Effects of Feeding Genetically Modified Cotton Seed Cake on Physiological Aspects and Meat Quality of the Rabbits (Oryctolagus Cuniculus)

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

Biosciences and Biotechnology (Biosafety)

Center of Biosciences and Biotechnology

Faculty of Engineering and Technology

May 2016

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

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

DEDICATION

To the Sudanese people and Humanity with gratitude, I dedicate this work.

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.

5 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.

دكخٕزاِ انفهسفت فٙ انعهٕو ٔانخمُٛت انبٕٛنٕجٛت (انساليت انحٕٚٛت ) يإٚ 1026

يسكص انعهٕو ٔانخمُٛت انبٕٛنٕجٛت

ملخص الدراسة

انكائُاث انًعدنت ٔزاثٛاً ْٙ حهك انكائُاث انخٙ حٕز انـ DNA فٛٓا خالل انُٓدست انٕزاثٛت. بٙ حٙ لطٍ أ انمطٍ انًحٕز ٔزاثٛاً اٚضاً حٕز انـ DNA فّٛ بإدخال جٍٛ يشفس بسى بكخسٚا انباسهس سٕزَجُٛٛسس نخمهٛم اإلعخًاد عهٗ انًبٛداث. اُجسٚج ْرِ اندزاست بٕالٚت انجصٚسة نخمٛٛى اثس برٔز انمطٍ انًحٕز ٔزاثٛاً عهٗ انُشاط انفسٕٛنٕجٙ نألباء ٔافساد انجٛم األٔل نألزاَب انًغرٚت برنك انكٛك. حٛث حى انحصٕل عهٗ األزاَب ٔانمطٍ انًحٕز ٔزاثٛاً ٔانمطٍ غٛس انًحٕز ٔزاثٛاً يٍ اسٕاق يدُٚت ٔديدَٙ. ٔحى حمسٛى األزاَب إنٗ ثالثت يجًٕعاث ٔ غٌرٚج بانمطٍ انًحٕز ٔزاثٛاً ٔانمطٍ غٛس انًحٕز ٔزاثٛاً ٔانسغٛف انجاف، ٔعُد َٓاٚت فخسة انخجسبت )ٚ 90ٕو( أُخرث عُٛاث يٍ دو االزاَب نخحهٛهٓا يٍ حٛث اندو انكايم )خالٚا اندو انبٛضاء، خالٚا اندو انحًساء، انصفائح انديٕٚت، ٔانُٕٛحسٔفٛم، انهًٛف( ٔنهكهٗ )انكسٚاحٍُٛٛ، انٕٛزٚا، انبٕحاسٕٛو، انصٕدٕٚو( ٔنهكبد )األنبٕٛيٍٛ، ٔاألنكانٍٛ فٕسفاث، اٜالٍَٛ أيُٕٛحساَس، اإلسبازحٛج أيُٕٛحساَس، يعدل اإلسبازحٛج أيُٕٛحساَس إنٗ اٜالٍَٛ أيُٕٛحساَس، انبٛهٛسٔبٍٛ انكايم( ثى شُسحج االزاَب نخمصٙ حانت األعضاء انداخهٛت كرنك اُجس٘ انخحهٛم انخمسٚبٙ نعُٛاث يٍ انهحٕو )يأخٕذة يٍ انعضالث(. حههج انبٛاَاث إحصائٛاً. ٔجدث اندزاست أٌ كٛكت برٔز انمطٍ انًحٕز ٔزاثٛاً لههج كسٚاث اندو انحًساء )يٍ 4.05 فٙ انعُٛت انشاْدة إنٗ 106µL×5.48( ٔٔشٌ انجسى )يٍ يخٕسظ 2654 فٙ انعُٛت انشاْدة إنٗ 2550جساو( ٔاٚضاً لههج يٍ كسٚاث اندو انبٛضاء )يٍ 8.68 فٙ انعُٛت انشاْدة إنٗ 103µL×4.80(. كٛك برٔز انمطٍ انًحٕز ٔزاثٛاً نٛس نّ حأثٛساث يعُٕٚت عهٗ ٔظائف انكبد )f= 0.95; f-crit=5.95( ٔانًكَٕاث انغرائٛت نهحٕو )اندٌْٕ، انبسٔحٍٛ، األنٛاف، انكسبْٕٛدزاث: f=0.006; f-crit= 4.46( ٔعُاصس حجهظ اندو (f= 1.03; f-crit=10.13) ٔعُاصس ٔظائف انكهٗ )f= 0.51; f-crit=161.45). فٙ انجٛم األٔل لهم كٛك برٔز انمطٍ انًحٕز ٔزاثٛاً يٍ كسٚاث اندو انبٛضاء ٔٔظائف انكهٗ ٔنٕاحك اندٌْٕ ٔيحخٕٖ انًعادٌ فٙ انهحٕو ٔشاد كسٚاث اندو انحًساء ٔعُاصس حجهظ اندو ٔيحخٕٖ انبسٔحٍٛ ٔاندٍْ فٙ انهحٕو. أٚضاً ال حٕجد فسٔق يعُٕٚت إحصائٛاً ٔنكٍ فسٕٛنٕجٛاً حٕجد فسٔق ٔاضحت بٍٛ االزاَب انًخغرٚت بكٛكت برٔز انمطٍ انًحٕز ٔزاثٛاً ٔ انعُٛت انشاْدة. حٕصٙ اندزاست بإجساء دزاساث يكثفت عهٗ انخاثٛس انفسٕٛنٕجٙ نكٛك برٔز انمطٍ انًحٕز ٔزاثٛاً عهٗ انثدٚاث انكبسٖ ٔأجٛانٓا انًخالحمت.

7 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

8 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

9 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

10 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

11 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

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

13 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

14 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

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

16 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:

17 To evaluate the effect of genetically modified (GM) cottonseed cake (as diets) on rabbits physiological aspect and meat quality.

18 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

19 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,

20 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

21 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).

22 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

23 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

24 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

25 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).

26 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,

27 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).

28 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

29 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

30 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).

31 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).

32 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).

33 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).

34 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

35 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,

36 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).

37 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 which began to be used circa 1400 AD ,لُطٍْ name derives from the Arabic (al) quṭ n (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

38 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

39 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).

40 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

41 121,500 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

42 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

43 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

44 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

45 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

46 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

47 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.

48 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

49 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

50 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

51 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.,

52 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

53 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).

54 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).

55 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).

56 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 37oC 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.

57 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.

58 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).

59 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%

60 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:

61 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

62 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

63 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).

64 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+

65 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.

66 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.

67 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 + +

68 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%).

69 Table (4.2): The Proximate Composition of GM and Non-GM Cotton Seeds

Components (%) GM Non-GM

Cotton 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

70 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.

71 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

72 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.

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 8.63 6.90 5.30 6.94 2.77 (WBC) 103/µl

Lymphocytes (Lymph) 3.17 3.70 2.90 3.26 0.17 103/µl

Intermediate Cells 0.90 0.60 0.45 0.65 0.053 (Mid) 103/µl

Neutrophilic- granulocytes (Gran) 4.87 2.60 1.95 3.14 2.35 103/µl

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

74 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).

75 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

6 Red Blood Cells (RBC) 10 \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) 68.08 66.55 69.10 67.91 1.647 fl

Mean Corpuscular Haemoglobin 20.95 20.93 21.75 21.21 0.219 (MCH) pg

Mean Corpuscular Haemoglobin 30.90 31.53 31.53 31.32 0.132 Concentration (MCHC) %

Red Cell Distribution Width 16.48 17.13 16.55 16.72 0.127 Coefficient of Variance (RDW-CV)

Red Cell Distribution Width 39.93 40.78 41.58 40.76 0.681 Standard Deviation (RDW-SD)

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

76 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.

77 Table (4.6): The Effects of GMCSC and Non-GMCSC on Blood Clotting Indices of the Rabbits After 90 days of feeding

Parameter Non- Control GM Average Variance GM

Platelets (PLT) 103/µl 293.0 262.5 281.0 278.83 236.08

Mean Platelets Volume (MPV) 6.53 8.33 6.65 7.17 1.01 fL

Platelet Distribution Width 15.33 16.45 15.13 15.64 0.51 (PDW)

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

78 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).

Table (4.7): The Effects of GMCSC and Non-GMCSC on Liver Functions Indices of the Rabbits After 90 days of Feeding

Non- Parameter Control GM Average Variance GM

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

80 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).

81 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

82 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),

83 Table (4.9): The Effect of GM Cottonseed Cake on Lipid Profile of the Rabbits After 90 Days of Feeding

Non- Blood parameter Control GM Average Variance GM

Cholesterol (mg\dL) 40.80 23.50 36.75 33.68 81.88

High density lipid cholesterol 11.30 12.50 11.75 11.85 0.37 (HDL-C) mg\dL

Low density lipid cholesterol 24.80 8.33 21.00 18.04 74.37 (LDL-C) mg\dL

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

84 133.25 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.

85 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

86 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).

87 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

88 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.

89 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

90 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.

91 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

92 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.

93 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) 8.15 8.10 8.13 0.001 103/µl

Lymphocytes (Lymph) 103/µl 2.15 2.00 2.08 0.01

Intermediate Cells (Mid) 0.55 0.70 0.63 0.01 103/µl

Neutrophilic-granulocytes 5.45 5.40 5.43 0.001 (Gran) 103/µl

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

94 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).

95 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

6 Red Blood Cells (RBC) 10 \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 27.5 27.9 27.7 0.08 (MCH) pg

Mean Corpuscular Haemoglobin 34.4 34.9 34.65 0.13 Concentration (MCHC) %

Red Cell Distribution Width 13.8 14.2 14 0.08 Coefficient of Variance (RDW-CV)

Red Cell Distribution Width 41.7 42.5 42.1 0.32 Standard Deviation (RDW-SD)

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

96 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.

97 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

98 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.

99 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

100 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.

101 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

102 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.

103 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) 4.5 5.0 4.8 0.1 mg\dL

Low density lipid cholesterol (LDL-C) 30.5 40.0 35.3 45.1 mg\dL

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

104 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.

105 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

106 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.

107 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

108 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.

109 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

110 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.

111 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

112 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.

113 References

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129

Appendices Appendix (1)

Rearing of the Rabbits.

130

Appendix-(2)

Rabbits feeding on GM and non-GM cottonseed cake.

131

Appendix (3):-

Rearing of the Young Rabbits of the First Generation.

132

Appendix (4):-

The Internal Organs of the Dissected Rabbit.

133

Appendix (5):-

Reserve the Internal Organs of the Rabbits in Special Container.

134

Appendix (6):-

Determining the Mineral Content by Flame Photometer.

135