mubarak abdelgabar abdalhameed haron
TRANSCRIPT
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Effects of Feeding Genetically Modified Cotton Seed Cake on
Physiological Aspects and Meat Quality of the Rabbits
(Oryctolagus cuniculus)
Mubarak Abdelgabar Abdalhameed Haron
B.Sc. in Science and Education, Faculty of Education,
University of Zalingei (2004)
Postgraduate Diploma in Biosciences and Biotechnology
University of Gezira (2008)
M.Sc. in Biotechnology, Faculty of Engineering and Technology
University of Gezira (2010)
A Thesis
Submitted to the University of Gezira in Fulfillment of the Requirements for
the Award of Doctor of Philosophy Degree
in
Biosciences and Biotechnology (Biosafety)
Center of Biosciences and Biotechnology
Faculty of Engineering and Technology
May 2016
2
Effects of Feeding Genetically Modified Cotton Seed Cake on
Physiological Aspects and Meat Quality of the Rabbits
(Oryctolagus cuniculus)
Mubarak Abdelgabar Abdalhameed Haron
Supervision Committee
Name position signature
Dr. Mutaman Ali A.Kehail Main Supervisor ……........……
Prof. Elnour Elamin Abdelrahman Co-Supervisor ....……..…….
Date: May 2016
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Effects of Feeding Genetically Modified Cotton Seed Cake on
Physiological Aspects and Meat Quality of the Rabbits
(Oryctolagus cuniculus)
Mubarak Abdelgabar Abdalhameed Haron
Examination Committee
Name position signature
Dr. Mutaman Ali A. Kehail Chairman ………….……
Prof. Mohammed A. A. Ahmed External Examiner ……………….
Prof. Mohamed Yousif Elbeeli Internal Examiner …………….…
Date of Examination: 16/ 5 /2016
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DEDICATION
To the Sudanese people and Humanity with gratitude, I dedicate this
work.
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ACKNOWLEDGEMENTS
Firstly, I am grateful to my God who enabled me to complete this
work. With great respect and gratitude, I thank the Management of the
University of Zalingei, the University of Geneina and the Ministry of High
Education and Scientific Research for the opportunity of the study, I am
deeply grateful to my supervisors Dr. Mutaman Ali A. Kehail and professor
Elnour Elamin Abdelrahman for their boundless help and guidance
throughout the study. Also with the gratitude I thank my family for the
unlimited aids and support throughout the study, I deeply thanks my aunt in
low Kareema Abdullah and my brother in low Maweia Mohamed
Abdurahman for his special aid and support.
I also thank the family of the faculty of Engineering and Technology,
with great thanks to staff in the laboratory especially to the technician
Ansari, Osama and Mubarak for their help and guidance throughout the tests.
I am thankful to the family of the Quality Medical Laboratory in Wad
Medani, especially the technician Nabeel Elnor Mohamed and the nurse
Khalid Abdalnor for their great help in the blood sampling and analysis.
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Effects of Feeding Genetically Modified Cotton Seed Cake on Physiological Aspects and
Meat Quality of the Rabbits (Oryctolagus cuniculus)
Mubarak Abdelgabar Abdalhameed Haron
Ph.D. in Biosciences and Biotechnology (Biosafety), May 2016
Center of Biosciences and Biotechnology
Abstract
A genetically modified organisms is that organisms have had DNA altered through
genetic engineering techniques. Bt cotton or genetically modified cotton also had altered their
DNA by inserting gene coding for Bacillus thuringiensis (Bt) toxin to reduce the heavy
reliance on pesticides. This study was conducted in Gezira State for evaluation the effects of
genetically modified cotton seed on the physiological activity of parent and individuals of the
first generation rabbit (Oryctolagus cuniculus) fed on its cake. The rabbits and the
genetically modified cottonseed (GMCS) and control cottonseed (C) were obtained from Wad
Medani markets. The rabbits were divided into three groups and fed on GMCS cake, C cake
and dried bread. At the end of experimental period (90 days), blood samples were taken to be
analyzed for whole blood counts (white cells, red cells, platelets, neutrophils and
lymphocytes) and for renal (creatinine, urea, Na+ and K+) and liver functions (albumin
number, alkalin phosphate (ALP), alanine aminotrans (ALT), aspartate aminotrans (AST),
AST\ALT ratio and total bilirubin), then the rabbits were dissected to monitor the condition of
the internal organs, also a proximate analysis for meat samples (from muscles). The obtained
data were statistically analyzed. It was found that, the GMCS cake decreases the red blood
cells (RBCs) (from 5.04 in control to 4.58 x106\µL) and body weight (from a mean of 1645 in
control to 1440 g), and also decreases the white blood cells (WBCs) (from 8.63 in control to
5.30 x103\µL). The GMCS cake do not had adversely affects on the liver function indices (f=
0.95; f-crit=5.95), the nutritional content of meats (fat, protein, fiber and carbohydrates:
f=0.006; f-crit= 4.46), blood clotting indices (f= 1.03; f-crit=10.13), renal function (f= 0.51; f-
crit=161.45). In the first generation, GMCS cake decreases the WBCs indices, renal function,
lipid profile and mineral content of meats, and increases the RBCs counts and blood clotting
and protein and fat contents of meat. Although that, there were no significant difference
―statistically‖ but ―physiologically‖, there was clear differences between GMCS cake fed
rabbit and limits of healthy and control rabbits. The study recommends running
comprehensive studies on the physiological effect of GMCS on other large mammals and their
subsequent generations.
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1026 يا )انسلايت انحت(انعهو انخمت انبنجت انفهسفت ف دكخزا
يسكص انعهو انخمت انبنجت
ملخص الدراسة
فا خلال اندست انزاثت. ب ح لط ا DNAنت زاثا حهك انكائاث انخ حز انـ انكائاث انعد
ف بإدخال ج يشفس بسى بكخسا انباسهس سزجسس نخمهم الإعخاد DNAانمط انحز زاثا اضا حز انـ
انشاط انفسنج مط انحز زاثا عهجسج ر اندزاست بلات انجصسة نخمى اثس برز اناعه انبداث.
حث حى انحصل عه الأزاب انمط انحز زاثا انمط انغرت برنك انكك. لأزابن افساد انجم الأل ءنلأبا
بانمط انحز زاثا رجغالأزاب إن ثلاثت يجعاث حى حمسى .ق يدت ديداساغس انحز زاثا ي
و( أخرث عاث ي دو الازاب 90انمط غس انحز زاثا انسغف انجاف، عد ات فخسة انخجسبت )
، انصفائح انديت، انحسفم، انهف(ندو انبضاء، خلاا اندو انحساءي حث اندو انكايم )خلاا اخحهها ن
و( نهكبد )الأنبي، الأنكان فسفاث، الا أيحساس، نهكه )انكساح، انزا، انبحاسو، انصد
سحج ثى شالإسبازحج أيحساس، يعدل الإسبازحج أيحساس إن الا أيحساس، انبهسب انكايم(
ة ي انعضلاث(. ذجس انخحهم انخمسب نعاث ي انهحو )يأخحانت الأعضاء انداخهت كرنك ا مصنخالازاب
ف 4.05)ي حههج انبااث إحصائا. جدث اندزاست أ ككت برز انمط انحز زاثا لههج كساث اندو انحساء
10×5.48إن عت انشادةان6µL)
جساو( اضا لههج 2550إن عت انشادةانف 2654انجسى )ي يخسظ ش
10×4.80إن عت انشادةان ف 8.68ي كساث اندو انبضاء )ي 3µLن (. كك برز انمط انحز زاثا نس
( انكاث انغرائت نهحو )اند، انبسح، الأناف، f= 0.95; f-crit=5.95عه ظائف انكبد )ت حأثساث يع
عاصس ظائف (f= 1.03; f-crit=10.13)( عاصس حجهظ اندو f=0.006; f-crit= 4.46انكسبدزاث:
. ف انجم الأل لهم كك برز انمط انحز زاثا ي كساث اندو انبضاء f= 0.51; f-crit=161.45)انكه )
يحخ حجهظ اندو عاصس حخ انعاد ف انهحو شاد كساث اندو انحساء يظائف انكه ناحك اند
حجد فسق اضحت ب الازاب فسق يعت إحصائا نك فسنجا حجد لا ح اند ف انهحو. أضاانبس
. حص اندزاست بإجساء دزاساث يكثفت عه انخاثس عت انشادةان انخغرت بككت برز انمط انحز زاثا
انفسنج نكك برز انمط انحز زاثا عه انثداث انكبس أجانا انخلاحمت.
8
List of Contents
Item page No.
Supervision Committee ……………………………………………….…… i
Examination Committee …………………………………..………………. ii
Dedication ……………………………………………………...…………. iii
Acknowledgment …………………………………………………..……… iv
English Abstract ………………………………………………...………….. v
Arabic Abstract ………………………………………………….………… vi
List of Contents ………………………………………...………………… vii
List of Tables …………………………………………......………………. xii
List of Appendices ...................................................................................... xiv
1. CHAPTER ONE; INTRODUCTION .............................……………………. 1
2. CHAPTER TWO; LITERATURE REVIEW ..............…………………… 3
2.1- Rabbits ……………………………………………………………..……………….. 3
2.1.1- Rabbit Habitat and Range ………..………….….……………..………………….. 3
2.1.2- Biology ………………………………………………………………............……. 3
2.1.3- Morphology ……………............……………………………….….……………… 4
2.1.4- Ecology ……………………...........……………………………….……………… 4
2.1.5- Digestive System (Gastrointestinal System) ……………...............….……..……. 5
2.1.6- Diet and Eating Habits …………………………………...……………………… 10
2.1.7- Rabbit Diseases ………...…...………………………………..…………………. 12
2.1.8- Rabbit Meat ……………………………………………...……...……………….. 13
9
2.1.9- Environmental Problems ..…………………………………….............…………. 13
2.1.10- Reproductive Traits ...…………………………………...............……………… 14
2.1.11- Clinical Pathology ...…………………………………………................………. 15
2.2- Cotton ...……………………………………………………………………………. 22
2.2.1- Types of Cotton …………………………………………….……………………. 22
2.2.2- Genetically Modified Cotton (GMC) ………………...………………………….. 23
2.2.3- GM Cotton in Sudan ...………………………………………………………….. 25
2.2.4- Cotton Genome ......……………………………………………………………… 26
2.2.5- Cotton Seed ...………...………………………………………………………….. 27
2.2.6- Uses of Cotton Seeds .........…………………………………………………….... 27
2.3- The Impacts of GM Crops ...…………………...………………………………….. 30
2.3.1- Environmental Impacts .......……………………………………………….…….. 30
2.3.2- Health Impacts ....………...……………………………….…….……………….. 32
2.4- The Effects of GM Cotton on other Living Organisms ....…………………..…….. 33
2.4.1- Effects of Bt Toxin on Various Tissues and Organs of Animals………………... 34
2.4.2- Effects of Bt Toxin on Lactating Animals ...…………………………………….. 34
2.4.3- Bt Toxins in Animal Excretion ...………...…………………………..…………. 35
2.4.4- Influence of Bt Cotton on Other Non-target Animals …………………………… 35
2.4.5- Effects of Bt Cotton on Human Health .........………………………...………….. 36
2.5- Genetically Modified Food Controversies ...…………..…………………….…….. 37
2.7- Biosafety ...…………………………………………………………..…………….. 38
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2.7.1- Toxicity, Allergenicity and Nutritional Assessment ……..….……………..……. 38
2.7.2- Nutritional Assessment of GM Feed …..…...…………………………….……… 39
2.8- Regulation of GM Crops ....………………………………………………..………. 39
2.9- Biosafety Regulations in Sudan ....……………..………………………………….. 40
3- CHAPTER THREE; MATERIALS AND METHODS ..……………… 41
3.1- The Experimental Animals ………...……………………………………………… 41
3.2- The Experimental Diets and Procedures ……………………..……………………. 42
3.3- Analysis of Nutritional Content for Cottonseeds (CS) ……...…………………….. 42
3.3.1- The phytochemical Screening of Cottonseed ……………………….…………… 42
3.3.2- Approximate Analysis of Cottonseed …………………………………………… 44
3.3.3- Determining of Minerals ………………………………………………………… 46
3.4- Body Weight Gain and Internal Organ Weight of Experimental Rabbits ……...…. 46
3.5- Sampling and Analysis of Rabbits Blood ………………………………….……… 47
3.5.1- Sampling Blood for Hematological and Biochemical Analysis …………..…..… 47
3.5.2- Whole Blood (WB) Mode (Hematological Analysis) ……..…………………..... 47
3.5.3- Blood Serum Analysis (Biochemical Analysis) ………..………………….…….. 47
3.5.3.1- Renal Function Test …..…...……………………………………….………….. 47
3.5.3.2- Liver Function Test …..……………………...………………………………… 48
3.5.3.3- Lipid Profile Test ...………..………………………………………….……….. 50
3.6- Dissection of the Rabbits and Weighing the Internal Organs …...……………..…. 50
3.7- Analysis of The Meat Nutrient Content ………………………………………...…. 50
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3.8- Data Analysis (Statistical Analysis) ……………………………………………….. 50
4- CHAPTER FOUR; RESULTS AND DISCUSSION ..…………………. 51
4.1- Phytochemical Characteristics of the Cotton Seed …...…...………………………. 51
4.2- The approximate Composition of GM and Non-GM Cotton Seeds ….………….... 53
4.3- The Mineral Content of Cotton Seeds ………...……………..……………………. 55
4.4- The Effects of GM and Non-GM Cotton Seed Cake on White Blood Cells (WBC)
Indices ……………………………………………………………………………….…. 57
4.5- The Effects of GM Cotton Seed Cake and Non-GM Cotton Seed Cake on Red Blood
Cells (RBCs) Indices …………………………………………………………………… 59
4.6- The Effects of GMCSC and Non-GMCSC on Blood Clotting ……………………. 61
4.7- The Effects of GMCSC and Non-GMCSC on Liver Functions …...…………….... 63
4.8- The Effect of GM cotton Seed on Renal Functions …….…………………...…….. 65
4.9- The Effect of GM cotton Seed on Lipid Profile ………..…………………..……… 67
4.10- The Effects of Cotton Seed Cake on Body Weight Gain …...….………………… 69
4.11- The Effect of Cotton Seed Cake on Internal Organ of Rabbits …….………….... 71
4.12- The Effect of GM Cotton Seed Cake on Approximate Composition of Meat
Nutrient Content ……………………………………..……………………………….… 73
4.13- The Effect of GMCSC on Mineral Content of The Rabbit Meat ………………… 75
4.14- The Effects of GMCSC on WBCs Indices of 1st Generation ………...………...… 77
4.15- The Effects of GMCSC on RBCs Indices of The 1st Generation …...………….… 79
4.16- The Effects of GMCSC on Blood Clotting Indices of the 1st Generation ...…...…. 81
4.17- The Effects of GMCSC on Liver Functions of 1st Generation .............................. 83
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4.18- The Effect of GMCSC on Renal Functions Indices of the 1st Generation …......… 85
4.19- The Effect of GMCSC on The Lipid Profile of the 1st Generation .....................… 87
4.20- The Effects of GMCSC on Body Weight of the 1st Generation ..........................… 89
4.21- The Effect of GMCSC on Internal Organ of The 1st Generation ............................ 91
4.22- The effect of GMCSC on Approximate Compositions of Meat Nutrient of The 1st
Generation …...............................................................................................................…. 93
4.23- The Effect of GMCSC on Meat Mineral Content of The 1st Generation ...........…. 95
5- CHAPTER FIVE; CONCLUSION AND RECOMMENDATION ... 97
5.1- Conclusions……………………………………...................................……………. 97
5.2- Recommendations …………..……………….............…………………………….. 97
References …...………………………...........…………………………………. 98
Appendices …….…………………………………..........……………………. 114
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List of Tables
Table page No.
4.1: The Phytochemical Characteristic of GM cotton and Non-GM cotton seeds …....... 52
4.2: The Approximate Composition of GM cottonseed and Non-GM cottonseed .......... 54
4.3: The Mineral Content percentage of GM cottonseed and Non-GM cottonseed ......... 56
4.4: The Effects of GM and Non-GM Cottonseed Cake on White Blood Cells (WBC)
Indices of the Rabbits After 90 Days of Feeding .........................................................… 58
4.5: The Effects of GM Cottonseed Cake and Non-GM cottonseed cake on Red Blood
Cells (RBCs) indices of the Rabbits After 90 Days of Feeding ....................................... 60
4.6: The Effects of GMCSC and Non-GMCSC on Blood Clotting Indices of the Rabbits
After 90 days of feeding .......................................................................................……… 62
4.7: The Effects of GMCSC and Non-GMCSC on Liver Functions Indices of the Rabbits
After 90 days of Feeding ........................................................................................……. 64
4.8: The Effect of GM Cottonseed Cake on Renal Functions Indices of the Rabbits After
90 days of feeding ........................................................................................................… 66
4.9: The Effect of GM Cottonseed Cake on Lipid Profile of the Rabbits After 90 Days of
Feeding ............................................................................................……………………. 68
4.10: The Effects of GM Cottonseed Cake on Body Weight Gain of the Rabbits After 90
days of Feeding ................................................................................................………… 70
4.11: The Relative Weight (%) of Internal Organs of the Rabbits After 90 days of
Feeding GMCSC and Non-GMCSC ............................................................................… 72
4.12: The proximate Composition (%) of Meat Nutrient Content of the Rabbits After 90
days of Feeding GMCSC and Non-GMCSC …..........................................................… 74
14
4.13: The Mineral Content % of The Rabbit Meat After 90 Days of Feeding GMCSC and
Non-GMCSC ................................................................................................................… 76
4.14: The Effects of Feeding GMCSC on WBCs Indices of Young Rabbits of 1st
Generation After 6 Weeks ............................................................................................… 78
4.15: The RBCs Indices of the Young Rabbits of The 1st Generation Fed on GMCSC for
6 Weeks …...............................................................................................................……. 80
4.16: The Blood Clotting Indices of the 1st Generation Fed on GMCSC for 6 Weeks..... 82
4.17: The Liver Functions Indices of the Young Rabbits of the 1st Generation Fed on
GMCSC for 6Weeks ....................................................................................................… 84
4.18: The Renal Function Indices of the Young Rabbits of the 1st Generation Fed on
GMCSC for 6 Weeks ...................................................................................................… 86
4.19: The Effect of GMCSC on Lipid Profile of the 1st Generation After 6 Weeks of
Feeding .........................................................................................................................… 88
4.20: Mean Body Weight (g) Gain of the 1st Generation Fed on GMCSC for 6 Weeks.. 90
4.21: The Relative Weight (%) of Internal Organ of the Young Rabbits of The 1st
Generation Fed on GMCSC for 6 Weeks .....................................................................… 92
4.22: Proximate Composition of Meat Nutrient Content of the Young Rabbits of The 1st
Generation Fed on GMCSC for 6 Weeks ......................................................................... 94
4.23: The Meat Mineral Content of the Young Rabbits of The 1st Generation Fed on
GMCSC for 6 Weeks ...................................................................................................… 96
15
List of Appendices
Appendix page No.
1- Rearing of the Rabbits ................................................................................................ 114
2- Rabbits Feeding on GM and Non-GM Cotton Seed Cake ......................................... 115
3- Rearing of the Young Rabbits of the First Generation .............................................. 116
4- The Internal Organs of the Dissected Rabbit ............................................................. 117
5- Reserve the Internal Organs of the Rabbits in Special Container ............................. 118
6- Determining the Mineral Content by Flame Photometer ........................................... 119
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CHAPTER ONE
INTRODUCTION
Genetically modified (GM) cotton was developed to reduce the heavy reliance on
pesticides. The bacterium Bacillus thuringiensis (Bt) naturally produces a chemical
harmful only to a small fraction of insects, most notably the larvae of moths and
butterflies, beetles, and flies, and harmless to other forms of life (Rose et al., 2007).
The gene coding for Bt toxin has been inserted into cotton, causing cotton,
called Bt cotton, to produce this natural insecticide in its tissues. In many regions, the
main pests in commercial cotton are lepidopteran larvae, which are killed by the Bt
protein in the transgenic cotton they eat. This eliminates the need to use large amounts of
broad-spectrum insecticides to kill lepidopteran pests (some of which have
developed pyrethroid resistance). This spares natural insect predators in the farm ecology
and further contributes to non insecticide pest management.
Bacillus thuringiensis (Bt) cotton is commonly grown in all over the world to
control wide range of pests. Bt cotton have several advantages over conventional
chemical fertilizers and biological control methods as it provide safe, quick, efficient and
long term resistance against diverse range of cotton insects. With the passage of time
several technical, socio-economical, ethical and biosafety issues arises with use of Bt
cotton. As Bt cotton adversely affects a variety of non targeted organisms including many
beneficial animals. Several researchers have reported that Bt toxins affect several
different species of animals such as cows, buffaloes, model mice, goats, pigs, chickens,
herbivores and human (Zia et al., 2015).
In Sudan Two Chinese Bt-cotton genotypes (G. hirsutum) carrying Cry 1A gene;
from Bacillus thuringiensis (Bt) a hybrid CN-C01 and an open pollinated CN-C02 were
approved for commercial production by Chinese National Authority in 2004 and 2008,
respectively. Animal feeding on crop residues and counts of major soil microorganism in
crop rizosphere were performed to assess the effect of the Bt gene on non-target
organisms. The Bt one tremendously out-yielded the non-Bt varieties with a difference of
17
over 5-6 times in sites with high bollworms infestation and overall increase of 129-166%
over the two check varieties in the combined field trails (El Wakeel, 2014).
The National Variety Release Committee approved the release of the two Bt
cotton genotypes; the hybrid CN-C01 and the open pollinated CN-C02 in the irrigated
and rain-fed production areas in Sudan for commercial production (El Wakeel, 2014).
Areas Planted with Bt cotton in Sudan in (2012) was 53,220 ha. Initially the
cultivation failed in Gezira and New Halfa regions due to water logging as a result of bad
land preparation. Controversy and Debate over Bt Cotton release in Sudan a raised. The
common debate issues on GMOs. A National Council for Biosafety was established later
after the release. The duration of confined greenhouse testing was inadequate. In most
cases, stakeholders (parliamentarians, civil society, farmers, …etc) are not really well
informed on biotechnology issues. Animal feeding testing was inadequate. The feed test
was for cotton foliage and not for seed cake. No cotton seed oil analyses. Time from
initial testing to release into the environment was very short (El Wakeel, 2014).
As over 80 % of the global cotton production is Bt cotton, we are certain to come
into daily contact with it, and primarily via our clothing. Only certified organic cotton is
free of Bt cotton. The evidences clearly reveal that acreage and popularity of Bt cotton is
increasing day by day as it plays a vital role to provide durable resistance against a wide
range of insect species. Bt cotton has played important role to sustain agriculture in all
over the world for their maximum yield and other agronomic practices as well. With the
passage of time, several biosafety and environmental issues arise with the use of different
Bt genes. Several researchers have reported the toxic effects of Bt proteins of cotton and
other crops on diverse range of non-target animal species including human being. Now it
is the responsibilities of the scientists to bring awareness in people to develop new Bt
cotton cultivars that assure no or very low toxicity on non-target organisms to minimize
risks associated with Bt cotton technology (Zia et al.,2015).
Research Problems:
The intensive production of GM cotton in Sudan will increase GM cotton seeds which
were used as diets for many livestock's animals including dairy and meat animals such as
sheep, cattle ,goats, beef and rabbits. Which might result in many biosafety issues.
Research Objectives:
18
To evaluate the effect of genetically modified (GM) cottonseed cake (as diets) on
rabbits physiological aspect and meat quality.
19
CHAPTER TWO
LITERATURE REVIEW
2.1. Rabbits
Rabbits are small mammals in the family Leporidae of the order Lagomorpha,
found in several parts of the world. There are eight different genera in the family
classified as rabbits, including the European rabbit (Oryctolagus cuniculus), cottontail
rabbits (genus Sylvilagus; 13 species), and the Amami rabbit (Pentalagus furnessi, an
endangered species on Amami Ōshima, Japan). There are many other species of rabbit,
and these, along with pikas and hares, make up the order Lagomorpha. The male is called
a buck and the female is a doe; a young rabbit is a kitten or kit (Wikipedia, 2015a).
2.1.1. Habitat and range
Rabbit habitats include meadows, woods, forests, grasslands, deserts and
wetlands. Rabbits live in groups, and the best known species, the European rabbit, lives in
underground burrows, or rabbit holes. A group of burrows is called a warren. More than
half the world's rabbit population resides in North America (Animal Habitats, 2009).
They are also native to southwestern Europe, Southeast Asia, Sumatra, some islands of
Japan, and in parts of Africa and South America. They are not naturally found in most of
Eurasia, where a number of species of hares are present. Rabbits first entered South
America relatively recently, as part of the Great American Interchange. Much of the
continent has just one species of rabbit, the tapeti, while most of South America's
southern cone is without rabbits. The European rabbit has been introduced to many places
around the world (Encyclopædia Britannica, 2007).
2.1.2. Biology
Because the rabbit's epiglottis is engaged over the soft palate except when
swallowing, the rabbit is an obligate nasal breather. Rabbits have two sets of incisor teeth,
one behind the other. This way they can be distinguished from rodents, with which they
are often confused (Brown, 2001). Carl Linnaeus originally grouped rabbits and rodents
under the class Glires; later, they were separated as the scientific consensus is that many
of their similarities were a result of convergent evolution. However, recent DNA analysis
and the discovery of a common ancestor has supported the view that they share a
20
common lineage, and thus rabbits and rodents are now often referred to together as
members of the super order Glires (Katherine and James, 2011).
2.1.3. Morphology
The rabbit's long ears, which can be more than 10 cm (4 in) long, are probably an
adaptation for detecting predators. They have large, powerful hind legs. The two front
paws have 5 toes, the extra called the dewclaw. The hind feet have 4 toes (Rabbits, 2010).
They are plantigrade animals while at rest; however, they move around on their toes
while running, assuming a more digitigrades form. Unlike some other paw structures of
quadruped mammals, especially those of domesticated pets, rabbit paws lack pads. Their
nails are strong and are used for digging; along with their teeth, they are also used for
defense (Wikipedia, 2015a).
Wild rabbits do not differ much in their body proportions or stance, with full, egg-
shaped bodies. Their size can range anywhere from 20 cm (8 in) in length and 0.4 kg in
weight to 50 cm (20 in) and more than 2 kg. The fur is most commonly long and soft,
with colors such as shades of brown, gray, and buff. The tail is a little plume of brownish
fur (white on top for cottontails) (Encyclopædia Britannica, 2007). Rabbits can see nearly
360 degrees, with a small blind spot at the bridge of the nose (Wikipedia, 2015a).
2.1.4. Ecology
Rabbits are hindgut digesters. This means that most of their digestion takes place
in their large intestine and cecum. In rabbits, the cecum is about 10 times bigger than the
stomach and it along with the large intestine makes up roughly 40% of the rabbit's
digestive tract (Wikipedia, 2015a). The unique musculature of the cecum allows the
intestinal tract of the rabbit to separate fibrous material from more digestible material; the
fibrous material is passed as feces, while the more nutritious material is encased in a
mucous lining as a cecotrope. Cecotropes, sometimes called "night feces", are high in
minerals, vitamins and proteins that are necessary to the rabbit's health. Rabbits eat these
to meet their nutritional requirements; the mucous coating allows the nutrients to pass
through the acidic stomach for digestion in the intestines. This process allows rabbits to
extract the necessary nutrients from their food (Navarre's, 1999).
Rabbits are prey animals and are therefore constantly aware of their surroundings.
For instances, in Mediterranean Europe, rabbits are the main prey of red foxes, badgers,
21
and Iberian lynxes (Fedriani et al., 1999). If confronted by a potential threat, a rabbit may
freeze and observe then warn others in the warren with powerful thumps on the ground.
Rabbits have a remarkably wide field of vision, and a good deal of it is devoted to
overhead scanning (Tynes, 2010). They survive predation by burrowing, hopping away in
a zig-zag motion, and, if captured, delivering powerful kicks with their hind legs. Their
strong teeth allow them to eat and to bite in order to escape a struggle (Davis and
DeMello, 2003). The expected wild rabbit lifespan is about 3 years (Wikipedia, 2015a).
2.1.5. Digestive system (Gastrointestinal System)
Rabbits are true non-ruminant herbivores. Their digestive reservoir permits and
increases the efficiency of utilization of fibrous diets. They have a large stomach and
well-developed cecum relative to other non-ruminant herbivores such as the horse (Cathy,
2006).
2.1.5.1. Stomach
The stomach of the rabbit holds approximately 15% of the volume of the entire
gastrointestinal tract (Harcourt-Brown, 2002). It is thinwalled, J-shaped, and lies to the
left of the midline. The well-developed cardiac sphincter is lined with non glandular
stratified squamous epithelium and prevents vomiting. The fundus contains parietal cells
that secrete acid and intrinsic factor as well as chief cells that secrete pepsinogen. The
pylorus has a well-developed, muscled sphincter. The adult rabbit stomach has a pH of 1–
2. The rabbit feeds frequently–up to 30 times per day of 2–8 g of food over 4-6 minute
periods. The stomach normally contains a mixture of food, hair, and fluid even after 24
hours of fasting (O‘Malley, 2005). The stomach pH of rabbits up until the time of
weaning falls into the range of 5.0–6.5.
Bacteria is kept in check during the first 3 weeks of life by the production of milk
oil containing octanoic and decanoic fatty acids produced by the enzymatic reaction of
the suckling rabbit‘s own digestive enzymes on the doe‘s milk. Young rabbits acquire gut
flora by consumption of the doe‘s cecotrophs beginning at 2 weeks of age. Milk oil
production ceases at 4–6 weeks of age. By this time, some ingested organisms have
colonized the cecum and hindgut fermentation can begin as the bunny weans (O‘Malley,
2005). Gastric transit time is approximately 3–6 hours. The bulk in the stomach effects
intestinal passage of digesta. The high voluntary feed intake (VFI) is at least 4 times
22
higher pro rata than a 250-kg steer. It is also associated with a low gut retention time of
17.1 hours in the rabbit compared with 68.8 hours in the bovine. High VFI together with
re-utilization of gut content by reingestion of cecal material supports the rabbit‘s high
nutrient requirement per unit of body weight and improves feed utilization for the rabbit
(Lowe et al, 2000). The bovine‘s main volatile fatty acid (VFA) produced by rumen
fermentation is propionic acid while the rabbit‘s main VFA is acetic acid with cecal
fermentation. The primary microflora of the rabbit is Bacteroides species while
Lactobacillus species is the primary microflora of the bovid (O‘Malley, 2005).
2.1.5.2. Small intestine
The small intestine is approximately 12% of the gastrointestinal volume in the
rabbit. The bile duct enters into the proximal duodenum. The right lobe of the pancreas is
situated in the mesoduodenum of the duodenal loop. The left lobe lies between the
stomach and transverse colon. There is a single pancreatic duct that opens at the junction
of the transverse and ascending loops of the duodenum. The duct drains both pancreatic
lobes. Technically this is the accessory pancreatic duct as the main pancreatic duct
connection to the duodenum disappears during embryonic development.1 The jejunum is
the longest section of small bowel and appears convoluted. Aggregates of lymphoid tissue
(Peyers patches) are present in the lamina propria with increasing prominence distally.
The distal end of the ileum has a spherical thick-walled enlargement known as the
sacculus rotundus. This marks the junction between the ileum, cecum, and colon. The
sacculus rotundus is often called the ―cecal tonsil‖ because of its lymphoid tissue and
macrophage composition. This organ is unique to rabbits. An ileocolic valve controls
movement of ingesta from the ileum into the sacculus and prevents reverse movement of
ingesta back up into the ileum. The ileocolic valve opens into the ampulla coli at the
junction of the ileum, colon, and cecum. There is a weak ileocecal valve that allows
chyme to pass into the cecum (O‘Malley, 2005).
Gastrointestinal smooth muscle is stimulated by motilin, a polypeptide hormone
that is secreted by enterochromaffin cells of the duodenum and jejunum. Motilin is
released in response to fat while carbohydrates inhibit release. Motilin activity is not
present in the cecum, but is present and stimulates smooth muscle in the colon and rectum
(Harcourt-Brown, 2002).
23
The stomach and small intestine in the rabbit function similarly to other
monogastric animals. Cecotroph digestion and some fermentation takes place during the
6–8 hours they remain in the gastric fundus. Cecotrophs contain microorganisms and
products of microbial fermentation including amino acids, volatile fatty acids, and
vitamins. A gelatinous mucous coating protects them from some of the stomach acid. As
the cecotrophs passed through the colon, lysozyme was incorporated. The lysozyme has
bacteriolytic activity that degrades microbial proteins for absorption in the small intestine.
Bacteria within the cecotroph produce amylase that converts glucose to carbon dioxide
and lactic acid. These products along with amino acids and vitamins are absorbed
primarily in the small intestest. Digestion in the stomach begins with hydrochloric acid
and pepsin and continues into the proximal small intestine. Amylase from the pancreas is
added, although amylase is also present from saliva and cecotrophs. The pancreas also
contributes proteolytic enzymes and chymotrypsin through the accessory duct as well as
most likely through small ducts connecting directly to the duodenum. Bicarbonate is
secreted by the proximal duodenum to neutralize the acidity of ingesta leaving the
stomach. The bicarbonate is absorbed in the jejunum. Transit time through the jejunum is
10–20 minutes and 30–60 minutes through the ileum (Harcourt-Brown, 2002).
2.1.5.3. Hindgut
The hindgut consists of the cecum and colon. The cecum of the rabbit is large and
may contain 40% of intestinal content. It has 10 times the capacity of the stomach.2 The
cecum is thin-walled and coiled in 3 gyral folds. It ends in a blind-ended tube called the
vermiform appendix. This appendix contains lymphoid tissue and secretes bicarbonate
that buffers the cecal acids, and water to form the cecal paste. In addition to Bacteroides
species, there may also be ciliated protozoa, yeasts, and small numbers of E coli and
clostridia species in the cecal flora (O‘Malley, 2005).
The fermentation process in the cecum results in volatile fatty acids that are
absorbed across the cecal epithelium. Cecal contents have an alkaline pH in the morning
and an acid pH in the mid afternoon, termed a ―transfaunation‖ as types of
microorganisms fluctuate. In addition the predominant VFA of acetate, butyrate, and
propionate are also produced (O‘Malley, 2005). The ascending colon is divided into 4
sections.1 The ampulla coli opens into the first section, approximately 10 cm long and
24
having 3 longitudinal flat bands of muscular tissue (taeniae) that separate rows of haustra
or sacculations (Harcourt-Brown, 2002). The mucosa of this section has small protrusions
approximately 0.5 mm in diameter that are termed ―warzen‖ or warts. This are unique to
lagomorphs and greatly increase the surface area of the colon for absorption. The warts
may also aid in mechanical separation of ingesta. The taeniae are innervated with
autonomic fibers from the myenteric plexus. The second section of colon has a single
taenia and fewer, smaller haustra. There are segmental and haustral contractions that
mechanically separates the ingesta into indigestible particles and liquid contents. As the
large pellets pass down the middle of the lumen, water is re-absorbed and they are
excreted as hard dry pellets. The third section is the fusus coli. It is a muscular area about
4 cm long, highly innervated, and vascular. Its mucosal surface has prominent
longitudinal folds and goblet cells. It opens into the fourth section of ascending colony
that is indistinguishable histologically from the transverse and descending colon.1 The
distal colon (sections distal to the fusus coli) ends at the rectum. Its mucosa has short
crypts with abundant goblet cells. It is thin-walled and usually contains hard fecal pellets
(Harcourt-Brown, 2002).
2.1.5.4. Cecotrophy
Cecotrophs are formed in the proximal colon and cecum. Rabbits begin
consuming them between 2 and 3 weeks of age as they begin to eat solid food. Fiber
material greater than 0.5 mm does not enter the cecum but transits to be formed and
passed as hard fecal pellets. The smaller particles and fluid remain in the cecum or are
returned to the cecum via antiperistalsis to form high nutrient particles that become
coated with mucus as they pass through the colon. They are usually passed 8 hours or so
after feeding, which coincides usually to nighttime. This mechanism requires high fiber
diets to function properly. Low fiber diets increase cecal retention time and promote
hypomotility of the entire gut, which further reduces the cecotrophs produced. Fiber in
the diet should be indigestible and at least 15%. A low protein diet increases a rabbit‘s
cecotroph ingestion. A high protein diet and low in fiber reduces consumption (O‘Malley,
2005). In crude fiber terms, diets that are less than 150 g/kg of feed will almost always
result in digestive upset while diets with greater than 200 g/kg crude fiber result in
increased incidence of cecal impaction and mucoid enteritis. A diet devoid of fiber has a
25
coefficient of apparent digestibility of organic matter of 0.90. This declines in a linear
fashion to 0.40 when the diet contains 350 g crude fiber per kilogram of feed. Increased
crude fiber of the diet increases the crude fiber of the cecal contents. This decreases the
protein content. Compounded, pelleted diets require the addition of hay in order to supply
a complete diet. In general, the recommendation that hay be supplied on a free-choice
basis as a rule of good husbandry of the pet rabbit should be emphasized (Lowe et al,
2000).
High carbohydrate diets cause several problems. Excessive glucose allows
Clostridium spiroforme and E coli to colonize. Excess VFAs produced drop the cecal pH,
that inhibits normal flora and allows pathogens to proliferate and colonize. Gas and toxins
can be produced by pathogenic bacteria, and motility and nutrient production and
absorption are interrupted. Fats such as full-fat soybeans, oilseeds can be used as a source
of energy without causing cecal hyperfermentation. However, feeding of vegetable fats
and seeds decrease the fiber content of the diet, and lead to motility and functional
depression. It is interesting to note that rabbits have a gall bladder and secrete about 7
times the amount of bile as a dog of similar weight. They secrete mainly biliverdin rather
than bilirubin. Rabbits have low levels of bilirubin reductase (O‘Malley, 2005).
Rabbits should be fed in a quiet place, preferably early in the morning and in the
evening. Rabbits do not like dusty food. A rabbit will selectively take concentrates if the
palatability of roughage is variable. This may result in diarrhea from consumption of too
much protein relative to hay. A well-fed rabbit masticates its food extensively whereas
when the rabbit is hungry, it doesn‘t chew to any great extent. The mastication of the fiber
is necessary for dental health and normal tooth wear (Cathy, 2006).
2.1.5.5. Gastrointestinal illness
Rabbits that are presented with or without malocclusion but with painful
abdomens, anorexia, diarrhea or lack of stool need treatment prior to correction of the oral
problems. Immediate administration of analgesics and fluids often results in the rabbit
beginning to eat and the gastrointestinal tract beginning to move. A detailed history and
physical examination including auscultation of the abdomen may allow the practitioner to
evaluate the stage of gastrointestinal distress the rabbit is in. Radiographs are useful to
determine ileus. Contrast series may be utilized to determine an impaction, although
26
barium introduced into cecums is problematic for function. It is prefer to utilize
endoscopy and/ or ultrasound, or an iodine-based contrast agent rather than a barium
series. Most trichobezoars will move once hydration is corrected and sufficient roughage
is available. Use of motility enhancers may be tried if no impaction is present. Once pain
is alleviated and hydration corrected, the rabbit may begin to walk around and nibble hay,
which will encourage gastrointestinal motility. While not proven, probiotics are often
administered per os or intrarectally. These are usually primarily lactobacillus spp. which
are not the primary microflora of the rabbit. Vitamin B complex may be given to
stimulate appetite. As hepatic lipidosis may be present and playing a role in anorexia, it is
advantageous to get some food into the anorexic rabbit as soon as possible. If the rabbit
does not immediately start eating hay, a gavage of diluted Critical Care (Oxbow Pet
Products, Murdock, NE, USA) is given. This commercial formulation can be mixed with
apple juice or flavored electrolyte solution to give directly orally. Many rabbits will take
hand feeding of this formula (Cathy, 2006).
Rarely is surgery necessary to relieve an impaction, but if a necrotic or ischemic
section of the gut is suspected, surgery may be necessary to resect the bowel. Prognosis is
guarded primarily because of endotoxins produced by Clostridium species present in most
herbivore gastrointestinal tracts. The anesthesia further decreases gastrointestinal motility,
again setting up the microflora to be altered and toxins produced. It may be necessary to
install an intraosseous or jugular intravenous catheter to administer antibiotics and fluids
perioperatively and postoperatively for several days in these cases. Restoration of gut
microbial flora and motility and postsurgery are priorities. Antibiotic choices in these
cases are a balancing act as a broad spectrum antibiotic with primarily gram negative and
efficacy against anaerobes should be used. Antimicrobials that primarily have a gram-
positive spectrum or that do not kill anaerobes are not recommended (Cathy, 2006).
2.1.6. Diet and Eating Habits
Rabbits are herbivores that feed by grazing on grass, forbs, and leafy weeds. In
consequence, their diet contains large amounts of cellulose, which is hard to digest.
Rabbits solve this problem via a form of hindgut fermentation. They pass two distinct
types of feces: hard droppings and soft black viscous pellets, the latter of which are
known as caecotrophs and are immediately eaten (a behaviour known as coprophagy).
27
Rabbits reingest their own droppings (rather than chewing the cud as do cows and many
other herbivores) to digest their food further and extract sufficient nutrients (Oak Tree
Veterinary Centre, 2010). Rabbits graze heavily and rapidly for roughly the first half hour
of a grazing period (usually in the late afternoon), followed by about half an hour of more
selective feeding. In this time, the rabbit will also excrete many hard fecal pellets, being
waste pellets that will not be reingested. If the environment is relatively non-threatening,
the rabbit will remain outdoors for many hours, grazing at intervals. While out of the
burrow, the rabbit will occasionally reingest its soft, partially digested pellets; this is
rarely observed, since the pellets are reingested as they are produced. Reingestion is most
common within the burrow between 8 o'clock in the morning and 5 o'clock in the
evening, being carried out intermittently within that period. Hard pellets are made up of
hay-like fragments of plant cuticle and stalk, being the final waste product after
redigestion of soft pellets. These are only released outside the burrow and are not
reingested. Soft pellets are usually produced several hours after grazing, after the hard
pellets have all been excreted. They are made up of micro-organisms and undigested plant
cell walls.
The chewed plant material collects in the large cecum, a secondary chamber
between the large and small intestine containing large quantities of symbiotic bacteria that
help with the digestion of cellulose and also produce certain B vitamins. The pellets are
about 56% bacteria by dry weight, largely accounting for the pellets being 24.4% protein
on average. The soft feces form here and contain up to five times the vitamins of hard
feces. After being excreted, they are eaten whole by the rabbit and redigested in a special
part of the stomach. The pellets remain intact for up to six hours in the stomach; the
bacteria within continue to digest the plant carbohydrates. This double-digestion process
enables rabbits to use nutrients that they may have missed during the first passage through
the gut, as well as the nutrients formed by the microbial activity and thus ensures that
maximum nutrition is derived from the food they eat (Encyclopædia Britannica, 2007).
This process serves the same purpose within the rabbit as rumination does in cattle and
sheep (Lockley, 1964). Rabbits are incapable of vomiting (Pop Quiz, 2011).
The recommended diet for a mature rabbit consists of unlimited grass hay; ¼ to ½
cup (timothy/oat if rabbit is hypercalcemic, older or obese; alfalfa only if underweight,
28
normocalcemic) pellets per 5–6 lbs (2.5–3 kg) of body weight. Fresh foods can be 1–2
cups of chopped vegetables (preferably a mix: beet greens, broccoli, carrot and carrot
tops, collard greens, mustard greens, parsley, pea pods (flat edible kind), romaine lettuce,
watercress, wheat grass. Other acceptable vegetables, but less Vitamin A content: alfalfa,
basil, bok choy, brussel sprouts, celery, cilantro, clover, dandelion greens and flowers
(not sprayed), endive, escarole/kale, green peppers, mint, peppermint leaves, raddichio,
radish tops, radish and clover sprouts, raspberry/blackberry leaves, and spinach. For treats
and only if the rabbit is not overweight and the owner is insistent on some sort of ―sweet
treat,‖ the following fruits are high in fiber and can be provided at 2 TBSP/3 kg (30 ml/3
kg) body weight daily: apple, melon, peach, plum, strawberry, blueberry, papaya,
pineapple, and raspberry. Rabbits evolved eating grass and herbs, not rich grains, alfalfa,
and fruits (O‘Malley, 2005).
Supplementation with vitamins and other treats is not necessary. Pellets are fed as
a larger portion of the diet to does in kindle starting approximately 10 days prior to
delivery, as well as to growing, young rabbits up to 10 weeks of age, then the amount of
pellets is scaled down to the adult amount. After weaning of the kits, the amount of
pellets for the doe is decreased until a non-breeding level of appetite is established.
Hypercalcemia and obesity are very commonly seen diseases with dietary etiologies
(O‘Malley, 2005).
2.1.7. Rabbit diseases
Rabbits can be affected by a number of diseases. These include pathogens that
also affect other animals and/or humans, such as Bordetella bronchiseptica and
Escherichia coli, as well as diseases unique to rabbits such as rabbit haemorrhagic
disease: a form of calicivirus, and myxomatosis (Cooke, 2014). Rabbits and hares are
almost never found to be infected with rabies and have not been known to transmit rabies
to humans (Centers for Disease Control and Prevention, 2012). Among the parasites that
infect rabbits are tapeworms such as Taenia serialis, external parasites like fleas and
mites, coccidia species, and Toxoplasma gondii (Boschert, 2013).
29
2.1.8. Rabbit Meat
World rabbit meat production increased up to 1.68 million tonnes in 2010
(FAOSTAT, 2012). Currently the leading producer of rabbit meat in the world is China
with 669.000 t/year, while, in Europe, the main producer is Italy (255.400 t/year),
followed by Spain (66.200 t/year), France (51.665 t/year), Czech Republic (38.500 t/year)
and Germany (37.500 t/year) (FAOSTAT, 2012).
In an efficient breeding, rabbits convert up to 20% of the protein consumed in
meat, more than for pigs (15-18%) and cattle (9-12%) (Suttle, 2010). Rabbit meat is high
in protein, low in calories and low in fat and cholesterol contents, being considered as a
delicacy and a healthy food product, easy to digest, indicated in feeding children and old
people (Zotte, 2000). Rabbit meat is one of the best white lean meats available on the
market, very tender and juicy. There is no religious taboo or social stigma regarding the
consumption of this meat. Content in calcium and phosphorus are higher than in other
meats as well as the nicotinic acid (13 mg/kg meat) (Williams, 2007). Also, rabbit meat
does not contain uric acid and has a low content of purines (Hernández et al., 2007). The
ash content is similar or higher than that of other livestock, while many studies shown
that rabbit meat is poor in potassium and phosphorus (Hermida et al., 2006).
Rabbit meat is a source of B vitamins (B2, B3, B5, B12) as reported by Combes
(2004). In rabbits, carcass quality, quantity and proportion of fatty acids in meat
composition and fat tissue are changing with diet and animal age (mainly intramuscular
fat content is increasing) (Cobos et al., 1993). Data regarding the chemical composition
of rabbit meat is variable especially in fat content for each section of carcass (Pla et al.,
2004).
2.1.9. Environmental Problems
Rabbits have been a source of environmental problems when introduced into the
wild by humans. As a result of their appetites, and the rate at which they breed, feral
rabbit depredation can be problematic for agriculture. Gassing, barriers (fences), shooting,
snaring, and ferreting have been used to control rabbit populations, but the most effective
measures are diseases such as myxomatosis (myxo or mixi, colloquially) and calicivirus.
In Europe, where rabbits are farmed on a large scale, they are protected against
myxomatosis and calicivirus with a genetically modified virus. The virus was developed
30
in Spain, and is beneficial to rabbit farmers. If it were to make its way into wild
populations in areas such as Australia, it could create a population boom, as those
diseases are the most serious threats to rabbit survival. Rabbits in Australia and New
Zealand are considered to be such a pest that land owners are legally obliged to control
them (Environment.gov.au., 2010).
2.1.10. Reproductive Traits
Rabbits are induced ovulators, which means that ovulation in female occurs after
mating. Females are going through periods of receptivity (estrus) that last about 7-10
days, followed with a "quiet" period (interestrus) for 1-2 days. Female rabbits can be very
aggressive toward males. Because of it, female taken to male for mating should be
watched closely. If female is receptive, they will mate twice within about 30 minutes,
after which they should be separated. Female builds a nest by collecting mouthfuls of
nesting material (usually dry, long grass), which she carries to an underground nesting
site. She lines the nest with her own fur, which she plucks from her own body. She closes
the nest by digging soil into the tunnel and then patting it down by alternate, downward
thrusts of the forepaws. She then deposits a few drops of urine and a few fecal pellets on
the top. The pattern can be sometimes observed in domesticated rabbits at the entrance of
their cage/pen nest box (Mullan and Main, 2007).
A breeding doe in captivity can provide 40 young per year (5 liters of 8, or 4 liters
of 10). Rabbits are more sexually active during long photoperiods (spring and summer).
In the late fall - early winter productivity decreases (Pitt and Carney, 1999).
Females can breed at any time of the year if there is sufficient feed available. The
main breeding season is determined by rainfall and the early growth of high-protein
plants. During this time, wild rabbits form territorial groups containing 1–3 males and 7–
10 females, led by a dominant pair. Wild rabbits can begin breeding at four months old
and may produce five or more litters in a year, with up to five young per litter. In less
favorable conditions they can still produce one or two litters each year (NSW, 2007).
Rabbits have a gestation time of 28–30 days. Young are born blind and hairless and open
their eyes after 7–10 days. They emerge from the warren weaned at about 18 days and
leave the nest at 23–25 days. Survival of young varies between years and with seasonal
31
conditions, and also depends on the incidence of diseases. Wild rabbits rarely survive past
six years of age (Williams et al, 1995).
2.1.11. Clinical Pathology
Over the past several years, the popularity of the domestic rabbit (Oryctolagus
cuniculus) as a pet has risen exponentially. As a result, the numbers of these animals
presented to the veterinary practitioner has grown as well. Unfortunately, this rise in
popularity has not been accompanied by an increase in the clinical pathology database as
it applies to the companion animal. Most of the reference values are still based upon
rabbits maintained within laboratory settings. To this date, many texts still comment on
the lack of current reference values for the rabbit.
In order for any laboratory data to be valid, the samples must be collected in a
fashion that is reproducible and avoids artifactural changes. Blood collection from the
rabbit depends upon the clinician's ability to adequately restrain the patient with minimal
stress placed upon it. In most instances, physical restraint is adequate; however, there are
occasions that may mandate judicious use of chemical restraint, typically inhalant
anesthesia (Murray, 2006).
2.1.11.1. Blood Collection From the Rabbit
The blood volume of the rabbit is estimated to be 55 to 65 ml/kg. In most cases,
one may safely collect 6 to 10% of the blood volume, or 3.3 to 6.5 ml/kg. Such volumes
will permit a variety of clinical laboratory procedures. It is preferly to collect blood for
complete blood counts (CBC) in EDTA tubes and samples for biochemical evaluation in
lithium heparin tubes. A variety of sites have been advocated for the collection of blood
from the rabbit. In the pet rabbit, the preferred sites are the cephalic vein, the lateral
saphenous vein and the jugular vein. While tremendous advances have been made in pet
rabbit medicine, much of the currently available reference data is based upon controlled
laboratory settings.. One must always take those steps necessary to control artifactural
changes that are within the control of the clinician. While not truly artifacts, one must
evaluate much of the laboratory data bearing in mind numerous intrinsic factors that will
affect various parameters. In the rabbit, age, sex, breed, and circadian rhythms all affected
the hemogram. As expected, young rabbits had significantly lower RBC and WBC
parameters than adults (Murray, 2006).
32
2.1.11.2. Complete Blood Count (CBC)
Complete blood count (CBC) also known as full blood count (FBC). CBC is a
laboratory test performed on a sample of blood included determination of rat HCT the
percentage of blood that consists of red blood cells. It is possible to use manual or semi-
automated or automated technique to determine various elements of blood cells (Gale
Encyclopedia of Medicine, 2008). Complete blood counts involve the following:
2.1.11.2.1. Hemoglobin
A complex protein iron compound in the blood that carries oxygen to the cell from
the lung and carbon dioxide away from the tissue to the lung .The hemoglobin
concentration may be estimation by several methods by measurement of its color, by its
power of combining with oxygen or carbon monoxide or by its iron content (Saunder,
2007). The normal range of hemoglobin in rabbits is 8.9 -15.5 g/dL (HewItt et al., 1989).
2.1.11.2.2. Red blood cell count (RBC)
The red blood count (RBC) is itself of use in diagnostic hematology, but it is also
importance because it permits the mean cell volume (MCV) and means cell hemoglobin
(MCH) to be calculated. However, a manual red cell count in which cell are count
visually is so time consuming and has such poor precision that both it and the red cell
indices derived from it are of limited use in the routine practice the reference method for
the (RBC) is an automated rather than a manual count (Berger, 2000). The normal range
of RBC in rabbits is 3.7- 7.5 106\µL (HewItt et al., 1989).
2.1.11.2.3. Hematocrit (HCT or PCV)
The packed cell volume (PCV) can be used as a simple screening test for anemia.
It can also be used as rough guide to the accuracy of haemoglobin measurement. PCV
as a percentage should be about three times the haemoglobin value (Dacie et al, 2006).
The normal range of hematocrit in rabbits is 26.7 – 47.2 % (HewItt et al., 1989). The
erythrocyte count, HB and HCT can be utilized in calculation to determine the
erythrocyte indices.
2.1.11.2.4. The Mean Corpuscular Volume (MCV)
The MCV indicate the average volume of a single erythrocyte in a given blood
sample the normal rang in rabbits is 58-79.6 fl (HewItt et al, 1989).
33
2.1.11.2.5. The Mean Corpuscular Hemoglobin (MCH)
The MCH indicates the mean weight of hemoglobin per erythrocyte: the normal
range is 19.2 – 29.5 pg (HewItt et al, 1989).
2.1.11.2.6. The Mean Corpuscular Hemoglobin Concentration (MCHC)
The MCHC indicates to average concentration of Hb in the erythrocyte in
specimen the normal range in rabbits is 31.1-37.0 % (HewItt et al, 1989).
2.1.11.2.7. White blood cell (WBC) count and differential
White blood cell or leucocytes are the cells of the immune system that are involve
in defending the body against both infectious disease and foreign material. It is counted
manually or automated. The normal range is 5.2 – 16.5 103/µl (HewItt et al., 1989). The
differential white cell count is usually performed by visual examination of blood film
which is prepared on slide by the spread technique (Dacie et al, 2006).
2.1.11.2.8. The platelet count
A minute, un-nucleated, disk-like cytoplasmic body found in the blood plasma of
mammals that is derived from a mega-karyocyte and function to promote blood clotting.
Also called thrombocyte blood (Dacie et al, 2006). The platelet count is important
component of the blood count. Platelets may be counted automated or manual (Anne et
al., 1998). Normal range of platelets in rabbits is 112 – 795 103/µl (HewItt et al, 1989).
Many of the concepts typically utilized in companion animal hematology are the
same for rabbits (Murray, 2006).
2.1.11.3. Serum Chemistry
The serum chemistry panel is subject to a variety of artifacturally induced
changes. For that reason, it is important that the clinician collect samples in such a
fashion as to assure minimal artifact and therefore provide reproducible results.
2.1.11.3.1. Alkaline phosphatase (ALP)
ALP is a cell membrane-associated enzyme with three isoenzymes in the rabbit.
Significantly higher levels are associated with osteoblasts, renal tubular epithelium,
intestinal epithelium, liver, and placenta. As a result of this wide distribution, elevations
of ALP are typically non-specific (Murray, 2006). The normal range in rabbits is 17 – 192
U\L (HewItt et al., 1989).
34
2.1.11.3.2. Alanine aminotransferase (ALT)
In most companion animals, ALT elevations are typically associated with
hepatocellular damage. In the rabbit, ALT is much less specific, as the liver contains less
ALT activity, and the enzyme is also present in cardiac muscle. Elevations are often seen
in cases of hepatocellular inflammation, hepatic lipidosis, Eimeria infection, and some
hepatic neoplasm. It has been observed that slight elevations of ALT in asymptomatic
rabbits may be associated with exposure to aromatics, such as those found in wood
shaving beddings (Murray, 2006). The normal range of ALT in rabbits is 12-67 U\L
(Research Animal Resources, 2003).
2.1.11.3.3. Aspartate aminotransferase (AST)
AST is found in a number of tissues in the rabbit, including liver, skeletal muscle,
kidney, and pancreas, the first two being the highest. Elevations of AST may be found in
cases of liver inflammation, skeletal muscle damage, or associated with physical exertion
(Murray, 2006). The normal range of AST in rabbits is 14-113 U\L (Research Animal
Resources, 2003). AST to ALT ratio can be very useful. When greater than 2.0, this
typically suggests alcoholic liver disease (Ahn, 2011).
2.1.11.3.4. Albumin
Albumin synthesis is an important function of the liver. Approximately 10 g is
synthesized and secreted daily. With Progressive liver disease serum albumin levels fall,
reflecting Decreased synthesis. Albumin levels are dependent on a number of other
factors such as the nutritional status, catabolism, hormonal factors, and urinary and
gastrointestinal losses. These should be taken into account when interpreting low albumin
levels. Having said that, albumin concentration does correlate with the prognosis in
chronic liver disease (Limdi and Hyde, 2003). The normal range in rabbits is 2.7- 4.6
g\dL (Research Animal Resource, 2003).
2.1.11.3.5. Bilirubin
The rabbit has low biliverdin reductase activity, and therefore the predominant
bile pigment excreted by the rabbit is biliverdin. Some bilirubin is, however, produced
(approximately 30% of the total), and elevations indicate cholestasis (Murray, 2006). The
normal range of Bilirubin in rabbits is 0- 1.0 mg\dL (Rosenthal, 2002).
35
2.1.11.3.6. Creatinine kinase (CK)
As in other mammals, CK is an enzyme associated with muscle, cardiac, skeletal,
and smooth, and brain. In general, elevations are noted in conjunction with myocyte
damage or inflammation (Murray, 2006). The normal range in the rabbits is 218- 2705
U\L (HewItt et al., 1989).
2.1.11.3.7. Lactate dehydrogenase (LDH)
LDH activity is widely distributed through a large variety of tissues in the rabbit,
and is therefore of limited diagnostic use in this species (Murray, 2006). The normal
range of LDH in rabbits is 59 – 389 U\L (HewItt et al., 1989).
2.1.11.3.8. Total protein (TP)
TP levels may vary depending upon the rabbit‘s age, reproductive state, and
breed. Elevations of TP generally indicate dehydration, chronic disease, or exotics
hyperthermia. Decreased values suggest loss, either via the urinary or digestive system,
nutritional disease (eg, malnutrition or starvation), or decreased liver production (Murray,
2006). The normal value in rabbits is 5.4 - 7.3 g\dL (Rosenthal, 2002).
2.1.11.3.9. Cholesterol
While changes in circulating cholesterol may suggest a variety of metabolic
aberrations in many species, such is not necessarily the case in the rabbit. A number of
normal physiologic variables may influence circulating cholesterol. First, males tend to
have a lower level than females. Second, there is a definite circadian fluctuation with
higher levels in late afternoon and evening. Finally, there is a significant postprandial
effect upon measured cholesterol. Unfortunately, cecotrophy makes collection of a
―fasting‖ sample problematic. Hypercholesterolemia may be associated with
arteriosclerosis, liver disease, hypothyroidism, and hypercortisolemia (Murray, 2006).
The normal range of cholesterol in rabbits is 10 – 80 mg\dL (Rosenthal, 2002).
2.1.11.3.10. Glucose
Interpretation of this serum chemistry parameter requires an understanding of the
normal physiology of the herbivore, particularly as it differs from that of the carnivore.
As a result, hypoglycemia is rarely seen in the rabbit. When seen, it is typically associated
with a poor prognosis in conditions such as hepatic lipidosis, sepsis, starvation, and
36
severe gastrointestinal disease. Hyperglycemia, on the other hand, may be associated with
a variety of conditions. Elevations may occur secondary to the effects of restraint and
handling. The diagnosis of diabetes mellitus in the rabbit has been controversial. For the
purposes of this discussion, suffice to say diagnosis cannot be based upon a single
sample. Other causes of hyperglycemia include hepatic disease, GI stasis, shock, and
hyperthermia (Murray, 2006). The normal range in rabbits is 75 -150 g\dL (Research
Animal Resources, 2003).
2.1.11.3.11. Blood Urea Nitrogen (BUN)
In traditional pet species, BUN elevations are associated with renal dysfunction,
either via renal disease or decreased perfusion. In the rabbit, urea concentrations may
vary depending upon a variety of physiologic factors. Circadian fluctuations are present
with highest levels found in late afternoon and early evening. Additionally, the quantity
and quality of protein in the diet may influence the BUN. In addition, there is an influence
on BUN exerted by cecal microflora, either catabolism or protein excesses. Therefore,
slight changes in BUN are difficult to interpret. In general, however, elevations may be
seen with dehydration, renal compromise, E. cuniculi infections, and urolithiasis.
Interpretations must be made with caution and in conjunction with other clinical
parameters (Murray, 2006). The normal range of BUN in rabbits is 10 – 33 mg\dL
(Rosenthal, 2002).
2.1.11.3.12. Creatinine
Creatinine, the product of muscle metabolism, is freely filtered through the
glomerulus without subsequent tubular resorption. As a result, elevations may be the
result of dehydration or renal disease (Murray, 2006). The normal range in rabbits is 0.8 –
1.8 mg\dL (Research Animal Resources, 2003).
2.1.11.3.13. Calcium
The rabbit‘s calcium metabolism is unique in domestic animal species in that most
of the dietary calcium is absorbed from the intestine independent of vitamin D. Therefore,
serum levels are directly related to dietary levels. The kidney plays a more significant role
in the elimination of calcium than in other species. Hypercalcemia may be associated with
high levels of dietary calcium, renal disease and subsequent compromise in the ability to
excrete calcium, and is often seen associated with thymoma. Hypocalcemia is uncommon,
37
but may be seen in lactating does (Murray, 2006). The normal rang in rabbits is 129 -150
mg\dL (HewItt et al., 1989).
2.1.11.3.14. Phosphorus
Interpretation of abnormalities of the circulating phosphorous levels is difficult, at
best, and should be made in conjunction with other clinical and laboratory parameters. In
general, there is little information regarding the interpretation of hyper/hypophosphatemia
in the rabbit (Murray, 2006). The normal range is 4.4 – 7.2 mg\dL (Rosenthal, 2002).
2.1.11.3.15. Triglycerides
This is the most common type of lipid formed in animals. Fat tissue is primarily
for the storage of this form of lipid. Triglyceride levels vary quite a bit over short time
periods. A meal high in sugar, fat, or alcohol can raise the triglyceride level drastically, so
the most repeatable measures of this lipid are taken after 12 hours of fasting. Even though
sugar and alcohol are not lipids, your body will convert any form of excess calories into
triglycerides for long-term storage. A value below the normal range indicates no
increased risk, within the normal range indicates a slight risk, and over normal range is a
high risk (Cholesterol Center, 2005).
2.1.11.3.16. LDL Cholesterol, or Low density lipoprotein
This is sometimes referred to as the ―bad cholesterol.‖ This form contains the
highest amount of cholesterol. The lowest the number the better (Cholesterol Center,
2005).
2.1.11.3.17. HDL Cholesterol, or High density lipoprotein
This is sometimes called ―good cholesterol.‖ The higher the number, the better.
HDL cholesterol is cholesterol that is packaged for delivery to the liver, where the
cholesterol is removed from the body (Cholesterol Center, 2005).
As one can readily appreciate, hematology and serum chemistry parameters are
valuable adjuncts to the diagnostic process in the rabbit. One must remember, however,
that much of the reference data is acquired from a laboratory setting, with rabbits of
limited genetic range, limited physical activity, and on a controlled diet. In addition, a
paradigm shift is required for the typical small animal clinician, as one is dealing with the
herbivorous rabbit, not the carnivore generally presented in the small animal practice
(Murray, 2006).
38
2.2. Cotton
Cotton is a soft, fluffy staple fiber that grows in a boll, or protective case, around
the seeds of cotton plants of the genus Gossypium in the family Malvaceae. The fiber is
almost pure cellulose. Under natural conditions, the cotton bolls will tend to increase the
dispersal of the seeds. The plant is a shrub native to tropical and subtropical regions
around the world, including the Americas, Africa, and India. The greatest diversity of
wild cotton species is found in Mexico, followed by Australia and Africa (Biologycotton,
2008). Cotton was independently domesticated in the Old and New Worlds. The English
name derives from the Arabic (al) quṭ n لط, which began to be used circa 1400 AD
(Metcalf, 1999). The fiber is most often spun into yarn or thread and used to make a
soft, breathable textile. The use of cotton for fabric is known to date to prehistoric times;
fragments of cotton fabric dated from 5000 BC have been excavated in Mexico and the
Indus Valley Civilization in Ancient India (modern-day Pakistan and some parts of
India). Although cultivated since antiquity, it was the invention of the cotton gin that
lowered the cost of production that led to its widespread use, and it is the most widely
used natural fiber cloth in clothing today. Current estimates for world production are
about 25 million tonnes or 110 million bales annually, accounting for 2.5% of the world's
arable land. China is the world's largest producer of cotton, but most of this is used
domestically. The United States has been the largest exporter for many years (Natural
Fibres, 2009). In US, cotton is usually measured in bales, which measure approximately
0.48 cubic metres (17 cubic feet) and weigh 226.8 kilograms (500 pounds) (National
Cotton Council of America, 2013).
2.2.1. Types of cotton
There are four commercially grown species of cotton, all domesticated in antiquity:-
Gossypium hirsutum – upland cotton, native to Central America, Mexico, the
Caribbean and southern Florida (90% of world production).
Gossypium barbadense – known as extra-long staple cotton, native to tropical
South America (8% of world production).
Gossypium arboreum – tree cotton, native to India and Pakistan (less than 2%).
Gossypium herbaceum – Levant cotton, native to southern Africa and the Arabian
Peninsula (less than 2%). The two New World cotton species account for the vast
39
majority of modern cotton production, but the two Old World species were widely
used before the 1900s. While cotton fibers occur naturally in colors of white, brown,
pink and green, fears of contaminating the genetics of white cotton have led many
cotton-growing locations to ban the growing of colored cotton varieties, which remain
a specialty product (Wikipedia, 2010).
2.2.2. Genetically modified (GM) cotton
Genetically modified (GM) cotton was developed to reduce the heavy reliance on
pesticides. The bacterium Bacillus thuringiensis (Bt) naturally produces a chemical
harmful only to a small fraction of insects, most notably the larvae of moths and
butterflies, beetles, and flies, and harmless to other forms of life (Mendelsohn et al,
2003). The gene coding for Bt toxin has been inserted into cotton, causing cotton,
called Bt cotton, to produce this natural insecticide in its tissues. In many regions, the
main pests in commercial cotton are lepidopteran larvae, which are killed by the Bt
protein in the transgenic cotton they eat. This eliminates the need to use large amounts of
broad-spectrum insecticides to kill lepidopteran pests (some of which have
developed pyrethroid resistance). This spares natural insect predators in the farm ecology
and further contributes to noninsecticide pest management. But Bt cotton is ineffective
against many cotton pests, however, such as plant bugs, stink bugs, and aphids;
depending on circumstances it may still be desirable to use insecticides against these. A
2006 study done by Cornell researchers, the Center for Chinese Agricultural Policy and
the Chinese Academy of Science on Bt cotton farming in China found that after seven
years these secondary pests that were normally controlled by pesticide had increased,
necessitating the use of pesticides at similar levels to non-Bt cotton and causing less profit
for farmers because of the extra expense of GM seeds (Lang, 2006). However, a 2009
study by the Chinese Academy of Sciences, Stanford University and Rutgers University
refuted this (Wang et al, 2009).
They concluded that the GM cotton effectively controlled bollworm. The
secondary pests were mostly miridae (plant bugs) whose increase was related to local
temperature and rainfall and only continued to increase in half the villages studied.
Moreover, the increase in insecticide use for the control of these secondary insects was
far smaller than the reduction in total insecticide use due to Bt cotton adoption. A 2012
40
Chinese study concluded that Bt cotton halved the use of pesticides and doubled the level
of ladybirds, lacewings and spiders (Carrington, 2012). The International Service for the
Acquisition of Agri-biotech Applications (ISAAA) said that, worldwide, GM cotton was
planted on an area of 25 million hectares in 2011 (ISAAA Brief, 2011). This was 69% of
the worldwide total area planted in cotton. GM cotton acreage in India grew at a rapid
rate, increasing from 50,000 hectares in 2002 to 10.6 million hectares in 2011. The total
cotton area in India was 12.1 million hectares in 2011, so GM cotton was grown on 88%
of the cotton area. This made India the country with the largest area of GM cotton in the
world (ISAAA Brief, 2011). A long-term study on the economic impacts of Bt cotton in
India, published in the Journal PNAS in 2012, showed that Bt cotton has increased yields,
profits, and living standards of smallholder farmers (Kathage and Qaim, 2012).
The U.S. GM cotton crop was 4.0 million hectares in 2011 the second largest area
in the world, the Chinese GM cotton crop was third largest by area with 3.9 million
hectares and Pakistan had the fourth largest GM cotton crop area of 2.6 million hectares
in 2011 (ISAAA Brief, 2011). The initial introduction of GM cotton proved to be a
success in Australia – the yields were equivalent to the non-transgenic varieties and the
crop used much less pesticide to produce (85% reduction) (Cottonaustralia.com.au, 2010).
The subsequent introduction of a second variety of GM cotton led to increases in GM
cotton production until 95% of the Australian cotton crop was GM in 2009 (GMO
Compass, 2010) making Australia the country with the fifth largest GM cotton crop in the
world. Other GM cotton growing countries in 2011 were Argentina, Myanmar, Burkina
Faso, Brazil, Mexico, Colombia, South Africa and Costa Rica (ISAAA Brief, 2011).
Cotton has been genetically modified for resistance to glyphosate a broad-
spectrum herbicide discovered by Monsanto which also sells some of the Bt cotton seeds
to farmers. There are also a number of other cotton seed companies selling GM cotton
around the world. About 62% of the GM cotton grown from 1996 to 2011 was insect
resistant, 24%stacked product and 14% herbicide resistant (ISAAA Brief, 2011). Cotton
has gossypol, a toxin that makes it inedible. However, scientists have silenced the gene
that produces the toxin, making it a potential food crop (Bourzac, 2006).
41
2.2.3. GM Cotton in Sudan
Cotton is one of the most important crops produced in Sudan. It was the main
foreign exchange earner contributing considerably to foreign exchange proceeds before
the oil. It is cultivated in clay soil in Gezira, Rahad, NewHalfa, Suki, Blue Nile, White
Nile, schemes . In silt soil in Tokar of Eastern Sudan and in heavy clay soil in Nuba
Mountains area of Western Sudan. Categorized by system of irrigation it is grown by
gravity and pumps in Gezira, Rahad, New Halfa (Girba), White Nile, Blue Nile, and Suki
Schemes, by flood in Tokar Delta and by rain in Nuba Mountains, and some areas of the
Blue Nile(Agdi). Chemical inputs applications vary from moderate in some places to zero
in others (El Wakeel, 2014).
Two Chinese Bt-cotton genotypes (G. hirsutum) carrying Cry 1A gene; from
Bacillus thuringiensis (Bt) a hybrid CN-C01 and an open pollinated CN-C02 were
approved for commercial production by Chinese National Authority in 2004 and 2008,
respectively. The two genotypes carry Cry1A gene which is specific toxin against
Lepidoptera larvae to protect cotton crop against bollworms. These genotypes were
evaluated for two seasons 2010/11 and 2011/12, and in open field trails in six
environments (three irrigated and three rainfed locations in Sudan with two local checks
fed (Abdin and Hamid).
Animal feeding on crop residues and counts of major soil microorganism in crop
rizosphere were performed to assess the effect of the Bt gene on non-target organisms.
The Bt one tremendously out-yielded the non-Bt varieties with a difference of over 5-6
times in sites with high bollworms infestation and overall increase of 129-166% over the
two check varieties in the combined field trails. The National Variety Release Committee
approved the release of the two Bt cotton genotypes; the hybrid CN-C01 and the open
pollinated CN-C02 in the irrigated and rain-fed production areas in Sudan for commercial
production (El Wakeel, 2014).
Areas Planted with Bt cotton in Sudan in (2012) is Totally 53,220 Ha. Initially the
cultivation failed in Gezira and New Halfa regions due to water logging as a result of bad
land preparation. Sudan has successfully introduced genetically modified (GM) cotton
technology in the country, in partnership with Brazil. Cotton planted in 2014 was
42
121,500 hectares of land in rain-fed areas, and on another 81,000 hectares under irrigation
(El Wakeel, 2014).
In 2012, Sudan became the fourth African country to commercialize a biotech
crop – Bt cotton. A total of 20 000 ha of Bt cotton were planted in the Sudan by about 10
000 smallholder farmers. The GM cotton variety planted is named ―Seeni 1‖and was
developed in China. The availability of GM cotton seed was a limiting factor in 2012, but
in 2013 the area tripled from 20,000 ha to 62,000 ha and is expected to expand even
further (David et al., 2014).
2.2.4. Cotton Genome
A public genome sequencing effort of cotton was initiated in 2007 by a
consortium of public researchers (Chen et al., 2007). They agreed on a strategy to
sequence the genome of cultivated, tetraploid cotton. "Tetraploid" means that cultivated
cotton actually has two separate genomes within its nucleus, referred to as the A and D
genomes. The sequencing consortium first agreed to sequence the D-genome relative of
cultivated cotton (G. raimondii, a wild Central American cotton species) because of its
small size and limited number of repetitive elements. It is nearly one-third the number of
bases of tetraploid cotton (AD), and each chromosome is only present once (Chen et al.,
2007). The A genome of G. arboreum would be sequenced next. Its genome is roughly
twice the size of G. raimondii's. Part of the difference in size between the two genomes is
the amplification of retrotransposons (GORGE). Once both diploid genomes are
assembled, then research could begin sequencing the actual genomes of cultivated cotton
varieties. This strategy is out of necessity; if one were to sequence the tetraploid genome
without model diploid genomes, the euchromatic DNA sequences of the AD genomes
would co-assemble and the repetitive elements of AD genomes would assembly
independently into A and D sequences respectively. Then there would be no way to
untangle the mess of AD sequences without comparing them to their diploid counterparts.
The public sector effort continues with the goal to create a high-quality, draft genome
sequence from reads generated by all sources. The public-sector effort has generated
Sanger reads of BACs, fosmids, and plasmids as well as 454 reads. These later types of
reads will be instrumental in assembling an initial draft of the D genome. In 2010, two
companies (Monsanto and Illumina), completed enough Illumina sequencing to cover the
43
D genome of G. raimondii about 50x. They announced that they would donate their raw
reads to the public. This public relations effort gave them some recognition for
sequencing the cotton genome. Once the D genome is assembled from all of this raw
material, it will undoubtedly assist in the assembly of the AD genomes of cultivated
varieties of cotton, but a lot of hard work remains (Chen et al., 2007).
2.2.5. Cottonseed
Cottonseeds are surrounded by fibres which grow from the surface of the seed.
This lint is removed and used to make cotton thread and fabric. Cottonseed is the seed of
the cotton plant. The mature seeds are brown ovoids weighing about a tenth of a gram. By
weight, they are 60% cotyledon, 32% coat and 8% embryonic root and shoot. These are
20% protein, 20% oil and 3.5% starch. Fibres grow from the seed coat to form a boll of
cotton lint. The boll is a protective fruit and when the plant is grown commercially, it is
stripped from the seed by ginning and the lint is then processed into cotton fibre. For unit
weight of fibre, about 1.6 units of seeds are produced. The seeds are about 15% of the
value of the crop and are pressed to make oil and used as ruminant animal feed. About
5% of the seeds are used for sowing the next crop (Smith, 2006).
2.2.6. Uses of Cottonseeds
Over 80 % of the global cotton cultivation now consists of Bt cotton. Bt cotton is
resistant to being eaten by certain insects, but the cotton fiber itself has not been altered.
In addition to cotton fiber, the cottonseed also has various uses. The oil that it contains is
used in cosmetic products and in certain food products. The ―pressed cake‖ that remains
after the oil has been extracted is sometimes processed in animal feed. Bt proteins can be
found in the seed and the pressed cake of Bt cotton, but not in the oil. The European
Union allows the import of certain varieties of Bt cotton and the processing in human
food and animal feed. If a product derived from Bt cotton is processed in human food or
animal feed, this must be stated on the label (Jo Bury, 2013).
2.2.6.1. Animal fodder
Animal fodders based on cottonseed stand out because of their high protein
content. However, the problem here remains the toxic substance gossypol. Non-ruminants
– such as chickens and pigs – experience the same problems as humans. Ruminants can
tolerate the substance, because it is broken down by the microorganisms in the stomachs
44
of these animals. Fodder based on cottonseed is therefore restricted to cattle and buffalo.
The use of cottonseed cakes as fodder – de-oiled or not – has been on the up for several
years in India. So strongly in fact, that cottonseed has now become the main ingredient
(33%) in processed fodder, followed in a distant second place by soy, rapeseed and
ground nuts (James, 2012).
Research by the Indian ―National Dairy Research Institute‖ has also revealed that
cows show no preference for cakes pressed from non-Bt cottonseed over Bt cottonseed
cakes. The researchers also found that there was no difference in digestion, milk
production, body condition and weight gain for animals fed Bt seed compared to non-Bt
seed. The Bt protein is completely harmless to cattle, as it is broken down to its basic
components just as all other proteins are. The Bt protein does not pass into the milk or
blood of the animals (Mohanta et al., 2010).
Researchers from the ―College of Veterinary Science‖ in Hyderabad confirm that
animal fodders based on Bt cotton are equal in quality and have no harmful effects on
animals. The blood values of sheep that received Bt cotton for three months did not differ
in any way from sheep that were fed non-Bt cotton (Anilkumar, 2008).
With the advancement of technology, the processing of food has become
considerably convenient. As a result, cottonseed has been able to flourish in new markets
such as feed products for livestock. Cottonseed is crushed in the mill after removing lint
from the cotton boll. The seed is further crushed to remove any remaining linters or
strands of minute cotton fibres. The seeds are further hulled and polished to release the
soft and high-protein meat. These hulls of the cottonseed are then mixed with other types
of grains to make it suitable for the livestock feed. Cottonseed meal and hulls are the most
abundantly available natural sources of protein and fibre used to feed livestock.
(Wikipedia, 2015c).
2.2.6.2. Cottonseed meal
Cottonseed meal is a good source of protein. The two types of meal extraction
processes are solvent extraction and mechanical extraction. Most of the meal is extracted
mechanically through cottonseed kernels. The flaked cottonseed kernels are put into high
pressure through a screw inside a barrel which is constantly revolving. The screw pushes
out the oil through the openings made in the barrel. The dry pieces left in the barrel are
45
preserved and ground into meal. During the solvent extraction process, the cottonseed
kernels are subjected to fine grinding by pushing them through an expander and then the
solvent is used to extract most of the oil. The solvent-extracted meals have a lower fat
content of 0.5% than the mechanically extracted meals with a fat content of 2.0%.
Cottonseed meal is considered to have more arginine than soybean meal. Cottonseed meal
can be used in multiple ways: either alone or by mixing it with other plant and animal
protein sources (GMO Compass, 2005).
2.2.6.3. Cottonseed hulls
The outer coverings of the cottonseed, known as cottonseed hulls, are removed
from the cotton kernels before the oil is extracted. Cottonseed hulls serve as an excellent
source of feed for the livestock as they contain about 8% of cotton linters which have
nearly 100% cellulose in them. They require no grinding and easily mix with other feed
sources. As they are easy to handle, their transportation cost is fairly low, as well. Whole
cottonseed is another feed product of cottonseed used to feed livestock. It is the seed left
after the separation of long fibres from cotton, and serves as a good source
of cellulose for ruminants. Whole cottonseed leads to high production of milk and fat, if it
is being fed to a high-producing dairy cow. It can be cost effective and provides nutrients
such as high protein value of about 23%, crude fibre value of 25%, and high energy value
of 20%. Whole cottonseed serves as a highly digestible feed which also improves the
reproductive performance in livestock. Pima cottonseed, which is free of linters by
default, and delinted cottonseed are other types of cottonseed feed products (Kelly, 2010).
2.2.6.4. Cottonseed oil
The seed oil extracted from the kernels, after being refined, serves as a good
edible and nutritious food. It can be used as a cooking oil, salad dressings. It is also used
in the production of shortening and margarine. Cotton grown for the extraction of
cottonseed oil is one of major crops grown around the world for the production of oil,
after soy, corn, and canola (Wikipedia, 2015c).
2.2.6.5. Fertilizer
The cottonseed meal after being dried can be used as a dry organic fertilizer, as it
contains 41% protein and contains other natural nutrients such as omega-9 fatty acids. It
can also be mixed with other natural fertilizers to improve its quality and use. Due to its
46
natural nutrients, cottonseed meal improves soil's texture and helps retain moisture. It
serves as a good source of natural fertilizers in dry areas due to its tendency of keeping
the soil moist. Cottonseed meal fertilizers can be used for roses, camellias, or vegetable
gardens (Organic Gardening for Life.com, 2013).
2.3. The Impacts of GM crops
2.3.1. Environmental impacts
Most genetically modified (GM) crops awaiting EU authorisation for cultivation
are either herbicidetolerant or pesticide-producing (or both). The environmental effects of
these crops are increasingly well documented, often from experience in North and South
America, where they are principally grown.
2.3.1.1: Effect of GM pesticide-producing crops
Kill specific pests, by secreting toxins known as Bt, which originate from a
bacterium. Peer-reviewed scientific evidence is mounting that these GM crops are:
a- Toxic to harmless non-target species
Long-term exposure to pollen from GM insectresistant maize causes adverse
effects on the behavior (Prasifka et al, 2007) and survival (Dively et al, 2004) of the
monarch butterfly, America‘s most famous butterfly. Few studies on European butterflies
have been conducted, but those that have suggest they would suffer from pesticide-
producing GM crops (Lang and Vojtech, 2006). These studies are all based on one type of
toxin, Cry1Ab, present in GM maize varieties Bt11 and MON810. Much less is known
about the toxicity of other types of Bt toxin (e.g. Cry1F, present in the GM maize 1507).
Cry1F is highly likely to also be toxic to non-target organisms (Lang and Otto, 2010).
B- Toxic to beneficial insects
GM Bt crops adversely affect beneficial insects important to controlling maize
pests, such as green lacewings (Obrist et al., 2006). The toxin Cry1Ab has been shown to
affect the learning performance of honeybees (Ramirez-Romero et al., 2008). The
environmental risk assessment under which current GM Bt crops have been assessed (in
the EU and elsewhere) considers direct acute toxicity alone, and not effects on organisms
higher up the food chain. But these effects can be important. The toxic effects to
47
beneficial lacewings came through the prey they ate. The single-tier risk assessment has
been widely criticised by scientists who (Andow and Zwahlen, 2006).
C- A threat to soil ecosystems
Many Bt crops secrete their toxin from their roots into the soil (Saxena et al.,
2002). Residues left in the field contain the active Bt toxin (Flores et al., 2005). The long-
term, cumulative effects of growing Bt maize are of concern (Icoz and Stotzky, 2008).
EU risk assessments so far fail to foresee at least two other impacts of Bt maize
(Greenpeac brief, 2011).
D- Risk for Aquatic Life
Leaves or grain from Bt maize can enter water courses (Cambers et al, 2010)
where the toxin can accumulate in organisms (Douville et al, 2009) and possibly exert a
toxic effect (Bøhn et al., 2008). This demonstrates the complexity of interactions in the
natural environment and underlines the shortcomings of the current risk assessment
(Greenpeac brief, 2011).
E- Swapping one pest for another
Several scientific studies show that new pests are filling the void left by the
absence of rivals initially controlled by Bt crops (Cloutier et al., 2008). Plant-insect
interactions are complex, are hard to predict and are not adequately risk assessed
(Greenpeac brief, 2011).
2.3.1.2: Effect of GM herbicide tolerant (HT) crops
HT are generally associated with one of two herbicides: glyphosate (the active
ingredient of Monsanto‘s herbicide Roundup used with Roundup Ready GM crops, also
sold by Monsanto), or glufosinate, used with Bayer‘s Liberty Link GM crops. Both
herbicides raise concerns, but many recent environmental studies have focussed on
glyphosate, which is associated with:
A- Toxic effects of herbicides on ecosystems
Several new studies suggest that Roundup is far less benign than previously
thought (Greenpeace and GM Freeze, 2011). For example, it is toxic to aquatic organisms
such as frog larvae (Relyea, 2005) and there are concerns that it could affect plants
essential for farmland birds (ACRE, 2004). Wider impacts may exist. Glyphosate is
48
associated with nutrient (nitrogen and manganese) deficiencies in GM Roundup Ready
soya, thought to be induced by its effects on soil microorganisms (Zobiole et al., 2011).
B- Increased weed tolerance to herbicide
Weed resistance to Roundup is now a serious problem in the US and South
America (Binimelis et al., 2009) where Roundup Ready crops are grown on a large scale
(Johnson et al., 2009). Increasing amounts of (Duke, 2005) glyphosate or additional
herbicides (Monsanto, 2008) are needed to control these ‗superweeds‘, adding to the
toxicity of food and the environment. Independent researchers complain about the lack of
seed material made available for tests on environmental effects (Waltz, 2009a) and are
seriously concerned because those finding adverse effects face persecution by the pro-
GM industry (Waltz, 2009b).
C- A decade of research fails to acquit GM crops
Contrary to GM industry spin, the publication ―A decade of EU-funded research‖
(European Commission, 2010) prepared by the Directorate-General for Research of the
European Commission, does not provide scientific evidence on the environmental safety
of GM plants. The vast majority of research referred to under the chapter Environmental
Impact of GMOs is mostly about the development of GM crops with plant protection
traits and has very little to do with assessing the environmental impacts (for example on
soil health or on butterflies and moths) of the pesticide-producing and herbicide-tolerant
GM crops awaiting an EU authorisation. The few projects that do examine environmental
safety raise concerns (Greenpeac brief, 2011).
2.3.2. Health Impacts
It is simply do not know if GM crops are safe for human or animal consumption.
This is reflected in the ongoing scientific controversy surrounding their safety assessment.
Independent scientific studies on the safety of GM crops for animals or humans are
severely lacking (Vain, 2007) and there is a tendency for studies conducted by researchers
with affiliations to the GM industry to give favourable results to GM crops (Diels et al.,
2011). GM crops do have the potential to cause allergenic reactions, more so than
conventional crops (Freese and Schubert, 2004). In Australia, for example, GM peas were
found to cause allergenic reactions in mice (Prescott et al., 2005). GM peas also made the
mice more sensitive to other food allergies.
49
Since the introduction of GM Bt (Cry1Ab) crops, both applicant companies and
the European Food Safety Authority (EFSA) have assumed that the Cry1Ab toxin
degrades rapidly in the human digestive system and is safe for human consumption
(Monsanto, 2002). However, new studies show there is a lack of degradation in the
human gut. This warrants further investigation as it may imply this toxin has a greater
potential to cause allergenic reactions than first thought (Guimaraes et al., 2010). Another
recent study found the Cry1Ab Bt toxin in the blood of pregnant women and their fetuses
showing that it can cross the placental boundary. This raises health concerns, although the
implications of this uptake and transference across the placenta are not yet known (Aris
and Leblanc, 2011). There are potential health risks associated with herbicides used with
GM crop cultivation. Studies indicate Roundup may be toxic to mammals (Paganelli et al,
2010) and could interfere with hormones (Richard et al., 2005). Evidence on the toxicity
of the herbicide glufosinate is so strong (EFSA, 2005) that it will have to be phased out
across Europe (E.U.Regulation, 2009). Almost all commercialised GM crops either
produce or tolerate pesticides (GM Crop Database, 2011). While pesticides are tested for
two years prior to European approval, the usual duration of safety tests for GM crops is
just 28 days, with the longest tests at 90 days, including for pesticide-producing GM
plants (Greenpeac brief, 2011).
2.4. The effects of GM cotton on other living organisms:-
Insect is one of the major plant enemies that damage about 15% of important
crops in the world (Kumar et al., 2008). Bacillus thuringiensis (Bt) is one of the important
genetic engineered gram positive bacterium that is used to control major crops pests. Bt
produced a specialize type crystalline proteins against a wide range of insects such as A,
D and E- endotoxins. The ä-Endotoxins (Cry toxins) that form a crystalline appearance
during sporulation time that cause death of insect larvae (Van Rie, 2000). Bt genes have
been transformed to many important crops including cotton that provide short and long
term tolerance against a large number of insects from order Lepidoptera, Diptera and
Coleoptera (James, 2006). Genetic engineered (Bt) crops have several advantages over
chemical pesticides as it is environmental friendly, remains for short time in soil and
provide durable resistance against wide range of insects (Krishna and Qaim, 2012). Bt
cotton plants have been widely adopted by many developed and developing countries of
50
world such as North and South America, Africa and Asia due to its quick and efficient
mode of action against a wide range of pests. Since last decades several technical, socio-
economical and environmental issues arise from the use of Bt crops as it affect a large
number of innocent non-target organisms including animals (Duan et al., 2010). Vertical
gene flow of Bt genes through pollen or seeds to non-target organism produce some
serious biosafety problems (Scorza et al., 2013). Therefore the present review provides a
baseline to describe the negative effects of Bt cotton on wide range of animal species. The
major effects of various Bt toxin alone or in combination on non-target organisms are
mentioned below.
2.4.1. Effects of Bt Toxin on Various Tissues and Organs of Animals
Gastro Intestinal Tract (GIT) is an important entry system for foreign molecules in
animals. The epithelial lining of GIT gives specific route to the foreign DNA and protein
fragments that comes from animal feeds (Aurora et al., 2011). The foreign DNA-
fragments of many important plant genes were found in blood, muscles tissues and many
other internal organs of many agriculture important animals such as broiler chickens,
calves, pigs and cattles (Tony et al., 2003). Two fragments of cry1Ab gene such as P35S
and cp4epsps, cry1Ab gene were found in liver, kidney, heart and muscle tissues of goats
(Swiatkiewicz et al., 2011). Sajjad et al. (2013) studied the presence of cry1AC gene of
cotton in digestive system of model animal mice. The mice were fed with normal feed
along with 50% mixture of crushed Bt cotton seeds. The tissue samples were taken from
stomach, intestines, blood, liver, kidney, heart and brain. The isolated DNA from all the
tested samples was screened through Polymerase Chain Reaction (PCR) with a set of
specific primer of cry1AC gene and tnos promoter. The targeted gene was found only in
intestinal tissues that affect the inner lining of intestine. They also reported that the acidic
medium of stomach degrade the foreign Bt DNA fragments.
2.4.2. Effects of Bt Toxin on Lactating Animals
Several researchers investigated the effects of Bt genes on nutrient utilization,
blood composition and other performance of dairy lactating animal that feeds on cotton
seeds. For example Mohanata et al. (2010) studied that effect of cry1Ac gene on
important nutrient utilization, blood biochemical composition and other performance of
lactating dairy cows. The tested animals were fed on both non-transgenic and transgenic
51
cotton seeds for 4 weeks. From the result they revealed that nutrient uptake, digestion
process, milk yield, composition, body physiology and blood composition were not varied
in control and non-control tested animals. The Bt protein (Cry1Ac) was not found in both
milk and plasma. They concluded that Bt protein (Cry1Ac) have no adverse effect on
qualitative and quantitative characters of lactating cows. Similar findings were noted by
Singhal et al. (2006) for lactating cows that fed on Bt cotton seeds. Singhal et al. (2006)
and Castillo et al. (2004) envisaged the effect of Cry1Ac alone or in combination with
Cry2Ab on lactating cows. The milk saturation content and milk quality was similar in
both control and treated experimental cows and no adverse morpho-physiological effects
were found. The milk and blood of ruminates, tissues of pigs and other poultry are free
from any Bt gene after feeding on Bt seeds, as it shows safer food for all animals (Huls et
al., 2008). Moreira et al. (2004) found no toxic effect of Bt toxins on digestion process of
animals. While, Sullivan et al. (2004) noted that low level of digestibility in lactating
cows feeding was similar or having higher level of Bt cotton seeds. Higher concentration
of haemoglobin and other serum compositions were noted in lactating buffalo feeding on
transgenic cotton seeds carrying Cry1Ac gene. Blood urea and creatinine concentrations
were also found similar in cows both controlled and experimental lactating cows groups
after feeding on Bt cotton seeds for 430 days (Coppock et al., 1985).
2.4.3. Bt Toxins in Animal Excretion
The lethal concentration of Cry1Ab toxin from animals faeces come to our
environment both directly and indirectly that affect target and non-target organisms.
Certain animals like pigs and cattle that feed on Bt crops to excrete toxic proteins in their
wastes by effecting targeted and non-targeted organisms (Chowdhury et al., 2003).
Foreign DNA fragments of Bt cotton was also found in the muscles of many types of
chickens (Einspanier et al., 2004).
2.4.4. Influence of Bt Cotton on Other Non-target Animals
Several researchers have studied the effect of Bt cotton on non-target herbivores.
Einspanier et al. (2001) studied the effect of Bt cotton on non-target Aphis gossypii that
feed on both Bt and non-Bt cotton. The enzyme-linked immunosorbant assay (ELISA)
was used to screen the presence of Bt proteins in A. Gossypii. Results showed that a
minute amount (=10 ng/g) of Bt protein was detected in Bt fed A. Gossypii. So, only small
52
amount of Bt protein was ingested during feeding on Bt-cotton. Zhang et al. (2012)
performed similar type of experiment by feeding A. gossypii on Bt cotton expressing
Cry1Ac protein. 11 out of 12 samples showed the presence of Bt antigen through ELISA.
Lawo et al. (2009) studied the effect of Bt and Cowpea trypsin inhibitor (CpTI) genes in
combination on Aphis gossypii. From the results they concluded that Bt gene along with
CpTI gene leads lower survival and reproductive rates in all tested organisms. But, in
second and third generation the aphid population gain immunity and fitness. Bt toxins
effect five major groups of herbivores species such as Spodoptera littorals, Apis
mellifera, monarch butterfly, spider mites Rhopalosiphum padi and two important
predators Chrysoperla carnea and coleomegilla maculate (Liu et al., 2005). The long
term application of Bt protein at pollen stage adversely affect the larvae of monarch
butterfly (Dorsch et al., 2002).
Many researchers proved that Bt cotton is safe for other living organisms. Dahi,
(2013) studied the effect of two Bt genes Cry 1Ac and Cry 2Ab of Egyptian Bt cotton on
non- target organisms i.e. arthropods (aphids, whiteflies, leafhopper green bugs and
spider mites) and other beneficial arthropods (green lacewing, ladybird coccinella, rove
beetle, Orius bugs and true spider). No significant differences were found in all tested
organisms after feeding on control and Bt cotton. Romeis et al, (2004) developed a new
method of direct application of Bt toxin to the larva of green lacewing (Chrysoperla
carnea). Their finding showed no toxic effects of Cry1Ab protein on C. carnea larvae.
Genetically engineered cotton plants have no adverse effects on non-targeted organisms
like coccinellids and spiders (Romeis et al, 2004) treated C. Carnea with Cryl Ab toxin at
higher concentration but no adverse effect was observed in all tested samples.
2.4.5. Effects of Bt Cotton on Human Health
Several antibiotics are used as marker gene to screen transgenic plants. Several
bacterial species tolerate antibiotics. So, it is a major concern to people who excessively
use antibiotic for controlling many lethal human diseases but on the other hand, it is used
in plant transformation experiments. If, these pathogens produce tolerance against
antibiotics so, it will no longer to be used for controlling human diseases. Similarly, the
horizontal transfer of marker genes or other lethal genes to other pathogens further
produce serious problem to human health and other non-target organism (Celis et al.,
53
2004). There are several reports that Bt genes cause some serious problem to human
health. Bhat et al., (2011) studied the cytotoxic and genotoxic effects of Cry1Ac toxin
from Bt cotton (RCH2) on human lymphocytes. The MIT test, cytokinesis blocked
micronucleus and erythrolysis tests showed that high dose of Cry1Ac toxin decreased the
cell survival ability up to 47.08% after 72 hours of incubation period. Only 2.52% of
micronuclei were found in test samples. The Cry1Ac toxin also showed lethal effect on
human leukocytes by their haemolytic action. It concluded that Cry1Ac toxin at higher
concentration have lethal cytotoxic and genotoxic effects on the human lymphocytes.
2.5. Genetically Modified Food controversies
GM foods are controversial and the subject of protests, vandalism, referenda,
legislation, court action and scientific disputes. The controversies involve consumers,
biotechnology companies, governmental regulators, non-governmental organizations and
scientists. The key areas are whether GM food should be labeled, the role of government
regulators, the effect of GM crops on health and the environment, the effects of pesticide
use and resistance, the impact on farmers, and their roles in feeding the world and energy
production.
Broad scientific consensus states that currently marketed GM food poses no
greater risk than conventionally produced food (Bett et al., 2010). No reports of ill
effects have been documented in the human population from GM food (Key et al.,
2008). Although GMO labeling is required in many countries, the United States Food and
Drug Administration does not require labeling, nor does it recognize a distinction
between approved GMO and non-GMO foods (Andrew Pollack, 2012).
Advocacy groups such as Greenpeace and the World Wildlife Fund claim that
risks related to GM food have not been adequately examined and managed, and have
questioned the objectivity of regulatory authorities and scientific bodies (Wikipedia,
2015b).
In Sudan Points raised against the Bt cotton release: The common debate issues on
GMOs. In addition to: The National Variety Release Committee is not Competent
Authority. A National Council for Biosafety was established later after the release. The
duration of confined greenhouse testing was inadequate. In most cases, stakeholders
(parliamentarians, civil society, farmers, …etc) are not really well informed on
54
biotechnology issues. Animal feeding testing was inadequate. The feed test was for cotton
foliage and not for seed cake. No cotton seed oil analyses. Time from initial testing to
release into the environment was very short (El Wakeel, 2014).
2.6. Genetic Modification unpredictable and risky method
There are fundamental reasons why GM organisms should not be released into the
environment. Genetic engineering inserts DNA sequences into a plant‘s genome in a
crude fashion, often causing unintended deletions and rearrangements of the plant‘s
DNA. Unexpected and unknown fragments of genetic material have been found in
commercial GM crops such as RR soya and MON810. Inserted genes can affect the
complex regulation of the genome, which is still poorly understood. Thus, scientists are
not able to predict exactly how inserted DNA will interact in the plant‘s genome. GM
crops therefore have the potential to produce unintended novel proteins or altered plant
proteins, raising concerns about their potential to cause allergies. This makes GM crops
prone to unexpected and unpredictable effects (Greenpeac brief, 2011).
2.7. Biosafety
The concept of biosafety involve assessing and monitoring the effects of possible
gene flow competitiveness and the effects on the other organisms as well as possible
deleterious effects of the products on the environment and human health and all the
products of modern biotechnology. The safety assessment is conducted in four steps such
as the description of the parent crop, the description of the transformation process, the
safety and allergenicity assessment of the gene products and metabolites, and the
combined safety and nutritional assessment of the whole plant (King et al., 2003).
2.7.1. Toxicity, Allergenicity and Nutritional Assessment
An assessment of any potential for toxicity and\or allergenicity to human and
animals or for modified nutritional value of crop should be provide idea for its risk. These
potential effects may arise from additive, synergistic or antagonistic effects of the gene
products or by these produced metabolites and may be particularly relevant where the
combined expression of the newly introduced genes has unexpected effects on
biochemical pathways. This assessment will clearly require a case-by-case approach
(European Food Safety Authority, 2007).
55
2.7.2. Nutritional assessment of GM feed
In case where composition of the GM plant differ significantly from the non GM
counterpart, a full range of physiological-nutritional study should be carried out on a
case-by-case basis with representative target animals. These study could include
digestibility, balance experiments or the determination of the nutritive value. For feeds, it
is recommended that comparative growth studies are conducted with the fast growing
livestock species such as broiler chick. Because of their rapid weight gain (The Scientific
Committee on plants, Food and Animal Nutrition, 2003).
2.8. Regulation of GM crops
The regulation of genetic engineering concerns the approaches taken by
governments to assess and manage the risks associated with the development and release
of genetically modified crops. There are differences in the regulation of GM crops
between countries, with some of the most marked differences occurring between the USA
and Europe. Regulation varies in a given country depending on the intended use of each
product. For example, a crop not intended for food use is generally not reviewed by
authorities responsible for food safety (Beckmann et al., 2011).
According to the 2013 ISAAA brief: "...a total of 36 countries (35 + EU-28) have
granted regulatory approvals for biotech crops for food and/or feed use and for
environmental release or planting since 1994... a total of 2,833 regulatory approvals
involving 27 GM crops and 336 GM events (NB: an "event" is a specific genetic
modification in a specific species) have been issued by authorities, of which 1,321 are for
food use (direct use or processing), 918 for feed use (direct use or processing) and 599 for
environmental release or planting. Japan has the largest number (198), followed by the
U.S.A. (165, not including "stacked" events), Canada (146), Mexico (131), South Korea
(103), Australia (93), New Zealand (83), European Union (71 including approvals that
have expired or under renewal process), Philippines (68), Taiwan (65), Colombia (59),
China (55) and South Africa (52). Maize has the largest number (130 events in 27
countries), followed by cotton (49 events in 22 countries), potato (31 events in 10
countries), canola (30 events in 12 countries) and soybean (27 events in 26 countries)
(ISAAA, 2013).
56
2.9. Biosafety regulations in Sudan
Sudan finalized the development of its NBF that was published in November
2005. In the framework the then Biological Safety Bill of 2005 was passed by the
National Assembly into law (http://bch.biodiv.org/default.aspx). The Act is meant ―to
ensure adequate level of protection in the field of safe transfer, handling and use of
GMOs resulting from modern biotechnology‖ with emphasis on the conservation and
sustainable use of biological diversity and human health. However, there is no known
research, field trials or commercial release of GMOs to date. Earlier on, Sudan had
banned the import of GM food in 2003 but issued a series of temporary waivers enabling
food aid shipments into the country to continue while alternatives were sort (Nang‘ayo,
2006).
57
CHAPTER THREE
MATERIALS AND METHODS
3.1. The Experimental Animals
Twelve Rabbits (12) and their first generation were used in this study, all rabbits
were mature aged (it was 7-10 months old). The species New Zealand white rabbit strain
that domesticated in Sudan were bought from Wad medani Market. Their average initial
body weight was 1350 g. The adult rabbits were divided into three groups A, B, and C,
four rabbits in each.
Each group of the adult rabbits were assigned to individual cages in a room of
6×4×3 meter with windows. The rabbits were maintained at an ambient temperature of
26o C at 9 Am minimum and 37
oC at 3 pm maximum with about 12 hours photoperiod.
The duration of the experiment was 90 days. Animal in group A were fed on GM cotton
seed cake (Seeni-1) considered as first treatment. The animals in group B were fed on
non-GM cotton seed cake (Hamid) considered as second treatment, while the animals in
group C were fed on natural food (dried bread) considered as control. The rabbit were
offered 15 g\day of cotton seed cake (CSC) for the group A and B, and equivalent amount
of dried breads for control group throughout the experimental period. Equivalent amounts
of supplementary hays and vegetables were offered to each group as fiber and other
nutrient supplement; also drinking water was offered adlibitum for each group.
The young rabbits of GM treated and non-GM treated were reared for period of 6
weeks (42 days) and considered as first generation. The young rabbits of the 1st
generation of the adult in group A that treated by GM cotton seed cake considered as
treatment while the young rabbits of the 1st generation of the adults in group B that treated
by non-GM cotton seed cake considered as control. The rabbit of the 1st generation were
offered 10g\day of cotton seed cake (CSC) for the treatment and control throughout the
experimental period. Equivalent amounts of supplementary hays and vegetables, and
adlibitum of drinking water were offered to each group.
58
3.2. The Experimental Diets and Procedures
A harvested GM cotton seed (CV-Seeni-1), and the relevant non-GM cotton seed
(CV- Hamid) were obtained from Almarkazi Market, Wad medani branch markets. The
two types of cottonseed were subjected to mechanical oil extraction to obtain the cotton
seed cake (CSC). The obtained cotton seed cake (CSC) were used as rabbit diet for group
A and B.
Supplementary foods were composites of hay, (e.g. Eleusineindica, mallow,
Fennel, Eroca and carrot tops), vegetables (e.g. Carrot and tops, Sweet pepper, Okra,
Tomato, Orange meal , Watermelon, cucumber, Eggplant and Botanical) and fruits (e.g.
Bananas and Orange), all of them were brought from Almarkazi market at each two days.
3.3. Analysis of Nutritional Content for Cotton Seeds (CS)
Samples of the obtained Cottonseeds were transferred directly to the laboratory of
the Faculty of Engineering and Technology (food technology lab) for analysis. The cotton
seed were grounded to fine particles (powder) using mortar and pestle. Two types of
analysis (the phytochemical screening and approximate analyses) were carried out on
cotton seed powders as follows:
3.3.1. The phytochemical Screening of Cottonseed
3.3.1.1. Glycosides
About 3.0 g of cottonseed powder were boiled with an aliquot of distilled water
(100 ml) and filtered. Aliquots (2 ml each) of the filtrate were tested for glycosides as
described by Sofowora (1993) and Trease and Evans (1989). The filtrate was dissolved in
2 ml of glacial acetic acid. To this solution two drops of ferric chloride solution were
added and mixed. The mixture was transferred to a narrow test tube. 2 ml conc H2SO4
were added carefully on the side of the tube using a pipette to form upper layer gradually
acquired a bluish green color which darkened on standing.
3.3.1.2. Flavonoids and Flavonones
About 2.0 g of cotton seed powder were macerated in 50 ml (1%) of hydrochloric
acid over knight filtered and the filtrate was subjected to the following tests:
a- about 10 ml from each filtrate was rendered alkaline with sodium hydroxide 10% w\v;
the yellow color indicated the presence of Flavonoids.
59
b- Shinodas tested; 5 ml of each filtrate were mixed with 1 ml concentrated HCl and
magnesium ions were added. The formation of red color indicated the presence of
Flavonoids, Flavonones and or flavonols (Harbone, 1998).
3.3.1.3. Saponins
About 2.0 g of the cotton seed powder of each were extracted with 20 ml ethanol
(50%) and filtered. Aliquot of the alcoholic extracts (10 ml) were evaporated to dryness
under reduced pressure. The residue was dissolved in distilled water (2 ml) and filtered.
The filtrate was vigorously shaken; if a voluminous was developed and persisted for
almost one hour, this indicated the presence of Saponins (Harbone, 1998).
3.3.1.4. Tannins
About 5.0 g of cotton seed powder were extracted with ethanol (50%) and
filtered. Ferric chloride reagent (5% w\v in methanol) was added. The appearance of
green colour which changed to bluish black colour or precipitate indicated the presence of
tannins (Harbone, 1998).
3.3.1.5. Sterols and\or Tri-terpenes
About 1.0 g of cotton seed powder of each sample was extracted with petroleum
ether (10 ml) and filtered. The filtrate was evaporated to dryness and the residue was
dissolved in chloroform (10 ml). Aliquots of chloroform extract (3 ml) were mixed with
concentrated acetic acid anhydride (3 ml), and a few drops of sulphuric acid were added.
The formation of a radish violet ring at the junction of the two layers indicated the
presence of unsaturated sterols and\or Triterpens (Harbone, 1998).
3.3.1.6. Alkaloids
About 5.0 g of cotton seed powder of each cottonseeds were extracted with
ethanol and filtered. 10ml of ethanolic extract were mixed with hydrochloric acid (10 ml;
10% v/v) and filtered. The filtrate was rendered alkaline with ammonium hydroxide and
extracted with successive portions of chloroform. The combined chloroform-extract was
evaporated to dryness. The residue was dissolved in hydrochloric acid (2 ml; 10% v/v)
and tested with mayers reagent, and dragendorffs reagent, respectively. The formation of
precipitate indicated the presence of alkaloids and\or nitrogenous bases (Harbone, 1998).
60
3.3.2. Proximate Analysis of Cotton seed
3.3.2.1. Moisture Content
Moisture content were carried out according to AACC (1983), whereby 5 g of
each sample was weighed into a pre-dried, clean weighed porcelain dish. The samples
were then placed in an air oven adjusted to 130oC for 3 hours, the samples were then
removed from the oven, and cooled in a desicator at room temperature and weighed.
Moisture and dry matter (D.M) content were calculated according to the following
formula:
Moisture content (M.C) =
D.M = 100- % moisture
3.3.2.2. Ash Content
The various samples were analyzed for their Ash content by the procedure
described by AOCS (1985). Five grams of ground cotton seed powder were weighed into
previously heated, pre-dried and pre-weighed crucible. This crucible with its contents was
then placed in muffle furnace at 550oC and maintained at this Temperature for 5 hours.
The crucibles were then transferred to desicator, cooled at room temperature and
reweighed. Ash content was then calculated on dry matter basis as follows:
Where:
a = weight of empty dish.
b = weight of dish with Ash.
M = weight of sample (gm).
F = moisture content of the sample .
3.3.2.3. Oil content
Oil contents of the various samples were determined according to AOCS (1985).
In this method, three grams of ground sample were weighed into filter paper folded in
such a way so as to prevent escape of the meal. apiece of absorbent cotton was placed on
the top of thimble to distribute the solvent as it drops on the sample. The wrapped sample
was then placed in the extraction tube of the soxhlet and then 150 mg of n-hexane (99%
61
purity) was poured in the extraction flasks before fixing the tubes, after 6 hours of
extraction the extraction flasks were disconnected. The hexane was recovered by
distillation under the vacuum. Last traces of Hexane were removed by putting the flask in
the oven. The flasks were then cooled at room temperature in desicator and weighed. Oil
content was calculated as follows:
Oil% =
Oil%(on D.M basis) =
3.3.2.4. Protein content
Protein content of each tested sample was determined according to AACC (1983) in
which one gram sample was digested using 25 ml of concentrated sulfuric acid for 6
hours. Then the digested sample was transferred to 100 ml volumetric flask. Five ml of
the clean digested sample was pipetted into distillation unit and then 10 ml of 40% NaOH
was poured in the funnel. The ammonia trapped in boric acid (2%) was titrated against
0.1 N HCl solution, a faint pink color was taken as end point.
The protein percentage was calculated as follows:
Crude protein % =
Protein% (on D M basis) =
3.3.2.5. Crude fiber content
Crude fiber contents were determined for the various samples according to AOCS
(1985). Three gram of the defatted samples were weighed into 600 ml beaker. Then
200ml of boiling 1.25% sulfuric acid and one drop of diluted antifoam agent were added.
The content were boiled under reflex for 30 minutes and filtrated through Buchner funnel.
The residue was then transferred back into the beaker using 200 ml of 1.25 % boiling
sodium hydroxide, and boiled under reflux for 30 minute. The content were again filtered
and transferred to a pre-dried and weighed dish. It was then dried at 100oC to constant
weight. The contents were then reweighed and ignited in muffle furnace at 550oC for 5
hours. The crude fiber content was calculated as follows:
Crude fiber % (on D.M. basis) =
Where:
62
a= weight of dish content before ashing
b= weight of dish content after ashing .
M= moisture content.
W= weight of sample.
3.3.2.6. Carbohydrates content
Carbohydrate of cottonseed were obtained by subtraction.
Carbohydrate Content = 100 - (protein % + oil % + fiber % + ash % + moisture %).
3.3.3: Determination of Minerals
Determination of minerals was done by flame photometer instruction as flowing:-
3.3.3.1. Sodium and Potassium
From the stock solution 10000 ppm for Na and K, a 5 concentration of 0.0 ppm,
25 ppm, 50 ppm, 75 ppm and 100 ppm were prepared. The 0.0 ppm and 100 ppm were
used to adjust the flame photometer and the rest of 25 ppm, 50 ppm and 75 ppm were
read for drawing the curve. The samples were read by flame photometer and the obtained
data were converted to concentration of ppm using drowned curve. The latest value were
converted to percentage using the flowing equation;-
3.3.3.2: Calcium:-
From the stock solution 100 ppm for Ca, a five concentrations of 0.0 ppm, 25
ppm, 50 ppm, 75 ppm and 100 ppm were prepared. The 0.0 ppm and 100 ppm were used
to adjust the flame photometer and the rest of 25 ppm, 50 ppm and 75 ppm were read for
drawing the curve. The samples were read by flame photometer and the obtained data
converted to concentration of ppm using drowned curve. The latest value were converted
to percentage using the flowing equation;-
3.4. Body weight gain and internal organ weight of experimental rabbits
A total of body weight gain were taken each 30 days started from the first day,
using electronic scale, to determine the effect on the total growth. A weight of internal
63
organs of the experimental rabbits were taken at the end of the experiment period to
determined the histological effects of the GM cottonseed cake on the internal organs.
3.5. Sampling and Analysis of Rabbits Blood
3.5.1. Sampling blood for hematological and Biochemical analysis
Blood samples of 5.0 ml were collected from the jugular vein of the rabbits after
90 days using microinjection. For hematology test about 2.0 ml of blood were poured in
clear container containing the anticoagulant EDTA so as to avoid clotting. For
biochemical tests about 3.0 ml of blood were taken and poured in container containing
lithium hebarin to avoid clotting.
3.5.2. Whole Blood (WB) Mode (Hematological analysis)
Blood sample of 2.5 ml was taken in a container containing the anticoagulant
EDTA to avoid clotting. Sysmex KX 21N model was used for counting blood cell, light
microscopy for morphology using immersion oil objective. The collected blood samples
were put on the hematology mixer machine that mixed the samples, and the samples were
given to the cell counter device to determine the blood cell: white blood cells (WBC), red
blood cells (RBC), platelets (PLT), packed Cell Volume (PCV), mean corpuscular
haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), mean
corpuscular volume (MCV) and neutrophils and lymphocytes, then a thin film was made
and air dried and rapidly placed on staining rack, flooded with lesihman stain and left for
one minute fixed and then added as twice as much of buffer distilled water (pH 7.2) and
left to stain for ten minutes and then washed off the stain with tap water. When measuring
blood analysis using Sysmex instrument, sample analysis can be executed when the
instrument is in the ready status, and tube setting was performed manually.
3.5.3. Blood Serum analysis (Biochemical analysis):
3.5.3.1: Renal function tests Renal function parameters were creatinine, urea, Na+ and K+. These tests in
addition to the liver function parameters and lipid profile were run at the Quality Medical
Laboratory, Wad Medani, Gezira State, Sudan.
Creatinine: The assay is based on reaction of creatinine with sodium picrate as described
by Jaffe method. Creatinine reacts with alkaline picrate forming a red complex. The time
64
interval chosen for measurements avoids interferences from other serum constituent. The
intensity of the colour formed is proportional to the creatinine concentration in the sample
(Spinreact, 2013).
Urea: Urea in the sample originates by means of the coupled reactions described below
and, which showed a coloured complex that can be measured (BioSystem, 2014).
Urea + H2O ------ urease -----> 2NH4+ + CO2
NH4+ + salicylate + NaCl -----nitroprusside----> indophenol
Sodium and Potassium ions procedure:
The 9180 electrolyte analyzer methodology is based on the ion selective electrode
(ISE). There are six different electrodes used in the 9180 electrolyte analyzer: sodium,
potassium, chloride, ionized calcium, lithium and a reference electrode. Each electrode
has an ion selective membrane that undergoes a specific reaction with the corresponding
ions contained in the sample being analyzed . the membrane potential, or measuring
voltage, which is built up in the film between the sample and the membrane. A galvanic
measuring chain within the electrode determines the difference between the two potential
values on either side of the membrane. The galvanic chain is closed through the sample
on one side by the reference electrode, reference electrolyte and the open terminal the
membrane, inner electrolyte and inner electrode close the other side. A difference in ion
concentrations between the inner electrolyte and the sample causes an electro-chemical
potential to form across the membrane of the active electrode. the potential is conducted
by a highly conductive, inner electrode to an amplifier. The reference electrode is
conducted to ground as well as to the amplifier. The ion concentration in the sample is
then determined by using a calibration curve determined by measured points of standard
solution with precisely known ion concentrations (9180 Electrolyte analyzer Abril, 1996).
Calibration procedure: A 2-point or a 3-point calibration is performed automatically
every 4 hours in ready mode and a 1-point calibration is automatically performed with
every measurement (9180 Electrolyte analyzer abril, 1996 ).
3.5.3.2. Liver function tests
Liver function parameters: Protein, albumin, bilirubin, ALP, ALT, and AST tests
were run following Spinreact, (2013) and BioSystem, (2014).
65
Total protein (TP): Proteins give an intensive violet-blue complex with copper salts in
an alkaline medium (iodide was included as antioxidant). The intensity of the colour
formed is proportional to the total protein concentration in the sample.
Albumin: Albumin in the presence of bromcresol green at a slightly acidic pH, produced
a colour change of the indicator from yellow-green to green-blue. The intensity of the
colour formed is proportional to the albumin concentration in the sample.
Bilirubin: Bilirubin was converted to coloured azobiliruin by diazotized sulfanilic acid
and measured photometrically. Of two fractions present in serum, bilirubin-glucuromide
and free bilirubin loosely bound to albumin, only the former reacts directly in aqueous
solution, while free bilirubin requires solubilization with dimethylsulfoxide to react. In
the determination of indirect bilirubin, the direct is also determined; the results were
corresponded to total bilirubin. The intensity of the colour formed is proportional to the
bilirubin concentration in the sample.
Alkaline phosphatase (ALP): Alkaline phosphatase catalyzes in alkaline medium the
transfer of the phosphate group from 4-nitrophenylphosphate to 2-amino2-methyl-1-
propanol (AMP), liberating 4-nitrophenol. The catalytic concentration was determined
from the rate 4-nitrophenol formation:
4-nitrophenylphosphate + AMP -----ALP ----> AMP -phosphate + 4-nitrophenol
Aspartate aminotransferase (AST/GOT): Aspartate aminotransferase catalyzes the
transfer of the amino group from aspartate to 2-oxoglutarate, forming oxalacetate and
glutamate. The catalytic concentration was determined from the rate of decrease of
NADH, measured at 340 nm, by means of malate dehydrogenase (MDH) coupled
reaction.
Aspartate + 2-oxoglutarate ------ AST ------ > Oxalacetate + Glutamate
Oxalacetate + NADH + H+ ----- MDH --- > Malate + NAD+ .
Alanine aminotransferase (ALT): Alanine aminotransferase catalyzes the transfer of the
amino group from alanine to 2- oxoglutarate, forming pyrovate and glutamate. The
catalytic concentration was determined from the rate of decrease of NADH measured at
340 nm, by means of lactate dehydrogenase (LDH) coupled reaction.
Alanine + 2 - Oxoglutarate ------ ALT ----- > Pyrovate + Glutamate
Pyrovate + NADH + H+ ----- LDH ----> Lactate + NAD+
66
3.5.3.3. Lipid Profile test
Plasma was separated by centrifugation at 3500 rpm for 15 minutes. Plasma total
cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein
cholesterol (LDL-C), triglyceride (TG) concentrations, were measured by commercial
enzymatic test kits according to the manufacturer‘s instructions (BioSystem, 2014) using
an automatic analyzer (Type 7170A, Hitachi, Tokyo, Japan).
3.6. Dissection of the Rabbits and weighting the Internal Organs
After the end of experimental period, the rabbits were dissected. During the
dissection, the abdomen was opened and the whole internal organs were removed out and
placed in special container containing saline water, then each organ like heart, lungs,
liver, spleen, and kidneys were removed and weighted immediately. The whole internal
organ were reserved in container containing 10% formalin. The relative organs weights
were calculated according to Farag (2006).
Organs weights% = (weight of organ ÷ live body weight) × 100
3.7. Analysis of The Meat Nutrient Content
Samples of the meat were taken from the rabbits muscles after the dissection, they
were then transferred directly to the laboratory of the Faculty of Engineering and
Technology (Food Technology Laboratory) where tests were done. The approximate meat
nutrient content and mineral were analyzed as mentioned above for cottonseed.
3.8. Data Analysis (Statistical Analysis)
Microsoft office, excel 2007 was used to analyze the data obtained. ANOVA two
factor without replication (f ; f-crit. for rows and columns levels) was used to describe the
observed variations between the control, non-GM treated and the GM treated samples.
The regression analysis was also used to describe the relation between the observed
increased in the growth parameters in accordance to the intervals of the test period. The
correlation coefficient R2 (which reflects the status of homogeneity), intercept (the
expected value corresponding to day zero), x-coefficient (the constant rate of
increase/day) and the standard error in X variable (SE-X) and in Y variable (SE-Y) were
found and recorded.
67
CHAPTER FOUR
RESULTS AND DISCUSSION
4.1: Phytochemical Characteristics of the Cottonseed:
From the result shown in Table (4.1) for the evaluation of the GM cottonseed
(GM) and non-GM cottonseed (non-GM) in term of phytochemical characteristics. The
results showed the presence of Alkaloids, glycosides, sterols and flavonoids in both type
of cottonseed (GM and non-GM). The presence of Alkaloids which act on a diversity of
metabolic systems in humans and other animals, they almost uniformly evoke a bitter
taste (Rhoades, 1979). The glycosides in animals and humans, often bound to sugar
molecules as part of their elimination from the body (Nic et al., 2006). The sterols
(corticosteroids), such as cortisol act as signaling compounds in cellular communication
and general metabolism (Lampe et al., 1983). The flavonoids may also act as chemical
messengers, physiological regulators, and cell cycle inhibitors (Galeotti et al., 2008).
On the other hand, the GM cottonseed and non-GM cottonseed have no saponins
and tannins. The saponins are used widely for their effects on ammonia emissions in
animal feeding. The mode of action seems to be an inhibition of the urease enzyme,
which splits up excreted urea in feces into ammonia and carbon dioxide (Zentner, 2011).
The tannins have traditionally been considered antinutritional for animals (Muller-Harvey
and McAllan, 1992).
It was clear that, both GM and non-GM cottonseed contain the same qualitative
phytochemical composition, irrespective of their quantitative values.
68
Table (4.1): The Phytochemical Characteristic of GM and Non-GM Cotton Seeds
Components GM Cotton Non-GM Cotton
Alkaloids + +
Glycosides + +
Saponin - -
Sterols or Triterpens + +
Tannins - -
Flavonoids and Flavonones + +
69
4.2: The Proximate Composition of GM and Non-GM Cotton Seeds:
The data obtained from the evaluation of the proximate composition of GM
cottonseed and non-GM cotton seed were presented in Table (4.2). The moisture content
was 7.75% in GM (the least value) and 8.41% in non-GM (the highest value). The fiber
contents were 2.99% in GM (the highest value) and 1.64% in non-GM (the least value).
The ash was 4.42% in GM and 4.43% in non-GM. The crude protein content was 21.59%
in GM (the highest value) and 20.13% in non-GM (the lowest value). The crude fat
content were 19.0 % in GM (the least value) and 27.89% in non-GM (the highest value).
The carbohydrates content were 45.25% in GM (the highest value) and 37.50% in non-
GM (the least value).
Although that, the variation in ash, moisture and fiber were relatively small, while
the variation in fat and carbohydrates were relatively high, but the statistical analysis
revealed a non significant difference (f= 0.006; f-crit= 6.61). It was clearly that the GM
cottonseed was rich in carbohydrates and protein than non-GM cottonseed. While the
non-GM cottonseed were rich in fat content. Adelola and Ndudi. (2012) found that, the
proximate compositions of cottonseed are Carbohydrate (57.06%), fat (13.30%), crude
fiber (0.5%), ash (1.5%), moisture content (7.21%), and crude protein (15.40%).
70
Table (4.2): The Proximate Composition of GM and Non-GM Cotton Seeds
Components (%) GM
Cotton
Non-GM
Cotton Average Variance
Moisture 7.75 8.41 8.08 0.22
Fiber 2.99 1.64 2.32 0.91
Ashes 4.42 4.43 4.43 5E-05
Protein 21.59 20.13 20.86 1.06
Fat 19.00 27.89 23.45 39.52
Carbohydrates 45.25 37.50 41.38 30.03
ANOVA
Source of Variation SS df MS F P-value F crit
Rows 2207.099 5 441.420 30.80 0.0009 5.05
Columns 0.083 1 0.083 0.005 0.9 6.61
71
4.3: The Mineral Content of Cotton Seeds:
The data obtained from the evaluation the mineral composition of GM cottonseed
and non-GM cottonseed were presented in Table (4.3). The Sodium (Na+) content was
0.00462% in GM (the highest value) and 0.00352% in non-GM (the lowest value). The
Potassium (K+) content were 0.00432% in GM (the lowest value) and 0.00556% in non-
GM (the highest value). The Calcium (Ca++
) content were 0.24% in GM (the lowest
value) and 0.30% in non-GM (the highest value). The Organisation for economic co-
operation and development, (2009) reported that the mineral content of Na, K and Ca in
cottonseed kernel roasted using reported moisture content of 4.65 % was 0.0262%, 1.417
and 0.105 respectively.
The statistical analysis reveal that, the variation in sodium, potassium and calcium
were very small (non significant difference; f = 1.01 ; f-crit = 18.51). The total sum of
sodium, potassium and calcium in the cottonseed were, 0.249 in GM (the lowest value),
0.309 in non-GM (the highest value). It was clear that, the GM cottonseed cake contained
lower mineral content than Non-GM cottonseed.
72
Table (4.3): The Mineral Content (%) of GM cottonseed and Non-GM Cottonseed
Element (%) GM Cotton Non-GM
Cotton Average Variance
Sodium (Na+) 0.00462 0.00352 0.004 6.05E-07
Potassium (K+) 0.00432 0.00556 0.005 7.69E-07
Calcium (Ca++
) 0.24 0.30 0.27 0.002
ANOVA
Source of Variation SS Df MS F P-value F crit
Rows 0.093984 2 0.047 78.41 0.01 19
Columns 0.000603 1 0.001 1.01 0.4 18.51
73
4.4: The Effects of GM and Non-GM Cottonseed Cake on White Blood
Cells (WBC) Indices:-
The data obtained for evaluation of the effect of the genetically modified
cottonseed cake (GMCSC) and non-genetically modified cottonseed cake (non-GMCSC)
that fed to rabbit for period of 90 days on white blood cells (WBCs) Indices were
presented in table (4.4). The WBCs number (in term of x103\µL) were 8.63 in control (the
highest value), 6.9 in non-GM and 5.30 in GM (the least value). The lymphocytes number
(lymph) count (in term of x103\µL) were 3.17 in control, 3.70 in non-GM (the highest
value) and 2.90 in GM (the least value). The intermediate cells number (Mid) in term of
x103\µL were 0.90 in control (the highest value), 0.60 in non-GM and 0.45 in GM (the
least value). The neutrophilic-granulocyte number (Gran) in term of x103\µL were 4.87 in
control (the highest value), 2.60 in non-GM and 1.95 in GM (the least value). All value
within reference range.
The statistical analysis revealed that, the variance in (lymph) and (Mid) were
relatively small, i.e. the variations (dispersion) in the lymph and mid were
correspondingly small, while the variance of WBC count and Gran was relatively large,
and hence, the variation in count was correspondingly large. The mean counts for WBC,
Lymph, Mid and Gran in the rabbit blood were, 4.39 in control group (the highest value),
3.45 in non-GM and 2.65 in GM (the lowest value).
It was clear that, the GM cottonseed cake decreased the WBC counts in the rabbit
after 90 days of feeding. This finding does not corresponding with Amao et al. (2012)
who found that, WBC and lymphocytes increased significantly (P<0.05) with increasing
level of cottonseed cake (CSC).
The statistical analysis also revealed that, there was significant difference in the
WBCs indices count (F= 26.32 ; F-.crit= 4.76 ; p = 0.00075), while there was a non-
significant difference between the tested groups (f= 3.97 ; f-crit= 5.14 ; p = 0.08) i.e. the
WBC counts in the rabbit fed on GM cottonseed cake were similar to that fed in non-GM
cottonseed cake. It was clear that, the two types of cottonseeds were similar in their
effects on WBC count as same as control.
74
Table (4.4): The Effects of GM and non-GM Cottonseed Cake on White Blood Cells
(WBC) Indices of the Rabbits After 90 Days of Feeding
Parameter Control Non-GM-t GM-treat Average Variance
White Blood Cells
(WBC) 103/µl
8.63 6.90 5.30 6.94 2.77
Lymphocytes (Lymph)
103/µl
3.17 3.70 2.90 3.26 0.17
Intermediate Cells
(Mid) 103/µl
0.90 0.60 0.45 0.65 0.053
Neutrophilic-
granulocytes (Gran)
103/µl
4.87 2.60 1.95 3.14 2.35
ANOVA
Source SS df MS F P-value F crit
Rows 60.50 3 20.17 26.32 0.0008 4.76
Columns 6.09 2 3.04 3.97 0.08 5.14
75
4.5: The Effects of GM Cottonseed Cake and Non-GM Cottonseed Cake
on Red Blood Cells (RBCs) Indices:
The data obtained for evaluation of the effect of the genetically modified
cottonseed cake (GMCSC) and non-genetically modified cottonseed cake (Non-GMCSC)
that fed to rabbit for period of 90 days on red blood cells (RBCs) indices were presented
in Table (4.5). The RBCs number (in term of x106\µL) were 5.04 in control (the highest
value), 4.79 in non-GM and 4.58 in GM (the lowest value). All of them within the
reference range of 3.7-7.5 106\µL reported by HewItt et al. (1989). The haemoglobin
(HGB) count (in term of g\dL) were 10.63 in control (the highest value), 10.03 in non-
GM and 9.98 in GM (the lowest value). The hematocrit (HCT or PCV) percent were
34.33 in control (the highest value), 31.75 in non-GM and 31.53 in GM (the lowest
value). The mean corpuscular volume (MCV) in term of fL were 68.08 in control, 66.55
in non-GM (the lowest value), and 69.10 in GM (the highest value). The mean
corpuscular haemoglobin (MCH) in term of pg were 20.95 in control, 20.93 in non-GM
(the lowest value) and 21.75 in GM (the highest value). The mean corpuscular
haemoglobin concentration (MCHC) percent were 30.90 in control and 31.53 equal in
non-GM and GM. The red cell distribution width coefficient of variation (RDW-CV)
were 16.48 in control (the lowest value), 17.13 in non-GM (the highest value) and 16.55
in GM. The red cell distribution width standard deviation (RDW-SD) were 39.93 in
control (the lowest value), 40.78 in non-GM and 41.58 in GM (the highest value). All
values of RBCs indices were within the reference range.
The statistical analysis revealed that, The variations in RBC, HGB, MCH, MCHC,
RDW-CV and RDW-SD were relatively small, i.e. the variations (dispersion) in RBC,
HGB, MCH, MCHC, RDW-CV and RDW-SD were correspondingly small, while in
HCT and MCV the variance was relatively large, and hence, the variation in count was
correspondingly large. The mean counts for RBCs indices in the rabbit blood were, 28.29
in control group, 27.94 in non-GM (the lowest value) and 28.33 in GM (the highest
value).
76
Table (4.5): The Effects of GM Cottonseed Cake and Non-GM cottonseed cake on Red
Blood Cells (RBCs) indices of the Rabbits After 90 Days of Feeding
Parameter Control Non-GM GM Average Variance
Red Blood Cells (RBC) 106\µL 5.04 4.79 4.58 4.80 0.053
Haemoglobin (HGB) g/dL 10.63 10.03 9.98 10.21 0.131
Hematocrit (HCT /PCV) % 34.33 31.75 31.53 32.54 2.424
Mean Corpuscular Volume (MCV)
fl 68.08 66.55 69.10 67.91 1.647
Mean Corpuscular Haemoglobin
(MCH) pg 20.95 20.93 21.75 21.21 0.219
Mean Corpuscular Haemoglobin
Concentration (MCHC) % 30.90 31.53 31.53 31.32 0.132
Red Cell Distribution Width
Coefficient of Variance (RDW-CV) 16.48 17.13 16.55 16.72 0.127
Red Cell Distribution Width
Standard Deviation (RDW-SD) 39.93 40.78 41.58 40.76 0.681
ANOVA
Source SS df MS F P-value F crit
Rows 8444.51 7 1206.36 1674.7 2.32E-19 2.76
Columns 0.74 2 0.37 0.52 0.607486 3.74
77
It was clear that, the GM cottonseed cake increased the RBC indices counts in the
rabbit after 90 days of feeding. This finding in RBC and the previous finding for WBC
agreed with Kranthi, (2012) who stated that: ―Interestingly feeding of Bt-cotton seed
increased RBC and decreased WBC in blood‖.
The statistical analysis also revealed that, there was significant difference in the
RBCs indices parameters (F= 1674.70 ; F-.crit= 2.76 ; p = 2.32E-19), while there was a
non-significant difference between the tested groups (f= 0.52; f-crit= 3.74 ; p = 0.61) i.e.
the RBCs indices counts in the rabbit fed on GM cottonseed cake were similar to that fed
in non-GM cottonseed cake. It was clear that, the two types of cottonseeds were similar in
their effects on RBCs indices count as same as control.
4.6: The Effects of GMCSC and Non-GMCSC on Blood Clotting Indices
The data obtained for evaluation of the effect of the genetically modified
cottonseed cake (GMCSC) and non-genetically modified cottonseed cake (Non-GMCSC)
that fed to rabbit for period of 90 days on clotting indices were presented in Table (4.6).
The platelet (PLT) number in term of 103/µl were 293.0 in control (the highest value),
262.5 in non-GM and 281.0 in GM (the lowest value) all of them within the reference
range of 112-795 103/µl reported by HewItt et al. (1989). The mean platelet volume
(MPV) count in term of fL were 6.53 in control (the lowest value), 8.33 in non-GM (the
highest value) and 6.65 in GM. The mean platelet distribution width (PDW) were 15.33
in control 16.45 in non-GM (the highest value) and 15.13 in GM (the lowest value). The
platelet hematocrit (PCT) percent in whole blood count were 0.20 in control (the highest
value), 0.13 in non-GM (the lowest value) and 0.17 in GM.
The statistical analysis revealed that, the variance in PDW and PCT were
relatively small, i.e. the variations (dispersion) in those were correspondingly small, while
in PLT the variance was very large, and hence, the variation in count was correspondingly
large. The mean counts for blood clotting indices in the rabbit blood were, 78.77 in
control group (the highest value), 71.85 in non-GM (the lowest value) and 75.74 in GM.
It was clear that, the GM cottonseed cake do not affect the clotting indices counts in the
rabbit after 90 days of feeding.
78
Table (4.6): The Effects of GMCSC and Non-GMCSC on Blood Clotting Indices of
the Rabbits After 90 days of feeding
Parameter Control
Non-
GM GM Average Variance
Platelets (PLT) 103/µl 293.0 262.5 281.0 278.83 236.08
Mean Platelets Volume (MPV)
fL 6.53 8.33 6.65 7.17 1.01
Platelet Distribution Width
(PDW) 15.33 16.45 15.13 15.64 0.51
Platelet Hematocrit (PCT) % 0.201 0.133 0.165 0.17 0.001
ANOVA
Source SS Df MS F P-value F crit
Rows 165816.6 3 55272.2 874.63 2.6E-08 4.76
Columns 96.0377 2 48.01885 0.76 0.5 5.14
79
The statistical analysis also revealed that, there was significant difference in the
clotting indices parameters (F= 874.63; F-.crit= 4.76 ; P-value= 2.6E-08), while there was
a non-significant difference between the tested groups (f= 0.76 ; f-crit= 5.14 ; p = 0.51)
i.e. the clotting indices counts in the rabbit fed on GM cottonseed cake were similar to
that fed in non-GM cottonseed cake. It was clear that, the two types of cottonseeds were
similar in their effects on clotting indices count as same as control. This finding agreed
with Yang et al. (2013) who reported that ―After 70 days of feeding of transgenic poplar
(Populus cathayana Rehd) leaves with binary insect-resistance genes and non transgenic
poplar, all hematological and biochemical parameters fell within normal ranges in both
the treated and control rabbits, and there was no significant difference between the 2
groups.
4.7: The effects of GMCSC and Non-GMCSC on Liver Functions
The data obtained for evaluation of the effect of the genetically modified
cottonseed cake (GMCSC) and non-genetically modified cottonseed cake (Non-GMCSC)
that fed to rabbit for period of 90 days on the liver function indices were presented in
Table (4.7). The Albumin number in term of g\dL were 4.68 in control (the lowest value),
5.55 in non-GM and 5.80 in GM (the highest value) this value were high than reference
range (2.7- 4.6 g\dL) reported by Research Animal Resources. (2003). The alkalin
phosphate (ALP) count in term of U\L were 88.0 in control (the highest value), 38.0 in
non-GM (the lowest value) and 61.5 in GM all values were within the reference rang (17
– 192 U\L) reported by HewItt et al. (1989). The Alanine aminotrans (ALT) in term of
U\L were 48.00 in control (the least value), 78.75 in non-GM (the highest value) and
67.75 in GM, the Non-GM and GM was increased above the reference range (12-67 U\L)
reported by Research Animal Resources (2003). The Aspartate aminotrans (AST) count
were 65.00 in control, 99.25 in non-GM (the highest value) and 59.25 in GM (the lowest
value), the values were within the reference rang (14-113 U\L) reported by Research
Animal Resources. (2003). The AST\ALT ratio were 1.35 in control (the highest value),
1.26 in non-GM and 0.88 in GM (the lowest value). The Total Bilirubin in term of mg\dL
were 0.13 in control (the highest value) and same as 0.08 in non-GM and GM (the least
value). The Total protein count in term of g\dL were 6.78 in control, 6.40 in non-GM (the
least value) and 7.13 in GM (the highest value).
80
Table (4.7): The Effects of GMCSC and Non-GMCSC on Liver Functions Indices of
the Rabbits After 90 days of Feeding
Parameter Control Non-
GM GM Average Variance
Albumin (g\dL) 4.68 5.55 5.80 5.34 0.35
Alkalin phosphate(ALP) U/L 88.0 38.0 61.5 62.5 625.75
Alanine aminotrans (ALT) U/L 48.00 78.75 67.75 64.83 242.77
Aspartate aminotrans (AST) U/L 65.00 99.25 59.25 74.5 467.69
AST\ALT ratio 1.35 1.26 0.88 1.16 0.06
Total Bilirubin (mg\dL) 0.13 0.08 0.08 0.1 0.001
Total protein (g\dL) 6.78 6.40 7.13 6.77 0.13
ANOVA
Source SS df MS F P-value F crit
Rows 21358.01 6 3559.67 16.29 3.93E-05 2.99
Columns 52.03 2 26.01 0.12 0.9 3.89
81
The statistical analysis revealed that, The variance in Albumin, AST\ALT ratio,
total Bilirubin and Total Protein were relatively small, i.e. the variations (dispersion) in
those were correspondingly small, while in ALP, AST and ALT the variance was very
large, and hence, the variation in count was correspondingly large. The mean counts for
liver function indices in the rabbit blood were, 30.56 in control group, 32.76 in non-GM
(the highest value) and 28.91 in GM (the lowest value).
It was clear that, the GM cottonseed cake decreased the liver function indices
counts in the rabbit after 90 days of feeding.
The statistical analysis also revealed that, there was significant difference in the
liver function indices parameters (F= 16.30 ; F-.crit= 3.00 ; p = 3.93E-05), while there
was a non-significant difference between the tested groups (f= 0.12 ; f-crit= 3.89 ; p =
0.89) i.e. the liver function indices counts in the rabbit fed on GM cottonseed cake were
similar to that fed in non-GM cottonseed cake. It was clear that, the two types of
cottonseeds were similar in their effects on liver function indices count as same as
control.
4.8: The Effect of GM Cottonseed on Renal Functions:-
The data obtained for evaluation of the effect of the genetically modified
cottonseed cake (GMCSC) and non-genetically modified cottonseed cake (Non-GMCSC)
that fed to rabbit for period of 90 days on the renal function indices were presented in
table (4.8). The Creatinine number in term of mg\dL were 1.11 in control (the highest
value), 1.07 in non-GM and 1.00 in GM (the lowest value), these values were within the
normal range. The Urea\Bun count in term of mg\dL were 28.25 in control (the least
value), 37.75 in non-GM (the highest value) higher than reference rang (10 – 33 mg\dL)
reported by Rosenthal (2002), and 32.00 in GM. The Sodium (Na+) in term of mmol\L
were 140.75 in control (the highest value), 139.25 in non-GM (the least value), and
139.75 in GM, all values were within the normal range of 130-150 m mol\L reported by
Research Animal Resources (2002). The potassium (K+) count were 4.38 in control (the
least value), 4.43 in non-GM (the highest value) and 4.40 in GM, all values were within
the normal range of 3.6 -7.5 m mol\L reported by Research Animal Resources (2002).
82
Table (4.8): The Effect of GM Cottonseed Cake on Renal Functions Indices of the
Rabbits After 90 days of feeding
Parameter Control Non-GM GM Average Variance
Creatinine mg\dL 1.11 1.07 1.00 1.06 0.003
Urea\Bun (mg\dL) 28.25 37.75 32.00 32.67 22.89
Sodium (Na+)
m mol\L 140.75 139.25 139.75 139.92 0.58
Potassium (K+)
m mol\L 4.38 4.43 4.40 4.40 0.001
ANOVA
Source SS df MS F P-value F crit
Rows 38217.43 3 12739.14 1977.91 2.25E-09 4.76
Columns 8.32 2 4.16 0.65 0.557071 5.14
83
The statistical analysis revealed that, The variance in Creatinine, Na + and K
+
were very small, i.e. the variations (dispersion) in those were correspondingly small,
while in Urea\BUN the variance was relatively large, and hence, the variation in count
was correspondingly large. The mean counts for renal function indices in the rabbit blood
were, 43.62 in control group (the lowest value), 45.63 in non-GM (the highest value) and
44.29 in GM.
It was clear that, the GM cottonseed cake do not affect the renal function indices
counts within groups in the rabbit after 90 days of feeding. The all values were within the
reference range except in urea\BUN the non-GM were higher than normal value, these
influence may where due to the quantity or quality of protein in Non-GMCSC diet.
The statistical analysis also revealed that, there was significant difference in the
renal function indices (F= 1977.91 ; F-.crit= 4.76 ; p-value= 2.25E-09), while there was a
non-significant difference between the tested groups (f= 0.65 ; f-crit= 5.14 ; p = 0.56) i.e.
the renal function indices counts in the rabbit fed on GM cottonseed cake were similar to
that fed in non-GM cottonseed cake. It was clear that, the two types of cottonseeds were
similar in their effects on renal function indices count as same as control.
The above finding for the effect of GMCSC on the liver and renal function was
disagreed with Antoniou, (2012) “There are evidently clear signs of toxicity especially
with respect to liver and kidney function‖.
4.9: The Effect of GM Cottonseed Cake on Lipid Profile
The data obtained for evaluation of the effect of the genetically modified
cottonseed cake (GMCSC) and non-genetically modified cottonseed cake (Non-GMCSC)
that fed to rabbit for period of 90 days on the lipid profile were presented in Table (4.9).
The Cholesterol in term of mg\dL were 40.80 in control (the highest value), 23.50 in non-
GM (the lowest value), and 36.75 in GM, all value within the reference range of 10 – 80
mg\dL reported by Rosenthal, (2002). The High density lipid cholesterol (HDL-C) in
term of mg\dL were 11.30 in control (the lowest value), 12.50 in non-GM (the highest
value) and 11.75 in GM. The low density lipid cholesterol (LDL-C) in term of mg\dL
were 24.80 in control (the highest value), 8.33 in non-GM (the least value), and 21.00 in
GM. The Triglycerides (in term of mg\dL) were 245.50 in control (the highest value),
84
Table (4.9): The Effect of GM Cottonseed Cake on Lipid Profile of the Rabbits After
90 Days of Feeding
Blood parameter Control Non-
GM GM Average Variance
Cholesterol (mg\dL) 40.80 23.50 36.75 33.68 81.88
High density lipid cholesterol
(HDL-C) mg\dL 11.30 12.50 11.75 11.85 0.37
Low density lipid cholesterol
(LDL-C) mg\dL 24.80 8.33 21.00 18.04 74.37
Triglycerides (mg\dL) 245.50 133.25 223.75 200.83 3543.89
ANOVA
Source SS Df MS F P-value F crit
Rows 73369.25 3 24456.42 32.85 0.0004 4.76
Columns 2933.51 2 1466.75 1.97 0.22 5.14
85
133.25 in non-GM (the least value) and 223.75 in GM.
The statistical analysis revealed that, The variance in HDL-C were relatively
small, i.e. the variations (dispersion) in HDL-C were correspondingly small, while in
Cholesterol, LDL-C and Triglycerides the variance was relatively large, and hence, the
variation in count was correspondingly large. The mean counts for lipid profile in the
rabbit blood were, 80.60 in control group (the highest value), 44.40 in non-GM (the
lowest value), and 73.31 in GM. It was clear that, the GM cottonseed cake do not affect
the lipid profile counts in the rabbit after 90 days of feeding.
The statistical analysis also revealed that, there was significant difference in the
lipid profile (F= 32.85; F-.crit= 4.76; p = 0.00041), while there was a non-significant
difference between the tested groups (f= 1.97 ; f-crit= 5.14 ; p = 0.22) i.e. the lipid profile
indices counts in the rabbit fed on GM cottonseed cake were similar to that fed in non-
GM cottonseed cake. It was clear that, the two types of cottonseeds were similar in their
effects on lipid profile indices count as same as control.
Those previous findings in WBC, RBC, Clotting indices, Liver function, renal
function and lipid profile were consistent with Rahman et al. (2015), who stated that:
“Similarly, no differences were observed in complete blood composition, liver enzymes,
random blood sugar or cholesterol.”
4.10: The Effects of Cottonseed Cake on Body Weight Gain:-
The data obtained for evaluation of the effect of the genetically modified
cottonseed cake (GMCSC) and non-genetically modified cottonseed cake (non-GMCSC)
that fed to rabbits for period of 90 days on the body weight gain were presented in table
(4.10). The mean initial body weight (g) were 1405 in control, 1428 in non-GM and 1255
in GM. The body weight after 30 days were 1409 in control (the lowest growth observe),
1434 in non-GM (highest growth observe) and 1300 in GM. The mean body weight after
60 days were 1516 in control (the highest growth value), 1483 in non-GM and 1320 in
GM (the lowest growth value). The mean body weight at the end (90 days) were 1645 in
control (the highest growth value), 1539 in non-GM (the lowest growth value) and 1440
in GM.
86
Table (4.10): The Effects of GM Cottonseed Cake on Body Weight Gain of the
Rabbits After 90 days of Feeding
Period \day Control Non-GM GM
0 1405 1428 1255
30 1409 1434 1300
60 1516 1483 1320
90 1645 1539 1440
Regression
Control Non-GM GM
R Square 0.89 0.91 0.88
Standard Error 46.01 18.57 33.07
Intercept 1369.7 1413.7 1242.5
Coefficients(x) 2.76 1.27 1.92
Standard Error(y) 38.50 15.54 27.67
Standard Error(x) 0.69 0.28 0.49
Significance F 0.057 0.044 0.060
87
The statistical analysis revealed that, The R squire was 0.89 , 0.91(highest value)
and 0.88 (lowest value) in control, non-GM and GM respectively. i.e. the homogeneity in
non-GM were relatively high than control and GM. It was clearly that the GM cottonseed
cake had no adversely affect on the homogeneity of growth rate after 90 days of feeding.
The coefficient (X) were 2.76 in control (highest value), 1.27 in non-GM (lowest
value) and 1.92 in GM. It was clear that the GMCSC had no adversely effects on rabbit
growth. This finding agreed with Rahman et al. (2015), who stated that: ―Bt cotton in the
diet has no adverse effect on growth and development of rabbits‖.
The statistical analysis also revealed that, the regression was significant in non-
GM (0.044), while there were no significant differences in control (0.057) and GM (0.06)
i.e. the weight gain in the rabbit fed on GM cottonseed cake were similar to that in
control. It was clear that, the GM cottonseeds were similar in their effects on weight gain
to control.
4.11: The Effect of GM Cottonseed Cake on Internal Organs of the
Rabbits:-
The data obtained for evaluation of the effect of the genetically modified
cottonseed cake and non-genetically modified cottonseed cake that fed to rabbit for period
of 90 days on the internal organ weight (in term of percentage) of the rabbits were
presented in Table (4.11). The mean Kidney weights were 0.29 in control (the least
value), 0.30 in non-GM and 0.37 in GM (the highest value). The mean heart weight %
were 0.33 in control (the highest value), 0.29 in non-GM (the least value) and 0.30 in
GM. The mean Lung weights % were 0.55 in control (the least value), 0.64 in non-GM
and 0.73 in GM (the highest value). The mean Spleen weights % were 0.04 same in
control and non-GM (the least value) and 0.05 in GM (the highest value). The Liver
weight % were 3.81 in control, 4.65 in non-GM and 5.10 in GM (the highest value).
The statistical analysis revealed that, The variance in Kidney, heart, Lung and
Spleen were very small, i.e. the variations (dispersion) in those were correspondingly
small, while in the Liver the variance was relatively large, and hence, the variation in
weight was correspondingly large. The mean percentage for internal organ weight in the
rabbit were, 1.00 in control group (the lowest value), 1.18 in non-GM and 1.31 in GM
(the highest value).
88
Table (4.11): The Relative Weight (%) of Internal Organs of the Rabbits After 90 days
of Feeding GMCSC and Non-GMCSC
Organ Control Non-GM GM Average Variance
Kidney 0.29 0.30 0.37 0.32 0.002
Hart 0.33 0.29 0.30 0.31 0.0004
Lung 0.55 0.64 0.73 0.64 0.008
Spleen 0.04 0.04 0.05 0.04 3.33E-05
Liver 3.81 4.65 5.10 4.52 0.43
ANOVA
Source SS df MS F P-value F crit
Rows 42.72 4 10.68 133.13 2.37E-07 3.84
Columns 0.24 2 0.12 1.47 0.3 4.46
89
It was clear that, the GM cottonseed cake increased the internal organ weight
percent in the rabbit after 90 days of feeding.
The statistical analysis also revealed that, there was significant difference in the
internal organ weight (F= 133.13 ; F-.crit= 3.84 ; p = 2.37E-07), while there was a non-
significant difference between the tested groups (f= 1.47 ; f-crit= 4.46 ; p-value = 0.29)
i.e. the internal organ weight in the rabbit fed on GM cottonseed cake were similar to that
fed in non-GM cottonseed cake. It was clear that, the two types of cottonseeds were
similar in their effects on internal organ weight as same as control. This finding were
similar to Joshi et al. (2001) who stated that: ―Survival, growth rate, feed intake, feed
conversion and carcass characteristics were not statistically different between boiler
chicks fed Bt cottonseed meal compared to broiler chicks fed non-Bt or conventional
cottonseed meal‖.
4.12: The Effect of GM Cottonseed Cake on Approximate Composition
of Meat Nutrient Content:-
The data obtained for evaluation of the effect of the genetically modified
cottonseed cake (GMCSC) and non-genetically modified cottonseed cake that fed to the
rabbit for period of 90 days on the approximate compositions of meat nutrient percentage,
were presented in table (4.12). The moisture percent (%) were 71.35 in control, 70.62 in
non-GM (the lowest value) and 73.98 in GM (the highest value). The ash % were 1.44 in
control, 2.09 in non-GM (the highest value) and 1.32 in GM (the lowest value). The
protein % were 25.33 in control, 26.05 in non-GM (the highest value) and 23.19 in GM
(the lowest value). The fat % were 0.99 in control (the highest value), 0.72 in non-GM
(the lowest value) and 0.84 in GM. The fiber content % were 0.89 in control (the highest
value), 0.52 in non-GM (the least value) and 0.67 in GM. These value was similar to that
reported by Tărnăuceanu et al. (2010) ―The average value of Basic chemical composition
in female rabbit meat was Moisture 74.49%, protein 22.13%, fat 1.40 % and mineral
substance 1,18%‖.
The statistical analysis revealed that, The variance in fat, fiber and ash were
relatively small, i.e. the variations (dispersion) in those were correspondingly small, while
the variance in moisture and protein was relatively large.
90
Table (4.12): The proximate Composition (%) of Meat Nutrient Content of the Rabbits
After 90 days of Feeding GMCSC and Non-GMCSC
Content (%) Control Non-GM GM Average Variance
Moisture 71.35 70.62 73.98 71.98 3.12
Ash 1.44 2.09 1.32 1.62 0.17
Protein 25.33 26.05 23.19 24.86 2.21
Fat 0.99 0.72 0.84 0.85 0.02
Fiber 0.89 0.52 0.67 0.69 0.03
ANOVA
Source of Variation SS df MS F P-value F crit
Rows 11409.81 4 2852.45 2051.85 4.49E-12 3.84
Columns 1.82E-12 2 9.09E-13 6.54E-13 1 4.46
91
It was clear that, the GM cottonseed cake decreased the protein percent in rabbit
meat rabbit after 90 days of feeding.
The statistical analysis also revealed that, there was significant difference in the
meat nutrient content (F= 2051.85 ; F-.crit= 3.84 ; p-value= 4.49E-12), while there was a
non-significant difference between the tested groups (f= 6.54E-13; f-crit= 4.46 ; p = 1.00)
i.e. the meat nutrient content in the rabbit fed on GM cottonseed cake were similar to that
fed in non-GM cottonseed cake and the control. It was clear that, the GM cottonseeds
were similar to the non-GM cottonseed in their effects on meat nutrient content as well as
control.
4.13: The Effect of GMCSC on Mineral Content of The Rabbit Meat
The data obtained for evaluation of the effect of the genetically modified
cottonseed cake (GMCSC) and non-genetically modified cottonseed cake that fed to the
rabbit for period of 90 days on the mineral content of meat percentage, were presented in
table (4.13). The sodium (Na+) percent (%) were 0.00271 in control (the lowest value),
0.00301 in non-GM (the highest value) and 0.00287 in GM. The potassium (K+) % were
0.00294 in control (the lowest value), 0.00315 in non-GM (the highest value) and
0.00308 in GM. The calcium (Ca++) % were 0.385 in control (the highest value), 0.355
in non-GM (the lowest value) and 0.375 in GM.
The statistical analysis revealed that, The variance in sodium. Potassium and
calcium were very small, i.e. the variations (dispersion) in those were correspondingly
small. The mean average were 0.130 in control (the highest value), 0.120 in non-GM (the
lowest value) and 0.127 in GM. It was clear that, the GM cottonseed do not affect the
mineral content percent in rabbit meat rabbit after 90 days of feeding.
The statistical analysis also revealed that, there was significant difference in the
meat mineral content (F= 1720.08 ; F-.crit= 6.94 ; p= 1.35E-06), while there was a non-
significant difference between the tested groups (f= 0.952753 ; f-crit= 6.94 ; p = 0.46) i.e.
the meat mineral content in the rabbit fed on GM cottonseed cake were similar to that fed
in non-GM cottonseed cake and the control. It was clear that, the GM cottonseeds were
similar to the non-GM cottonseed in their effects on meat mineral content as well as
control.
92
Table (4.13): The Mineral Content % of The Rabbit Meat After 90 Days of Feeding
GMCSC and Non-GMCSC
Element (%) Control Non-GM GM Average Variance
Sodium (Na+) 0.0027 0.003 0.0029 0.003 2.25E-08
Potassium (K+) 0.0029 0.0032 0.0031 0.003 1.14E-08
Calcium (Ca++) 0.39 0.36 0.38 0.37 0.0002
ANOVA
Source SS Df MS F P-value F crit
Rows 0.27 2 0.136 1720.08 1.35E-06 6.94
Columns 0.0002 2 7.53E-05 0.95 0.46 6.94
93
4.14: The Effects of GMCSC on WBCs Indices of 1st Generation:-
The data obtained from evaluation of the effect of the genetically modified
cottonseed cake that fed to young rabbit of 1st generation for period of 6 weeks on white
blood cells (WBCs) Indices were presented in table (4.14). The WBCs number (in term of
x109\L) were 8.15 in control (the highest value) and 8.1 in GM (the least value). The
lymphocytes number (lymph) count (in term of x109\L) were 2.15 in control (the highest
value) and 2.00 in GM (the least value). The intermediate cells number (Mid) (in term of
x109\L) were 0.55 in control (the least value) and 0.70 in GM (the highest value). The
neutrophilic-granulocyte number (Gran) in term of x109\L were 5.45 in control (the
highest value) and 5.4 in GM (the least value).
The statistical analysis revealed that, the variance in WBC indices were very
small, i.e. the variations (dispersion) in the WBC indices were correspondingly small. The
mean counts for WBC indices in the rabbit Kits blood were 4.08 in control group (the
highest value) and 4.05 in GM (the lowest value).
It was clear that, the GM cottonseed cake decreased the WBC indices counts in
the young rabbit of 1st generation after 6 weeks of feeding. This finding consists with that
in their parents.
The statistical analysis also revealed that, there was significant difference in the
WBC indices count (F= 2873.84 ; F-.crit= 9.28 ; P = 1.1E-05), while there was a non-
significant difference between the tested groups (f= 0.16 ; f-crit= 10.13; p = 0.72) i.e. the
WBC counts in the rabbit fed on GM cottonseed cake were similar to that fed in non-GM
cottonseed cake. It was clear that, the GM cottonseed cake (GMCSC) and control (non-
GMCSC) were similar in their effects on WBC indices count of the young rabbits of 1st
generation.
94
Table (4.14): The Effects of Feeding GMCSC on WBCs Indices of Young Rabbits of
1st Generation After 6 Weeks
Blood Parameter Control GM-treat Average Variance
White Blood Cells (WBC)
103/µl
8.15 8.10 8.13 0.001
Lymphocytes (Lymph) 103/µl 2.15 2.00 2.08 0.01
Intermediate Cells (Mid)
103/µl
0.55 0.70 0.63 0.01
Neutrophilic-granulocytes
(Gran) 103/µl
5.45 5.40 5.43 0.001
ANOVA
Source SS Df MS F P-value F crit
Rows 68.25 3 22.75 2873.84 1.1E-05 9.28
Columns 0.001 1 0.00125 0.16 0.7 10.13
95
4.15: The Effects of GMCSC on RBCs Indices of The 1st Generation
The data obtained from the evaluation of the effect of the genetically modified
cottonseed cake (GMCSC) that fed to rabbit kits for period of 6 weeks on red blood cells
(RBCs) indices were presented in table (4.15). The RBCs number (in term of x106\µL)
were 4.41 in control (the highest value) and 4.37 in GM (the lowest value) all of them
within the reference range. The haemoglobin (HGB) count (in term of g\dL) were 12.15
in control (the lowest value) and 12.20 in GM (the highest value). The hematocrit (HCT
or PCV) percent were 35.25 in control (the highest value) and 34.9 in GM (the lowest
value). The mean corpuscular volume (MCV) in term of fL were 80.0 equally in control
and GM. The mean corpuscular haemoglobin (MCH) in term of pg were 27.5 in control
(the least value) and 27.9 in GM (the highest value). The mean corpuscular haemoglobin
concentration (MCHC) percent were 34.4 in control (the lowest value) and 34.9 in GM
(the highest value). The red cell distribution width coefficient of variation (RDW-CV)
were 13.8 in control (the lowest value) and 14.2 in GM (the highest value). The red cell
distribution width standard deviation (RDW-SD) were 41.7 in control (the lowest value)
and 42.5 in GM (the highest value).
The statistical analysis revealed that, the variance in RBCs indices were relatively
small, i.e. the variations (dispersion) in RBCs indices were correspondingly small. The
mean counts for RBCs indices in blood of the young rabbits of 1st generation were, 31.15
in control group (the lowest value) and 31.37 in GM (the highest value).
It was clear that, the GM cottonseed cake increased the RBCs indices counts in
the young rabbits of 1st generation after 6 weeks of feeding. This finding also was consists
to that in their parents.
The statistical analysis also revealed that, there was significant difference in the
RBCs indices (F= 16498.65 ; F-.crit= 3.79 ; p-value= 3.23E-14), while there was a non-
significant difference between the tested groups (f= 2.86 ; f-crit= 5.59 ; p-value = 0.14 )
i.e. the RBCs indices counts in the rabbit fed on GMCSC were similar to that in control
(Non-GMCSC).
96
Table (4.15): The RBCs Indices of the Young Rabbits of The 1st Generation Fed on
GMCSC for 6 Weeks
Blood parameter Control GM –t Average Variance
Red Blood Cells (RBC) 106\µL 4.41 4.37 4.39 0.0008
Haemoglobin (HGB) g/dL 12.15 12.20 12.18 0.001
Hematocrit (HCT /PCV) % 35.25 34.90 35.08 0.06
Mean Corpuscular Volume (MCV) fl 80.0 80.0 80 0
Mean Corpuscular Haemoglobin
(MCH) pg 27.5 27.9 27.7 0.08
Mean Corpuscular Haemoglobin
Concentration (MCHC) % 34.4 34.9 34.65 0.13
Red Cell Distribution Width
Coefficient of Variance (RDW-CV) 13.8 14.2 14 0.08
Red Cell Distribution Width
Standard Deviation (RDW-SD) 41.7 42.5 42.1 0.32
ANOVA
Source SS Df MS F P-value F crit
Rows 7831.91 7 1118.84 16498.65 3.23E-14 3.79
Columns 0.19 1 0.19 2.85 0.134947 5.59
97
4.16: The Effects of GMCSC on Blood Clotting Indices of the 1st
Generation
The data obtained from evaluation of the effect of the genetically modified
cottonseed cake (GMCSC) that fed to rabbit kits for period of 45 days on Blood clotting
indices were presented in table (4.16). The platelet (PLT) number in term of 103/µl were
93.0 in control (the lowest value) and 111.0 in GM (the highest value) all value lower
than the reference range (112-795), these values was not issued in young rabbits. Murray,
(2006) said that, ―As expected, young rabbits had significantly lower RBC and WBC
parameters than adults‖. The mean platelet volume (MPV) count in term of fL were 9.7 in
control (the lowest value) and 10.0 in GM (the highest value). The platelet distribution
width (PDW) in term of FL were 15.8 in control (the highest value), and 15.7 in GM (the
lowest value). The platelet hematocrit (PCT) percent in whole blood count were 0.09 in
control (the lowest value) and 0.11 in GM (the highest value).
The statistical analysis revealed that, The variance in MPV, PDW and PCT were
relatively small, i.e. the variations (dispersion) in those were correspondingly small, while
in PLT the variance was very large, and hence, the variation in count was correspondingly
large. The mean counts for blood clotting indices in the rabbit kits were, 29.65 in control
group (the lowest value) and 34.20 in GM (the highest value).
It was clear that, the GMCSC increases the blood clotting indices counts in the
young rabbit of 1st generation after 6 weeks of feeding. This finding consists to that of
their parent.
The statistical analysis also revealed that, there was significant difference in the
clotting indices (F= 110.70 ; F-.crit= 9.28 ; p = 0.0014), while there was a non-significant
difference between the tested groups (f = 1.03 ; f-crit = 10.13 ; p = 0.38) i.e. the blood
clotting indices counts in the young rabbit of 1st generation fed on GMCSC were similar
to that in control (fed in non-GMCSC). It was clear that, the GMCSC and control were
similar in their effects on blood clotting indices count.
98
Table (4.16): The Blood Clotting Indices of the 1st Generation that Fed on GMCSC for
6 Weeks
Parameter Control GM –t Average Variance
Platelets (PLT) 103/µl 93.0 111 102 162
Mean Platelets Volume (MPV) fL 9.7 10.0 9.85 0.05
Platelet Distribution Width (PDW) 15.8 15.7 15.75 0.005
Platelet Hematocrit (PCT) % 0.090 0.111 0.10 0.0002
ANOVA
Source SS Df MS F P-value F crit
Rows 13344.48 3 4448.16 110.69 0.0014 9.28
Columns 41.50 1 41.50 1.03 0.3843 10.13
99
4.17: The Effects of GMCSC on Liver Functions of 1st Generation
The data obtained from evaluation of the effect of the genetically modified
cottonseed cake (GMCSC) that fed to the young rabbits of 1st generation for period of 6
weeks on the liver function indices were presented in table (4.17). The Albumin number
in term of g\dL were 3.25 in control (the lowest value) and 3.40 in GM (the highest
value). The Alkaline phosphate (ALP) count in term of U\L were 172.0 in control (the
lowest value) and 228.0 in GM (the highest value) higher than normal range which non-
specific for hepatic diseases in young growing rabbits. The Alanine aminotrans (ALT) in
term of U\L were 95.0 in control and 88.0 in GM. The Aspartate aminotrans (AST) count
were 117.5 in control (the least value) and 125.0 in GM (the highest value) these value
higher than normal range. The AST\ALT ratio count were 1.24 in control (the lowest
value) and 1.42 in GM (the highest value). The Bilirubin-direct count in term of mg\dL
were 0.2 in control and 0.0 in GM. The Total Bilirubin in term of mg\dL were 2.15 in
control (the highest value) and 1.10 in GM (the lowest value). The Total protein count in
term of g\dL were 5.55 in control and 5.70 in GM (the highest value).
The statistical analysis revealed that, the variance in Albumin, AST\ALT ratio,
Bilirubin-direct, total Bilirubin and Total Protein were relatively small, i.e. the variations
(dispersion) in those were correspondingly small, while in ALP, AST and ALT the
variance was very large, and hence, the variation in count was correspondingly large. The
mean counts for liver function indices in the rabbit blood were, 49.61 in control group(the
lowest value) and 56.58 in GM (the highest value).
It was clear that, the GM cottonseed cake increases the liver function indices
counts in the young rabbit of 1st generation after 6 weeks of feeding. These slight
elevation in liver enzymes non-specific for hepatocellular damage or hepatic diseases
when the AST\ALT ratio > 2.0.
The statistical analysis also revealed that, there was significant difference in the
liver function indices (F= 56.85 ; F-.crit= 3.79 ; p-value= 1.22E-05), while there was a non-
significant difference between the tested groups (f= 0.95 ; f-crit= 5.59 ; p = 0.361685) i.e. the
liver function indices counts in the rabbit kits fed on GMCSC were similar to control (that
fed in non-GMCSC). It was clear that, the two types of feeds were similar in their effects
on liver function indices count.
100
Table (4.17): The Liver Functions Indices of the Young Rabbits of the 1st Generation
Fed on GMCSC for 6Weeks
parameter Control GM Average Variance
Albumin (g\dL) 3.25 3.40 3.3 0.01
Alkalin phosphate(ALP) U/L 172.0 228.0 200 1568
Alanine aminotrans (ALT) U/L 95.0 88.0 91.5 24.5
Aspartate aminotrans (AST) U/L 117.5 125.0 121.3 28.1
AST\ALT ratio 1.24 1.42 1.3 0.02
Total Bilirubin (mg\dL) 2.15 1.10 0.1 0.02
Total protein (g\dL) 5.55 5.70 1.6 0.55
ANOVA
Source SS Df MS F P-value F crit
Rows 81137.65 7 11591.09 56.85 1.22E-05 3.79
Columns 194.11 1 194.11 0.95 0.4 5.59
101
4.18: Effect of GMCSC on Renal Functions Indices of the 1st Generation
The data obtained from evaluation of the effect of the GMCSC that fed to rabbit
kits for period of 45 days on the renal function indices were presented in table (4.18). The
Creatinine number in term of mg\dL were 0.45 in control (the least value) and 0.70 in GM
(the highest value). The Urea\Bun count in term of mg\dL were 17.5 in control (the
highest value) and 16.0 in GM (the least value).
The statistical analysis revealed that, The variance in Creatinine were relatively
small, i.e. the variations (dispersion) in creatinine were correspondingly small, while in
Urea\BUN the variance was relatively large, and hence, the variation in count was
correspondingly large. The mean counts for renal function indices in blood of the rabbit
kits were, 8.98 in control group (the highest value) and 8.35 in GM (the lowest value).
It was clear that, the GMCSC decreases the renal function indices counts in the
rabbit kits after 45 days of feeding.
The statistical analysis also revealed that, there was significant difference in the
renal function indices (F= 341.72 ; F-.crit= 161.45 ; p-value= 0.03), while there was a
non-significant difference between the tested groups (f= 0.51 ; f-crit= 161.45 ; p-value =
0.61) i.e. the renal function indices counts in the rabbit fed on GM cottonseed cake were
similar to that in control (fed in non-GMCSC). It was clear that, the two types of diets
(GMCSC and non-GMCSC) were similar in their effects on renal function indices count.
102
Table (4.18): The Renal Function Indices of the Young Rabbits of the 1st Generation
Fed on GMCSC for 6 Weeks
Blood parameter Control GM -t Average Variance
Creatinine (mg\dL) 0.45 0.70 0.58 0.03
Urea\Bun (mg\dL) 17.5 16.0 16.8 1.1
ANOVA
Source SS Df MS F P-value F crit
Rows 261.63 1 261.63 341.72 0.0344 161.45
Columns 0.39 1 0.39 0.51 0.6051 161.45
103
4.19: The Effect of GMCSC on The Lipid Profile of the 1st Generation
The data obtained from evaluations of the effect of the genetically modified
cottonseed cake (GMCSC) that fed to rabbit kits for period of 6 weeks on the lipid profile
were presented in table (4.19). The Cholesterol number in term of mg\dL were 35.5 in
control (the highest value) and 45.0 in GM (the least value). The HDL-C count in term of
mg\dL were 4.5 in control (the least value) and 5.0 in GM (the highest value). The LDL-
C in term of mg\dL were 30.5 in control (the least value) and 40.0 in GM (the highest
value). The Triglycerides count were 230.0 in control (the highest value) and 190.0 in
GM (the least value).
The statistical analysis revealed that, The variance in HDL-C were relatively
small, i.e. the variations (dispersion) in HDL-C were correspondingly small, while in
Cholesterol, LDL-C and Triglycerides the variance was relatively large, and hence, the
variation in count was correspondingly large. The mean counts for lipid profile in blood
of the rabbit kits were, 75.13 in control group (the highest value) and 70.0 in GM (the
lowest value).
It was clear that, the GM cottonseed cake decreases the lipid profile counts in the
rabbit kits after 45 days of feeding.
The statistical analysis also revealed that, there was significant difference in the
lipid profile (F= 61.88 ; F-crit= 9.28 ; P-value= 0.0034), while there was a non-significant
difference between the tested groups (f= 0.19 ; f-crit= 10.13 ; p-value = 0.69) i.e. the lipid
profile indices counts in the rabbit fed on GM cottonseed cake were similar to that in
control (fed in non-GMCSC). It was clear that, the two types of diets were similar in their
effects on lipid profile indices count.
104
Table (4.19): The Effect of GMCSC on Lipid Profile of the 1st Generation After 6
Weeks of Feeding
Blood parameter Control GM Average Variance
Cholesterol (mg\dL) 35.5 45.0 40.3 45.1
High density lipid cholesterol (HDL-C)
mg\dL 4.5 5.0 4.8 0.1
Low density lipid cholesterol (LDL-C)
mg\dL 30.5 40.0 35.3 45.1
Triglycerides (mg\dL) 230.0 190.0 210 800
ANOVA
Source SS df MS F P-value F crit
Rows 51847.84 3 17282.61 61.88 0.003 9.28
Columns 52.53 1 52.53 0.19 0.7 10.13
105
4.20: The Effects of GMCSC on Body Weight of the 1st Generation
The data obtained from evaluation of the effect of the genetically modified
cottonseed cake (GMCSC) that fed to the young rabbits of the 1st generation for period of
6 weeks on the body weight gain were presented in Table (4.20). The mean initial body
weight (g) at newly birth were 52 g equally in control and GM. The body weight after 4
weeks (weaning 28 day) were 227 g in control (the lowest growth observe) and 306g in
GM. The mean body weight after 5 weeks were 273 g in control (the highest growth
value) and 373 g in GM (the lowest growth value). The mean body weight at the end (6
weeks) were 318 g in control (the lowest growth value) and 464 g in GM (the highest
growth value).
The statistical analysis revealed that, The R square (R2) was 1.00 in control
(highest value) and 0.99 in GM (lowest value). i.e. the homogeneity in control were
relatively high than GM. It was clearly that the GM cottonseed cake decreases the
homogeneity of growth rate after 6 weeks of feeding to young rabbit of 1st generation.
The coefficient (X) were 45.5 in control and 79.0 in GM. The intercept were 45.17
in control and -14 in GM.
The statistical analysis also revealed that, the regression was significant in control
(0.004), while there was a non-significant in GM (0.056) i.e. the weight gain in the rabbit
fed on GM cottonseed cake were similar to that in control. It was clear that, the GM
cottonseeds had adverse effects on weight gain of the young rabbits of 1st generation
after 6 weeks of feeding.
106
Table (4.20): Mean Body Weight (g) Gain of the 1st Generation Fed on GMCSC for 6
Weeks
Period\weak Control GM
4 227 306
5 273 373
6 318 464
Regression
R- Square 0.99996 0.992368
Standard Error 0.4 9.8
Intercept 45.2 -14
Coefficients (x) 45.5 79
Standard Error (y) 1.5 35.1
Standard Error (x) 0.3 6.9
107
4.21: The Effect of GMCSC on Internal Organ of The 1st Generation
The data obtained from evaluation of the effect of the genetically modified
cottonseed cake (GMCSC) that fed to the first generation of the rabbit for period of 45-75
days on the internal organ weight (in term of percentage) were presented in Table (4.21).
The mean Kidney weight % were 0.35 in control (the least value) and 0.54 in GM (the
highest value). The mean Hart weight% were 0.34 in control (the highest value) and 0.42
in GM. The mean Lung weight % were 0.76 in control (the least value) and 0.80 in GM
(the highest value). The mean Spleen weight % were 0.06 in control (the least value) and
0.08 in GM (the highest value). The Liver weight % were 6.28 in control (the least value)
and 6.33 in GM (the highest value).
The statistical analysis revealed that, the variance in Kidney, Hart, Lung, Spleen
and Liver were from relatively to very small, i.e. the variations (dispersion) in those were
correspondingly small. The mean percentage for internal organ weight % in the rabbit
were, 1.56 in control group (the lowest value) and 1.63 in GM (the highest value).
It was clear that, the GM cottonseed cake increases the internal organ weight
percent in the 1st generation of the rabbit kits after 45 days of feeding.
The statistical analysis also revealed that, there was significant difference in the
internal organ weight % (F= 6174.98 ; F-.crit= 6.39 ; p-value= 7.86E-08), while there was
a non-significant difference between the tested groups (f= 6.38 ; f-crit= 7.71 ; p-value =
0.065) i.e. the internal organ weight in the rabbit kits fed on GM cottonseed cake were
similar to that in control (fed in non-GM cottonseed cake). It was clear that, the GM
cottonseeds were similar to the control in their effects on internal organ weight.
108
Table (4.21): The Relative Weight (%) of Internal Organ of the Young Rabbits of The
1st Generation Fed on GMCSC for 6 Weeks
Organ Non-GM GM Average Variance
Kidney 0.35 0.54 0.45 0.02
Hart 0.34 0.42 0.38 0.003
Lung 0.76 0.80 0.78 0.0008
Spleen 0.06 0.08 0.07 0.0002
Liver 6.28 6.33 6.31 0.001
ANOVA
Source SS Df MS F P-value F crit
Rows 55.95 4 13.99 6174.98 7.86E-08 6.39
Columns 0.014 1 0.014 6.38 0.07 7.71
109
4.22: Effect of GMCSC on Proximate Compositions of Meat Nutrient of
The 1st Generation
The data obtained from evaluation of the effect of the genetically modified
cottonseed cake (GMCSC) that fed to the first generation of the rabbit for period of 45
days on the approximate compositions of meat nutrient percentage, were presented in
table (4.22). The moisture percent (%) were 75.09 in control (the highest value) and 74.30
in GM (the least value). The ash % were 1.47 in control (the highest value) and 1.39 in
GM. The protein % were 21.84 in control (the least value) and 22.64 in GM (the highest
value). The fat % were 0.77 in control (the highest value) and 0.74 in GM (the least
value). The fiber content % were 0.83 in control (the least value) and 0.93 in GM (the
highest value).
The statistical analysis revealed that, the variance in fat, ash and fiber were
relatively small, i.e. the variations (dispersion) in those were correspondingly small,
while the variance in moisture and protein was relatively large.
It was clear that, the GM cottonseed cake increases the protein percent in meat of
the 1st generation of the rabbit kits after 45 days of feeding.
The statistical analysis also revealed that, there was significant difference in the
meat nutrient content (F= 12727.8 ; F-.crit= 6.39; p-value= 1.85E-08), while there was a
non-significant difference between the tested groups (f= 0.0 ; f-crit= 7.71 ; p-value =
1.00) i.e. the meat nutrient content in the rabbit kits fed on GM cottonseed cake were
similar to that in control (fed in non-GM cottonseed cake). It was clear that, the GM
cottonseeds were similar to the control in their effects on meat nutrient content.
110
Table (4.22): Proximate Composition of Meat Nutrient Content of the Young Rabbits
of The 1st Generation Fed on GMCSC for 6 Weeks
Content (%) Control GM Average Variance
Moisture 75.09 74.30 74.69 0.3
Ash 1.47 1.39 1.43 0.003
Protein 21.84 22.64 22.24 0.32
Fat 0.77 0.74 0.76 0.0005
Fiber 0.83 0.93 0.88 0.005
ANOVA
Source SS df MS F P-value F crit
Rows 8154.7 4 2038.68 12727.8 1.85E-08 6.39
Columns 0 1 0 0 1 7.71
111
4.23: Effect of GMCSC on Meat Mineral Content of The 1st Generation
The data obtained from evaluation of the effect of the genetically modified
cottonseed cake (GMCSC) that fed to the young rabbits of 1st generation for period of 6
weeks on the mineral content of meat in term of percentage, were presented in table
(4.23). The sodium (Na+) percent (%) were 0.00325 in control (the highest value) and
0.00310 in GM (the lowest value). The potassium (K+) % were 0.00396 in control (the
highest value) and 0.00318 in GM (the lowest value). The calcium (Ca++) % were 0.42 in
control (the highest value) and 0.34 in GM (the lowest value).
The statistical analysis revealed that, the variance in sodium. Potassium and
calcium were very small, i.e. the variations (dispersion) in those were correspondingly
small. The mean mineral count within group were 0.142 in control group (the highest
value) and 0.115 in GM. It was clear that, the GM cottonseed decreases the mineral
content in young rabbit meat of 1st generation after 6 weeks of feeding.
The statistical analysis also revealed that, there was significant difference in the
meat mineral content (F= 89.69 ; F-.crit= 19 ; P= 0.01), while there was a non-significant
difference between the tested groups (f= 1.04 ; f-crit= 18.51 ; P = 0.42) i.e. the meat
mineral content in the rabbit fed on GM cottonseed cake were similar to that fed in non-
GM cottonseed cake. It was clear that, the GM cottonseeds were similar to the non-GM
cottonseed in their effects on meat mineral content.
112
Table (4.23): The Meat Mineral Content of the Young Rabbits of The 1st Generation
Fed on GMCSC for 6 Weeks
Element % Control GM Average Variance
Sodium (Na+) 0.0033 0.0031 0.0032 1.13E-08
Potassium (K+) 0.004 0.0032 0.0036 3.04E-07
Calcium (Ca++) 0.42 0.34 0.38 0.003
ANOVA
Source SS df MS F P-value F crit
Rows 0.189 2 0.09 89.69 0.01 19
Columns 0.001 1 0.001 1.04 0.4 18.51
113
CHAPTER FIVE
CONCLUSIONS AND RECOMMENDATIONS
5.1: Conclusions
The GM cottonseed had a varied proximate property for rabbits feeding, i.e. it
contains high level of protein and carbohydrates, and low level of Fat than the non-GM
cottonseed. The GM cottonseed contains high level of sodium, and low level of calcium
and potassium. It was clear that the GM cottonseed have high nutritive value than non-
GM cottonseed.
The GM decreases the WBCs counts, increased the RBCs and blood clotting
indices. In the serum biochemical the GM cottonseed decreases the liver function indices,
and had no adverse effect on the renal function indices and lipid profile indices in mature
rabbits, while it is increased the liver function indices and decreased the renal function
and lipid profile indices in the young rabbits of 1st generation.
The GM cottonseed decreases the homogeneity of growth rate and increase the
internal organ weight percent in the rabbit.
The GM cottonseed cake decreases the protein content and increase the moisture
content in the mature rabbits meat, while it is increases the protein and fat content, and
decreases the moisture content in the young rabbits meat of the first generation. Also the
GM cottonseed had no adverse effect on the mineral content in the rabbit meat.
The statistical analysis showed that, there was no-significant differences between
groups in all testes. It was clear that the GM cottonseed cake and non-GM cottonseed
cake was similar in their effect on the rabbits feeding as same as control. although that,
there were an obvious variations from the standards.
5.2: Recommendations
It is recommended that more comprehensive study should be done on the effects
of GM cottonseed on the other living organisms including diary animals and subsequent
generations.
114
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Appendices
Appendix (1)
Rearing of the Rabbits.
131
Appendix-(2)
Rabbits feeding on GM and non-GM cottonseed cake.
132
Appendix (3):-
Rearing of the Young Rabbits of the First Generation.
133
Appendix (4):-
The Internal Organs of the Dissected Rabbit.
134
Appendix (5):-
Reserve the Internal Organs of the Rabbits in Special Container.
135
Appendix (6):-
Determining the Mineral Content by Flame Photometer.