Effect of Supplementation of Antioxidants on the Quality of Chilled and Cryo-Preserved Ram Semen

166 Pages·2016·1.71 MB·English

Cairo University
Faculty of Veterinary Medicine
Department of Theriogenology

Effect of Supplementation of Antioxidants on
the Quality of Chilled and Cryo-Preserved Ram
Semen

A Thesis Presented by
Hossam Elsherbiny Ramadan Elsherbiny
(B. V. Sc., Cairo University; 2009)
For obtaining the degree of M. V. Sc.
(Theriogenology)

Under Supervision of
Prof. Dr. A. M. Ghallab Prof. Dr. M. M. Abou-Ahmed
Professor of Theriogenology Professor of Theriogenology
Faculty of Veterinary Medicine Faculty of Veterinary Medicine
Cairo University Cairo University
Cairo University
Faculty of Veterinary Medicine
Department of Theriogenology

APPROVAL SHEET
This to certify that the dissertation presented by Hossam Elsherbiny Ramadan
Elsherbiny to Cairo University, for master degree in Veterinary Medical Sciences
(Theriogenology) has been approved by the examination committee.

Prof. Dr. Farouk Hamed El-Ghanam
Professor of Theriogenology
Faculty of Veterinary Medicine
Zagazig University

Prof. Dr. Kamal El-Din Mahmoud Zaki
Professor of Theriogenology
Faculty of Veterinary Medicine
Cairo University

Prof. Dr. Mostafa Mohamed Mostafa Abou-Ahmed
Professor of Theriogenology
Faculty of Veterinary Medicine
Cairo University
(Supervisor)

Prof. Dr. Abdel-Raouf Moursy Ghallab
Professor of Theriogenology
Faculty of Veterinary Medicine
Cairo University
(Supervisor)
Cairo University
Faculty of Veterinary Medicine
Department of Theriogenology

Name: Hossam Elsherbiny Ramadan Elsherbiny
Date & place of birth: 13/9/1987. Egypt
Nathionality: Egyptian.
Specialization: Theriogenology
Thesis title: Effect of some antioxidant additives on the viability of
cryopreserved ram semen.
Supervisors: 1. Prof. Dr. Mostafa Mohamed Mostafa Abou-Ahmed
Professor of Theriogenology
Faculty of Veterinary Medicine, Cairo University

2. Prof. Dr. Abdel-Raouf Moursy Gallab
Professor of Theriogenology
Faculty of Veterinary Medicine, Cairo University

Abstract
The objective of this study was to determine the effect of cysteine, glutamine,
melatonin, sucrose and bovine serum albumin on the sperm progressive motility,
alive sperm percentage, functional membrane integrity by HOST, percentage of
intact acrosome and viability index of cryopreserved and chilled ram semen. Three
mature fat-tailed rams were used in this study. Semen collection occurred twice
weekly, once a day. Collected semen transferred to the water bath at 37 oC, the
semen samples were pooled and divided into 16 aliquots, each aliquot was diluted
using different diluents, egg yok-Tris diluents containing cysteine (5, 10 and 15
mM), egg yok-Tris diluents containing glutamine (2.5, 5 and 7.5 mM), egg yok-Tris
diluents containing melatonin ( 0.5, 1 and 1.5 mM), egg yok-Tris diluents containing
sucrose ( 25, 50 and 75 mM), egg yok-Tris diluents containing bovine serum
albumin (5, 10 and 15 mg/ml) an egg yolk-Tris diluents without any additives
(control). The diluted semen containing antioxidant was cooled to 5 oC and
processed into 0.5 ml straws or processed in 0.1 ml pellets. The best results based on
the protection of semen characteristics were obtained from cysteine (5 and 10 mM),
glutamine (2.5 and 5 mM), melatonin (0.5 and 1 mM) and bovine serum albumin (10
and 15 mg/ml). No significant effect was noticed by addition of sucrose to freezing
media. Only slight increase in the percentage of intact acrosome was repoted for
sucrose addition in chilled ram semen.
CONTENTS
1. INTRODUCTION 1
2. LITERATURE OF REVIEW 5
2.1 . Oxidative stress 5
2.1.1. Cryopreservation/ Freezing-thawing oxidative stress 5
2.2. Free radicals 7
2.3. Reactive oxygen species(ROS) 8
2.3.1. Types of (ROS) 8
2.3.2. Sources of ROS in male reproductive system 9
2.4. Lipid peroxidation (LPO) 10
2.4.1. Detrimental effects of (LPO) on sperm function 11
2.5. Antioxidants 11
2.5.1. Types of antioxidants 12
2.5.1.1. Enzymatic antioxidants (natural antioxidants) 12

2.5.1.2. Nonenzymatic antioxidants (synthetic antioxidants) 13
2.5.1.2.1. Amino acids and proteins 13
2.5.1.2.2. Sugars 14
2.5.1.2.3. Vitamines 15
2.6. Ram semen freezability and lipid peroxidation 15

2.7. Effect of different antioxidants on frozen semen 18
2.7.1. Cysteine hydrochloride 18
2.7.1.1. Effect of cysteine on post-thaw sperm motility 19
2.7.1.2. Effect of cysteine on post-thaw viability index 21
2.7.1.3. Effect of cysteine on structural membrane integrity 21
2.7.1.4. Effect of cysteine on functional membrane integrity 23
2.7.1.5. Effect of cysteine on acrosome integrity 25
2.7.2. Glutamine 26
2.7.2.1. Effect of glutamine on the post-thaw sperm motility 27

2.7.2.2. Effect of glutamine on structural and functional
sperm membrane integrity 28
2.7.2.3. Effect of glutamine on sperm acrosome integrity 29
2.7.3. Melatonin 30
2.7.3.1. Effect of melatonin on the post-thaw sperm motility 32

2.7.3.2. Effect of melatonin on the structural sperm
membrane integrity 33

2.7.3.3. Effect of melatonin on post-thaw sperm viability 33

2.7.4. Sucrose 33

2.7.4.1. Effect of sucrose on post-thaw sperm motility 35
2.7.4.2. Effect of sucrose on the structural and functional sperm

membrane integrity 36

2.7.4.3. Effect of sucrose on sperm acrosome integrity 37

2.7.4.4. Effect of sucrose on sperm viability 37
2.7.5. Bovine Serum Albumin (BSA) 38
2.7.5.1. Effect of (BSA) on post-thaw sperm motility 38
2.7.5.2. Effect of (BSA) on structural sperm membrane integrity 40

2.7.5.3. Effect of (BSA) on functional sperm membrane integrity 41

2.7.5.4. Effect of (BSA) on sperm acrosome integrity 42

3. MATERIALS AND METHODS 44
3.1. Experimental animals 44
3.2. Tested antioxidants 44
3.2.1. Cysteine Hydrochloride 44
3.2.2. Glutamine 45
3.2.3. Melatonin 45
3.2.4. Sucrose 46
3.2.5. Bovine serum albumin 46
3.3. Experimental procedures 47
3.3.1. Semen collection 47
3.3.2. Semen evaluation 47
3.3.2.1. Evaluation of freshly collected semen 47
3.3.2.2. Evaluation of chilled (5 oC) semen 48
3.3.3. Semen extention, cooling and freezing 48
3.3.3.1. Semen diluent 49
3.3.3.2. Semen cooling 49
3.3.3.3. Semen freezing 49
3.3.3.3.1. Freeing in 0.5 ml straws 49
3.3.3.3.2. Freeing in 0.1 pellet 50

3.3.4. Thawing procedures 50

3.3.5. Evaluation of frozen semen 51

3.3.5.1. Post-thaw motility 51

3.3.5.2. Viability index 51

3.3.5.3. Acrosome integrity 51

3.3.5.4. Structural membrane integrity 52

3.3.5.5. Functional membrane integrity 52

3.4. Statistical analyses 53

4. RESULTS 54

4.1. Effect of cysteine on the studied

parameters of chilled (5oC) ram semen 54

4.2. Effect of glutamine on the studied parameters
of chilled (5oC) ram semen 57

4.3. Effect of melatonin on the studied parameters
4.4. of chilled (5oC) ram semen 60

4.5. Effect of sucrose on the studied
parameters of chilled (5oC) ram semen 63
4.6. Effect of bovine serum albumin (BSA) on the studied
4.7. parameters of chilled (5 oC) ram semen 66
4.8. Effect of cysteine on the studied parameters
of frozen thawed ram semen 69
4.8.1. Processed in 0.5 ml straws 69
4.8.2. Processed in 0.1 ml pellets 72
4.9. Effect of glutamine on the studied parameters of

frozen thawed ram semen 75

4.9.1. Processed in 0.5 ml straws 75
4.9.2. Processed in 0.1 ml pellets 78

4.10. Effect of melatonin on studied parameters

of frozen thawed ram semen 81

4.10.1. Processed in 0.5 ml straws 81
4.10.2. Processed in 0.1 ml pellets 84

4.11. Effect of sucrose on the studied parameters

of frozen thawed ram semen 87

4.11.1. Processed in 0.5 ml straws 87
4.11.2. Processed in 0.1 ml pellets 90

4.12. Effect of BSA on studied parameters
of frozen thawed ram Semen 94

4.12.1. Processed in 0.5 ml straws 93
4.12.2. Processed in 0.1 ml pellets 96
5. DISCUSSION 99
6. SUMMERY 114
7. CONCLUSION 117
8. REFERENCES 118
ARABIC SUMMERY
List of tables
Table Page
1. Effect of different concentrations of cysteine on the studied 55
parameters of chilled (5oC) ram semen (Mean±SEM)
2. Analysis of variance for the effect of cysteine addition to egg yolk- 55
Tris diluent on the studied parameters of chilled (5 oC) ram semen
3. Effect of different concentrations of glutamine on the studied 58
parameters of chilled (5oC) ram semen (Mean±SEM)
4. Analysis of variance for the effect of glutamine addition to egg 58
yolk-Tris diluent on the studied parameters of chilled (5 oC) ram
semen
5. Effect of different concentrations of melatonin on the studied 61
parameters of chilled (5oC) ram semen (Mean±SEM)
6. Analysis of variance for the effect of melatonin addition to egg 61
yolk-Tris diluent on the studied parameters of chilled (5 oC) ram
semen
7. Effect of different concentrations of sucrose on the studied 64
parameters of chilled (5oC) ram semen (Mean±SEM)
8. Analysis of variance for the effect of sucrose addition to egg yolk- 64
Tris diluent on the studied parameters of chilled (5 oC) ram semen
9. Effect of different concentrations of BSA on the studied 67
parameters of chilled (5oC) ram semen (Mean±SEM)
10. Analysis of variance for the effect of BSA addition to egg yolk-Tris 67
diluent on the studied parameters of chilled (5 oC) ram semen
11. Effect of different concentrations of cysteine on the studied 70
parameters of frozen thawed ram semen processed in 0.5 ml straws
(Mean±SEM)
12. Analysis of variance for the effect of cysteine addition to egg yolk- 70
Tris diluent on the studied parameters of frozen thawed ram semen
13. Effect of different concentrations of cysteine on the studied 73
parameters of frozen thawed ram semen processed in 0.1 ml pellets
(Mean±SEM)
14. Analysis of variance for the effect of cysteine addition to egg yolk- 73
Tris diluent on the studied parameters of frozen thawed ram semen
processed in 0.1 ml pellets
15. Effect of different concentrations of glutamine on the studied 76
parameters of frozen thawed ram semen processed in 0.5 ml straws
(Mean±SEM)
16. Analysis of variance for the effect of glutamine addition to egg 76
yolk-Tris diluent on the studied parameters of frozen thawed ram
semen processed in 0.5 ml straws
17. Effect of different concentrations of glutamine on the studied 79
parameters of frozen thawed ram semen processed in 0.1 ml pellets
(Mean±SEM)
18. Analysis of variance for the effect of glutamine addition to egg 79
yolk-Tris diluent on the studied parameters of frozen thawed ram
semen processed in 0.1 ml pellets
19. Effect of different concentrations of melatonin on the studied 82
parameters of frozen thawed ram semen processed in 0.5 ml straws
(Mean±SEM)
20. Analysis of variance for the effect of melatonin addition to egg 82
yolk-Tris diluent on the studied parameters of frozen thawed ram
semen processed in 0.5 ml straws
21. Effect of different concentrations of melatonin on the studied 85
parameters of frozen thawed ram semen processed in 0.1 ml pellets
(Mean±SEM)
22. Analysis of variance for the effect of melatonin addition to egg 85
yolk-Tris diluent on the studied parameters of frozen thawed ram
semen processed in 0.1 ml pellets
23. Effect of different concentrations of sucrose on the studied 88
parameters of frozen thawed ram semen processed in 0.5 ml straws
(Mean±SEM)
24. Analysis of variance for the effect of sucrose addition to egg yolk- 88
Tris diluent on the studied parameters of frozen thawed ram semen
processed in 0.5 ml straws
25. Effect of different concentrations of sucrose on the studied 91
parameters of frozen thawed ram semen processed in 0.1 ml pellets
(Mean±SEM)
26. Analysis of variance for the effect of sucrose addition to egg yolk- 91
Tris diluent on the studied parameters of frozen thawed ram semen
processed in 0.1 ml pellets
27. Effect of different concentrations of BSA on the studied 94
parameters of frozen thawed ram semen processed in 0.5 ml straws
(Mean±SEM)
28. Analysis of variance for the effect of BSA addition to egg yolk-Tris 94
diluent on the studied parameters of frozen thawed ram semen
processed in 0.5 ml straws
29. Effect of different concentrations of BSA on the studied parameters 97
of frozen thawed ram semen processed in 0.1 ml pellet
(Mean±SEM)
30. Analysis of variance for the effect of BSA addition to egg yolk-Tris 97
diluent on the studied parameters of frozen thawed ram semen
processed in 0.1 ml pellets
List of figures
Figure Page
1. Effect of different concentrations of cysteine (mM) on the 56
studied parameters of chilled (5 oC) ram semen
2. Effect of different concentrations of glutamine (mM) on the 59
studied parameters of chilled (5 oC) ram semen
3. Effect of different concentrations of melatonin (mM) on the 62
studied parameters of chilled (5 oC) ram semen
4. Effect of different concentrations of sucrose (mM) on the 65
studied parameters of chilled (5 oC) ram semen
5. Effect of different concentrations of BSA (mg/ml) on the 68
studied parameters of chilled (5 oC) ram semen
6. Effect of different concentrations of cysteine (mM) on the post 71
thaw parameters of frozen thawed ram semen processed in 0.5
ml straws
7. Effect of different concentrations of cysteine (mM) on post thaw 74
parameters of frozen thawed ram semen processed in 0.1 ml
pellets
8. Effect of different concentrations of glutamine (mM) on post 77
thaw parameters of frozen thawed ram semen processed in 0.5
ml straws
9. Effect of different concentrations of glutamine (mM) on post 80
thaw parameters of frozen thawed ram semen processed in 0 .1
ml pellets
10. Effect of different concentrations of melatonin (mM) on post 83
thaw parameters of frozen thawed ram semen processed in 0.5
ml straws
11. Effect of different concentrations of melatonin (mM) on post 86
thaw parameters of frozen thawed ram semen processed in 0.1