1

MOLECULAR CHARACTERIZATION OF INDIAN BREED KADAKNATH CHICKENS

Thesis submitted in partial fulfillment for the award of Degree of Doctor of

Philosophy in

BIOTECHNOLOGY

By

DEEPAK PRAKASH SAXENA, M.Sc., M.PHIL,

Under the Guidance of

DR. R. STEPHAN, M.Sc., M.C.A., M.TECH., Ph.D.,

Assistant Professor, Department of Botany,

Government Arts College, Ariyalur-13

VINAYKA MISSION UNIVERSITY SALEM, TAMILNADU, INDIA

SEPTEMBER -2014

2

VINAYAKA MISSION UNIVERSITY

DECLARATION

I DEEPAK PRAKASH SAXENA declare that the thesis entitled

“MOLECULAR CHARACTERIZATION OF INDIAN BREED

KADAKNATH CHICKENS” submitted by me for the Degree of Doctor of

Philosophy in Biotechnology is the record of work carried out by me

during the period from October 2008-2014 under the guidance of

DR.R.STEPHAN,M.Sc.,M.C.A.,M.Tech.,PhD., and has not formed the

basis for the award of any Degree, Diploma, associate ship, fellowship,

titles in this or any other university or other similar institutions of higher

learning.

Place:

Date: Signature of the Student

3

VINAYAKA MISSION UNIVERSITY

CERTIFICATE BY THE GUIDE

I DR.R.STEPHAN, M.Sc., M.C.A., M.Tech., PhD., Certify that the thesis

entitled “MOLECULAR CHARACTERIZATION OF INDIAN BREED

KADAKNATH CHICKENS” submitted for the degree of Doctor of

Philosophy in Biotechnology by Mr. Deepak Prakash Saxena is the

record of research of work carried out by him during the period from

2008- 2014 under my guidance and supervision and that this work has

not formed the basis for the award of any degree, diploma

associateship,fellowship or other titles in this university or any other

university or institutions of higher learning.

Signature of the

Supervisor with Designation

Place:

Date:

4

ACKNOWLEDGEMENT

On the accomplishment of the present study it is with deep sense

of reverence and gratitude, I deem it a proud privilege and feel immense

pleasure to acknowledge all those who have helped me during the

pursuit of my present study.

At the outset, I sincerely thank authorities of the Vinayaka

Mission University, Salem. For kindly permitting me to register for PhD,

Degree and granting permission to complete my research studies.

I express my sincere thanks to Dr.A.Shanmuga Sundaram,

Chairman, Vinayaka Mission University, Salem who gave an opportunity

to do this dissertation work.

First and foremost I find great pleasure in expressing my deep

sense of gratitude heartfull thanks to my research advisor

Dr.R.Stephan,M.Sc.,M.C.A..,M.Tech.PhD.,Lecturar Department of

Botany ,Government Arts College ,Ariyalur.who supported encouraged

and guided to my work with at most interest ,enthusiasm and patience

,discussion with him guided me in developing new ideas and inferences

with ease.

I would like to express my sincere thanks to the Dr.K.Rajedran,

Dean of Research, Vinayaka Mission University, Salem for having

given me an opportunity to do this dissertation work.

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I wish to place on record person who never showed prosaic

attitude in helping me at the best of their skills, Dr. D.P. Singh, Principal

Scientist & I/c Desi Fowl Unit, CARI, Izatnagar , I am sincerely highly

thankful to him for his kind cooperation and support.

I feel great pleasure to express my deep respect for Dr. Deepak

Sharma, Dr. S.K. Mishra CARI for their constructive suggestions and

guidance to accomplish this assignment.

I feel a great sense of elation and fulfillment to submit this tiny drop

of research in the ocean of human knowledge. Though this was a short

episode in the long journey of life, a thousand hands were there to help,

thousand smiles to make it joyful and thousand caring hearts to make it

memorable.

I am highly thanks to my dear Sir Dr.Ajay Kumar, Scientist, IVRI,

without him help I am not able to do this biggest opportunity in my life.

I lack to say words of reverence, gratification and affection for my

venerated parents who brought me to this stage and because of whom I

was able to pursue my higher study.

Friends are forever no matter they are far or away and we owe

them something for shaping us. At this moment I have no words to

thanks to my dearest, closest and beloved friend Dr. Sanjeev Kumar

Shukla and Dr.Kuldeep Shivalaya whose love, affection, wish, blessing,

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courage and moral support was always with me and with god’s grace the

care and concern shown by her will remain forever.

I apologize for faux pas of the persons who have extended the

help to me in completion of this work in a way or other and deserved

such thanks.

Least but not last I records my thanks for giving me patience and

strength to overcome the difficulties and remain stoic throughout my stay

away from home.

Faith remains as the surest and strongest hope during darkest

days and the Almighty God Lord Krishna leads us to light. Faith in Him

has been the prime support that leads me to this position in life.

(DEEPAK PRAKASH SAXENA)

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CONTENTS

CHAPTER NO. TITLE PAGE

NO.

1. INTRODUCTION 8-11

2. REVIEW OF LITERATURE 12-31

3. AIM AND OBJECTIVES 32-33

4. MATERIALS AND METHODS 34-55

5. RESULTS 56-91

6. DISCUSSION 92-103

7. SUMMARY AND CONCLUSION 104-108

8. REFERENCES

109-128

9. LIST OF PUBLICATION -----

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1- INTRODUCTION

__________________________________________________

Increasing population of India, increased demand of nutritious food and

shrinkage of land has drawn the attention of people towards the

livestock production and poultry farming. Due to high nutritive value and

less input cost, the poultry farming has grown up nicely. Poultry is

considered to be the best experimental animal because of its small size,

shorter generation interval, low feed intake, easy handling and fast

metabolism. Poultry production has been increasing world over because

of affordable prices, health consciousness and successful use of

scientific knowledge in poultry enterprises. Eggs and poultry meat are

protective food, are the cheapest source of quality animal protein, and

have wide acceptability in India.

Consistent with the increase in production and productivity, the per

capita availability of eggs and poultry meat in India has also increased to

44 eggs and 1.76 kg poultry meat per annum, which is still lower than

the recommended levels of 180 eggs and 11 kg of poultry meat, as per

the Nutrition Advisory Committee to Government of India (Indian Poultry

Industry Year Book., 2004).

Poultry industry has made tremendous growth during last three

decades and it has expanded multifold. Commercial strains of poultry

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have attained almost the plateau of production traits because of

maximum exploitation of available genetic variations; hence further

improvement in production performance through conventional breeding

seems to be impractical. Advanced genetic selection has improved the

economic performance parameters, but reduced the immune status

(Yegani et al., 2005).

The term poultry includes a number of avian species such as chicken,

ducks, turkeys, geese, guinea fowls and pea fowls which have been

domesticated, but is very often used as synonymous to chicken. Chicken

alone account for about 90% of the total poultry. We have a fairly good

knowledge and understanding with regards to its breeding and

husbandry practices.

The common country hen, the desi, is as a rule the best mother for

hatching. She is a good forager. Some of the Indian fowls resemble the

Leghorn in size and shape, but have poor laying qualities. They are

found in various colours. One variety found in India resembles the

Sussex or Plymouth Rock in shape, but is smaller. These birds lay fairly

well and are more common in the eastern parts of the country. The

Indian birds are mostly non-descript, and are of very little value as

layers. There are only 4 pure breeds of fowls indigenous to India. They

are the Chttagong, the Aseel, the Karaknath and the Busra.

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In India, the Kadaknath breed of the Jhabua District of Madhya Pradesh

is known to be well adapted to the harsh environment, which is

characterized by extreme climatic conditions and poor management,

housing and feeding. Because, long ago, the Kadaknath breed was

reared by tribal people, and following many years of selection mainly for

plumage characteristics, a recent study of their growth potential

recorded a daily weight gain of 6.2 grams from 0 to 20 weeks of age –

based on growth in the breed’s usual production environment with very

little supplemental feed.

The colour of the day old chicks is bluish to black with irregular dark

stripes over the back. The adult plumage varies from silver to gold

spangled to blue black without any spangling. The skin, beak, shank,

toes and soles of feet of males and females are dark gray colour. Even

the comb, wattles and tongue also show a purplish hue. The shining

blue tinge of the ear-lobes adds to its unique features. The peculiarity of

this breed is that most of the internal organs show the characteristic

black pigmentation which is more pronounced in trachea, thoracic and

abdominal air sacs, gonads, elastic arteries, at the base of the heart and

mesentery. The black colour of muscles and tissues is due to the

deposition of melanin pigment, a genetic condition called

"Fibromelanosis".

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The immune system is the natural means by which animals resist

infection, and immunological parameters may reflect the immuno-

competence of the immune system and, in turn, the ability of the animal

to resist infection. So far, only a few genes have been identified in

influencing disease resistance. Heritability of antibody response of

chickens to an inoculation of SRBC is moderate and the trait responds to

divergent selection (Siegel and Gross., 1980; van der Zijppand Leenstra,

1980; Martin et al., 1990; Pinard et al., 1992). The literature is

inconsistent concerning the role of nonadditive genetic variation in

antibody responses to SRBC (Siegel et al., 1982; Ubosi et al., 1985;

Pinard and van der Zijpp., 1993; Boa-Amponsem et al., 1997).

In a study, chickens from different ecotypes were immunized with

the non-pathogenic multi determinant SRBC and breeds from India

showed the highest humoral response to SRBC, suggesting higher

immunocompetence (Baelmans et al., 2005).

Owing to the recent development in molecular genetics and

biotechnology, the immune system is an ideal component for

multidisciplinary analysis and efforts to improve immunocompetence in

chicken, which would be helpful to economize poultry production.

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2-REVIEW OF LITERATURE

_________________________________________________________

2.1. IMMUNOCOMPETENCE

Several workers have studied different immunocompetence traits

in selection experiments based on immune response to a single antigen

such as Sheep red blood cells (SRBC) in chicken (Siegel and Gross.,

1980; Van der Zijpp., 1983; Dunnington et al., 1984; Van der Zijpp et al.,

1986; Scott et al., 1988; Boa-Amponsem et al., 1997, and 2000;

Shivakumar., 2003; Sivaraman., 2004; Singh et al., 2008; Kumar et al.,

2007; and Jaiswal et al., 2008). Nucleotide polymorphism in important

candidate genes viz., Natural resistance-associated macrophage protein

1(NRAMP1) and Prosaposin (PSAP), Annexin A1 (ANXA1), Osteoclast

Stimulating Factor (OSF), Thrombospondin-4 (THBS4), Programmed

Cell Death Proteins (PDCD), Follistatin (FST), Growth Hormone

Receptor (GHR), Interferon (IFN) alpha and beta (Georgina and Aggrey.,

2005), etc. results in varied response of individuals to variety of

pathogens/antigens. The divergently selected groups or lines have been

evaluated for DNA polymorphism for such genes having bearing on

immune response by DNA methods viz., PCR-RFLP. The available

literature on the proposed objectives has been reviewed.

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2.1.1. Evaluation of Immunocompetence status

The Immunocompetence status can be evaluated by assessing

important parameters related to various facets of immunity such as

antibody response to SRBC; cell mediated immune response to PHA-P,

serum Lysozyme activity and serum IgG level etc. Various genetic

groups/ varieties/breeds/species have also shown significant differences

in antibody titres against SRBC, serum lysozyme level, CMI response to

PHA-P and serum immunoglobulin (IgG) level (Toro et al., 1997;

Saxena., 1993; Saxena et al., 1997; Sarker et al., 1998; Santosh., 1999;

Shivakumar., 2003; Singh et al., 2003; Singh., 2005; Kumar., 2006 and

Jaiswal et al., 2008).

Genetic differences in immune response have been observed by

several workers (Balcarova et al., 1973; Van der Zijpp., 1983; Sivaraman

et al., 2005). The capacity of antibody formation is inherited as a

dominant trait (Van der Zijpp., 1983).

2.1.2. Antibody response to SRBC

Sheep red blood cell (SRBC) is a non-pathogenic, non-specific,

natural multideterminant antigen. It provides good indication of innate

humoral immune response status of an individual because of its broad

immune response characteristics, thus valid for multiple pathogens.

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The use of antibody responses to SRBC in a multitrait selection

programme is a well-known concept in avian immunology that reveals

various aspects of immune responses and their genetic basis. (Kean et

al., 1994).Several workers have studied different immunocompetence

traits in selection experiments based on immune response to a single

antigen such as Sheep red blood cells (SRBC) in chicken (Gross et al.,

1980; Van der Zijpp., 1983; Dunnington et al., 1984; Van der Zijpp et al.,

1986; Scott et al., 1988; Pinard et al., 1992; Boa-Amponsem et al., 1997,

and 2000; Shivakumar 2005; and Sivaraman., 2004).

Bi-directional selection experiment based on antibody response to

Sheep RBC in Cornell random bred chicken and their crosses were

carried out. The response to selection was immediate as highly

significant differences in the titre values between the High and Low

SRBC lines were observed. The response in first generation was 1.27

for males and 1.21 for females in first generation and progressively

increased to 1.77 and 2.42 in second generation. They observed that

the mean 5 dpi antibody titres of the crosses intermediate of the parental

lines were significantly greater than the mid parent values and

suggested the influence of non-additive gene action on antibody titres.

They also observed the response to selection for persistence and non-

persistence of antibodies at 5 dpi and 21 dpi in the same population is

highly significant between the lines (Siegel and Gross., 1980).

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(Van der Zijpp and Leenstra., 1980) observed highest mean total

Ab titre on 7 dpi as 5.2 using 2% SRBC in HA. Pullets generally showed

higher titres than the cockerels and from day 3 onward these differences

were highly significant.

(Van der Zijpp and Leenstra., 1980) observed highest mean total

titre (5.2; log2 values) on 7 dpi and females showed significantly higher

response than male chicken. The h2 estimates (Full sibs) for total

antibody titre, MES and MER antibody titre ranged from 0-0.5. The

genetic correlation between 2-β-ME resistant and susceptible antibodies

to SRBC ranged between -0.3 and -0.9.

(Yamamoto and Glick., 1982) could not observe any significant

difference between sexes within line during primary Ab response against

SRBC in the Hampshire birds having different sizes of bursa. (Van der

Zijpp., 1983) evaluated humoral immune response to SRBC in White

Plymouth Rock and White Leghorn (WL) chickens, with emphasis on

dose, genetic origin, interaction and correlation between primary and

secondary immune responses and also between humoral and cell-

mediated immune responses. They demonstrated significant differences

in total antibody titres among genetic groups.

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(Ubosi et al., 1985) analyzed the age dependency of antibody

response to SRBC in divergently selected lines for this trait. There were

differences among populations for frequency of responders at 7 days of

age. The serological maturity was reached at 14 days of age. After the

attainment of serological maturity, the high SRBC line had significantly

higher antibody titre than low line and the reciprocal crosses were

intermediate to their parental lines. The weight of bursa and spleen was

more in the high line and the thymus weight was less than those in low

line.

(Gross, 1986) compared the doses required to get optimum

response in the divergently selected lines for SRBC response and

reported that the high line responded with low dosages of SRBC,

whereas low line could not respond to that dose. (Van der Zijpp and

Nieuwland, 1986) reported that in ISA Warren fowl line, selection for

high and low anti-SRBC titre did not affect the cell mediated immunity.

(Kim et al., 1987) estimated the correlation between the

immunological and production traits in S1 White Leghorn line. The

correlations (0.21-0.31) among response to SRBC and body weights in

all the ages were significant.

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In HA line chickens; the heterozygous for MHC had higher titres

than either homozygous subclass. These findings suggested that the

response to SRBCs was dependant on MHC haplotypes as well as on

the background genotype. (Kreukniet et al., 1990) studied the effect of

SRBC dosage on the humoral responses of chicken lines selected for

high (H) and low (L) antibody production against SRBC and observed

that the H line had higher and early peak titre response than L line after

immunization. Further, the response was higher for pullets than

cockerels in H line.

(Martin et al., 1990) studied the correlation between antibody titre,

growth and reproduction traits in lines selected for high and low

response to SRBC. The genetic correlations were moderate among

most of the traits. They observed that the females of low lines for SRBC

response were heavier and had high egg number as compared to those

of high line. (Pinard et al., 1990) estimated the h2 for SRBC response in

fowl selected divergently for 8 generations. The estimates of the realized

heritability for response to SRBC in high line were 0.15 and 0.16 when

estimated by regression and animal model, respectively. The

corresponding values for low line were 0.22 and 0.26.

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(Chao and Lee, 1991) reported higher immune response to SRBC

in country chicken than White Leghorn in Taiwan. Significantly higher

(P<0.01) Ab titre in females than did males in high, controls and low

response lines of SRBC response was reported by Pinard et al., (1992).

The Ab titres for females and males in 9th generation were 11.12, 10.11;

6.60, 5.87 and 2.35, 1.53 in high, control and Low lines, respectively.

(Miller et al., 1984) reported that additive genetic variation was

important in inheritance of both primary and secondary response to

antigen. Reciprocal differences and heterosis also influenced the

secondary response.

(Boa-Amponsen et al., 1997) measured SRBC antibody titre in

embryos and chicks of high and low SRBC lines. Maternal antibody was

detected earlier in high line as compared to low line chicks.

(Kundu ,1997) studied SRBC response in Aseel, Kadaknath,

Naked neck, Frizzle, Dahlem Red, White Leghorn, SDL broilers and

Naked Neck broilers and revealed that all genetic groups showed

highest titre on 5dpi except broilers which revealed peak titre on 12 days

post immunization (dpi). (Saxena et al., 1997) estimated the response to

SRBC in Guinea fowl, Kadaknath and broilers as 1.520±0.487,

1.525±0.068, and 1.386±0.119, respectively. Significant differences

among varieties and sire families were observed in guinea fowl.

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(Parmentier et al., 1998) administered SRBC, LPS and BSA

intramuscularly in the fowl divergently selected for antibody response to

SRBC. Levels of antibody responses to SRBC and BSA were higher in

the High line than in the control and low line.

In different genetic groups of broiler chickens, (Nath, 1999) did not

observe any significant effect of sex on response to SRBC. Whereas,

(Kundu et al., 1999) reported that females had lower antibody titres in

Indian native and white leghorn chickens. The apparent differences

between sexes were not significant but interaction of breed and sex

were significant at 5 and 19 days post immunization.

(Santosh, 1999) evaluated the response to SRBC in White

Leghorn, Aseel, Kadaknath, and Dahlem Red (DR) and their crosses,

and reported significant genotype differences for SRBC response. The

DR birds showed the maximum titre. (Boa-Amponsem et al., 2000)

studied the primary and secondary antibody responses to different

dosages of SRBC, at different days of inoculation, in divergently

selected lines for SRBC response. They observed different patterns of

antibody response to SRBC dosages in different lines, which revealed

interactions between line x dosage x days. Antibody responses to the

booster inoculations differed between lines with a dosage effect for low

line chicks but not for high line chicks. They also observed greater

anamnesis response to SRBC in low lines than in high SRBC lines.

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The females had higher antibody titres to SRBC response at 5 dpi

in divergent SRBC lines than males. The total antibody titre for females

was 10.1±0.5 and males had 5.8±0.7 (Boa-Amponsem et al., 2001).In

broiler chicken, (Ahmed, 2001) developed divergent lines based on

antibody response to SRBC at Central Avian Research Institute,

Izatnagar (India). Significant differences in the antibody titre even after

only one generation of selection were observed between lines.

(Shivakumar, 2003) estimated humoral immune response in

divergently selected (high & low antibody response) IWG & IWJ lines of

WLH chickens and observed that the SRBC response was higher in high

line than in low line. The respective least squares means in S1

generation were 8.89±0.29 and 8.63±0.19. (Sivaraman et al., 2003)

observed non-significant sex difference in the antibody response to

SRBC on 5 dpi in broiler chicken.( Sivaraman et al., 2004) observed

wide range of variability in the base population of SDL broiler chicken for

response to SRBC with their least squares mean as 6.289±0.246.

(Singh, 2005) observed non-significant effect of hatch and sex on

HA titre in IWG-WLH chicken. (Kumar, 2006) observed significant effect

(P<0.05) of age and sex on HA titre in Aseel native chicken. Jaiswal et

al. (2008) observed effect non-significant effect (P>0.05) of sire and sex

on HA titre in Kadaknath native chicken.

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2.1.3. Serum Immunoglobulin-G (IgG) level

Humoral immunity is one of the most important body’s defense

mechanisms. Humoral immunity is the primary function/effect through

body fluid molecules, particularly the heterogeneous group of

immunoglobulins (IgG and IgM) and many other immunologically

relevant effecter substances. IgG is the most abundant antibody and it

constitutes approximately 80% of the total serum immunoglobulins

(Kuby, 1997) and is traceable in all body fluids.

In most cases, correlations between anti-SRBC response and

serum IgG level has been found to be positive but of low magnitude

(Chao and Lee, 2001). By moderating the humoral and cell mediated

immune responses, this correlation magnitude can be enhanced many

folds (Saxena et al., 1997). Similarly the phenotypic correlation (rp)

between HA titre and serum IgG level also been found to be positive but

lower in magnitude (Sivaraman et al., 2003).(Rees and Nordskog, 1981)

analyzed the serum IgG level of ten different inbred chicken lines with a

mean ranging from 6.6±0.33 to 13.5±0.68 mg/ml. The heavy breed line

W showed the lowest (6.6±0.33 mg/ml) and Leghorn HN showed the

highest (13.5±0.68 mg/ml) level.

(Sato et al., 1986) reported that IgG levels in high and low lines for

IgG in Plymouth Rock chicken, were 1200-1600 mg/ml and 500-600

mg/ml. The significant differences observed between high and low lines

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were ascribed to the genetic factor or factors included in selection.

(Ahrestani et al., 1987) estimated the serum IgG levels in different

breeds of chicken by Single Radial Immunodiffusion (SRID) method.

Aseel (20.51±0.22 mg/ml) had significantly higher level than White

Leghorn (7.53±0.61 to 15.99±2.2 mg/ml).

(Scott et al., 1988) analyzed serum IgG levels in two lines i.e. large

bursal line (LBL) and small bursal line (SBL) of chicken and observed

that LBL line had higher serum IgG level (105+50.7 to 834.4+201.6mg)

than SBL line (3.8+7.9 and 576.3+122.7 mg).

(Saxena, 1993) studied serum Ig-G levels in Guinea fowl,

Kadaknath and broilers and estimated overall mean of serum Ig-G was

12.19+0.10, 10.01+0.4 and 8.1+0.4 mg/ml, respectively. The high Ig-G

levels were observed during first two weeks of age (15.7+0.49 and

14.3+0.32 mg/ml), which declined rapidly till fourth week of age and

finally attained a static level.

(Toro et al., 1997)) Studied the levels of lacrimal IgA and serum

IgG response to IBV vaccine by different doses, and routes of

immunization in different groups of chicken. Higher IgG levels were

detected throughout the experimental period after a vaccination by

ocular route as compared with vaccination via drinking water.

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(Sarker et al., 1998) studied the effect of chicken MHC (B

complex) on IgM and IgG production, cell mediated immune response

and immunity against different disease agents in two pairs of B congenic

chicken lines. The MHC type was shown to have significant effect on

serum IgM and IgG production. Heterozygotic advantage of B haplotype

on IgM and IgG production was also observed.

(Sarker et al., 1999) studied the direct and correlated response to

divergent selection for serum IgM and IgG level in chicken and found

that the total antibody titres to SRBC were significantly (P<0.01) higher

in low IgG line than in the high IgG line at both 7 and 14 dpi. From the

results, it was suggested that selection of chicken on the basis of serum

immunoglobin isotypes might change antibody producing cells as well as

other immunocompetent cells that modulate the immune response of

selected lines.

(Chao-ChingHsein et al., 2000) studied the reproductive and

immunological traits in two Taiwan country chicken lines (H and L lines)

that had been divergently selected for high and low serum gamma-

globulin percentage at 34 weeks of age. The H lines had significantly

higher serum gamma-globulin percentage than that of L line. The h² of

serum gamma globulin estimated from sire variance component and

parent offspring regression was in the range of 0.218 - 0.529. Estimate

of realized h² for serum gamma globulin was 0.330.

24

(Sivaraman et al., 2001) recorded mean serum IgG level of

7.81±0.17 mg/ml in synthetic dam line of broiler birds. (Shivakumar,

2003) estimated mean serum IgG concentration in IWG and IWJ

genotypes of WLH chickens as 10.66±0.22 mg/ml and 10.52±0.16

mg/ml, respectively. High SRBC response line had significantly higher

titres than low SRBC line in both the genotypes.

(Singh, 2005) observed non-significant effect (P>0.05) of hatch

and sex on IgG level in IWG-WLH chicken. (Kumar, 2006) observed no

significant effect (P>0.05) of age and sex on IgG level in Aseel native

chicken. (Jaiswal et al., 2008) observed non-significant effect (P>0.05) of

sire and sex on IgG level in Kadaknath native chicken.

2.2. GENERAL GROWTH PERFORMANCE OF KADAKNATH

CHICKEN

The domestic chicken (Gallus gallus, 2n = 78) is believed to have

descended from the wild Indian and Southeast Asian red jungle fowl.

The evolutionary history of the domestic fowl can be divided into three

phases. The first phase started with the evolution of the genus Gallus,

followed by the emergence of the domestic fowl from its progenitors and

lastly the appearance of the large number of the current breeds,

varieties, strains and lines. The conservation and systemic study of this

breed using modern technologies is essential for assessment of its

25

productive and reproductive potential along with other traits as a pure

breed.

The KN breed reveals appreciable degree of resistance to

diseases compared with other exotic breeds of fowl in its natural habitat

in free range. These birds are also resistant to extreme climatic

conditions like summer heat and cold winter stress and thrive very well

under adverse environment like poor housing, poor management and

poor feeding (Thakur et al., 2006). However there is very little

information available regarding description, native breeding areas, and

geographical, demographical, morphological and productive traits of the

KN breed of poultry.

2.2.1. Growth traits

Growth involves simultaneous deposition of bones, muscle and fat;

each exhibiting an individual’s pattern of development. when based on

the percentage increase over the weight at the end of the pre-laying

phase, the most rapid growth or weight gain are made when the chick is

young.

(Mohammed et al., 2005; Devi and Reddy, 2005; Chatterjee et al.,

2007) reported that Body weight is the direct reflection of growth and it

influences the production and reproduction traits of birds. The significant

effect of genetic group on bodyweights of chicken was reported by many

workers similar to the present study.

26

2.2.1.1. Body Weight (BW)

(Singh et al., 1996) also observed better growth performance in

Nana chickens compared to their full feathered counterparts in India.

Naked neck chickens perform well in productive adaptability at hot-

humid climate, because of their association with the reduction in feather

coverage, which can increase heat loss, and so indirectly increase feed

intake and productive adaptability (Merat, 1986; Islam and Nishibori,

2009).The diversity of the local chickens reported so far is mostly on

phenotypes including adult bodyweight, egg weight, reproduction

performance and immune responses to various diseases (Gueye, 1998;

Msoffe et al., 2001; 2004).

2.2.1.2. Confirmation Traits

Growth and production traits of a bird indicate its genetic

constitution and adaptation with respect to the specific environment

(Ahmed and Singh, 2007).

(Vasu et al., 2004) reported lower body weights of 1413.96±7.97 at

40 weeks and 1405.79±9.47 at 64 weeks in White Leghorn control

populations which were lower than the present estimates. The C1 cross,

being developed as a dual purpose bird for backyard farming is excelling

Vanaraja in body weights and egg production.

27

(Niranjan and Singh, 2005) observed higher body weights, 1860

and 2773 g at 20 and 40 week of age in Gramapriya birds respectively.

They agreed that Gompertz and Logistic functions were more

appropriate function for chickens in general. These authors, however,

used only body weight-age data. Using other characteristics such as

body length, shank length, breast width and breast circumference helps

increase accuracy and detail for defining growth. The shape of growth

curves change depending on the species, gender, investigated

characteristic, time (age) and the environment (Ersoy et al., 2007).

The genetic components of body weight are mainly additive in

nature. Significant and positive effect of heterosis on body weight in

broiler was reported in literature (Iraqi et al., 2005). (Yalcin et al., 2000)

observed negative heterosis for body weight in chicken while (Nestor et

al., 2004) reported negative and significant heterosis for body weight at

8, 16 and 20 week of age in male and female turkey for fitness traits like

live ability in crossbred Breeding of rural chicken is important for small

farmers to produce more income and also to conserve genetic variation

of native breeds.

(Kiani-Manesh, 2000) showed that age at sexual maturity, number

of eggs, egg weight and body weight at eight weeks of age are the most

important traits for improving the economic efficiency of rural

chickens.The average day old weight was highest in RIR, intermediate in

28

Desi and lowest in Fayoumi. Similar trend was observed by (Farooq et

al., 2001). (Mostageer et al., 1975) indicated that live weight at hatching

averaged 28.5 and 34.5 g for the Fayoumi and RIR, with non significant

sex difference for the two breeds.(Halima et al., 2006) reported that day-

old weight, final body weight, body weight gain and mortality rate in RIR

were 35.2, 1394, 1359 g and18.3%, respectively.

The age at sexual maturity of indigenous Irani chicken were

reported 157.190.8 days and for three genetic groups of indigenous

chickens such as Naked Neck, Marandy and Public, sexual maturity age

were 23, 25 and 22 weeks, respectively (Nasrollah, 2008).

2.2.1.3. Heritability

The heritability is ratio of additive genetic variance to phenotypic

variance and therefore estimates of haritability vary from time to time

with change in additive and phenotypic variance .The importance of

haritability in breeding experiments lies in its predictive role expressing

the reliability of phenotypic value as a guide of the breeding value.

2.3. DNA POLYMORPHISM IN CHB6, CASPASE-1, IAP-1, AND ZOV3

GENES

Candidate gene analysis is a powerful approach to detect

variations in the genes controlling traits of economic importance in farm

animals, such as immune response (Rothschild and Soller., 1997).

Candidate genes chosen for studying immune response traits may have

29

known physiological functions with immune response or be in regulatory

or biochemical pathways affecting immune response. An amplified 215-

bp fragment of ChB6 gene showed a C→A substitution at base 470,

which caused a predicted amino acid change from Gln (Leghorn line) to

Lys (Fayoumi line) (Zhou and Lamont., 2003).

For the Caspase-1 gene, a 1,070-bp amplified fragment showed a

T→C substitution at −368 bp between the Leghorn and Fayoumi lines

(Zhou and Lamont., 2003). For the IAP-1 gene, a394-bp fragment

showed a T→A substitution from the Leghorn to the Fayoumi lines, and

a PCR-RFLP assay was developed to identify a Bgl I SNP to

characterize the polymorphism at Ala157 (Zhou and Lamont, 2003). For

ZOV3, the amplified 320-bp product showed a T→G SNP, which caused

a predicted amino acid change from Cys157 to Phe157 (Zhou and

Lamont., 2003).

2.3.1. Association among Immunocompetence traits

(Van der Zijpp, 1983) evaluated humoral immune response of

White Plymouth Rock and White Leghorn (WL) chickens to SRBC, but

did not observe any correlation between antibody titres to SRBC, on 3,

7, 14 dpi and PHA-P response, neither overall nor within groups of

different genotypes. It was suggested that the selection for general

immune responsiveness should include parameters of antibody and cell

mediated immune responses.

30

(Cheng and Lamont, 1988) conducted a detailed study for the

phenotypic correlations among immunological traits. Wherein, the

correlation among antibody response, phagocytosis and T-cell mediated

immunity were mostly found to be non-significant (0.14-0.17) but the

difference in correlation coefficient in males and females were

significantly different. Their results indicated the relative independence

of genetic control of these components.

(Saxena et al., 1997) observed the phenotypic correlation of anti-

SRBC antibodies in Guinea fowl to be 0.22±0.27, 0.62±0.46 and

0.27±0.25 with phagocytic index (PI), IgG and wing index (WI),

respectively.

The serum IgG concentration had a low rp with anti- SRBC

response (-0.043) in Taiwan country chicken (Chao and Lee, 2001).

(Sivaraman et al., 2003) reported positive (0.06) but significant rp

between HA titre and serum lysozyme level but observed a negative

(-0.01) and non-significant correlation between HA titre and serum IgG

level. The rp between serum lysozyme and IgG was positive (0.04) and

non-significant.

(Singh, 2005) observed highly positive genetic correlations among three

IC-traits viz., haemagglutination (HA), lysozyme (Lyso) and

immunoglobin-G (IgG) in WLH chicken. The rP among them was also

31

positive but very low in magnitude. (Kumar, 2006) observed genetic

correlations among three IC-traits viz., haemagglutination (HA),

lysozyme (Lyso) and immunoglobin-G (IgG) in Aseel native chicken. The

rP among them was also positive and low to medium in magnitude.

32

3-AIM & OBJECTIVES

__________________________________________________

The genetic resistance in poultry is controlled by immune system that

plays an important role in maintaining the normal health. Breeding

chickens for higher immunocompetence (IC) and disease resistance

provides a viable approach for commercial poultry production.

Indigenous chicken (IC) are mainly raised in resource poor rural

households of developing countries. Various economic traits viz. juvenile

body weights and body measurement sat 5 week-intervals from day old

to 20weeks of age, 4 weekly egg production, egg weight and feed

efficiency from 20to 72 weeks of age were studied for 29genetic groups

comprising purebreds, with the other native breeds and their reciprocals.

Juvenile body weights and body measurements revealed significant

breed differences at different ages. Genetic parameters were estimated

using animal model fitted with common environmental effects for growth

traits and ignoring common environment for egg production traits. The

aim of this study was to estimate heritabilities and genetic and

phenotypic correlations for growth and egg production traits to

understand which traits should be included in breeding programs for

Kadaknath chickens.

33

To evaluate the Immunocompetence status of Kadaknath.

To Measure the phenotypic traits of kadaknath.

To study DNA polymorphism of Kadaknath using PCR-RFLP

analysis.

34

4-MATERIALS AND METHODS

_________________________________________________________

4.1. IMMUNOCOMPETANCE

In the present investigation, chicks belonging to the selected lines

of Kadaknath chickens were used for the study of immunological traits

viz., humoral response to sheep RBCs, and serum IgG concentrations.

Phenotypic traits and growth rate of Kadaknath of different stages and

DNA polymorphism at candidate genes viz., ChB6, Caspase-1, IAP-1,

and ZOV3 genes. The statistical analysis was performed by using SPSS

10 (newer version) and ANOVA software for evaluating various

immunological parameters. Details of the materials used and the

procedures employed including various statistical models are given

below-

4.1.1. Selection of Parents

The birds were ranked from highest to lowest based on

immunocompetence index. 115 Randomly chicken were selected as the

parents of the first generation of high SRBC line.

35

4.1.2. Experimental Animals - Kadaknath Chickens

4.1.3. Birds and their genetic background

The current study involved approximate 115 chickens of Kadaknath

breed (indigenous chickens), randomly selected male and female from

a flock of 300 birds being maintained at experimental farm of CARI,

Izatnagar, India under standard management conditions will be used for

studying the genetic and phenotypic parameters. There are three main

varieties of Kadaknath breed, like Jet Black, Pencilled and Golden

Kadaknath. In all the three varieties of Kadaknath breed, most of the

internal organs exhibit intense black coloration which is due to the

deposition of Melanin pigment in the connective tissue of organs and in

the dermis.

36

Fig. 1.Experimental Birds of Kadaknath Native Chicken

37

4.1.4. Sheep

Healthy Muzaffarnagari breed of sheep maintained at Sheep and

Goat farm of Livestock Production Research Centre, Indian Veterinary

Research Institute, Izatnagar were used for collection of blood from the

Jugular vein to prepare SRBC suspension. Which was used in humoral

immune response studies.

4.2. Assaying of Immunocompetence traits

The Immunocompetence status of the birds can be assessed by

analyzing various components of immune system. A few important

facets of immunity response were evaluated in this investigation. Total

IgG antibody titre and humoral immune response manifested by its

components like antibody titres against an antigen were estimated. Non-

specific immunity conferred by humoral immune response to sheep

RBC and IgG concentration through SRID method were evaluated in this

investigation.

4.2.1. Humoral Response to Sheep RBC

The immune response to SRBC was assessed through HA test as

per Van der Zijpp and Leenstra (1980) using following procedure:-

4.2.1.1. Preparation of Sheep RBC antigen

Approximately 10 ml of heparinized (20 IU/ml) blood was collected

from jugular vein of healthy sheep. It was centrifuged at 40C 120C on 3-5

38

thousand rpm for 10 minutes to settle down the RBCs. The RBCs were

then washed thrice with PBS/NSS by mixing and centrifuging it to

remove other serum components. Finally, 1% sheep RBC suspension

was prepared by mixing 1 ml of packed sheep RBCs and 99 ml of PBS

which was then used for injection in the birds as antigen.

4.2.1.2. Administration of antigen

0.1 ml of 1% sheep RBC suspension was injected into the jugular

vein of each bird with tuberculin syringe. Jugular vein was the choice of

injection as it led to minimum bleeding in comparison to other veins like

brachial vein etc.

4.2.1.3. Harvesting of immune sera from SRBC sensitized birds

Two ml of blood was collected in sterilized glass tubes at 5 days

post immunization (5 dpi) and allowed to clot for 2-3 hours at 370C. The

hyper immune sera oozed out of the clot or the clot was broken. Sera

were collected by centrifuging the tube at 1000 rpm for 3-4 min. and

stored at -200C till further analysis.

4.2.1.4. Estimation of antibody titer against sheep RBCs

The antibody response to SRBC was assessed using

haemagglutination test (Vander Zijpp and Leenstra, (1980) as mentioned

below-

39

The test was performed in round bottom (U shaped) micro titer

plates.50 l of phosphate buffered saline (PBS) was added in each

well. Then, 50 l of serum was added in first well of each row

except the last row where 50 l of PBS was added, that acted as

control.

After thorough mixing thoroughly, the sera were two fold serially

diluted by taking 50 l from each of the well and adding it to the

subsequent wells, mixing thoroughly and it was continued like this

till last column, which was discarded. Equal volume (50l) of 1%

SRBC suspension was added in all the wells and was thoroughly

mixed with sera samples.

The plates were then incubated at 370C for 1 hour. The highest

dilution that gives complete agglutination (button shaped clumping

of RBCs indicated haemagglutination reaction) and it was recorded

as titer and was expressed as log2n.

4.2.2. Estimation of serum IgG by Single Radial Immuno-diffusion

(SRID) Assay

Chicken serum IgG neutralizes the anti chicken IgG. Agarose gel was

used as a solidifying base to assay IgG concentrations through Single

40

Radial Immunodiffusion (SRID) assay (Mancini et al., 1965). The

procedure is given below.

Clean and sterilized glass plate was placed on leveled horizontal

surface. About 50 ml of 0.1 M Tris-HCl was divided equally into

two halves. In first half, of 3% Agarose concentration was added

@ 3% (w/v) and boiled. In second half, 1.750 ml of anti-chicken

IgG (Sigma, USA) was added and after thorough mixing, it was

kept at 50ºC in a water bath.

The temperature of first half (boiled and cooled) was brought down

to about 50ºC and second half was mixed. The standards of IgG

(Sigma, USA) viz., 25g/ml, 12.5g/ml, 6.25 g/ml, 3.125 g/ml

and 1.5625 g/ml, prepared by serial dilution of stock solution were

loaded in the wells to plot standard curve.5 l of unknown sera

were diluted to 4 times with 0.1 M Tris and then loaded the wells.

The plate was incubated at 37ºC for 24 h in humid chamber. The

diameters of the ring around standard as well as unknown samples

were measured with the help of Digital Vernier Calipers.

The serum IgG concentrations in unknown samples were

determined with the help of regression equation obtained by

41

plotting log2 concentrations of IgG standards against diameter of

the precipitation ring.

4.2.2.1. Statistical analysis

The data generated on immunological traits were analyzed by LS

ANOVA using following Statistical model: -

Y= µ + Li + Li:Sij + Hk + Sxl + eijklm

Where, Y = value of a trait measured on ijklmth individual

µ = Overall mean

Li = Effect of line (i = 1, 2)

Li:Sj = Random effect of jth sire in ith line

Hk = effect of kth hatch

Sxl = Effect of lth sex (l = 1, 2)

eijklm = random error associated with mean ‘0’ and variance

σ2

42

4.3. BODY WEIGHT

The data utilized in this study were obtained from Desi Breeds for

present investigations were collected from the records of the indigenous

chicken maintained at Central Avian Research Institute, Izatnagar.

4.3.1. Management of Flock and Traits Measured

Chicks each of the breeds were obtained in more than one hatch

with seven to ten dams’ difference between the hatches. The selected

and controlled populations were maintained under uniform conditions of

feeding, management and disease control as far as possible over year’s

.following trait were measured in each of the generation in both the lines.

4.3.1.1. Pedigree Mating by Artificial Insemination

After identification of the parents of divergent lines, four females

were allotted to each male within each line. The semen from the cock

was collected by gentle massaging at the back and groin region of the

bird. The collected semen was diluted with normal saline and each of the

four females allotted to a particular male were inseminated with 0.2 ml of

the diluted semen.

4.3.1.2. Incubation and Hatching of eggs

Pedigreed eggs were marked for their sire as well as dam. They

were collected twice a day for 30 days, at 10 days interval and stored in

a cold chamber before setting. Cracked and grossly abnormal eggs were

43

discarded. Fumigation was done as per the standard procedure prior to

setting. The fertility was checked by candling of eggs on day 18 and the

fertile eggs were transferred to hatcher on 18th day of incubation by

placing the eggs in pedigree boxes transferred to hatching trays. On

22nd day, chicks were taken out from the hatcher after proper drying

and wing banded.

4.3.1.3. Fertility

Fertility of the birds was calculated as the percent age of eggs that

were found to be fertile after candling.

No. of Fertile eggs

Fertility (%) = X 100

Total number of egg set

4.3.1.4. Hatchability

Hatchability percentage were calculated on the basis of total eggs

set (TES) as well as fertile eggs set (FES) as given below-

Hatchability (TES) % = No. of chicks hatched/ Total no. of eggs set X

100

Hatchability (FES) % = No. of chicks hatched/ Total no. of fertile eggs

set X 100

All chicks of the S1 generation divergent lines were hatched in two

hatches.

44

4.3.1.5. Body Weight: Body weight at 4, 6, 8, 10 and 12 weeks of age

were recorded for individual birds. Gains in weight between 4 – 6

weeks, 6 – 8 weeks, 8 – 10 weeks, and 10 – 12 weeks of age were

recorded.

4.3.1.6. Age at Sexual Maturity: The age in days at the time of laying

its first egg was taken as measured of age at sexual maturity.

4.3.1.7. Egg Weight: The egg weight was recorded to nearest of 1g

accuracy. The average weight of three consecutive eggs at 12 weeks of

age was taken as average egg weight of egg for individual pullet.

4.3.1.8. Part Period Egg Production: it was measured as the number

of egg laid up to 280 days of age for each bird.

4.3.1.9. Statically Data Analysis

The collected data were analyzed using the general linear model

of SPSS 11.0 with sex and plumage colour as fixed factors. Significant

means were separated by the Duncan’s multiple range tests. Correlation

between measurements was determined by the Pearson’s Correlation

Coefficient.

Separate models (Linear and Multiple) for body measurements

singly and combined were enumerated. The regression model adopted

was as follows:

45

Y = a + b1 X

1 + b

2 X

2 + b

3 X

3

Where Y = body weight (kg)

X1 to X

3 = body measurements

a = Intercept

b (1-3) = regression coefficients of Y on X (i = 1, 2, 3).

4.4. DNA Polymorphism in ChB6, Caspase-1, IAP-1, and ZOV3

genes by PCR-RFLP

Nucleotide polymorphism at ChB6, Caspase-1, IAP-1, and ZOV3

genes were studied with PCR-RFLP.

4.4.1. DNA Sampling

Twenty four genomic DNA samples were isolated, twelve from

each breed and six from high IC group and six from low IC group in both

the breeds, using Phenol extraction method (Kagami et al., 1990).

Brief methodology is presented below-

0.1 ml of blood was collected from jugular vein in heparinized tube.

Centrifugation at 3000 rpm for 5 min. with PBS (for compositions

refer to the annexure) in refrigerated centrifuge for washing of

cells.

The supernatant was discarded and lysis buffer (composition is

given in annexure) was added. RBCs were suspended in the lysis

46

buffer and kept at room temperature for 15-20 min. Then

Proteinase K (200µg/ml) and SDS (0.5%) was added and it was

kept overnight for incubation.

In this mixture equal volume of Tris-saturated Phenol was added

and mixed gently for 10 minutes. Two phases were separated by

centrifuging at 5000 rpm for 5 min and the upper aqueous phase

was transferred to a new microfuge tube and this step was

repeated twice.

The aqueous phase was then extracted twice with equal volume of

Phenol: Chloroform: Isoamyl Alcohol (25:24:1).Lastly, the aqueous

phase was extracted twice with equal volume of Chloroform:

Isoamyl Alcohol (24:1).

DNA was precipitated from the aqueous phase by adding 2-2.5 ml

volume of chilled absolute ethanol and gentle mixing. The

precipitated DNA was centrifuged at 10000 rpm for 10 min and the

DNA pellet was washed twice with 70% ethanol. It was then air

dried and dissolved in sufficient volume of autoclaved distilled

water.

47

4.4.2. Checking of concentration, purity and quality of DNA

The concentration of DNA was calculated spectrophotometrically

by taking optical density (OD) at 260 nm using the following formula:

DNA concentration (µg/ml) = OD 260 x Dilution factor x 50

The purity of DNA was assessed by calculating ratio of optical

densities at 260 and 280 nm. The samples with OD ratio (260nm/

280nm) ranging from 1.7 to 1.9 were used in subsequent experiments.

Quality of DNA was assessed through 0.7% horizontal submarine

agarose gel electrophoresis as below-

The gel casting plate was sealed with adhesive tape and placed on

a leveling table. Agarose (0.7% w/v) was boiled in 1 X TBE (Refer

annexure for composition) buffer then cooled to 55°C, then

ethidium bromide (0.5 µg/ml) was added. The gel was poured into

the casting tray.

After solidification, the comb and adhesive tape were removed.

The gel casting tray was submerged in electrophoresis tank having

1 X TBE buffer.

48

DNA samples were prepared by mixing 3µl of genomic DNA, 7µl of

D.W. and 2µl of Bromo Phenol Blue dye (Refer Annexure for

composition). It was carefully loaded in the well. Electrophoresis was

performed at 2-3 volts/cm for one hour and then gel was visualized

under UV transilluminator.

4.4.3. PCR optimization

The reaction mixture and PCR programs for ChB6, Caspase-1, IAP-1,

and ZOV3 genes were optimized as per Zhou and Lamont (2003) with

slight modifications.

4.4.4. Optimized PCR reaction mixture

For ChB6, Caspase-1, IAP-1, and ZOV3 genes, the optimum

combination of various reaction components for each 25 µl PCR reaction

mix was 25 ng genomic DNA, 0.8 mM of each primer, 0.2 mM each

dNTPs and 1 U Taq DNA polymerase.

4.4.5. Optimization of PCR programme

To optimize the PCR conditions different annealing temperatures were

used. In case of all the four genes i.e., ChB6, Caspase-1, IAP-1, and

ZOV3 genes in Kadaknath breed of native chickens, initial denaturation

at 94˚C for 5 min, 35 cycles of (a) Denaturation at 96˚C for 1 min. in

case of ChB6, Caspase-1, and ZOV3 whereas in case of IAP-1, it was

49

94˚C, (b) Annealing at 53˚C, 65˚C, 62˚C, and 52˚C for ChB6, Caspase-

1, IAP-1, and ZOV3, respectively for 1 min., (c) Extension at 72˚C for 1

min. and final extension at 72˚C for 10 min. for ChB6, Caspase-1, and

ZOV3 whereas in case of IAP-1, it was 72˚C for 15 min. produced

distinct and robust amplified product.

The genomic DNA samples having good quality (Intact without

smearing) were used for further analysis.

4.4.6. PCR-RFLP Analysis

PCR-RFLP is a simple and reliable technique for studying

nucleotide polymorphism in nucleotide sequences in genes. It involves

designing of a set of primers for the locus of interest which are used for

PCR amplification of that segment of DNA, followed by restriction

enzyme digestion of the PCR product and visualization of restriction

fragments in gel.

4.5. Primer preparation

Following primer sequences were got synthesized utilizing the

published information and used in this study. The sequences for forward

and reverse primer for ChB6, Caspase-1, IAP-1, and ZOV3 genes were

obtained from Zhou and Lamont (2003) and presented below in table

no.1.

50

The upstream and downstream primers used to amplify ChB6,

Caspase-1, IAP-1, and ZOV3 were expected to yield a 215, 1070, 394,

and 320 bp product, respectively (Zhou and Lamont, 2003).

Table no. 1. Upstream and Downstream primer and their Sequences.

Name of

genes

Name of Primer Sequence

ChB6

Upstream primer

Downstream primer

5’-GCTTCCCCAATGGAACTG-3’

5’-GAGCACAATGGGCCTAGTC-3’

Caspase-1

Upstream primer

Downstream primer

5’-CCATGCTTGGGCTCTCAGTG-3’

5’-GGTCCCGCAGATCCCAGTG-3’

IAP-1

Upstream primer

Downstream primer

5’-TCACCATCTCTACGTTCCAT-3’

5’-CATTGAAACTTGGTTGGTCT-3’

ZOV3

Upstream primer

Downstream primer

5’-GCTTGGACCTGGTATATGAC-3’

5’-GGCTAAAGTAGGTCAAGTGAC-3’

4.5.1. PCR reaction mixture

The PCR was carried out in a total reaction volume of 25l. The

components and annealing temperature were optimized. The optimized

reaction that was finally used for amplification was as below in table.2.

51

Table. 2. PCR reaction mixture for ChB6, Caspase-1, IAP-1, and ZOV3

4.5.2. PCR amplification Program

PCR amplification was carried out in a thermal cycler (PTC-200,

MJ Research, USA) as per Zhou and Lamont (2003) using following

cyclic conditions.

S. No. Reaction component Volume Final conc.

1. Template (Genomic DNA) 1.0 l 25 ng

2. Up stream primer 1 l 0.8µM

3. Down stream primer 1 l 0.8µM

4. 10 X PCR buffer (without MgCl2) 2.5 l 1.5 mM

5. MgCl2 1.5 l 1.5 mM

6. dNTP mix 2 l 0.2 mM

7.. Autoclaved triple glass distilled water 15.8 l

8. Taq DNA polymerase 5U/l 0.2 l 1 U

Total 25 l

52

Table. 3. PCR Amplification Program.

ChB6 Caspase-1 IAP-1 ZOV3 1. Initial

denaturation at

94ºC for 5 min. 2. 35 cycles of a). Denaturation

at 96ºC for 1

min. b). Annealing at

53ºC for 1 min. c). Extension at

72ºC for 1 min. 3. Final extension

at 72ºC for 10

min.

1. Initial

denaturation at

94ºC for 5 min. 2. 35 cycles of a). Denaturation at

96ºC for 1 min. b). Annealing at

65ºC for 1 min. c). Extension at

72ºC for 1 min. 3. Final extension

at 72ºC for 10 min.

1. Initial

denaturation at

94ºC for 5 min. 2. 35 cycles of a). Denaturation

at 94ºC for 1 min. b). Annealing at

62ºC for 1 min. c). Extension at

72ºC for 1 min. 3. Final extension

at 72ºC for 15min.

1.Initial

denaturation at

94ºC for 5 min. 2. 35 cycles of a). Denaturation

at 96ºC for 1 min. b). Annealing at

52ºC for 1min. c). Extension at

72ºC for 1 min. 3. Final extension

at 72ºC for10min.

4.5.3. Documentation of PCR products by Agarose Gel

electrophoresis

Approximately, 8 l of PCR product was added with 2 l triple

distilled water and 2 l Bromophenol blue dye for loading in a 1.4%

agarose gel containing ethidium bromide (0.5 µg/ml). The

electrophoresis was done at 2-5 V/cm. 100 bp and low range DNA

ladder (Bangalore Genei, India) was used as molecular size marker for

identification of the desired product. The amplified product was

examined under UV illumination and photographed for documentation.

53

4.5.4. Restriction enzyme digestion

One restriction enzyme was used for each amplified product to

study RFLP. The RE digestion was carried out in 20µl under the

manufacturer’s recommended conditions. RE Pvu II, Hsp 92 II, Bgl I and

SnaB I were employed for ChB6, Caspase-1, IAP-1, and ZOV3 genes,

respectively (Zhou and Lamont, 2003).

Table .4. Pvu II RE digestion of 215 bp of ChB6 gene

Reaction components Amount (1x)

Pvu II (10U/µl) 0.1µl

10X buffer B 2 µl

Autoclaved triple distilled water 7.9µl

PCR product 10 µl

Total 20 µl

The Pvu II digestion was carried out for overnight at 37ºC in a

water bath. After digestion, the digested products were kept in

refrigerator at 4ºC till further study.

Table.5.Hsp 92 II RE digestion of 1070 bp of Caspase-1 gene

Reaction components Amount (1x)

Hsp 92 II (1U/µl) 1µl

10X buffer R 2µl

Autoclaved triple distilled water 7.0µl

PCR product 10 µl

Total 20 µl

54

The Hsp 92 II digestion was carried out for overnight at 37ºC in a

water bath. After digestion, the digested products were kept in

refrigerator at 4ºC till further study.

Table.6.Bgl I RE digestion of 394 bp of IAP-1 gene

Reaction components Amount (1x)

Bgl I (10U/µl) 0.1µl

10 X buffer R 2µl

Autoclaved triple distilled water 7.9µl

PCR product 10 µl

Total 20 µl

The Bgl I digestion was carried out for overnight at 37ºC in a water

bath. After digestion, the digested products were kept in refrigerator at

4ºC till further study.

Table .7.SnaB I RE digestion of 320 bp of ZOV3 gene

Reaction components Amount (1x)

SnaB I (5U/µl) 0.2µl

10 X buffer R 2µl

Autoclaved triple distilled water 7.8µl

PCR product 10 µl

Total 20 µl

The SnaB I digestion was carried out for overnight at 37ºC in a

water bath. After digestion, the digested products were kept in

refrigerator at 4ºC till further study.

55

4.5.5. Determination of molecular sizes of digests and recording of

RFLP pattern

The molecular sizes of the digests were determined with the help

of Quantity One Software of BioRad provided with the Gel Doc System

(BioRad, USA).

4.5.6. Statistical analysis

On the basis of molecular sizes of digests, genotypes were

grouped according to the reports of Zhou and Lamont (2003). Standard

statistical method was followed to calculate gene and genotypic

frequencies.

56

5-RESULTS

___________________________________________________________________

5.1. EVALUATION OF IMMUNOLOGICAL TRAITS

The Immunocompetence status can be assessed by analyzing various

components of immune system viz., antibody titres against SRBC,

serum IgG level.

Accordingly two important traits viz., humoral response to sheep

RBCs, and serum IgG level were analyzed in Kadaknath breeds of

native chicken.

The least square analysis of variance for these traits of Kadaknath

is presented in table no. 8 and their factor-wise least squares mean are

presented in table 9.

5.1.1. Antibody response to Sheep RBC

The antibody titres against sheep RBCs were measured through

HA test on 5th dpi. HA titres ranged from 2-15 in Kadaknath .Average HA

titers were 8.32±0.13 in Kadaknath.

57

Fig .2. Humoral Immune response against SRBC titer in kadaknath chicken.

HA

12.011.010.09.08.07.06.0

40

30

20

10

0

Std. Dev = 1.45 Mean = 8.3

N = 114.00

58

Fig .3.Effect of IGG against SRBC in Kadaknath Chicken.

IGG

3.002.001.00

Cou

nt

120

100

80

60

40

20

0

The wide variations observed in the present study might be due to

the reason that no artificial selection has been applied on this breed of

chicken for immune response or production traits. Kadaknath breed of

native chicken demonstrated higher HA titre than most of the other

breeds.

59

5.1.2. Serum IgG concentrations

Serum IgG is the most abundant antibody and constitutes

approximately 80% of the total immunoglobulins. The bird’s ability to

mount antibody responses to other antigens is primarily revealed by

serum IgG concentration. The average serum IgG concentration was

10.07±0.20 mg/ml in Kadaknath breeds, respectively.

60

Fig .4 .Effect of Heamagglutination on Kadaknath chicken.

Fig .5.Effect of IgG serum Concentration on Kadaknath Chicken.

61

5.2. GENERAL PERFORMANCE OF KADAKNATH CHICKEN

The data utilized in this study were obtained from the Indian native breed

kadaknath .The average weekly body weight from 4th weeks to 12th of

age for both the sexes of these breeds along with their slandered

deviation. The observed results are listed in the following tables and

graphs by using SPSS10 software of data Analysis for evaluating

various statistical parameters of Kadaknath native Chickens. Trends for

breeding values and phenotypic performance were obtained by

regression of average breeding values and phenotypic least squares

means, respectively, on generation number.

5.2.1. Growth Traits

5.2.1.1. Body Weight

Body weights of the birds were controlled every week. The difference in

day old body weight of different genotypes might be due to difference in

weight of egg obtained from different female lines whose body weight

differ significantly due to the difference in breed characteristics. The

body weight of female followed similarly pattern to that of males. Males

were heavier then the females in all the breeds .No significant difference

however could be observed for day old weight due breeds and sex.The

hatch effects and breed-hatch interaction effects were significant.

62

5.2.1.2. Mean and Slandered Errors

The mean value for body weight at four weeks of age followed

similar trend to that of body weight at 2 weeks of age. Effect due to

breed, sex hatch and hatch-breed interaction were highly significant.

Mean along with the slandered errors and coefficient of variation of

the body weight and confirmation traits at various age are shown in

tables. Least-squares analysis of variance revealed significant difference

(p<0. 01) for general combining ability (GCA) in body weight at all age

groups. Significance of GCA indicates importance of additive genetic

effect for inheritance of juvenile body weight.

The average mean body weight at 4, 6, 8, 10, and 12, weeks of age

were 162.92, 175.71, 340.88, 391.75 and 500.04 gm. respectively. there

was a study increase in the body weight from 4th day to 12th weeks of

age .Growth was accelerated up to 12 week of age .The gain in the body

weight was highest during 8 to 12 weeks of age. The average body

weight week’s difference from 4th week to 12 weeks is 113. The average

slandered error of mean from the 4th, 6th, 8th, 10th and 12th weeks of age

is continuously increasing as 2.50, 3.43, 6.43, 7.68 and 9.88gm

respectively. The genetic components of body weight are mainly additive

in nature. Since the body weight is not directly related with fitness trait;

therefore in general it was observed that all crosses in a diallel

experiment did not express positive heterosis. It is concluded that using

63

Gompertz and linear growth models would be sufficient in modelling the

growth, based on the 7 traits determined. At four weeks of age the

kadaknath indigenous chickens had the lowest and highest mean body

weight gain of all the strains with average daily growth rate of 3.30 g and

4.20 g per bird per day in the starter growth phase, respectively.

The study of least square of analysis of slandered deviation of

kadaknath chicken from 4th week of body weight to 12th weeks of body

weight is increasing like 26.73, 36.64, 68.67, 82.00 and 105.56

respectively.

The mean body weight gain at the age of twelve weeks from the

on-farm experiment in management system is comparable with mean

body weight gain at eight weeks of age under favourable management

conditions.

The probable reason for such differences in the ranking of body

weight might have been due to genetics diversity of the stock used in

different places or genotype environment interaction.

The general trend of coefficient of variation indicated an increase

in the variation during growing period. The high coefficient of variation

appears to be the consequences of the expression of the differentials

rates of growth during this period .The sudden exposer of the keets to

the external environment in the post hatching periods i .e. brooder

house, feeding condition and other managemental factors combined with

64

the inherent genetics factors could be the cause for the general raise in

the coefficient of variation.

5.2.1.3. Phenotypic correlation among by body weight at various

stages

The Phenotypic correlation between body weights at various ages.

Day old body weight was positively correlated with body weight at later

ages. The genotypic correlation mostly varied from low to medium. The

magnitude of genetic correlation gradually increase up to 16 week of age

and then decrease .the phenotypic correlation between these traits were

small and positive and follow similar trend.

Body weight at 4 week of age was positively correlated with body

weight at 8, 10, and 12 week of age. The phenotypic correlation of Body

weight 4, 6, 8, 10, and 12 was 1.000, 0.843, 0.738, 0.729 and 0.563

respectively. The magnitude of such phenotypic correlation gradually

decrease with increase in age. The phenotypic correlation although

followed similar trend .12 week body weight showed high Pearson

correlation with 10 week of body weight .The phenotypic correlation

between 10 week and 12 week body weight were almost similar age of

body weight. Phenotypic correlation among body weights at different

ages ranged from low to high .The keets weight at hatching is not

supposed to bear ay genetic relationship with subsequent body weights.

Low phenotypic correlation between 0 day body weight and body weight

65

at later ages indicates that Body weight at 0 day of ages is not suitable

criterion for selection for improving market weight. Body weights to 16

weeks of age were used to characterize the growth of chicken in this

study. The phenotypic correlation among body weight at other ages

were high and possitive.As the time interval between two body weight

increased there was corresponding reduction in the magnitude of these

correlation .This may be because of the fact that the body weight of two

successful stage are more closely related and perhaps governed by

more common gene than the weight with large time interval.

66

Fig.6.Analysis of Standard Deviation & Mean of Body weight 4 of Kadaknath

Chicken.

BW4

230.0220.0

210.0200.0

190.0180.0

170.0160.0

150.0140.0

130.0120.0

110.0100.0

90.0

30

20

10

0

Std. Dev = 26.73 Mean = 162.9

N = 114.00

Fig .7. Analysis of Standard Deviation & Mean of Body weight 6 of KN Chicken.

BW6

300.0280.0

260.0240.0

220.0200.0

180.0160.0

140.0120.0

100.080.0

30

20

10

0

Std. Dev = 36.64 Mean = 175.7

N = 114.00

67

Fig .8. Analysis of Standard Deviation & Mean of Body weight 8 of KN Chicken.

BW8

580.0560.0

540.0520.0

500.0480.0

460.0440.0

420.0400.0

380.0360.0

340.0320.0

300.0280.0

260.0240.0

220.0200.0

20

10

0

Std. Dev = 68.67 Mean = 340.9

N = 114.00

Fig.9. Analysis of Standard Deviation & Mean of Body weight 10 of KN

Chicken.

BW10

650.0625.0

600.0575.0

550.0525.0

500.0475.0

450.0425.0

400.0375.0

350.0325.0

300.0275.0

250.0225.0

200.0

20

10

0

Std. Dev = 82.00 Mean = 391.8

N = 114.00

68

Fig.10. Analysis of Standard Deviation of Body weight 12 of KN Chicken.

BW12

800.0750.0

700.0650.0

600.0550.0

500.0450.0

400.0350.0

300.0250.0

14

12

10

8

6

4

2

0

Std. Dev = 105.57 Mean = 500.0

N = 114.00

69

5.3. DNA POLYMORPHISM IN CHB6, CASPASE-1, IAP-1, AND ZOV3

GENES

DNA polymorphism at ChB6, Caspase-1, IAP-1, and ZOV3 genes

were studied using PCR-RFLP.

DNA isolation and its evaluation for quality, purity and concentration

Genomic DNA were isolated from 24 blood samples (6 each at

high and low extremes of HA titers from both the breeds) using Phenol

extraction method.

5.3.1. Agarose Gel Electrophoresis

2% Horizontal Submarine Agarose gel was prepared for

documentation of RE digested products.

5.3.1.1. Procedure

20µl of the digestion mixture was kept in water bath for digestion at

37ºC for overnight.4µl Bromophenol blue dye was added in the

entire digested product. Uncut sample was made by mixing 10µl of

the PCR product and 2µl Bromophenol blue dye was added to it.

Marker was made by adding 7µl triple distilled water, 2 µl

Bromophenol blue dye, and 1µl ladder (100 bp in case of ChB6,

IAP-1, and ZOV3 gene whereas in case of Caspase-1, low range

70

DNA marker was used).The gel casting plate was sealed with

adhesive tape and placed on a leveled table surface.

2% Agarose (w/v) (Biogene, USA) was boiled in 1 X TBE (Refer

Annexure for composition) buffer. After boiling it was cooled to 55ºC

and then ethidium bromide (0.5 µg/ml) was added. The gel was

gently poured into the casting tray avoiding bubble formation and

was allowed to solidify at room temperature.

After solidification, the comb and adhesive tape were removed. The

gel casting tray was submerged in gel tank of electrophoresis unit

having 1 X TBE buffer.

Digested DNA samples, uncut sample, and marker sample were

carefully loaded in the wells. Electrophoresis was performed at 2-5

volts/cm for two to three hour and then gel was visualized and

photographed under UV transilluminator.

The quality of genomic DNA was assessed by 0.7% horizontal Agarose

gel electrophoresis and found to be good. All samples gave intact bands

without smearing on 0.7% Agarose. Gel was visualized under UV

transilluminator.

71

Purity and concentration of genomic DNA was determined by

optical density (OD) determination at 260 nm and 280 nm in a UV

Spectrophotometer. Ratio of OD260 to OD280 of most of the DNA samples

were lying in the range of 1.7 to 1.9 indicating satisfactory purity of DNA

samples.

Concentrations of DNA ranged from 258.8–321.7 ng/µl in

Kadaknath breed. DNA samples were diluted with triple glass distilled

water to obtain a final concentration of approximately 50 ng/µl. These

diluted DNA samples were used in PCR reactions.

The optimized PCR reaction mixture and PCR programmes gave

satisfactory amplification of all the genes in Kadaknath breed of native

chicken.

5.3.2. DNA Polymorphism at ChB6 gene

For ChB6 gene, a 215 bp amplicon was obtained in Kadaknath

native chickens on PCR amplification. The Pvu II RE digestion of PCR

product generated fragments of 215 bp in Kadaknath. Smaller fragments

could not be resolved properly. In present investigation revealed in the

case of Kadaknath, only two genotype i.e. AA and BB with fragment size

of 147 and 215 bp fragments, respectively could be observed. Genotypic

and gene frequencies for ChB6 are presented below-

72

Table. 34. Gene and Genotypic frequencies of PCR-RFLP alleles of ChB6 gene

in Kadaknath.

Genotype Gene

AA AB BB A B

Frequency 0.5 0.0 0.5 0.5 0.5

Thus, PCR-RFLP of 215 bp amplicon of ChB6 gene with Pvu II

restriction enzyme revealed polymorphic banding pattern in the

Kadaknath native chicken.

5.3.3. DNA Polymorphism at Caspase-1 gene

For Caspase-1 gene, 1070 bp amplicon was obtained on PCR

amplification in Kadaknath native chickens. The Hsp 92 II RE digestion

of PCR product generated fragments of 109, 122, 227, 244, 316, and

421 bp in Kadaknath. Smaller fragments could not be resolved properly.

On the basis of presence and absence of restriction fragments,

Kadaknath demonstrated only one genotype i.e. AB in all the birds.

Genotypic and gene frequencies for Caspase-1 are presented below-

73

Table.35.Gene and Genotypic frequencies of PCR-RFLP alleles of Caspase-1 gene in Kadaknath.

Genotype Gene

AA AB BB A B

Frequency 0.0 1.0 0.0 0.5 0.5

Thus, PCR-RFLP of 1070 bp product of Caspase-1 gene with Hsp

92 II restriction enzyme revealed polymorphic banding pattern in

Kadaknath native chicken.

5.3.4. DNA Polymorphism at IAP-1 gene

For IAP-1 gene, a 394 bp amplicon was obtained in Kadaknath

native chickens on PCR amplification. The Bgl I RE digestion of PCR

product generated fragments of 140, 254, and 394 bp in Kadaknath. In

Kadaknath breeds, present investigation revealed two genotypes i.e. AA

and AB, in all the birds analysed. In Kadaknath, AA genotype had the

254 and 140 bp fragments, whereas AB genotype had the fragments i.e.

140, 254, and 394 bp fragments.

Genotypic and gene frequencies for IAP-1 are presented below-

74

Table.36. Gene and Genotypic frequencies of PCR-RFLP alleles of IAP-1 gene

in Kadaknath

Genotype Gene

AA AB BB A B

Frequency 0.38 0.62 0.0 0.69 0.31

Thus, PCR-RFLP of 394 bp product of IAP-1 gene in Kadaknath

with Bgl I restriction enzyme revealed polymorphic banding patterns in

Kadaknath native chickens.

5.3.5. DNA Polymorphism at ZOV3 gene

For ZOV3 gene, 320 bp amplicon was obtained on PCR

amplification in Kadaknath. The SnaB I RE digestion of PCR product

generated fragments of 270 and 320 bp in Kadaknath breeds of native

chicken. Smaller fragments could not be resolved properly. On the basis

of presence and absence of restriction fragments, Kadaknath birds

resolved into two genotypes i.e. BB and AB. In Kadaknath, BB genotype

had the fragments viz., 320 bp whereas AB genotype had the fragments

viz., 270, and 320 bp.

75

Genotypic and gene frequencies for ZOV3 are presented below-

Table.37. Gene and Genotypic Frequencies of PCR-RFLP alleles of ZOV3 gene in Kadaknath.

Genotype Gene

AA AB BB A B

Frequency 0.0 0.6 0.4 0.3 0.7

Thus, PCR-RFLP of 320 bp product of ZOV3 gene with SnaB I

restriction enzyme revealed polymorphic banding pattern in Kadaknath

native chicken.

76

(A)

(B)

Fig .11. PCR-RFLP analysis of ChB6 gene in Kadaknath. (A) Amplified product. (B) Pvu II PCR-RFLP patterns in Kadaknath. bp1 and bp2 indicate molecular sizesofspecificamplicon/REdigestsandDNAmolecularsizemarkers,respectively. Various genotypes have been indicated above the lane numbers.L-IC= Low Immunocompetence birds; H-IC= High Immunocompetence birds.

L-IC H-IC

1 2 3 M 5 6 7 8 bp2

300

100

200

147

bp1

1000

215

AA BB UC BB BB AA AA

bp1

L-IC H-IC 1 2 3 M 5 6 7

300

100

200 215

bp2

1000

77

(A)

(B)

L-IC H-IC

Fig .12. PCR-RFLP analysis of Caspase-1 gene in Kadaknath native chickens.(A) Amplified product of 1070 bp (B) Hsp 92 II PCR-RFLP patterns in Kadaknath. bp1 and bp2 indicate molecular sizes of specific amplicon/ RE digests and DNA molecular size markers, respectively. Various genotypes have been indicated above the lane numbers. L-IC= Low Immunocompetence birds; H-IC= High Immunocompetence birds.

1 2 3 M 5 6 7 8

bp2

300

100

200

123

bp1 bp2

1000

112

600

232 249 316 421

AB AB UC AB AB AB AB

L-IC H-IC

1 2 3 M 5 6 7 8

bp2

300

100

200

bp1

1000 1070

600

78

(A)

(B)

Fig .13. PCR-RFLP analysis of IAP-1 gene in Kadaknath native chickens.(A) Amplified product of 394 bp. (B) Bgl I PCR-RFLP patterns indicates various genotypes in Kadaknath. bp1 and bp2 indicate molecular sizes of specific amplicon/ RE digests and DNA molecular size markers, respectively. Various genotypes have been indicated above the lane numbers.L-IC= Low Immunocompetence birds; H-IC= High Immunocompetence birds.

L-IC H-IC

bp1

1 2 3 4 5 M 5 6 7 8

bp2

300

100

200

1000

394 400

254

140

AB AB AA AA UC AB AA AB AB

bp2

L-IC H-IC

1 2 3 4 M 5 6 7

bp1

300

100

200

1000

394 400

79

(A)

(B)

Fig .14. PCR-RFLP analysis of ZOV3 gene in Kadaknath native chickens. (A) Amplified product of 320 bp (B) SnaB I PCR-RFLP indicates various genotypes. bp1 & bp2 indicate molecular sizes of DNA markers and specific amplicon (A) or RE digests (B), respectively. Various genotypes have been indicated above the lane numbers.

L-IC= Low Immunocompetence birds; H-IC= High Immunocompetence birds.

L-IC H-IC

1 2 3 M 5 6 7

8 bp2

300

100

200

bp1

1000

320

270

AB BB UC BB AB AB

BB

1 2 3 M 5 6 7 8

bp1

300

100

200

bp2

1000

L-IC H-IC

320

80

Table .8. Least Square means and Standard Error for Humoral immune

response against SRBC and IgG for Kadaknath native chicken.

S.N. SEX HA IgG

1- Male Mean 8.1596 1.9255

Std. Error of Mean .1713 0.098

2- Female Mean 7.0714 1.8000

Std. Error of Mean .2028 0.066

3- Total Mean 7.6951 1.8720

Std. Error of Mean .1371 0.063

Means in a column with different superscripts differ significantly (P<0.05) *P ≤ 0.05, **P≤0.01 (samples were taken in three replicates). Table.9.Least squares analysis of variance of important immunological traits in Kadaknath breed of native chicken for Descriptive Statistics.

N Mean Std. Deviation

Minimum Maximum

HA 114 8.3246 1.4482 6.00 12.00

IgG 114 2.0439 .3854 1.00 3.00

(Note - Samples has been taken three replicates.)

81

Table .10.Least square analysis of Chi-Square Test- Frequencies of Heam-agglutination against SRBC of Kadaknath Chicken.

Frequency Observed N Expected N Residual

6.00 13 16.3 -3.3 7.00 20 16.3 3.7 8.00 32 16.3 15.7 9.00 27 16.3 10.7

10.00 11 16.3 -5.3 11.00 10 16.3 -6.3 12.00 1 16.3 -15.3 Total 114

Table .11. Least square analysis of Chi-Square Test- Frequencies of IgG against SRBC of Kadaknath chicken.

Observed N Expected N Residual

1.00 6 38.0 -32.0

2.00 97 38.0 59.0

3.00 11 38.0 -27.0

Total 114

Table .12.Least Square analysis of Test Statistics of HA and IgG of Kdaknath Chicken.

HA IgG Chi-Square 42.211 137.737

df 6 2 Asymp. Sig. .000 .000

A. 0 cells (.0%) have expected frequencies less than 5. The minimum expected cell frequency is 16.3. B. 0 cells (.0%) have expected frequencies less than 5. The minimum expected cell frequency is 38.0.

82

Table. 13. Least Square Pearson Correlations of HA and IgG of Kadaknath Chicken.

HA IgG HA Pearson Correlation 1.000 .006

Sig. (2-tailed) . .950

N 114 114 IGG Pearson Correlation .006 1.000

Sig. (2-tailed) .950 .

N 114 114

Table .14.Least Square analyses of Frequencies of Statistic mean of HA and IgG of Kadaknath Chicken.

HA IgG

N Valid 114 114

Missing 0 0

Mean 8.3246 2.0439

Samples were taken three replicates.

83

Table 15. Least Square analysis of Frequency Table of Heamaglutination.

N

Frequency Percent Valid Percent Cumulative Percent

Valid 6.00 13 11.4 11.4 11.4

7.00 20 17.5 17.5 28.9

8.00 32 28.1 28.1 57.0

9.00 27 23.7 23.7 80.7

10.00 11 9.6 9.6 90.4

11.00 10 8.8 8.8 99.1

12.00 1 .9 .9 100.0

Total 114 100.0 100.0

Table .16. Least Square analysis of Frequency Table of IgG of kadaknath chicken.

N Frequency Percent Valid Percent Cumulative Percent

Valid 1.00 6 5.3 5.3 5.3

2.00 97 85.1 85.1 90.4

3.00 11 9.6 9.6 100.0

Total 114 100.0 100.0

84

Table .17. Least Square analysis of One way descriptive analysis of IgG of kadaknath Chicken.

Valid N Mean Std. Deviation Std. Error 95% Confidence Interval for Mean

Lower Bound

6.00 13 2.0769 .2774 7.692E-02 1.9093

7.00 20 2.0000 .3244 7.255E-02 1.8482

8.00 32 2.0313 .4004 7.077E-02 1.8869

9.00 27 2.0741 .5495 .1058 1.8567

10.00 11 2.0909 .3015 9.091E-02 1.8884

11.00 10 2.0000 .0000 .0000 2.0000

12.00 1 2.0000 . . .

Total 114 2.0439 .3854 3.609E-02 1.9724

(Note-samples have been taken four replicates.) Table .18. Least Square analysis of Case Processing Summary of Means of HA, IgG & POP. Of kadaknath chicken.

Cases

Included Excluded Total

N Percent N Percent N Percent HA * POP 114 100.0% 0 .0% 114 100.0% IgG * POP 114 100.0% 0 .0% 114 100.0%

85

Table .19.Least Square Mean and Std. Error report of HA and IgG of Kadaknath Chicken.

POP HA IgG

1.00 Mean 8.3246 2.0439

Std. Error of Mean .1356 3.609E-02

Total Mean 8.3246 2.0439

Std. Error of Mean .1356 3.609E-02

Samples were taken four replicates. Means in a column with different superscripts differ significantly

(P<0.05)

Table .20.Least Square of T-Test of One-Sample Statistics of HA and IgG of kadaknath chicken.

N Mean Std. Deviation Std. Error Mean

HA 114 8.3246 1.4482 .1356 IgG 114 2.0439 .3854 3.609E-02

Samples were taken three replicates. Table .21.Least Square analysis of One-Sample Test of mean difference of HA and IgG of kadaknath chicken.

Test Value = 0 t Df Sig. (2-

tailed) Mean

Difference 95%

Confidence Interval of the

Difference

Lower Upper HA 61.374 113 .000 8.3246 8.0558 8.5933 IgG 56.629 113 .000 2.0439 1.9724 2.1154

Sample were taken four replicates

86

Table .22.Least Square mean and Standard errors of one way Descriptive analysis of HA of Kadaknath chicken.

N Mean Std. Deviation

Std. Error 95% Confidence Interval for Mean

Minimum Maximum

Lower Bound

Upper Bound

1.00 6 8.3333 .8165 .3333 7.4765 9.1902 7.00 9.00 2.00 97 8.3196 1.5176 .1541 8.0137 8.6254 6.00 12.00 3.00 11 8.3636 1.1201 .3377 7.6112 9.1161 6.00 10.00 Total 114 8.3246 1.4482 .1356 8.0558 8.5933 6.00 12.00

Samples were taken three replicates. Table .23. Least Square Analysis of ANOVA of Sum Square of mean of HA of Kadaknath chicken.

Sum of Squares

Df Mean Square F Sig.

Between Groups

1.966E-02 2 9.828E-03 .005 .995

Within Groups 236.972 111 2.135

Total 236.991 113

Table .24. Least Square Analysis of ANOVA of mean square of IgG of Kadaknath chicken.

Sum of Squares

Df Mean Square F Sig.

Between Groups

.128 6 2.132E-02 .137 .991

Within Groups 16.653 107 .156

Total 16.781 113

Samples were taken four replicates

87

Table .25. Least Square analysis of Descriptive Statistics of NPar Tests body weight of KN Chicken.

Samples were taken four replicates.

Table.26. Least Square Statically Analysis of Chi-Square Test of Body Weight of KN Chicken.

BW4 BW6 BW8 BW10 BW12

Chi-Square 40.947 28.807 24.965 21.842 20.105

df 63 73 88 88 90

Asymp. Sig. .986 1.000 1.000 1.000 1.000

a 64 cells (100.0%) have expected frequencies less than 5. The minimum expected cell frequency is 1.8. b 74 cells (100.0%) have expected frequencies less than 5. The minimum expected cell frequency is 1.5. c 89 cells (100.0%) have expected frequencies less than 5. The minimum expected cell frequency is 1.3. d 91 cells (100.0%) have expected frequencies less than 5. The minimum expected cell frequency is 1.3.

N Mean Std. Deviation Minimum Maximum

BW4 114 162.9298 26.7333 86.00 233.00

BW6 114 175.7193 36.6404 71.00 299.00

BW8 114 340.8860 68.6741 190.00 570.00

BW10 114 391.7544 82.0032 204.00 659.00

BW12 114 500.0439 105.5695 249.00 803.00

88

Table.27.Least Square Analysis of Correlations of Body Weight of KN Chicken.

BW4 BW6 BW8 BW10 BW12

BW4 Pearson Correlation

1.000 .843 .738 .729 .563

Sig. (2-tailed)

. .000 .000 .000 .000

N 114 114 114 114 114

BW6 Pearson Correlation

.843 1.000 .713 .714 .558

Sig. (2-tailed)

.000 . .000 .000 .000

N 114 114 114 114 114

BW8 Pearson Correlation

.738 .713 1.000 .765 .651

Sig. (2-tailed)

.000 .000 . .000 .000

N 114 114 114 114 114

BW10 Pearson Correlation

.729 .714 .765 1.000 .796

Sig. (2-tailed)

.000 .000 .000 . .000

N 114 114 114 114 114

BW12 Pearson Correlation

.563 .558 .651 .796 1.000

Sig. (2-tailed)

.000 .000 .000 .000 .

N 114 114 114 114 114

* Correlation is significant at the 0.01 level (2-tailed). Table. 28. Descriptive Statistics Analysis of mean and Std. Error of Body Weight of KN Chicken.

N Mean

Statistic Statistic Std. Error

BW4 114 162.9298 2.5038

BW6 114 175.7193 3.4317

BW8 114 340.8860 6.4319

BW10 114 391.7544 7.6803

BW12 114 500.0439 9.8875

Samples were taken four replicates.

89

Table .29. Least Square analysis of Case Processing Summary of Body Weight of Kadaknath Chicken.

Cases

Included Excluded Total

N Percent N Percent N Percent

BW4 * POP 114 100.0% 0 .0% 114 100.0%

BW6 * POP 114 100.0% 0 .0% 114 100.0%

BW8 * POP 114 100.0% 0 .0% 114 100.0%

BW10 * POP

114 100.0% 0 .0% 114 100.0%

BW12 * POP

114 100.0% 0 .0% 114 100.0%

Table.30.Least square Analysis of Mean and Standard Error Report of kadaknath chicken.

POP BW4 BW6 BW8 BW10 BW12

1.00 Mean 162.9298 175.7193 340.8860 391.7544 500.0439

Std. Error of Mean

2.5038 3.4317 6.4319 7.6803 9.8875

Total Mea n

162.9298 175.7193 340.8860 391.7544 500.0439

Std. Error of Mean

2.5038 3.4317 6.4319 7.6803 9.8875

Samples have been taken three replicates.

90

Table .31.Least Square analysis of One-Sample Statistics of Mean and Standard .Deviation of kadaknath chicken.

N Mean Std. Deviation Std. Error Mean

BW4 114 162.9298 26.7333 2.5038

BW6 114 175.7193 36.6404 3.4317

BW8 114 340.8860 68.6741 6.4319

BW10 114 391.7544 82.0032 7.6803

BW12 114 500.0439 105.5695 9.8875

Samples have been taken three replicates. Table.32.Least Square Analysis of One-Sample Test of body Weight of KN Chicken.

Test Value = 0

t df Sig. (2-tailed)

Mean Difference

95% Confidence Interval of the

Difference

Lower Upper BW4 65.073 113 .000 162.9298 157.9693 167.8903

BW6 51.205 113 .000 175.7193 168.9205 182.5181

BW8 52.999 113 .000 340.8860 328.1432 353.6288

BW10 51.008 113 .000 391.7544 376.5383 406.9705

BW12 50.573 113 .000 500.0439 480.4550 519.6328

Samples were taken three replicates.

91

Table.33.Least Square Analysis of ANOVA for Sum Square and Mean Square. Sum of Squares df Mean

Square F Sig.

BW4 Between Groups

245274.431 63 10036.100 2.569 .001

Within Groups

412300.450 50 6052.009

Total 657574.876 113 BW6 Between

Groups 136043.351 63 2159.418 6.894 .000

Within Groups

15661.667 50 313.233

Total 151705.018 113 BW8 Between

Groups 437405.034 63 6942.937 3.634 .000

Within Groups

95518.483 50 1910.370

Total 532923.518 113 BW10 Between

Groups 599554.706 63 9516.741 2.968 .000

Within Groups

160316.417 50 3206.328

Total 759871.123 113 BW 12 Between

Group 947274.331 63 15036.100 2.409 .001

Within Groups

312100.450 50 6242.009

Total 1259374.781 113 Samples were taken four replicates.

92

6-DISCUSSION

___________________________________________________________________________

6.1. IMMUNOLOGICAL TRAITS

Antibody titers against SRBC and serum IgG level act as indicators for

humoral immune response, bacteriolytic activity of serum lysozyme

acts as indicator for non-specific immune response and CMI response

to PHA-P injection is a thymus cell dependent cutaneous basophil

hypersensitivity (CBH) in chickens (Corrier and Deloach, 1990) .

6.1.1. Antibody Response to Sheep RBC

Singh (2003) reported a titre of 7.4520.279 and 6.8700.254 in

black and white varieties of Turkeys on 5dpi.

Shivakumar (2003) reported a titre of 8.890.29 in the base

population of IWG of WLH chicken.

Sivaraman et al. (2005) observed a mean HA titre of 6.2890.246 in

SDL broiler chickens.

Singh (2005) estimated an average HA titre of 8.23±0.60 in IWG

genotype and 12.00±1.48 and 4.46±0.68 in high and low line,

respectively in IWG-WLH chicken. It reveals that third generation of

divergent lines differs significantly for humoral response to sheep

erythrocytes.

93

Kumar (2006) estimated the least squares mean of HA titre as

12.38±0.60 in Aseel native chicken. In males, the average was

12.80±0.74 whereas in case of females, it was 12.350.62.

Jaiswal et al. (2008) estimated the least squares mean of HA titre as

7.46±0.23 in Kadaknath native chicken. In male, the average was

7.49±0.31 whereas in case of female, it was 7.440.36.

Hatch effect was significant (P<0.05) on all the traits. Chicks in the

first hatch generally demonstrated highest magnitude of the traits

(P<0.05).

It has been reported that advanced genetic selection for economic

performance parameters reduced the immune status (Yegani, et al.,

2005).

Besides, genetic factors (Gyles et al., 1986, Saxena et al., 1997,

Sivaraman et al., 2003) other factors have also been reported to

influence the response to sheep RBCs. Higher dose elicited higher

response (Ubosi et al., 1985, Van der Zijpp, 1983a & Boa-Amponsem et

al., 2000); intravenous route elicited higher responses than other routes

(Boa-Amponsem et al., 2001).

94

In Kadaknath also the least squares mean indicated significant

(P<0.05) differences between all the three hatches, in which the first

hatch birds were having highest antibody titres (8.44±0.05) whereas the

second hatch had the lowest titres (6.61±0.39).

Van der Zijpp and Leenstra (1980) reported significant hatch

differences on HA titres at various dpis and attributed it to the difference

in the climatic conditions during different hatches.

The dietary feed intake also influenced the antibody titres (Siegel

et al., 1982) and genetic environmental interactions influencing antibody

responses in chickens (Gross et al., 1980), which might also contributed

in the present study as these factors may not be precisely same in all

the hatches.

Shivakumar (2003) also observed significant effect of hatch on

antibody response to SRBC.

Contrary to the present finding, non-significant effect of hatch was

noticed for antibody titres at 5 dpi in broiler chickens (Sivaraman et al.,

2003).

Singh (2003) also observed non-significant effect of hatch on

antibody response to SRBC in Black variety of turkeys but not in White

variety.

95

Singh (2005) also observed non-significant effect of hatch on

antibody response to SRBC in IWG-WLH chicken (Singh, 2005).

6.1.2. Serum IgG concentrations

Previous reports on IgG concentrations revealed 7.53±0.61 to

15.99±2.2 mg/ml IgG concentration in White leghorn chicken and

20.51±0.22 mg/ml in indigenous Aseel (Ahrestani et al., 1987).

The serum IgG concentrations in broiler & indigenous birds were

reported to be 8.01±0.4 and 10.01±0.4 mg/ml, respectively (Saxena,

1993). Another finding revealed IgG concentration of 13.5±0.68 mg/ml in

WLH (Rees & Norskog, 1981).

Singh (2003) estimated serum IgG concentrations of 0.672±0.105

mg/ml and 0.930±0.096 mg/ml in black and white varieties of Turkeys,

respectively.

Shivakumar (2003) estimated serum IgG concentrations of 5.65 ±

0.17 mg/ml and 5.24 ± 0.15 mg/ml in IWG and IWJ genotype of WLH

chicken, respectively.

Sivaraman (2004) estimated serum IgG concentrations of

7.361±0.53 mg/ml and 7.137±0.53 mg/ml in the high and low index line,

respectively in broilers.

96

Singh (2005) estimated serum IgG concentration of 55.59±7.37

mg/ml and an average of 61.73 ±15.48 mg/ml and 49.44±6.07 mg/ml in

high and low line of IWG-WLH chicken, respectively i.e. it remain

unaffected through divergent selection.

Kumar (2006) estimated serum IgG concentration of 69.87±6.57

mg/ml in Aseel native chickens. In males, the average was 74.09±8.13

mg/ml whereas in case of females, it was 65.64±7.05 mg/ml.

Jaiswal et al. (2008) estimated an average of serum IgG

concentration of 12.62±0.58 mg/ml in Kadaknath native chicken. In male,

the average was 13.35±0.75 μg/ml whereas in case of female, it was

11.89±0.89 μg/ml.

In Kadaknath also the least squares mean indicated significant

differences (P<0.01) among all the three hatches, in which the first hatch

birds were having highest values i.e. 10.69±0.41mg/ml) whereas the

third hatch had the values (8.98±0.32 mg/ml). There was not much

difference between the values of first and second hatch but the influence

of hatch on IgG values were statistically significant.

Van der Zijpp & Leenstra (1980) obtained significant hatch

differences for MES titres at 3,7,10 and 13 dpis.

Singh et al. (2003) also obtained non-significant hatch differences

in Black variety of Turkey but not in white variety of Turkey. Contrary to

97

the present findings, the IgG concentration of the broiler birds was not

significantly affected by hatches (Sivaraman et al. 2003).

Shivakumar (2003) observed non-significant effects of hatch on

MES antibody titres in both genotypes of WLH chicken.

Singh (2005) observed non-significant (P>0.05) hatch effect on

IgG concentrations in Aseel native chicken.

It was evident that IgG antibodies are significantly affected by non-

significantly factors like hatch, unlike IgM antibodies (Shivakumar, 2005).

6.2. General Performance of Kadaknath Chicken

A recent study of their growth potential recorded a daily weight gain of

6.2 grams from 0 to 20 weeks of age – based on growth in the breed’s

usual production environment, with very little supplemental feed. Similar

trend was observed by Farooq et al. (2001), who reported higher day-old

chick weight in RIR (35.3290.86 g), in comparison to Desi (33.8490.67

g) and Fayoumi chicken (30.7490.72 g.

6.2.1. Growth Traits

6.2.2. Body Weight

The hatch effects and breed-hatch interaction effects were significant.

(Williams et al., 2002). Yalcin et al. (2000)observed negative heterosis

for body weight in chicken while Nestor et al. (2004) reported negative

98

and significant heterosis for bodyweight at 8, 16 and 20 week of age in

male and female turkey.

6.2.3. Mean and Slandered Errors

Significant and positive effect of heterosis on body weight in broiler was

reported in literature (Iraqi et al., 2005). Analysis of slandered errors of

mean revealed highly significant differences for six body weight due to

breed, hatch hatch-breed interaction and sex. Similar results were

reported by Hoque et al. (1975). For instance, growth curves may enable

us to forecast the future growth of an animal and, thereby, select the

animals for breeding purposes when they are young (Akbag, 1995).

Also, with the help of growth curves, it is possible to revise growing

systems in effect at early stages (Ersoy et al,, 2006; 2007; Dincer et al.,

2007). It is concluded that using Gompertz and linear growth models

would be sufficient in modeling the growth, based on the 7 traits

determined. These findings are in agreement with the earlier literature

stating “growth curve models differ based on the trait under

investigation” (Ersoyet al., 2006; 2007; Dinver et al., 2007). At four

weeks of age the kadaknath indigenous chickens had the lowest and

highest mean body weight gain of all the strains with average daily

growth rate of 3.30 g and 4.20 g per bird per day in the starter growth

phase, respectively.

99

The result of this study is similar to the work reported by

Shanawany (1987) who stated that differences in hatching weight may

be attributed to differences in the age of the breeder flock, which have

been reported to affect the subsequent growth performance (North,

1984). It was also reported that at egg laying stage, the mean

bodyweight of Thai indigenous hens reared under intensive

management systems was 1.45 kg (Bansidhi et al., 1988;

Gongrattananun et al., 1992). These results also indicated the presence

of a substantial amount of variation in growth rate among and between

the indigenous chicken populations.

The mean body weights of Horro chicken were generally within the

ranges reported for unselected indigenous populations in north western

Ethiopia (Halima et al. 2007)and many other countries of Africa (Gueye

1998).

6.2.4. Phenotypic correlation among by body weight at various

stages

The mean body weights of Horro chicken were generally within the

ranges reported for unselected indigenous populations in north western

Ethiopia (Halima et al. 2007)and many other countries of Africa (Gueye

1998).

100

Phenotypic correlation among body weights at different ages ranged

from low to high .The keets weight at hatching is not supposed to bear

ay genetic relationship with subsequent body weights. Low phenotypic

correlation between 0 day body weight and body weight at later ages

indicates that Body weight at 0 day of ages is not suitable criterion for

selection for improving market weight. Body weights to 16 weeks of age

were used to characterize the growth of chicken in this study. Selection

for rapid early growth at a market age (40–50 days) has been the most

common approach in broiler chicken breeding programs (Emmerson

2003).

6.3. DNA Polymorphism in ChB6, Caspase-1, IAP-1, and ZOV3

genes.

6.3.1. PCR optimization

The reaction mixture and PCR programs for ChB6, Caspase-1,

IAP-1, and ZOV3 genes were optimized as per Zhou and Lamont (2003)

with slight modifications.

6.3.2. Optimized PCR reaction mixture

For ChB6, Caspase-1, IAP-1, and ZOV3 genes, the optimum

combination of various reaction components for each 25 µl PCR

reaction mix was 25 ng genomic DNA, 0.8 mM of each primer, 0.2 mM

each dNTPs and 1 U Taq DNA polymerase.

101

6.3.3. Optimization of PCR programme

To optimize the PCR conditions different annealing temperatures

were used. In case of all the four genes i.e., ChB6, Caspase-1, IAP-1,

and ZOV3 genes in Kadaknath breed of native chickens, initial

denaturation at 94˚C for 5 min, 35 cycles of (a) Denaturation at 96˚C for

1 min. in case of ChB6, Caspase-1, and ZOV3 whereas in case of IAP-

1, it was 94˚C, (b) Annealing at 53˚C, 65˚C, 62˚C, and 52˚C for ChB6,

Caspase-1, IAP-1, and ZOV3, respectively for 1 min., (c) Extension at

72˚C for 1 min. and final extension at 72˚C for 10 min. for ChB6,

Caspase-1, and ZOV3 whereas in case of IAP-1, it was 72˚C for 15 min.

produced distinct and robust amplified product.

The optimized PCR reaction mixture and PCR programmes gave

satisfactory amplification of all the genes in Kadaknath breed of native

chicken.

6.3.4. DNA Polymorphism at ChB6 gene

For ChB6 gene, a Previous reports revealed the presence of sites

in the 215 bp of ChB6 which generated the fragments of 147 and 68 bp

(LL Genotype) and 215 bp (FF Genotype) (Zhou and Lamont, 2003). In

present investigation revealed in the case of Kadaknath, only two

genotype i.e. AA and BB with fragment size of 147 and 215 bp

fragments, respectively could be observed.

102

A 215 bp fragment of ChB6 gene showed a C→A substitution at

base 470, which caused a predicted amino acid change from Gln

(Leghorn line) to Lys (Fayoumi line)(Zhou and Lamont, 2003).

6.3.5. DNA Polymorphism at Caspase-1 gene

For Caspase-1 gene, Previous reports revealed the presence of

Hsp 92 II RE sites in the 1070 bp of Caspase-1 which generated the

fragments of 421, 244, 227, 122, and 56 bp (LL Genotype) and 312,

244, 227, 122, 109, and 56 bp (FF Genotype) (Zhou and Lamont, 2003).

For the Caspase-1 gene, a 1,070-bp amplified fragment showed a

TC substitution at 368 bp between the Leghorn and Fayoumi lines. A

PCR-RFLP assay was developed with Hsp 92 II (Zhou and Lamont,

2003).

6.3.6 DNA Polymorphism at IAP-1 gene

For IAP-1 gene, Previous reports revealed the presence of Bgl I

RE sites in the 394 bp of IAP-1 gene which generated the fragments of

254 and 140 bp (LL Genotype) and 394 bp (FF Genotype) (Zhou and

Lamont, 2003).

A 394-bp fragment showed a TA substitution from the Leghorn

to the Fayoumi lines for the IAP-1 gene, and a PCR-RFLP assay was

103

developed to identify a Bgl I SNP to characterize the polymorphism at

Ala157 (Zhou and Lamont, 2003).

6.3.7. DNA Polymorphism at ZOV3 gene

For ZOV3 gene, Previous reports revealed the presence of SnaB I

RE sites in the 320 bp of ZOV3 which generated the fragments of 270

and 50 bp (LL Genotype) and 320 bp (FF Genotype) (Zhou and Lamont,

2003).

An aamplified 320-bp product of ZOV3 demonstrated a TG SNP,

which caused a predicted amino acid change from Cys157 in the

Leghorn line to Phe157 in the Fayoumi lines. The SnaB I RE produced

fragment sizes of 270 and 50 bp for the Leghorn lines, whereas the

Fayoumi lines had a 320-bp fragment. Because ZOV3 maps to the Z

chromosome, no heterozygous pattern was generated from the hens

(Zhou and Lamont, 2003).

104

7-SUMMERY & CONCLUSIONS

___________________________________________________________________________

Several elite layer line have been developed in our country which

have resulted into bringing the country 4th rank in the world. Still there

exists a gap between availability and requirements; hence further growth

in the egg production sector is very much needed. Besides feed, health

coverage takes the major chunk of the input cost. Owing to wide variety

of pathogens and the poultry rearing by diversified group across the

country, development of lines for general higher imuunocompetent

seems more feasible. Immune response to sheep erythrocytes (SRBC)

is one of such parameter which is used to develop general

immunocompetance lines. Periodical evaluation of these lines for

various immunological traits is an essential feature. Molecular analysis

of genes that have bearing on immune response would add the

knowledge of understanding the mechanism of disease resistance.

Indian breeds of chicken are well known for their resistance to diseases

and tropical adaptability.

In present investigation, was undertaken to estimate genetic and

phenotypic parameter of Kadaknath breed of native chicken were

evaluated for immunocompetence traits viz., antibody response to

SRBC, and serum IgG level, Body Weight and DNA polymorphism at

105

ChB6, Caspase-1, IAP-1, and ZOV3 genes along with associations

among immunocompetence traits. Growth is multi-factorial and

multidimensional phenomenon. Many genes and gene products play

key role in development and growth of an individual.

One hundred and fifteen birds of Kadaknath breed of native

chicken were used in the investigation for evaluation of

immunocompetence traits. Humoral immune response to SRBC was

measured through HA test. Immunization was done by injecting 1 ml of

1% SRBC intravenously. Sera samples were collected 5th day post

immunization. The antibodies were titrated against 1% SRBC

suspension. Single radial immunodiffusion assay was used to estimate

the total IgG in serum. Diameter of precipitation ring of samples was

compared with that of standard to determine the concentration of serum

IgG. The antibody titres against sheep RBCs were measured through

HA test on 5th dpi. HA titers ranged from 2-15 in Kadaknath .Average HA

titers were 7.69±0.13 in Kadaknath. Serum IgG is the most abundant

antibody and constitutes approximately 80% of the total

immunoglobulins. The bird’s ability to mount antibody responses to other

antigens is primarily revealed by serum IgG concentration. The average

serum IgG concentration was 10.07±0.20 mg/ml in Kadaknath breeds,

respectively.

106

Least-squares analysis of variance revealed significant difference

(p<0. 01) for general combining ability (GCA) in body weight at all age

groups. The gain in the body weight was highest during 8 to 12 weeks of

age.

On the basis of HA titer birds were grouped high and low titre

group. Polymorphism in these genes were analyzed by PCR-RFLP

technique using Pvu II, Hsp 92 II, Bgl I and SnaB I RE, for ChB6,

Caspase-1, IAP-1, and ZOV3 genes, respectively. On the basis of

presence and absence of restriction fragments, birds were screened for

their genotypes i.e. AA, BB, and AB. In Kadaknath, only two genotype

was present i.e. AA and BB. AA genotype had 147 bp fragments

whereas BB genotype had 215 bp fragments.

Overall, the present study demonstrated that divergent selection

based on response to SRBC gave direct response but did not influence

other immunological and early layer traits, hence HA titer may be

considered as selection criterion along with production traits for

improving production and protection status of Kadaknath native chicken.

Kadakanath demonstrated relatively higher humoral response to

Sheep erythrocytes than other chicken breeds. The differences

between HA titers in males and females were not significant. Older

bird’s higher response than younger group.

107

Serum lysozyme level was also relatively higher in Kadakanath. It

did not differ between sexes or among age groups.

Serum IgG level did not vary significantly (P>0.05) between sexes

or age groups; although older birds demonstrated higher values.

Overall, the present study demonstrated that Kadakanath breed of

native chicken have high immunocompetence status. Varied levels

of humoral immune response in Kadakanath can be exploited for

development of higher immune tolerant birds through selective

breeding.

The Kadaknath birds attain 1 kg body weight between 6 to 7

months of age and the birds reached around 1.5 kg by 1 year of

age.

The growth trends in both sexes showed linear increase in body

weights; however the rate of increase in body weights were higher

from 6 to 52 weeks of age in males as compared to females, thus

showing clear sex-dimorphism.

The differences in body weights of male and females were

significant (P>0.05).

BB genotype of ChB6 had high frequency than the heterozygotes

in Kadaknath. Heterozygotes were more in Kadaknath for

Caspase-1 and IAP-1 gene. Heterozygotes were equal to BB in

Kadaknath for ZOV3 gene.

108

The unique characteristics of native chicken of various countries

are well established by research findings. Hence it is essential to

conserve the precious genetic resources and every effort from the

government and the public needs to be taken to conserve them for

the present and future. On the other hand, newer genomic tools

could be applied to utilize the potential of native chickens for the

betterment of mankind.

109

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