pH and

bicarbonate contents than those producing eggs having thick shell.

Plasma K+ concentrations were found to be decreased in birds exposed to heat stress.

Increase in excretion of K+ appears to be predominated in chronic heat stress, whereas its

increase in uptake by the cells is manifested during acute heat stress (Berne and Levy, 1993).

43

Naseem et al. (2005) observed the effect of sodium bicarbonate on serum potassium and

serum bicarbonate levels in birds. Hyperthermia in these birds led to significant decrease in

serum potassium and serum bicarbonate level. However, dietary inclusion of NaHCO3 at

levels of 0.5% during heat stress has shown to increase serum potassium and bicarbonate

level in blood of the birds.

The effect of heat stress on blood potassium concentration has also been found to be

similar in different species of birds such as in broilers (Mushtaq et al., 2005), layers

(Ghorbani and Fayazi, 2009) and quails (Keskin and Durgan, 1997). Moreover, dietary

supplementation of different levels of sodium bicarbonate has shown to prevent any decrease

in blood potassium level in heat stressed birds (Ghorbani and Fayazi, 2009).

It can be concluded and is also well documented that the level of blood potassium is

reduced (P<0.05) in the heat stress birds (Borges et al., 2004; Takahashi and Akiba, 2002).

However, addition of sodium in the diet of birds may prevent the blood K+ concentration

when reared under hot weather conditions (Ahmad et al., 2006; Mushtaq et al., 2005).

However, Kurtoglu et al. (2007) have found decreased plasma potassium concentration in

layers fed NaHCO3 containing diets as compared to those fed diets containing either NaCl or

KCl.

2.14.14 Serum lipids, hormones and enzymatic profile

Heat stress shows negative impact on production performance also different

biochemical processes including lipids, hormone and enzymatic status of birds

(Anjum, 2000). Important factors which affect cholesterol level in the body are: sex, age,

ration, hyperthermia and starvation. High serum cholesterol concentration in birds kept at

high temperature has also been reported by Haazele et al . (1991); Takahashi et al . (1991);

Sahota et al . (1993) and Sahota and Gilani, (1994).

Response of birds to dietary inclusion of sodium bicarbonate with respect to growth

hormones has not been studied much. However, Hussan et al. (2011) have observed a

noteworthy (P<0.05) decrease in blood (plasma) T3 and T4 hormones concentration in laying

birds supplementing diet with sodium bicarbonate. Similarly Attlla et al. (2002) found that

supplementation of NaHCO3 and KCl in drinking water, decreased concentration of T3

hormone in layer birds kept at higher ambient temperature (34 °C) for the period of fours

constant hours daily followed by a normal temperature (22° to 24 °C) throughout three

44

months experimental period. Genedi (2000) also reported pronounced effect of NaHCO3 on

plasma triiodothyronine hormone (T3) in Matrouh hens reared in heat stress conditions,

however, the effect was non-significant in Leghorn hens. Findings of Badran, (2003) have

also shown that the level of plasma T3 hormone in egg laying birds due to the addition of

different level of sodium bicarbonate (2, 3 and 4%) remained unaffected.

The investigations presented above have reported some changes in lipids, hormones

and enzymatic profile of poultry birds under the influence of various temperatures. However,

there is a dearth of research regarding the effect of dietary inclusion of sodium bicarbonate

on serum lipids, hormones and enzymatic profile of poultry birds exposed to heat stress.

Therefore, it can be envisaged that there is a dire need to explore the behavior of layers

regarding the variation of these parameters as a result of dietary inclusion of sodium

bicarbonate.

2.14.15 Immune response

Heat stress is known to increase lymphocyte and may severely affect the immunity in

poultry birds (Anjum, 2000; El-Gendy et al., 1995; Savic et al. 1993; Thaxton and Siegel,

1972). Dietary inclusion of sodium bicarbonate, however, has been shown to reduce effects

of heat stress (Ahmad, 1997; Ahmad et al., 2007). Deficiency of sodium has been reported to

suppress immune response and reduction in peripheral gustatory function (Guagliardo et al.,

2009). Use of NaHCO3 may be attributed to the partial correction in acid-base balance, which

may play a vital role in increasing immune response against some diseases. An increase in

dietary electrolyte balance has shown to cause a decrease in heterophil to lymphocyte ratio in

blood, leading to increase in antibody titer (Borges et al., 2003).

Santin et al. (2003) reported an increase in immune response against Newcastle

disease (ND) with increase in dietary electrolyte balance (40, 140, 240, 340mEq/kg) in birds.

Use of sodium bicarbonate in the diet of birds during hot weather significantly increased

antibody titters against Newcastle disease and Avian Influenza in poultry birds (Hussan et

al., 2011). These results are compatible to the results of the research conducted by Genedi

(2000) who found that use of anti-stressors (NaHCO3, KCl and NaHCO3 + KCl) may

improve immune response in Leghorn and Matrouh layers.

Hoshi et al . (1995) studied the effect of oral administration of formalin inactivated

virus suspended in buffered containing sodium bicarbonate in chicken. It was observed that

45

this form of antigen when administered orally, stimulate a serum defense response against

Gumboro disease virus (IBDV) in birds.

Fletcher et al . (1993) used a rinse process using NaHCO3 on recovery of pathogens

(bacteria) from the carcass of chicken. They implemented a three step rinsing procedure with

2% sodium bicarbonate and reported that the process was effective in reducing pathogen

recovery after seven days of the treatment. Findings of Khatak et al. (2012) have revealed

higher haemaglutination inhibition titer against NDV in birds consuming diets containing

sodium bicarbonate.

2.14.16 Digestibility of nutrients

High ambient temperature may exert a significant influence on digestion and

absorption of nutrients and their metabolism (Macleod, 2004; Puvadolpirod and Thaxton,

2000). Heat stress decreases blood flow towards digestive tract (Wolfenson, 1986).

Consequently it may reduce proteolytic activities of the respective enzymes in upper part of

digestive system (Haiet al.,2000) and hence may lead toa decrease in digestibility of protein.

Factors, which may influence digestibility of nutrients include; ambient temperature

(Macleod, 2 004; Hai et al., 2000 ; Puvadolpirod and Thaxton, 2000), level of feed intake and

passage rate of digesta (Ravindran et al., 2008; Ahmad et al., 2007), anti-nutritional factors

present in feed ingredients (Hughes and Choct, 1999), age and physiological status of the

bird (Batal and Parsons, 2002; Huang et al., 2007; Garcia et al., 2007) and composition of

the diet (Leeson and Summer, 2001a).To improve digestibility of feed ingredients different

nutritional manipulations have been used such as adding enzymes in feed (Bryden et

al.,2009; Selle et al., 2010), heat treatment and processing of feed ingredients and (Friedman,

1996; Amerah et al., 2007) addition of electrolytes in feed/water during hot weather

(Ravindran et al., 2008).

NaHCO3 in the diet of poultry birds exposed to heat stress may improve nutrient

digestibility by increasing sodium ions concentration (Fethiere et al., 1994); improving

electrolyte balance in the diet (Borges et al., 2003) and decreasing the production losses

caused by heat stress (Gorman and Balnave, 1994). Supplementation of either sodium or

potassium in drinking water may also increase water intake, which is associated with a

reduction in body temperature (Smith and Teeter, 1989) and an increase in nutrients

utilization. Gous (2004) suggested that producers should encourage the birds to drink more

46

water by adding mineral salts in to their drinking water (2 g NaHCO3/L water) or using

NaHCO3 (up to 16 g/kg) in the feed as a sodium source, or both.

Use of saline solutions has been a common practice to stimulate nutrient digestion

and absorption during heat stress (Jeukendrup et al., 2009) in birds. Electrolyte balance can

influence appetite and the metabolism of certain nutrients (Patience, 1990).Dietary

electrolyte balance (DEB) has also been reported to influence the absorption of

monosaccharides and amino acids (Johnson and Karunajeewa, 1985; Ravindran et al., 2008).

Therefore, an optimum level of dietary electrolyte balance can enhance digestion of nutrients

whereas a significant deviation to either side of optimum DEB may decrease digestibility of

nutrients (Ravindran et al., 2008).

Dietary addition of sodium in birds has been shown to improve digestibility by

increasing sodium ions concentration (Fethiere et al., 1994). Sodium containing compounds

such as sodium bentonites have been successfully used in sorghum containing diets to

prevent deleterious effects of tannins present in it, on nutrient digestibility (Pasha et al.,

2008; Banda-Nyirenda and Vohra, 1990). Santurio et al. (1999) and Salari et al. (2006) also

observed an increase in nutrient digestibility due to addition of sodium bentonite in broiler

diets.

Dietary inclusion of sodium containing compound such as NaHCO3 increase the

dietary electrolyte balance, which favors the proper digestibility of nutrients (Drinah et al.,

1990; Borges et al., 2003; Mahmud et al., 2010; Ahmad, 1997). However, there is dearth of

information in the literature where the significance of dietary inclusion of sodium

bicarbonate during hot weather has been evaluated.

It may be concluded that among many factors which influence digestibility of

nutrients in birds, heat stress is the most important factor, which has shown a pronounced

effect on digestibility of most of the key nutrients. Although, many research workers have

used different substances (mentioned above) in the diets of broilers during hot weather to

improve digestibility of nutrients, however, information regarding dietary inclusion of

sodium bicarbonate to see its effect on nutrient digestibility in layers during heat stress is still

scanty and therefore, is required to be explored.

47

CHAPTER-3

MATERIALS AND METHODSPrimary objective of this study was to explore the effect of varying levels of sodium

bicarbonate (NaHCO3) on production performance, immune response, blood profile and

nutrient digestibility in caged layers during summer. Two separate experiments (performance

trial and digestibility trial) were carried out for this study. The first experiment was designed

to probe the question of cause and effect when diets containing different levels of NaHCO3

were fed to caged layers during summer. Whereas, the second experiment was carried out to

investigate the effect of different levels of dietary NaHCO3 on digestibility of crude protein,

crude fiber and ether extract, as well as absorption of some minerals (Na, K, Ca, P and Fe) in

the layers.

3.1 Performance Trial

The details of the experiment regarding the effect of various level of sodium

bicarbonate on production performance, egg quality characteristics, immune response and

blood profile of caged layers are as follows

3.1.1 Experimental birds

One hundred sixty, 24 weeks White Leghorn layers were employed as experimental

birds for the performance trial. These birds were maintained in individual cages in a Poultry

House of the Department of Parasitology, Faculty of Veterinary Sciences, University of

Agriculture, Faisalabad (Pakistan) for a period of 12 weeks.

3.1.2 Allocation of the birds to the cages

Initially the experimental layer birds were kept in a group and were fed layer ration

during adaptation period (25th week of age). After the adaptation period, at the start of 26 th

week, all the birds were separately weighed, leg banded and allocated to the treatments using

a Completely Randomized Design (CRD). The cages were supplied with feeder and drinker

lines. The dimension of each cage was 41, 39 and 37cm, length, width and height

respectively. The birds were indiscriminately (randomly) divided into 20 experimental

units/replicates having 8 birds in each unit. These units (layer birds) were further allotted to

five experimental diets (4 replicates/treatment). The birds were provided experimental diets

48

throughout the study period (26-37 week of age).

3.1.3 Management of the experimental birds

These birds were maintained in a thoroughly cleaned and disinfected poultry house,

where they were given Erythromycin (1 table spoon/8 liter of water) for a week (25 th week of

age) as prophylactic measure to lessen the probability of any disease outbreak. A weighed

amount (110 g/bird) of the experimental diets was fed two times a day (morning and

evening). Fresh and clean water was offered to the birds. Daily, 17 hours light was

maintained to the birds throughout the study. The data on weekly feed consumption and body

weight were recorded during the experimental period.

3.1.4 Experimental diets, groups and their feeding plans

Diets used for the in vivo trials were iso-nitrogenous (CP, 17%) and isocaloric (ME,

2700 Kcal/kg diet). Five diets i.e. A, B, C, D and E were formulated with or without addition

of sodium bicarbonate (Table 3.1). Diet A, was without sodium bicarbonate and considered

as control whereas, diets B, C, D and E contained 0.5, 1.0, 1.5 and 2.0% sodium bicarbonate,

respectively. All the diets were formulated according to the NRC (1994) requirements. Each

experimental diet was fed for 12 weeks (26-37 weeks of age).

Before the start of experiment, all the diets were analyzed for their proximate

composition as shown in table 3.2, according to the technique described by AOAC (2010), in

the Analytical Laboratory of the Institute of Animal Sciences, Faculty of Animal Husbandry

(FAH), University of Agriculture, Faisalabad (Pakistan).

3.2 Data CollectionDuring this study, following data were recorded.

3.2.1 Initial body weight of the birds

The layer birds of all the experimental groups were individually weighed to have their

initial body weight on very first day of the experimental period.

3.2.2 Weight gain

The experimental birds were weighed every week in order to get weekly weight gain

of the birds and the weight records were maintained accordingly.

49

Table 3.1: Proportion of the ingredients used in the experimental diets

Ingredients

(%)

A

Basal diet

B

0.5% NaHCO3

C

1% NaHCO3

D

1.5% NaHCO3

E

2% NaHCO3

Maize 31.50 28.00 29.00 30.60 30.60

Rice broken 30.20 30.00 30.00 30.00 30.00

Fish meal 3.60 5.50 7.00 7.00 7.00

Soybean meal 17.00 1.50 0.00 2.00 4.40

Canola meal 4.50 14.00 13.60 11.60 8.40

Rapeseed meal 3.10 3.00 3.00 3.00 3.00

Guar meal 0.00 2.50 3.00 3.00 3.00

Corn-gluten 60% 0.00 2.00 2.00 2.00 2.00

Rice polishing 0.00 2.20 2.00 0.00 0.00

Dicalcium

phosphate

0.50 0.00 0.00 0.00 0.00

Limestone 9.00 9.00 8.70 8.70 8.70

Mineralsand

vitamin Premix

0.30 0.30 0.30 0.30 0.30

DL-met 0.13 0.08 0.07 0.07 0.09

Lys sulphate 65% 0.00 0.15 0.14 0.13 0.12

Salt 0.23 0.00 0.17 0.17 0.18

Sodium

bicarbonate

0.00 0.50 1.00 1.50 2.00

Allzymea 0.015 0.015 0.015 0.015 0.015

Lincomixb 0.02 0.02 0.02 0.02 0.02

a: a naturally fermented product with multiple enzyme activities including carbohydrase,

protease and phytase.

b:an antibiotic that provides consistent disease control for necrotic enteritis caused by

Clostridium perfringens

50

Table 3.2: Calculated chemical composition of the experimental diets

Nutrients A

Basal diet

B

0.5% NaHCO3

C

1%

NaHCO3

D

1.5% NaHCO3

E

2% NaHCO3

ME Kcal/Kg 2700 2700 2700 2700 2700

Crude protein (%) 17.00 17.00 17.00 17.00 17.00

Crude fiber (%) 3.29 3.80 3.74 3.44 3.39

Crude fat (%) 3.27 3.9 3.9 3.67 3.65

Crude ash (%) 11.97 11.9 12.01 11.85 11.89

Calcium (%) 3.9 3.9 3.9 3.9 3.9

Available Phosphorus (%) 0.37 0.37 0.38 0.38 0.39

Sodium (%) 0.17 0.23 0.43 0.57 0.7

Potassium (%) 0.70 0.53 0.51 0.52 0.52

Chloride (%) 0.22 0.15 0.20 0.20 0.20

Metabolizable Lys (%) 0.80 0.80 0.80 0.80 0.80

Metabolizable Met (%) 0.44 0.41 0.42 0.42 0.47

Metabolizable Thr (%) 0.55 0.56 0.56 0.57 0.57

MetabolizableTrp (%) 0.16 0.16 0.16 0.16 0.16

Dietary Electolyte

Balance (DEB) mEq/kg

210 210 262 325 388

51

3.2.3 Feed consumption

A weighed amount of feed was provided to each experimental unit twice a day,

throughout each week and left-over (remained) feed was weighed back to determine weekly

feed consumption of each replicate.

3.2.4 Egg production

Record of daily egg production of each replicate was used to calculate average

number of eggs produced/bird/week.

3.2.5 Egg mass

Total number of eggs produced by each replicate were weighed daily to calculate

weekly egg mass produced per replicate.

3.2.6 Feed conversion ratio

Feed conversion ratio (FCR) was calculated in two ways;

a) The kilograms of feed consumed to produce one dozen of eggs.

FCR /dozeneggs= Feed consumed(kg)No . of eggs produce

b) The kilograms of feed consumed to

produce one kilogram of egg mass.

FCR /Kg eggs mass= Feed consumed (kg)Egg mass produced (kg)

3.2.7 Water consumption

A measured quantity of fresh water was offered to each group, in the morning and

evening time. At each time, the residual water was again measured and recorded. Then daily

water intake of each replicate was summed up to calculate water consumption/group.

3.2.8 Rectal temperature and respiration rate

On the last day of each experimental week, rectal temperature and respiration rateof

three birds from each replicate were recorded at 6:00 am, 12:00 noon and 6:00 pm and

thereafter average of these observations was calculated to be used in the statistical analysis.

3.2.9 Ambient temperature and humidity index

A dry bulb thermometer was installed in the middle of the house to manually record

daily ambient temperature. Whereas, daily records of relative humidity inside the poultry

house were maintained by using a digital hygrometer.

52

3.3 Egg quality characteristicsFrom the start of production, at weekly interval, five eggs from each replicate were

selected randomly. These eggs were broken down individually (one by one) and their

material (content) was transferred into a separate Petri dish to find out the following

characters in relation to measurement of the quality of the eggs produced.

3.3.1 Egg weight

All the eggs produced by the birds of each experimental unit were numbered and

weighed daily. At the last day of each experimental week average egg weight was

determined for that particular week.

3.3.2 Shell thickness

Egg shell thickness was measured with a Micrometer Screw Gauge, accurately up to

0.01mm from the samples (eggs) used for meat and blood spots. Shell membranes of the egg

shells were removed by hand and then shell thickness of each egg was measured by taking

three readings (one reading each from broader end, narrow end and girth of the egg).

Average of these three values, however, was taken as the final reading.

Shell thickness (mm)=a+b+c3

Where

a = reading (mm) taken at the broader end of the egg

b = reading (mm) taken at the narrow end of the egg

c = reading (mm) taken at the girth of the egg

3.3.3 Specific gravity of eggs

Specific gravity of intact eggs was determined by the method described by Hamilton

(1982). The detail of the method used is as follows:

Procedure1. Nine beakers having 500ml water each; containing different concentration of salt

solution and having a known specific gravity were used in this process. Specific

gravity of different beakers is described below.

53

Beaker

No.

Conc. Of salt for 3litre H2O

(g)

Conc. Of salt for 500ml

H2O (g)

Specific gravity of

beaker

1. 276 276/6=46.00 1.06

2. 298 298/6=49.66 1.065

3. 320 320/6=53.34 1.07

4. 342 342/6=57.00 1.075

5. 365 365/6=60.84 1.08

6. 390 390/6=65.00 1.085

7. 414 414/6=69.00 1.09

8. 438 438/6=73.00 1.095

9. 462 462/6=77.00 1.1

2. The salt was weighed via a digital balance according to the amount of water in each

beaker. The appropriate amount of salt (as mentioned in the above table) was added

to each beaker and was dissolved properly by thorough manual stirring.

3. Firstly, the eggs were lowered one by one, into a beaker having pure water (without

the addition of salt), considered as pre-dip solution.

4. Thereafter, each egg was carefully lowered down one by one into the 1 st beaker, with

the help of a spoon and sliding down slowly, with the wall of the beaker. The egg was

kept in this state for 15-20seconds. The egg was taken out, when it started floating in

the beaker i.e. broke the surface of water.

5. The egg, if did not float in the 1st beaker, was taken out and dipped for few seconds in

the pre-dip solution again; it was then lowered down in the 2nd beaker having different

salt concentration.

6. The process was continued up to the 9th beaker and/or till the egg floated by itself in

some beaker.

54

7. The value of specific gravity of an egg was taken as that of known value of the

solution in the beaker, into which the egg floated.

3.3.4 Albumen Height

A tripod micrometer was used to determine the height (mm) of the albumen. The

tripod stand was positioned in midway between the yolk and edge of the albumen. Probe of

tripod micrometer was then lowered down on the surface of the albumen so that it made just

touch with it. The point when needle made contact with the surface of the albumin, the

reading was noted.

3.3.5 Yolk Height

A tripod micrometer was used to determine the height (mm) of yolk. The tripod stand

was placed on middle of the yolk. The needle of the micrometer was lowered even it

contacted. The reading was then noted.

3.3.6 Yolk Diameter

Yolk diameter was determined by using digital Verniercaliper. The jaws of Vernier-

caliper were placed around the yolk of the egg and thereafter were brought close to the yolk.

When the jaws just contacted the edges of the yolk, the reading was noted.

3.3.7 Blood and meat spots

The eggs, which were broken to check the egg quality parameters, were carefully

checked for the presence of blood or meat spots, if any.

3.3.8 Yolk index

Yolk index of egg was calculated using the following formula:

Yolk index= Yolk heightYolk diameter 3.3.9 Haugh unit score

The observations obtained for albumen height were used to determine Haugh unit

score of the eggs by the following formula.

Haugh unit = 100 log {H+7.6 – 1.7 W0.37}

Here,

55

H = albumen height (mm)

W = weight of eggs (g)

3.3.10 Yolk and albumen pH

The pH values of albumen and yolk of the eggs broken for the determination of egg

quality parameters were determined by digital pH meter.

3.3.11 Egg yolk cholesterol

Principle

The value of cholesterol in egg yolk was determined after enzymatic (esterase)

hydrolysis and oxidation. The end products of the reaction were cholesterol and fatty acids. It

was converted into cholestene-3-one and hydrogen peroxide by the action of oxidase

enzyme. The indicator quinoneimine was formed from H2O2 and 4-aminoantipyrine in the

presence of phenol and peroxides.

Procedure

For the estimation of cholesterol, Enzymic endpoint of Randox Kit was used

following the procedure of Roeschlau et al. (1974) as described below.

Pipetted out, 10µl standard solution in a standard tube and 10µl of egg yolk in other

separate sample test tube. Thereafter, 1000 µl of the reagent was added in a standard blank,

as well as in the sample tubes. The tubes were then gently shaken and incubated at 25°C for

25 minute. Absorbance of the standard and those of the samples was measured by

spectrophotometer model Meterek Sp-851 at 500nm wavelength. Calculations were made as

follows:

Conc .of Cholesterol(mg /egg)=( A ˚ Sample) /(A ˚ Standard) ˟ Conc. of standard

Where,

A0 sample = absorbance of sample

A0 standard = absorbance of standard

3.4 MortalityA complete record of mortality, if any, was maintained throughout the experiment.

Post-mortem examination of the dead birds was conducted immediately after their death to

find out the cause of death, if any.

3.5 Hematological profile

56

At the last day of 37th week blood samples from two birds (randomly selected from

each replicate) were taken separately in 10 ml test tubes. The tubes containing blood samples

were kept in a test tube rack for 2 hours to obtain blood serum of each sample. After that

supernatant of each tube was separated with micropipette and was put into 2 ml plastic

tubes. The tubes containing the supernatant (serum) were stored at -20 °C till further analysis

for the blood profile.

3.5.1 Glucose

Principle

Serum glucose was determined after enzymatic oxidation (in the presence of glucose

oxidase). The hydrogen peroxide thus formed reacts, under the catalysis of peroxidase, with

phenol and 4-amino-phenazone to form a red-violet quinoneirnine dye as indicator.

Following specifications were applied while using the method of Randox kit (Barham and

Trinder, 1972) for the determination of glucose.

Wavelength 500nm, Hg 546nm

Cuvette 1 cm path length

Temperature 15-25 °C

Measurement against reagent blank

Procedure

1. Pipetted out various necessary solutions into test tubes as under:

Macro Semi Micro

Standard or sample Reagent blank Standard

SampleReagentBlank

Standard or sample

Reagent 20µl2000µl

--------2000µl

10µl-------

1000 µl--------1000µl

2. Mixed the contents thoroughly and then incubated the above mentioned solutions for 10

minutes at 37 °C.

3. Measured the absorbance of the sample solution and standard solution and against the reagent

blank within 60 minutes.

57

4. Glucose concentration was calculated by the formula:

Glucose conc .( mgdl )= Absorbancesample

Absorbance standard×Conc .Standard

3.5.2 Hemoglobin

Hemoglobin (Hb) concentration was determined by using the Sahli’s instrument.

Erythrocytes were leaked in dilute hydrochloric acid to form acid hematin with the specific

color appearance and then the color was matched with color standards provided in the Sahli’s

instrument. Finally, the hemoglobin level was read from the scale where it matched with the

standard color.

3.5.3 Erythrocyte Sedimentation Rate (ESR)

For ESR determination, Westergren tube method (mm/hour) was used. Westergen

tube is 2mm in diameter and has graduation from 0 to 200 at one mm gap. The bottom of

the tube was dipped in the blood and drawn into the tube up to the “0” mark by aspiration.

The tube was then set in an upright position in a rack with a soft rubber cushion at the

bottom. The drop/fall (mm) of the red blood cells in the tube was noted after one hour.

3.5.4 Packed Cell Volume (PCV)

PCV was determined by the method described by Benjamin, (1978). One third of the

micro-haematocrit tube was filled with the blood (well mixed heparinized) and the open end

of the tube was sealed. Placed this tube in a centrifuge machine in such a position that its

sealed end remained away from the center, taking care to balance it against another tube of

the same size. The tube was covered with a screw top and centrifuged from 5-7 minutes at

12000 rpm. Similarly, contents of all the sample tubes were centrifuged. After removing

from the machine, percent values of PCV were recorded directly from the graphic

haematocrit tubes such that each tube was held against the liner chart, with the top of the

liquid exactly at the top line and the bottom of the tube against the bottom line.

3.5.5 Total leucocyte count (TLC)

Total leucocyte count was determined using fresh heparin mixed blood by the method

of Benjamin (1978). The White blood cell counting pipette was filled with the blood up to

mark 0.5 and sucked 3% acetic acid up to mark 11 above the bulb of the pipette. Closed the

tip of the pipette and brought it in horizontal position. Rotated the pipette gently, so that

leucocytes were dissolved in diluting solution. Discarded 1/3 contents of the pipette. Placed a

58

drop of diluted blood to the engraved area of the counting chamber and allowed to settle for

one minute. Set the microscope and counted the white blood cells in the respective squares

and calculated total number of leucocytes in 1 cu.mm by the following formula.

Total number of leucocytes in 1 cu.mm = F/4 x 20 x 10

Where:

F = No. of cells in four fields.

20 = Diluting factor

10 = Factor for converting 1/10.

3.5.6 Red blood cells (RBCs) count

Following are the requisites for enumeration of red blood cells;

Counting chamber with cover glasses

A diluting pipette

Diluting fluid

Hand tally counting

Reagents

Gower solution

Sodium sulphate 12.5g

Glacial acetic acid 33.3ml

Distilled water 200ml

Procedure

1. Blood sample was taken and thoroughly mixed for 3-5 minutes on mechanical mixer

or by inverting the tube for at least 20 times.

2. Diluted the blood 1:200 in a solution of 10 ml 40% formalin in a liter of 32g/l

trisodium citerate

3. Filled the counting chamber as discussed in the protocol for determination of WBC.

4. Let the cells settle (for 2-3 minutes) in a wet chamber (Petri dish with a small piece

of damp blotting paper).

5. Then counted the RBC at the 40X of microscope. An ample number of RBCs were

counted to minimize errors due to uneven cell distribution.

Total erythrocytes count ¿X80

× 40×200×10 ¿X1012

59

Where,

X¿Cells counted in 80small squares

80¿ Subdivisions of 5 small squars

400¿Total numbers of small squares

200¿Dilution 1:200

10¿ Depth of chamber (0.1mm)

3.6 Serum proteinsSamples of blood serum were analyzed for their protein composition including:

1) Total serum protein concentration

2) Albumin concentration

3) Globulin concentration

3.6.1 Total serum protein concentration

Principle

Cupric ions interact with protein peptide bonds in an alkaline medium resultantly

forming a color complex.

Procedure

Total protein was assayed by the Burette method (Henry et al., 1974) as described

below:

A volume of 0.02ml of distilled water, standard and serum was taken in reagent

blank, standard and samples tubes, respectively. Then add 1.0ml of reagent solution in all

the tubes. Mixed thoroughly and incubated the above mentioned solutions for 30 minutes at

25 °C. Then read absorbance of the standard (A° standard) and of the samples (A0 samples)

against the reagent blank at wavelength of 500nm within 60 minutes on spectrophotometer.

Finally total protein concentration was calculated by the following expression:

Total protein conc .( gdl )= Absorbancesample

Absorbance standard×Conc . standard

3.6.2 Serum albumen concentration

Principles

60

The determination of serum albumin is based on its quantities binding to the indicator

3, 3, 5, 5 tetra-bromocresol sulphonphthalen (Bromocresol green BCG).

Procedure

Bromocresol green Randox kit method was used following the procedure of Doumes

et al. (1971). The details of the procedure are described below.

Wavelength Hg 578nm

Cuvette 1cm light path

Incubation Temperature 20-25 °C

Measurement against reagent blank

A volume of 0.01 ml of distilled H2O, serum sample and standard was taken into

the test tubes marked as reagent blank, standard and samples, respectively, and then added

3.0 ml BCG reagent in each tube. Mixed thoroughly and then incubated the above

mentioned solutions for 5 minute at 25 °C. Read the absorbance of standard (A0 standard)

and of the sample (A0 sample) against the reagent blank on spectrophotometer.

Albumin concentration was calculated by the following formula:

AlbuminConc .( gdl )= Absorbance sample

Absorbance standard× Conc . standard

3.6.3 Serum globulin concentration

Serum globulins were determined by deducting albumin from the total concentration

of serum proteins.

Serum globulin (g/dl) = Conc. of total serum protein Conc. of albumin

3.7 Serum lipids profile3.7.1Serum cholesterol concentration

Principle

Serum cholesterol level was assayed by enzymatic (esterase) hydrolysis and

oxidation. The end products of the reaction were cholesterol and fatty acids. It was

converted into cholestene-3, 1 and hydrogen peroxide by the action of enzyme oxidase.

61

The indicator quinoneimine was formed from H2O2 and 4-aminoantipyrine in the presence

of phenol and peroxides.

Procedure

For the estimation of cholesterol, Enzymic endpoint of Randox Kit was used,

following the procedure of Roeschlau et al. (1974) as described below.

10µl standard solution was pipetted out into standard tube and 10µl of serum

sample was taken in another sample tube. Thereafter, 1000µl of reagent was added in both

tubes. The tubes were gently shaken and incubated at 25 °C for 25 minute. Using the

spectrophotometer model Meterek Sp-851, absorbance of standard and samples were

measured at 500nm wavelength and calculation was made as under:

Conc .of Cholesterol(mgdl )= Absorbance sample

Absorbance standard×Conc . standard

3.7.2 Serum triglyceridesSerum triglyceride concentration was estimated by enzymatic GOP-Pap method

(Trinder, 1969) by using kit manufactured by Human.

Principal

The principle involved is determination of triglycerides after enzymatic hydrolysis

with lipases. Indicator is quinoneimine formed from H2O2. 4-amino antipyrine and 4-cholro-

phenol under the catalytic action.

Triglycerides Lipases→

Glycero+fatty acid

Glcerol+ ATP GK→

GLycerol 3 Phosphate+ ADP

Glcerol3 Phosphate+O2 GPO→

dihydroxyacetone phosphate+ H 2O 2

H 2O 2+4−aminoantipyrine POD→

quinoneimine+HCl+2 H 2O+4chlorophenol

Reagents composition

1. Buffer solution (500 ml)

62

PIPES buffer (pH 7.5) 40mMol/L

4-chlorophenol 5mMol/L

Magnesium ions 4.7mMol/L

Sodium-azide 0.05 %

2. Buffer solution (15×7 or 10×50ml )

4-aminoantipyrine 0.4mMol/L

Glycerol-3-phosphate oxidase ≥1.5U/ml

ATP 1mMol/1

Lipases ≥0.2U/ml

Peroxidase ≥0.5U/ml

Glycerol Kinase ≥0.4U/ml

3. 3ml standard

Triglyceride 200g/dl or 2.28mMol/1

Reagent stability

The reagents were stable up to the stated expiry date when stored at 2-8°C (if

contamination avoided). The working reagent was stable for 6 weeks at 2-8°C for days at

15-25°C (if protected from light).

Instrument:

Micro lab 300 made by Merck international

Assay procedure

Wave length 500nm, Hg 546nm

Optimal path 1cm

Temperature 20-25 oC or 37oC

Measurement Against reagent blank

Pipetting scheme

Pipette into Cuvettes Sample/standard Working reagent

63

Reagent blank ----- 1000µl

Sample /Standard 10µl 1000µl

Mixed the contents and incubated these for 10 minutes at 20-25°C or for 5 minutes at 37°C

and then measured the absorbance of the standard and sample (∆ Asample).

Concentration of triglycerides were calculated as under;

With factor

Wave length C (mg/dl) C (mMol/L)

546 nm 1048 x ∆ A 11.95 x ∆ A

With standard`

C (mg/dl) = 200 x ∆ A sampe∆ A standard or

C (mMol/L) = 2.28 x ∆ A sampe∆ A standard

3.7.3 HDL cholesterol

High density lipoproteins (HDL) cholesterol was estimated by enzymatic CHOD-

PAP method as described by Schettler and Nussel (1975) by using the kit made by

DiaSys (Diagnostic System International).

Principle

Chylomicrons, VLDL (Very low density lipoproteins) and LDL are precipitated by

adding Phosphotungstic acid and Magnesium ions to the sample, centrifugation process left

only the HDL in the supernatant and the cholesterol contents can be determined

enzymatically.

Reagent preparation and stability

The precipitant for macro assays was stable up to the end of the indicated expiry date

and was prepared to be used. Reagents were kept contamination free and stored at 15-25 °C.

Precipitation for semi micro assays

The100 ml line was filled with distilled water. The reagent was stable up to the end of

the mentioned expiry date. Contaminations were kept away from and stored at 15-25 °C.

Specimen

64

Serum, Heparinized or EDTA plasma

Serum Tooke apart from the blood clot as quickly as possible

Component and concentration in the test

Mono Reagent

Magnesium Chloride 25mMol/L

Phosphotungstic acid 0.55mMol/L

Instruments

Micro Lab-300 made by Merck international

Procedure

Precipitation

Contents Macro Semi macro

Sample 500µl 200µl

HDL reagent undiluted 1000µl ---------

HDL reagent Diluted ----------- 500µl

Reaction components were mixed well and then permitted to stand for 1 minute at

room temperature (25 °C), then centrifuged at 10000rpm for 2 minutes. Clear supernatant

was separated from the precipitate within one hour. HDL concentration was determined

using the DiaSys (Diagnostic systems) cholesterol FS reagent.

Cholesterol determination

Wavelength 500nm, Hg 546nm

Optical path 1cm

Temperature 20-25 °C

Measurement against reagent blank

65

Pipetted scheme

Contents Blank Sample/standard

Dist. Water 100µl ----------

Supernatant ------------ 100µl

Reagent 1000µl 10000µl

Mixed well, incubated five minutes at 37 °C, finally read the absorbance against

reagent blank within one hour.

Calculation of HDL cholesterol

Cholesterol calibrator of DiaSystem (content 200mg/dl or 5.2mMol/L was used

like a sample in the precipitation step

HDL cholesterol (mg/dl) ¿∆ A sample

∆ A standard× Conc . Std( mg

dl)

Calculation of LDL cholesterol

LDL cholesterol was determined by calculation method using Friede-Wald et al.

(1972) formula:

LDL cholesterol (mg/dl) ¿Total cholesterol Triglyceride5

−HDL cholesterol

3. 8 Plasma electrolytes (i.e. Na+, K+, Cl-, HCO3-) and mineral (Ca and P)

profile

3.8.1 Estimation of plasma pH

For the determination of plasma pH,a digital pH meter was used. The bulb of the

electrode was completely dipped in the blood taken in a glass test tube and the final reading

was read out on the pH meter screen. The bulb of the electrode was washed with distilled

water after every consecutive reading.

3.8.2 Estimation of Sodium (Na+) and/or potassium (K+)

Na+and K+ were determined by using clinical flame photometer model 410-C, U.S.A.

66

The sodium concentration was calculated by the following formula.

Na+or K+(mEq/lit) = T/S x CS

Where,

T = Flame photometer displacement reading of test

S = Flame photometer displacement reading of standard.

CS= Concentration of standard

3.8.3 Estimation of chloride

For the estimation of chloride two reagents were used.

Reagent 1 = Mercury 2 mEq/lit

Thiocyanate iron 20 mEq/lit

Nitrate nitric acid 45 m Eq/lit

Reagent 2 = Chloride standard 100 m Eq/lit.

The spectrophotometer tubes were arranged as under.Blank Standard Sample

Reagent 1 1ml 1ml 1mlReagent 2 — 10µlSample — — 10µl

The displacement was read at 480nm by adjusting the spectrophotometer at zero

through running the blank. Concentration of Cl-was calculated as:

Chloride mEqL

= Absorbance sampleAbsorbance standard

× Conc . of standard

3.8.4 CalciumPrinciple

Calcium present in the sample reacts with arsenazo III forming a colored complex

which can be quantized using the spectrophotometer.

Reagent preparation

Reagent and Standard were made ready to use.

Equipments

Analyzer, spectrophotometer able to read at 650 ± 20nm.

Samples

Serum, heparinized plasma was collected by standard procedures. Calcium in plasma

was stable for 10 days at 2-8ºC.

67

Procedure

1. Took the reagent to room temperature.

2. Pipetted out various test solution into labeled spectrophotometer test tubes as detailed

below.

Blank Standard Sample

Calcium standard (S) ----- 15ul -----

Reagent 1ml 1ml 1ml

3. Mixed thoroughly and let these tubes stand at room temperature for 2 minutes.

4. Read the absorbance (A) of the Sample and Standard at 650 nm against the blank.

Calculations

Concentration of calcium (Ca) in the sample was quantized by the following formula:

Calcium conc .(mgdl )= Absorbance sample

Absorbance standard× Conc . std .× sampledilution factor

3.8.5 Phosphorus

Principle

Inorganic phosphorus (P) in the sample reacts with molybdate in acid medium

forming a phosphor-molybdate complex which can be quantized by spectrophotometer.

Contents and composition

A. Reagent: 3 x 40ml. Sulfuric acid 0.36Mol/L, sodium chloride 154mMol/L.

B. Reagent: 1 x 50ml. Sulfuric acid 0.36mmol/L, sodium chloride 154mMol/L,

ammonium molybdate 3.5mMol/L.

C. Phosphorus Standard: 1 x 5mL. Phosphorus 5mg/dl (1.61mMol/L) and aqueous

primary standard. For further warnings and precautions, see the product safety data

sheet (SDS).

Reagent preparation

Standard (S) was provided ready to use. Working Reagent: Mix thoroughly in the

68

proportion: 7mL Reagent A + 3mL Reagent B. It remains stable for 12 months at 15-30ºC.

Equipment

Analyzer spectrophotometer able to read at 340 ± 20nm.

Samples

Serum, heparinized plasma collected by standard procedures. Phosphorus in plasma was

stable at 2-8ºC for 7 days. Centrifuged the samples and diluted 1/10 with distilled water

before measurement.

Procedure

1. Pipetted various solutions into labeled spectrophotometer test tubes as detailed below.

Reagent blank Sampe blank Sample Stardand

Distilled water 10ul ------- ------- -------

Sample ----- 10ul 10ul -------

P standard ------ ---------

1 Mixed carefully and let the tubes to stand for 5 minutes at room temperature.

2 Read the absorbance (A) of the Sample Blanks at 340nm against distilled water.

4 Read the absorbance (A) of the Samples and of the Standard at 340nm against the

Reagent Blank.

Calculations

The phosphorus concentration in the sample was calculated using the following general

formula:

Phos . conc .( mgdl )= Absorbancesample

Absorbance standard×Conc . standard× sample dilution factor

3.8.6 Estimation of HCO3-

Plasma HCO3̶level was determined in a gasometer. For this a known volume of

serum was used to be reacted with liberating reagent (C3H6O3). The liberating reagent

69

converted the HCO3- to CO2. In the presence of an indicator solution column (C6H5COOH),

after running the standard solution of HCO3- (NaHCO3), the displacement of pressure

produced by CO2 was read out. The HCO3- content was calculated as under:

HCO3- mEq/L = T/S x C.S.

Where,

T = Displacement reading of the test (serum)

S = Displacement reading of the standard.

C.S. = Concentration of standard.

3.9 Hormono-enzymatic determination

Concentrations of different hormones were determined by using the Radio-

immunoassay (RIA) kits manufactured by ICN Pharmaceuticals, Inc. California. The analysis

relies upon the capability of antibody to bind its antigen. To quantify the antigen, radioactive

and non-radioactive type of the antigens vies for binding sites on a specific antibody. Since

more non radioactive antigen is added, less radioactive antigen leftovers bound until

equilibrium establishes between the free and antibody bound antigens.

3.9.1 Assay for Triiodothyronine (T3) determination

Principle

Radioimmunoassay based on the capability of an antibody to bind the antigen. To

quantify the antigen, radioactive and non-radioactive type of the antigens vies for binding

sites on a particular antibody. Since more non radioactive antigen is added, less radioactive

antigen leftovers bound until equilibrium establishes between the free and antibody bound

antigens.

Procedure

Blood serum T3 was assayed by the method used by Abraham (1981). A brief

description of the method is given below.

1. Brought all standard, samples, coated tubes and Triiodothyronine 125I at room temperature

before to use.

70

2. All the tubes were labeled accordingly.

3. Placed the required number of anti-Triiodothyronine coated tubes in test tube frams/racks.

Placed the sample labeled coated tubes in stands.

4. Allowed them to stand to attain room temperature.

5. Pipetted 100µl each of T3 standard, control and samples to their respective tubes.

6. Added one ml of T3 125Ito all tubes, vortexed all the test tubes carefully and incubated at 37

°C for 120 minutes.

7. Thereafter decanted the supernatant from the tubes.

8. Finally, subjected the liquid for counting in a gamma counter, already calibrated for 125I. The results were calculated using RIA data reduction system.

3.9.2 Assay procedure for Thyroxin (T4)

Principle

The antibodies used have alike affinity both for standard and the analyte, which is

present in the serum sample. The unlabeled analyte vies with labeled analyte for the

existing antibody binding sites, thus falling the quantity of the labeled analyte attached to

antibody. Hence the level of radioactivity bound is thus, inversely interrelated to the

quantity of analyte in the sample or standard. Following an adequate incubation period,

the bound and free fractions are separated. Blood serum T4 was assayed by following the

method of Abraham (1981). A brief description of it is given as under.

1. Brought all standard, samples coated tubes and thyroxin to room temperature prior

to use. All the tubes were labeled accordingly.

2. Pipetted 100µl each of T4 standard, control and samples to their respective tubes.

Added 1.0 ml of T4125I to all tubes, vortexed all the tubes thoroughly for a minute

and incubated in a water bath at 37 °C±1 for 60 minutes.

3. Ensured the level of water bath being above the level of the reaction mixture in the

tubes.

4. Aspirated liquid from the tubes, and subjected all the empty tubes for counting in a

71

gamma counter already calibrated for 125I.

5. The results were calculated using the RIA reduction system.

3.9.3 Assay procedure for Cortisol

Radioimmunoassay (RIA) depends upon the capability of an antibody to attach its

antigen. To quantize the antigen, the radioactive and nonradioactive types of the antigen

compete for binding sites on its exact antibody. Since more nonradioactive antigen is

appended, less radioactive antigen leftovers bound until/unless equilibrium establishes

between the free and antibody bound antigen. Blood serum Cortisol was assayed by

following the procedure of Oelkers et al. (1992). A brief description of the method is as

under.

1. Brought all standards, samples, coated tubes and Cortisol 125| to room

temperature before use. Placed the requisite number of anti-Cortisol coated test

tubes in a test tube rack/frame.

2. Resealed the unused tubes in the plastic bag along web desiccant and

refrigerated. In the end, added all solutions in the quantity specified directly

from the reagent vials.

3. Pipetted out 25µl each of standard, control and samples into their respective

coated test tubes. Added 1.0 ml of Cortisol 125ǀ to all test tubes and vortexed for

a short while.

4. Incubated all test tubes in a water bath, set at 37 ± 1 °C for 45 minutes.

5. Decanted the contents of the tubes and then counting in a gamma counter,

already calibrated for 125ǀ

3.9.4 Assay procedure for Estrogen

Principle

In the ImmuChem total estrogen Assay, the reaction follows the Law of Mass Action.

The labeled and non-labeled analytes unite to the antibody in quantity to their relative

quantity. The amount of radioactive analyte which binds is inversely proportional to the

concentration of unbound analyte in the sample. In view of the fact of the inverse relationship

between the quantity of the analyte and the counts bound, counts bound decrease whilst

rising concentration of non-labeled analyte. Blood serum Oestrogen was assayed following

72

the method of Buster and Abraham (1975). A brief account of it is as under.

1. Added 0.6 ml of diluent buffer to the tube number 1 and 2, and0.5 ml to tubes 3

and 4, labeled four tubes 1, 2, 3, and 4.

2. Pipetted 0.6 ml of diluent buffer to test tube number 1 and 2 and 6.5 ml to tubes 3

and 4. Placed 0.5 ml of total Oestrogen standard to tubes 5-16 and took 0.5 ml of

reconstituted samples to tube number 17.

3. Treated all the assay tubes, except 1 and 2 with 0.1 ml of anti-total Oestrogen.

Thereafter, put 0.1 ml of 17 β-oestradiol125I to all the assay tubes mixed the

contents and incubated them for 1.5 hour at room temperature.

4. Finally added 0.1 ml of second antibody tr. all the tubes and incubated at room

temperature for 60 minutes.

5. After incubation centrifuged the tubes at 2300-2500rpm for 15 minutes and

aspirated the supernatant.

6. Subjected the residue of tubes for counting in gamma counter. Calculated the

results using the RIA data reduction system.

3.9.5 Assay procedure for progesterone

Principle

Radioimmunoassay based upon the capability of an antibody to bind its antigen.

In order to quantize the antigen, a radioactive antigen and a non-radioactive antigen

struggle for a limited numbers of binding sites on a specific antibody. As more non-

radioactive antigen is introduced into the system, less binding sites are accessible for

the radioactive antigen creating a method for quantitation. Method of Abraham et al.

(1972) was followed for the assay of progesterone. A brief description of the method is

as follow.

1. Brought all standards, samples, coated tubes and progesterone 125l to room

temperature before use.

2. Then placed the required number of anti- progesterone coated tubes in a test

tube rack/frame. Resealed the unused tubes in the plastic bag together with

the desiccant and refrigerated.

73

3. Pipetted 100 µl each of standard, sample and control into the respective

tubes. Added 1.0 ml of progesterone 125I to all the tubes and vortexed briefly.

4. Incubated the tubes in a water bath, Set at 37±1 °C for 120 minutes.

5. It was ensured that the level of water bath was above the level of reaction

mixture in the tubes.

6. Calculated the results using the RIA data reduction system.

3.9.6 Serum Glutamic Pyruvic Transaminase (SGPT)

Serum Glutamic Pyruvic Transaminase (SGPT) was determined by observing the

amount of pyruvate hydrazone formed with 2, 4-dinitro phenylhydrazine (DNP).

Assay Procedure

The Randox Kit method was used for the assay of GPT following the

procedure of Reitman et al. (1957) as described below.

Wavelength Hg 546nm (530-550nm)

Cuvette 1 cm light path

Incubation temperature 37 °C

1. Measured against reagent blank and pippeted into a test tube 0.1ml of sample.

Added 0.5ml of buffer, each in reagent blank and sample tubes but 0.1ml of

distilled water was put in reagent blank tube only.

2. The content of every test tube were thoroughly mixed and incubated for exactly

half an hour at 37 °C. Then added 0.5 ml of 2, 4-DNP in reagent blank and

sample, respectively.

3. Thoroughly mixed and then permitted to stand for exactly 30 minutes at 25 °C.

Then added 5.0ml of sodium hydroxide in reagent blank and sample, respectively.

Mixed and read the absorbance of sample (A° sample) against the reagent blank

after 5 minutes. The activity of GPT in the serum was obtained from the table by

comparing the absorbance of spectrophotometer.

74

3.9.7 Serum Glutamic-Oxaloacetic Transaminase (SGOT) Principle

Glutamic Oxalocetic transaminase was determined by monitoring the concentration

of pyruvate hydrazine formed with 2,4- dinitro phenylhydrazine (DNP).

Assay procedure

The Randox Kit method was used for the assay of GPT following the procedure of

Reitman et al. (1957) as described below.

Wavelength Hg 546nm (530-550nm)

Cuvette 1cm light path

Incubation temperature 37 °C

4. Measured against reagent blank and pippeted into a test tube 0.1ml of sample.

Added 0.5ml of buffer, each in reagent blank and sample tubes but 0.1ml of

distilled water was put in reagent blank tube only.

5. The content of each test tube were thoroughly mixed and incubated for exactly 20

minutes at 25 °C. Then added 0.5 ml of sodium hydroxide each in reagent blank

and sample, respectively.

6. Thoroughly mixed and then permitted to stand for exactly 30 minutes at 25 °C.

Then mixed 5.0ml of sodium hydroxide in reagent blank and sample, respectively.

7. Mixed and read the absorbance of sample (A° sample) against the reagent blank

after 5 minute.

8. The activity of GOT in the serum was found from the table by comparing the

absorbance of spectrophotometer.

3.10 Serum metabolites3.10.1 Urea

Principle

Urea is hydrolyzed in the occurrence of water and urease to produce ammonia and

CO2, the NH4 formed in the first reaction with α-oxoglutarate and NADH in the presence of

glutamate- dehydrogenase to yield glutamate and NAD+.

Procedure

75

Urea in the serum was estimated by enzymic kinetic method described in the Randox

Kits, following the UV method (Kassirer, 1971).

Wavelength 340nm (Hg 334nm or Hg 365nm)

Cuvette 1cm light path

Incubation Temp. 37 °C

Measurement against reagent blank.

Pipetted out 10µlof each of standard solution and serum into test tubes for standard

and sample. Working reagent 1.0ml was added in each tube (Reagent blank, standard,

sample). Shacked and incubated the above mentioned solutions for at least 3 minutes at 37

°C. Added 200µlreagent 2 in reagent blank, standard and sample, respectively. Shackedand

incubated for at least 5 minutes at 37 °C. Measured the standard (A standard) and the

sample (A sample) against the reagent blank within 2 hours.

Concentration of urea in serum sample was calculated by the formula;

UreaConc .(mg /dl)= Absorbance sampleAbsorbancestandard

×Conc . standard

3.10.2 Serum creatinine concentration

Creatinine produces an orange-red colored compound in an alkaline solution

with picric acid. The absorbance of this complex is directly proportional to the amount

of creatinine in the sample.

Creatinine + Picric acid→ Creatinine-picric acid complex

Reagents

Components and Concentrations

Picric acid (Rl): 70mMol/L

NaOH (R2): 1.6mMol/L

Standard (R3): 176.8 pMol/L (2mg/dl)

Assay

Temperature: +25 °C, +30 °C, +37 °C

Wavelength: Hg 492nm (480-520nm)

Optical path: 1cm

Measurement: against the air

76

Procedure

1. Working reagent was prepared by mixing of one part of reagent number 1 and

one part of reagent number 2.

2. Up to 100µl sample was taken and 1000µl working reagent was added and in it

then mixed carefully. Absorbance was noted/read after 20 seconds and again

after 80 seconds for both, test sample and standard.

Creatinine Conc .(mg /dl)= Absorbance sampleAbsorbance standard

× 2

3.10.3 Uric acid

Principle

Uric acid in the sample originates, by means of the coupled reactions described

below, a colored complex which can be quantified by spectrophotometer.

Uric acid + oxygen + 2 H2O →Alantoin + carbon dioxide + hydrogen peroxide

2 H2O2 + 4 – Aminoantipyrine + DCFS →Quinoneimine + 4 H2O

Composition

A. Reagent: Phosphate 100mMol/L, detergent 1.5 g/L, dichlorophenolsulfonate 4mMol/L,

uricase > 0.12 U/mL, ascorbate oxidase > 5 U/mL, peroxidase > 1 U/mL, 4-aminoantipyrine

0.5mMol/L, pH 7.8.

Serum uric acid standard: Uric acid 6mg/dl (357µMol/L). Aqueous primary standard.

Reagent preparation

Reagent and Standard solutions were made ready to utilize.

Equipments

Water bath set at 37ºC, analyzer and spectrophotometer able to read at 520 ± 10nm

Procedure

1. Took the reagent to room temperature and pipetted then into test tubes having labeled.

2. Pipetted into labeled test tubes:

3. Mixed carefully and incubated the contents of test tubes for for 5 minutes at 37ºC.

4. Measured the absorbance of the standard solution and the Sample solution at 520nm

against the blank.

77

The spectrophotometer tubes were arranged as underBlank Standard Sample

Distilled water 25ul -------- --------

Uric acid standard (S) ------- 25ul --------

Sample --------- --------- 25ul

Reagent 1ml 1ml 1ml

CalculationThe concentration/quantity of uric acid in the sample was calculated by the following general

formula:

Uric acid conc .( mgdl )= Absorbance sample

Absorbance standard× Conc . standard ×sample dilution factor

3.10.4 Alkaline Phosphatase Principle2-amino-2-methyl-1 -propanol + p-nitrophenvlophosphate + H2O ↔4-nitrophenol + 2

amino-2-methyl-1 -propanol phosphate

Reagents/chemicalsComponents and Concentrations

Enzyme/Buffer Reagent2-amino-2-methyl-1 -propanol 350mMol/L

Zn+2 1.0mMol/L

Mg+2 2.0mMol/L

Substratep- Nitrophenyl phosphate 16.0mMol/L

EDTA 2.0mMol/L

AnalysisWavelength: 405nm

Optical path: 1cm

Temperature: 30 °C, 37 °C

Measurement: ‘ against air

ProtocolWorking reagent was get ready d by adding 4 parts of 1-ALP reagent and 1 part of 2-

78

ALP reagent. Firstly 20μl serum sample was taken after that 1000μl of buffer was added in it

then after 1 minute and again after 1, 2 and 3 minutes absorbance was read.

ResultFrom the absorbance readings, ∆A/ minute was determined and the result was

multiplied by the factor i.e. 3433.

3.11 Determination of Antibody titer against Newcastle disease virus (NDV).Five ml of whole blood was collected from healthy adult birds (wing-web) in screw-top test

tube having 1mg/ml EDTA as anticoagulant as described by Allan et al. (1978). The test tube

was gently rotated for the mixing of blood and anticoagulant, but great care was taken to

avoid hemolysis.

Washing of Red Blood Cells (RBC)

a. The blood containing anticoagulant was centrifuged at 1500rpm for 5 minutes.

b. The plasma and buffy coat was separated via sterile pasture pipette.

c. Physiological saline was added to the sediment and the cells were re-suspended

by gentle shaking of test tube.

d. Again centrifuged at 1500rpm for 5 minute.

d. The cells were given 3 washing in this way.

e. A 2 % suspension of washed erythrocytes was prepared in Phosphate Buffer

Saline (PBS).Source of Virus

Newcastle disease virus (Mukteswar strain) was obtained in allantoic fluid from

the Department of Veterinary Microbiology University of Agriculture Faisalabad

(UAF), Pakistan.

Micro Haemagglutination Test (for ND virus)Procedurea. A 50Μl of sterilized physiological saline (pH 7.2) was distributed/dispensed in

all wells of rows A to H of micro titer plate with the help of micro diluter.

b. To well one of each row 50 Μl of NDV in allontoic fluid was added and mixed

with physiological saline.

c. Using micro diluter 2-fold serial dilution of each NDV was prepared up to 11th

well of each row.

79

d. A 50ΜL of 1% RBC’s was added to each of the wells of micro titer plates

e. Well number 12 was considered as control since merely the diluents and red

blood cells control suspension was added to this well.

f. The micro titer plate was quaked/shaken lightly to let mixing of contents in the

wells then incubated at room temperature afterwards. The results were

recorded/documented after 15-20 minutes, when the RBC’s in well number 12

were settled down in the form of a button at the bottom.

The results of HA test were interpreted as follows.

Positive: The bottom of the well covered by the thin layer of finally clumped RBC’s.

Negative: A small sharply outlined button of RBC’s (Bead formation) on the bottom of

the well.

Doubtful: A ring formed by the un-agglutinated RBC’s disrupting the thin layer of

clumped cell coating the bottom of the well. The HA titer was the reciprocal of the

highest dilution exhibiting haemagglutination.

Note: The highest dilution of the virus showing HA was considered as one HA unit. The

4 HA titer was calculated by dividing highest dilution showing HA titer by four.

Collection of serum samples

a. Blood was collected through wing vein by inserting 24 gauge needle fitted with 3cc

syringe.

b. After taking 3ml blood in syringe, it was taken in sterilize glass tubes and kept in

slanting position.

c. Upon clotting, it was kept in refrigerator for 2 hours.

d. Serum oozing out was collected in clean dry plastic vials and stored at -20 °C till

use.

e. Serum samples were collected at 10 days post first, second and third

vaccination.

Haemagglutinion Inhibition (HI) Test for determination of serum antibody titer in test

serum

Heamagglutination inhibitions (HI) test for determination of serum antibody titer described

by Beared (1976).

a. Micro titration plates (8 rows and 12 column of well) were used. Sterile tips were

80

used for micro titration dispenser.

b. All the wells in the plate were filled with 50µl of the normal saline solution.

c. Serum sample of 50µl were placed in the first well of all the rows.

d. With the help of multichannel micro-titration dispenser, the mixture in the first

well of each row was mixed properly and 50µl of the mixture was transferred to 2nd

well of the respective rows thus diluting each sample as 2- fold. Dilutions of serum

samples were done up to the well No. 10.

e. With the help of micro dispenser 50µl of the 4 HA unit of NDV was added to each

well upto well number 11. The plates were incubated at room temperature for 30

minutes.

f. Chickens RBC’s suspension (50µl of 1 % RBC’s) was added with the help of

micro-titration dispenser into each well of the plate from well 1 to 12 of each row.

g. The plates were then agitated/disconcerted backward and forward and from side to

side to make sure even suspension of RBC’s.

h. The plates were then set aside undisturbed/serene at room temperature until/unless

a clear pattern of haemagglutinion inhibition button formation was observed.

Maximum dilution of each serum sample which caused haemagglutination inhibition

was the endpoint. The titer of each serum sample expressed as reciprocal of the highest

serum dilution, which gave positive result (Lin, 1997).

3.12 Digestibility trialA digestibility trial was conducted during the experiment at 38 week of age. For this

purpose a separate group of thirty pullets obtained from the same batch as used for

performance trial, was reared in individual metabolism cages to be used in digestibility trial.

These layers were randomly allotted to five treatments (6 birds/treatment) such that each bird

served as a replicate. These pullets were fed rations mixed with cellite (acid insoluble ash) at

1% as a marker.

The birds of these groups were fed their respective rations for five days (start of 38 th

week of age) to assure that the passage of marker (AIA) in the feces of the birds was

stabilized (Sales and Janssens, 2003) and during this period no feces were collected. After

stabilization of the marker in the feces (week 38), all the birds were offered same amount of

their respective rations. The feed offered to the birds was divided in to two equal portions

81

and half of the feed was given at 9:00 am, and the rest at 9:00 pm. The feed not eaten was

removed from the feeders and weighed at the end of the digestibility period.

Excreta collections, which started at the 6th experimental day, were made for a period

of 48 hours (2 consecutive/uninterrupted days) at the interval of 2 hours. Excreta samples

were immediately/instantly frozen after every collection. The sample thus obtained were

dried, finely ground and then analyzed for the determination of digestibility of dry matter

(DM), crude protein (CP), ether extract (EE) and crude fiber (CF) contents using the AO AC

(2010) methods. The samples collected were also analyzed for their mineral contents (Ca, P,

Na, K, Fe and Mg) using atomic absorption spectrophotometer.

Digestibility of the nutrient was calculated by the following formula:

D(% )=100−( Acid insoluble ash (AIA )∈feedAcid insoluble ash( AIA)∈ feces

× Nutrient∈fecesNutrient∈ feed

×100)3.13 Proximate composition

Proximate analysis of feed and excreta samples were carried out for calculation of

nutrients digestibility for the determination of digestibility of DM, CP, EE and CF contents

by the method described by AOAC (2010). The description of these procedures is given

below,

2.13.1Dry matter

Dry matter contents of the samples (feed and/or excreta) were determined by drying

the samples in hot air oven at 65 °C for 48 hours having no further moisture. The dried

samples were transferred into desiccators for thirty minutes to attain constant weight.

Weights of the samples were recorded prior and after drying. The DM was calculated using

the following formula!

W1 - W2

Dry matter % =––––––––––––––––––––––x 100 W1

Where,

W1= weight (g) of sample before drying

W2= weight (g) after drying

3.13.2 Crude protein

Nitrogen contents of the samples (feed and/or excreta) were determined by the

Kjeldahl method. One gram (gm) of dried and ground sample was taken in digestion flask

82

with 5 gram of digestion mixture containing K2SO4, CuSO4 and FeSO4 (90:9:1) and 25 ml of

concentrated H2SO4. Then digestion flask was placed/put on heater until a clear solution was

attained. Thereafter the contents of flask were cooled and diluted with distilled water up to

250ml in a volumetric flask. Ten ml of this solution was transferred/shifted to a micro

Kjeldahl distillation apparatus and distilled with 10ml of 40% sodium hydroxide solution.

The ammonia (NH3), produced, was accumulated in a beaker having 10ml of 2 % boric acid

solution containing two drops of indicator containing methyl red. Distillate so obtained was

titrated against 0.1 N H2SO4 to end point (pink) and the percentage (%age) of nitrogen was

calculated using the formula given below:

Volume used N/10 x 250 x 0.0014 x l00N2 (%) = –––––––––––––––––––––––––––––––––––––

Weight of sample x 10CP (%) = % N2 x 6.25

3.13.3 Ether extract

Crude fat (ether extract) was determined by Soxhlet’s continuous extraction

apparatus using fat solvent. Five grams of moisture free sample (feed and/or excreta)

was taken into the dried extraction thimble. The thimble was placed in the glass jacket

which is fixed under the condenser for the extraction apparatus. About 150 ml diethyl

ether (BP 40-60 °C) was poured in the already weighed receiving flask of the apparatus.

The water and heater were turned on and extraction was continued for 10 hours at the

condensation rate of 90drops/minute. Thereafter, thimble was removed and the diethyl

ether was collected in the glass jacket until the receiving flask contained about 20ml

ether and fat extracted. Then the receiving flask was heated to evaporate the solvent.

The percentage of ether extract was calculated by the following formula:

Ether extract (%)¿Lossof weight of sample

weight of sample∗100

3.13.4 Crude fiber

Two grams of moisture free sample (feed and/or excreta) was made fat free

(ether extracted) and was taken in a 600ml beaker provided with a reflux condenser.

The sample (feed and/or excreta) was digested at simmering temperature ( 80 °C) with

200 ml of 1.25 % H2SO4 solution for 30 minutes, which hydrolyzed the protein and

carbohydrate. The volume of simmering medium was kept constant by frequent addition

83

of hot water. The contents left were filtered immediately/instantly under vacuum and

the residue remaining after filtration was washed with H 2O and transferred to 600ml

glass beaker. The residue was digested with 1.25 % NaOH simmering solution for

exactly 30 minutes. Again the residue left was washed and filtered in similar way. The

contents were shifted to an oven at l00oC for drying to a constant weight. The dried

residue was ignited in a muffle furnace at a temperature of 550 oC for 20 minutes.

Residue left after ignition was weighed and loss of weight of original sample is reported

as crude fiber. Crude fiber calculated by using the following formula.

Weight of residueCrude fiber (%) = ––––––––––––––––––––– x 100

Weight of sample

3.13.5 Acid Insoluble Ash (AIA)

The AIA was determined using the method of Van Keuien and Young (1977) as follows:

Five gram dried ground sample of feed and/orexcreta were washed overnight at 450

°C.

The ash was transferred/shifted into a conical flask and 100ml of 2N HCl was added.

The mixture obtained was thus boiled for 5 minutes on a crude fiber digestion

apparatus. A condenser was attached to the flask to avoid the loss of HCl.

The hot hydrolysate was then filtered and washed free of acid with hot distilled water.

The ash and filter paper were then transferred/shifted back into the crucible and kept

for conversion into ash, overnight at 450oC.

The crucibles, along with its contents were cooled in a desicator to room temperature/

weighed having ash and reweighed instantly after emptying.

The percentage of AIA was calculated using the following formula!

Wf - We AIA% = ––––––––––––––––––––––––– × 100 WsWhere,

Wf = weight of crucible with ash

We = weight of empty crucible

84

Ws = weight of sample:

3.14 Mineral analysisBefore the estimation of minerals (Ca, P, Na, K, Mg and Fe), the fecal and/or feed

samples were dried for 24 hours. The dried samples were ground in Willy mill and sieved

with stainless steel sieve and stored in air tight containers.

3.14.1 Wet digestion

One gram of dried and ground excreta and/or feed sample was taken into 150 ml

Pyrex beaker, and soaked thoroughly with 10 ml of concentrated Nitric acid (HNO3). Then

3ml of 60 percent per chloric acid (HClO4) was added. The contents were heated slowly on a

hot plate, until frothing ceased and then heated gently with another 10ml of HNO3. The

heating continued/persistent until brown fumes of nitric acid came to an end and white fumes

of HClO 4appeared. The beaker was cooled and the contents were dissolved in 10ml of 6 N

HCl and transferred to 100ml volumetric flask quantitatively and volume was made up with

de-ionized water. Concentration of various mineral elements were determined/found as

described by Smith et al. (1979) by atomic absorption spectrophotometer model 170-10

(Hitachi Ltd.) using air acetylene flame at spectral line 213.9nm. While, sodium and

potassium were determined on Flame Photometer using corning (EEL Model) flame

photometer according to the method as described by AOAC (20100).

3.14.2 Determination of calcium

Calcium was estimated by titration method using micro burette (EDTA-verenuate).

Digested eggshell sample was titrated against 0.01 N Ethylinediaminetetraacetate (EDTA)

solution (Allison et al . , 1954). 0.01 N EDTA was prepared by dissolving 2gm of sodium

salt of EDTA in 1 liter of distilled water and added one crystal of MgCl2. 2.5gm of digested

material was taken in a china dish and added 20ml of distilled water along with 8-10 drops of

4N NaOH. Stirred it with glass rod and added 50gm of ammonium purpurate i.e.(4.5gm

ammonium purpurate and 100gm K2SO4 were ground to form homogeneous mixture) stirred

with glass rod. Pink color was obtained. Titrated it against 0.01 N EDTA up to purple color

end point and then recorded the reading of EDTA used for calculations.

Calculation Volume of sample (feed and/or excreta) solution taken = 10 ml

85

Strength of calcium/L = N× Eq. weight, = Bgm/L

1000ml of sample solution contain (Ca ) = Bgm/L

100 ml of calcium solution contain calcium = Bgm/1000 ×100 = A%

3.14.3 Determination of phosphorus

Three test tubes were taken and these solutions were treated with reagents as

illustrated.

86

Pipetted scheme

Reagents Test (ml) Standard (ml) Blank (ml)

Sample solution (Wet

digested)

1.0 ------ -------

Standard solution of P1 ---------- 1.0 --------

Ammonium molibidate 1.0 1.0 1.0

Aminonapthol

sulfonate 3

0.4 0.4 0.4

Distilled water 7.6 7.6 8.6

Standard phosphateThe contents of test tubes were finely quivered/shaken and then permitted to settle for

the minutes, when blue color appeared in them, the absorbance was read on HITACIII U

1100 spectrophotometer at 720 nm after setting the zero with blanks.

The amount of total Phosphorousin feed and/or feces was calculated by using the following

formula:Phosphorusmg /100 ml=A / B× 0.08

Where,

A = absorbance of sample solution

B= absorbance of standard solution

3.14.4 Determination of Sodium and Potassium by Flame PhotometerAfter wet digestion of the sample(feed and/or excreta), a standard solution of sodium

or potassium was prepared for running in flame analyzer. Recorded the readings of the

standard and sample solutions by the flame analyzer and made the calculations accordingly.

Calculation Reading of standardsolution = X

Reading of sample solution = Y

X reading is due to presence of = 10mg/100ml

Y reading is due to presence of = 10X

×Y = A mgof sodium∨potassium

100 ml

500 mg of feed/or feces contain = A mg of sodium or potassium

% of sodium or potassium in feed/or feces = A

500×100

87

CHAPTER-4

RESULTS AND DISCUSSIONEFFECT OF DIETARY INCLUSION OF SODIUM BICARBONATE ON

PRODUCTION PERFORMANCE AND BLOOD PROFILE OF CAGED

LAYERS DURING SUMMER

4 RESULTS

4.1 Production performanceMeans and their standard deviation values of initial body weight, final body weight,

weight gain and feed consumption of the birds fed diets with or without addition of different

levels of sodium bicarbonate are shown in table 4.1.

4.1.1 Live body weight

Mean values for body weight gain of the layers were found to be 166, 187, 199, 177

and 169g for treatment groups A, B, C, D and E, respectively. Weight gain of the layers was

markedly influenced due to dietary addition of sodium bicarbonate in the layer diets.

Statistical analysis of the data showed that birds using diets containing 0.5 and 1% sodium

bicarbonate gained significantly (P<0.05) more weight as compared to those of groups D, E

and control. Disparity in weight gain among the treated groups was also found to be

significant. Birds of group C, which were fed diet having 1% sodium bicarbonate, exhibited

maximum body weight when compared to the birds of other treated groups, followed by

those of group B, D and E. However, difference in weight gain between groups A, D and E

was non-significant. Similarly, differences in weight gain between treated groups B and C

were also found to be non-significant.

4.1.2 Feed consumption

Mean weekly feed consumption values were 743, 770, 780, 766 and 755 g/bird for

treatment A, B, C, D and E, respectively. Feed consumption of layer birds was markedly

influenced due to dietary addition of sodium bicarbonate. Statistical analysis of the data

showed that birds receiving diets containing sodium bicarbonate consumed significantly

(P<0.05) more feed than those of control (group A). Differences in feed consumption among

treated group were also found to be significant. Birds of group C, which were fed diet

containing 1% sodium bicarbonate showed maximum

88

Table: 4.1 Effect of dietary inclusion of sodium bicarbonate (NaHCO3) on weight gain

and feed consumption caged layers

Variables

Treatment

A

Control

B

0.5%NaHCO3

C

1%NaHCO3

D

1.5%NaHCO3

E

2%NaHCO3

Initial body

weight (g)1328±14.3 1310 ±7.0 1318 ±16.4 1324 ±11.0 1312 ±12.1

Final body weight

(g) 1494±60.9 1497±53.4 1517±36.7 1501±24.6 1481±61.0

Weight gain (g) 166±11.4c 187±6.4ab 199±15.1a 177±9.3bc 169±13.5c

Feed

consumption (g)743±12.90 770±10.45b 780±14.84a 766±10.51b 755±12.16c

Values within the same row having different superscripts are significantly (P<0.05) different.

89

feed intake followed by those of group B and E among treated groups. Whereas, the lowest

feed intake was observed in the layers of control group. On the other hand, difference in feed

consumption was found to be non-significant between treated groups B and D.

4.1.3 Egg production

Means and their standard deviation values of weekly egg production, egg weight, egg

mass, FCR per kg egg mass produced and FCR per dozen eggs produced by the birds fed

diets with or without addition of different levels of sodium bicarbonate are presented in table

4.2.

Mean values of weekly egg production were 5.33, 5.63, 5.86, 5.51 and 5.42 eggs/bird

for treatment A, B, C, D and E, respectively. Egg production of layers was markedly

influenced due to dietary inclusion/addition of sodium bicarbonate in the layer diets.

Statistical analysis of the data showed that birds consuming diets containing sodium

bicarbonate produced more eggs (P<0.05) as compared to those of control group.

Differences in egg production among treated group were also found to be significant. Layer

birds which were fed diet containing 1% sodium bicarbonate, showed maximum egg

production when compared to the birds of other treated groups. However, differences in egg

production between treated groups D and E were non-significant (P>0.05).

4.1.4 Egg weight

Mean values for egg weight were 50.9, 54.0, 58.2, 56.0 and 55.2 g, for treatment A,

B, C, D and E, respectively. Egg weight of layers was significantly influenced due to dietary

inclusion of sodium bicarbonate in the layer diets. Statistical analysis of the data showed that

birds consuming diets containing sodium bicarbonate produced significantly (P<0.05)

heavier eggs as compared to those of control group. Birds of group C, which were fed diet

containing 1% sodium bicarbonate, showed maximum egg weight when compared to the

birds of other treated groups. However, the differences in egg weight among treated groups D

and E were found to be non significant.

4.1.5 Egg massMean values for weekly eggs mass produced were found to be 267, 303, 339, 307 and

299 g for treatments A, B, C, D and E, respectively. Statistical analysis regarding egg mass

90

produced by each replicate showed that inclusion of NaHCO3 in the diet of layers has

91

Table 4.2: Effect of dietary inclusion of sodium bicarbonate on production

performance of caged layers

Variables

Treatment

A

Control

B

0.5%NaHCO3

C

1%NaHCO3

D

1.5%NaHCO3

E

2%NaHCO3

Egg Prod.(Nos./week)

5

.33±0.20

5.63±0.21b 5.8

6±0.36a5.51±.12c 5.4

2±0.51c

Average egg weight

(g) 50.9±4.0 54.0±3.5c58.

2±1.50a 56.0±1.1b 55.2±1.6b

Egg mass produced

(g/week)

267±10.8 303±13.0b 339±12.6a 307±11.2b 299±17.5b

FCR/ dozen eggs 1

.70±0.052

1.6

4±0.050c

1.6

0±0.051d1.67±0.020b 1.6

7±0.021b

FCR/kg egg mass 2.78

±0.012a

2.54

±0.025b

2.3

0±0.015c2.48 ±0.051b 2.5

2±0.051b

Values within the same row bearing dissimilar superscripts are significantly different (P<0.05)

92

significant positive effect (P<0.05) on egg mass produced by the addition of sodium bicarbonate

in the diet. Difference in mean values of egg mass produced between sodium bicarbonate treated

groups was significant. The birds in treatment group C, consuming diet containing sodium

carbonate at 1% level produced more egg mass than the other treatment groups. However, the

difference in egg mass produced among treated groups B, D and E was found to be non-

significant.

4.1.6 Feed efficiency1. FCR/dozen eggs

Mean feed conversion ratio on the basis of per dozen eggs produced was found to

be1.70, 1.64, 1.60, 1.67 and 1.67 for treatments A, B, C, D and E, respectively. Feed

conversion ratio on the basis of per dozen eggs produced was significantly affected due to

dietary inclusion of NaHCO3 in the diets. Statistical analysis of the data showed that birds

consuming diets containing sodium bicarbonate showed significantly (P<0.05) improved feed

conversion ratio on the basis of per dozen eggs produced as compared to those of control

group. Differences in FCR/ dozen eggs produced among treated groups were also found to be

noteworthy (P<0.05). Birds of group C, which were fed diet containing 1% sodium

bicarbonate, showed better FCR/ dozen eggs produced when compared to the birds of other

treated groups. Whereas, the poor FCR was noted in the birds of control group. However,

differences in the FCR values among treated groups D and E were found to be inconsistent

(P>0.05).

2. FCR/Kg egg mass producedMeans feed conversion ratios on the basis of per kg eggs mass produced were found

to be 2.78, 2.54, 2.30, 2.48 and 2.52 for treatments A, B, C, D and E, respectively. Feed

conversion ratio on the basis of per kg egg mass produced by the layers was significantly

influenced by the dietary addition of NaHCO3. Statistical analysis of the data showed that

birds consuming diets containing sodium bicarbonate showed significantly (P<0.05)

93

improved FCR/Kg egg mass produced than those of control group. The birds in treatment

group C (1% sodium bicarbonate added to the diet) utilized their feed more efficiently than

the other treatment groups. Difference in mean values of FCR/Kg egg mass produced

between sodium bicarbonate treated groups was also significant. Whereas, the poorest

FCR/Kg egg mass produced was observed in the layers of group A (control). However, the

differences in FCR/Kg egg mass produced among treated groups B, D and E were found to

be non-significant.

4.2 Egg qualityMeans and their standard deviation values, of specific gravity, shell thickness,

albumen height, Haugh unit score, yolk diameter, yolk height, yolk index, yolk cholesterol,

yolk pH and albumen pH are shown in table 4.3.

4.2.1 Specific gravity

Mean specific gravity values of the eggs produced by the layers were 1.077, 1.081,

1.086, 1086 and 1.079 for treatment A, B, C, D and E, respectively. Specific gravity values

of the eggs produced by the birds were significantly influenced due to dietary inclusion of

sodium bicarbonate in their diets. Statistical analysis of the data showed that birds consuming

diets containing sodium bicarbonate produced eggs, which had more specific gravity values

(P<0.05) than those of control group. However, the differences in specific gravity values

among treated groups C, D; B, E and A, E were found to be non-significant. Specific gravity

of the eggs produced by the birds of group C, which were fed diet containing 1% sodium

bicarbonate, was found to be higher followed by those of treated groups D, E and B.

4.2.2 Shell thickness

Mean values pertaining to shell thickness of the eggs produced by the layers were

found to be 0.307, 0.335, 0.334, 0.336 and 0.34 for treatment groups A, B, C, D and E,

respectively. Shell thickness values of the eggs produced by the birds were markedly

influenced due to the dietary inclusion of NaHCO3 in their diets. Statistical analysis revealed

that shell thickness of the eggs produced by the birds consuming diets containing sodium

bicarbonate increased (P<0.05) when compared to those of control group. However,

differences in shell thickness among treated groups B, C, D and C, D, E were found to be

non-significant. Birds of group C, which were fed diet containing 1% sodium bicarbonate,

apparently but non-significantly showed maximum shell thickness when compared to the

94

birds of other treated groups. The eggs produced by the layer of control group showed the

minimum value of shell thickness of their eggs than all those of treated groups.

Table 4.3: Effect of dietary inclusion of NaHCO3 on egg quality characteristics of caged

layers

Variables

Treatment

A

Control diet

B

0.5%NaHCO3

C

1%NaHCO3

D

1.5%NaHCO3

E

2%NaHCO3

Specific gravity 1.077±0.0301.081±0.012b 1.086±0.020a 1.086±0.020a 1.079±.02

8bc

Shell thickness (mm)

0

.307±0.0132

0.335±0.010

4b0.334±0.011

9ab0.336±.010

9ab0.34±0.011

0a

Albumen height (mm) 4.39±0.1074.71±0.101b 4.97±0.123a 4.6±0.150bc 4.46±0.13

0cd

Haugh unit 66.9±1.25 68.7±0.73 ab 70.2±0.97a 68±1.29bc 67.2±1.15bc

Yolk diameter (mm) 33.4±1.00 34.5±1.14b 36.6±1.55a 33.9±1.20b 33.4±1.02b

Yolk height (mm) 13.3±0.37 14.2±0.26a 14.5±0.28a 13.8±0.20b 13.49±0.1

4bc

Yolk index0.404±0.01380.401±0.0141 0.393±0.0140 0.408±0.0108 0.401±0.010

1

Yolk Cholesterol

mg/egg

243±5.9a 214±13.7b 193±15.0c 191±17.0c 185±6.4c

Yolk pH 6.8±0.22b 6.8±0.56b 7.1±0.18ab 7.3±0.16a 7.4±0.17a

Albumen pH 6.9±0.32c 7.3±0.29bc 7.7±0.26ab 7.6±0.24ab 7.8±0.21a

95

Values within the same row which have different superscripts letter are significantly different (P<0.05)

4.2.3 Albumen height

Mean values pertaining to albumen height were found to be 4.39, 4.71, 4.97, 4.6 and

4.46 mm for treatment groups A, B, C, D and E, respectively. Albumen height of the eggs

produced by the birds was significantly influenced due to dietary inclusion of NaHCO3 in

their diets. Statistical analysis of the data showed that birds using diets containing sodium

bicarbonate produced eggs having significantly (P<0.05) more albumen height as compared

to those of control group.

Differences in albumen height among treated group were also found to be significant.

Birds of group C, which were offered diet containing 1% sodium bicarbonate, showed

maximum albumen height when compared to the eggs produced by the birds of other treated

groups. However, the differences in albumen height among groups A, E; B, D and D, E were

not observed (P>0.05).

4.2.4 Haugh unit

Mean values pertaining to Haugh unit score were found to be 66.9, 68.7, 70.2, 68.0

and 67.2 for the treatment groups A, B, C, D and E, respectively. Haugh unit score of the

eggs produced by the birds was significantly influenced due to dietary addition of sodium

bicarbonate in their diets. Statistical analysis of the data showed that birds using diets

containing sodium bicarbonate produced eggs having significantly (P<0.05) greater Haugh

unit score as compared to those of control group. Differences in Haugh unit score among

treated groups were also found to be significant. Birds of group C, which were fed diet

containing 1% sodium bicarbonate, showed maximum Haugh unit score when compared to

the eggs produced by the birds of other treated groups. However, differences in Haugh unit

score between treated groups, B, C and D, E were found to be inconsistent (P>0.05).

Differences in Haugh unit values among groups A, D and E were also found to be

inconsistent (P>0.05).

4.2.5 Yolk diameter

96

Mean values pertaining to yolk diameter were found to be 33.4, 34.5, 36.6, 33.9 and

33.4 mm for the treatment groups A, B, C, D and E, respectively. Yolk diameter of the eggs

produced by the birds was significantly influenced due to dietary inclusion of NaHCO3 in

their diets. Statistical analysis of the data showed that birds using diets containing sodium

bicarbonate produced eggs having significantly (P<0.05) more yolk diameter as compared to

those of control group. Differences in yolk diameter among treated groups were also found to

be significant. Birds of group C, which were fed diet containing 1% sodium bicarbonate,

showed maximum yolk diameter when compared to the eggs produced by the birds of other

treated groups. However, the differences in yolk diameter among treated group, B, D and E

were found to be non-significant (P>0.05).

4.2.6 Yolk height

Mean values pertaining to yolk height were found to be 13.3, 14.2, 14.5, 13.8 and

13.49 mm for the treatment A, B, C, D and E, respectively. Yolk height values of the eggs

produced were significantly affected due to dietary inclusion of sodium bicarbonate in the

layer diets. Statistical analysis of the data showed that birds using diets containing sodium

bicarbonate produced eggs having higher (P<0.05) yolk height values as compared to those

of control group. Differences in yolk height among treated groups were also found to be

significant. Birds of group C, which were fed diet containing 1% sodium bicarbonate,

showed maximum yolk height values when compared to the birds of other treated groups.

However, the differences in these values among treated groups D and E were found to be

non-significant (P>0.05). Similarly, differences in yolk heights between group A and E were

found to be non-significant.

4.2.7 Yolk Index

Mean values pertaining to yolk index of the eggs produced were found to be 0.404,

0.401, 0393, 0.408 and 0.401 for the treatments A, B, C, D and E, respectively. Yolk index of

eggs was non-significantly influenced due to dietary inclusion of sodium bicarbonate in the

layer diets. Statistical analysis of the data showed that birds using diets containing sodium

bicarbonate produced eggs having apparently but inconsistently (P>0.05) slightly higher yolk

index as compared to those of control group.

97

4.2.8 Egg yolk cholesterol

Mean values regarding egg yolk cholesterol concentration for treatment groups A, B,

C, D and E, were found to be 243, 214, 193, 192 and 185 mg/egg, respectively.

Concentration of yolk cholesterol in the eggs produced by the birds of treated group was

significantly influenced due to addition of NaHCO3 in their diets. Statistical analysis of the

data showed that birds using diets containing sodium bicarbonate produced eggs having

lower (P<0.05) yolk cholesterol as compared to those of control group. Differences in yolk

cholesterol concentration among treated groups fed diets having various levels of sodium

bicarbonate were also found to be significant. Birds of group E, which were fed, diet

containing 2% sodium bicarbonate, produced eggs having lowest concentration of yolk

cholesterol when compared to those of other treated groups. However, difference in egg yolk

cholesterol between treated groups C, D and E was found to be non-significant.

4.2.9 Yolk pHMean values regarding egg yolk pH for treatment groups A, B, C, D and E, were

found to be 6.8, 6.8, 7.1, 7.3 and 7.4, respectively. Yolk pH of the eggs produced by the birds

in treated groups was significantly influenced due to addition of NHCO3 in their diets.

Statistical analysis of the data showed that birds using diets containing sodium bicarbonate

produced eggs having higher (P<0.05) egg yolk pH as compared to those of control group.

Differences in yolk pH values among treated groups fed diets containing different levels of

sodium bicarbonate were also found to be significant. Birds of group E which were fed diet

containing 2.0% sodium bicarbonate, produced eggs having maximum yolk pH value when

compared to those of other treated groups. The pH value in the treated group showed a linear

increase with increase in the level of sodium bicarbonate used. However, difference in egg

yolk pH value between treated groups B and C and among those of C, D, and E were not

found to be significant (P>0.05). The eggs produced by the birds of control group exhibited

the lowest pH value in their yolks.

98

4.2.10 Albumen pHMean values regarding egg albumen pH for treatment groups A, B, C, D and E, were

found to be 6.9, 7.3, 7.7, 7.6 and 7.8, respectively. Albumen pH of the eggs produced by the

birds in treated groups was significantly influenced due to addition of NaHCO3 in their diets.

Statistical analysis of the data showed that birds using diets containing sodium bicarbonate

produced eggs having higher (P<0.05) albumen pH as compared to those of group A

(control). The pH values in the treated groups showed a linear increase with increase in the

level of sodium bicarbonate used in the diets. However, differences in albumen pH among

treated groups were also found to be significant. Birds of group E, which were fed diet

containing 2% sodium bicarbonate, showed maximum albumen pH values when compared to

the birds of other treated groups. However, the differences in albumen pH values among treated

groups C, D and E and those of B, C and D were found to be non-significant.

99

4.2.11 Meat and blood spotsThe percentage of meat and blood spots observed in the eggs laid by the birds of

control was found to be 2.5% and 1.25%, respectively. Blood and meat spots were found to

be present only in the eggs laid by the birds of control group, which were fed diet without

addition of sodium bicarbonate, whilst treated groups were devoid of these. It has been

observed that higher ambient temperature is positively correlated with the incidence of meat

and blood spots in laying hens (Anjum, 2000). However, results of this study did not exhibit

presence of any blood or meat spots in the birds fed diet containing different levels of sodium

bicarbonate.

4.3 Rectal temperature, respiration rate and water consumption

Means and their standard deviation values of rectal/body temperature,

respiration rate and water consumption of layers are presented in table 4.4.4.3.1 Rectal temperature

Mean values regarding rectal temperature of the birds kept under treatment groups A,

B, C, D and E, were found to be 106.7, 106.39, 106.37, 106.41 and 106.38oC, respectively.

Rectal temperature of the birds kept under treated groups was significantly influenced due to

addition of sodium bicarbonate in their diets. Statistical analysis of the data revealed that

birds using diets containing sodium bicarbonate exhibited significantly (P<0.05) lower rectal

temperature as compared to those of control group. However, difference in rectal temperature

among the birds of treated groups fed diets containing different levels of sodium bicarbonate

was found to be non-significant. Rectal temperature of the birds kept under treated groups

exhibited a linear decrease with increase in the level of sodium bicarbonate used. Birds of

group C which were fed diet containing 1% sodium bicarbonate apparently experienced

lowest rectal temperature when compared to those of other treated groups. However, the

highest rectal temperature was observed in the birds of control group.

4.3.2 Respiration rate

Mean values pertaining to respiration rate of the layers for treatment groups A, B, C, D and E

were found to be 57.04, 53.46, 47.74, 51.3 and 50.5 respiration/minute, respectively.

100

Table 4.4: Effect of dietary inclusion of sodium bicarbonate on rectal temperature,

respiration rate and water consumption of caged layers

Variables

Treatment

A

Control

B

0.5%NaHC

O3

C

1%NaHC

O3

D

1.5%NaHC

O3

E

2%NaHCO3

Rectal

temperature

(0F)106.75±5.81106.39±4.43b

106.20±4.

2b

106.41±3.14

b

106.38±2.10

b

Respiration

rate (per

minute)57.04±1.7 53.46±3.18b 47.74±3.9d 51.30±4.2bc 50.5±4.1c

Water intake

(ml/day)333±9.51d 380±14.6 c 427±9.7 b 446±10 ab 467±8.7a

Values within the same row having different superscripts letter are significantly different (P<0.05)

101

Finding of the study depicted that respiration rate of the birds was significantly

influenced due to dietary inclusion of NaHCO3 in their diets. Statistical analysis of the data

revealed that birds using diets containing sodium bicarbonate exhibited lower respiration rate

(P<0.05) when compared to those of group A (control). Differences in respiration rate among

the birds of treated groups were also found to be significant. Birds of group C, which were

fed diet containing 1% sodium bicarbonate, showed minimum respiration rate values when

compared to the birds of other treated groups. Similarly the differences in respiration rate

among treated groups D and E was also non-significant (P>0.05). Heart rate quality of all the

birds was found to be normal and satisfactory and no abnormal sound was observed.

4.3.3 Water intakeMean values pertaining to daily water consumption were found to be 333, 380, 427,

446 and 467 ml/bird/day for treatment A, B, C, D and E, respectively. Water consumption of

the layers was significantly influenced due to dietary inclusion of NaHCO3 in their diets.

Statistical analysis of the data showed that birds using diets containing sodium bicarbonate

took more water (P<0.05) as compared to those of control group. There was a linear increase

in water consumption of the birds with increase in the level of NaHCO3 in the diets. Birds of

group E, which were fed diet containing (2%) sodium bicarbonate, took maximum water as

compared to the birds of other treated groups. Differences in water intake among sodium

bicarbonate treated groups were also found to be significant. However, differences in water

consumption among treated groups D and E, and those of groups C and D were found to be

non-significant.

4.4 MortalityIncidence of mortality was zero in all groups. It may probably be due to the reason

that the experiment was conducted under the best possible controlled hygienic conditions

except ambient temperature and the birds were kept well observed.

4.5 Hematological profileMeans and their standard deviation values, regarding serum glucose level, packed cell

102

volume (PCV), blood hemoglobin (Hb), erythrocyte sedimentation rate (ESR), red blood

cells count (RBCs) and white blood cells count (WBCs) are given in table 4.5.

Table 4.5: Effect of dietary addition of sodium bicarbonate on hematological profile of

caged layers

Variables

Treatment

A

Control

B

0.5%NaHCO3

C

1%NaHCO3

D

1.5%NaHCO3

E

2%NaHCO3

Glucose

(mg/dl)

21

2.7±17.84a

200.2±17.4

0b195.3±5.91b 186.6±5.88c

174.7±10.5

1d

PCV (%) 33±3.56 32.5±2.46 32.7±2.99 32±2.16 32.7±2.63

Hb (mg/dl) 9.72±0.84610.53±0.89

6ab 11.37±0.675a10.58±1.16

0ab

10.75±1.320

ab

ESR

(mm/hour)3.93±0.29 3.75±0.44 3.7±1.04 3.73±0.62 3.7±0.52

RBCs

count

(106/mm3)

2.53±0.522.73±0.45 2.81±0.53 2.69±0.67 2.66±0.18

WBCs

count

(103/mm3)

26.5±1.21 22.5±2.80b 24±2.73ab 25±2.51ab 26.2±1.00a

Means within the same row having dissimilar superscripts letter are significantly different (P<0.05)

103

104

4.5.1 Serum glucose

Mean values pertaining to serum glucose level of the layers for treatment groups were

found to be 212.7, 200.2, 195.3, 186.6 and 174.7 mg/dl, respectively. Findings of the study

depicted that glucose level of birds was significantly affected due to dietary

addition/inclusion of sodium bicarbonate in the layer diets. Statistical analysis of the data

revealed that birds consuming diets containing sodium bicarbonate had reduced (P<0.05)

serum glucose level when compared to those of control group. Disparity in glucose level due

to the use of different levels of sodium bicarbonate was also found to be significant among

the birds kept under treated groups. The birds of group E, which were fed diet containing 2%

sodium bicarbonate, showed minimum serum glucose level when compared to the birds of

other treated groups. Yet, the difference in glucose level between groups B and C were non-

significant. Similarly the differences in glucose level between groups C and D were also

known to be non-significant. Maximum level of serum glucose was noted in the blood of the

layers kept under control group.

4.5.2 Packed cell volume (PCV)

Mean values of PCV in blood of the layers were found to be 33, 32.5, 32.7, 32.0 and

32.7% for treatment A, B, C, D and E, respectively. Statistical analysis of the data exposed

insignificant difference among the birds of all groups, indicating no effect on packed cell

volume of the layers due to dietary inclusion of NaHCO3 in their diets. However, birds of

group E, which were fed diet containing 2% sodium bicarbonate, apparently but non

significantly exhibited minimum packed cell volume among the birds of all treatment groups.

4.5.3 Blood hemoglobin

Mean values pertaining to blood hemoglobin of the layers were found to be 9.72,

10.53, 11.37, 10.58 and 10.75 mg/dl for treatment A, B, C, D and E, respectively. Statistical

analysis of the data showed significant difference in hemoglobin values among the birds of

all treatment groups, indicating improved effect on blood hemoglobin value of the layers due

to inclusion of NaHCO3 in their diets. Birds of group C, which were offered feed containing

1% sodium bicarbonate, significantly showed maximum concentration of hemoglobin among

the birds of all treatment groups. However, the difference in glucose level between treated

groups B and C, D and E were found to be insignificant (P>0.05). Similarly the differences in

glucose level between groups A, B, D and E were also found to be insignificant.

105

4.5.4 Erythrocytes sedimentation rate

Mean values regarding to erythrocytes sedimentation rate (ESR) of the layers were

found to be 3.93, 3.75, 3.7, 3.73 and 3.7 mm/ hour for treatments A, B, C, D and E,

respectively. Statistical analysis of the data showed non-significant difference in hemoglobin

values among the birds of all treatment groups, indicating no effect on erythrocytes

sedimentation rate of the layers due to dietary inclusion of NaHCO3 (sodium bicarbonate) in

their diets. However, birds of group A (control) apparently but non significantly exhibited

higher erythrocytes sedimentation rate among the birds of all other groups.

4.5.5 Red blood cells count

Mean values pertaining to red blood cells count in blood of the layers were found to

be 2.53, 2.73, 2.81, 2.69 and 2.66 ×106/mm3for treatment A, B, C, D and E, respectively.

Statistical analysis of the data showed insignificant difference among the birds of all groups,

indicating no effect on red blood cells count of the layers due to dietary inclusion of sodium

bicarbonate in their diets. However, layers of group C, which were offered, diet containing

1% sodium bicarbonate, apparently but non significantly showed maximum red blood cells

count among the birds of all treatment groups.

4.5.6 White blood cell count (WBCs)

Mean values for white blood cells count were 26.5, 22.5, 24, 25 and 26.2 ×103/mm3

for treatment A, B, C, D and E, respectively. White blood cells count was affected due to

dietary inclusion of sodium bicarbonate. Statistical analysis of the data showed that birds

using diets containing sodium bicarbonate had significant (P<0.05) effect on white blood

cells WBCs) count as compared to those of group A. Birds of group B, which were fed diet

containing 0.5% sodium bicarbonate, exhibited minimum white blood cells (WBC) count

when compared to the birds of other treated groups. However, the difference in the result of

white blood cells count among groups A, C, D and E were insignificant. Similarly the

differences in white blood cells count among groups B, C and D were also found to be

insignificant.

4.6 Serum metabolitesMeans and their standard deviation values regarding serum urea, serum uric acid, serum

creatinine and serum alkaline phosphatase levels are presented in table 4.6

106

4.6.1 Serum urea

Mean values relevant to serum urea level were 12.33, 8.96, 8.58, 9.57 and 11.35

mg/dl for treatment A, B, C, D and E, respectively. Serum urea level values of layers were

significantly affected due to dietary inclusion of sodium bicarbonate in the diets. Statistical

analysis of the data showed that birds using diets containing NaHCO3 had less (P<0.05)

serum urea level as compared to those of control group. Variations in serum urea level

among treated group were also found to be noteworthy (P<0.05). Birds of group C, which

were fed diet containing 1% sodium bicarbonate, showed minimum serum urea level when

compared to the birds of other treated groups. However, the differences regarding the serum

urea level among groups B, C and D were found to be insignificant. Similarly the disparity in

serum urea level among groups A and E were found to be insignificant (P>0.05).

4.6.2 Serum uric acid

Mean values regarding serum uric acid level were found to be 5.9, 6.52, 6.62, 7.6 and

7.8mg/dl for treatment A, B, C, D and E, respectively. Serum uric acid values of layers were

significantly affected due to dietary addition/inclusion of NaHCO3 in the diets. Statistical

analysis of the data showed that birds using diets containing sodium bicarbonate had

significantly (P<0.05) less serum uric acid level as compared to those of control group.

Difference in serum uric acid level among groups was also found to be noteworthy (P<0.05).

Birds of group E, which were fed diet containing 2% sodium bicarbonate, showed maximum

serum uric acid level when compared to the birds of other treated groups. However, the

differences in serum uric acid level among groups A, B and C were found to be non-

significant. Similarly the differences in serum uric acid level among groups D and E were

also found to be insignificant (P>0.05).

107

Table 4.6: Effect of dietary inclusion of sodium bicarbonate on serum metabolites of

caged layers

Variables

Treatment

A

Control

B

0.5%NaHCO3

C

1%NaHCO3

D

1.5%NaHCO3

E

2%NaHCO3

Serum urea

mg/dl12.33±.718.96±1.31b 8.58±0.92b 9.57±1.02b 11.35±0.81a

Serum uric

acid

mg/dl

5.9±0.706.52±0.62b 6.62±0.45b 7.6±0.56a 7.8±0.78a

Serum

creatinine

mg/dl

0.77±0.050.76±0.05 0.69±0.09 0.72±0.08 0.75±0.04

Alkaline

Phosphatase

mg/dl

14.0±0.66 13.62±0.47 13.12±0.62 13.17±0.88 13.77±0.26

Mean values within the same row with dissimilar superscripts are significantly different (P<0.05)

108

4.6.3 Serum creatinine

Mean values pertaining to creatinine concentration in blood of the layers were found

to be 0.77, 0.76, 0.69, 0.72 and 0.75mg/dl for treatment A, B, C, D and E, respectively.

Statistical analysis of the data showed insignificant (P>0.05) difference among the birds of

all treatment groups, indicating no effect on creatinine concentration of the layers due to

dietary inclusion of sodium bicarbonate in their diets. However, birds of group C, which

were fed diet containing 1% sodium bicarbonate, apparently but non significantly showed

minimum creatinine concentration among the birds of all treatment groups.

4.6.4 Serum alkaline phosphatase

Mean values pertaining to serum alkaline phosphatase level in blood of the layers

were found to be 14.0, 13.62, 13.12, 12.77 and 14.77mg/dl for treatment A, B, C, D and E,

respectively. Statistical analysis of the data revealed non-significant difference among the

birds of all treatment groups, indicating no effect on alkaline phosphatase level of the layers

due to dietary inclusion of sodium bicarbonate in their diets. However, birds of group C,

which were fed diet containing 1% sodium bicarbonate, apparently but non significantly

showed minimum alkaline phosphatase level among the birds of all treatment groups.

4.7 Serum proteins analysisMeans and their standard deviation values regarding serum total proteins, serum

albumen and serum globulin concentration are presented in table 4.7.

4.7.1 Total proteins

Mean values regarding total protein were found to be 4.97, 6.13, 6.53, 6.02 and

5.05mg/dl for treatment A, B, C, D and E, respectively. Total protein concentration was

affected due to dietary addition/inclusion of sodium bicarbonate in the layer diets. Statistical

analysis of the data showed that birds using diets containing sodium bicarbonate had

significant (P<0.05) effect on total protein as compared to those of control group. Birds of

group C, which were fed diet containing 1% sodium bicarbonate, showed apparently

maximum total protein when compared to the birds of other treated groups. However, the

differences in

109

Table 4.7 Effect of dietary inclusion of sodium bicarbonate on serum proteins

concentration of caged layers

Variables

Treatment

A

Control

B

0.5%NaHCO3

C

1%NaHCO3

D

1.5%NaHCO3

E

2%NaHCO3

Total protein

(mg/dl) 4.97±0.59 6.13±0.65a 6.53±0.58a 6.02±0.50ab 5.05±.49b

Albumin

(mg/dl)3.35±0.26 3.82±.26ab 3.9±0.53a 3.5±0.15ab

3.41±0.2

9ab

Globulin

(mg/dl)1.62±0.18 2.31±0.32 2.63±0.62 2.52±0.15 1.63±0.13

Values within the same row which have unlike superscripts are different significantly (P<0.05)

110

serum total protein level among the groups B, C and D were found to be insignificant

(P>0.05). Similarly the differences in serum total proteins among treated groups A and E

were also found to be insignificant P>0.05).

4.7.2 Albumin

Mean values pertaining to serum albumin concentration were found to be 3.35, 3.82,

3.9, 3.5 and 3.41 mg/dl for treatment A, B, C, D and E, respectively. Albumin concentration

was affected due to dietary addition/inclusion of sodium bicarbonate in the layer diets.

Statistical analysis of the data showed that birds using diets containing sodium bicarbonate

had significant effect (P<0.05) on albumin as compared to those of group A.

Birds of group C, which were provided diet containing 1% sodium bicarbonate,

showed apparently maximum serum albumin concentration when compared to the birds of

other treated groups. However, the differences in serum albumin protein level among groups

B, C, D and E were found to be non-significant.

4.7.3 Globulin

Mean values pertaining to globulin concentration in blood of the layers were found to

be 1.62, 2.31, 2.63, 2.52 and 1.63 mg/dl for treatment A, B, C, D and E, respectively. Data

obtained when statistically analyzed revealed non-significant difference among the birds of al

groups, indicating no effect on globulin concentration of the layers due to dietary

addition/inclusion of sodium bicarbonate in their diets. However, the birds of group C, which

were provided diet containing 1% sodium bicarbonate, apparently but non significantly

showed maximum globulin concentration among the birds of all treatment group.

4.8 Plasma electrolytes, minerals and serum pHMeans and their standard deviation values of plasma sodium, potassium, chloride,

bicarbonates, calcium, phosphorus and serum pH are given in table 4.8.

4.8.1 Plasma sodium

Mean values for plasma sodium were 132.7, 141.3, 149.6, 155.2 and 154.7mMol/L

for treatment A, B, C, D and E, respectively. Plasma sodium level was affected due to dietary

inclusion of NaHCO3 in the layer diets. Birds receiving diets containing sodium bicarbonate

exhibited significant (P<0.05) effect on plasma sodium concentration than those of group A

(control group). Birds of group E, which were fed diet containing 2% sodium bicarbonate,

111

Table 4.8: Effect of dietary inclusion of sodium bicarbonate supplementation on

plasma electrolytes and serum pH of caged layers

Variables

Treatment

A

Control

B

0.5%NaHCO3

C

1%NaHCO3

D

1.5%NaHCO3

E

2%NaHCO3

Plasma Sodium

(mMol/L)

132.7±9.3

5d 141.3±8.70149.6±11.6

2b 155.2±9.10154.7±8.6

9a

Plasma potassium

(mMol/L)3.92±0.314.97±0.20a 4.87±0.22a 4.86±0.54a 4.1±0.26b

Plasma chloride

(mMol/L)

13

6.46±1.84a

118.21±5.9

5b

102.11±3.8

4c 94.63±3.26d77.46±3.0

7e

Plasma HCO3

(mMol/L)

2

1.86±1.30d25.83±2.13c

27.58±

1.50b 27.81±1.54b28.9

3±1.43a

Plasma Calcium

(mg/dl)

10.7±0.51 11.10±0.64 11.30±0.35 11.00±0.45 10.70±0.39

Plasma

Phosphorus

(mg/dl)

7.90±0.43 8.10±0.47 7.80±0.35 7.50±0.76 7.40±0.17

Serum pH 7.65±0.247.42±0.15ab 7.38±0.22ab 7.22±0.19b 7.32±0.2

5b

Values within the same row with different superscripts are different significantly (P<0.05)

112

showed maximum plasma sodium concentration when compared to the birds of other treated

groups except those of group D, where the differences in plasma sodium level were found to

be non-significant.

4.8.2 Plasma Potassium

Mean values for plasma potassium were 3.92, 4.97, 4.87, 4.86, and 4.1mMol/L for

treatment A, B, C, D and E, respectively. Plasma potassium was affected (P<0.05) due to

dietary inclusion of sodium bicarbonate in the layer diets. Statistical analysis of the data

showed that birds using diets containing sodium bicarbonate had significant (P<0.05) effect

on plasma potassium as compared to those of group A (control group). Birds of group B,

which were fed diet having 0.5% sodium bicarbonate, showed maximum plasma potassium

when compared to the birds of other treated groups. However, the differences in plasma

potassium protein level among groups B, C and D were found to be non-significant.

Similarly the differences in plasma potassium among treated groups A and E were also found

to be insignificant (P>0.05). A probable explanation of increased plasma potassium level in

treated group may be that treated groups were fed diets containing varying levels of

NaHCO3, therefore as the levels of sodium were increased in diets, it increased sodium in

plasma of birds and decreased plasma chloride ions, therefore level of potassium was

increased in plasma of treated groups.

4.8.3 Plasma chloride

Mean values pertaining to plasma chlorides were 136.46, 118.21, 102.11, 94.63 and

77.46mMol/L for treatment A, B, C, D and E, respectively. Plasma chlorides concentration

was affected due to dietary inclusion of sodium bicarbonate in the layer diets. Statistical

analysis of the data showed that birds using diets containing sodium bicarbonate had

significant (P<0.05) effect on plasma chlorides as compared to those of group A (control).

Birds of group E, which were fed diet containing 2% sodium bicarbonate, showed apparently

maximum plasma chlorides concentration when compared to the birds of other treated

groups.

4.8.4 Plasma bicarbonate

Mean values of plasma bicarbonate were 21.86, 25.83, 27.58, 27.81 and

28.93mMol/L for treatment A, B, C, D and E, respectively. Plasma bicarbonate was affected

due to inclusion of sodium bicarbonate in the diets of layers. Statistical analysis of the data

113

showed that birds using diets containing sodium bicarbonate exhibited significant (P<0.05)

effect on plasma bicarbonate compared to those of group A. Birds of group E, which were

fed diet containing 2% sodium bicarbonate, showed apparently maximum plasma

bicarbonate concentration when compared to the birds of other treated groups. However, the

differences in plasma bicarbonate level among groups C and D were found to be non-

significant.

4.8.5 Plasma calcium

Mean values regarding plasma calcium concentration of the layers were found to be

10.7, 11.1, 11.3, 11 and 10.6mg/dl for treatment A, B, C, D and E, respectively. The data

obtained when analyzed statistically showed non-significant difference among the birds of all

treatment groups, indicating no effect on plasma calcium concentration of the layers due to

dietary inclusion of NaHCO3 in their diets. However, birds of group C, which were provided,

diet containing 1% sodium bicarbonate, apparently but non significantly showed maximum

plasma calcium concentration among the birds of all treatment groups.

4.8.6 Plasma phosphorus

Mean values pertaining to plasma phosphorus concentration in blood of the layers

were found to be 7.9, 8.1, 7.8, 7.5 and 7.4mg/dl for treatment A, B, C, D and E, respectively.

Data obtained when statistically analyzed revealed non-significant difference among the

birds of all treatment groups, indicating no effect on plasma phosphorus concentration of the

layers due to dietary inclusion of NaHCO3 in their diets. However, birds of group B, which

were fed diet having 0.5% sodium bicarbonate, apparently but non significantly showed

maximum plasma phosphorus concentration among the birds of all treatment groups.

4.8.7 Serum pH

Mean values of serum pH were found to be 7.65, 7.42, 7.38, 7.22 and 7.32 for

treatment A, B, C, D and E, respectively. Serum pH was affected due to inclusion of sodium

bicarbonate in the layer diets. Statistical analysis of the data showed that birds using diets

containing sodium bicarbonate exhibited significant (P<0.05) effect on serum pH compared

to those of group A. Birds of group A (control) showed maximum serum pH when compared

to the birds of treated groups. However, the differences in serum pH level among treated

groups B, C, D and E were found to be non-significant.

114

4.9 Serum lipids profile

Means and their standard deviation values, of serum cholesterol, serum high density

lipoprotein (HDL) and low density lipoprotein (LDL) are given in table 4.9.

4.9.1 Serum cholesterol

Mean values pertaining to serum cholesterol concentration of the layers were found to

be 161.25, 149.50, 141.63, 158.1 and 163.13mg/dl for treatment A, B, C, D and E,

respectively. Serum cholesterol was affected due to inclusion of sodium bicarbonate in the

layer diets. Statistical analysis of the data showed that birds using diets containing sodium

bicarbonate exerted significant (P<0.05) result on serum cholesterol concentration when

compared to those of group A. Birds of group C showed the lowest serum cholesterol when

compared to the birds of other groups. However, differences in serum cholesterol level

among groups A, B, D and E were noted as non-significant (P>0.05). Similarly the

differences in serum cholesterol between treated groups B and C were also found to be non-

significant.

4.9.2 Serum triglyceride

Mean values regarding serum triglyceride concentration of the layers were found to

be296,226, 163, 203 and 185mg/dl for treatment A, B, C, D and E, respectively. Serum

triglyceride concentration was markedly affected due to inclusion of sodium bicarbonate in the

layer diets. Statistical analysis of the data showed that birds using diets containing sodium

bicarbonate exhibited significant effect (P<0.05) on serum triglyceride as compared to those of

control group. Birds of group C showed the lowest serum triglyceride when compared to the

birds of other groups. However, the differences in serum triglyceride level among groups B, D

and E were found to be insignificant (P>0.05). Similarly the differences in serum triglyceride

among treated groups C, D and E were also found to be non-significant.

4.9.3 Serum high density lipoprotein

Mean values pertaining to serum high density lipoprotein concentration of the layers

were found to be 121, 130, 138, 141 and 154mg/dl for treatment A, B, C, D and E,

respectively. Serum high density lipoprotein concentration was significantly affected due to

inclusion of sodium bicarbonate in the layer diets. Statistical analysis of the data revealed

that birds using diets containing sodium bicarbonate exhibited significant effect (P<0.05) on

the serum high density lipoprotein as compared to those of control group. Birds of

115

Table 4.9: Effect of dietary inclusion of sodium bicarbonate on serum lipids profile

of caged layers

Variables

Treatment

A

Control

B

0.5%NaHCO3

C

1%NaHCO3

D

1.5%NaHCO3

E

2%NaHCO3

Serum

cholesterol

(mg/dl)

161.25±9.2

0a

149.50

±2.71ab

141.63

±5.62b

158.1

±9.20a

163.13

±6.73a

Serum

triglyceride

(mg/dl)

296±27.0 a 226±19.0 b 163±8.0 c 203±20.0 bc 185±18.0 bc

Serum HDL

(mg/dl)121±9.9 b 130±10.3ab 138±13.7a 141±4.5a 154±9.1a

Serum LDL

(mg/dl)62.6±6.4228.2±2.29b 28.6±2.34b 27.1±2.0b 31.3±1.43b

Values within the same row having different superscripts are differed significantly (P<0.05)

116

control group showed the lowest serum high density lipoprotein concentration when

compared to the birds of treated groups. Birds of group E, which were provided diet

containing 2% sodium bicarbonate, showed highest serum high density lipoprotein

concentration when compared to the birds of other treated groups. However, the differences

in Serum high density lipoprotein level among all treated groups were found to be non-

significant.

4.9.4 Serum low density lipoprotein

Mean values of serum low density lipoprotein (LDL) concentration of the layers were

found to be 61.9, 28.3, 28.1, 27.0 and 31.3mg/dl for treatment A, B, C, D and E, respectively.

Serum low density lipoprotein was affected due to inclusion of sodium bicarbonate in the

layer diets. Statistical analysis of the data showed that diets containing sodium bicarbonate

had significant (P<0.05) effect on serum low density lipoprotein. Birds of control group

showed highest serum low density lipoprotein concentration when compared to the birds of

treated groups. Birds of group D, which were fed diet containing 1.5% sodium bicarbonate,

showed highest serum low density lipoprotein concentration when compared to the birds of

other treated groups. The differences in Serum low density lipoprotein level among all

groups were found to be significant.

4.10 Hormones and enzymesMeans and their standard deviation values of serum tri-iodothyronine (T3), thyroxin

(T4), cortisol, estrogen progesterone, serum glutamic-oxaloacetic transaminase (SGOT) and

glutamic pyruvic transaminase (SGPT) are presented in table 4.10.

4.10.1 Tri-iodothyronine (T3) and Thyroxin (T4)

Mean values regarding serum T3 concentration of the layers for treatment groups A,

B, C, D and E, were found to be 2.87, 3.07, 3.27, 3.25 and 2.99 ng/ml, respectively. Findings

of the study depicted that serum T3 concentration of the birds was significantly influenced

due to dietary inclusion of sodium bicarbonate in their diets. The data obtained when

analyzed statistically showed that birds using diets containing sodium bicarbonate exhibited

higher serum T3 concentration (P<0.05) when compared to those of control group. The

differences in serum T3 concentration among the birds of treated group were also found to be

significant. Birds of group C, which were fed diet containing 1% sodium bicarbonate,

exhibited maximum serum T3 concentration when compared to those of other treated groups.

117

Table 4.10: Effect of dietary inclusion of sodium bicarbonate on serum hormones and

liver enzymes of caged layers

Variables

Treatment

A

Control

B

0.5%NaHCO3

C

1%NaHCO3

D

1.5%NaHCO3

E

2%NaHCO3

T3 (ng/ml) 2.87±0.163.07±0.15ab 3.27±0.20a 3.25±0.12a 2.99±0.14b

T4 (ng/ml) 1.79±0.06 1.90±0.01c 2.08±0.03a 1.79±0.03c 1.93±0.03b

Cortisol

(ng/ml)

7

1.25±2.22a 69.25±2.75ab 65.77±2.03b 66.50±1.91b 70.19±2.84a

Estrogen

(pg/ml)

143.3±8.00

b 168.6.±5.34a 171.3±3.42 168.3±4.82a 166.8±9.80a

Progesterone

(ng/ml) 0.89±0.101.08±0.14a 1.18±0.05a 1.16±0.03a 1.16±.02a

SGOT (U/L)11

5.50±5.65a 101.75±8.80b 93.88±7.31c 99.00±7.03bc98.13±6.9

5bc

SGPT (U/L) 71.70±2.4067.60±2.56 65.70±3.10 69.50±6.80 63.80±5.27

Means within the same row having different superscripts are differed significantly (P<0.05)

118

However, differences in T3 among treated groups B, C, and D were noted to be non-

significant. Similarly, differences in serum T3 concentration among treated groups A, B and

E, were also found to be insignificant (P>0.05).

Mean values of serum T4 concentration of the layers for treatment groups A, B, C, D

and E, were found to be 1.788, 1.898,2.075, 1.79 and 1.925ng/ml, respectively. Findings of

the study depicted that serum T4 concentration of the birds was significantly influenced due

to dietary inclusion of sodium bicarbonate in their diets. Statistical analysis of the data

revealed that birds using diets containing sodium bicarbonate exhibited significantly

(P<0.05) higher serum T4 concentration when compared to those of control group.

Differences in serum T4 concentration among the birds of treated group were also found to be

significant. Birds of group C, which were fed diet containing 1% sodium bicarbonate,

showed maximum serum T4 concentration when compared to the birds of other treated

groups. However, the differences in serum T4 concentration among treated groups B, C and

D, were found to be non-significant. Similarly the differences in serum T4 concentration

among treated groups A, B and E, were also found to be non-significant.

4.10.2 OestrogenMean values regarding serum estrogen concentration of the layers for treatment

groups A, B, C, D and E, were found to be 143.3,168.6, 171.3, 168.3 and 166.8 pg/ml,

respectively. Findings of the study depicted that serum estrogen concentration of the birds

was significantly influenced due to dietary inclusion of sodium bicarbonate in their diets. The

data obtained when analyzed statistically showed that birds using diets containing sodium

bicarbonate exhibited significantly higher (P<0.05) serum estrogen concentration when

compared to those of control group. Differences in serum estrogen concentration among the

birds of treated group were also found to be significant. Birds of group C, which were fed

diet containing 1% sodium bicarbonate, showed maximum serum estrogen concentration

when compared to the birds of other treated groups. However, the differences in serum

estrogen concentration among treated groups B, C, D and E, were noted to be non-

significant.

4.10.3 ProgesteroneMean values of serum progesterone concentration of the layers for treatment group A,

group B, group C, group D and group E, were noted to be 0.89, 1.08, 1.18, 1.16 and

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1.16pg/ml, respectively. Findings of the study depicted that serum progesterone

concentration of the birds was significantly influenced due to addition/inclusion of NaHCO3

in their diets. (P<0.05) that birds using diets containing sodium bicarbonate exhibited

significantly higher serum progesterone concentration (P<0.05) when compared to those of c

group A. However, differences in serum progesterone concentration among the birds of

treated group (B, C, D and E) were found to be non-significant. Birds of group C, which

were fed diet containing 1% sodium bicarbonate, showed maximum serum progesterone

concentration when compared to the birds of control group.

4.10.4 CortisolMean values pertaining to serum cortisol concentration of the layers for treatment

groups A, B, C, D and E, were found to be 71.25, 69.25, 65.77, 66.50 and 70.19ng/ml,

respectively. Findings of the study depicted that serum cortisol concentration of the birds was

significantly influenced due to dietary inclusion of sodium bicarbonate in their diets. The

data obtained when analyzed statistically showed that birds using diets containing sodium

bicarbonate exhibited significantly (P<0.05) lower serum cortisol concentration when

compared to those of control group. Differences in serum cortisol concentration among the

birds of treated group were also found to be significant. Birds of group C, which were fed

diet containing 1% sodium bicarbonate, showed minimum serum cortisol concentration when

compared to the birds of other treated groups. However, differences in serum cortisol

concentration among treated group B, group C and group D were found to be non significant.

Similarly the differences in serum cortisol concentration among treated groups A, B and E

were also found to be non significant.

4.10.5 Serum Glutamic-Oxaloacetic Transaminase (SGOT) and Serum Glutamic Pyruvic Transaminase (SGPT)Mean values pertaining to SGOT concentration of the layers for treatment groups A,

B, C, D and E, were found to be 115.5, 101.75, 93.88, 99.0and 98.13ng/ml, respectively.

Findings of the study depicted that serum SGOT concentration of the birds was significantly

influenced due to dietary inclusion of sodium bicarbonate in their diets. The data obtained,

when analyzed statistically, showed that birds using diets containing sodium bicarbonate

exhibited significantly (P<0.05) lower serum SGOT concentration when compared to those

of control group.

Differences in serum SGOT concentration among the birds of treated group were also

found to be significant. Birds of group C, which were fed diet containing 1% sodium

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bicarbonate, showed minimum serum SGOT concentration when compared to the birds of

other treated groups. However, differences in serum SGOT concentration among treated

groups C, D and E, were non significant. Similarly, differences in serum SGOT

concentration among treated groups B, D and E, were also found to be non-significant

Mean values of SGPT concentration of the layers for treatment groups A, B, C, D and

E, were found to be 71.7, 7, 67.6, 65.7, 69.5 and 63.8U/L, respectively. Findings of the study

depicted that serum SGPT concentration of the birds was not influenced due to inclusion of

sodium bicarbonate in their diets. The data obtained when analyzed statistically showed that

birds using diets containing sodium bicarbonate exhibited non-significantly (P>0.05) lower

serum SGPT concentration when compared to those of control group.

4.11 Immune responseMean values of antibody titer against Newcastle disease virus (NDV) 10 days post

vaccination during 1st, 2nd and 3rd month are presented in table 4.11.

Mean values pertaining to antibody titer against NDV of the layers for treatment groups A,

B, C, D and E, were found to be 43, 86, 167, 145 and 118, respectively. Findings of the study

depicted that serum antibody titer against Newcastle disease virus of the birds was significantly

influenced due to dietary addition/inclusion of sodium bicarbonate in their diets. Statistical

analysis of the data revealed that birds using diets containing sodium bicarbonate exhibited

significantly (P<0.05) higher serum antibody titer against NDV when compared to those of

control group. Differences in serum antibody titer against NDV among the birds of treated

groups were also found to be significant. Birds of group C, which were fed diet containing

1% sodium bicarbonate, showed maximum serum antibody titer against NDV when

compared to the birds of other treated groups. However, the differences in serum antibody

titer against Newcastle disease virus between treated groups B and E, were found to be non-

significant. Similarly, differences in serum antibody titer against Newcastle disease virus

between treated groups C and D were also found to be non-significant.Mean values regarding antibody titer against Newcastle disease virus,10 days post 2 nd

vaccination, of the layers for treatment groups A, B, C, D and E, were found to be 135, 154,

237, 263 and 205, respectively. Findings of the study depicted that serum antibody titer

against NDV of the birds was significantly influenced due to dietary inclusion of sodium

bicarbonate in their diets. Statistical analysis of the data revealed that birds using diets

containing sodium bicarbonate exhibited significantly (P<0.05) higher serum antibody titer

against NDV when compared to those of control group.

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122

Table 4.11: Effect of dietary inclusion of sodium bicarbonate on immune response of

caged layers

Variables

Treatment

A

Control

B

0.5%NaHCO3

C

1%NaHCO3

D

1.5%NaHCO3

E

2%NaHCO3

1st

Vaccination43 d 86 cd 167a 145 ab 118 bc

2nd

Vaccination135 b 154 b 237 a 263 a 205 ab

3rd

Vaccination208 b 272 ab 368 a 288 ab 272 ab

Mean values within the same row with unlike superscripts are significantly different (P<0.05)

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Differences in serum antibody titer against NDV among the birds of treated groups

were found to be significant. Birds of group D, which were fed diet containing 1.5% sodium

bicarbonate, showed maximum serum antibody titer against NDV when compared to the

birds of other treated groups. However, the differences in serum antibody titer against NDV

between treated groups B and E were found to be non-significant. Similarly, differences in

serum antibody titer against NDV between treated groups C and D, were also found to be

non-significant.

Mean values relevant to antibody titer against Newcastle disease virus, 10 days post

3rd vaccination, of the layers for treatment groups A, B, C, D and E, were found to be 208,

272, 368, 288 and 272, respectively. Findings of the study depicted that serum antibody titer

of the birds against NDV was significantly influenced due to dietary inclusion of sodium

bicarbonate in their diets. Statistical analysis of the data revealed that birds using diets

containing sodium bicarbonate exhibited significantly (P<0.05) higher serum antibody titer

against NDV when compared to those of control group. Differences in serum antibody titer

against NDV among the birds of treated groups were also found to be significant.

Birds of group C, which were fed diet containing 1% sodium bicarbonate, showed

maximum serum antibody titer against NDV when compared to the birds of other treated groups.

However, the differences in serum antibody titer against NDV among treated groups were

also found to be non-significant.

4.12 Economic AppraisalEconomics of production of the caged layers calculated on the current values of

various commodities has been described in table 4.12. The table showed that net profit

obtained from the layers of groups A, B, C, D and E, was found to be Rs: 40.64, 60.92,

104.57, 77.12 and 73.16, respectively. These findings revealed that profit margin obtained

from the experimental birds was found to be influenced due to dietary inclusion of sodium

bicarbonate in their diets. The birds of group C, which were fed diet containing 1% sodium

bicarbonate, exhibited maximum profit (Rs. 104.57) followed by those of group D (1.5%

NaHCO3) and E (2% NaHCO3).Better profit margin, from the layers of group C was because

of their higher egg production and efficient utilization of feed containing sodium bicarbonate

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Table: 4.12 Economics of production of the layers fed different levels of sodium

bicarbonate, calculated for 12 weeks of production (26th -37th week of age)

Variables

TreatmentA

Control diet

B0.5%NaHC

O3

C1%NaHC

O3

D1.5%NaHC

O3

E2%NaHC

O3Cost of pullets 400 400 400 400 400Feed consumed

(Kgs)/bird 8.91 9.24 9.36 9.19 9.06

Feed cost @36Rs/kg 320.76 332.64 336.99 330.84 326.16

Housing (equipped)@Rs

2/month6.00 6.00 6.00 6.00 6.00

Labor charges@Rs

2/month6.00 6.00 6.00 6.00 6.00

Vaccination and medication Rs3/month

9.00 9.00 9.00 9.00 9.00

Miscellaneous expenses (Rs) 5.00 5.00 5.00 5.00 5.00

Total expenses 746.76 758.64 762.99 756.84 752.16Sales of table eggs @ Rs.

8/egg482.4 514.56 562.56 528.96 520.32

Sales of culled birds 300 300 300 300 300

Sales of dropping as

manure5 5 5 5 5

Total cash received 787.4 819.56 867.56 833.96 825.32

Net profit 40.64 60.92 104.57 77.12 73.16

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as compared to those of other experimental groups. Moreover, reduction in body temperature

due to the use of sodium bicarbonate might have provided comfortable physiological

conditions to the layers, suitable for efficient egg production as compared to those of control

groups.

However, minimum profit (Rs 40.64/hen) was obtained from the layers of control

group. Lower return from control birds may probably be due to their lower production

performance because of high ambient temperature. Anjum (2000) and Ahmad et al. (1993)

while justifying their findings have also attributed lower egg production of layers to high

ambient temperature. They concluded that high ambient temperature was a major

contributing factor affecting the economics of poultry production.

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DISCUSSIONNumerous acid-base imbalances have been studied in broilers kept under heat-

stressed condition, which have resulted in the incidence of respiratory alkalosis in the birds

(Dibartola, 1992; Carlson, 1997; Ahmad et al., 2005). However, dietary supplementation of

certain minerals such as sodium bicarbonate appears to be helpful in alleviating the effect of

heat stress in broilers (Ahmad et al., 2008) by improving their feed consumption and water

intake.

4.13 Performance4.13.1 Live body weight

Body weight gain of the birds consuming diets containing sodium bicarbonate was

found to be significantly affected due to the inclusion of NaHCO3 in their diets. The birds

receiving diets containing sodium bicarbonate exhibited higher weight gain as compared to

those of untreated group (control). A probable explanation of increased live body weight of

the layers in treated groups may be the higher feed intake of the birds receiving sodium

bicarbonate. Similar results have been observed by Balnave and Gorman (1993) who

reported improved weight gain because of inclusion of NaHCO3 in the diets of birds. Genedi

(2000) also reported that adding anti-stressors like NaHCO3 in to drinking water offered to

Leghorn and Matrouh hens increased their weight gain under heat stress condition. Another

possible explanation of these results may be the response of inclusion of NaHCO3, which

depends upon existence or absence of factors influencing acid-base balance of the birds.

Presence of metabolizable anions (Na+) in poultry diets has shown to exhibit a significant

improvement in the body weight gain of broilers (Ruiz-Lopez and Austic, 1993).

Opposing to the results of the present study, Junqueira et al. (2003) and Osman et al.

(2015), found no effect due to dietary addition of different levels of sodium bicarbonate on

weight gain of poultry birds. Comparable, results are also reported by Hayat et al. (1999) and

Wideman et al. (2003), where inclusion of NaHCO3 in the diets of birds did not exhibit any

significant increase in body weight gain of birds. Saedi and Khajali (2010) found that body

weight of broilers remained unaffected because of dietary addition of sodium bicarbonate.

Moreover, Peng et al. (2013) found a significant decrease in final body weight in

broilers fed NaHCO3 added diets under summer conditions of relatively high temperature.

Similarly, a decrease in body weight of the birds due to the addition of sodium bicarbonate in

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their diets has been reported by Squires and Julian, (2001) who observed that addition of

NaHCO3 (0.42%) reduced body weight gain in broilers. Wideman et al. (2003) have also

observed 7% reduction in final body weight of broilers fed a diet containing of 1% NaHCO3.

Differences among the results of different experiments might have been due either to the

difference in genetic makeup (Kassim and Brence, 2001) of the birds or difference in the

levels of sodium bicarbonate used in these studies.

4.13.2 Feed consumption

Birds receiving diets containing sodium bicarbonate exhibited more feed

consumption as compared to those of untreated group (control). Increase in feed consumption

of the treated groups may be due to more sodium ions concentration in the rations containing

sodium bicarbonate (Puron et al., 1997; Mc Dowell, 1992). Similar effect of increased

sodium ions concentration in broilers has been observed by Fethiere et al. (994) who

concluded that feed intake is reliant upon Na+ level in the ration and high sodium level could

cause more feed consumption. The results of presents study supports the findings of Ahmad

et al. (2006), and Balnave and Gorman (1993) who observed a significant (P<0.05) increase

in feed consumption in broilers fed diet added with sodium bicarbonate as compared to

control group. Puron et al. (1997) also observed a significant improvement in feed intake by

adding sodium bicarbonate (0.5%) in the diets of poultry birds.

Feed intake of the birds was also found to be significantly affected, among the birds

of treated groups, due to different levels of sodium bicarbonate (0.5-2.0%) used in their diets.

These results are comparable to those reported by Gongruttananun and Ratana (2005) who

reported a significant increase in feed consumption of Thai native hens due to the addition of

different levels of sodium bicarbonate (1.0-1.5%) in their diets. Similarly, Yoruk et al. (2004)

examined the effect of different levels of inclusion of NaHCO3 (0.1%-.4%) in the diet on

feed consumption of layers during their late laying period and observed a marked

improvement in feed consumption of the birds.

However, Bonsembiante and Chiericato (1990) have observed a non significant

difference in feed intake (P<0.05) of birds receiving rations with or without supplemented

sodium bicarbonate. Parallel results are also reported by Senkoylu et al. (2005) who explored

the effect of inclusion of different levels of NaCl, NaHCO3 and K2CO3 in poultry diets, on

feed consumption of layers during peak production and did not observe any effect due to the

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dietary inclusion of these compounds on feed intake of the birds. Findings of Balnave and

Muheereza (1997) and Waldroup et al. (2005) have also depicted that feed intake of birds fed

diets with or without sodium bicarbonate remained unaffected. Contrary to the outcomes of

current study, Fuentes et al. (1997) found no effect of different levels of sodium bicarbonate

(0.6, 1.2, 1.8 and 2.4%) in diet on feed consumption in guinea fowl reared at high ambient

temperature.

Moreover, Khattak et al. (2012) observed some reduction in feed consumption of

birds fed diet supplemented with sodium bicarbonate during summer. The discrepancy in the

results of these research results may probably be due to the variations in experimental

conditions maintained, strain/species of birds or levels of sodium bicarbonate added in the

diets (Nayak et al., 2015).

4.13.3 Egg Production

Birds receiving diets containing sodium bicarbonate exhibited more egg production

when compared to those of untreated group (control). Increase in egg production of the

treated groups may be due to either more feed consumption (Dai et al., 2009) or increased

sodium ions concentration or both in the rations containing sodium bicarbonate (Puron et al.,

1997a; Mc Dowell, 1992). Observations of the research are in line with the results of Okan,

(1999) who reported an increase in egg production by supplementation of NaHCO3 in layer

diets. Similar effect has also been observed by Ghorbani and Fayazi (2009) who studied the

effect of inclusion of NaHCO3 in the feed of layers on egg production during chronic heat

stress and found considerable increase (P<0.05) in egg production due to dietary inclusion of

sodium bicarbonate. The results of this study are also compatible with the observations of

Hassan et al. (2011) who found that inclusion of sodium bicarbonate used at 0.25% and

0.50% in poultry diet showed a significant increase in egg production of layers during hot

weather. Improved egg production has also been observed by Balnave and Muheereza (1997)

because of inclusion of NaHCO3 (1%) in layers diet.

The difference in egg production of the birds was also found to be significant among

the birds of treated groups due to different levels of sodium bicarbonate (0.5-2.0%) used in

the diets. Similar results were also reported by Ghorbani and Fayazi (2009) who studied the

effect of addition of NaHCO3 and rearing system on production performance of layers kept

under chronic heat stress. They observed that dietary levels of sodium bicarbonate from

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0.5%-1.5% in laying hens diet improved their egg production. Similarly, Yoruk et al. (2004)

studied the effect of different levels of NaHCO3 (0.1%-0.4%) on egg production of layers.

Results showed that inclusion of different levels of sodium bicarbonate (0.1%-0.4%) in

laying hens diet improved their egg production.

Contrary to the results of present study, Gongruttananun and Ratana (2005) found

non-significant effect of adding varying levels of NaHCO3 on feed consumption in layers.

They fed diets supplemented with 1.0-1.5% sodium bicarbonate to Thai native hens and

observed that differences in egg production between treated and non-treated birds, even due

to different levels of sodium bicarbonate were non-significant. Similar results pertaining to

egg production are reported by Grizzle et al. (1992); Gongruttananun et al. (1999) and

Waldroup et al. (2005) due to the inclusion of sodium bicarbonate in layer diets. Egg

production has also been found unaffected due to the inclusion of different levels of NaCl,

NaHCO3 and K2CO3 in the diets of layers (Senkoylu et al., 2005), during peak production

period.

Moreover, significant reduction of egg production in control group due to heat stress

is also in accord with the studies of Melesse, (2011); Mashaly et al. (2004); Peguri and Coon,

(1991). Furthermore, heat stress not merely lessens feed consumption but is also reported to

decrease absorption of various components of the diet (Bonnet et al., 1997). However,

inclusion of sodium bicarbonate may result in increased absorption and availability of

nutrients and hence results in increased egg production.

4.13.4 Egg weight

Birds receiving diets containing sodium bicarbonate had heavier eggs as compared to

untreated group (control). Increase in egg weight of the treated groups may probably be due

to better utilization of digested proteins, amino acids, monosaccharide and energy due to

metabolic effect of sodium present in sodium bicarbonate containing rations (Murkami et al.,

2001). Another possible reason of better egg weight in treated groups may be higher feed

intake of the birds, as has been observed in the results of present study (see section 4.1.2).

The results of this study are in accordance with the findings of Balnave and Muheereza

(1997) who fed either basal diet or diets containing 1% sodium bicarbonate, 0.05% Zinc

methionine or 0.04% vitamin C, to layers kept under high ambient temperature and found

significantly higher weight of eggs produced by the birds fed diet supplemented with 1%

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sodium bicarbonate. Similar effects of sodium bicarbonate on egg weight have also been

reported by Ghorbani and Fayazi (2009) in layers.

The difference in egg weight was also known to be considerable among the birds fed

diets containing different levels of sodium bicarbonate (0.5-2.0%). These results are in line

with the results of Yoruk et al. (2004) who observed the effect of different levels of sodium

bicarbonate (0.1%-0.4%) on egg weight of layers during late laying period and observed that

inclusion of different levels of sodium bicarbonate (0.1%-0.4%) in the feed of layers

improved their egg weight. Identical results have also been observed by Ghorbani and Fayazi

(2009) who studied the effect of different levels of sodium bicarbonate (0.5%-1.5%) and

rearing systems on egg weight of layers kept under chronic heat stress and found that dietary

levels of sodium bicarbonate (0.5%-1.5%) in laying hens diet improved their egg weight,

where hens fed diet supplemented with 1.5% sodium bicarbonate produced heavier eggs.

Contrary to the results of present study, Senkoylu et al. (2005) who tested the effect

of inclusion of different levels of NaCl, NaHCO3 and K2CO3 in poultry diets, on egg weight

in layers during peak production, and found no effect due to dietary inclusion of these

compounds on egg weight of layers. Akin results have also been observed by

Gongruttananun and Ratana (2005) who found non-significant effect of sodium bicarbonate

(1.0-1.5%) on egg weight of Thai native hens. Similarly, Waldroup et al. (2005) observed a

non-significant effect of adding sodium bicarbonate (1%) in the diet on mean egg weight of

layers. The discrepancy in the results of these studies might be due to varying environmental

conditions of the experiment, strain/species of birds used in these studies (Nayak et al., 2015)

or difference in the levels of sodium bicarbonate added in the experimental diets.

4.13.5 Egg mass

A significant reduction in egg mass/size of control group was observed due to

hyperthermia. High ambient temperature not merely reduces feeding activity but also has

reported to lessen absorption of various nutrients of the feed (Bonnet et al., 1997). Therefore,

it is quite possible that metabolic machinery of the birds may have been used for homeostasis

regulation rather than to be used for production (Carlson, 1997). However, inclusion of

sodium bicarbonate has been known to ameliorate hyperthermia effects due to beneficial

effect of sodium and bicarbonate ions, by increasing absorption of the nutrients present in the

diets along with their availability and hence resulting in the increase of egg mass.

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Birds receiving diets containing sodium bicarbonate exhibited significantly (P<0.05)

more egg mass than those of untreated group. Moreover, the birds receiving sodium

bicarbonate also exhibited higher feed intake and produced more eggs when compared to

those of control group (see table 4.2). Therefore, higher egg mass production of the eggs by

the layers getting treated ration may possibly be due to more feed consumption coupled with

its efficient utilization by these birds as has also been observed by Dai et al. (2009). Another

possible reason of increased egg mass of the treated groups may be the increase in sodium

ions concentration in the diets containing sodium bicarbonate (Kurtoglu et al., 2007). Yoruk

et al. (2004) also found beneficial effect of sodium bicarbonate on egg mass due to its

addition in the diets of turkey. Better feed utilization of layers receiving sodium bicarbonate

added diets and improved electrolyte balance in these diets might have created favorable

physiological conditions for an improvement in egg mass (Drinah et al., 1990). Results of the

present study are in accord with the findings of Balnave and Muheereza (1997) who fed

either basal diet or diets containing sodium bicarbonate (1%), 0.05% Zinc methionine or

0.04% vitamin C to layers kept under high ambient temperature and exhibited a significant

improvement in egg mass produced by the bird fed diet supplemented with 1% sodium

bicarbonate.

The difference in FCR/Kg egg mass produced by the layers was also found to be

significant among the birds of treated groups due to different levels of sodium bicarbonate

(0.5-2.0%) used in the diets. These results coincide with the findings of Ghorbani and Fayazi

(2009) who studied the effect of dietary addition of NaHCO3 and rearing system on the

performance of layers kept under chronic heat stress. They found that dietary levels of

sodium bicarbonate (0.5%-1.5%) in laying hens diet improved their egg weight/egg mass.

Findings of the current research are also in according with those observed by Yoruk et al.

(2004). They studied the effect of different levels of NaHCO3 (0.1%-.4%) on egg weight/egg

mass of layers during late laying period and observed that inclusion of different levels of

sodium bicarbonate in the diets of laying hens improved their egg weight/egg mass.

Contrary to the results of current study, Senkoylu et al. (2005) reported a non

significant effect due to the inclusion of different levels of NaHCO3 in the diets, on egg mass,

in layers during peak production. The discrepancy in results of these experiments may

probably be due to the differences in stage of production of the birds used in these studies.

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4.13.6 Feed efficiency

a) FCR/dozen eggs

Birds fed diets supplemented with sodium bicarbonate utilized their diets more

efficiently when compared to those of untreated group (control). A probable explanation of

better FCR/dozen eggs produced in the birds of treated groups may be more synthesis of

tissue proteins (Borges et al., 2003) as a result of higher feed consumption. Similar results

regarding efficiency of feed utilization have also been reported by Barton (1996) due to the

inclusion of NaHCO3 in turkey feeds. Another probable explanation of better efficiency of

feed utilization in the birds receiving NaHCO3 may be the improved electrolyte balance in

the diets, which might have created some favorable conditions for improvement in the feed

efficiency (Drinah et al., 1990). Better FCR may also be attributed to better digestion and

absorption of nutrients due to incorporation of sodium carbonate, which ultimately may have

resulted in improved egg production; a vital factor involved in the calculation of feed

efficiency.

Efficiency of feed utilization of the layers, calculated on the basis of per dozen eggs

produced was also found to be due affected due to the inclusion of different levels of sodium

bicarbonate (0.5-2.0%) in layer diets. A probable explanation of better utilization of feed

containing different levels of sodium bicarbonate may be either due to improved feed

consumption or improved egg production and/or both, by the birds of treated groups. These

results are in line with the results of Yoruk et al. (2004) who studied the effect of different

levels of sodium bicarbonate (0.1%-0.4%) on feed conversion ratio of layers during late

laying period. Their findings revealed that dietary inclusion of different levels of sodium

bicarbonate improved feed conversion ratio of the birds, calculated on the basis of one dozen

eggs produced.

b) FCR/Kg egg mass produced

The results regarding efficiency of feed utilization calculated on the basis of per kg

egg mass produced, revealed that the birds receiving diets containing sodium bicarbonate

utilized their feeds more efficiently when compared to those fed diet without addition of

sodium bicarbonate (control). Better FCR/kg eggs mass produced in the treated groups may

probably be due to their higher feed consumption, resulting in increased availability of

nutrients after fulfilling the maintenance requirements of the birds and ultimately leading to

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more egg production and heavier eggs (see table 4.2). Improvement in efficiency of feed

utilization in birds receiving sodium bicarbonate may also be due to the improved electrolyte

balance, better digestion and absorption of nutrients, enzymatic reactions and synthesis of

tissue proteins in the diet by creating favorable conditions for an improvement in feed

efficiency (Drinah et al., 1990; Borges et al., 2003). The results of the present study are in

line with the findings of Keskin and Durgan (1997) who have reported an improved FCR in

quails fed diet supplemented with NaHCO3, KCl, CaCl2, NH4Cl and CaSO4.

The difference in FCR/Kg egg mass produced of the layers was also found to be

significant among the birds of treated groups due to different levels of sodium bicarbonate

(0.5-2.0%) used in the diets. These results are in line with the findings of Yoruk et al. (2004)

who studied the effect of different levels of sodium bicarbonate (0.1%-0.4%) on feed

conversion ratio of layers during late laying period. They reported that inclusion of different

levels of sodium bicarbonate in laying hens diet improved their feed conversion ratio.

Contrary to the results of present study, Senkoylu et al. (2005) reported non-

significant effect of inclusion of different levels of NaCl, NaHCO3 and K2CO3 in the diet on

FCR (g of feed/g of egg) in layers during peak production. Fuentes et al. (1998) have also

observed non-significant effect of adding different levels of sodium bicarbonate (0.6, 1.2,

1.8, and 2.4%) on FCR values calculated on the basis of per kg egg mass in guinea fowls

raised at high ambient temperatures. Contradictions in the findings of these studies may

probably because of difference in the species of poultry birds used in these studies.

4.14 Egg quality4.14.1 Specific gravity

Birds receiving diets containing sodium bicarbonate exhibited more specific gravity

value as compared to those of untreated group (control). Increase in specific gravity (SG) of

the eggs laid by the birds of treated groups may probably be due to better utilization of

calcium because of metabolic effect of sodium/bicarbonate ions, present in sodium

bicarbonate containing (Keskin and Durgan, 1997, Squire and Julian, 2001), and leading to

the production of thick shell eggs. On the other hand, shell thickness of an egg is closely

correlated with SG of a newly laid egg. That’s why SG measurements are used to find out

shell quality.

Findings of this study are in accordance with those stated by Yoruk et al. (2004) who

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studied the effect of different dietary levels of NaHCO3 (0.1%-0.4%) on specific gravity of

eggs in layers, during late laying period and found that inclusion of NaHCO3 in laying hens

diet increased specific gravity of their eggs. They also reported that higher levels of sodium

bicarbonate (0.2%-.4%) in the diet decreased the SG of eggs produced by birds. However,

Grizzle et al. (1992) did not notice any such effect on specific gravity of eggs because of

dietary addition of 1% NaHCO3 in layers. Similarly, Makled and Charles (1987) also found

no change in specific gravity of eggs in hens fed diet supplemented with 0.5% sodium

bicarbonate during peak production period.

4.14.2 Shell thickness

Shell thickness of eggs produced by the birds consuming diets containing sodium

bicarbonate was found to be significantly higher than those of untreated group (control).

Addition of sodium bicarbonate in the diets has been shown to increase calcium retention in

layers (Ferguson et al., 1974). Therefore, increase in egg shell thickness of the treated groups

may probably be due to more utilization of calcium because of some positive metabolic

effects of sodium and bicarbonate ions in sodium bicarbonate containing diets (Keskin and

Durgan, 1997; Squire and Julian, 2001). Better shell thickness (ST) of the eggs may also be

the result of relatively higher bicarbonate level, which ultimately reduced panting in the birds

using diets containing sodium bicarbonate than those of control group. Production of thick

shell eggs due to the dietary inclusion of sodium bicarbonate level also coincides with better

production performance of the birds, as has been observed in the present study.

El-Boushy and Raternick, (1993) observed that the birds exposed to high

environmental temperature produced eggs with poor ST, which is quite in agreement with the

findings of present study where birds fed diet without adding sodium bicarbonate produced

thin shelled eggs. Decrease in ST of eggs produced by the birds exposed to heat stress has

been related either to low feed intake (Balnave and Muheereza, 1997), which may lead to

ultimate reduction in calcium intake, a necessary element required for shell formation

(Karimian et al., 2004) or to decrease in calcium level in blood (Hassan et al., 2003).

However, shell thickness of eggs produced by the birds fed diet supplemented with 1%

sodium bicarbonate was significantly improved (Balnave and Muheereza, 1997). Therefore, a

probable explanation of production of thinner shelled eggs by the birds under the influence of

heat stress and fed diet without addition of sodium bicarbonate might be due to lower feed

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intake and bicarbonate level in their blood as compared to those fed diets containing different

levels of sodium bicarbonate.

The results observed in this study are in line with the findings of Hayat et al. (1999)

who observed a significant improvement in egg shell thickness in layers fed

NaHCO3supplementeddiet. Similar beneficial effects of including sodium bicarbonate in

poultry diets have also been reported on egg shell thickness by Davison and Wideman

(1992). Makled and Charles (1987) observed an improvement in egg shell thickness in hens

fed diets supplemented with 0.5% sodium bicarbonate. Gongruttananum and Ratna (2005)

also reported markedly higher shell thickness of eggs in birds fed diet supplemented with

sodium bicarbonate. Increasing NaHCO3 level in the diets also increased the DEB level of

these diets (see table 3.2), however, birds fed diet containing 1% sodium bicarbonate

(DEB=262) laid thicker shelled eggs when compared to other groups. Ghasemi et al. (2014)

have also reported that, under tropical conditions, using a DEB of 250mEq/Kg achieved a

correction of the lay-induced metabolic acidosis and resulted in thicker shelled eggs.

Therefore, increase in shell thickness of the eggs may be ascribed to the inclusion of sodium

bicarbonate in the diet, which might have led to an improvement in eggshell thickness.

In contrary to the results of the present study, Kaya et al. (2004) observed

insignificant improvement in shell thickness of the eggs produced by geese layers, offered

diet containing 0.5% NaHCO3. Yoruk et al. (2004) also observed non-significant effect of

adding different levels of sodium bicarbonate (0.1%, o.2%, 0.4%) on egg shell thickness of

laying hens. Contradiction in the results/outcomes of these researches may be due either to

varying level of dietary addition/inclusion of sodium bicarbonate or difference in the poultry

species used (layer vs geese) in these trials, or both.

4.14.3 Albumen height

Eggs produced by the birds receiving diets containing sodium bicarbonate exhibited

more albumen height as compared to those of untreated group (control). An increased

albumen height of the eggs produced by the birds of treated groups may probably be due to

higher feed consumption and better absorption of nutrients present in the experimental diets

containing sodium bicarbonate when compared to those of control group. Moreover, the birds

receiving sodium bicarbonate also exhibited higher serum albumen concentration than those

of control group (see table 4.7). Therefore, higher albumen height of the eggs produced by

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the birds consuming treated ration may possibly be due to more serum albumen

concentration. Inclusion of sodium bicarbonate in feed has also shown potential benefits on

egg characteristics (Balnave and Muheereza, 1997; Kaya et al., 2004) in poultry birds during

heat stress period. Similar results are quoted by Yoruk et al. (2004) who studied the effect of

different levels of NaHCO3 (0.1%-0.4%) on albumen index and Haugh unit score of layers

during late laying period.

The albumen height is essential constituents for calculation of Haugh units and is

known to be affected by the temperature during storage. The quality of albumen and yolk

deteriorates with the increasing storage time and temperature (North and Bell, 1990).

However, in the present study the eggs used were fresh. Therefore, probably albumen quality

of the eggs produced by the birds of control group deteriorated only because of high

environmental temperature. However, according to the findings of present study, dietary

supplementation of sodium bicarbonate improved albumen quality of the eggs produced by

the birds.

Contradictory results have been reported by Ghorbani and Fayazi (2009) and

Gongruttananum and Ratna (2005) who studied the effect of dietary dietary and system of

rearing on egg quality characteristics in layers kept under chronic heat stress. They reported

that dietary levels of sodium bicarbonate (0.5%-1.5%) in the diets of laying hens did not

show any improvement in the albumen quality of the eggs produced by these birds.

4.14.4 Haugh unit

Haugh unit score was found to be significantly better in the birds fed diets containing

sodium bicarbonate as compared to those fed diets without its addition. The difference in

Haugh unit score of the birds was also found to be significant among the birds of treated

groups due to different levels of sodium bicarbonate (0.5-2.0%) used in the diets. An

increased Haugh unit score of the treated groups may probably be due to higher feed

consumption and better absorption of nutrients of experimental diets when compared to those

of control group, as has been reflected in the findings of present study. Moreover, the birds

receiving sodium bicarbonate also exhibited higher serum proteins concentration than those

of control group (see table 4.7), which may have caused an increase in Haugh unit value of

eggs produced by these layers. These results are matched with those reported by Kaya et al.

(2004) who observed that inclusion of sodium bicarbonate in feed has shown potential

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benefits on egg characteristics in poultry birds during heat stress period.

Contradictory to the findings of present study, Ghorbani and Fayazi (2009) did not

observe any effect due to dietary inclusion of sodium bicarbonate and rearing system on

Haugh unit score of the eggs produced by the layers kept under chronic heat stress. Their

findings depicted that neither addition of sodium bicarbonate nor its various levels (0.5%-

1.5%) exhibited any effect on Haugh unit score in the layers. These type of findings have

also been quoted by Yoruk et al. (2004) who also did not examine any effect of dietary

inclusion of different levels of sodium bicarbonate (0.1%-0.4%) on Haugh unit score of the

eggs produced by layers, during their late laying period. Findings of Gongruttananum and

Ratna (2005) have also revealed that dietary levels (1%-1.5%) of sodium bicarbonate in diets

did not improve Haugh unit score of the egg laid by the hens. A probable explanation of

these contradictory findings may be the difference in ambient conditions in which the birds

were kept during these experiments.

4.14.5 Yolk diameter

Effect of dietary addition/inclusion of NaHCO3 on yolk diameter of the eggs

produced by the layers was found to be significant. The eggs produced by the birds, which

used diets containing sodium bicarbonate exhibited more yolk diameter as compared to the

controls. Increase in yolk diameter of the eggs of treated groups may probably be due to

higher feed ingestion and better absorption of nutrients present in the experimental diets, as

has been depicted in the results of this study. Inclusion of sodium bicarbonate in feed has

shown potential benefits on egg characteristics (Balnave and Muheereza, 1997; Kaya et al.,

2004) in poultry birds during heat stress period.

However, findings of Ghorbani and Fayazi (2009) did not showed any effect on yolk

diameter of the eggs produced by the hens due to dietary addition of NaHCO3 and rearing

system in which the birds were kept under chronic heat stress. Similarly, Yoruk et al. (2004)

who studied the effect of different levels of NaHCO3 (0.1%-0.4%) on yolk diameter of eggs

produced by layers during late laying period, did not find any improvement in yolk diameter

of the eggs . Findings of Gongruttananum and Ratna (2005) reported that yolk diameter of

eggs due to different dietary levels of sodium bicarbonate (1%-1.5%) in laying hens

remained unaffected. Probably these contradictory findings may be due to the difference in

experimental conditions maintained during these studies

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4.14.6 Yolk height

The eggs produced by the birds, which used diets containing sodium bicarbonate

exhibited more yolk height as compared to those fed untreated diet. Reasons of increase in

yolk height of the eggs produced by the birds in treated groups may probably be the same as

have been advocated in the previous section (yolk diameter). The results pertaining to egg

yolk height in this trial are in line with those observed by Yoruk et al. (2004). They studied

the effects of dietary supplementation of different levels of sodium bicarbonate (0.1%-0.4%)

on yolk quality of the eggs produced by the layers during their late laying period and found a

significant improvement in yolk quality of eggs because of these treatments. Addition of

NaHCO3 in feed has also shown potential benefits on various egg characteristics in poultry

birds during heat stress period (Davison and Wideman, 1992; Kaya et al., 2004)

Contrary to the results of this study, findings ofGongruttananum and Ratna, (2005)

and Ghorbani and Fayazi, (2009) have also revealed that yolk quality remained unaffected in

the eggs laid by the layers fed diets containing different levels of sodium bicarbonate (1-

1.5%). The reasons of these contradictory results may be the same as have been reported in

the previous section (4.15.4).

4.14.7Yolk Index

The difference in yolk index of eggs produced by the birds fed diets with or without

addition of sodium bicarbonate was found to be non-significant. These findings are in

accordance with those observed by Ghorbani and Fayazi (2009) who studied the effect of

dietary NaHCO3 and rearing system, on yolk index of layers kept under chronic heat stress.

The results of their study revealed that neither dietary inclusion of sodium bicarbonate nor its

levels (0.5%-1.5%) exhibited any effect on yolk index of the eggs produced by the hens.

Similarly, Gongruttananum and Ratna (2005) have also observed that dietary levels of

sodium bicarbonate (1%-1.5%) in laying hens diet did not improve their yolk quality.

However, contradictory results are reported by Yoruk et al. (2004) who studied the

effect of different levels of sodium bicarbonate (0.1%-0.4%) on yolk index of layers during

late laying period. They found significant improvement in yolk index of eggs by dietary

inclusion of sodium bicarbonate at 0.1% level. The differentiation in the results of these

experiments might be due to variation in the levels of sodium bicarbonate incorporated in the

diets used in these studies.

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4.14.8 Egg yolk cholesterol

Addition of NaHCO3 in the diets of layers reduced (P<0.05) the concentration of

cholesterol in their eggs as compared to those produced by the control birds. A probable

explanation of decrease in egg cholesterol may be that sodium bicarbonate might have

stimulated the production of bile acids, which utilized cholesterol for their synthesis

(Naviglio et al., 2011); hence ultimately resulting in decreased concentration of yolk

cholesterol in the birds of treated groups. Another possible reason for decrease in egg

cholesterol in layers offered sodium bicarbonate added diet may be the inhibition of enzyme

“squalene epoxidase” which is vital for the production of cholesterol (Angelovicova, 1997).

Reduction in egg cholesterol perhaps may reduce the chances of cardiovascular

diseases due to the use of chicken eggs, in human beings. Recent research has shown that

intake of eggs having low cholesterol, does not increase cholesterol in patients suffering from

cardiovascular diseases (Harman et al., 2008; Spence et al., 2010).However, reduction of

cholesterol in hatch able eggs received from breeder flocks is questionable because

cholesterol is vital for yolk formation and embryo growth (Griffin, 1992). Hence, strict

cholesterol fall in egg yolk can stop of egg production and increase in embryonic deaths.

4.14.9Yolk pH

Diets containing sodium bicarbonate when fed to the layers, exhibited significantly

higher yolk pH (P<0.05) value of the eggs produced by them than those fed control diet. A

probable explanation of significantly higher pH value in the yolk of the eggs produced by the

birds receiving diets containing sodium bicarbonate may be the increase in concentration of

bicarbonate ions in their guts, which might have given rise to yolk pH of eggs produced by

the birds. Addition of NaHCO3 in the diet of layers has also been reported to support

maintenance of venous blood pH (Kaya et al. (2004). Moreover, it has also been observed

that increase in blood bicarbonate ions concentration can compensate the ill effects of

chloride ions contributed by heat stress (Fethiere et al., 1994). Results of the present

experiment are compatible with the findings of Balnave and Gorman, (1993) who observed

increase in blood pH in birds due to increase in HC03- ions concentration in their diets.

4.14.10 Albumen pH

Dietary addition/inclusion of sodium bicarbonate revealed a significant increase in

albumen pH (P<0.05) of the eggs produced by them than those of untreated group (control).

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The findings are compatible to the results of Kaya et al. (2004) who reported that inclusion

of NaHCO3 in the diet of layers may support to maintain the blood pH of birds. Therefore,

increase in the albumen pH may probably be due to increase in blood bicarbonate ions

concentration of the birds. Addition of NaHCO3 in the diet of layers has been reported to

support the maintenance of venous blood pH (Kaya et al., 2004). Similar results have been

observed by Glahn et al., 1988; Squires and Julian, 2001, who found that dietary use of

NaHCO3 increased blood pH of birds. Increase in albumen pH also coincides with the

findings of Balnave and Gorman, (1993) who observed increase in blood pH in birds due to

increase in HC03- ions concentration in their diets.

4.14.11 Blood and meat spots

The blood and meat spots were observed only in the eggs produced by the birds fed

ration without dietary addition of sodium bicarbonate, while those of treated groups were

devoid of these. The exact mechanism of how dietary inclusion of different levels of sodium

bicarbonate prevented the occurrence of blood spots in the eggs produced by the birds of

treated groups is still not known. However, probably reduction in body temperature of the

layers receiving sodium bicarbonate added diets may have resulted in less stress upon the

reproductive tract of the birds of treated groups.

The findings of this study are in accordance with those stated by North and Bell

(1990), that incidence of meat spots are reported to be affected by the ambient temperature

along with other factors such as genetics and age of the birds. As the layers used in this

experiment were of the same age and strain, therefore no genetic variation or age affect can

be expected. Therefore, production of eggs without any blood or meat spots, by the layers fed

diet containing various levels of sodium bicarbonate may be attributed to less physiological

stress on reproductive system of the birds than those fed control diet without containing

sodium bicarbonate (control).

4.15 MortalityIncidence of mortality was zero in all groups. It may probably be due to the reason

that the experiment was conducted under the best possible controlled hygienic conditions,

(except ambient temperature) and the birds were kept well observed. Resultantly, no bird

died from any group (treated or untreated) during the experimental period. The results of the

present study are in conformity with the findings of Mushtaq et al. (2005) who observed no

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mortality in broilers fed diet supplemented with sodium bicarbonate (0.025% Na+). However,

generally high environmental temperature has been ascribed to more mortality of the birds

(Branton et al., 1986).

4.16 Rectal temperature, respiration rate and water consumption4.16.1 Rectal temperature

Birds receiving diets containing sodium bicarbonate exhibited lower rectal

temperature (P<0.05) when compared to those of untreated group (control). A probable

reason of decrease in rectal temperature of the birds in treated groups may be increased

sodium ions concentration in their diets, which might have resulted in increased water

consumption (Ahmad, 1997; Ahmad, 2007). Increased water consumption in birds has shown

to cause an increase in body heat loss through evaporation (Belay and teeter, 1993).

Therefore, birds in positive water balance (treated groups) were capable to sustain their

internal body temperature to optimum level. Results of the study are in agreement with the

findings of Ahmad et al. (2005) who studied the influence of sodium bicarbonate

supplementation on rectal temperature of heat stressed broilers and observed a significant

(P<0.05) reduction in body temperature of the birds fed diet having sodium bicarbonate. The

birds which were supplied sodium bicarbonate containing diets also exhibited lower

respiration rate and thus produced less heat for this physiological norm. This probably could

be the reason, for significantly lower rectal temperature in this case.

Layers fed diet without addition of sodium bicarbonate (control group) manifested a

rise in their rectal temperature in response to increase in ambient temperature during the

experimental period. However, layers of treated groups revealed a decrease in their rectal

temperature with increase in levels of sodium bicarbonate throughout the period of heat

stress. Higher relative humidity in combination with severe temperature was possibly a

contributing factor for upholding the initial rise in rectal temperature.

Mean values of rectal temperature of the layers fed diet containing 1% sodium

bicarbonate were found to be lower than those of its counterparts. The reduction in

temperature may have been, probably due to comfortable physiological environment of the

birds because of sodium/bicarbonate ions, which might have favored lowering of their body

temperature than its other counterparts.

Contrary to the results of current study, Mushtaq et al. (2007) did not find any

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correlation between rectal temperature of poultry birds and dietary sodium levels in broilers.

Dissimilarity in the results of these researches may be either because of difference in the

levels of sodium bicarbonate incorporated in the diets or due to the different species of the

birds (broilers vs layers) used in these studies, or both.

4.16.2 Respiration rateThe birds fed diets without adding sodium bicarbonate (control) exhibited faster

respiration rate when compared to those of treated groups, which is quite in line with the

findings of Angiletta et al. (2010) who noted increased respiration rate in birds exposed to

hyper-thermal environment. The changes in respiration rate per minute, in general are

correlated with the variations in the ambient temperature and relative humidity. In fact, when

ambient temperature goes beyond thermo-neutral zone, coupled with high humidity,

chemical reactions speed up in the body, heat is generated and body temperature of birds

rises (North and Bell, 1990). Moreover, as there are no sweat glands in birds hence they

dissipate their body heat mainly through respiration (Nillipour and Melog, 1999). Therefore,

when environmental temperature goes higher than the thermo-neutral zone, respiration rate

increases up to 10 times, from a normal rate of 25 breaths/minute (Remus, 2001), which in

turn causes a raise in the pH and thus results in respiratory alkalosis. In such conditions

sodium bicarbonate can be used as a buffering agent to ameliorate the problem (Whiting et

al., 1991) hyperthermia.

It has been observed that birds kept under heat stress spent less time for feeding, more

time for drinking water and panting (Mack et al., 2013). However, birds utilize multiple ways

to maintain their body temperature and homeostasis when subjected to heat stress, which

include increased radiation, convection and evaporation along with the heat loss through

vasodilatation (Mustaf et al., 2009). Birds also have an additional mechanism (air sacs) for

exchange of heat between their body and the ambient environment. Air sacs help air

circulation on surfaces adding increase in gaseous exchange with air and evaporative loss of

heat (Fedde et al., 1998).

Dietary inclusion of sodium bicarbonate has shown to decrease respiration rate of the

birds in treated groups as compared to those of untreated group (control). A probable

explanation of decrease in respiration rate of the birds may be the increase in bicarbonate

ions concentration in the blood of the treated birds. The birds which were fed sodium

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bicarbonate containing diets exhibited lower respiratory rate and thus produced less heat for

this physiological norm. This probably could be the reason for significantly lower rectal

temperature in this case.

4.16.3Water intakeResults of the preent study revealed that birds offered diets containing different levels

of sodium bicarbonate exhibited more water consumption as compared to those of untreated

group (control). An increased water consumption of the treated groups may probably be due

to higher dietary electrolyte balance of experimental diets and more feed consumption by

these birds when compared to those of control group. At high environmental temperatures,

the stimulus for more water intake and increased rate of water exchange in the body of birds

can be useful (Borges et al., 2003). A raise in water intake helps in lowering the body

temperature of broilers exposed to higher ambient temperature (Ahmad et al., 2005). Increase

in water consumption of the treated groups was compatible with the findings of Balnave and

Gorman (1993) and Teeter and Belay (1996) who reported that sodium ions induced increase

in water intake which might have been helpful in heat dissipation.

Water consumption of the birds exhibited a linear increase with increase in the level

of addition of sodium bicarbonate in the experimental diets. Borges et al. (2003) reported that

water consumption of birds increased linearly as the dietary electrolyte balance increased.

They observed that birds fed diets supplemented with NaHCO3 (DEB, 360mEq/kg)

consumed more water. Vieites et al. (2005) compared 0 to 350mEq/kg of DEB in diets and

found the lowest litter moisture contents at 138 and 147mEq/kg, whilst Oliveira et al. (2010)

recommended a DEB of 200mEq/kg for the best litter moisture and bone development. A

significant increase in water intake in sodium bicarbonate supplemented groups during

summer was in accordance with the results observed by Sayed and Scott (2008).

Findings of Ahmad (1997) haverevealed maximum water consumption (176

ml/bird/day) in birds kept on 75% supplemented sodium from NaCl. Whereas, minimum

water consumption (201ml/bird/day) was recorded in the birds kept on 75% supplemented

Na+ from NaHCO3. Contradictory to results of the present study, Fowler (1990) reported an

increase in water intake of birds fed ration containing 100% sodium from NaCl. Similar

findings have also been stated by Hooge (1995) in a review paper that complete replacement

of NaCl with NaHCO3 in broiler diets reduced water consumption by 3.04%.

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4.17 Hematological profile4.17.1 Serum glucose

The birds fed diet without addition of sodium bicarbonate (control group), exhibited

the highest glucose level in their blood as compared to those of treated groups (B, C, D and

E). As the ambient temperature was very high during the experimental period, therefore, the

birds experienced a continuous heat stress. In response, to combat this heat stress the birds in

control group might have secreted higher level of hormones like glucocorticoid, adrenaline

and nor-adrenaline, which might have caused gluconeogenesis, ultimately leading to rise in

blood glucose concentration (Yang et al . , 1992). Similar results have also been reported by

Borges (2001) in the birds exposed to heat stress.

On the other hand, the birds which used diets containing sodium bicarbonate

exhibited lower blood glucose level than those of control group. Decrease in blood glucose

level of the birds kept in treated groups may probably be because of decrease in heat stress

upon the birds used in the present study. That’s why the treated birds have exhibited

significantly lower rectal temperature as compared to those of group A. The results of this

study are close to the findings of Ahmad et al. (2005) who observed a decrease (P<0.05) in

glucose level in broilers fed diet containing sodium bicarbonate those of controls. Al-Hassani

et al. (2001) also reported a decrease (P<0.05) in glucose level in Hisex brown layers

subjected to heat stress when fed diet containing sodium bicarbonate.

In contradiction to the findings of present study, Koelkebeck and Odom (1995) did

not observe effect of high ambient temperature on blood glucose concentration in layers.

Similarly, Zakaria et al. (2009) also did not discover any outcome of dietary addition of

sodium bicarbonate on glucose level in chickens. Discrepancy in the results of these research

experiments may be due either to the variation in the levels of sodium bicarbonate

incorporated in the diets used or temperature stress to which the birds were exposed in these

studies, or both.

4.17.2 Packed cell volume (PCV), Erythrocyte sedimentation rate (ESR) and Red blood

cells count (RBCs)

PCV, ESR and RBCs count of all the experimental birds remained unaffected (P>0.05) due

to the dietary addition/inclusion of NaHCO3. Conflicting results have so far been reported

regarding the effect of dietary addition/inclusion of NaHCO3 on packed cell volume of

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poultry birds. A significant decrease in packed cell volume has been observed by Oladele et

al. (2001) in birds exposed to high environmental temperature. They attributed this increase

to high ambient temperature and nutritional stress, which impaired the production of blood

cells in the birds. Whereas, Mubarak et al. (1999); Al-Hassani et al. (2001) and Ahmad et al.

(2005) have observed an increase in hematocrit values in birds treated with sodium

bicarbonate and these findings are quite in contrast to the findings of present study. Similarly,

contradictory to the findings of this study, Ekanayake et al. (2004); Mubarak and Sharkawy,

(1999) have reported increase in RBCs count in birds fed diets containing sodium

bicarbonate.

In fact, increase in water consumption due to the use of sodium bicarbonate may

result in peripheral vasodilation, which has been known to cause an influx of extracellular

fluid in to vascular space (Boulahsen, 1989), thus causing decrease in packed cell volume of

the birds. However, results of this study did not reflect such effects even water consumption

of the birds receiving sodium bicarbonate has shown a significant increase (see section

4.3.3). However, hematological profile of birds varies with age, sex, environmental factors

including season and stress to which the animals have been exposed (Olayemi and Arowolo,

2009).

4.17.3 Blood hemoglobin

Birds receiving diets containing sodium bicarbonate exhibited significantly higher

hemoglobin concentration in their blood as compared to those of untreated group (control). In

the present study, decline in hemoglobin concentration at higher environmental temperature

in the birds of control group also coincides with the findings of Yahav et al. (1997). Sahota

and Gilani, (1995) and Vecerek et al. (2002) have also observed a decreased hemoglobin

concentration in layers kept at high ambient temperature.

However, findings of Genedi (2000) have shown that addition of anti-stressors

(NaHCO3) in drinking water of Leghorn and Matrouh hens markedly increased their

hemoglobin concentration (%), even under heat stress conditions. Therefore, increased

hemoglobin concentration in sodium bicarbonate treated groups may probably be due to

increased nutrient uptake and reduction in body temperature, which may have led to

improved physiological performance of the layers. Results of the current study are in

accordance with report of Ahmad et al. (2005) who noted an increase in hemoglobin

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concentration in birds due to inclusion of sodium bicarbonate in their diet.

However, the highest level of sodium bicarbonate (2%) included in the diet of the

experimental birds did not show any significant difference on their hemoglobin concentration

when compared to those of control group. A probable explanation of this result may be the

decrease in feed intake (see section 4.1.2), which might have quenched the thirst due to

increase in sodium intake, probably because of higher blood osmotic pressure of the birds

(Borges et al., 2003) and ultimately resulted in increased dilution of blood/blood contents.

On the other hand, excessive water intake probably improved feed passage rate and diluted

the enzymes of digestive tract resulting in reduced nutrient uptake (Ahmad et al., 2009;

Ravindran et al., 2008). Ultimately it might have reduced hemoglobin concentration in the

birds fed higher level of sodium carbonate.

4.17.4 White blood cell count (WBCs)

Concentration of WBCs was known to be higher in the birds fed diet without

inclusion of sodium bicarbonate (control group)) when compared to those of treated groups.

Higher WBCs count in the control birds may probably be due to high environmental

temperature to which these birds were exposed. Results of the present study are compatible

to those examined by Khattak et al. (2012) who found increase in WBCs count (3.2×104µl)

in the birds exposed to 38-40oC as compared to those fed sodium bicarbonate containing diet

(1.8× 104µl) at the same temperature. An increased white blood cells count at higher

environmental temperature has also been reported by Anjum (2000).

The birds fed diets containing sodium bicarbonate exhibited a decrease (P<0.05) in

WBCs count than those of control group. The reduction in WBCs count coincides with

reduction in bogy temperature of the birds of various groups (see section 4.3.1) due to the

treatments. Therefore, reduction in WBCs count due to the dietary inclusion of sodium

bicarbonate may be credited to the reduction in body temperature of the birds which might

have decreased heat stress and thus resulting in decrease in WBCs count (Maxwell et al.,

1992). The other factors, which may influence this parameter (hematological profile) in birds

include age, sex, season and stress to which the animals have been exposed (Olayemi and

Arowolo, 2009).

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4.18 Serum metabolites4.18.1Serum urea

Concentration of serum urea was discovered to be higher in the birds fed diet without

inclusion of sodium bicarbonate (control group) when compared to those of treated groups.

Higher concentration of serum urea in the control birds may probably be due to high

environmental temperature to which these birds were exposed. These results are matched to

those reported by Anjum et al. (2000) who observed an increase in serum urea concentration

in layers kept at higher ambient temperature as compared to those reared under heat

combating systems. Results of the current research are also in accordance with the findings

of Yang et al . (1992) who found significantly higher serum urea concentration in birds kept

at low temperature (12 °C). However birds kept at relatively high ambient temperatures (23

and 28 °C) showed lower serum urea concentration.

The birds fed diets containing sodium bicarbonate exhibited a decrease (P<0.05) in

their serum urea concentration as compared to those of control group. The reduction in serum

urea concentration also coincides with the reduction in body temperature of the birds of

various groups (see table 4.4) due to the treatments. Therefore, reduction in serum urea

concentration due to the dietary inclusion of sodium bicarbonate may be attributed to the

reduction in body temperature of the birds which may have decreased heat stress and hence

resulting in decrease in serum urea concentration (Maxwell et al. (1992). Olayemi and

Arowolo, (2009) have also reported that environmental factors and stress to which the

animals have been exposed, may affect their hematological profile.

4.18.2 Serum uric acid

The birds fed diets containing sodium bicarbonate exhibited a significant decrease in

their serum uric acid concentration as compared to those of control group. A possible

explanation of these results may be that excessive intake of minerals like sodium may cause

synergistic effect on other minerals i.e. excess of one can reduce the availability of another.

Excessive intake of sodium may cause hypernatremia (Mc Dowell, 1992; Oviedo-Ronden et

al., 2001) and thus may cause excessive water intake, diarrhea and increase in serum uric

acid (Davison and Wideman, 1992) in poultry birds.

However, Kurtoglu et al. (2007) did not observe effect of sodium bicarbonate on uric

acid level in Brown-Nick layer hens. Similarly, Koelkebeck and Odom (1995) also reported

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different results that heat stress had no effect on serum uric acid concentration of laying

birds. Dissimilarity in the results of these studies may probably be because of the differences

in the levels of sodium bicarbonate used.

4.18.3 Serum creatinine

Addition of different levels of sodium bicarbonate in the diets of layer did not give

any significant effect on serum creatinine values of the birds when compared to those fed diet

without inclusion of sodium bicarbonate (control group). These results are in line with the

research results of Koelkebeck and Odom (1995) who found that layers kept under acute heat

stress had no effect on their serum creatinine levels.

4.18.4 Serum alkaline phosphatase

Addition of different levels of sodium bicarbonate in the diets of layer did not affect

(P>0.05) serum alkaline phosphatase level of the birds. These results are in line with those

reported by Bogin et al. (1981) who reported that broilers subjected to heat stress for two

hours showed non-significant effect on their blood serum alkaline phosphatase level. Results

of the present study are also in line to those reported by Koelkebeck and Odom (1995) who

observed that acute heat stress had no effect on alkaline phosphatase enzyme of laying hens.

4.19 Serum proteins analysis4.19.1 Total proteins

Dietary inclusion of different levels of sodium bicarbonate showed a significant

increase in total protein concentration in blood serum of layers when compared to those fed

diet without any addition of sodium bicarbonate (control group). Results of the present study

are in accordance with the findings of Kurtoglu et al. (2007) who reported significant

(P<0.05) effect due to dietary inclusion of sodium bicarbonate on total protein in blood

serum of Brown-Nick layers indicating that increase in sodium ions concentration may

improve synthesis of total proteins in layers.

4.19.2 Albumin

The birds fed control diet exhibited a significantly lower albumin concentration in

their blood than those fed diet containing different levels of sodium bicarbonate (treated

groups). Lower albumen concentration in the birds of control group may probably be due to

decrease in digestion and absorption of protein contents of the diet because of heat stress

(Koh and Macleod, 1 999 ;), which possibly may also have exerted negative effect on protein

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synthesis, ultimately resulting in reduced albumin concentration. Findings of this study are in

accordance with those of Geraert et al. (1996) and Yahav et al. (1997) who observed

decreased protein level in birds reared in high ambient temperature (heat stress).

Inclusion of NaHCO3 in the diets of birds, however, showed an increase in their blood

albumen concentration probably because of lowering down body temperature of the birds,

which also had a positive effect on digestion and absorption of nutrients. Another probable

explanation of his fact may be the increased feed intake of the birds fed sodium bicarbonate

added diets. These findings coincide with those observed by Kurtoglu et al. (2007) who

found an increase (P<0.05) in blood albumen concentration of Brown-Nick layers fed diet

containing sodium bicarbonate.

In contrast to the findings of present study, Anjum (2000) noted increased serum albumin

concentration in heat stressed birds. Contradictions in these results may be due to difference in

environmental factors like season and type of stress to which the birds were exposed. Change

in albumin concentration of birds due to these factors (season and type of stress) has also

been reported by Olayemi and Arowolo, (2009).

4.19.3 Globulin

Addition of different levels of sodium bicarbonate in the diets of layer did not cause

any significant effect (P>0.05) on globulin concentration of the birds when compared to

those fed diet without inclusion of sodium bicarbonate (control group). Information regarding

the effect of inclusion of sodium bicarbonate on blood globulin concentration is very scanty.

However, decline in serum globulin of birds is observed by Yang et al. (1992); Geraert et al.

(1996); Anjum, (2000) due to heat stress. They also reported decreased protein level in the

heat exposed birds. Discrepancy in results of these research studies may perhaps be because

of difference in environmental factors like season and type of stress to which the birds were

affected. Olayemi and Arowolo, (2009) have also observed change in albumen concentration

of birds due to these factors (season and type of stress).

4.20 Plasma electrolytes, minerals and serum pH4.20.1Plasma sodium

Dietary inclusion of different levels of sodium bicarbonate depicted a significant

increase in plasma sodium concentration of layers when compared to those fed diet without

addition of sodium bicarbonate (control group). Increase in plasma sodium concentration in

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may be due to the fact that the treated birds were being fed more sodium in their diets with

sodium bicarbonate. However, birds fed diet without addition of sodium bicarbonate (control

group) showed low plasma sodium concentration. A probable explanation of lower sodium

concentration in blood plasma of heat stressed birds (control) may be due to increase in

urinary excretion of this essential mineral (Gorman et al., 1997). Results of the study are in

line to those observed by Borges et al. (2004) who reported decreased blood sodium level in

broilers kept under heat stress. A decline in blood sodium ions level in birds kept under heat

stress has also been observed by Takahashi and Akiba (2002).

On the other hand, increased plasma sodium ion concentration in the birds of treated

groups may be related to the dietary addition of different levels of sodium bicarbonate,

because a linear increase in sodium ion concentration has been observed with increase in its

level of inclusion. These results are compatible to those observed by Ahmad et al. (2006).

They observed a rise in plasma Na+ concentration in birds due to the supplementation of

sodium bicarbonate in their diets. Similarly, Mushtaq et al. (2005) found an increased serum

sodium concentration because of dietary addition of different levels of sodium.

In contrast to the findings of present study, Bonsembiante and Chiericato (1990) did

not observe effect (P>0.05) of dietary addition/inclusion of sodium bicarbonate on sodium

ion concentration in meat type turkeys. Contradictions in these results may either be due to

the difference in species of the birds (layers vs turkeys) used in these studies or to the degree

of heat stress to which the birds were exposed (Olayemi and Arowolo, 2009; Teeter et al.

1985), or both.

4.20.2Plasma Potassium

Concentration of plasma potassium was found to be lower in the birds fed diet

without inclusion of sodium bicarbonate (control group) when compared to those of treated

groups. A probable explanation of lower potassium ion concentration in blood plasma of heat

stressed birds (control) is increase in urinary excretion of this essential mineral (Gorman et

al., 1997). These results are in line to those observed by Borges et al. (2004) who reported

decreased blood potassium concentration in broilers kept under heat stress. A decline in the

level of potassium ions in birds kept under heat stress has also been observed by Takahashi

and Akiba (2002).

However, birds receiving diets containing sodium bicarbonate exhibited increased

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plasma potassium level as compared to untreated group (control). Results of the present study

supports the findings of Ahmad et al. (2006) who observed that blood K+, was significantly

increased by the addition of sodium sources (sodium carbonate, NaHCO3 or sodium sulfate)

in the diet. Similar effect has also been observed by Ghorbani and Fayazi (2009) who studied

the dietary effect of NaHCO3 on plasma potassium concentration of layers kept under

constant/chronic heat stress and found an increase (P<0.05) in plasma potassium

concentration due to dietary inclusion of sodium bicarbonate. Similarly Mushtaq et al. (2005)

reported that serum K+ level of birds was significantly affected (P<0.05) by inclusion of

varying dietary sodium levels.

Contrary to the results of current research, Kurtoglu et al. (2007) found a decreased

plasma potassium concentration in layers offered diets having NaHCO3 as compared to those

fed diets containing either NaCl or KCl. Similarly, Keskin and Durgan (1997) found

decreased plasma potassium concentration in quails fed diets containing sodium bicarbonate

(1%) kept at high ambient temperature when compared to those kept at thermo-neutral zone.

Dissimilarity in results of these experiments may possibly be due either to difference in

environmental factors like ambient temperature or difference in species of the birds (layers vs

quails) used in these studies.

4.20.3 Plasma chloride

Birds receiving diets containing sodium bicarbonate exhibited decreased plasma

chloride level as compared to those of untreated group (control). Exchange of Na+/H+ takes

place in kidneys of the birds. When level of sodium is increased in blood, the excretion of

chloride in urine is also increased (Pech-Waffenschmidt et al., 1995), ultimately resulting in

reduction of its level in blood plasma. Therefore, decrease in plasma chloride concentration

in the birds fed various levels of sodium bicarbonate may be attributed to increased level of

sodium in their blood, as has been depicted in the results of present study. These findings are

compatible to the findings of Ahmad et al. (2006) who reported that blood Cl-, was decreased

by the supplementation of sodium sources (sodium carbonate, NaHCO3 or sodium sulfate) in

the diet of poultry birds.

Contrary to the results of present research, Ahmad et al. (1997) reported that blood

Cl- concentration was not affected due to the addition of dietary sodium bicarbonate in the

diet of broilers. Contradictory results have also been reported by Bonsembiante and

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Chiericato, (1990) who observed non-significant effect (P>0.05) of dietary addition/inclusion

of NaHCO3, on blood chloride in meat type turkeys. Differences in these results may be due

to the various species of the birds or levels of sodium bicarbonate used in these studies or

both.

4.20.4 Plasma bicarbonate

Concentration of plasma bicarbonate (HCO3-) was known to be higher in the birds fed

diet containing sodium bicarbonate (treated groups) as compared to those of control group.

Increase in plasma HCO3- concentration in treated birds may possibly be due to the inclusion

of sodium bicarbonate in their diets, which led to increased availability of these ions to the

birds. Generally, a decrease in plasma Cl– increases HCO3– re-absorption by the kidneys (Ait-

Boulahsen et al., 1989), leading to higher concentration of HCO3– in blood plasma. Results of

the current study supports the findings of Keskin and Durgan (1997) who found an increase

(P<0.05) in plasma bicarbonate level due to the inclusion of NaHCO3 (1%) in the diet of

birds exposed to heat stress. Similar findings have also been reported by Squire and Julian

(2001) who observed a significant rise in blood HC03- of the layers receiving ration

supplemented with sodium bicarbonate.

Whereas, plasma bicarbonate level was significantly decreased in birds kept under

heat stress (control) probably because of increased respiration rate (Teeter et al.,1985).

Moreover, heat stress can induce increase in metabolic requirements of this essential anion

(Gorman and Balnave, 1994). However, this situation can be improved as has been depicted

in the results of this study, by the dietary inclusion sodium bicarbonate. On the other hand,

excessive level of NaHCO3 in diet may cause increase in water consumption, diarrhea and

urine pH (Mert, 1991; Davison and Wideman, 1992). Therefore, based upon the discussion

above, it may be concluded that sodium bicarbonate if used at an optimum level in the ration,

can increase HC03- and hence may lead to higher blood pH (Austic and Keshavarz, 1988) of

broilers resulting in their better rate of growth.

4.20.5 Plasma calcium and phosphorus

The difference in plasma calcium and phosphorus concentration in birds fed diets

with or without addition of sodium bicarbonate was found to be non-significant. These

findings are in accordance with those observed by Ghorbani and Fayazi (2009) who studied

the effect of varying dietary levels NaHCO3 and rearing system on plasma calcium of layers

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kept under chronic heat stress. Results of their study revealed that neither dietary inclusion of

sodium bicarbonate nor its levels (0.5%-1.5%) exhibited any effect on plasma calcium

concentration in layers. Findings of the present research also coincide with those observed by

Kurtoglu et al. (2007) who found no effect (P>0.05) of NaHCO3 on plasma calcium

concentration in layers. Similarly, Keskin and Durgan (1997) found non-significant

differences in blood calcium concentration between thermo-neutral group and sodium

bicarbonate supplemented (1% NaHCO3 in diet) group of quails exposed to heat stress.

4.20.6 Serum pH

Diets containing sodium bicarbonate when fed to experimental birds, exhibited a

decrease in their serum pH as compared to those of untreated group (control). High ambient

temperature during summer (heat stress) has shown to increase heat production in birds

(Teeter & Belay, 1996; Macleod et al., 1984). To get rid of this excessive body heat, birds

start panting which may increase their respiratory rate, resulting in excessive loss of CO2.

This loss causes an increase in blood pH of birds and may lead to respiratory alkalosis. Under

such conditions sodium bicarbonate can be used as a buffering agent, which is known to

normalize blood pH by supplying bicarbonate ions (Whiting et al., 1991). Therefore, it may

be inferred that the birds fed diets containing sodium bicarbonate may have low serum pH

value (optimum physiological range), as has been investigated in the current study.

Findings of the present research are compatible to the findings of Ahmad et al. (2005)

who observed that blood pH was decreased by the supplementation of sodium sources

(sodium carbonate, sodium bicarbonate or sodium sulfate) in the diets of birds. Similarly

Mushtaq et al. (2005) investigated that blood pH was decreased (P<0.05) by increasing

dietary addition of sodium levels 0.20% vs 0.25%. Similar results are also observed by

Fuentes et al. (1998) who reported a decrease in blood pH in guinea fowl fed diets containing

different levels (0.6-2.4%) of sodium bicarbonate. Khattak et al. (2012) also observed

decreased pH (8.04) in broilers kept at 38-40 0C as compared to those fed sodium bicarbonate

containing diet (8.34 pH) at the same temperature.

However, Keskin and Durgan (1997) found a significant increase in blood pH of

quails exposed to heat stress when fed diet containing 1% NaHCO3. Disparity in results of

these experiments may possibly be due to variation in ambient temperature or difference in

species of the birds (layers vs quails) used in these studies (Olayemi and Arowolo, 2009).

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4.21 Serum lipids profile

Birds receiving diets containing sodium bicarbonate exhibited significantly less

serum lipids profile i.e. low density lipoproteins (LDL) and triglyceride in their blood when

compared to those of control group. A probable explanation of decrease in this parameter

may be that sodium bicarbonate might have stimulated the synthesis of bile acids from

cholesterol, leading to decreased level of blood cholesterol in this group (Naviglio et al.,

2011). Another possible reason for reduction in serum cholesterol may be the inhibition of

“squalene epoxidase” an enzyme which is necessary for production of cholesterol

(Angelovicova, 1997). The reduction of serum low density lipoprotein to some extent may

reduce the chances of cardiovascular diseases.

Birds receiving diets containing sodium bicarbonate exhibited less serum triglyceride

level as compared to untreated group (control). Layers in group C which were fed diet

containing 1% sodium bicarbonate showed the lowest serum triglyceride level in their blood.

A possible explanation of lower level of triglyceride in treated groups may be inhibition of

fatty acid synthesis and production of bile acids from cholesterol (Naviglio et al., 2011),

leading to decreased concentration of serum triglyceride in this group. Moreover, inhibition

of an enzyme called “squalene epoxidase” may also cause reduction in serum triglyceride

(Angelovicova, 1997).

Probable explanation of decreased serum triglycerides level in the birds fed sodium

bicarbonate containing diets may be decrease in cortisol level of the birds, as has been

observed in the results of present study (see section 4.11.4). A similar relationship between

serum triglyceride and cortisol level has been observed by Sahin et al. (2002) who observed a

linear rapport between serum triglycerides and ACTH level, due to its catabolic effect, in

birds kept under high ambient temperature.

4.22 Hormones and enzymes4.22.1Tri-iodothyronine (T3) and Thyroxin (T4)

Addition of different levels of sodium bicarbonate in the diets of layer showed

significant effect on serum T3 and T4 concentration of the birds when compared to those fed

diet without inclusion of sodium bicarbonate (control group). The birds fed diets without

adding sodium bicarbonate, exhibited the lowest level of these growth hormones (T3 and T4)

in their blood. Secretion rate of thyroxin (T3 and T4) is known to be influenced by

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environmental temperature (Wright et al., 1952) and these hormones are secreted at their best

when the birds are free from all types of stress, particularly heat stress. Moreover, fall in

temperature results in increased thyroid secretion. As incorporation of sodium bicarbonate in

the diets has shown to reduce heat stress in the present study, therefore, higher concentration

of these hormones in the birds of treated groups may probably be due to the heat stress

combating ability of dietary sodium bicarbonate.

Hypothalamus and pituitary receive stimulus of high environmental temperature and

in turn causes a decline in the secretions of growth hormones (Anjum, 2000). However,

dietary inclusion of sodium bicarbonate during hot period was found to be accompanied by

reduction in heat stress (Remus, 2001). Hence, dietary addition of bicarbonate might have

caused an increase in concentration of growth hormones in the blood of treated birds. Higher

concentration of these hormones corresponded to maximum performance as has also been

observed by Anjum, (2000).

Findings of the present research coincide with the research results of Sahin et al.

(2001) who investigated a decrease (P<0.05) in serum T4 and T3 concentration in birds kept

under heat stress. Decrease in concentration of these hormones in birds reared under heat

stress has also been reported by Honog et al . (1995) and Anjum, (2000).

4.22.2 Estrogen

Difference in serum estrogen concentration in birds fed diets with or without addition

of sodium bicarbonate was found to be significant. The birds fed diets without adding sodium

bicarbonate (control) exhibited lower serum estrogen concentration when compared to those

of treated groups. Estrogen plays a pivotal role in the reproductive efficiency of fowls (Lile,

1976), therefore, its decreased concentration in control birds consequently adversely affected

the performance of the birds. In fact, when ambient temperature goes beyond thermo-neutral

zone, chemical reactions speed up in the body, heat is generated and body temperature of

birds rises (North and Bell, 1990) which probably may have been the cause of adverse effect

on normal synthesis of this hormone. It has been observed that under such conditions sodium

bicarbonate can acts as a buffering agent to mitigate the heat stress (Whiting et al., 1991).

Results of this study are compatible to the research results of Anjum (2000) who reported

significant decrease in the level of estrogen in layers kept under heat stress conditions.

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4.22.3 Progesterone

Concentration of progesterone was found to be lower in birds kept at high ambient

temperature when compared to those of treated groups. A possible explanation of this result

may probably be that high ambient temperature may have depressed ovarian function, posing

hindrance against the release of leutinizing hormone, subsequently resulting in decreased

progesterone concentration. These results are in line with the research results observed by

Anjum (2000) who reported a significant decrease in progesterone concentration in layers

exposed to heat stress. Furthermore the results of the current research are also in concord

with the earlier findings of Novero et al . (1991) and Chostesangasa (1992), where higher

environmental temperature has been reported to inhibit growth and sexual maturity,

respectively, as a result of decreased secretion of progesterone in birds.

4.22.4 Corticosterone

Dietary inclusion of different levels of sodium bicarbonate depicted significantly

lower concentration of corticosterone hormone in layers when compared to those fed diet

without sodium bicarbonate. In fact, when ambient temperature goes beyond its thermo-

neutral zone, chemical reactions speed up in the body, heat is generated and body

temperature of birds rises (North and Bell, 1990), which causes heat stress in birds. In such

conditions sodium bicarbonate can be used as an anti-stress agent to ameliorate the effect of

heat stress (Whiting et al., 1991). Therefore, a probable explanation of decrease in cortisol

level of the birds may probably be due to increase in bicarbonate ions concentration in the

blood of the treated birds.

Reduction in serum corticosterone concentration in the layers fed diets containing

sodium bicarbonate also corresponds to decrease in their rectal temperature, as has been

depicted in the results of this study. Whereas, the highest level of this hormone was noted in

the serum of layers of group A (control), which were offered diet without addition of sodium

bicarbonate. An increase in corticosterone level of birds, with increase in environmental

temperature has also been observed by Sahin et al. (2002), Teukam et al. (1996), Bains

(1996) and Yang et al. (1992),.

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4.22.5 Serum Glutamic-Oxaloacetic Transaminase and Serum Glutamic Pyruvic

Transaminase

Birds receiving diet without addition of sodium bicarbonate (control group) exhibited

higher concentration of liver enzyme Serum Glutamic-Oxaloacetic Transaminase (SGOT),

when compared to those of treated groups. Heat stress has shown to increase plasma cortisol

concentration (Sahin, et al., 2002) in birds. This increase may cause an increase in catabolic

effect in liver, which exerts maximum stress on it, leading to a raised level of serum SGOT

of birds exposed to heat stress. Therefore, increase in serum SGOT concentration in the birds

of control group may probably be because of stress on their liver due to higher body

temperature of these birds as compared to those of treated groups. These results are similar to

those reported by Anjum, (2000) who observed an increase in SGOT in layers kept/reared

under heat stress. Reduction in concentration of serum SGOT in layers kept under heat

combating systems was also noted when compared to those kept at high ambient temperature.

Another possible explanation of higher concentration in blood serum of heat stressed

birds may be the deviation in blood pH of birds exposed to heat stress as a result of

respiratory alkalosis. Though pH influences many aspects of cell structure and functions, yet

catalytic activity of the enzymes is specifically sensitive to it (Lehninger, 1970). Each

enzyme has maximum activity at a specific pH, called optimum pH, and the activity reduces

sharply on either side of the optimum pH. Therefore, biological control of pH of cells and

body fluids is of utmost importance for metabolism and cellular functions.

On the other hand, the birds fed diets containing sodium bicarbonate exhibited a

significant reduction in SGOT concentration. A possible explanation of this fact may

probably be the reduction in body temperature of birds, which might have reduced stress on

their liver and hence led to a decrease in SGOT concentration in the birds of treated groups.

Based upon these findings, it can be envisaged that dietary inclusion of sodium bicarbonate

may help alleviate heat stress by decreasing body temperature and concentration of plasma

cortisol. These findings are compatible to those observed by Ahmad et al. (2005) who

reported that dietary inclusion of sodium bicarbonate can decrease body temperature of heat

stressed birds.

In contrast to the results of present study, Ozbey et al. (2004) observed that blood

SGOT concentration was not affected due to heat stress in quails. Dissimilarity in results of

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these researches, however, may likely be due to variation in ambient temperature maintained

or difference in species of the birds (layers vs. quails ), used in these studies. However,

concentration of Serum Glutamic Pyruvic Transaminase (SGPT) remained unaffected in the

birds due to the inclusion of NaHCO3 in their diets.

4.23 Immune responseDietary inclusion of different levels of sodium bicarbonate depicted an increase

(P<0.05) in antibody titer against NDV in layers when compared to those fed diet without its

addition (control group). Environmental stressors have been known to affect immunity and

innate resistance of the host directly or indirectly (Robertson, 1998). Therefore, increase in

antibody titer against NDV in birds offered diets having varying levels of sodium bicarbonate

may probably be due either to less heat stress upon these birds because of reduction in their

body temperature or lower cortisol concentration as compared to those of group A (control),

or both. Results of the present experiment are compatible to the observations of Khatak et al.

(2012) who investigated higher haemaglutination inhibition titer against NDV in birds

consuming diets containing sodium bicarbonate.

Borges et al. (2003) have investigated that increase in DEB may cause decrease in

heterophil to lymphocyte ratio in blood, leading to increase in antibody titer. Similarly,

Santin et al. (2003) have reported a linear increase (P<0.05) in NDV antibody titers with

rising DEB (40, 140, 240, 340mEq/kg), receiving NaCl, NaHCO3, and NH4Cl as

supplements, Therefore, it may safely be concluded that dietary addition of sodium

bicarbonate may improve antibody titter against NDV in birds.

On the other hand, birds receiving diets without sodium bicarbonate (control)

exhibited reduced antibody titer against NDV comparing to those of treated groups. Control

of antibody mediated immunity at various environmental temperatures hasbeen studied by

El-Gendy et al. (1995) and they found that birds exposed to heat stress had significantly

depressed agglutinin levels. Moreover, exposure of layer birds to stressors like heat stress

have shown to cause a decrease (P<0.05) in lymphocytes and increase (P<0.05) in

heterophils (Borges et al., 2004), resulting in reduced immunity. Therefore, outcomes of

present study correspond with the results of El-Gendy et al. (1995) who observed that

antibody titer against NDV was lower in heat stressed broilers as compared to those kept

under normal temperatures. Similar effects have also been reported by Anjum (2000) who

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observed decrease in antibody titer against Newcastle disease virus, probably because of

increase in total leukocyte count (Maxwell, 1993), in birds exposed to heat stress. Bains et al.

(1996) have observed an effect (P<0.05) on immune system of turkey breeder hens when

exposed to heat stress. Comparable findings were also noted by Savic et al. (1993) who

exposed the birds to heat stress at different intervals and found lower antibody titer against

Lasota strain virus in heat stressed birds.

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CHAPTER-5

DIGESTIBILITY TRIALEFFECT OF DIETARY INCLUSION OFNaHCO3ON NUTRIENT

DIGESTIBILITY IN CAGED LAYERS DURING SUMMER

5.1 IntroductionSodium bicarbonate (NaHCO3) is a white solid crystalline compound soluble in

water, which is commonly used as an antacid to treat acid indigestion (Thomas and

Stone, 1994). It is generally supplemented as a simple solution for reinstate the pH of

water which has a high concentration of chlorine (Teeter et al., 1985; Whiting et al.,

1991). Sodium bicarbonate in feed or water has also shown potential benefits on production

performance (Ahmad et al., 2005; Khatak et al., 2012), egg characteristics (Kaya et al.,

2004), blood profile (Kurtoglu et al., 2007) and nutrient digestibility (Ahmad, 2007), in

poultry birds reared under heat stress.

Sodium bicarbonate in the diet of layers may improve nutrient digestibility by

increasing sodium ions concentration (Fethiere et al., 1994); improving electrolyte balance in

the diet (Borges et al., 2003); meeting the requirements for the HCO3- ions (Gorman and

Balnave, 1994) and decreasing the losses caused by heat stress (Gorman and Balnave, 1994;

Mirsalimi and Julian, 1993; Braton et al., 1989). It is cheap, easily available and easy to

handle, therefore, can be safely incorporated in poultry diets to ameliorate the adverse effects

caused by heat stress.

Therefore, the present trial was conducted to study the effects of dietary inclusion of

sodium bicarbonate on in vivo digestibility of dry matter (DM), crude protein (CP), crude

fibers (CF) and ether extract (EE). The effect of addition of this compound in poultry diets

was also studied on absorption of some minerals i.e. calcium, phosphorus, sodium, potassium

and iron, in caged layers during summer.

5.2 Materials and methodsThe digestibility trial (in vivo) was conducted in 36 weeks old layers. Thirty layers

having similar body weight were obtained from the same batch which was used for

performance trial. All the experimental layer birds were maintained/kept in individual

metabolic cages. These layer birds were randomly allotted/allocated to five experimental

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diets/rations in such a way that each diet was offered to 6 layers so that each bird served as a

replicate. At the end of 38th week of age, fecal samples were collected for two days at the

interval of 3 hours. The birds in all the groups were fed same amount of feed during the

collection period.

5.2.1 Experimental diets

Five experimental diets i.e. A (control, without Sodium bicarbonate), B (0.5%Sodium

bicarbonate), C (1 % Sodium bicarbonate), D (1.5% Sodium bicarbonate), E (2% Sodium

bicarbonate) used in the performance trial were also used in the digestibility trial, however

acid insoluble ash (1%) was included/incorporated as an indigestible marker in the diets to be

used in the digestibility trial (See table 3). These diets/feeds were formulated according to the

NRC (1994) recommendations for nutrient requirements of layers. All the diets were iso-

nitrogenous (CP 17 %) and iso-caloric (ME 2700 Kcal/Kg diet).

5.2.2 Chemical analysis of feed/excreta

Feed and or excreta samples were analyzed for DM, CP, CF, EE, calcium,

phosphorus, sodium, potassium, iron and AIA marker determination as described by AOAC

(2010). The nutrients digestibilities were calculated receiving the equations outlined in

chapter 3.

5.2.3 Statistical analysis

The data collected were subjected to statistical analysis for interpretation of results

using completely randomized design (CRD). Treatment means were compared by the Least

Significance Differences (Steel et al., 1997) test.

5.3 ResultsMean values regarding digestibility of DM, CP, CF and EE in birds fed diets with or

without dietary inclusion of sodium bicarbonate are shown in table 5.1.

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Table 5.1: Effects of dietary inclusion/addition of NaHCO3 on nutrient

digestibility coefficient in layers

Variables

Treatment

A

Control

B

0.5%NaHCO3

C

1%NaHCO3

D

1.5%NaHCO3

E

2%NaHCO3

Dry matter (%)70.4±

6.09c 73.4±5.89b 77.1 ±5.79a 74.1± 6.34b 71.6±2636c

Crude protein

(%)

68.6±5.7

2c 72.7±4.65a 75±3.27a 72.2±3.38ab69.

2±5.44bc

Crude fibers %)29.

0±2.81b 33.9±2.36ab 40.7 ±2.85a 33.2 ±2.1ab 30.9±2.95b

Ether extract

(%)

82.

0±5.57b 89.4± 4.02a 93.7± 5.46a 84.8±7.78b82.7±

4.67b

Values within the same row with unlike superscripts are significantly different (P<0.05)

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5.3.1 Dry matter

Mean values of dry matter (DM) digestibility for treatments A, B, C, D and E, were

found to be 70.4, 73.4, 77.1, 74.1 and 71.6%, respectively. The results revealed an effect

(P<0.05) on DM digestibility due to the addition/ inclusion of NaHCO3 in the diets of layer

when compared to those of control group. Statistical analysis of the data depicted that the

birds of treated groups, receiving diets containing sodium bicarbonate showed higher

(P<0.05) DM digestibility than those of group A (control). The differences in DM

digestibility values were also found to be significant among the treated groups. Birds

receiving diet containing 1% sodium bicarbonate exhibited maximum digestibility followed

by those of group D and B whereas, the lowest dry matter digestibility was recorded in

groups A (control) and E. However, differences in the digestibility values of groups B and D,

and those between A and E were found to non-significant (P>0.05).

5.3.2 Crude protein

Mean values of crude protein (CP) digestibility for treatment A, B, C, D and E, were

found to be 68.6, 72.7, 75.0, 72.2 and 69.2%, respectively. The results revealed a significant

effect on CP digestibility due to the inclusion of NaHCO3 in the diets of layer when

compared to those of control group. Statistical analysis of the data depicted that the birds of

treated groups, receiving diets containing sodium bicarbonate showed higher (P<0.05) CP

digestibility than those of group A. The difference in CP digestibility values was also

significant among the treated groups. Birds receiving diet containing 1% sodium bicarbonate

exhibited maximum digestibility followed by those of group B, D and E. whereas, the lowest

CP digestibility was recorded in the control group. However, differences in the digestibility

values of groups B, C and D, and those between A and E and between D and E were found to

be non-significant (P>0.05).

5.3.3 Crude fiber

Mean values of crude fiber (CF) digestibility for treatment A, B, C, D and E, were

found to be 29.0, 33.9, 40.7, 33.2 and 30.9%, respectively. The results revealed a significant

effect on CF digestibility due to the addition/inclusion of NaHCO3 in the diets of layers when

compared to those of control group. Statistical analysis of the data depicted that the birds of

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treated groups, receiving diets containing sodium bicarbonate showed higher CF (P<0.05)

digestibility than those of group A. The difference in CF digestibility values was also

significant among the treated groups. Birds receiving diet containing 1% sodium bicarbonate

exhibited maximum digestibility followed by those of group B, D and E, whereas, the lowest

CF digestibility was recorded in the control group. However, differences in the digestibility

values of groups B, C and D, and those among A, B, D and E were found to non-significant.

5.3.4 Ether extract

Means values of ether extract (EE) digestibility for treatment A, B, C, D and E, were

found to be 82.1, 89.4, 93.7, 82.3 and 82.8%, respectively. The results revealed a significant

effect on EE digestibility due to the inclusion of NaHCO3 in the diets of layer when

compared to those of control group. Statistical analysis of the data depicted that the birds of

treated groups, receiving diets containing sodium bicarbonate showed higher (P<0.05) EE

digestibility than those of control group. The difference in EE digestibility values was also

significant among the treated groups. Birds receiving diet containing 1% sodium bicarbonate

exhibited maximum digestibility followed by those of group B, D and E. whereas, the lowest

EE digestibility was recorded in the group A. However, differences in the digestibility values

of groups B and C, and those among A, D and E were found to non-significant (P>0.05).

5.4 Minerals Mean values pertaining to the absorption of minerals i.e. calcium, phosphorous, iron,

sodium, and potassium in birds fed diets with or without dietary inclusion of sodium

bicarbonate are shown in table 5.2.

5.4.1 Calcium

Means values of regarding digestibility of calcium (Ca), for treatment A, B, C, D and

E, were found to be 56.1, 58.0, 60.3, 58.1 and 54.8, respectively. The results revealed a

significant effect on digestibility of Ca due to the inclusion of NaHCO3 in the diets of layer

when compared to those of control group. Statistical analysis of the data depicted

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Table 5.2: Effect of dietary inclusion of NaHCO3 on absorbability of minerals in

layers

Variables

Treatment

A

Control

B

0.5%NaHCO3

C

1%NaHCO3

D

1.5%NaHCO3

E

2%NaHCO3

Calcium 56.1± 3.29c 58.3±3.10b 60.3±3.20a 58.1± 3.28b 54.8 ±3.19c

Phosphorus 50.8±2.32c 53.5±3.09b 57.6±3.27a 53±3.33b 49.6±2.40c

Iron50.5 ±

2.36d 53.1±1.38c 60.8 ±3.34a 55.6 ±3.2b 50±2.27d

Sodium 51.6± 2.1c 54.1± 1.3b 59.2± 1.1a 53.5±1.11b51.6.±

1.913b

Potassium 51.8±2 c 53.5±2.47 b 56.3±3.64 a 53.5±3.64 b 52±3.47 c

Values within the same row with unlike superscripts are significantly different (P<0.05)

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that the birds of treated groups, receiving diets containing sodium bicarbonate showed

significantly (P<0.05) higher Ca digestibility as compared to those of control group. The

difference in Ca digestibility values was also significant among the treated groups. Birds

receiving diet containing 1% sodium bicarbonate exhibited maximum digestibility followed

by those of group B, D and E. whereas, the lowest Ca digestibility was recorded in the

control group. However, differences in the digestibility values of groups B and D, and those

between A, and E were found to non-significant (P>0.05).

5.4.2 Phosphorous

Means values of regarding digestibility of phosphorous (P), for treatment A, B, C,

D and E, were found to be 50.8, 53.5, 57.6, 53.0 and 49.6, respectively. The results

revealed a significant effect on digestibility of P due to the inclusion of NaHCO 3 in the

diets of layer when compared to those of control group. Statistical analysis of the data

depicted that the birds of treated groups, receiving diets containing sodium bicarbonate

showed higher (P<0.05) P digestibility than those of control group. The difference in P

digestibility values was also significant among the treated groups. Birds receiving diet

containing 1% sodium bicarbonate exhibited maximum digestibility followed by those of

group B, D and E. Whereas, the lowest P digestibility was recorded in the control group.

However, differences in the digestibility values of groups B and D, and those between A,

and E were found to non-significant (P>0.05).

5.4.3 Iron

Means values of regarding digestibility of Iron, for treatment A, B, C, D and E, were

found to be 50.5, 53.1, 60.8, 55.6 and 50.0, respectively. The results revealed a significant

effect on digestibility of Iron due to the inclusion of sodium bicarbonate in the diets of layer

when compared to those of control group. Statistical analysis of the data depicted that the

birds of treated groups, receiving diets containing sodium bicarbonate showed significantly

(P<0.05) higher Iron digestibility as compared to those of control group. The difference in

the values of digestibility of Iron also found to be significant among the treated groups. Birds

receiving diet containing 1% sodium bicarbonate exhibited maximum digestibility followed

by those of group D, B and E, whereas the lowest Iron digestibility was recorded in the

control group. However, difference in the digestibility values of groups A, and E was found

to non-significant.

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5.4.4 SodiumMeans values of regarding digestibility of sodium (Na), for treatment A, B, C, D and

E, were found to be 51.6, 54.1, 59.2, 53.5 and 51.6, respectively. The results revealed a

significant effect on digestibility of Na due to the inclusion of NaHCO3 in the diets of layer

when compared to those of control group. Statistical analysis of the data depicted that the

birds of treated groups, receiving diets containing sodium bicarbonate showed higher

(P<0.05) Na digestibility than those of control group. The difference in Na digestibility

values was also significant among the treated groups. Birds receiving diet containing 1%

sodium bicarbonate exhibited maximum digestibility followed by those of group B, D and E.

Whereas, the lowest Na digestibility was recorded in the control group. However, differences

among the digestibility values of groups B, D and A were found to non-significant.

5.4.5 PotassiumMeans values of regarding digestibility of potassium (K), for treatment A, B, C, D

and E, were found to be 51.8, 53.5, 56.3, 53.5 and 52.0, respectively. The results revealed a

significant effect on digestibility of K due to the inclusion of NaHCO3 in the diets of layer

when compared to those of control group. Statistical analysis of the data depicted that the

birds of treated groups, receiving diets containing sodium bicarbonate showed higher

(P<0.05) K digestibility than those of control group. The difference in K digestibility values

was also significant among the treated groups. Birds receiving diet containing 1% sodium

bicarbonate exhibited maximum digestibility followed by those of group B, D and E. whilst,

the lowest digestibility of K was recorded in the control group. However, differences in the

digestibility values of group B and group D, and those between group A, and group E were

found to be non-significant (P>0.05).

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5.5 Discussion5.5.1 Dry matter

Diets containing sodium bicarbonate exhibited better digestibility of dry matter in

layers. Increase in digestibility of dry matter of the treated groups may be due to more

sodium ions concentration in rations containing sodium bicarbonate. Similar effect of

increased sodium ions concentration in broilers has been observed by Fethiere et al. (1994).

Dietary inclusion of sodium bicarbonate might have improved the DEB by forming favorable

circumstances for improving the digestibility of nutrient (Borges et al., 2003; Mahmud et al.,

2010). Gorman and Balnave (1994) reported that high ambient temperature could lead to a

metabolic need for the HCO3- ions. The pancreatic juices which involve digestion of most of

the nutrients primarily contain H2O, NaCl, and NaHCO3. The function of NaHCO3 in

pancreatic juice is to neutralize high acidity (pH) of chyme and raise it to be alkaline to

prepare the chyme for digestion and absorption. This process takes place in the small

intestine (Leeson and Summer, 2001). Therefore, decreased digestibility of dry matter in

control group may have been due to lower bicarbonate and sodium levels, which probably

occurred due to increase in respiration rate.

Heat stress may exert a negative influence on digestion and/or absorption of dietary

nutrients and their metabolism (Macleod, 2004; Puvadolpirod and Thaxton, 2000), as have

been observed in the birds of control group. Presence of sodium bicarbonate in the diets of

treated birds might have improved their digestibility and prevented losses caused by heat

stress (Mirsalimi and Julian, 1993; and Braton et al., 1989). However, beneficial effects of

NaHCO3 can be achieved only when its recommended/optimum levels are incorporated in

the diets. An excessive level of this chemical compound in the diet has been reported to be

toxic in White Leghorn layers (Davison and Wideman, 1992). Therefore, it might be the

possible reason of reduced digestibility of dry matter in group E, which were fed a diet

containing 2% sodium bicarbonate. Another reason for decrease in digestibility of dry matter

in group E (2% NaHCO3) might be increased passage rate of digesta (Ravindran et al., 2008).

However, Ahmad (1997) observed that dry matter digestibility in broilers was not influenced

due to dietary inclusion of sodium bicarbonate.

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5.5.2 ProteinThe birds fed diet without inclusion of sodium bicarbonate (control) exhibited the

lowest digestibility of protein. At ambient temperature above 30 °C, thermoregulatory

system is activated and causes an increase in blood flow to upper respiratory tract and other

organs associated in heat excretion i.e. combs and wattles, which causes a decrease in blood

flow to the digestive tract (Wolfenson, 1 986 ). Consequently, activities of proteolytic

enzymes may be decreased in the upper gastrointestinal tract (Hai et al., 2 000 ), ultimately

leading to decrease in protein digestibility.Considering the fact that heat stressed birds use

glucogenic amino acids for glucose production (Nelson et al., 2000) during the process of

gluconeogenesis inbirds, which is metabolically expensive process (Nelson et al., 2000),

provision of sodium bicarbonate in their diets can decrease glucose production from amino

acids, which may lead to improved digestibility of protein during stress.

Addition/inclusion of NaHCO3 in the diet of layers exhibited more digestibility of

protein in these bids as compared to those of control group. Protein ingested by the birds is

broken down by the action certain enzymes to its constituent amino acids (a.a.) in the

gastrointestinal tract prior to absorption, and most of these a.a. require sodium (Leeson and

summer, 2001) for this process. Therefore, increase in digestibility of protein of the treated

groups may probably be due to the presence of more sodium ions concentration in the rations

containing sodium bicarbonate. Sodium containing compounds such as sodium bentonites

have been successfully used in sorghum containing diets to prevent deleterious effects of

tannins present in it, on digestibility of protein (Pasha et al., 2008).

The results of present experiment are in accordance with the investigation of Ahmad

(1997) who found maximum digestibility of protein in birds kept on 75% supplemented

sodium from sodium bicarbonate whereas, minimum digestibility of protein was noted in

birds kept on 100% supplemented sodium from sodium chloride. Banda-Nyirenda and Vohra

(1990) found significant improvement in apparent protein digestibility by treating sorghum

by sodium bicarbonate. The results are also in agreement with the observations of Choi and

Han (1982) who observed improved protein digestibility due to dietary inclusion of sodium

bicarbonate in broiler ration having low crude protein level.

Gorman and Balnave (1994) investigated that heat stress could lead to a metabolic

needs for the bicarbonate ions. Therefore, decreased digestibility of protein in control group

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might be due to lower bicarbonate and sodium levels which probably occurred due to

increased respiration. Sodium bicarbonate in the diet of broilers has shown to improve

digestibility and decrease losses caused by heat stress (Mirsalimi and Julian, 1993; Braton et

al., 1989). An improvement in protein digestibility and nitrogen retention was observed in

broilers offered sorghum grains treated with alkaline solution (Mohammed and Ali., 1988).

Sodium salts and electrolytes may respond differently to amino acid during heat

stress. Chen et al. (2005) investigated that respond to synthetic a.a. is affected by DEB.

Brake et al. (1998) found that increasing the Arginine: Lysine ratio with low levels of NaCl

in the diets of broilers reared at 31 °C, enhanced their body weight and efficiency of feed

utilization. However, Balnave and Brake (2001) observed that NaHCO3 enhanced the

performance with high Arg: Lys ratio in broilers. Gonzales-Esquerra and Leeson (2006)

observed that the Arg: Lys ratio, methionine source and time of contact to heat stress can

disturb the protein utilization in birds reared at higher ambient temperature.

5.5.3 Crude fiber Dietary inclusion of different levels of sodium bicarbonate depicted a significant

increase in digestibility of crude fibers in layers when compared to those fed diet without

addition of sodium bicarbonate. Increase in digestibility of crude fibers of the treated groups

may be due to availability of more sodium ions and bicarbonate ions concentration in rations

containing sodium bicarbonate. Dietary inclusion of sodium bicarbonate has also shown to

improve DEB by creating physiological conditions better for enhancing the digestibility of

nutrients. Pasha et al. (2008) used different levels of sodium bentonite in broiler rations and

found an improvement in nutrient digestibility as compared to the control group (without

sodium bentonit). Similarly, Santurio et al. (1999) and Salari et al. (2006) have also observed

improvement in nutrient digestibility due to addition of sodium bentonite in broiler diets.

High ambient temperature has shown a significant influence on feed consumption,

digestion, nutrients absorption and their metabolism (Macleod, 2004). As ambient teperature

shoots up, feed intake of the birds is reduced (Ain-Baziz et al., 1 996 ) to avoid the

thermogenic effect of heat increment associated with nutrient utilization, absorption and

assimilation (Koh and Macleod, 1999 ). However, at ambient temperature above 30 °C,

thermoregulatory system is activated and exhibits increase in blood flow to upper respiratory

tract and other organs associated in heat excretion i.e. combs and wattles, which intern causes

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a decrease in digestibility ofcrude fibers.

Glucose is the main energy source for poultry birds. Absorption/assimilation of

glucose from the gastro intestinal tract requires sodium. Galactose also needs sodium for

transport system as glucose. Absorption of glucose, galactose, and amino acids require

transporters in the gastro intestinal tract that require sodium to be transported along with

these, as has been stated by Nelson and Cox, (2000). They have also pointed out that sodium

may remarkably be reduced in birds during high ambient temperature due to its loss via

excretion, which may reduce the process of digestion and absorption. However, addition of

NaHCO3 in such situation can improve digestibility of nutrients.

Sodium bicarbonate is associated in increasing sodium ions concentration (Fethiere et

al., 1994); improving electrolyte balance in the diet (Borges et al., 2003), decreasing the

losses caused by heat stress (Gorman and Balnave, 1994) and reduction in body temperature

(Smith and Teeter, 1989). As all these factors are related to the performance, therefore, it is

expected that inclusion of NaHCO3 in the diet may result in better performance of birds,

including better digestibility of crude fiber. Use of saline solutions has been a common

practice to stimulate nutrient digestion and absorption during heat stress (Jeukendrup et al.,

2009) in birds.

The birds fed diet without inclusion of NaHCO3 (control) exhibited the lowest

digestibility of crude fiber in the present study. Gorman and Balnave, 1994) have reported

that high ambient temperature (heat stress) may lead to an increase in metabolic requirements

for HCO3- ions. Keeping this fact in view, a possible explanation of decreased digestibility of

crude fibers in control birds may be the less availability of bicarbonate ions (HCO3-), which

probably occurred due to increased respiration. Therefore, presence of enough HCO3- ions

because of adding NaHCO3 in the diets of broilers might have improved digestibility and

decreased losses caused by heat stress (Mirsalimi and Julian, 1993; Braton et al., 1989).

On the other hand, presence of excessive levels/addition of NaHCO3 in the diets has

been reported to be toxic in White Leghorn layers (Davison and Wideman, 1992). This may

be the probable reason of decreased digestibility of crude fiber in the birds of group E, which

were fed a diet containing 2% NaHCO3. Another possible explanation of decrease in

digestibility of crude fibers in group E (2% NaHCO3) was because of accelerated passage of

feed (Ravindran et al., 2008).

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5.5.4 Ether extractDigestibility of ether extract was found to be significantly better in the birds receiving

diets containing sodium bicarbonate as compared to those of untreated group. Hyperthermia

seems to be the most possible contributing factor for decreased digestibility andabsorption of

ether extract in the birds fed diet without sodium bicarbonate as have been observed by Koh

and Macleod, ( 1999 ). Leeson and summer, (2001), while discussing the factors affecting

digestibility of fats, have also stated that fat digestibility is negatively affected in the birds

exposed to heat stress. These results are coincided to those reported by Bonnet et al. (1997)

in birds maintained under hyperthermic (heat stress) conditions.

On the other hand, the results have shown an increase in digestibility of ether extract

contents in layers fed diets containing different levels of sodium bicarbonate. Increase in

digestibility of ether extract of the treated groups may probably be due to more Na+ and

HCO3- concentration in the diets containing sodium bicarbonate (Gormanand Balnave, 2004).

Findings of our study have also exhibited that digestibility of ether extract was affected due

to the level of feed intake of birds as has been reported by Ravindran et al.(2008).

Bile salts also reported to have an important role in the digestibility and absorption of

fats (Leeson and summer, 2001)). These are sodium containing salts like sodium

glycocholate and sodium taurocholate, which emulsify fats and thus rendering them

digestible. Therefore, it is quite possible that addition of NaHCO3 might have improved the

production of bile salts in the birds receiving diets containing different levels of NaHCO3,

which increased the digestibility of ether extract of the diets. Moreover, buffers like NaHCO3

when added to diets may increase or stabilize the pH, improving enzymatic activity (Paggi et

al., 1999), hence result in better digestion of the nutrients. Contribution of better electrolyte

balance in the birds fed diets containing different levels of NaHCO3 has already been

discussed in the previous section (5.5.2) and the same is expected in this particular case. In

contrast to the findings of present study, Ahmad (1997) observed that digestibility of ether

extract in broiler was not affected by dietary inclusion of NaHCO3. This contradiction in the

results might because of varying species of the birds used in these studies.

5.6 MineralsDietary inclusion of sodium bicarbonate significantly influenced the absorption of all

minerals (Ca, P, Fe, Na and K) studied in this trial. Birds receiving diets containing sodium

bicarbonate exhibited better absorption of these minerals as compared to those of untreated

group (control). Minerals and trace elements are essential for optimum performance (Leeson

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and summers, 2001) of poultry birds. Therefore, increased mineral absorption/assimilation in

NaHCO3 fed birds may either be due to more accessibility of minerals because of increased

feed intake (Karimian et al., 2004) or improved DEB (Borges et al., 2003), or both.

Another possible reason of increased absorption of the minerals by the birds receiving

sodium bicarbonate in their diets may be increased availability of sodium ions (Fethiere et

al., 1994), leading to more uptake of the minerals. Body temperature record of the

experimental birds of treated groups has also shown a decrease in the body temperature of

birds probably because of enough availability of minerals and HCO3- ions to meet body

requirements (Gorman and Balnave, 1994), thus repairing/decreasing the losses of these

elements caused by heat stress (Mirsalimi and Julian, 1993; Braton et al., 1989).

The birds of control group, which were fed diet without inclusion of sodium

bicarbonate exhibited lower feed intake as compared to their counterparts, resulting in less

availability of bicarbonate ions. This situation may have caused increased respiration rate and

ultimately led to heat stress. Moreover, heat stress in birds may lead to increase in metabolic

requirements for the HCO3 ions (Gorman and Balnave, 1994) and less availability of these

ions to the birds fed control diet might have ultimately resulted in low absorption of the

minerals. Therefore, decreased absorption of minerals in control group may be attributed to

less feed intake of the birds.

Absorption of mineral ions may be accelerated by dietary sodium salts, which can

enhance function of digestive and absorptive enzymes and passive permeability (Nelson and

Cox, 2000). Increased influx of dietary sodium salts may also cause changes in fluid

absorption and/or secretion, and could also stimulate intestinal HCO3- secretion. It has been

reported that phytates and fiber present in plant ingredients reduce the availability of calcium

and other minerals (Champagne, 1989; Sugiura et al., 1998), however, absorbability of these

key minerals can be improved by dietary addition of sodium bicarbonate.

Minerals absorption may decrease under hot conditions (Smith and Teeter, 1987). In

addition to effect on specific nutrients, gastrointestinal size is also reported to be decreased in

heat-exposed chickens (Savory, 1986; Mitchell and Carlisle, 1992). In all of these studies, the

high ambient temperature also caused some reduction in feed intake. Therefore, decreased

mineral absorption in the birds kept under heat exposure may have been somewhat due to

decrease in feed consumption. Reduced minerals absorption in birds exposed to heat stress

174

had also been observed by Smith and Teeter (1987) and Belay et al.(1992). However,

improvement in absorption of minerals in the birds of treated groups may be obtained by

dietary addition of sodium bicarbonate (table 5.2).

In fact, a big proportion of dietary minerals is absorbed/assimilated from the small

intestine receiving a Na+ reliant transport system (Leeson and Summer, 2001), therefore,

transport of mineral ions and sodium must be at the same time. However, presence of an

optimum level of NaHCO3in the diet is important for its maximum utility/efficiency

(Ravindran et al., 2008). Therefore, a possible reason for decrease in absorption of these

minerals in the birds fed diet containing higher level of sodium bicarbonate (2%)when

compared to those of other treated groups, may be its (NaHCO3) less effectiveness/utility

because of increased passage rate of digesta of the birds.

An other possible reason of decreased absorption of minerals in the birds of control

group may be decreased blood flow towards their digestive tracts under the influence of high

ambient temperature. At higher ambient temperature, thermoregulatory system of the birds

might have been activated and caused an increased blood flow to their upper respiratory

tracts and organs associated in heat dissipation (Wolfenson, 1 986 ).Obviously this situation

might have decreased blood flow to the digestive tracts andconsequently resulted in reduced

absorption of minerals in control birds as compared to those of NaHCO3 treated groups.

ConclusionsIn making final assessment of the study, inclusion of NaHCO3 in the diet of layers

during summer improved production performance, egg quality characteristics, blood profile,

immune response, and digestibility of proteins, fats and carbohydrates as well as absorption

of some minerals, in caged White Leghorn layers. Addition of NaHCO3 @1% (DEB= 262) in

the diet of the layers was found to be the most effective in ameliorating/mitigating the effect

of heat stress upon the performance of the caged layers.

RecommendationsDietary inclusion of sodium bicarbonate @ 1% is recommended to be used for better

efficiency of production performance, blood profile, immune response against Newcastle

disease virus, digestibility of nutrients and absorption of some minerals, in the caged layers

during summer.

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Practical Implications1- Sustainable productivity of layers during summer receiving 1% sodium bicarbonate in

diet.

2- Reduction in expenditures to overcome heat stress using mechanical devices.

3- Reduction in economic losses due to mortality and reduced production performance

of layers reared in heat stress conditions.

Future work1- Further research may be conducted for a longer periods to compare production

performance of layers receiving NaHCO3 during first and second production cycles.

2- The effect of NaHCO3 at different ages and seasons can also be probed along with

economics of production.

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CHAPTER-6

SUMMARYPakistan is situated in the subtropical zone of Northern Hemisphere of the world

where temperature usually remains well beyond the higher side of thermo-neutral zone (25-

370C) for greater part of the year. The optimum temperature for efficient performance is 19-

220C for laying birds; however, ambient temperature especially on higher side is very

disruptive and may reduce survival rate and production. Egg production declines drastically,

thereby adversely affecting the economics of poultry production. Hence, low egg production

may lead to excessive culling of layers from a flock.

Different techniques are being used in poultry production to combat heat stress,

which include nutritional manipulations such as dietary addition of oils reduction in protein

level of feed, supplementation of feed with limiting amino acids and management practices

like intermittent feeding, feeding the birds in cool hours of the day, time limit feeding,

sprinkling of water, evaporative cooling, improved ventilation and supplementation of

electrolytes. Many studies have reported beneficial effects of supplementing drinking water

and/or diets of broilers with sodium bicarbonate as sodium source. However, scientific

information regarding the use of NaHCO3 in layer diet is still scarce. Therefore, this study

was planned to investigate the effect of dietary inclusion of NaHCO3 on production

performance, nutrient digestibility and blood profile of caged layers, during summer.

One hundred sixty commercial layers (24 weeks old) were purchased from a poultry

farm and were reared in a group for one week (adaptation period).At the start of 26 th week of

age, these layers were divided into 20 experimental units/replicates (8 layers/replicate),

which were further allotted to five treatment groups (4 replicate/treatment).Five diets (A, B,

C, D and E) were prepared with or without addition of sodium bicarbonate. Diet A, was

without sodium bicarbonate and served as control whereas, diets B, C, D, and E contained

0.5, 1.0, 1.5 and 2.0% sodium bicarbonate, respectively. All the diets were iso-nitrogenous

(CP 17%) and iso-caloric (ME 2700 Kcal/Kg) and were formulated according to the

requirements prescribed by NRC (1994). The diets were fed to the experimental birds ad

libitum, for 12 weeks (26-37 weeks of age).

Data on feed consumption, number of eggs, egg weight and egg mass laid by the

177

birds were recorded. From these observation feed conversion ratios on the basis of per dozen

eggs and per kg egg mass produced, were calculated. Five eggs from each replicate were

checked weekly for their shell thickness, yolk index, albumen index, Haugh unit yolk pH,

albumen pH, specific gravity and yolk cholesterol. At the last day of each week, rectal

temperature and respiration rate of three layers from each replicate were recorded. At the end

of the experiment (37th week of age), blood sample of two birds/replicate were collected for

hematological analysis, hormonal profile (T3, T4, Cortisol, Progesterone and Estrogen), serum

proteins, lipids profile, plasma electrolytes (HCO3–,Cl Na+ and K+), plasma minerals analysis

(Calcium and Phosphorus).Blood samples were also collected from two birds from each

replicate 10 days post vaccination of 1st, 2nd, and 3rd vaccination to the immune response.

Digestibility of dry matter, crude protein, crude fiber, ether extract and absorption of

minerals (calcium, phosphorus, sodium, potassium and iron)was also determined in a

separate trial, during the last week (38th week) of experiment. For this purpose 30 layers

obtained from the same batch, as were used for the performance trial, were put into

individual cages. Each bird acted as a replicate. These birds were allotted to the treatment

groups (A, B, C, D and E) such that each treatment received 6 birds (replicate). The diets

used in this trail were the same, which were used in the performance trial except that 1%acid

insoluble ash was added in these diets, as an indigestible marker. Economics of each

treatment was calculated at the end of experiment. The data thus collected were statistically

analyzed under completely randomized design.

The results of the study revealed that dietary inclusion of sodium bicarbonate

significantly (P<0.05) improved feed intake, weight gain, feed efficiency, egg production and

egg weight of the birds. The birds of Group C which were fed diet containing 1% NaHCO3

performed better than those of its counterparts and exhibited better feed consumption, weight

gain, egg production and egg mass production. Moreover, the birds fed diets containing

sodium bicarbonate utilized their feed more efficiently than those fed diet without any such

addition (control).

Egg quality characteristics such as specific gravity, shell thickness, albumen height,

haugh unit score, yolk height and yolk diameter were also improved by dietary inclusion of

sodium bicarbonate. Yolk cholesterol was found to be the lowest in the eggs produced by the

birds of group C, which were fed diet containing 1% NaHCO3. Whilst, pH values of yolk and

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albumen were found to be the highest in the birds of group E which were fed diet containing

2% NaHCO3.

Dietary inclusion of sodium bicarbonate significantly reduced rectal temperature and

respiration rate of layers; however, their water intake was significantly increased. Serum

glucose and WBCs count was found to be higher in birds of control group. Birds of group C,

which were fed diet containing 1% sodium bicarbonate, showed maximum concentration of

hemoglobin in their blood. However, red blood cells count, PCV and ESR values were not

affected due to the dietary treatments. Serum urea concentration was found to be the highest

in the layers of control group whereas, serum uric acid concentration was found to be the

highest in birds of group E (2% NaHCO3). However, values of serum creatinine and alkaline

phosphatase were not affected due to the treatments. Dietary inclusion of sodium bicarbonate

significantly increased serum total protein and albumen concentration of the birds. Birds fed

diets containing 1% sodium bicarbonate exhibited higher concentration of these proteins as

compared to those of other groups. However, serum globulin contents of birds remained

unaffected due to the dietary inclusion of sodium bicarbonate.

Plasma sodium level showed a linear increase with increase in the level of dietary

inclusion of sodium bicarbonate. Serum potassium and bicarbonate also showed increasing

trend due to the dietary treatments. Whereas, birds fed diets containing sodium bicarbonate

exhibited decreased levels of serum chlorides and serum pH. However, plasma calcium and

potassium levels remained unaffected due to sodium bicarbonate treatments. Serum

cholesterol, triglycerides and LDL were decreased by the dietary inclusion of sodium

bicarbonate and birds fed diets containing 1% NaHCO3 exhibited lower concentration of

these lipids as compared to those of other treated groups. However, concentration of HDL

was found to be increased in the birds fed diets containing 1% sodium bicarbonate.

The birds of group C (1% NaHCO3) showed a marked increase in the concentrations

of estrogen, progesterone, T3 and T4 hormones, whereas, serum cortisol was higher in birds

exposed to higher temperature and fed diet without NaHCO3).Serum SGPT concentration

was not affected due to the dietary treatments. While, serum SGOT concentration was

significantly decreased due to the use of NaHCO3 in the diets during heat stress. Dietary

inclusion of NaHCO3 has also shown a significant (P<0.05) increase in immune response

against Newcastle disease virus in the layers when compared to those of control group.

179

Digestibility of DM, crude protein, crude fiber and ether extract was found to be

higher in the birds fed diets containing sodium bicarbonate than those of control group. The

best digestibility of these nutrients was observed in the birds fed diet containing 1% sodium

bicarbonate among those of treated groups. Similarly, absorption of the minerals studied

(calcium, phosphorus, sodium, potassium and iron)was also found to higher in the treated

birds as compared to untreated ones.

Findings of this study clearly indicated that dietary inclusion of sodium bicarbonate

exhibited beneficial effect on production performance, digestibility of nutrients and

biochemical profile of the layers during summer, addition of 1% NaHCO3 being the best. It

was, therefore, concluded that dietary inclusion of 1% NaHCO3 may be used for efficient and

economical production performance of layers during summer season. However, effects of

dietary sodium bicarbonate on production performance of birds at different ages and during

different seasons of the years can be addressed, as future research work.

180

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