TNV PT 08002 PDF

EVALUATION OF BULL SEMEN QUALITY IN RELATION TO
FERTILITY ASSOCIATED PROTEINS, LIPID PEROXIDATION

AND IN VITRO SPERM CHARACTERS
M.KARUNAKARAN
I.D.No. DPV 08002
DEPARTMENT OF ANIMAL REPRODUCTION,GYNAECOLOGY

AND OBSTETRICS,
MADRASVETERINARYCOLLEGE,
TAMILNADU VETERINARY AND ANIMALSCIENCESUNIVERSITY,
CHENNAI – 600 007
2011
EVALUATION OF BULL SEMEN QUALITY IN RELATION TO
FERTILITY ASSOCIATED PROTEINS, LIPID PEROXIDATION
AND IN VITRO SPERM CHARACTERS
M . KARUNAKARAN
I.D.No. DPV 08002
Thesis submitted in partial fulfilment of

the requirements for the Degree of
DOCTOR OF PHILOSOPHY
IN
ANIMAL REPRODUCTION,
GYNAECOLOGY AND OBSTETRICS
to the
Tamilnadu Veterinary and AnimalSciencesUniversity

Chennai
DEPARTMENT OF ANIMAL REPRODUCTION,

GYNAECOLOGY AND OBSTETRICS,
MADRASVETERINARYCOLLEGE,
TAMILNADU VETERINARY AND ANIMALSCIENCESUNIVERSITY
CHENNAI – 600 007
2011
TAMILNADU VETERINARY AND ANIMAL SCIENCES UNIVERSITY
Department of Animal Reproduction,

Gynaecology and Obstetrics,
MadrasVeterinaryCollege,
Chennai – 600 007
CERTFICATE
This is to certify that the thesis entitled “EVALUATION OF BULL SEMEN
QUALITY IN RELATION TO FERTILITY ASSOCIATED PROTEINS,
LIPID PEROXIDATION AND IN VITRO SPERM CHARACTERS” submitted
in partial fulfilment of the requirements for the degree of DOCTOR OF
PHILOSOPHY in ANIMAL REPRODUCTION, GYNAECOLOGY AND
OBSTETRICS to the Tamilnadu Veterinary and Animal Sciences University,
Chennai, is a record of bonafide research work carryout out by
M.KARUNAKARANunder my supervision and guidance and that no part of the
thesis has been submitted for the award of any other degree, diploma, fellowship or
other similar titles or prizes and that the work has not been published in part or full
in any scientific or popular journal or magazine.
Date: (Dr. T. G. DEVANATHAN)

Place: Chennai-7 Chairman
APPROVED BY
Chairman : (Dr. T. G. DEVANATHAN)
Members : (Dr. K. KULASEKAR)
(Dr. P. SRIDEVI)
(Dr. K. THILAK PON JAWAHAR)
Date: (EXTERNAL EXAMINER)

CURRICULUM VITAE
Name of the Candidate : M. KARUNAKARAN

Date of Birth : 21.03.1975
Place of Birth : Gobichettipalayam
Major filed of specialization : Animal Reproduction,
Gynaecology and Obstetrics
Educational Status : Completed B.V.Sc., in 1998 at
VeterinaryCollege and Research Institute,
Namakkal.
Completed M.V.Sc., in 2001 at
MadrasVeterinaryCollege
Chennai – 600 007
Joined Ph.D., Programme in 2008-2009 at
MadrasVeterinaryCollege
Chennai – 600 007
Professional Experience : Working as Scientist since 2003 at
ICAR-RC- for Goa
Old Goa
Marital Status : Married
Permanent Address : 246, Asiriyar Nagar,
Kullampalayam,
Gobichettipalayam,
Erode- 638 476.
Publication made : Research articles
International : 14
National :4
Membership of Professional : Life member of Indian Society for the Study
Society of Animal Reproduction
Life member of Tamilnadu Veterinary
Council
Life member of Indian society for Hill
Farming
Dedicated To
Shri Maragathavalli Nayaki Sametha

Shri Lakshmi Narasimhar Swamy
Perambakkam
ACKNOWLEDGEMENT
With men it is impossible; but to God all things are possible. I thank the
almighty God for his abundant blessings.
With sincere gratitude and indebtedness, I express my deep sense of

obligation and gratefulness to the chairman of the advisory committee,
Dr.T.G.Devanathan, Professor, Department of Animal Reproduction, Gynaecology
and Obstetrics, MVC, Chennai for his meticulous guidance, constant support,
tremendous patience, constructive criticism and encouragement throughout the study
to ensure the quality in the piece of work.
I express my heartfelt gratitude to members of advisory committee

Dr.K.Kulasekar, Professor, Department of Animal Reproduction, Gynaecology and
Obstetrics, Dr. P. Sridevi, Associate Professor, Department of Clinics, MVC and
Dr. K. Thilak Pon Jawahar, Associate Professor, University Research Farm,
Madhavaram Milk Colony for their valuable suggestions, encouragement, support
and care throughout the research work.
I express my profound and deep sense of gratitude and personal indebtedness

to Dr. C. Veerapandian, Professor and Head, Department of Animal Reproduction,
Gynaecology and Obstetrics, MadrasVeterinaryCollege for his support throughout
the work.
No words can ever express my sincere gratitude to Dr. S. Selvaraju,

Scientist, NIANP (ICAR), Bangalore, for his generous support, priceless help,
words of inspiration and persuasion rendered during the whole course of study.
I am grateful to Dr. B.P. Bhatt, then Joint Director, Dr. S.V. Ngchan,
Director and Dr. Azad Thakur, Director (i/c), ICAR- RC- NEH Region for
granting study leave to pursue Ph.D. programme.
I express my sincere gratitude to Dr. K. Manimaran, Assistant Professor,

Central University Laboratory, Madhavaram, for his generous support and help
rendered during the whole course of study. I register my profound gratitude to
Dr.Anand Chitra Assistant Professor, Dr. Vajravel Jayakumar, Associate
Professor (Retd), Dr. Ravikumar, Professor, Ms. Indumathi and Ms. Monashri,
Leptospirrosis Research Laboratory, Madhavaram for providing laboratory space
and guidance in carrying out protein work.
I record my sincere, profound and deep sense of gratitude to Dr. Kunju, then
DGM, TCMPFL, Madhavaram, Dr. Raju, Dr.A.C. Kuppan, Dr. Dhanasekaran,
NJF, Ooty and Dr. Suresh Kumar, BFSS, Erode for providing semen samples for
the research work.
I am much obliged to Dr. A. Subramanian, Professor (Retd), Dr. Cecilia

Joseph, Professor, Dr. S. Balasubrmanian, Associate Professor,
Dr.T.Sathiamoorthy, Associate Professor, Dr. S. Rangasamy, Assistant Professor,
Dr. N. Arunmozhi, Assistant professor, Department of Animal Reproduction,
Gynaecology and Obstetrics, Madras Veterinary College for their constant support
throughout the study.
No words can express the deep sense of gratitude and indebtedness I have to
Dr. A. Dhali, Senior Scientist, NIANP, Bangalore for his support in statistical
analysis, priceless help and hospitality rendered during the study. I thank
Dr.K.T.Sampath, Director, Dr. Ravindra, Principal Scientist, NIANP, Bangalore
for granting permission to use the laboratory facilities.
My special thanks are due to Dr. K. Loganathasamy, Assistant Professor,

Department of Bio-chemistry, MadrasVeterinaryCollege, for his help and support
rendered during the study.
I thank Dr. S. A. Asokan, Professor and Head and Dr.R.C. Rajasundaram,

Professor, Dr. D. Reena, Assistant Professor, Dr. Sarath, Assistant Professor,
Department of Clinics, MadrasVeterinaryCollege for all their support rendered
through out the study.
I register my profound gratitude to Dr. M. Selvaraju, Associate Professor,

Dr. Palanisamy, Assistant professor Dr. S. Manokaran, Assistant professor,
Dr.K.Prabhakaran, Assistant professor, Department of Animal Reproduction,
Gynaecology and Obstetrics, VCRI, Namakkal for their kindness and support.
I avail this opportunity to express my affectionate to Dr. Kulanthaivel,

Dr.R. Vinodh Kumar, Scientist (CSWRI), Dr. Krishnan, Scientist (NRC- Y), Dr.
Umesh Boopalan, Dr. Lidiya, Dr. Raja, Dr. Thirumugan, Dr. Vijay Dhomble,
Shri. Thiruvenkatasami and Shri.Sivasankaran for all their help and support. I
acknowledge the cooperation rendered by the non-technical staff of the Department
of ARGO, MVC.
My special thanks are due to Shri. T.P. Kurian, Shri. Thia Kaba Aao,
Shri. Bikas Chandra Sharma, ICAR- RC- NEH Region, Nagaland Centre for all
their help and support during the study.
I am indebted to all who provided me with information, guidance and

support. This research study could not have been carried out without the liberal and
generous contribution of several people with varied skills and experiences. “Thank
You” is a humble word with tremendous meaning and appreciation in it. I would like
to thank all the people who made this endeavor a fruitful one.
The love, affection and encouragement of my parents, sisters and brother are
what I am today. No words can express the indebtedness, and regards I have for
them. As we always save the best for the last, I express thanks to my wife and my
sons for their immense love, patience, forgiveness, fervent prayers, which made me
to sail against all the odds and to reach the shore successfully.
M. KARUNAKARAN
ABSTRACT
Title : Evaluation of bull semen quality in relation

to fertility associated proteins, lipid
peroxidation and in vitro sperm characters
Name and Degree : M. KARUNAKARAN

Ph.D. in Animal Reproduction,
Gynaecology and Obstetrics
Name of the Chairman : Dr. T.G. DEVANATHAN, Ph.D.,

Professor,
Department of Animal Reproduction,
Gynaecology and Obstetrics,
Madras Veterinary College,
Vepery, Chennai – 600 007.
University : Tamilnadu Veterinary and Animal Sciences

University
Year : 2011
Fresh semen samples were collected from 22 breeding bulls to screen for the
presence of fertility associated proteins. Seminal plasma proteins revealed 15
numbers of protein bands. The fertility related proteins such as 15/14 kDa and 28
kDa were present in all the 22 bulls (100 %), while 26 kDa protein was present in 18
bulls (81.82 %) and 55 kDa protein was present in 14 bulls (63.3 %). SDS- PAGE of
sperm membrane protein revealed a total of 14 protein bands. Proteins related with
bull fertility such as 15/14 kDa protein was observed in all the 22 bulls (100 %), 28
kDa protein was present in 21 bulls (95.45 %), 26 kDa protein was present in 14
bulls (63.64 %) and 55 kDa protein was present in only 11 bulls (50.00 %).
Heparin binding proteins of the seminal plasma revealed 7 protein bands, the
fertility related proteins such as 15/14 kDa protein was present in all the 22 bulls
(100 %) and 28 kDa protein was present in 18 bulls (81.82 %). 10 protein bands
were observed in the heparin binding proteins of the sperm membrane. 15/14 kDa
fertility related protein was present in all the bulls, while 28 kDa protein was present
in 11 bulls (50.00 %). Based on the presence of 28 kDa heparin binding protein in
sperm membrane, bulls in the present study were categorized into group I bulls
which were positive for the protein and group II bulls negative for the protein.
To study the effect of oxidative stress and role of fertility associated proteins
on in vitro sperm characters, frozen thawed semen of the bulls evaluated for their in
vitro sperm characters after treating with H2O2 and with fertility associated proteins.
In group I bulls, frozen thawed semen samples treated with 25µg of fertility
associated protein had significantly (P < 0.01) lower level of MDA than the control
at 60 min ((1.86 ± 0.17 vs 2.67 ± 0.19), at 120 min (2.25 ± 0.19 vs 2.79 ± 0.24) and
at 180 min (2.81 ± 0.26 vs 3.77 ± 0.41).In group IIbulls, semen samples treated with
fertility associated protein also had significantly lower level of MDA than the
control at 60 min (2.03 ± 0.12 vs 3.04 ± 0.15), at 120 min (2.55 ± 0.13 vs 3.61 ±
0.28) and at 180 min (3.16 ± 0.14 vs 4.84 ± 0.46).In H2O2 treatment, bulls in group I
had significantly (P < 0.05) lower MDA level than group II bulls at 30 min of
incubation (2.85± 0.16 vs 3.26 ± 0.12) and at 60 min of incubation (3.04 ± 0.16 vs
3.51 ± 0.12).
Bulls in group I when treated with fertility associated proteinhad

significantly more number of sperm cells with intact plasma membrane than the
control at 120 min (36.02 ± 2.10 vs 28.22 ± 3.10) and at 180 min (26.69 ± 2.06 vs
15.27 ± 2.11) of incubation. Similarly, bulls in group II when treated with fertility
associated protein had significantly more number of sperm cells with intact plasma
membrane than the control at 120 min (32.50 ± 3.03 vs 25.96 ± 3.32) and at 180 min
(23.36 ± 2.17 vs 15.06 ± 2.16) of incubation.In H2O2 treated semen samples, group I
bulls had significantly (P < 0.01) more number of sperm cells with intact plasma
membrane than the group II bulls at 10 min (45.75 ± 3.44 vs 31.79 ± 3.34) and at 30
min(26.03 ± 2.53 vs 17.54 ± 1.59) post thaw incubation periods.
Bulls in group I and II did not differ with each other in the per cent of sperm
cells with HMMP at 10 min of incubation with H2O2 (15.30 ± 0.92 vs 14.86 ± 0.77).
When the semen samples were treated with fertility associated proteins, bulls in
group I had significantly (P< 0.01) more per cent of sperm cells with HMMP than
the group II during different points of incubation; 60 min (17.51 ± 0.90 vs 13.57 ±
0.85), at 120 min (14.54 ± 0.58 vs 9.58 ± 0.76) and at 180 min (10.54 ± 0.55 vs 7.49
± 0.89) incubation periods.
In H2O2 treated samples, group I bulls had significantly (P < 0.05) more
number of DNAintact sperm cells than the group II bulls at 60 min of incubation
(94.01 ± 0.51 vs 92.26 ± 0.56). In H2O2 treatment, group I bulls had significantly (P
< 0.01) less number of apoptotic cells (10.53 ± 1.35 vs 13.90 ± 1.52)than group II at
30 min of incubation. When the samples were treated with fertility associated
protein, group I bulls had significantly (P <0.05) less number of apoptotic cells than
group II during incubation.
Significant reductions in motility and velocity parameters were observed

during incubation in control as well as in fertility associated protein treated samples.
Treatment with fertility associated protein caused significant reductions in motility
parameters when compared to the untreated control.
In group Ibulls, the per cent of sperm cells with B pattern in heparin and
heparin with fertility associated proteins treated groups were significantly (P < 0.01)
higher than the control group at 60 min (62.37 ± 1.92, 63.24 ± 1.55 and 50.86 ±
1.82), at 120 min (69.79 ± 1.97, 69.78 ± 1.41 and 53.10 ± 1.76) and at 180 min post
thaw incubation (74.48 ± 1.98, 74.29 ± 1.33 and 54.79 ± 2.08). But B pattern cells in
heparin treated group and heparin with fertility associated proteins treated group did
not differ significantly at any point of incubation.
When the bulls were ranked by analyzing the in vitro sperm characters
during incubation from immediate post thaw to 180 min in the frozen thawed semen
samples by Duncan Multiple Range Test, bulls in the group I had 5 bulls each in
rank 1 and rank 2, while only one bull was categorized under rank 3. Group II bulls
had 4 bulls each in rank 1 and 2 and 3 bulls in rank 3.
Key words: Bull semen, fertility associated proteins, lipid peroxidation, sperm
characters
CONTENTS
Chapter Page
Title
No. No.
LIST OF TABLES
LIST OF PLATES
I INTRODUCTION 1
II REVIEW OF LITERATURE 4
2.1 MOLECULAR INDICATORS OF FERTILITY 4
2.1.1 Seminal plasma and sperm membrane proteins 4
2.1.2 Seminal proteins and sperm function 5
2.1.3 Electrophoretic profile of seminal plasma proteins 7
BOVINE SEMINAL PLASMA AND SPERM 8
2.2
MEMBRANE PROTEINS
2.2.1 BSP proteins 8
2.2.2 Osteopontin 9
2.2.3 Heparin binding proteins 11
2.2.4 Fertility associated antigen 12
2.2.5 Prostaglandin D- synthase 13
2.2.6 Type 2 Tissue inhibitor of metallo proteinases 14
2.2.7 Spermadhesin Z13 15
2.2.8 Clusterin 15
2.2.9 Phospholipase A2 15
2.2.10 Heat shock protein 16
2.2.11 Acrosin 16
2.3 OXIDATIVE STRESS 16
2.3.1 Origin of ROS in male reproductive system 17
2.3.2 Positive and negative effects of ROS 18
2.3.3 Lipid peroxidation 18
2.3.4 Cryopreservation and oxidative stress 19
2.4 EVALUATION OF IN VITRO SPERM CHARACTERS 20
Chapter Page
Title
No. No.
2.4.1 Sperm morphology 21
2.4.2 Plasma membrane integrity 21
2.4.3 Functional membrane integrity 23
2.4.4 Mitochondrial membrane potential of sperm cells 23
2.4.5 DNA integrity 24
2.4.6 Apoptosis 25
2.4.7 Motility 27
2.4.8 Capacitation status of sperm cells 28
2.4.8.1 Acrosome integrity 30
2.4.8.2 Induction of capacitation 31
III MATERIALS AND METHODS 33
3.1 Experimental animals and source of semen 33
3.2 Collection of semen 33
3.3 Extraction of seminal plasma and sperm membrane proteins 34
3.4 Isolation of heparin binding proteins 34
Characterization of seminal plasma and sperm membrane 35
3.5
proteins by electrophoresis
3.5.1 Sample preparation 35
3.5.2 Electrophoretic run 36
3.6 Bull grouping 36
3.7 Assessment of in vitro sperm characters 36
3.7.1 Sperm cell morphology 37
3.7.2 Estimation of lipid peroxidation 37
Simultaneous assessment of plasma membrane integrity 38
3.7.3
and mitochondrial membrane potential
3.7.4 Functional membrane integrity 38
3.7.5 DNA integrity 39
3.7.6 Assessment of sperm cell apoptosis 39
Chapter Page
Title
No. No.
3.7.7 Sperm motility 40
Effect of fertility associated proteins on in vitro sperm 40
3.8
characters
3.9 Induction of lipid peroxidation 41
3.10 Assessment of capacitation status 41
3.10.1 Induction of capacitation with heparin 42
Effect of fertility associated proteins on induction of 42
3.10.2
capacitation
3.11 Data analysis 43
IV RESULTS 45
4.1 ISOLATION AND CHARACTERIZATION OF 45
SEMINAL PLASMA AND SPERM MEMBRANE
PROTEINS
4.1.2 Electrophoretic profile of sperm membrane proteins 47
4.1.3 Electrophoretic profile of heparin binding proteins of 49
bovine seminal plasma
4.1.4 Electrophoretic profile of heparin binding proteins of 51
bovine sperm membrane
4.2 EVALUATION OF IN VITRO SPERM CHARACTERS 54
4.2.1 Sperm morphology 54
4.2.2.1. Correlation between MDA levels and other in vitro sperm 56
characters during the incubation at 37° C for 180 min
4.2.3. Plasma membrane integrity 58
4. 2. 3.1. Correlation between plasma membrane integrity and other 60
in vitro sperm characters during the incubation at 37° C for
180 min
4. 2. 4. Functional membrane integrity 61
4.2.4.1. Correlation between functional membrane integrity and 63
other in vitro sperm characters during the incubation at
37°C for 180 min
Chapter Page
Title
No. No.
4.2.5. Mitochondrial membrane potential of sperm cells 63
4.2.5.1. Sperm cells with high mitochondrial membrane potency 63
4.2.5.2. Correlation between sperm cells HMMP and other in vitro 66
sperm characters during the incubation at 37° C for 180 min
4.2.5.3. Sperm cells with low mitochondrial membrane potency 66
4.2.5.4. Correlation between sperm cells LWMMP and other in 68
vitro sperm characters during the incubation at 37° C for
180 min
4.2.5.5. Sperm cells with lost mitochondrial membrane potency 69
4.2.5.6. Correlation between sperm cells LTMMP and other in vitro 71
4.2.6. DNA integrity 72
4.2.6.1. Correlation between sperm cell DNA integrity and other in 74
180 min
4.2.7. Assessment of sperm cell apoptosis 75
4.2.7.1 Viable sperm cells 75
4.2.7.2. Correlation between viable sperm cells and other in vitro 77
4.2.7.3. Necrotic sperm cells 77
4.2.7.4. Correlation between necrotic sperm cells and other in vitro 79
4.2.7.5. Apoptotic sperm cells 80
4.2.7.6. Correlation between apoptotic sperm cells and other in vitro 82
4.2.8. Motility and velocity parameters of sperm cells 83
4.2.8.1. Motility and velocity parameters of sperm cells- bull 83
group I
4.2.8.2. Motility and velocity parameters of sperm cells- bull 86
group II
Chapter Page
Title
No. No.
4.2.8.3. Motility and velocity parameters of sperm cells- treatment 87
with H2O2
4.2.8.4. Correlation between sperm cell motility parametersand 89
other in vitro sperm characters during the incubation at 37°
C for 180 min
4.2.8.4.1. Progressive forward motility 89
4.2.8.4.2 Static sperm cells. 90
4.2.8.4.3. Sperm velocity parameters 90
4.3. CAPACITATION STATUS OF SPERM CELLS 91
4.3.1. F pattern cells 91
4.3.2. Correlation between F pattern sperm cells and other in vitro 93
4.3.3. B pattern cells 94
4.3.4. Correlation between B pattern sperm cells and other in vitro 96
4.3.5. AR pattern cells 97
4.3.6. Correlation between AR pattern sperm cells and other in 99
180 min
4.4. RANKING OF BULL 100
V DISCUSSION 104
CHARACTERIZATION OF SEMINAL PLASMA AND 104
5.1
SPERM MEMBRANE PROTEINS
5.1.2 Electrophoretic profile of sperm membrane proteins 106
5.1.3. Heparin binding proteins of seminal plasma 107
5.1.4. Heparin binding proteins of sperm membrane 107
5.2. EVALUATION OF IN VITRO SPERM CHARACTERS 108
5.2.1. Sperm morphology 108
Chapter Page
Title
No. No.
Correlation between MDA levels and other in vitro sperm 112
5.2.2.1.
characters
5.2.3. Plasma membrane integrity 113
Correlation between plasma membrane integrity and other 115
5.2.3.1.
in vitro sperm characters
5.2.4. Functional membrane integrity 116
Correlation between functional membrane integrity and 118
5.2.4.1.
other in vitro sperm characters
5.2.5. Mitochondrial membrane potential of sperm cells 118
Correlation between mitochondrial membrane potential of 120
5.2.5.1.
sperm cellsand other in vitro sperm characters
5.2.6. DNA integrity 121
Correlation between sperm cell DNA integrity and other in 122
5.2.6.1.
vitro sperm characters
5.2.7. Assessment of sperm cell apoptosis 123
Correlation between apoptotic sperm cells and other in vitro 126
5.2.7.1.
sperm characters
5.2.8. Motility and velocity parameters of sperm cells 126
Correlation between sperm cell motility parametersand 130
5.2.8.1.
other in vitro sperm characters
5.3. CAPACITATION STATUS OF SPERM CELLS 131
Correlation between B pattern sperm cells and other in vitro 134
5.3.1.
sperm characters
5.4. RANKING OF BULL 134
VI SUMMARY 136
REFERENCES 140
LIST OF TABLES
Table Page
Title
No. No.
1 Electrophoretic profile of bovine seminal plasma proteins 46
assessed by SDS-PAGE
2 Electrophoretic profile of bovine sperm membrane proteins 48
assessed by SDS-PAGE
3 Electrophoretic profile of heparin binding proteins of bovine 50
seminal plasma assessed by SDS-PAGE
4 Electrophoretic profile of heparin binding proteins of bovine 52
sperm membrane assessed by SDS-PAGE
5 MDA level (µ mol/ml) in frozen thawed bull semen samples 55
treated with fertility associated protein and hydrogen peroxide
6 Correlation among in vitro sperm characters 57
7 Per cent of sperm cells with intact plasma membrane in frozen 59
thawed bull semen samples treated with fertility associated
protein and hydrogen peroxide
8 Per cent of sperm cells with functional membrane integrity in 62
frozen thawed bull semen samples treated with fertility
associated protein and hydrogen peroxide
9 Per cent of sperm cells with high mitochondrial membrane 65
potential in frozen thawed bull semen samples treated with
fertility associated protein and hydrogen peroxide
10 Per cent of sperm cells with low mitochondrial membrane 67
11 Per cent of sperm cells with lost mitochondrial membrane 70
12 Per cent of sperm cells with intact DNA in frozen thawed bull 72
semen samples treated with fertility associated protein and
hydrogen peroxide
Table Page
Title
No. No.
13 Per cent of viable sperm cells in frozen thawed bull semen 76
samples treated with fertility associated protein and hydrogen
peroxide
14 Per cent of necrotic sperm cells in frozen thawed bull semen 78
peroxide
15 Per cent of apoptotic sperm cells in frozen thawed bull semen 81
peroxide
16 Motility and velocity parameters of frozen thawed bull semen 84
samples treated with fertility associated protein
16a Velocity parameters of frozen thawed bull semen samples 85
treated with fertility associated protein
17 Motility and velocity parameters of frozen thawed bull semen 88
samples treated with H2O2
18 Per cent of F pattern sperm cells in frozen thawed bull semen 92
samples treated with heparin and heparin with fertility
associated protein
19 Per cent of B pattern sperm cells in frozen thawed bull semen 95
associated protein
20 Per cent AR pattern sperm cells in frozen thawed bull semen 98
associated protein
21 In vitro sperm characters after incubation for 180 min in 101
bull group I
22 In vitro sperm characters after incubation for 180 min in 103
bull group II
LIST OF PLATES
Plate Title Between

No. pages
1 SDS – PAGE apparatus 35-36
2 SDS – PAGE power pack assembly 35-36
3 Electrophoretic pattern of seminal plasma proteins 36-37
4 Electrophoretic pattern of sperm membrane proteins 36-37
5 Sperm cell morphology assessment 37-38
6 Plasma membrane integrity and mitochondrial membrane 37-38

potential assessment
7 Functional membrane integrity assessment 38-39
8 DNA integrity assessment 38-39
9 Apoptosis assay 40-41
10 CASA- sperm motility patterns 40-41
11 CASA- sperm velocity parameters 40-41
12 Capacitation status – F pattern cells 42-43
13 Capacitation status – B pattern cells 42-43
14 Capacitation status – AR pattern cells 42-43

CHAPTER - I
INTRODUCTION
Artificial insemination (AI) is the reproductive biotechnology that has

made possible the safe use of semen from selected sires in a breeding female
population. Application of AI as a tool for dissemination of semen from superior
sires has contributed to the improvement of the genetic quality of breeding herds.
This improvement has been exponential in dairy cattle, in which use of frozen semen
is most common. A prerequisite for the best use of this genetic material is to obtain
acceptable fertility after AI apart from achieving a high milk production. For this
reason, both screening of the semen for normality and fertility are essential.
Accurate evaluation of the bull fertility is important because it influences the
reproductive potential of the present and future herd. The aim of the breeding
industry is to identify genetically superior bulls and maximize the number of
offspring produced by these bulls. The fertility of the selected bull is important in
achieving this aim.
Currently breeding soundness examination (BSE) is carried out before

introducing a bull into the semen collection programme. BSE involves evaluation of
the bull for its physical health with special reference to rear leg conformation, proper
development of reproductive organs, scrotal circumference, libido and mating ability
of the bull. Semen from the bull is evaluated for its volume, sperm cell
concentration, motility, viability, morphology and ability to withstand freezing and
thawing procedures. Frozen semen samples from bulls that have passed through
BSE and possessing quality standards of semen industry were found to yield fertility
rates that differed by 20 – 25 per cent among bulls. The variations in the fertility rate
among the bulls were not addressed by the routine semen evaluation parameters
(Larson and Miller, 2000).
The most accurate method for testing the bull fertility is the insemination
of many fertile females, butthis method is time consuming, expensive for routineuse
and only allows a limited number of bulls to be tested at any given time (Barth and
Oko, 1989). Consequently, it would be of great benefit to the cattle industry to
develop a simple, accurate and reliable method of assessing the potential fertility of
bulls based on analysis of semen. Subsequently attention is now being directed
towards the assessment of other aspects of semen quality as predictors of bull
fertility. Proteins present in the seminal plasma and sperm have been reported as
markers of bull fertility. Seminal plasma, a complex mixture of secretions from
testis, epididymis and accessory sex glands contained factors that modulated the
fertilizing ability of sperm.Several studies provided direct evidence that seminal
proteins were adsorbed to the surface of sperm and affected its function and
properties (Yanagimachi, 1994). It has been suggested that seminal proteins mediate
the binding of sperm cells to oviductal epithelium and preserve membrane integrity
by exerting inhibiting effects on the mitochondrialactivity and metabolism to
conserve energy needed until fertilization as well as to minimize the production of
reactive oxygen species and lipid peroxidation of sperm membrane (Schoneck et al.,
1996). Proteins would also have activities inanti-apoptosis and cell survival
(Rangaswami et al., 2006; Chakraborty et al., 2006). It has been suggested that
proteinswould promote capacitation of sperm cells by increasing the number of
heparin binding sites on the sperm surface and stimulating cholesterol release
(Therein et al., 1998). Seminal proteins would mediate sperm–oocyte interaction and
fertilization (Moura et al., 2006). Proteins such as osteopontin, prostaglandin D
synthase, bovine seminal plasma proteins (BSP A1, A2, A3)and heparin binding
proteinshave been reported as indicators of bull fertility (Killian et al., 1993; Bellin
et al., 1994; Gerena et al., 1998; Sprott et al., 2000; McCauley et al., 2001; Moura et
al., 2006). 28 – 30 kDa heparin binding protein of sperm membrane was known as
fertility associated antigen (FAA) and it was considered as one of the genetic marker
for male fertility and heritable character. The bulls positive for the protein had 9 –
40 per cent more conception than the negative bulls. It was likely that up to 50 per
cent of bulls in a herd might lack the fertility associated protein (Bellin et al., 1994;
Sprott et al., 2000; McCauley et al., 2001; Ax, 2004).
Reactive oxygen species (ROS) generated by spermatozoa play an

important role in normal physiological processes such as, sperm capacitation,
acrosome reaction and maintenance of fertilizing ability (Desai et al., 2010). When
the ROS were generated in excess, they caused oxidative stress to sperm cells
(Bansal and Bilaspuri, 2007). Mature spermatozoa have little capacity for repairing
oxidative damage because their cytoplasm contains low concentrations of
scavenging enzymes (Alvarez and Storey, 1989). Seminal plasma is endowed with
antioxidant capacity (Jimenez et al., 1990) and should be capable of scavenging
ROS and protect spermatozoa against oxidative stress. Semen dilution with
extenders reduces the potential protective capacity of seminal plasma (Maxwell and
Stojanov, 1996). The freezing and thawing procedures also causes severe damages
and/or death to certain per cent of sperm cells, which generate ROS in excess via an
aromatic amino acid oxidase catalyzed reaction (Sariozkan et al., 2009).This results
in impaired cell functions and cell death (Bucak et al., 2010). Thus reactive oxygen
species are independent markers of male infertility factor.
India possess one fifth of worlds total cattle population and contributes
significantly to the national economy. Infertility among dairy cattle population is
one of the major problems which affect the growth of dairy industry. Reduced
fertility potential of the bull semen used for insemination might be a contributing
factor in the infertility problem. To address the issue, the present study was therefore
undertaken with the following objectives,
i) To study the presence of fertility associated proteins, status of lipid

peroxidation and functional integrity of sperm membrane and
organelles in the semen samples from bulls included in the breeding
programme.
ii) To determine role of fertility associated proteins and lipid

peroxidation on the in vitro sperm characters.
iii) To rank the bulls included in the breeding programme based on the
spermatozoal attributes.
CHAPTER II
REVIEW OF LITERATURE
2.1. MOLECULAR INDICATORS OF FERTILITY
Fertility associated proteins such as osteopontin, prostaglandin D

synthase, bovine seminal plasma proteins (BSP A1, A2, A3), heparin binding
proteins, fertility associated antigen, phospholipase A2, sperm adhesion Z13,
clusterin, heat shock proteins and others have been reported as indicators of fertility
(Killian et al.,1993; Bellin et al., 1994; Rolf et al., 1996;Gerena et al., 1998; Cancel
et al.,1999; Fouchecourt et al., 2000; Sprott et al., 2000; McCauley et al., 2001;
Moura et al., 2006). Some of these molecules were probably related to fertility
because of their vital function, while others may be more general indicators of
problems during spermatogenesis or spermatozoal maturation. A battery of
molecular tests/ indicators would be more valuable to detect the fertility potential in
semen samples.
2.1.1. Seminal plasma and sperm membrane proteins
In the recent years several proteins had been linked in some way or
another to fertilizing capacity of sperm. While most of these proteins were
associated with the seminal plasma, some were identified in sperm membrane. The
seminal plasma, a complex mixture of secretions from testis, epididymis and
accessory sex glands contained factors that modulated the fertilizing ability of sperm
(Killian et al., 1993; Yanagimachi, 1994; Henault et al., 1995; Bellin et al., 1996;
Amann et al., 1999). These secretions were considered “accessory” because the
spermatozoa present in cauda epididymis, had not been exposed to seminal plasma
proteins and were not sterile (Holt and Smidt, 1976). Depletion of these accessory
glands caused reduction in embryonic development and numbers (Chen et al., 2002),
suggesting that components of accessory sex glands had a potential influence on
fertilization and post fertilization events.
Immature spermatozoa newly formed in the seminiferous tubules were
transported through the epididymis where in they became motile and underwent a
series of events that included cytoskeleton rearrangement, changes in the
composition of membrane lipids and proteins (Olson et al., 2002; Gatti et al., 2004).
The epididymal epithelium secreted proteins that potentially affected not only sperm
maturation (Dacheux and Dacheux, 2002), but also other aspects of physiology
while these cells were stored in the cauda compartment. It has been well established
that important attributes of the sperm such as motility, oocyte binding and
penetrating capacity were acquired during epididymal transit (Amann and Griel,
1974).
2.1.2 Seminal proteins and sperm function
The role of seminal plasma proteins in the regulation of sperm function

was highly complex and was manifested during different molecular events. Several
studies provided direct evidence that proteins of seminal plasma were adsorbed to
the surface of sperm (Desnoyers and Manjunath, 1992) and affected its function and
properties (Yanagimachi, 1994). Some of these proteins were probably adsorbed
onto the surface so tightly that they would be inseparable or indistinguishable from
the ‘intrinsic’ membrane proteins, while others may be removed by simple washing
(Russell et al., 1985). The proteins were topographically reorganized into specific
regions of the sperm surface (Wolf and Voglmayr, 1984) and changed the properties
of the sperm membrane by binding to it and / or modifying the structure or the
arrangement of the existing membrane molecules. It was suggested that these
proteins maintained the stability of membrane up to the process of capacitation.
Capacitation and subsequent acrosome reaction reduced the content of proteins on
the sperm surface and approximately 35 per cent of them only remained on the
spermatozoa after acrosomal reaction (Desnoyers and Manjunath, 1992; Barrios et
al., 2005; Caballero et al., 2006).
Immunocytochemical analysis showed that membrane alterations induced

by capacitation and the acrosome reaction accounted for the redistribution of
proteins to the equatorial and post-equatorial regions of the sperm head, which
indicated their contribution to capacitation and gamete interactions (Yanagimachi,
1994; Therien et al., 2001; Caballero et al., 2006). The protein remodeling during
capacitation and the acrosome reaction probably contributed to the exposure and
binding of recognition residues, or the proteins could even act as direct
intermediaries in fusion of the spermatozoon with the zona pellucida.
Seminal plasma proteins protected sperm from peroxidative damage

(Schoneck et al., 1996) and prevented premature capacitation of sperm (Roberts et
al., 2003). Ram seminal plasma proteins RSVP 14 or RSVP 20 were able to increase
the resistance of spermatozoa to cooling and preserved membrane integrity (Barrios
et al., 2005). The cold shock damage on ram sperm membrane was reverted by
seminal plasma proteins getting adsorbed onto the cold-shocked ram sperm surface
partially repairing the membrane alterations induced by cold shock (Pascual et al.,
1999; Barrios et al., 2000; Perez-Pe et al., 2001). Scanning electron microscopy
confirmed that the repairing effect of seminal plasma proteins on the cold shocked
ram sperm membrane was concomitant with the restoration of intact-membrane cells
as assessed by non-penetration of the nuclear membrane by propidium iodide. This
protein adsorption was a concentration- dependent process that induced cell surface
restoration in relation to the amount of protein in the incubation medium (Barrios et
al., 2000). The reversal of the cold-shock damage by the dilution of frozen–thawed
ram semen with seminal plasma was also reflected in increased fertility of
spermatozoa (Rebolledo et al., 2007).
Blanco (2008) reported that the addition of seminal plasma proteins to

ram spermatozoa before the cold-shock treatment prevented sperm membrane
damage and maintained viability. Both the protective and repairing effects were
highly species specific because neither seminal plasma proteins from bull nor bovine
serum albumin had the ability to restore ram sperm membrane integrity. Moreover,
thermal denaturation of seminal plasma proteins removed these effects. Seminal
plasma lipoproteins were not involved in the recovering effect, because the addition
of lipoproteins isolated from ram seminal plasma were unable to reverse the cold-
shock-induced damage (Barrios et al., 2000). However, the fractionation of ram
seminal plasma proteins by exclusion chromatography provided three fractions that
were able to repair the damage (Barrios et al., 2000).
The addition of seminal plasma to frozen thawed ram sperm improved
motility, viability and mitochondrial respiration (Ollero et al., 1997; Maxwell et al.,
2007; Rebolledo et al., 2007). Addition of seminal plasma also increased the
resistance to spermatozoa of bull (Garner et al., 2001), ram (Ollero et al., 1997) or
red deer (Martinez-Pastor et al., 2006) to cryo-injury.
Henault and Killian (1996) reported that the fertility of sub-fertile

spermatozoa was improved by combining with seminal plasma from highly fertile
semen sample. Proaposin, a poly peptide derived from seminal plasma was found to
increase the fertility of bovine spermatozoa (Amann et al., 1999). Studies have
reported correlations between non return rates of bulls and seminal plasma proteins
(Killian et al., 1993; Gerena et al., 1998; McCauley et al., 2001).
2.1.3 Electrophoretic profile of seminal plasma proteins
Seminal plasma proteins partly originated from the blood plasma by

exudation through the lumen of the male genital tract, and partly synthesized and
excreted by testes (Kato et al., 1985), epididymis (Turner and Reich, 1987), vas
deferens and seminal vesicles (Manjunath et al., 1994). At least 4-6 proteins of the
seminal plasma were antigenically similar to those in the blood stream, and at least
6-7 proteins in buffalo and 5-6 proteins in cattle were specific to the seminal plasma
(Kulkarni, 1985).
Arora and Jain (1995) observed 6 protein components in seminal plasma

of buffalo. The molecular weights of these proteins were in the range of 7 to 71 kDa,
as found out by Sephadex G- 200 column chromatography. Seminal plasma of
buffalo and cattle were reported to contain 19 to 20 protein bands in the molecular
weight range of 11 to 92 kDa with varying intensities as observed in SDS- PAGE. In
cattle 21 kDa proteins were higher in concentration, whereas in buffaloes 66 and 11
kDa proteins were higher in concentration (Kulkarni et al., 1996). Arangasamy etal.
(2005) reported eight major heparin binding proteins in the molecular weight range
of 13 to 71 kDa and seven major gelatin binding proteins of 13 to 61 kDa in the
buffalo seminal plasma.
Comparative sequence analysis revealed strong similarities between
certain seminal plasma proteins identified in several species. The sequence of the
ram seminal plasma proteins RSVP14 fragment determined by N-terminal automatic
sequencing (Barrios et al., 2005) showed a high homology with several seminal
plasma proteins of other species, particularly bovine PDC-109 (Esch et al., 1983)
and goat GSP-14 / 15 kDa (Villemure et al., 2003).
2.2. BOVINE SEMINAL PLASMA AND SPERM MEMBRANE

PROTEINS
2.2.1. BSP proteins
Family of major proteins of seminal plasma designated as BSP- A1, BSP-

A2, BSP-A3 and BSP - 30 were collectively called as bovine seminal plasma (BSP)
proteins (Manjunath, 1984). The BSP- A1 and BSP-A2 were known as PDC-109
(Calvete et al., 1994). The apparent molecular masses of BSP- A1, BSP-A2, BSP-A3
proteins ranged from 15 to 17 kDa and of BSP 30 protein ranged from 28 to 30 kDa.
These proteins were secretory products of seminal vesicle and ampulla and their
biochemical characters were well described (Moura et al., 2006). All these BSP
proteins bound to sperm at ejaculation via their interaction with phospholipids
(especially phosphodidylcholine, plasmalogen and spingomyelin) of sperm
membrane (Desnoyers and Manjunath, 1992) and appeared to be immunologically
ubiquitous in mammalian species (Calvete et al., 1994).
Studies suggested that BSP proteins mediated sperm binding to the

oviductal epithelium, helping to preserve sperm viability and motility while in the
oviductal reservoir (Gwathmey et al., 2006). Souza et al. (2006) reported that
intensity of BSP proteins were observed in the acrosome and mid piece of the
spermatozoa. In acrosome reacted sperm the intensity of BSP proteins significantly
decreased in the acrosome region after the sperm came in contact with both isthmic
and ampullary oviductal fluids. Such modifications were probably the result of
fusion of the acrosome membrane caused by the acrosome reaction and/or
rearrangements of the sperm membrane triggered by movement of cholesterol and
phospholipids (Souza et al., 2006).
Intense immunoreaction of anti-BSP A1/A2 (PDC 109) at the mid piece
has been reported for ejaculated bovine sperm (Aumuller et al., 1988). It was
suggested that localization of BSP proteins in the mid piece closeto the mitochondria
was indicative of the fact that BSP proteins affected sperm motility. Although
protein receptors for BSP proteins were not yet identified, BSP A1/A2 was shown to
stimulate sperm motility and membrane-bound calcium ATPase activity in bull
sperm (Sanchez-Luengo et al., 2004). The functional connection between binding of
BSP proteins to the mid piece and stimulation of mitochondrial activity and sperm
motility suggested multifaceted role for BSP proteins as sperms were prepared for
fertilization. Although it has not been tested, BSP proteins probably influenced
hyperactivation of sperm.
2.2.2. Osteopontin
The 55 kDa protein in seminal plasma identified as osteopontin (OPN)

was an acidic glycoprotein rich in aspartic acid, glutamic acid, and serine (Sørensen
and Petersen, 1994). Reverse transcription polymerase chain reaction studies
identified OPN mRNA in the testis and epididymis and immunofluorescence studies
confirmed the presence of OPN in the sperm tail (Siiteri et al, 1995). Rodriguez et
al. (2000) established that the OPN present in bull seminal plasma originated from
the ampulla and seminal vesicles, with the majority of the protein being synthesized
by the epithelial cells of the ampulla. Killian et al. (1993) and Cancel et al. (1997)
reported a high correlation (r = 0.89) between presence of OPN proteins and actual
fertility data indicating that OPN was more prevalent in the seminal plasma of bulls
with higher fertility.
OPN mediated sperm–oocyte interaction and fertilization (Moura, 2005;

Moura et al., 2006). In addition to sperm binding, it also had theability to interact
with the zona pellucida and oolema of bovine oocytes. However, the oviductal fluid
also had OPN (Gabler et al., 2003), which may attach to the zona before
spermatozoa even reached the site of fertilization. Sperm-OPN connected to the
OPN already bound to the zona because OPN was capable of forming bonds with
other OPN molecules with high affinity (Goldsmith et al., 2002).After entering the
periviteline space, OPN attached to the post-equatorial segment and mediated the
interaction of sperm with the oolema, also through integrins and/or CD44 (Souza et
al., 2008). The binding OPN to sperm and oocytes through integrins and CD44 was
supported by observations that α5 integrins had been identified in cattle (Erikson et
al., 2003) and human spermatozoa (Fusi et al., 1996; Reddy et al., 2003), as well as
on the oolema (D’Cruz, 1996). The CD44 transmembrane glycoproteins were also
present in bovine sperm and oocyte (Schoenfelder and Einspanier, 2003) and OPN
had been reported to interact with integrins and CD44 in several other cell types
(Mazzali et al., 2002; Rangaswami et al., 2006).
Souza et al. (2008) reported that anti-osteopontin antibody binding was

present on the post-equatorial segment and acrosomal cap of ejaculated sperm, with
less fluorescence on the midpiece. Incubation of sperm with isthmic oviductal fluid
increased the fluorescence on post-equatorial segment, but without alterations on the
acrosomal cap or mid piece. Oviductal fluid contained OPN (Gabler et al., 2003)
and it was possible that such OPN bound to sperm, causing the increase in
fluorescence seen in the post-equatorial region. However, reductions on acrosome
fluorescence and intensification in equatorial fluorescence in acrosome reacted
sperm in the isthmic and ampullary oviductal fluid, as compared with acrosome
intact cells, were obviously caused by other mechanisms. These changes might be
the consequence of membrane fusion and remodeling which occurred between the
plasma and outer acrosomal membranes prior to the acrosome reaction (Souza et al.,
2008).
The importance of OPN in reproduction was also demonstrated by

Goncalves et al. (2003) in experiments using in vitro fertilization. Incubation of
oocytes with OPN led to increases in cleavage rates at day 4 (from 78.10 ±1.30 to
85.80 ±1.40 %), blastocyst development at day 8 (from 24.20 ±1.20 to 33.80 ±1.40
%) and hatched blastocysts at day 11 (from 10.60 ±1.60 to 18.50 ±1.40 %). Bull
semen frozen with different concentrations of OPN induced greater in vitro
fertilization rates (85.00 ± 4.00 vs 75.00 ± 4.00 %) and blastocyst development at
day 8 (45.00 ± 2.90 vs 33.00 ± 2.30 %) in comparison with untreated semen
(Goncalves et al., 2003). The fact that OPN had effects on post-fertilization events
was indeed exciting but how it happened remains to be elucidated. OPN had known
activities in anti-apoptosis and cell survival through activation of integrins and
CD44 membrane receptors and signal transduction mechanisms, including activation
of Map kinases, phosphoinositide (PI) 3-kinase/Akt-dependent NFkB, IKK/ERK-
mediated pathways, which stimulated uPA-dependent MMP-9 activation and PLC-
g/PKC/PI 3-kinase pathways (Wai and Kuo, 2004; Rangaswami et al., 2006;
Chakraborty et al., 2006; Khodavirdi et al., 2006; Lee et al., 2007). Numerous
intracellular signals were activated in the egg (such as those involving Src kinases
and PKC) after sperm–egg fusion occured. Such intracellular messengers were
thought to play important roles in the resumption of meiosis after fertilization and
consequent development of the zygote (Eliyahu et al., 2001; Eliyahu et al., 2002;
Halet, 2004; McGinnis et al., 2007).
2.2.3. Heparin binding proteins
This group represented five classes of heparin binding proteins (HBPs)

with molecular weights ranging from 14 to 31 kDa (Miller and Ax, 1989). HBPs
were secreted from the prostate, seminal vesicles and bulbourethral glands into
seminal fluid and bind to sperm at ejaculation (Nass et al., 1990). Bulls with
increased fertility produced sperm with greater affinity to bind heparin like complex
sugars that were commonly found in the reproductive tract of females (Marks and
Ax, 1985). The larger HBPs such as HBP- 21.5, HBP- 24 and HBP- 30 were known
to have greatest heparin affinity.
HBPs promoted capacitation of sperm cells by increasing the number of

heparin binding sites on the sperm surface (Therien et al., 1995) and stimulating
cholesterol release from membrane (Therien et al., 1998). In female reproductive
tract, HBP bound sperm interacted with oviductal components like high density
lipoproteins which stimulated a second cholesterol efflux resulting in capacitation
(Therien et al., 1998). Thus, the positive effect of HBPs on fertility could be linked
to its ability to mediate these events, which were crucial for successful fertilization.
HBPs potentiated heparin induced capacitation and acrosomal reactions in
epididymal sperm cells (Miller and Ax, 1989). Bulls with lower fertility produced
sperm cells that displayed a poorer ability to undergo capacitation in response to
heparin (Ax et al., 1985; Ax and Lenz, 1987).
2.2.4. Fertility associated antigen
HBP with molecular weight of 28 to 31 kDa in the sperm membrane were

named as fertility associated antigen (FAA). FAA was a non-glycosylated, basic
protein and the FAA cDNA sequence displayed 88 per cent identity to DNase-I like
3 (DNase1L3), a gene cloned from human liver expressed sequence tags (Rodriguez
et al., 1997a). Expression of FAA originated in the seminal vesicles, prostate and
bulbourethral glands. FAA has been detected in semen from bulls, boars, rams,
bucks and humans and has been localized primarily to the acrosomal region of the
sperm head (Dawson et al., 2002).
Hypothesis was that FAA was involved in regulation of sperm

capacitation and /or induction of acrosome reaction due to its heparin binding
characteristics. The response of sperm in vitro to heparin supplemented media was
characterized by a dose response increase in acrosome reactions upon exposure to an
appropriate inducer (Ca2+ ionophore or a fusogenic agent such as
lysophosphodidylcholine), and the ability of sperm to undergo acrosome reactions
under such conditions was positively correlated to fertility of bulls (Ax and Lenz,
1987).
Bull semen with the presence of FAA in sperm membranes had increased
fertility by 9 to 40 per cent points under natural service in beef breeds namely, Red
Angus, Gelbvieh, Santa Gertrudis and crossbred Santa Gertrudis X Gelbvieh (Bellin
et al., 1996). When beef cattle heifers and cows were artificially inseminated with
FAA positive spermatozoa, pregnancy rates were about 15 per cent higher than in
females inseminated with FAA negative spermatozoa (Sprott et al., 2000).
Spermatozoal FAA was shown to be a significant marker for fertility, even if bulls
displayed similar behavioural serving capacities (Bellin et al., 1998). Bellin et al.
(1998) reported that the percentage of bulls that were FAA negative among 44 herds
ranged from zero to 50 per cent (average, 12 %; n = 2,191 bulls). Given this wide
range, the chance of having FAA negative bulls may be relatively high in some
herds. Thus screening semen samples for FAA after a breeding soundness
examination for all bulls was prudent whether natural mating or AI will be
employed. FAA negative bulls were to be eliminated regardless of whether they
were used in natural or artificial breeding programme (Sprott et al., 2000).
2.2.5. Prostaglandin D- synthase
Gerena et al. (1998) identified a 26 kDa protein associated with high

fertility that was present in bull seminal plasma, as lipocalin-type prostaglandin D
synthase. Prostaglandin D synthase was a member of the lipocalin super family
(Nagata et al., 1991; Urade et al., 1995), which was a group of proteins that bound
to small hydrophobic molecules and specific cell surface receptors.
Studies indicated that lipocalin-type prostaglandin D synthase, also known

as beta-trace, played a role in male reproductive function. High concentrations of
beta-trace were detected in human seminal fluid and reduced concentrations of beta-
trace were found in semen from azoospermic men (Olsson, 1975; Tokugawa et al.,
1998). These data indicated that a reduction in the concentrations of lipocalin type
prostaglandin D synthase in semen may be associated with human infertility.
Northern blotting revealed that prostaglandin D synthase mRNA was

present in human testes, epididymides and prostate glands. Furthermore,
immunohistochemical studies using anti-lipocalin type prostaglandin D synthase
antibody detected prostaglandin D synthase in the Leydig cells of the testes, the
epididymal epithelium and the secretory elements of the prostate gland (Tokugawa
et al., 1998). The concentration of prostaglandin D synthase in the caput
epididymides was 6 and 80 times greater than in the brain and testes, respectively,
and indicated that prostaglandin D synthase gene expression was dependent on
androgens, as well as other testicular factors (Sorrentino et al., 1998). Prostaglandin
D synthase was found in fluid from the rete testis and cauda epididymis of bulls, as
well as in tissue extracts and histological sections from the testes, and caput, corpus
and cauda epididymis (Gerena et al., 1998; Rodríguez et al., 1998).
As a lipocalin, prostaglandin D synthase was probably involved in the
transport of bioactive lipophilic substances, which was analogous to the function of
other lipocalins, such as retinol binding protein and beta lactoglobulin. Prostaglandin
D synthase bound all trans- and 9-cis-retinoic acids with the same affinity as other
retinoid transporters (Tanaka et al., 1997). Retinoid derivatives of vitamin A played
an important role in the male reproductive system including induction of epithelial
differentiation, testicular function and maintenance of spermatogenesis (Tanaka et
al., 1997). In the epididymis, prostaglandin D synthase probably supplied retinoic
acid for the maintenance of epithelial tissue and normal epididymal function,
including sperm maturation.
It was also possible that prostaglandin D synthase was involved in the

transfer of other lipophilic substances, such as androgens. Production of testosterone
by the Leydig cells could raise the intra-testicular concentrations of testosterone to
300 ng which was necessary for maintenance of spermatogenesis (Amann, 1989).
Lipocalin type prostaglandin D synthase might aid in the transport and concentration
of androgens near germ cells at specific stages of development. Likewise,
prostaglandin D synthase in fluid from the rete testis and cauda epididymis (Gerena
et al., 1998) might be involved in the transport of androgens from the testis to the
efferent ducts and the caput epididymis, thus contributing to the marked differences
in steroid profiles of fluids entering and leaving the epididymis of bulls.
2.2.6. Type 2 Tissue inhibitor of metallo proteinases
Type 2 tissue inhibitor of metallo proteinases (TIMP-2) was a normal

constituent of semen from bulls (Calvete et al., 1994; McCauley et al., 2001),
humans (Baumgart et al., 2002), rats and rams (Metayer et al., 2002). TIMP 2 was
produced in various cell types including the testis and accessory sex glands. TIMP 2
bound to sperm traversing the urogenital tract during ejaculation. In a retrospective
analysis Dawson et al. (2002) reported that bulls which possessed TIMP-2 in
detergent extracts of sperm were 13 % more fertile than TIMP-2 negative bulls.
TIMP-2 was a heparin binding protein (McCauley et al., 2001) that inhibited the
catalytic activity of matrix metallo proteins (MMPs). TIMP-2 preferentially
regulated MMPs by inhibiting the cleavage or conversion of inactive pro-MMPs
zymogen to its active form (Nagase and Woessner 1999; Brew et al., 2000). MMPs
were mediators of various reproductive processes including prostate and testicular
function (Hulboy et al., 1997) and were localized to the acrosome and middle piece
of sperm (Buchman Shaked et al., 2002).
2.2.7. Spermadhesin Z13
Spermadhesin Z13 was a peptide that displayed 50 and 43 per cent

homology with the acidic seminal fluid protein and seminal plasma motility
inhibitor (SPMI), respectively (Tedeschi et al., 2000). The former had positive
effects on bovine sperm in vitro characters when at average concentrations, but
inhibited both sperm motility and mitochondrial activity when at high levels
(Schoneck et al., 1996).
2.2.8. Clusterin
Clusterin, an acidic heterodimeric glycoprotein was found in the

epididymal fluid of a number of species (Fouchecourt et al., 2000; Ibrahim et al.,
2000). Epididymal cells in the proximal corpus secreted clusterin abundantly,
allowing the concentrations to remain high throughout the epididymis. Clusterin
mediated protein precipitation (Ibrahim et al., 1999; Wilson and Easterbrook-Smith,
2000), agglutination of abnormal spermatozoa (O’Bryan et al., 1990; O’Bryan et al.,
1994) and complement induced sperm lysis (Ibrahim et al., 1999). Clusterin bound
to the plasma membrane of spermatozoa but was normally undetectable on
ejaculated spermatozoa (Ibrahim et al., 2000). Semen samples with many clusterin
positive spermatozoa had lower fertility, as determined by nonreturn-to-estrus rates
and many morphological abnormalities (Ibrahim et al., 2000). The presence of
clusterin in ejaculated sperm may indicate improper spermatogenesis or irregular
epididymal maturation.
2.2.9. Phospholipase A2
Phospholipase A2 (PLA2) associated with sperm membranes appeared to

participate in the acrosome reaction (Breitbart and Spungin, 1997; Chen et al., 2005)
and sperm-egg fusion (Riffo and Parraga, 1997), a notion supported by the report
that a PLA2 β-gene knockout mouse had sperm with reduced capacity to fertilize
oocytes both in vitro and in vivo (Bao et al., 2004).
2.2.10. Heat shock protein
Heat shock protein (HspA2) was a 70 kDa molecular weight protein and it
was not a form of creatin kinase as thought previously (Huszar et al., 2000). HspA2
content per spermatozoa was positively correlated with morphological defects in
spermatozoa and negatively correlated (r = - 0.70) with spermatozoal concentration
in human semen samples (Gergely et al., 1999). It was proposed that larger amounts
of HspA2 were associated with immature sperm cells that had not extruded their
cytoplasm during spermatogenesis and not completed plasma membrane remodeling
during epididymal maturation (Huszar et al., 2000).
2.2.11. Acrosin
Acrosin was a serine protease that was stored in the acrosome as a

zymogen (proacrosin) and then cleaved to acrosin during the acrosome reaction
(Yanagimachi, 1994). Proacrosin can bind to carbohydrates within the zona
pellucida, leading to the hypothesis that this protease was involved in spermatozoal
penetration through the zona pellucida (Jones, 1991). The possible function of
proacrosin /acrosin in fertilization had made it a target of study as a laboratory assay.
In human studies, acrosin enzyme activity was lower in ejaculates with low
penetration rates using zona-free hamster eggs (Francavilla et al., 1994). Shimizu et
al. (1997) found that decreased acrosin was correlated with poor IVF success. In
bovine studies, a high correlation between zona penetration and the presence of
acrosin indicated that spermatozoa have not undergone a premature acrosome
reaction and acrosin was available for penetration of zona pellucida (De los Reyes
and Barros, 2000).
2.3. OXIDATIVE STRESS
The term oxidative stress was generally applied when oxidants

outnumbered antioxidants. The imbalance between the production of reactive
oxygen species and a biological systems ability to detoxify the reactive
intermediates or easily repair the resulting damage was known as oxidative stress
(Agarwal et al., 2003). Oxidative stress was believed to be the underlying cause for
numerous cell dysfunctions. Gametes were susceptible to ROS attack.
Reactive oxygen species (ROS) represented a broad category of molecules

that indicated the collection of radicals (hydroxyl ion, superoxide, nitric oxide,
peroxyl, etc.), nonradicals (ozone, single oxygen, lipid peroxides, hydrogen
peroxide) and oxygen derivatives (Agarwal et al., 2005).
ROS were formed as necessary by-products during the normal enzymatic

reactions of inter and intra cellular signaling. Mammalian spermatozoa represented a
growing list of cell types that exhibited a capacity to generate ROS when incubated
under aerobic conditions. Due to their highly reactive nature, they combined readily
with other molecules, directly causing oxidation that lead to structural and functional
changes and result in cellular damage (Agarwal et al., 2005).
2.3.1. Origin of ROS in male reproductive system
In male, two ROS generating systems were possibly involved, a

hypothetical NADH oxidase at the level of sperm membrane and low sperm
diphorase (mitochondrial NADH-dependent oxidoreductase; Gavella and Lipovac,
1992). In bovine semen, ROS were generated primarily by dead spermatozoa via an
aromatic amino acid oxidase catalyzed reaction (Sariozkan et al., 2009). Leukocytes
and immature spermatozoa were the two main sources of ROS (Garrido et al.,
2001). Leukocytes particularly neutrophils and macrophages were associated with
excessive ROS production and they ultimately caused sperm dysfunction
(Ochsendorf, 1999).
Mature spermatozoa had little capacity for repairing oxidative damage

because their cytoplasm contained low concentrations of scavenging enzymes
(Alvarez and Storey, 1989). Seminal plasma was endowed with low molecular non-
enzymatic and enzymatic antioxidant capacity (Jimenez et al., 1990) being capable
of scavenging ROS to act as an additional protection of spermatozoa against
oxidative stress. The antioxidant enzyme system that comprised of superoxide
dismutase (SOD), glutathione reductase (GR), glutathione peroxidase (GPx) and
catalase (Alvarez and Strorey, 1989) was shown in bull (Beconi et al., 1993) and
ram semen (Marti et al., 2003). Studies on lipid peroxidation and antioxidant
enzymes in male infertility concluded that the increase in SOD and GPx activity
represented an attempt to overcome the ROS insult (Dandekar et al., 2002). Two of
the main factors contributing to ROS accumulation in vitro were the absence of
endogenous defence mechanism and second exposure of gametes and embryos to
various manipulation techniques as well as environment that could lead to
generation of oxidative stress (Agarwal et al., 2005).
2.3.2. Positive and negative effects of ROS
ROS generated by spermatozoa played an important role in normal

physiological processes such as, sperm capacitation, acrosome reaction, maintenance
of fertilizing ability and stabilization of the mitochondrial capsule in the mid-piece
in bovine (Desai et al., 2010). When in excess, ROS caused adverse effects on the
sperm plasma membrane, DNA and physiological processes, thereby affecting the
quality of spermatozoa. Sperm cells were sensitive to ROS attack which resulted in
decreased sperm motility, presumably by a rapid loss of intracellular ATP leading to
axonemal damage, decreased sperm viability, and increased mid-piece sperm
morphological defects with deleterious effects on sperm capacitation and acrosome
reaction (Bansal and Bilaspuri, 2007). Thus, ROS were independent markers of male
factor infertility (Sikka et al., 1995).
2.3.3. Lipid peroxidation
The mechanism of ROS-induced damage to spermatozoa included an

oxidative attack on the sperm membrane lipids leading to initiation of lipid
peroxidation (LPO) cascade (Sharma and Agarwal, 1996). The susceptibility of
ruminant spermatozoa to oxidative stress was a consequence of the abundance of
PUFAs in sperm plasma membrane and the presence of double bonds in these
molecules made them vulnerable to free radicals attack and the initiation of LPO
cascade. This resulted in a subsequent loss in membrane and morphological
integrity, impaired cell function, along with impaired sperm motility and induction
of sperm apoptosis (Bucak et al., 2010).
Concerning the chemistry of LPO in spermatozoa, it implied that once this

process was initiated, its propagation was impeded, leading to accumulation of lipid
peroxides in the sperm plasma membrane (Sharma and Agarwal, 1996).
Peroxidation of PUFAs in sperm cell membrane was an autocatalytic, self-
propagating reaction, which probably gave rise to cell dysfunction associated with
the loss of membrane functions and integrity.
2.3.4. Cryopreservation and oxidative stress
Given that high levels of antioxidants in seminal plasma contributed to the

preservation of spermatozoa, semen dilution or washing reduced the potential
protective capacity provided by the plasma (Maxwell and Stojanov, 1996). During
cryopreservation, semen was exposed to cold shock and atmospheric oxygen, which
in turn increased the production of ROS and decreased antioxidant level (Bucak et
al., 2010). The loss of antioxidant enzyme activity by cryoinjury in ram (Marti et al.,
2008) and human (Lasso et al., 1994) spermatozoa also has been described. Both
freezing and thawing caused tremendous alterations in cell water volume.
Spermatozoa discarded most of their cytoplasm during the terminal stages of
differentiation and lacked the significant cytoplasmic component containing
antioxidants that counteracted the damaging effect of ROS and LPO (Bucak et al.,
2010). Due to this, spermatozoa were highly susceptible to LPO during
cryopreservation and thawing (Bucak et al., 2010), which confered considerable
mechanical stress on the cell membrane. Excessive production of ROS during
cryopreservation was associated with reduced post thaw motility, viability,
membrane integrity, sperm functions and fertility.
Interestingly, the addition of seminal plasma proteins preserved not only

the enzyme activity levels but also the distribution of antioxidant enzymes on the
ram spermatozoa surface (Marti et al., 2008), which clearly verified the protective
effect of these compounds. High seminal plasma antioxidant activity correlated
positively with semen quality and low levels of both DNA damage and lipid
peroxidation (Alvarez and Storey, 1989). Likewise, a decreased antioxidant ability
of seminal plasma was correlated with lipid peroxidation of sperm membranes and
resulted in infertility (Chen et al., 2001). Negative relationship between semen
quality parameters, lipid peroxidation and glutathione peroxidase activity in ram
spermatozoa was reported (Kasimanickam et al., 2006).
2.4. EVALUATION OF IN VITRO SPERM CHARACTERS
Part of the complexity dealing with the spermatozoa came from the fact
that each spermatozoon was a multi-compartmental cell that must possess different
attributes to be able to fertilize an oocyte. Each spermatozoon must possess motility,
active mitochondria to supply the energy necessary for motility, intact acrosomal
membranes that were capable of undergoing capacitation changes thereby permitting
the acrosome reaction to occur, plasma membranes that permitted fusion with the
oolemma and a nucleus that was capable of proper decondensation and nuclear
reorganization to maintain zygotic and embryonic development.
The traits of a semen sample that were important for fertility were divided
into compensable and uncompensable. Compensable traits were those that did not
affect fertility if high numbers of spermatozoa were used during insemination
(Ballachey et al., 1988). For example, spermatozoa with defects in compensable
traits (i.e., motility and morphology) might have difficulty crossing the barriers of
the female reproductive tract to reach the site of fertilization. Because an excessive
number of spermatozoa were usually inseminated, compensable traits were not as
closely related to fertility as uncompensable traits. Uncompensable traits were those
that were not overcome by increasing the number of spermatozoa in the inseminate
because these defects affected the function of spermatozoa during later stages of
fertilization and embryonic development. Nuclear vacuoles and defective chromatin
structure were examples for uncompensable traits (Ballachey et al., 1988).
2.4.1. Sperm morphology
Sperm cell abnormalities were classified based on the location of defects

(head, tail, mid piece) or site of origin (primary: testis; secondary: epididymis;
tertiary: accessory glands/post ejaculation). The significance of specific sperm
abnormalities were better understood from the results of mating trials, analysis of
non-return rates to artificial insemination and in vitro fertilization with semen
containing high percentages of sperm with individual classes of abnormalities.
Accurate morphological screening of the ejaculates allowed elimination of

bulls with a potential low fertility, prior to the entrance of bulls to progeny testing
program and the preservation of semen, thus contributing to a major saving for AI
enterprises (Padrick and Jaakma, 2002; Esteso et al., 2006).. There was undoubtedly
a correlation between motility and fertility as well as for morphology and fertility;
however these correlations were reduced concomitantly with an increase in the
lower limit set to accept an ejaculate for further processing. When the acceptable
range for these parameters was narrow, motility and gross morphology only had
limited value for separating ejaculates in respect to the expected fertility of the
ejaculate.
2.4.2. Plasma membrane integrity
Structural and functional integrity of sperm outer membrane

(plasmalemma) was essential for sperm metabolism, capacitation, ova binding and
acrosome reaction. Membrane exerted role in the maintenance of the sperm
fertilizing capability (Oura and Toshimori, 1990). The plasma membrane was
responsible for the mechanism of maintaining the cell osmotic equilibrium, acting as
a barrier between intra and extra cellular mediums. Damages in this structure
conduced to homeostasis loss, leading to cellular death. Consequently, plasma
membrane integrity was crucial to sperm survival inside the female reproductive
tract (Oura and Toshimori, 1990).
The sperm plasma membrane was the primary site where lesions occured
during freezing-thawing of semen (Hammerstedt et al., 1990). It was recognized
during the last several decades that one of the major features discriminating dead
from live cells was a loss of the transport function and physical integrity of the
plasma membrane. For example, since the intact membrane of live cells excluded a
variety of charged dyes, such as trypan blue or propidium iodide (PI), incubation
with these dyes resulted in selective labeling of dead cells, while live cells showed
no or minimal dye uptake. A combination of supra vital staining dyes such as trypan
blue/giemsa, eosin/aniline blue and some other classical dyes were widely used for
differential live/dead staining of spermatozoa. For light microscopic evaluation a
relatively high concentration of the dye (in mg/ml) was required. At these
concentrations, eosin and many other dyes were toxic, which can lead to under
estimation of the proportion of live cells (Woelders, 1991).
The development of staining technology using fluorophores for nucleic

acid, intra cytoplasmic enzymes or membrane potential provided new tools for
assessing the functionality of frozen-thawed spermatozoa. Single fluorophores or in
combinations were used to determine sperm membrane integrity. The combination
of carboxyfluoroscein diacetate (CFDA) with propidium iodide (PI), or CYBR-14
with PI was commonly used. PI was membrane-impermeant red fluorescent
molecule that entered the nucleus of a cell in which the plasma membrane was
damaged. CFDA was a membrane-permeant colourless substrate that was rapidly
converted by intracellular esterases into a membrane-impermeant green fluorescent
derivative (Garner et al., 2001; Colenbrander et al., 2003). Used in combination,
cells with damaged membranes fluoresced red as PI entered the cell, and
intracellular esterases leaked from the cell, therefore, CFDA was not converted into
its green derivative. Cells with intact membranes fluoresced green as the membranes
prohibited PI entry and the retained esterases converted CFDA into a green
derivative. In contrast, SYBR-14 was a membrane-permeant green fluorescent probe
that bound to the DNA in the nucleus of all cells, both membrane intact and
membrane compromised. When used in combination with PI, cells with intact
plasma membranes only stained with SYBR-14 and fluoresced green, while cells
with damaged plasma membranes stained with both SYBR- 14 and PI and
fluoresced red, or red-orange as the fluorescence of the PI was brighter than that of
the SYBR-14 (Garner and Johnson, 1995).
2.4.3. Functional membrane integrity
Vital staining of sperm evaluated the structural integrity of the plasma

membrane which indicated whether the sperm cell was viable or dead. But in the
viable cells the functional integrity of plasma membrane had to be evaluated since
sperm required an active membrane during fertilization and will fail to fertilize
ovum if plasma membrane was physically intact but biochemically / functionally
inactive. Functional integrity of the plasma membrane was evaluated by measuring
the resistance of sperm membranes to swelling in a hypo-osmotic medium. This
much simpler method was based on the ability of the membranes to allow passage of
water in order to establish equilibrium between the fluid compartment within the
spermatozoon and the external surroundings (Drevius and Eriksson, 1996). It was
suggested that the ability of spermatozoa to swell in the presence of hypo-osmotic
medium reflected normal water transport across the sperm membrane, which was a
sign of normal membrane integrity and functional activity (Jeyendran, et al., 1984).
Spermatozoa with compromised or inactive membranes were unable to regulate
water influx and remain not swollen. Brito et al. (2003) concluded that HOST was
the only plasmalemma functional evaluation method that significantly contributed to
conventional sperm quality tests in predicting in vitro fertilization rate. The use of
this inexpensive and simple assay was recommended as an additional fertility
indicator to be incorporated in the routine semen analysis.
2.4.4. Mitochondrial membrane potential of sperm cells
Energy was stored in the mitochondria as a proton concentration gradient

and an electric potential gradient across the membrane. These gradients were
generated by electron transport maintained by the inner mitochondrial membrane
and drive the synthesis of ATP. Membrane permeable lipophilic cations
accumulated in the mitochondria and exhibited a negative interior membrane
potential. The lipophilic cationic fluorescent carbocyanine dye, JC-1, was used to
differentially label mitochondria with high and low membrane potential. When JC-1
formed monomers in mitochondria with low potential, the JC-1 stain emited a green
fluorescence at 510-520 nm when the JC-1 formed multimers known as J-aggregates
after accumulation in mitochondria with high membrane potential, the JC-1 stain
emited a bright red-orange fluorescence at 590 nm. Any changes in mitochondrial
membrane potential were a good indicator of sperm motility. These dyes were used
to study the effect of cryopreservation on bovine sperm organelle function and
viability (Thomas et al., 1998). They showed that fluorometric measurement of
mitochondrial function after thawing correlated with SYBR-14-assessed sperm
viability and with microscopic assessment of motility.
2.4.5. DNA integrity
Sperm chromatin integrity was vital for successful pregnancy and

transmission of genetic material to the offspring. The nuclear chromatin of
mammalian sperm had a peculiar organizational status characterized by a
remarkable process of remodeling or condensation (Govin et al., 2004; Caron et al.,
2005). The sperm DNA was organized in a specific way that kept the chromatin
compact and stable in the nucleus. It was packed into a tight, almost crystalline
condition and occupied nearly the whole nucleus (Fuentes-Mascorro et al., 2000).
This process of condensation occured in two main phases. The first phase, which
occurred in the testis, involved the substitution of somatic histones by testis-specific
protamines (Caron et al., 2005). The protamines contained numerous cysteine
residues, which generated disulphide cross-links between adjacent protamine
molecules during the condensation of the chromatin. The formation of large
numbers of disulphide cross-links between protamine molecules occurred in the
second main phase of chromatin condensation, when the sperm left the caput
epididymis and were en route to the cauda epididymis (Caron et al., 2005).
Sperm DNA fragmentation resulted from aberrant chromatin packaging

during spermatogenesis (Gorczyca et al., 1993; Manicardi et al., 1995; Sailer et al.,
1995), defective apoptosis before ejaculation (Sakkas et al., 1999; Sakkas et al.,
2002), or excessive production of reactive oxygen species (ROS) in the ejaculate
(Kodama et al., 1997; Aitken and Krausz, 2001; Moustafa et al., 2004). Exposure to
environmental or industrial toxins, genetics, oxidative stress, etc, was known to
cause sperm DNA fragmentation and infertility (Wang et al., 2003). The sperm
DNA integrity was reported to be both unaltered (Evenson et al., 1994; Evenson et
al., 2002; Van der Schans et al., 2000) and impaired after cryopreservation
(Hamamah et al., 1990; Bochenek et al., 2001; Baumber et al., 2000; Peris et al.,
2004).
Several methods were developed to evaluate the DNA integrity. The

sperm chromatin structure assay (SCSA), which was considered the most efficient
and successful, used flow cytometric analysis (Evenson et al., 2002). TUNEL and
Comet assays were also used to measure DNA fragmentation (Duty et al., 2002;
Sakkas et al., 2002; Evenson and Wixon 2006). The acridine orange staining was a
simple microscopic procedure based on the same principle as the SCSA (Chohanet
al., 2004). Acridine orange intercalated into native DNA and fluoresced green when
exposed to blue light and fluoresced red when associated with single stranded DNA.
Sperm with chromatin abnormalities were associated with reduced fertility

or abortions (Chemes and Rawe 2003). Fertilization by sperm with fragmented DNA
resulted in poor embryonic development, decreased implantation, lower pregnancy
rates, and recurrent pregnancy losses in human (Henkel et al, 2003; Virro et al,
2004) and reduced fertility in bulls (Kasimanickam et al., 2006).
2.4.6. Apoptosis
Cell death in general occurred through two distinct ways, necrosis and
apoptosis. Necrosis was a passive process that resulted from injury and caused cell
swelling and membrane rupture. During necrosis, the cellular contents were released
uncontrolled into the cell’s environment and resulted in damage of surrounding cells
and a local inflammatory response; in contrast, apoptosis was an active reaction that
followed a sequence of controlled steps leading to locally and temporally defined
self-destruction without causing an inflammatory reaction (Lockshin, 1964; Kerr et
al., 1972; Wyllie et al., 1980).
Apoptosis was a complex phenomenon that was divided into three phases:
induction, execution, and degradation. Mitochondria were known to play a central
role during the execution phase. After induction of apoptosis, mitochondrial pores
were opened, characterized by decreased mitochondrial membrane potential.
Opening of mitochondrial pores lead to the release of proapoptotic factors from the
mitochondria (Ravagnanet al., 2002). In the cytoplasmic compartment, the
proapoptotic factors—for example, different proteases related to the caspases family
(cysteine proteases with aspartate specificity) were subsequently activated, leading
to the degradation phase. During this phase, changes at both the cell surface and the
nucleus occurred. Phosphatidylserine (PS), ordinarily sequestered in the plasma
membrane inner leaflet, appeared in the outer leaflet, where it triggered
noninflammatory phagocytic recognition of the apoptotic cell (Bratton et al., 1997)
In the apoptotic cells, inter nucleosomal cleavage of DNA by specific endonucleases
produced ;180-base pair DNA fragments (Kerr et al., 1972).
Apoptosis was characterized by distinct ultra structural and biochemical

changes in cells including chromatin aggregation, cytoplasmic aggregation,
cytoplasmic condensation, and indentation of nuclear and cytoplasmic membranes,
which occurred on the cell surface and in the DNA. During early apoptosis, a cell
lost its membrane asymmetry. Phosphatidylserine, normally present on the inner
cytoplasmic leaflet of the plasma membrane of healthy cells, was translocated and
exposed on the outer leaflet (Martin et al., 1995). The externalization of
phosphatidylserine (PS) from inner to outer leaflet of cell plasma membrane was one
of the hallmarks of apoptosis. The disturbance of membrane function was detectable
as an increase in the cell ability to bind to the calcium-dependent binding of
annexin-V to the outwardly translocated PS (Andree et al., 1990). It was
demonstrated that PS translocation were used as a marker for disturbed
(deteriorating) membrane function of post-thaw human (Duru et al., 2001), and
bovine (Anzar et al., 2002) spermatozoa.
Use of annexin-V in combination with propidium iodide (PI, supravital

fluorescent dye) allowed simultaneous detection of apoptotic and/or necrotic cells or
cells with compromised plasma membrane. Annexin V was a Ca2+-dependent,
phospholipid binding protein that had a high affinity for PS and bound to cells with
exposed PS (Koopman et al., 1994; Vermes et al., 1995). Annexin V conjugated to
fluorescein isothiocyanate (FITC) fluorochrome retained its high affinity for PS and,
therefore, served as a sensitive probe that was used for detection of cell death.
Different categories of apoptotic, necrotic and viable cells were then sorted out
through flow cytometer or visually evaluated using fluorescent microscope (Van
Engeland et al., 1998).
Germ cell apoptosis appeared necessary for normal spermatogenesis to

develop, reducing germ cells to a number that was effectively supported by the
existing population of Sertoli cells (Rodriguez et al., 1997b; Blanco-Rodriguez and
Garcia 1999; Kierszenbaum, 2001). Normal spermatogenesis depended on the
efficiency of apoptosis. Approximately 25–75% of germ cells degenerated and died
in the adult mammal testis. Ejaculated spermatozoa were shown to exhibit certain
characteristics of apoptotic somatic cells such as DNA fragmentation or
phosphatidylserine translocation, especially in human (Gorczyca et al., 1993) and
bull (Anzar et al., 2002) semen. Likewise, Martin et al. (2004) analyzed the PS
translocation and caspase-3 and -7 activities in ram sperm samples washed by
different methods. The reasons why apoptotic sperm were present in ejaculated
semen was not very clear. Some authors attributed it to the existence of immature
sperm (Paasch et al., 2004), others to a phenomenon of abortive testicular apoptosis
(Sakkas et al., 2004), and some to pathologic causes (Oehninger et al., 2003).
Whatever the cause, the presence of apoptotic spermatozoa in seminal doses was
responsible for poor fertility, as reported in humans (Taylor et al., 2004; Said et al.,
2006). Furthermore, apoptosis in sperm would be activated as a mechanism of
elimination of abnormal spermatozoa, or in response to environmental stress. Thus,
some authors described certain features of frozen–thawed spermatozoa as an
apoptosis-like phenomenon (Martin et al., 2004).
2.4.7. Motility
Motility and gross morphology estimated by light microscopy were by

now most used parameters for semen quality assessment, especially in AI
laboratories. The evaluation of sperm motility provided important information on the
energy status of mammalian sperm (Quintero-Moreno et al., 2004). Furthermore, the
motility function played an important role once spermatozoa reached the uterotubal
junction, which might act as barrier to sperm with poor motility (Mortimer, 1997).
Variations of 30–60 per cent have been reported in subjective microscopic

evaluations of motility characters of human and animal semen in the same ejaculates
(Amann, 1989; Auger et al., 1993). Despite a close match between subjective and
objective evaluations of sperm motility, subjective estimation of motility was
affected by numerous factors (Verstegenet al., 2002; Rijsselaere et al., 2003).
The development of computer-assisted semen analysis (CASA), using

software that analyzed every sperm track characteristics, had strongly improved the
semen evaluation. The availability of data recorded by CASA facilitated the
comparison of results and made it possible to find subtle differences between bulls
or treatments (Verstegenet al., 2002). Furthermore, CASA systems appeared to have
high accuracy and repeatability (Farrellet al., 1995). CASA was an objective method
that gave extensive information about the kinetic property of the ejaculate based on
measurements of the individual sperm cells. Using CASA, motility and movement
characteristics of spermatozoa were correlated to in vivo fertility (Holt et al., 1997).
However, CASA were not ready-to-use devices, thus results depended largely on the
expertise of the user and the technical settings (Mortimer et al., 1995). Numerous
variables like the frequency of frame acquisition, number of fields analyzed, sample
concentration and dilution affected motility resulted in semen evaluation even with
the same CASA device (Rijsselaere et al., 2003).
Correlation coefficients estimated between post thaw motility and in vitro

fertilization rate were of very low magnitude. The lower correlations may be
because motility reflected the ability of sperm to reach fertilization site, but not its
ability to undergo capacitation, acrosome reaction and oocyte penetration (Graham
et al.,1987; Brahmkshtri et al., 1999).
2.4.8. Capacitation status of sperm cells
The process of capacitation was originally recognized by Austin (1952)

and Chang (1951) independently, who reported that spermatozoa had to reside in the
female reproductive tract for acquisition of fertilizing competence. Capacitation was
a continuous biochemical change associated with the functional and structural
changes in the sperm. Removal of cholesterol from sperm membrane increased
membrane fluidity (Langlais et al., 1988), resulted in increased calcium influx
(Singh et al., 1978), cAMP level (White and Aitken, 1989) and changed some
enzymatic activities such as protein kinase C (Furuya et al., 1993). These
biochemical modifications lead to a transient change in the pattern of sperm motility
called hyperactivation (Yanagimachi and Usui, 1974). The preparation process
ended with an exocytotic event called the acrosome reaction, an essential stage for
oocyte fertilization (O’Flaherty et al., 1999). The physiological mammalian
acrosome reaction was experienced only by sperm that had been previously
capacitated.
Cryopreservation resulted in a loss of lipids from the sperm membranes

and a rearrangement of lipids and proteins within the membrane. Destabilization of
sperm membranes following cooling resembled that of the physiological sperm
capacitation (Collin et al., 2000) and resulted in a ‘‘precapacitated’’ spermatozoa
with a reduced fertilizing lifespan. Therefore, assays to evaluate sperm capacitation
were used to evaluate the normalcy of spermatozoa after freezing and thawing.
The destabilization of sperm membranes were evaluated by tracking the

distribution of Ca2+ in spermatozoa. There were two basic classes of Ca2+ indicators.
The main example of the first class was antibiotic chlortetracycline (CTC), which
accumulated in organelles containing high concentrations of Ca2+. The second class
consisted of molecules that resided in aqueous compartments such as cytosol and
changed their spectra when bound to Ca2+. Indicators of cytosolic free Ca2+
concentrations are quin-2, fura-2, indo-1 and fluo-3 (Tsien, 1989). Neutral
uncomplexed CTC easily crossed the membranes where it ionized to an anion and
chelated Ca2+. The latter complex bound preferentially to hydrophobic site, such as
membrane and showed increased fluorescence as a result. The extent of the binding
to membranes depended on the surface-to-volume ratio of the vesicle and the
properties of the lipid. Due to the compartmentalization of the plasma membrane of
spermatozoa, several distinct staining patterns were evaluated which were associated
with a functional status of spermatozoon (Fraser et al., 1995). A number of
investigators found that human, mouse, bull (Cormier et al, 1997), boar and several
other species exhibited similar patterns of CTC staining. The three patterns were F
Pattern, with uniform fluorescence on the head that indicated uncapacitated,
acrosome intact spermatozoa; B Pattern, with a fluorescence-free band on the post
acrosomal region that indicated capacitated, acrosome intact spermatozoa; and AR
pattern with uniformly fluorescence-free head and with a fluorescence band on the
equatorial region that indicated acrosome reaction. Several studies (Thomas et al.,
1997) were performed aiming to find the key to the hypothesis that freezing/thawing
destabilized sperm membranes and the extent of destabilization was inversely
related to the fertility values.
2.4.8.1.Acrosome integrity
The acrosome, a large lysosome- like vesicle overlying the sperm nucleus
contained large array of powerful hydrolyzing enzymes including hyaluronidase and
acrosin (Zaneveld and De Jonge, 1991). The acrosome of spermatozoa was to be
maintained intact up to the time it bound to zona pellucida of the oocyte and
underwent the acrosome reaction to release acrosomal enzymes (Grahamet al.,
1987). Therefore an intact acrosome was a must before and during the transit of the
sperm to the isthmus until zona binding was accomplished.
Two types of acrosome reaction were described for mammalian

spermatozoa; a true acrosome reaction that consisted of progressive vesiculation of
the outer acrosome membrane and overlying plasma membrane, and a false
acrosome reaction in which the acrosome was lost following cell death. Procedures
for the detection of true acrosome reaction in spermatozoa included triple stain
technique (trypan blue, bismark brown and rose bengal stains), lectins labeled with
flurochromes (Cummins et al., 1986) and antibiotic chlortetracycline (CTC) which
stained the sperm surface differently depending upon the stage of capacitation (Kim
and Gerton, 2003). The use of Coomassie blue G- 250 staining was another reliable
method for the assessment of acrosomal status in variety of mammalian species
(Larson and Miller, 1999) by simple microscopy.
2.4.8.2.Induction of capacitation
Heparin belonged to a class of molecules called glycosaminoglycans

(GAGs). A number of GAGs have been identified in uterine fluid, cytoplasm of
oocytes and were probably important factors associated with the process of in vivo
capacitation of sperm cells and fertilization (Lenz et al., 1983). Among the
conditions used for sperm capacitation in vitro, heparin appeared superior in terms
of repeatability and flexibility to accommodate bull differences (Parrish et al.,
1986). In vitro fertilization frequency achieved by in vitro capacitated spermatozoa
with heparin differed between bulls (Parrish et al., 1986; Totey et al., 1993). The
differences in the IVF results between bulls were related to the ability of their
spermatozoa to undergo in vitro capacitation with heparin.
Normally sperm underwent acrosomal reactions only after the completion

of capacitation. Ejaculated sperm cells required 9 h of incubation with GAGs (at
physiological concentrations) to undergo capacitation and acrosomal reaction,
whereas epididymal spermatozoa required 22 h (Handrow et al., 1982; Lenz et al.,
1982). Exposure of epididymal sperm to seminal plasma for 20 min reduced the time
required for the completion of acrosome reaction to 9 h as like that of ejaculated
semen (Lee at al., 1985).
Ca2+ ionophore, liposomes, lysophosphodidylcholine and solublized zona

pellucida were commonly used to induce acrosomal reactions in the sperm cells
capacitated with heparin. If solublized zona pellucidae was used to induce acrosome
reaction in spermatozoa treated with heparin for 4 h, ejaculated sperm responded
with acrosome reactions but epididymal sperm did not (Florman and First, 1988).
The exposure of epididymal spermatozoa to seminal plasma in vitro enabled those
sperm to be capacitated by heparin and respond to zonae pellucidae with an increase
in acrosome reactions in a manner similar to ejaculated spermatozoa (Florman and
First, 1988).
Graham et al., (1987) induced acrosome reaction with liposomes in

freshly collected bull spermatozoa and observed that high fertility bulls required less
lipid to induce the acrosome reaction than the lower fertility bulls. Similar inference
was drawn in another study on relationship of sire fertility to acrosome reacted
spermatozoa with liposomes in frozen thawed bull spermatozoa (Davis and Foote,
1987). Blotter et al. (1990) demonstrated a correlation between acrosome reactions
and in vitro as well as in vivo fertilization rates. These findings were confirmed by
Whitefield and Parkison (1992) who found a significant correlation between the
fertility of bulls predicted on the basis of acrosome reaction induced by heparin and
90 days non return rate.
The amount of heparin bound to spermatozoa was associated with fertility

in bulls; bulls exhibiting non return rates greater than 71 per cent had a greater
affinity for heparin than bulls with lower fertility (Bellin et al., 1996). It was
believed that heparin binding proteins played a role in fertilization by attaching to
the sperm surface, enabling heparin like GAGs in the female tract to induce
capacitation (Miller and Ax, 1989). Direct measurement of heparin binding was also
investigated for in vitro assessment of sperm fertility (Bellin et al., 1996). In buffalo
seminal plasma, eight major heparin binding proteins were isolated in the range of
13- 71 kDa. The addition of these seminal proteins improved the in vitro sperm
functions of buffalo caudal spermatozoa (Arangasamy et al., 2005). It is now evident
that sperm from different bulls varied in their heparin binding capacity and this
could be used as a fertility evaluation method in cattle and buffalo bulls.
CHAPTER III
MATERIALS AND METHODS
3.1. Experimental animals and source of semen
Semen ejaculates were obtained from 22 bulls (10 Jersey bulls, 10 Jersey
crossbred and 2 Holstein Friesian bulls) maintained by Tamil Nadu Co-operative
Milk Producer’s Federation Limited, Nucleus Jersey and Stud Farm,
Udhagamandalam and Semen Bank, Department of Animal Genetics and Breeding,
Madras Veterinary College, Chennai. Bulls were given with the following
identification numbers by the bull stations as NJ 612, NJ 621, NJ 624, NJ 626, NJ
627, NJ 657, NJ 658, NJ 662, NJ 670, 4049, JL 31, JS 149, 4072, 5011, 4021, 5038,
5049, 5052, 5055, 2208, HF 29 and HF 30. All the bulls were in regular semen
collection programme for industrial use and maintained under standard management
conditions.
3.2 Collection of semen
Semen was collected by artificial vagina method and semen samples that
fulfilled the quality criteria of the industry were used in the study. The seminal
plasma and sperm cells were separated immediately after collection by
centrifugation (560g for 10 minat 5° C). The sperm cells were washed with 2 ml of
TC buffer (40 mM Tris, 2 mM CaCl2 and 0.01% sodium azide, pH 7.3) by
centrifugation (560g for 5 min at 5°C) to remove the left over seminal plasma, if
any. The sperm cells were re-suspended with 1 ml of TC buffer containing protease
inhibitor (1 mM phenyl methyl sulfonyl fluoride) and washed thrice by
centrifugation (560g for 10 min at 5° C). The sperm pellet and the seminal plasma
weretransported in ice to the laboratory and stored at – 80° C until extraction of
protein.
3.3 Extraction of seminal plasma and sperm membrane proteins
Proteins in the seminal plasma were precipitated by adding ice-cold

ethanol 9 times the volume of seminal plasma and incubating at refrigeration
temperature overnight (Asadpour et al., 2007). Protein precipitates were separated
by centrifugation (8950g for 10 min at 5°C), air-dried and resuspended in milli-Q
water. Protein concentration was estimated using spectrophotometer (Nanodrop,
ND-1000, USA) and stored at – 80°C.
Sperm membrane proteins were extracted as per the method described by

Nass et al. (1990) with slight modifications. The sperm pelletswere thawed to room
temperature and washedtwice with 1 ml of TC buffer at 5°C. Washedpellets were
resuspended in 1 ml of TC buffer containing Triton X-100 (0.1 % v/v)and incubated
for 2 h at 5°C with vortexing at 15 min interval. After completion of 2 hincubation,
the suspension was centrifuged (8950g for 10 min at 5°C) to remove cellular debris.
The supernatant containing sperm membrane protein was recovered and the proteins
were precipitated by adding ice-cold ethanol 9 times the volume of supernatant and
incubating at refrigeration temperature overnight with intermittent vortexing for
initial 2 h.Protein precipitates were separated by centrifugation (8950g for 10 min at
5°C), air-dried and resuspended in milli-Q water. Protein concentration was
estimated using spectrophotometer and stored at – 80°C.
3.4 Isolation of heparin binding proteins
Heparin binding proteins in the sperm membrane and seminal plasma

proteins were isolated using heparin-sepharose affinity chromatography (Heparin-
CL agarose prepacked column, Bangalore Genei, India) as per the method described
by Manaskovaet al. (2002) with slight modifications. The column was equilibrated
with TC buffer five times the bed volume. One ml of seminal plasma/ sperm
membrane protein was added on a heparin sepharose column. Once the sample
entered the column the flow was stopped for 30 min to allow the proteins to interact
with the gel. The column was washed with TC buffer 10 times the bed volume to
remove unadsorbed proteins. The bound proteins were eluded with 0.7 M and 1.0 M
NaCl in TC buffer. The heparin binding fractions were pooled, dialyzed against
sodium phosphate buffer to remove NaCl and precipitated with ice cold ethanol.
Heparin binding proteins air dried, resuspended in milli-Q water and stored at
– 80° C.
3.5 Characterization of seminal plasma and sperm membrane proteins

by electrophoresis
Discontinuous sodium dodecyl sulphate polyacrylamide gel

elecrophoresis (SDS-PAGE) was performed according to Laemmli (1970). Glass
plates with 1.5 mm spacers were assembled in gel casting mould. The spacers were
inserted between plates. The notched plate was marked one cm below the comb
teeth for stacking gel. The resolving gel solution (12 %) was prepared (as described
in annexure) and poured between the glass plates up to the mark made earlier. The
gel surface was layered with water saturated butanol immediately and the gel was
allowed to polymerize for 30 min. After the polymerization of resolving gel was
complete, the water saturated butanol was decanted. The stacking gel (5 %) was
prepared (as described in annexure) and poured on the resolving gel. Comb was
inserted between the plates to create wells and the gel was allowed to polymerize for
60 min. After polymerization of the stacking gel, the comb was removed carefully.
The plates with the gel were then taken out of the casting mould and put into
electrophoresis apparatus (Plate No.1). Electrode buffer was poured in upper and
lower tanks of the apparatus and it was connected to the power supply pack
(Pharmacia, Model EPS 500/400).
3.5.1 Sample preparation
Stored protein samples (seminal plasma proteins, sperm membrane

proteins, heparin binding proteins of seminal plasma and sperm membrane) were
thawed to room temperature. 100 µg of sample protein (seminal plasma protein/
sperm membrane protein/ heparin binding proteins) from each bull and standard
marker proteins (Broad range marker 3 to 205 kDa, Bangalore Genei, India) were
prepared by mixing protein solution with sample buffer (4:1) and boiling in water
bath at 60-70° C for 10 min. Then the samples were loaded into each well in the
stacking gel with a help of Hamilton syringe.
3.5.2 Electrophoretic run
The electrophoresis was carried out at room temperature in constant

voltage mode at 70 volts till the dye front entered the resolving gel, thereafter
electrophoresis was carried out at 85 volts (Plate No.2). The power supply was
disconnected after the dye front reached the bottom of the gel. The gel plates were
separated apart and the gel was carefully removed and stained with coomassie
brilliant blue R- 250 (0.15 %) including 50 per cent methanol and 10 per cent acetic
acid for 10 – 12 hand de-stained in a mixture of methanol (25 %) and acetic acid
(10 %) in distilled water until no background was detectable.The apparent molecular
mass was determined by using molecular weight markers (Plate No. 3 and 4) and
Gel Documentation and Analysis System (Gel-Doc. Bio- Rad, UK) and the gels
were stored in acetic acid (7 %).
3.6 Bull grouping
Based on the presence/absence of 28- 30 kDa heparin binding protein in

the sperm membrane, bulls were categorized into two groups as group I and group
II. Bulls in the group I were positive for 28 – 30 kDa heparin binding protein in
sperm membrane and bulls included in this group were NJ 621, NJ 627, NJ 662, NJ
670, 4072, 5011, JS 149, 4049, 2208, HF 29 and HF 30. Bulls in the group II were
negative for 28 – 30 kDa heparin binding protein in sperm membrane and bulls
included in this group were NJ 612, NJ 624, NJ 626, NJ 657, NJ 658, JL 31, 4021,
5038, 5049, 5052 and 5055.
3.7 Assessment of in vitro sperm characters

Frozen semen samples (French mini straws) were procured for all the 22
bulls for the assessment of in vitro sperm characters. Frozen semen straws were
thawed at 37°C for 60 s. The contents were emptied into an eppendorf tube, mixed
thoroughly and maintained at thawing temperature in a water bath. Morphological
evaluations of sperm cells were carried out immediate post thaw to ascertain the per
cent of abnormal sperm cells present in the frozen thawed semen samples. Other in
vitro sperm characters namely, sperm motility, functional membrane integrity,
plasma membrane integrity, mitochondrial membrane potency, DNA integrity,
apoptosis (membrane modifications) assayand lipid peroxidation status were
assessed at immediate post thaw, 60 min, 120 min and 180 min post thaw incubation
(Gil et al., 2000; Bollwein et al., 2008).
3.7.1 Sperm cell morphology
Morphological evaluations of sperm cells were carried out by using rose

bengal stain. Briefly after thawing, the contents of frozen semen straws were
emptied into an eppendorf tube. Two drops of 3 per centrose bengal stain and 1000
μl of Tris buffer were added to semen samples. The contents were mixed and kept at
37° C for 10 min. Then the sperm cells were washed twice in Tris buffer by
centrifugation (560g for 5 min) and sperm cellssuspended in Tris buffer were placed
on a glass slide and covered with cover slip. A minimum of 200 cells were analyzed
at 100X using a microscope (Plate No.5).
3.7.2 Estimation of lipid peroxidation
Lipid peroxidation level of spermatozoa was estimated in semen samples

by measuring the malondialdehyde (MDA) production, using thiobarbituric acid
(TBA) as per the method described by Suleiman et al. (1996) with slight
modifications in sperm concentration and incubation time. The semen was thawed
and washed twice in Tris buffer by centrifugation (500g for 5 min). Then the sperm
pellet was re-suspended in 1 ml of PBS (pH 7.2) or a variable volume of PBS to
obtain a sperm concentration of 30×106/ml. Lipid peroxide level was measured in
spermatozoa after the addition of 2 ml of TBA-TCA reagent (15% w/v Trichlor
acetic acid and 0.375% w/v TBA in 0.25N HCl) to 1 ml of spermatozoa suspension.
The mixture was kept in a boiling water bath for 45 min. After cooling, the
suspension was centrifuged at 500g for 15 min. The supernatant was separated and
the absorbance measured at 535 nm under UV spectrophotometer (Cecil CE 2021,
2000 series). The MDA concentration was determined by the specific absorbance
coefficient (1.56×105/molcm-3).
MDA (µmol/ml) = OD × 106 × total volume (3ml) = OD × 30

1.56 × 105 × test volume (1ml) 1.56
3.7.3 Simultaneous assessment of plasma membrane integrityand
mitochondrial membrane potential
Mitochondrial membrane potential was assessed by using JC-1 (5, 5’, 6,

6’-tetrachloro-1, 1’, 3, 3’-tetraethylbenzimidazolylcarbocyanine iodide) and
plasmalemma integrity was assessed by carboxy fluoresin diacetate (CFDA) and
propidium iodide (PI).1.53 mM of JC-1 in DMSO, 8.69 mM of CFDA in DMSO
and 0.4 mMof PI in phosphate buffer were prepared and stored at -20°C in dark. 2 µl
of JC-1 and 10 µl of CFDA solutions were added to 100 µl of semen sample. The
semen sample was incubated at room temperature for 30 min in dark. The sperm
nuclei were counterstained by adding 10 µl of PI stock solution and incubated in
dark for 10 min. Then the sperm cells were washed in PBS by centrifugation (560g
for 5 min). Sperm cellssuspended in PBS were placed on a glass slide and covered
with cover slip. A minimum of 200 cells were analyzed at 400X using an
epifluorescence microscope (Nikon Eclipse 50i) equipped with a CFDA filter set
(excitation filter 510– 560 nm, barrier filter 505 nm). Sperm cells exhibiting orange
to red fluorescence in the mid-piece were considered as those having high
mitochondrial membrane potential and yellowish fluorescence considered as low
potential (Plate No. 6). Cells showing complete green fluorescence in the plasma
membrane were considered plasmalemma intact and those showing partial or
complete red nuclei were classified as dead (Selvaraju et al., 2008).
3.7.4 Functional membrane integrity
Functional membrane integrity was assessed using osmotic resistance test

(hypo-osmotic swelling test - HOST) by incubating an aliquot (100 µl) of semen
sample with one ml of 150 mOsm hypo-osmotic and 300 mOsm iso-osmotic
(control) solutions at 37°C for 30 min (Jeyendran et al., 1984). After incubation, one
drop of the well mixed sample is placed on a glass slide and covered with cover slip.
A minimum of 200 spermatozoa were observed for tail coiling (Plate No. 7).
3.7.5 DNA integrity
Integrity of sperm DNA was assessed by using acridineorange staining

(Chohan et al., 2004). Semensamples were smeared on glass slides and air-dried.
Then the smears were fixed with Carnoy’s solution (1 part glacialacetic acid: 3 parts
methanol) for 2 h. After fixation, smears were air-dried and stained with freshly
prepared acridineorange stain (0.19 mg/ml) for 5min in the dark. After staining,
smears were washed with distilled water and immediately evaluated under a
fluorescent microscope at excitation wavelengthof 450–490 nm. An average of 200
sperm was counted on each slide and the duration of evaluation was 40 s per field.
Sperm with normal DNA content showed green fluorescence whereas sperm with
damaged DNA content gave a spectrum of yellow-greento red (Plate No. 8).
3.7.6 Assessment of sperm cell apoptosis
Annexin-V-FITC apoptosis detection kit (Sigma – Aldrich, Saint Louis,

USA) wasused to detect the translocation of membrane phospholipid
phosphatidylserine (PS). The staining procedure was conducted according tothe
protocol recommended by the manufacturer, with slight modifications. In order to
differentiate between viable cells with or without PS translocation CFDA was used
along with Annexin V. The non-fluorescent CFDA enters the cell and was converted
to green fluorescent compound carboxyfluorescein. This conversion was a function
of the esterases present only in living cells. After thawing,spermatozoa were washed
twice at 500 X g, 10 min, in DBPS. The sperm pellet was then resuspended in
Annexin-V binding buffer (10 mM Hepes / NaOH [pH 7.5], containing 140 mM
NaCl and 2.5 mM CaCl2) to a concentration of 1 X 106 spermatozoa/ml. Aliquots of
washed and extended semen (500 µl) were transferred to a 2 ml eppendorf tubes
andsupplemented with 5 µl of CFDA (1mM in DMSO), 5 µl Annexin-V-FITC and
10 µl of PI (50 mg/ml). The tubes were gentlymixed and further incubated at room
temperature for 10 min in dark. Each sample was placed on a slide and analyzed by
epifluorescence microscopy. Threepatterns of fluorescence were observed: 1)
CFDA+ /Annexin V- Live cells without PS exposure—this subpopulation was
labeled ‘‘viable cells’’; 2) CFDA+/Annexin V+: Live cells with PSexposure—this
subpopulation was labeled ‘‘apoptotic cells’’;and 3) CFDA-/Annexin V+: ‘‘necrotic
cells’’- this subpopulationshowed Annexin V labeling in the entire cell (Plate No. 9).
3.7.7 Sperm motility
Motility analysis was conducted using a computer-assisted semen

analyzer (Sperm Class Analyzer, Microptic, Barcelona, Spain). Analysis was based
on examination of 25 consecutive digitalized images per second using a 5 X
negative phase contrast objective. The parameters set were cell size (range 5–70
µm2), spermatozoa concentration (5–8×106 /ml), motile cells (>10 µm/s at 37 ºC),
progressive forward motility (>60% straightness index, STR; Plate No. 10 ); circular
(<50% linearity index, LIN), the straight-line velocity (VSL, in µm/s is the average
path velocity of the spermatozoa head along a straight line from its first to last
position: Plate No.11), curvilinear velocity (VCL, in µm/s is the average path
velocity of the spermatozoa head along its actual trajectory), the percentage of
linearity (LIN, is the ratio between VSL and VCL) and the lateral head displacement
(ALH, in µm/s is the average value of the extreme side-to-side movement of the
spermatozoa head in each beat cycle). Motility analysis was carried out in 5µl of
semen samples placed onto a pre-warmed (37 ºC) microscopic slide covered with
22mm X 22mm cover slip (Blue star, Mumbai, India). A minimum of 500
spermatozoa from at least two different drops was analyzed for each sample. The
final analysis was done after removing the number of objects incorrectly identified
as spermatozoa (Selvaraju et al., 2008).
3.8 Effect of fertility associated proteins on in vitro sperm characters

To assess the effect of fertility associated proteins on in vitro sperm
characters heparin binding proteins isolated from neat semen of bulls positive for
28- 30 kDa protein in the sperm membrane were used. Frozen semen straws were
thawed at 37°C for 60 s. Then the sperm cells were washed in PBS by centrifugation
(560g for 5 min). 25 µg of heparin binding protein was added to the semen and
incubated at 37°C (Schoneck et al., 1996). In vitro sperm characters
namely, sperm motility, plasmalemma integrity, functional membrane

integrity, mitochondrial membrane potency, DNA integrity, sperm cell apoptosis
and lipid peroxidation status were assessed at 60, 120 and 180 min of incubation.
3.9 Induction of lipid peroxidation
Lipid peroxidation was induced by adding 0.1mM hydrogen peroxide

(H2O2)to the semen samples immediately after thawing (Martinez-Pastor et al.,
2009). The exogenous addition of H2O2caused immediate changes on the in vitro
sperm characters and they were assessed at 10, 30 and 60 min. after addition of
H2O2. In lipid peroxidation induced samples the development of particulate matter
after 10 min of incubation interfered with the computer- assisted semen analysis;
hence sperm motility was assessed manually.
3.10 Assessment of capacitation status
Capacitation status of sperm cells was assessed by chlortetracycline

fluorescence assay as described previously by Fraser et al. (1995) with
modifications. As a counter stain EthD-1 (CTC/EthD-1) was used in order to
consider only intact spermatozoa. Chlortetracycline (CTC) staining was made
freshly by dissolving chlortetracycline and L-cysteine in a chilled, 20 mM Tris
buffer supplemented with 130 mM NaCl to produce final concentrations of 7.50 mM
CTC and 5 mM L-cysteine, respectively. The pH of the final solution was adjusted
to 7.8, and it was kept in the dark at 4° C until it was used.
Two numbers of frozen semen straws from each bull were thawed and
washed twice with 3 ml of modified Tyrode’s solution (mTALP, as described in
annexure) by centrifugation (400g for 10 min at room temperature). The sperm
pellet thus obtained was resuspended in 400 µl mTALP and incubated in CO2
incubator (5 % CO2 and 38.5° C) for 180 min. Samples were evaluated for
capacitation status at 0, 60, 120, 180 min of incubation.
10 µl of EthD-1 (23.3 µM) was added to 100 µl of sperm suspension and
incubated at room temperature for 10 min. Thereafter 100 µl of CTC solution was
added and incubated in dark for 30 min. 10 µl of CTC added sperm suspension was
placed on a warm slide and a drop of 0.22 M 1, 4-diaza-byciclo (2.2.2) octane in
glycerol (9:1 glycerol: PBS) was added to retard fluorescence fading. Next, the
droplet was covered with a cover slip and the slide was gently but firmly pressed
under two folds of a tissue paper to absorb any excess fluid. To avoid evaporation
and CTC fading, the prepared slide was then stored in wet chamber in dark until it
was analyzed (within 2 h of preparation). The slide was examined with a microscope
equipped with epifluorescence optics and violet blue (420 – 490 nm excitation, 510
nm emission) and green filters (530 – 560 nm excitation, 580 nm emission).
Sperm cells were classified according to their acrosomal staining

patterns: Pattern F - bright fluorescence over the entire sperm head and positive mid-
piece of the tail-non-capacitated, acrosome-intact sperm; Pattern B - prominent
fluorescent positive equatorial segment, mid-piece of the tail and fluorescence free
dark band in the post acrosomal region- capacitated, acrosome –intact sperm; Pattern
AR - low fluorescent signal thorough out the sperm head, with remaining positive
signal in the equatorial segment and mid piece- acrosome reacted sperm (Plate No.
12, 13 and 14). Sperm cells with a non specific or intermediate fluorescent signal
status were not selected for analysis.
3.10.1 Induction of capacitation with heparin
Frozen semen straws were thawed and washed with mTALP as described
above and the sperm pellet thus obtained was resuspended in 400 µl mTALP
containing heparin (10 µg/ml) and incubated in CO2 incubator for 180 minto induce
capacitation. Samples were evaluated for the capacitation status at 60, 120, 180 min
of incubation.
3.10.2 Effect of fertility associated proteins on induction of capacitation

To study the effect of fertility associated proteins on in vitro sperm
capacitation, 25 µg of HBP was added to sperm cells suspended in mTALP with
heparin (10µg/ml) and incubated in CO2 incubator. Samples were evaluated for
capacitation status at 60, 120, 180 min of incubation.
3.11 Data analysis
All statistical analyses were carried out using the Statistical Package for
Social Sciences programme (SPSS), version 15.00 software for windows (SPSS Inc.
Chicago, IL, USA). Statistical analysis was performed after arcsine transforming the
percentage values. Statistical significance was set at 0.05 probability level. If the
effect was found significant, comparison of means was done by Duncan Multiple
Range Test (DMRT). Results are expressed as Mean ± Standard Error of Mean.
The effect of different duration of incubation on the different in vitro

sperm characters was analyzed by one way ANOVA for control group (immediate
post thaw, 60 min, 120 min and 180 min post thaw incubation), for fertility
associated protein treated group (60 min, 120 min and 180 min incubation) and for
hydrogen peroxide treated group (10 min, 30 min and 60 min incubation). The
following model was used
Yij = μ + Pi + eij
Yij = observation at ith time of incubation
μ = over all mean
Pi = effect of ith time of incubation
eij = error
The comparison between control and fertility associated protein treated

group at each point of incubation for different invitro sperm characters was analyzed
by one way ANOVA. The following model was used
Yij = μ + Pi + eij
Yij = observation at ith treatment
μ= over all mean
Pi = effect of ith treatment
eij = error
The difference between bull group I and group II in different invitro

sperm characters at each point of incubation was analyzed by one way ANOVA for
control, fertility associated protein treated and hydrogen peroxide treated groups.
The following model was used
Yij = μ + Pi + eij
Yij = observation for ith bull group
μ= over all mean
Pi = ith bull group
eij = error
The association among the different in vitro sperm characters in control

group following 180 min of incubation was analyzed by Pearson correlation
analysis. To rank the bulls used in the study, the in vitro sperm characters in control
group during incubation (immediate post thaw to 180 min) was analyzed by Duncan
Multiple Range Test (DMRT). Following the DMRT test bulls were categorized
under different subsets for each in vitro character and suitable rank was given to the
bulls that were categorized under same subsets. However, if a bull was categorized
under more than one subset, the best available rank was assigned to that bull
(Bewick et al., 2004).
CHAPTER IV
RESULTS
4.1 ISOLATION AND CHARACTERIZATION OF SEMINAL

PLASMA AND SPERM MEMBRANE PROTEINS
Protein bands in the molecular weight ranging from 3 to 205 kDa were
observed in the SDS-PAGE of bovine seminal plasma protein (Table1). Protein
bands of varying intensities were observed in the gel with dense bands at 15/14 and
28 kDa. Protein bands of 15/14 kDa, 28 kDa, 36 kDa, 66 kDa and 160 kDa were
present in all the 22 bulls.
55 kDaprotein (osteopontin)was present in 6 Jersey bulls (bull No. 621,

627, 662, 670, 657, 4049), 6 Jersey crossbred bulls (bull No. JS 149, 4021, 4072, JL
31, 5011, 2208) and 2 Holstein Friesian bulls (bull No. HF 29, HF 30). Over all 14
out of 22 bulls (63.63 %) were positive for 55 kDa protein.
26 kDa protein (lipocalin type prostaglandin D synthase) was present in 8

Jersey bulls (bull No. 621, 662, 627, 670, 624, 626, 658, 4049), 8 Jersey crossbred
bulls(bull No. JS 149, 4021, 4072, 5049, 5052, 5011, 2208, 5038) and 2 Holstein
Friesian bulls (bull No. HF 29, HF 30). Over all 18 out of 22 bulls (81.82 %) were
positive for 26 kDa protein. 28 kDa protein (BSP- 30) and 15/14 kDa protein (BSP-
A1/A2 and BSP-A3) were present in all the 22 bulls.
205 kDa protein band was observed in 2 Jersey and 4 Jersey crossbred
bulls. None of the Holstein Friesian bulls were positive for this protein. Over all 6
out of 22 bulls (27.27 %) were positive for 205 kDa protein. 97 kDa protein band
was observed in 7 Jersey and 7 Jersey crossbred bulls. None of the Holstein Friesian
bulls were positive for this protein. Over all 14 out of 22 bulls (63.64 %) were
positive for 97 kDa protein.
Table1: Electrophoretic profile of bovine seminal plasma proteins assessed by SDS-PAGE
Number of bulls positive for protein fractions

Protein
molecular weight
(kDa) Jersey (n = 10) Jersey crossbred (n =10) Holstein Friesian (n =2) Total bulls (n = 22)
205 2 (20.00) 4(40.00) 0(0) 6 (27.27)

160 10 (100.00) 10 (100.00) 2 (100.00) 22 (100.00)
97 7 (70.00) 7 (70.00) 0 (0) 14 (63.64)
66 10 (100.00) 10 (100.00) 2 (100.00) 22 (100.00)
55 6 (60.00) 6 (60.00) 2 (100.00) 14 (63.64)
43 2 (20.00) 10 (100.00) 2 (100.00) 14 (63.64)
41 7 (70.00) 1 (10.00) 0 (0) 8 (36.36)
36 10 (100.00) 10 (100.00) 2 (100.00) 22 (100.00)
28 10 (100.00) 10 (100.00) 2 (100.00) 22 (100.00)
26 8 (80.00) 8 (80.00) 2 (100.00) 18 (81.82)
25 9 (90.00) 6 (60.00) 2 (100.00) 17 (77.27)
17 4 (40.00) 8 (80.00) 2 (100.00) 14 (63.64)
15/14 10 (100.00) 10 (100.00) 2 (100.00) 22 (100.00)
6.5 2 (20.00) 8 (80.00) 2 (100.00) 12 (54.55)
3 1 (10.00) 3 (30.00) 0 (0) 4 (18.18)
Figures in parentheses indicate percentage to total
43 kDa protein band was observed in 2 Jersey, 10 Jersey crossbred and 2
Holstein Friesian bulls. Over all 14 out of 22 bulls (63.64 %) were positive for 43
kDa protein. 41 kDa protein band was observed in 7 Jersey and 1 Jersey crossbred
bulls. None of the Holstein Friesian bulls were positive for the protein. Over all 8

kDa protein. 17 kDa protein band was observed in 4 Jersey, 8 Jersey crossbred and 2
kDa protein. 6.5 kDa protein band was observed in 2 Jersey, 8 Jersey crossbred and
2 Holstein Friesian bulls. Over all 12 out of 22 bulls (54.55 %) were positive for 6.5
kDa protein. 3 kDa protein band was observed in 1 Jersey and 3 Jersey crossbred
bulls. None of the Holstein Friesian bulls were positive for the protein. Over all 4
4.1.2 Electrophoretic profile of sperm membrane proteins
Protein bands in the molecular weight ranging from 3 to 205 kDa were
observed in the SDS- PAGE of bovine sperm membrane protein (Table 2). Protein
bands of varying intensities were observed in the gel with dense bands at 15/14 kDa.
Protein bands of 66 and 15/14 kDa were present in all the 22 bulls.
55 kDa protein (osteopontin)was present in 5 Jersey bulls (bull No. 621,

627, 662, 670, 4049), 4 Jersey crossbred bulls (bull No. JS 149, 4072, 5011, 2208)
and 2 Holstein Friesian bulls (bull No. HF 29, HF 30). Over all 11 out of 22 bulls
(50.00 %) were positive for 55 kDa protein.
26 kDa protein (lipocalin type prostaglandin D synthase) was observed in

6 Jersey (bull No. 621, 627, 662, 670, 657, 4049), 6 Jersey crossbred bull No. JS
149, 4021, 4072, JL 31, 5011, 2208 and 2 Holstein Friesian bulls. Over all 14 out of
22 bulls (63.64 %) were positive for 26 kDa protein. 15/14 kDa protein (BSP-A1/A2
and BSP-A3) was present in all the 22 bulls. 28 kDa protein (BSP- 30) was present
in 21 out of 22 bulls.
Table 2: Electrophoretic profile of bovine sperm membrane proteins assessed by SDS-PAGE

Protein
molecular weight
Jersey (n = 10) Jersey crossbred (n =10) Holstein Friesian (n =2) Total bulls (n = 22)
(kDa)
205 5 (50.00) 9 (90.00) 2 (100.00) 16 (72.73)

160 1 (10.00) 8 (80.00) 2 (100.00) 11 (50.00)
97 5 (50.00) 10 (100.00) 2 (100.00) 17 (77.27)
80 0 (0) 2 (20.00) 0 (0) 2 (9.09)
66 10 (100.00) 10 (100.00) 2 (100.00) 22 (100.00)
55 5(50.00) 4(40.00) 2(100.00) 11(50.00)
43 8 (80.00) 3 (30.00) 2 (100.00) 13 (59.09)
36 4 (40.00) 6 (60.00) 2 (100.00) 12 (54.55)
28 9(90.00) 10(100.00) 2(100.00) 21(95.45)
26 6(60.00) 6(60.00) 2(100.00) 14(63.64)
17 2(20.00) 3(30.00) 0(0) 5(22.73)
15/14 10(100.00) 10(100.00) 2(100.00) 22(100.00)
6.5 6 (60.00) 4 (40.00) 0 (0) 10 (45.45)
3 0 (0) 1 (10.00) 0 (0) 1 (4.55)
kDa protein.160 kDa protein band was observed in 1 Jersey, 8 Jersey crossbred and
2 Holstein Friesian bulls. Over all 11 out of 22 bulls (50.00 %) were positive for 160
kDa protein.

kDa protein.80 kDaband was observed only in 2 Jersey crossbred bulls and not
observed in Jersey and Holstein Friesian bulls.

kDa protein.36 kDa protein band was observed in 4 Jersey, 6 Jersey crossbred and 2
kDa protein.

bulls. None of the Holstein Friesian bulls had this protein. Over all 5 out of 22 bulls
(22.73 %) were positive for 17 kDa protein. 6.5 kDa protein band was observed in 6
Jersey and 4 Jersey crossbred bulls. None of the Holstein Friesian bulls had this
protein. Over all 10 out of 22 bulls (45.45 %) were positive for 6.5 kDa protein.3
kDa protein band was observed in only one Jersey crossbred bull.
4.1.3 Electrophoretic profile of heparin binding proteins of bovine seminal

plasma
Protein bands in the molecular weight ranging from 15/14 to 205 kDa
were observed in the SDS- PAGE of heparin binding proteins of bovine seminal
plasma (Table 3). Dense band was observed at 15/14 kDa.
Heparin binding protein with molecular weight 15/14 kDa was present in
all the 22 bulls (100.00 %). Heparin binding protein with molecular weight 28 kDa
was present in 18 out of 22 bulls (8 Jersey bulls 621, 624, 626, 627, 662, 670, 657,
Table 3: Electrophoretic profile of heparin binding proteins of bovine seminal plasma assessed by SDS-PAGE

Protein
molecular weight
(kDa) Jersey (n = 10) Jersey crossbred (n =10) Holstein Friesian (n =2) Total bulls (n = 22)
1 2 1 4
205
(10.00) (20.00) (50.00) (18.18)
5 9 2 16
160
(50.00) (90.00) (100.00) (72.73)
5 4 0 9
66
(50.00) (40.00) (0) (40.91)
4 5 1 10
43
(40.00) (50.00) (50.00) (45.45)
2 1 0 3
36
(20.00) (10.00) (0) (13.64)
8 8 2 18
28
(80.00) (80.00) (100.00) (81.82)
10 10 2 22
15/14
(100.00) (100.00) (100.00) (100.00)

Table 4: Electrophoretic profile of heparin binding proteins of bovine sperm membrane assessed by SDS-PAGE

Protein
molecular weight
Jersey (n = 10) Jersey crossbred (n =10) Holstein Friesian (n =2) Total bulls (n = 22)
(kDa)
8 8 2 18
205
(80.00) (80.00) (100.00) (81.82)
9 6 2 17
160
(90.00) (60.00) (100.00) (77.27)
9 6 0 15
97
(90.00) (60.00) (0) (68.18)
8 4 0 12
66
(80.00) (40.00) (0) (54.55)
0 0 2 2
55
(0) (0) (100.00) (9.09)
8 4 2 14
43
(80.00) (40.00) (100.00) (63.64)
4 0 2 6
36
(40.00) (0) (100.00) (27.27)
5 4 2 11
28
(50.00) (40.00) (100.00) (50.00)
0 0 2 2
26/25
(0) (0) (100.00) (9.09)
10 10 2 22
15/14
(100.00) (100.00) (100.00) (100.00)
4049; 8 Jersey crossbred bulls 2208, 5038, JS 149, 5011, 5049, 4072, 5055, JL 31
and 2 Holstein Friesian bulls HF 29, HF 30).
kDa protein. 160 kDa protein band was observed in 5 Jersey, 9 Jersey crossbred and
kDa protein.

bulls. Over all 9 out of 22 bulls (40.91 %) were positive for 66 kDa protein. 43 kDa
protein band was observed in 4 Jersey, 5 Jersey crossbred and 1 Holstein Friesian
bulls. Over all 10 out of 22 bulls (45.45 %) were positive for 43 kDa protein. 36 kDa
protein band was observed in 2 Jersey and 1 Jersey crossbred bulls. Over all 3 out of
22 bulls (13.64 %) were positive for 36 kDa protein.
4.1.4 Electrophoretic profile of heparin binding proteins of bovine sperm

membrane
Protein bands with molecular weight ranging from 15/14 to 205 kDa
were observed in the SDS- PAGE of heparin binding proteins of bovine sperm
membrane (Table 4).
28 kDa protein (fertility associated antigen) was present in 5 Jersey (bull

No. 621,627, 662, 670, 4049), 4 Jersey crossbred (bull No. 4072, 5011, JS 149,
2208) and 2 Holstein Friesian bulls (bull No. HF 29, HF 30).Over all 11 out of 22
bulls (50.00 %) were positive for 28 kDa protein.Dense band was observed at 15/14
kDa and was present in all 22 bulls.
kDa protein. 160 kDa protein band was observed in 9 Jersey, 6 Jersey crossbred and
kDa protein.
97 kDa protein band was observed in 9 Jersey and 6 Jersey crossbred bulls.
Over all 15 out of 22 bulls (68.18 %) were positive for 97 kDa protein. 66 kDa
protein band was observed in 8 Jersey and 4 Jersey crossbred bulls. Over all 12 out
of 22 bulls (54.55 %) were positive for 66 kDa protein. 55 kDa protein band was
observed only in 2 Holstein Friesian bulls. 43 kDa protein band was observed in 8
Jersey, 4 Jersey crossbred and 2 Holstein Friesian bulls. Over all 14 out of 22 bulls
(63.64 %) were positive for 43 kDa protein.
36 kDa protein band was observed in 4 Jersey and 2 Holstein Friesian

bulls. Over all 6 out of 22 bulls (27.27 %) were positive for 36 kDa protein. 26/25
kDa protein band was observed only in 2 Holstein Friesian bulls.
28 kDa heparin binding protein in sperm membrane is known as Fertility

Associated Antigen (FAA), 55 kDa protein is known as osteopontin, 28 kDa protein
as BSP 30,26 kDa protein as lipocalin-type prostaglandin D synthase and 15/14 kDa
proteins as BSP A1/ A2 and BSP A3. These above four proteins and 15/14 kDa and
28 kDa heparin binding proteins in seminal plasma and sperm membrane were
associated with bull fertility.
Bulls which were positive for 28 kDa heparin binding proteins in sperm
membrane were also positive for the other fertility associated proteins in seminal
plasma and sperm membrane such as 55 kDa, 28 kDa, 26 kDa, 15/14 kDa proteins
as well as 15/14 kDa, 28 kDa heparin binding proteins in seminal plasma and 15/14
kDa heparin binding proteins in sperm membrane.
Hence, based on the presence of 28 kDa heparin binding proteins in

sperm membrane, bulls in the present study were categorized into group I bulls those
were positive for the protein fraction and group II bulls those were negative for the
protein fraction.Over all 11 out of 22 bulls (50.00 %) were positive for the fertility
associated protein.The bulls under group I were 5 Jersey bulls (bull No. 621, 627,
662, 670, 4049); 4 Jersey crossbred bulls (bull No. 4072, 5011, JS 149, 2208) and 2
Holstein Friesian bulls (bull No. HF 29, HF 30) and the bulls under the group II
were 5 Jersey bulls (612, 624, 626, 657, 658) and 6 Jersey crossbred bulls (JL 31,
4021, 5038, 5049, 5052, 5055).
Table 5: MDA level (µ mol/ml) in frozen thawed bull semen samples treated with
Post thaw MDA levels (µ mol/ml)

Treatment Bull group
Immediate 60 min 120 min 180 min
Group I 1.60± 0.15a 2.67± 0.19b p 2.79± 0.24 b p i 3.77± 0.41 c p i
Control
Group II 1.73± 0.13a 3.04± 0.15b x 3.61± 0.28 b X ii 4.84± 0.46 c x ii
Treatment with fertility Group I 1.60± 0.15a 1.86± 0.17ab q 2.25± 0.19b q 2.81± 0.26b q
associated protein Group II 1.73± 0.13a 2.03± 0.12ab y 2.55± 0.13b Y 3.16± 0.14c y
Bull group Immediate 10 min 30 min 60 min
Treatment with hydrogen
Group I 1.60± 0.15a 1.79± 0.14a 2.85± 0.16b1 3.04±0.161c
peroxide
Group II 1.73± 0.13a 1.91± 0.14a 3.26± 0.12b2 3.51± 0.122c
Data shown all mean ± SEM (n = 11)

Means with different superscripts a, b, c in a row differ significantly at P< 0.01
Means with different superscripts i, ii and 1, 2 in a column for particular treatment differ significantly at P< 0.01 and P < 0.05, respectively
Means of group I bulls with different superscripts p, q in a column differ significantly at P< 0.01
Means of group II bulls with different superscripts x, y and X, Y in a column differ significantly at P< 0.01 and P<0.05, respectively
Table 7: Per cent of sperm cells with intact plasma membrane in frozen thawed bull semen samples
Treatment Bull group Sperm cells with intact plasma membrane during post thaw incubation (%)
Control Group I 58.37±2.79 a 43.30±2.80 b 28.22±3.10 c p 15.27±2.11d p

Group II 52.67±4.12 a 38.98±3.48 b 25.96±3.32 c x 15.06±2.16 d x
Treatment with fertility Group I 58.37±2.79a 47.13±2.39b 36.02±2.10c q 26.69±2.06d q
associated protein
Group II 52.67±4.12a 44.24±3.38b 32.50±3.03c y 23.36±2.17d y
Treatment with hydrogen Bull group Immediate 10 min 30 min 60 min
peroxide
Group I 58.37±2.79a 45.75±3.44 b i 26.03±2.53 c i 0
Group II 52.67±4.12a 31.79±3.34 b ii 17.54±1.59 c ii 0
Data shown all mean ± SEM (n = 11).

Means with different superscripts a, b, c, d in a row differ significantly at P< 0.01
Means with different superscripts i, ii in a column for particular treatment differ significantly at P< 0.01.
Means of group I bulls with different superscripts p, q in a column differ significantly at P< 0.01.
Means of group II bulls with different superscripts x, y in a column differ significantly at P< 0.01.
4.2 EVALUATION OF IN VITRO SPERM CHARACTERS
4.2.1 Sperm morphology
Sperm cell morphology in frozen thawed semen samples was assessed at

immediate post thaw by using rose bengal stain. In bulls ofgroup I and II, the per
cent of abnormal / detached heads were 5.30 ± 0.12 and 5.60± 0.26, abnormal mid
piece were 1.50± 0.40 and 1.80± 0.36, abnormal tails (coiled / bent / detached tails)
were 10.90 ± 2.56 and 8.50 ± 1.50, total abnormal cells were 17.70 ± 2.50 and 15.90
± 1.25 and normal sperm cells were 82.70 ± 2.50 and 84.10± 1.50,
respectively.There was no significant difference between the group I and II bulls in
the sperm cell morphology.
4.2.2 Lipid peroxidation
The MDA level in frozen thawed semen samples of group I and II bulls
with different treatments (treated with H2O2 and with fertility associated protein) are
presented in Table 5.
MDA level increased significantly (P < 0.01) during different periods of

incubation in control as well as in treatment groups. In control group though it was
not significant, bulls in group I had low level of MDA than group II at immediate
post thaw (1.60 ± 0.15 vs 1.73 ± 0.13) and at 60 min post thaw incubation (2.67 ±
0.19 vs 3.04 ± 0.15). But the MDA level was significantly lower (P < 0.01) in group
I bullsthan group II at 120 min post thaw (2.79 ± 0.24 vs 3.61 ± 0.28) and at 180 min
post thaw (3.77 ± 0.41 vs 4.84 ± 0.46).
In H2O2 treatment, bulls in group I had significantly (P < 0.05) lower

MDA level than group II bulls at 30 min of incubation (2.85± 0.16 vs 3.26 ± 0.12)
and at 60 min of incubation (3.04 ± 0.16 vs 3.51 ± 0.12).
In fertility associated protein treatment, though it was not significant,

bulls in group I had low level of MDA than group II at different periods of
incubation; 1.86 ± 0.17 vs 2.03 ± 0.12 at 60 min, 2.25 ± 0.19 vs 2.55 ± 0.12 at 120
min and 2.81 ± 0.26 vs 3.16 ± 0.14 at 180 min post thaw respectively.
In group I bulls, semen samples treated with fertility associated protein had
significantly (P < 0.01) lower level of MDA than the control at 60 min ((1.86 ± 0.17
vs 2.67 ± 0.19), at 120 min (2.25 ± 0.19 vs 2.79 ± 0.24) and at 180 min (2.81 ± 0.26
vs 3.77 ± 0.41). Similarly the fertility associated protein treatment had significantly
(P <0.01) lower level of MDA than the hydrogen peroxide treatment at 60 min (1.86
± 0.17 vs 3.04 ± 0.16).
In group IIbulls, semen samples treated with fertility associated protein

had significantly lower level of MDA than the control at 60 min (2.03 ± 0.12 vs 3.04
± 0.15), at 120 min (2.55 ± 0.13 vs 3.61 ± 0.28) and at 180 min (3.16 ± 0.14 vs 4.84
± 0.46). Similarly the fertility associated protein treatment had significantly (P
<0.01) lower level of MDA than the hydrogen peroxide treatment at 60 min of
incubation (2.03 ± 0.12 vs 3.51 ± 0.12).
4.2.2.1. Correlation between MDA levels and other in vitro sperm characters
during the incubation at 37° C for 180 min (Table 6)
MDA had highly significant (P< 0.01) negative correlation with sperm
cell viability (- 0.559), plasma membrane integrity (- 0.562), functional membrane
integrity (- 0.575), sperm cells with high mitochondrial membrane potency (- 0.689),
progressive forward motility (- 0.342), straight line velocity of sperm cells (- 0.286)
and F pattern uncapacitated- acrosome intact sperm cells (- 0.274). MDA had
significant (P < 0.05) negative correlation with average path velocity of sperm cells
(- 0.239), linearity of sperm cell motility (- 0.227) and straightness of sperm cell
motility (- 0.250).
MDA had non significant negative correlation with non progressive

forward motility (- 0.089), sperm cells with low mitochondrial membrane potency
(- 0.199), curvilinear velocity of sperm cells (- 0.098), wobble (- 0.109), amplitude
of lateral head displacement (- 0.105), beat cross frequency (- 0.079) and apoptotic
sperm cells (- 0.017).
MDA had highly significant (P< 0.01) positive correlation with static
sperm cells (0.285), sperm cells with lost mitochondrial membrane potency (0.384)
and necrotic sperm cells (0.617).MDA had significant (P< 0.05) positive correlation
with B pattern capacitated- acrosome intact sperm cells (0.251), non significant
positive correlation with AR pattern capacitated acrosome reacted sperm cells
(0.177).
Plasma membrane integrity assessed by fluorogenic stain propidium

iodide and carboxy fluoresin diacetate in frozen thawed semen samples of group I
and II bulls with different treatments (treated with H2O2and withfertility associated
protein) are presented in Table 7.
Per cent of sperm cells with intact plasma membrane decreased

significantly during different periods of incubation in control as well as in treatment
groups. In the untreated control though the sperm cells with intact plasma membrane
did not differ significantly, bulls in group I had more number of intact sperm cells
than group II at immediate post thaw (58.37 ± 2.79 vs 52.67 ± 4.12), at 60 min
(43.30 ± 2.80 vs 38.98 ± 3.38), at 120 min (28.22 ± 3.10 vs 25.96 ± 3.32) and at 180
min (15.27±2.11 vs 15.06±2.16) incubation periods.
In H2O2 treated semen samples, group I bulls had significantly (P < 0.01)
more number of sperm cells with intact plasma membrane than the group II bulls at
10 min (45.75 ± 3.44 vs 31.79 ± 3.34) and at 30 min(26.03 ± 2.53 vs 17.54 ± 1.59)
post thaw incubation periods.
In the fertility associated protein treated semen samples as like that of

control, though the sperm cells with intact plasma membrane did not differ
significantly between bull groups, bulls in group I had more number of intact sperm
cells than group II at 60 min (47.13 ± 2.39 vs 44.24 ± 3.38), at 120 min (36.02 ±
2.10 vs 32.50 ± 3.03) and at 180 min (26.69 ± 2.06 vs 23.36 ± 2.17) incubation
periods.
Bulls in group I when treated with fertility associated proteinhad non

significantly more number of sperm cells with intact plasma membrane than the
Table 8: Per cent of sperm cells with functional membrane integrity in frozen thawed bull semen samples
Sperm cells with functional membrane integrity during post thaw incubation (%)
Control Group I 55.61± 2.70a 40.69± 2.75b P 25.46± 2.95c p 11.59± 2.24d p
Group II 49.12± 3.98a 36.29± 3.20 b x 22.13± 3.18c x 11.52± 2.07d x
Treatment with fertility Group I 55.61± 2.7a 45.45± 2.59 b,1 Q 33.91± 2.31c,1 q 23.94± 2.08d q
associated protein
Group II 49.12± 3.98a 40.86± 2.30 b, 2 y 28.64± 3.03c,2 y 20.37± 2.12d y
peroxide
Group I 55.61± 2.7a 41.60± 3.06 b i 21.76± 2.27 c i 0
Group II 49.12± 3.98a 28.63± 3.32 b ii
14.08± 1.46 c ii 0

Means with different superscripts i, ii and 1, 2 in a column for particular treatment differ significantly at P< 0.01 and P < 0.05, respectively.
Means of group I bulls with different superscripts p, q and P, Q in a column differ significantly at P< 0.01 and P<0.05, respectively.
Means of group II bulls with different superscripts x, y in a column differ significantly at P< 0.01
Table 9: Per cent of sperm cells with high mitochondrial membrane potential in frozen thawed bull semen
samples treated with fertility associated protein and hydrogen peroxide
Sperm cells with high mitochondrial membrane potential during post thaw
Treatment Bull group incubation (%)
Control Group I 19.65±1.17 Aa 16.72±1.02 B i 12.32±1.04 b i p 8.31±0.78 c i p
Group II 19.13± 0.99 a 12.71± 0.99 b ii 8.39± 1.02 c ii 5.19±1.00 d ii x
Treatment with fertility Group I 19.65±1.17a 17.51± 0.90i ab 14.54± 0.58i b q 10.54± 0.55ic q
associated protein
Group II 19.13± 0.99 a 13.57± 0.85 ii b 9.58± 0.76 ii c 7.49±0.89 ii d y
peroxide
Group I 19.65±1.17a 15.30± 0.92b 0 0
Group II 19.13± 0.99 a 14.86± 0.77b 0 0

Means with different superscripts A, B in a row differ significantly at P< 0.05
Means with different superscripts i, ii in a column for particular treatment differ significantly at P< 0.01
.
Table 10: Per cent of sperm cells with low mitochondrial membrane potential in frozen thawed
bull semen samples treated with fertility associated protein and hydrogen peroxide
Sperm cells with low mitochondrial membrane potential during post thaw
Control Group I 52.96±2.50a 44.35± 3.95b 39.75± 5.07bc 32.03± 4.50c
Group II 55.38± 1.73a 45.68± 3.35b 35.34± 3.67c 33.58± 4.24c
Treatment with fertility Group I 52.96±2.50a 46.94± 3.64b 42.23± 4.73bc 38.51± 4.01c
associated protein
Group II 55.38± 1.73a 47.01± 3.06b 39.84±3.58c 36.05± 4.11c
peroxide
Group I 52.96±2.50a 16.03± 3.17b 10.63± 1.43c 0
Group II 55.38± 1.73a 13.36± 2.36b 12.87± 1.33b 0

Means with different superscripts (a, b, c, d) in a row differ significantly at P< 0.01.
control at 60 min (47.13 ± 2.39 vs 43.30 ± 2.80) and significantly (P <0.01) more
number of intact cells at 120 min (36.02 ± 2.10 vs 28.22 ± 3.10) and at 180 min
(26.69 ± 2.06 vs 15.27 ± 2.11) of incubation. Similarly, bulls in group II when
treated with fertility associated protein had non significantly more number of sperm
cells with intact plasma membrane than the control at 60 min (44.24 ± 3.38 vs 38.98
± 2.80) and significantly (P <0.01) more number of intact cells at 120 min (32.50 ±
3.03 vs 25.96 ± 3.32) and at 180 min (23.36 ± 2.17 vs 15.06 ± 2.16) of incubation.
4.2.3.1. Correlation between plasma membrane integrity and other in vitro

(Table 6)
Plasma membrane integrity of the sperm cells assessed by fluorogenic

staining had highly significant (P <0.01) positive correlation with functional
membrane integrity (0.997), sperm cells with high mitochondrial membrane potency
(0.606), sperm cells with low mitochondrial membrane potency (0.628), F pattern
uncapacitated- acrosome intact sperm cells (0.838), progressive forward motility
(0.697), curvilinear velocity of sperm cells (0.465), straight line velocity of sperm
cells (0.665), average path velocity of sperm cells (0.614), linearity (0.283),
straightness of sperm cell motility (0.295), amplitude of lateral head displacement
(0.418) and DNA integrity (0.360).Plasma membrane integrity of the sperm cell had
non significant positive correlation with non progressive forward motility (0.100),
wobble (0.158) and beat cross frequency (0.208).
Plasma membrane integrity of the sperm cell had highly significant (P

<0.01) negative correlation with MDA (- 0.562), sperm cells with lost mitochondrial
membrane potency (- 0.706), static sperm cells (- 0.548), B pattern capacitated-
acrosome intact sperm cells (- 0.764), AR pattern capacitated acrosome reacted
sperm cells (- 0.638) and necrotic cells (- 0.701).Plasma membrane integrity of the
sperm cell had non significant negative correlation with apoptotic sperm cells
(- 0.140).
4. 2. 4. Functional membrane integrity
Functional membrane integrity assessed by hypo osmotic swelling test in

frozen thawed semen samples of bulls in group I and II with different treatments
(treated with H2O2and withfertility associated protein) are presented in Table 8.
Per cent of sperm cells with functional membrane integrity decreased

significantly during different periods of incubation in control as well as in treatment
groups. In control though the percent of sperm cells with intact functional membrane
did not differ significantly between bull groups I and II during incubation, bulls in
group I had more number of functional membrane intact sperm cells than group II
bulls at immediate post thaw (55.61 ± 2.70 vs 49.12 ± 3.98), at 60 min (40.69 ± 2.75
vs 36.29 ± 3.20), at 120 min (25.46 ± 2.95 vs 22.13 ± 3.18) and at 180 min (11.59±
2.24 vs 11.52± 2.07) incubation periods.
In H2O2 treated semen samples bulls in group I had significantly (P <

0.01) more number of sperm cells with functional membrane integrity than group II
bulls at 10 min (41.60 ± 3.06 vs 28.63 ± 3.32) and significantly (P < 0.01) more cells
at 30 min (21.76 ± .2.27 vs 14.08 ± 1.46) incubation periods.
In the fertility associated protein treated semen samples, bulls in group I

had significantly (P < 0.05) more number of sperm cells with functional membrane
integrity than group II bulls at 60 min (45.45 ± 2.59 vs 40.86 ± 2.30) and at 120 min
(33.91 ± 2.31 vs 28.64 ± 3.03) incubation periods.
Bulls in group I when treated with fertility associated protein had

significantly more number of sperm cells with functional membrane integrity than
the control at 60 min (45.45± 2.59 vs 40.69 ± 2.75) and at 120 min (33.91± 2.31 vs
25.46 ± 2.95) and at 180 min of incubation (23.94 ± 2.08 vs 11.59 ± 2.24).
Similarly, bulls in group II when treated with fertility associated protein
hadsignificantly more number of sperm cells with functional membrane integrity
than the control at 60 min (40.86 ± 2.30 vs 36.29 ± 3.20) and at 120 min (28.64 ±
3.03 vs 22.13 ± 3.18) and at 180 min of incubation (20.37 ± 2.12 vs 11.52 ± 2.07).
Table 11: Per cent of sperm cells with lost mitochondrial membrane potential in frozen thawed
bull semen samples treated with fertility associated protein and hydrogen peroxide
Sperm cells with lost mitochondrial membrane potential during post thaw
Control Group I 27.39± 2.35a 38.92± 3.65b 47.90± 5.05c 59.79± 4.45d
Group II 25.49± 1.35a 41.62± 3.13b 56.32± 3.92c 61.13± 4.56d
Treatment with fertility Group I 27.39± 2.35a 35.55± 3.38b 43.23± 4.53b 53.53± 3.78c
associated protein
Group II 25.49± 1.35a 39.42± 2.84b 51.14± 3.86c 56.46± 4.45c
peroxide
Group I 27.39± 2.35a 68.69± 3.39b 89.37± 1.42c 100.00d
Group II 25.49± 1.35a 71.78± 2.53b 87.13± 1.33c 100.00d

Table 12: Per cent of sperm cells with intact DNA in frozen thawed bull semen samples treated
with fertility associated protein and hydrogen peroxide
Sperm cells with intact DNA during post thaw incubation (%)
Control Group I 94.94± 0.61 94.66± 0.56 93.80±0.661 93.64± 0.661
Group II 93.55±0.70a 93.69±0.76ab 92.17±0.502b 91.99±0.432b
Treatment with fertility Group I 94.94± 0.61 94.72± 0.58 93.95± 0.641 93.84± 0.691
associated protein
Group II 93.55±0.70 93.71±0.72 92.24±0.502 92.09± 0.452
peroxide
Group I 94.94± 0.61 94.82± 0.76 94.80± 0.80 94.01±0.511
Group II 93.55±0.70 93.30±0.82 93.35±0.60 92.26± 0.562

Means with different superscripts 1, 2 in a column differ significantly at P < 0.05.
Table 13: Per cent of viable sperm cells in frozen thawed bull semen samples treated
Per cent of viable sperm cells during post thaw incubation

Control Group I 60.20± 3.93a 40.99± 4.66b P 25.34±5.29c p 13.39± 2.68d p
Group II 50.51± 6.89a 35.78±6.08b 23.84± 5.84c 15.56±5.67d
Treatment with fertility Group I 60.20± 3.93a 50.36± 4.20b Q 38.36± 4.72c q 30.24±2.621d q
associated protein
Group II 50.51± 6.89a 40.42± 4.75b 30.46± 5.20c 20.68± 5.042d
peroxide
Group I 60.20± 3.93a 22.92± 4.27ib 0 0
Group II 50.51± 6.89a 11.54± 1.38iib 0 0

Means with different superscripts i, ii and 1, 2 in a column for particular treatment differ significantly at P< 0.01
and P < 0.05, respectively
Means of group I bulls with different superscripts p, q and P, Q in a column differ significantly at P< 0.01
and P<0.05, respectively
4.2.4.1. Correlation between functional membrane integrity and other in
vitro sperm characters during the incubation at 37° C for 180 min
(Table 6)
Functional membrane integrity of the sperm cells assessed by hypo

osmotic swelling test showed highly significant (P <0.01) positive correlation with
plasma membrane integrity (0.997), sperm cells with high mitochondrial membrane
potency (0.620), sperm cells with low mitochondrial membrane potency (0.632), F
pattern uncapacitated- acrosome intact sperm cells (0.822), progressive forward
motility (0.702), curvilinear velocity of sperm cells (0.467), straight line velocity of
sperm cells (0.666), average path velocity of sperm cells (0.608), linearity (0.283),
straightness of sperm cell motility (0.296), amplitude of lateral head displacement
(0.421) and DNA integrity (0.362).Functional membrane integrity of the sperm cell
showed non significant positive correlation with non progressive forward motility
(0.103), wobble (0.154) and beat cross frequency (0.209).
Functional membrane integrity of the sperm cell showed highly

significant (P <0.01) negative correlation with MDA (- 0.575), sperm cells with lost
mitochondrial membrane potency (- 0.714), static sperm cells (- 0.551), B pattern
capacitated- acrosome intact sperm cells (- 0.743), AR pattern capacitated acrosome
reacted sperm cells (- 0.643) and necrotic cells (- 0.713). Functional membrane
integrity of the sperm cell showed non significant negative correlation with
apoptotic sperm cells (- 0.151).
4.2.5.1. Sperm cells with high mitochondrial membrane potency
Sperm cells with high mitochondrial membrane potency (HMMP)

assessed by using JC-1 (5, 5’, 6, 6’-tetrachloro-1, 1’, 3, 3’-
tetraethylbenzimidazolylcarbocyanine iodide) and carboxy fluoresin diacetate
(CFDA) in frozen thawed semen samples of group I and II bulls with different
treatments (treated with H2O2and withfertility associated protein) are presented in
Table 9.
Per cent of sperm cells with HMMP decreased significantly during

different periods of incubation in control as well as in treatment groups. In untreated
control, bulls in group I had significantly (P<0.01) more number of sperm cells with
high mitochondrial membrane potency than the group II at 60 min (16.72 ± 1.02 vs
12.71 ± 0.99), at 120 min (12.32 ± 1.02 vs 8.39 ± 1.02) and at 180 min (8.31 ± 0.78
vs 5.19 ± 1.00) post thaw incubation periods.
Bulls in group I and II did not differ with each other in the per cent of
sperm cells with HMMP at 10 min of incubation with H2O2 (15.30 ± 0.92 vs 14.86 ±
0.77). When the semen samples were treated with fertility associated proteins, bulls
in group I had significantly (P< 0.01) more per cent of sperm cells with HMMP than
the group II during different points of incubation; 60 min (17.51 ± 0.90 vs 13.57 ±
0.85), at 120 min (14.54 ± 0.58 vs 9.58 ± 0.76) and at 180 min (10.54 ± 0.55 vs 7.49
± 0.89) incubation periods.
Bulls in group I, when treated with fertility associated protein had

significantly (P < 0.01) more per cent of sperm cells with HMMPthan the control at
120 min (14.54 ± 0.58 vs 12.32 ± 1.04) and at 180 min (10.54 ± 0.55 vs 8.31 ± 0.78)
and non significantly more cells at 60 min (17.51±0.90 vs 16.72±1.02) post thaw
incubation.
Bulls in group II, when treated with fertility associated protein had
significantly (P < 0.01) more per cent of sperm cells with HMMPthan the control at
180 min of incubation (7.49 ± 0.89 vs 5.19 ± 1.00) and non significantly more cells
observed at 60 min (13.57 ± 0.85 vs 12.71 ± 0.99) and at 120 min (9.58 ± 0.76 vs
8.39 ± 1.02) post thaw incubation.
4.2.5.2. Correlation between sperm cells HMMPand other in vitro sperm
characters during the incubation at 37° C for 180 min (Table 6)
Sperm cells with high mitochondrial membrane potency had highly

significant (P <0.01) positive correlation with sperm cell viability (0.774), plasma
membrane integrity (0.606), functional membrane integrity (0.620), sperm cells with
low mitochondrial membrane potency (0.386), progressive forward motility (0.592),
straight line velocity of sperm cells (0.422), average path velocity of sperm cells
(0.410) and F pattern uncapacitated- acrosome intact sperm cells (0.306).
Sperm cells with high mitochondrial membrane potency had significant

(P <0.05) positive correlation with non progressive forward motility (0.229),
curvilinear velocity of sperm cells (0.230), linearity (0.233), straightness of sperm
cell motility (0.246) and amplitude of lateral head displacement (0.228).Sperm cells
with high mitochondrial membrane potency had non significant positive correlation
with wobble (0.124), beat cross frequency (0.010) and DNA integrity (0.102).
Sperm cells with high mitochondrial membrane potency had highly

significant (P <0.01) negative correlation with MDA (- 0.689), sperm cells with lost
mitochondrial membrane potency (- 0.652), static sperm cells (- 0.554), AR pattern
capacitated acrosome reacted sperm cells (- 0.374) and necrotic sperm cells (-
0.814).Sperm cells with high mitochondrial membrane potency had significant (P
<0.05) negative correlation with B pattern capacitated- acrosome intact sperm cells
(- 0.226) and non significant negative correlation with apoptotic sperm cells (-
0.204).
4.2.5.3. Sperm cells with low mitochondrial membrane potency
Sperm cells with low mitochondrial membrane potency (LWMMP)in

frozen thawed semen samples of bulls in group I and II with different treatments
Per cent of sperm cells with LWMMP decreased significantly during

incubation in control as well as in treatment groups. In untreated control, bulls in
Table 14: Per cent of necrotic sperm cells in frozen thawed bull semen samples treated with
Per cent of necrotic sperm cells during post thaw incubation

Control Group I 32.30± 2.96a 50.15±4.17b P 64.56± 4.24c p 77.02± 2.32d p
Group II 39.37±5.27a 53.65±5.37b 64.83±6.18b 73.70±4.06c
Treatment with fertility Group I 32.30±2.96a 41.22±3.40b Q 50.60± 4.20c q 59.32± 4.24c q
associated protein
Group II 39.37±5.27a 48.80±4.62ab 60.42±5.63bc 69.75±5.21c
peroxide
Group I 32.30± 2.96a 66.55± 3.90ib 100 100
Group II 39.37±5.27a 74.56± 1.55 ii b 100 100

Means of group I bulls with different superscripts p, q and P, Q in a column differ significantly at P< 0.01
and P<0.05, respectively
Table 15: Per cent of apoptotic sperm cells in frozen thawed bull semen samples treated
Per cent of apoptotic sperm cells during post thaw incubation

Control Group I 7.50±1.07 8.86±1.23 10.10±1.37 9.58±1.21
Group II 10.12± 1.67 10.57±1.29 11.33±1.36 11.99±1.15
Treatment with fertility Group I 7.50±1.07 8.00±1.041 8.14±1.241 8.36±1.101
associated protein
Group II 10.12±1.67 11.20±1.202 12.36±1.382 12.90±0.802

peroxide
Group I 7.50±1.07a 10.53±1.35ib 0 0
Group II 10.12± 1.67a 13.90±1.52iib 0 0

Means with different superscripts a, b in a row differ significantly at P< 0.01
Means with different superscripts i, ii in a column differ significantly at P< 0.01
Means with different superscripts 1, 2 in a column differ significantly at P< 0.05
Table 18: Per cent of F pattern sperm cells in frozen thawed bull semen samples treated
with heparin and heparin with fertility associated protein
Per cent of F pattern sperm cells during post thaw incubation

Control Group I 51.73± 2.11a 43.19± 2.061b p 40.38± 2.011b p 37.48± 2.211b p
Group II 48.19± 2.39a 37.11± 2.112b x 34.48± 2.142b x 31.44± 2.282b x
Treatment with heparin Group I 51.73± 2.11a 31.03±1.841b q 22.18±1.911c q 15.76±1.871d q
Group II 48.19± 2.39a 37.43±2.162b x 27.81±1.962c y 21.48± 1.592d y
Treatment with heparin Group I 51.73± 2.11a 30.19± 1.43b q 21.87± 1.60c q 15.63± 1.49d q
and fertility associated
protein Group II 48.19± 2.39a 33.32±1.17b y 24.07±1.99c z 17.60±1.54d z

Means of group II bulls with different superscripts x, y, z in a column differ significantly at P< 0.01
group I and II did not differ each other in the per cent of sperm cells with LWMMP
starting from immediate post thaw (52.96 ± 2.50 vs 55.38 ± 1.73), at 60 min (44.35
± 3.95 vs 45.68 ± 3.35), at 120 min (39.75 ± 5.07 vs 35.34 ± 3.67) and at 180 min
(32.03 ± 4.50 vs 33.58 ± 4.24) post thaw incubation periods.
When the semen samples were treated with fertility associated protein,
bulls in group I and II did not differ each other in the per cent of sperm cells with
LWMMP during different points of incubation at 60 min (46.94 ± 3.64 vs 47.01 ±
3.06); at 120 min (42.23 ± 4.73 vs 39.84 ± 3.58) and at 180 min post thaw
incubation (38.51 ± 4.01 vs 36.05 ± 4.11). Similarly, when the semen samples were
treated with H2O2, bulls in group I and II did not differ each other in the per cent of
sperm cells with LWMMP at 10 min (16.03 ± 3.17 vs 13.36 ± 2.36) and at 30 min
post thaw incubation with H2O2 (10.63 ± 1.43 vs 12.87 ± 1.33).
Though it was not statistically significant, bulls in group I when treated

with fertility associated protein had more sperm cells with LWMMP than the control
at 60 min (46.94 ± 3.64 vs 44.35 ± 3.95), at 120 min (42.23 ± 4.73 vs 39.75 ± 5.07)
and at 180 min (38.51 ± 4.01 vs 32.03 ± 4.50). Similarly, bulls in group II also had a
non significantly more number of sperm cells with LWMMP when treated with
fertility associated proteinthan the control at 60 min (47.01 ± 3.06 vs 45.68 ± 3.35),
at 120 min (39.84 ± 3.58 vs 35.34 ± 3.67) and at 180 min of incubation (36.05 ±
4.11 vs 33.58 ± 4.24).
4.2.5.4. Correlation between sperm cells LWMMPand other in vitro sperm

Sperm cells with low mitochondrial membrane potency had highly

significant (P <0.01) positive correlation with sperm cell viability (0.467), plasma
membrane integrity (0.628), functional membrane integrity (0.632), sperm cells with
high mitochondrial membrane potency (0.386), progressive forward motility
(0.946), non progressive forward motility (0.333), curvilinear velocity of sperm cells
(0.725), straight line velocity of sperm cells (0.699), average path velocity of sperm
cells (0.759), amplitude of lateral head displacement (0.670), F pattern
uncapacitated- acrosome intact sperm cells (0.584) and DNA integrity (0.514).
Sperm cells with low mitochondrial membrane potency had non significant positive
correlation with straightness of sperm motility (0.017) and beat cross frequency
(0.031).
Sperm cells with low mitochondrial membrane potency had highly

significant (P <0.01) negative correlation with static sperm cells (- 0.840), sperm
cells with lost mitochondrial membrane potency (- 0.948), B pattern capacitated-
sperm cells (- 0.581), apoptotic sperm cells (- 0.338) and necrotic sperm cells (-
0.448). Sperm cells with low mitochondrial membrane potency had non significant
negative correlation with MDA (- 0.199), linearity (- 0.031) and wobble (- 0.122).
4.2.5.5. Sperm cells with lost mitochondrial membrane potency
Sperm cells with lost mitochondrial membrane potency (LTMMP)in

frozen thawed semen samples of group I and II bulls with different treatments
Per cent of sperm cells with LTMMP increased significantly during

different periods of incubation in control as well as in treatment groups. In untreated
control, though it was not significant group I bulls had comparatively low percent of
sperm cells with LTMMP than the bulls of group II at 60 min (38.92 ± 3.65 vs 41.62
± 3.13), at 120 min (47.90 ± 5.05 vs 56.32 ± 3.92) and at 180 min (59.79 ± 4.45 vs
61.13 ± 4.56) post thaw incubation periods.
though it was not significant, bulls in group I had comparatively low percent of
sperm cells with LTMMP than group II at 60 min 35.55 ± 3.38 vs 39.42 ± 2.84; at
120 min 43.23 ± 4.53 vs 51.14 ± 3.86; at 180 min post thaw incubation 53.53 ± 3.78
vs 56.46 ± 4.45.
When the samples were treated with H2O2, bulls in group I and II did not differ with
each other in the per cent of sperm cells with LTMMP at 10 min(68.69 ± 3.39 vs
71.78 ± 2.53) and at 30 min post thaw incubation with H2O2 (89.37 ± 1.42 vs 87.13
± 1.33).
Bulls in group I when treated with fertility associated protein had non
significantly lower per cent of sperm cells with LTMMP than the control at 60 min
(35.55 ± 3.38 vs 38.92 ± 3.65), at 120 min (43.23 ± 4.53 vs 47.90 ± 5.05) and at 180
min (53.53 ± 3.78 vs 59.79 ± 4.45) of incubation. Similarly, bulls in group II when
treated with fertility associated protein had non significantly lower per cent of
sperm cells with LTMMP than the control at 60 min (39.42 ± 2.84 vs 41.62 ± 3.13),
at 120 min (51.14 ± 3.86 vs 56.32 ± 3.92) and at 180 min of incubation (56.46 ±
4.45 vs 61.13 ± 4.56).
4.2.5.6. Correlation between sperm cells LTMMPand other in vitro sperm

Sperm cells with lost mitochondrial membrane potency had highly

significant (P <0.01) positive correlation with MDA (0.384), static sperm cells
(0.887), B pattern capacitated- acrosome intact sperm cells (0.457), AR pattern
capacitated acrosome reacted sperm cells (0.602), apoptotic sperm cells (0.343) and
necrotic sperm cells (0.632). Sperm cells with lost mitochondrial membrane potency
had non significant positive correlation with wobbliness of sperm motility (0.066).
Sperm cells with lost mitochondrial membrane potency had highly

significant (P <0.01) negative correlation with plasma membrane integrity (- 0.706),
functional membrane integrity (- 0.714), sperm cells with high mitochondrial
membrane potency (- 0.652), sperm cells with low mitochondrial membrane potency
(- 0.948), progressive forward motility (-0.971), non progressive forward motility (-
0.379), curvilinear velocity of sperm cells (- 0.673), straight line velocity of sperm
cells (- 0.710), average path velocity of sperm cells (- 0.758), amplitude of lateral
head displacement (- 0.632), F pattern uncapacitated- acrosome intact sperm cells
Table 19: Per cent of B pattern sperm cells in frozen thawed bull semen samples treated
Per cent of B pattern sperm cells during post thaw incubation

Control Group I 44.12± 1.86 a 50.86± 1.821 b p 53.10±1.761 b p 54.79± 1.701 b p
Group II 46.30± 1.88 a 55.73±1.762 b x 56.971.69 2 b x 58.43±1.58 2 b x
Treatment with heparin Group I 44.12± 1.86a 62.37± 1.921b q 69.79± 1.971c q 74.48± 1.98ic q
Group II 46.30± 1.88a 54.68±1.652b x 62.30±2.662c y 66.28±2.16ii c y
Treatment with heparin Group I 44.12± 1.86 a 63.24±1.55b q 69.78± 1.41c q 74.29± 1.33 c q
protein Group II 46.30± 1.88 a 59.61± 1.87 b y 66.57± 1.71c z 71.21± 1.23 d z

Means of group II bulls with different superscripts x, y, z in a column differ significantly at P< 0.01
Table 20: Per cent AR pattern sperm cells in frozen thawed bull semen samples treated
Per cent of AR pattern sperm cells during post thaw incubation

Control Group I 4.15±0.57a 6.09±0.78 b 6.51±0.87 bc P
7.76±0.711 c P
Group II 5.50±0.72a 7.14±0.82b 8.55±0.90bc 10.14±0.732c x

Treatment with heparin Group I 4.15±0.57a 6.60±0.81b 8.03±1.08 bc PQ
9.76±0.80 c Q
Group II 5.50±0.72a 7.89±0.71b 9.90±0.73bc 12.32±0.94c y

Treatment with heparin Group I 4.15±0.57a 6.60±0.72 ab 8.54±0.77 b Q
10.08± 0.60 c Q

protein Group II 5.50±0.72 a 7.07±0.59 ab 9.37±0.45 b 11.15± 0.63c xy

Means of group I bulls with different superscripts P, Q in a column differ significantly at P< 0.05
(- 0.566), DNA integrity (- 0.439), sperm cell viability (- 0.633). Sperm cells with
lost mitochondrial membrane potency had non significant negative correlation with
linearity (- 0.043), straightness of motility (- 0.086) and beat cross frequency (-
0.036).
Sperm cells with intact DNA assessed by acridine orange stainingin

frozen thawed semen samples of group I and II bulls with different treatments
In untreated control, there was no significant reduction in per cent of

sperm cells with intact DNA during 180 min of incubationin group I bulls. But
group II bulls showed a significant reduction at 120 min of incubation. When the
bulls in group I and II were compared, in untreated control, the per cent of sperm
cells with intact DNA did not differ between bull groups at immediate post thaw and
at 60 min of post thaw incubation. But group I bulls had significantly (P < 0.05)
more number of intact cells than the group II at 120 min (93.80 ± 0.66 vs 92.17 ±
0.50) and 180 min post thaw (93.64 ± 0.66 vs 91.99 ± 0.43).
In H2O2 treated samples, group I bulls had significantly (P < 0.05) more
number of DNAintact sperm cells than the group II bulls at 60 min of incubation
(94.01 ± 0.51 vs 92.26 ± 0.56). When the samples were treated with fertility
associated protein, there was no significant reduction in DNA intact cells during
incubation of 180 min in both the bull groups. But group I bulls had significantly (P
< 0.05) more number of DNA intact cells than the group II at 120 min (93.95 ± 0.64
vs 92.24 ± 0.50) and 180 min post thaw (93.84 ± 0.69 vs 92.09 ± 0.45).
In group Ibulls, the per cent of sperm cells with intact DNA at 60 min of
incubation in control, treatment with fertility associated protein and with hydrogen
peroxide did not differ significantly and the values were 94.66 ± 0.56 vs 94.72 ±
0.58 vs 94.01± 0.51. The values for control and fertility associated protein treated
groups at 120 min were 93.80 ± 0.66 vs 93.95 ± 0.64 and at 180 min of incubation
were 93.64 ± 0.66 vs 93.84 ± 0.69.
In group IIbulls, the per cent of sperm cells with intact DNA were
significantly (P < 0.05) lower in hydrogen peroxide treated group at 60 min (92.26 ±
0.56) than the control (93.69 ± 0.76) and fertility associated protein treated group
(93.71 ± 0.72). There was no significant difference between control and fertility
associated protein treated groups at 60 min (93.69± 0.76 vs 93.71± 0.72), at 120 min
(92.17 ± 0.50 vs 92.24 ± 0.50) and at 180 min (91.99 ± 0.43 vs 92.09 ± 0.45) of
incubation.
4.2.6.1. Correlation between sperm cell DNAintegrity and other in vitro

(Table 6)
Sperm cells with intact DNA had highly significant (P <0.01) positive
correlation with plasma membrane integrity (0.360), functional membrane integrity
(0.362), sperm cells with low mitochondrial membrane potency (0.514), progressive
forward motility (0.495), curvilinear velocity of sperm cells (0.462), straight line
velocity of sperm cells (0.468), average path velocity of sperm cells (0.472),
amplitude of lateral head displacement (0.356) and F pattern uncapacitated-
acrosome intact sperm cells (0.368). Sperm cells with intact DNA had significant (P
<0.05) positive correlation with sperm cell viability (0.236) and non significant
positive correlation with linearity (0.103), straightness (0.061), beat cross frequency
(0.035), wobble (0.123), sperm cells with high mitochondrial membrane potency
(0.102) and MDA (0.086).
Sperm cells with intact DNA had highly significant (P <0.01) negative
correlation with static sperm cells (- 0. 356), sperm cells with lost mitochondrial
membrane potency (- 0.439), B pattern capacitated- acrosome intact sperm cells (-
0.346), AR pattern capacitated acrosome reacted sperm cells (- 0.300), apoptotic
sperm cells (- 0.365) and non significant negative correlation with non progressive
forward motility (- 0.051) and necrotic cells (- 0. 199).
4.2.7. Assessment of sperm cell apoptosis
4.2.7.1. Viable sperm cells
Per cent of viable sperm cells in frozen thawed semen samples of group I
and II bulls with different treatments (treated with H2O2and withfertility associated
The per cent of viable cells decreased significantly during different

periods of incubation in control as well as in treatment groups. Though it was not
significant, in untreated control, bulls in group I had more number of viable sperm
cells than the group II during the incubation. The per cent of viable sperm cells in
group I and II bulls were 60.20 ± 3.93 vs 50.51 ± 6.89 at immediate post thaw, 40.99
± 4.66 vs 35.78 ± 6.08 at 60 min, 25.34 ± 5.29 vs 23.84 ± 5.84 at 120 min and 13.39
± 2.68 vs 15.56 ± 5.67 at 180 min post thaw incubation periods.
In H2O2 treatment, bulls in group I had significantly (P < 0.01) more

number of viable cells (22.92 ± 4.27 vs 11.54 ± 1.38) than group II at 30 min of
incubation.In fertility associated protein treatment, bulls in group I had significantly
(P<0.05) high per cent of viable sperm cells than group II at 180 min of incubation
(30.24 ± 2.62 vs 20.68 ± 5.04) and non significantly more number of viable cells at
60 min (50.36 ± 4.20 vs 40.42 ± 5.60) and at 120 min incubation (38.36 ± 4.72 vs
30.46 ± 5.20).
In group I bulls, the per cent of viable sperm cells were significantly
higher in fertility associated protein treated group than the control at 60 min (50.36 ±
4.20 vs 40.99 ± 4.66), at 120 min (38.36 ± 4.72 vs 25.34 ± 5.29) and at 180 min
(30.24 ± 2.62 vs 13.39) of incubation. But when treated with H2O2 viable cells
significantly reduced from 60.20 ± 3.93 at immediate thaw to 22.92 ± 4.27 within 30
min of incubation.
In group II bulls, though it was not significant, the per cent of viable
sperm cells were higher in fertility associated protein treated group than the control
at 60 min ( 40.42 ± 4.75 vs 35.78 ± 6.08), at 120 min (30.46 ± 5.20 vs 23.84 ± 5.84)
and at 180 min of incubation (20.68 ± 5.04 vs 15.56 ± 5.67).But when the samples
were treated with H2O2, viable cells were reduced significantly from 50.51±6.89 at
immediate thaw to 11.54 ± 1.38within 30 min of incubation.
4.2.7.2. Correlation between viable sperm cells and other in vitro sperm
Viability of sperm cell had highly significant (P <0.01) positive

correlation with plasma membrane integrity (0.661), functional membrane integrity
(0.674), sperm cells with high mitochondrial membrane potency (0.774), sperm cells
with low mitochondrial membrane potency (0.467), progressive forward motility
(0.596), F pattern uncapacitated- acrosome intact sperm cells (0.397), curvilinear
velocity of sperm cells (0.318), straight line velocity of sperm cells (0.490), average
path velocity of sperm cells (0.441) and straightness of sperm cell motility (0.284).
Viability of sperm cell had significant (P <0.05) positive correlation with

linearity (0.272), amplitude of lateral head displacement (0.272), and DNA integrity
(0.236). Viability of sperm cell had non significant positive correlation with non
progressive forward motility (0.134) and wobble (0.151).
Viability of sperm cell had highly significant (P <0.01) negative

correlation with MDA (- 0.559), sperm cells with lost mitochondrial membrane
potency (- 0.633), static sperm cells (- 0.502), B pattern capacitated- acrosome intact
sperm cells (- 0.315), AR pattern capacitated acrosome reacted sperm cells (- 0.440),
apoptotic sperm cells (- 0.555) and necrotic cells (- 0.980). Viability of sperm cell
had non significant negative correlation with beat cross frequency (- 0.066).
4.2.7.3. Necrotic sperm cells
Per cent of necrotic sperm cells in frozen thawed semen samples of group
I and II bulls with different treatments (treated with H2O2and withfertility associated
The per cent of necrotic cells increased significantly during different

periods of incubation in control as well as in treatment groups. In untreated control,
the per cent necrotic cells did not differ between bulls of group I and II during
incubation. The values were 32.30 ± 2.96 vs 39.37 ± 5.27 at immediate post thaw,
50.15 ± 4.17 vs 53.65 ± 5.37 at 60 min, 64.56 ± 4.24 vs 64.83 ± 6.18 at 120 min and
77.02 ± 2.32 vs 73.70 ± 4.06 at 180 min post thaw incubation periods.
In H2O2 treatment, group I bulls had significantly (P < 0.01) less number
of necrotic cells (66.55 ± 3.90 vs 74.56 ± 1.55) than the group II at 30 min of
incubation. When the semen samples were treated with fertility associated proteins,
though it was not statistically significant, group I bulls had less per cent of necrotic
cells than the group II. The values were 41.22 ± 3.40 vs 48.80 ± 4.62 at 60 min,
50.60 ± 4.20 vs 60.42 ± 5.63 at 120 min and 59.32 ± 4.20 vs 69.75 ± 5.21 at 180 min
post thaw incubation periods.
In group Ibulls, the per cent of necrotic cells were significantly lower in
fertility associated protein treated group than the control at 60 min (41.22 ± 3.40 vs
50.15 ± 4.17), at 120 min (50.60 ± 4.20 vs 64.56 ± 4.24) and at 180 min of
incubation (59.32 ± 4.24 vs 77.02 ± 2.32). In group IIbulls, though it was not
significant, fertility associated protein treated group had low per cent of necrotic
cells than the control group at 60 min (48.80 ± 4.62 vs 53.65 ± 5.37), at 120 min
(60.42 ± 5.63 vs 64.83 ± 6.18) and at 180 min of incubation (69.75 ± 5.21 vs 73.70 ±
4.06).
4.2.7.4. Correlation between necrotic sperm cells and other in vitro sperm
Necrotic sperm cells had highly significant (P <0.01) negative correlation

with plasma membrane integrity (- 0.701), functional membrane integrity (- 0.713),
sperm cells with high mitochondrial membrane potency (- 0.814), sperm cells with
low mitochondrial membrane potency (- 0.448), progressive forward motility (-
0.594), curvilinear velocity of sperm cells (- 0.303), straight line velocity of sperm
cells (- 0.481), average path velocity of sperm cells (- 0.436), linearity (- 0.276),
straightness of sperm cell motility (- 0.282), F pattern uncapacitated- acrosome
intact sperm cells (- 0.432) and sperm cell viability (- 0.980). Necrotic sperm cells
were having significant (P <0.05) negative correlation with amplitude of lateral head
displacement (- 0.265) and non significant negative correlation with non progressive
forward motility (- 0.161), wobble (- 0.160) and DNA integrity (- 0.199).
Necrotic sperm cells had highly significant (P <0.01) positive correlation

with MDA (0.617), sperm cells with lost mitochondrial membrane potency (0.632),
static sperm cells (0.520), B pattern capacitated- acrosome intact sperm cells
(0.352), AR pattern capacitated acrosome reacted sperm cells (0.455) and apoptotic
sperm cells (0.403).
4.2.7.5. Apoptotic sperm cells
Per cent of apoptotic sperm cells in frozen thawed semen samples of

group I and II bulls with different treatments (treated with H2O2and withfertility
associated protein) assessed by Annexin-V-FITC apoptosis detection kit are
There was no significant change in the per cent of apoptotic cells during
different periods of incubation in control as well as in treatment with fertility
associated protein. In untreated control though it was not significant, group I bulls
had less number of apoptotic cells than group II during incubation. The values were
7.5 ± 1.07 vs 10.12 ± 1.67 at immediate post thaw, 8.86 ± 1.23 vs 10.57 ± 1.29 at 60
min, 10.10 ± 1.37 vs 11.33 ± 1.36 at 120 min and 9.58 ± 1.21 vs 11.99 ± 1.15 at 180
min post thaw incubation periods.
In H2O2 treatment, group I bulls had significantly (P < 0.01) less number
of apoptotic cells (10.53 ± 1.35 vs 13.90 ± 1.52)than group II at 30 min of
incubation. When the samples were treated with fertility associated protein, group I
bulls had significantly (P <0.05) less number of apoptotic cells than group II during
incubation. The values were 8.00 ± 1.04 vs 11.20 ± 1.20 at 60 min, 8.14 ± 1.24 vs
12.36 ± 1.38 at 120 min and 8.36 ± 1.10 vs 12.90 ± 0.80 at 180 min post thaw
incubation periods.
In group Ibulls, there was no significant difference between the fertility

associated protein treated group and the control group in per cent of apoptotic cells
during different periods of incubation- 60 min (8.00 ± 1.04 vs 8.86 ± 1.23), 120 min
(8.14 ± 1.24 vs 10.10 ± 1.37) and 180 min (8.36 ± 1.10 vs 9.58 ± 1.21). Similarly, in
group IIbulls, there was no significant difference between the fertility associated
protein treated group and the control group in per cent of apoptotic cells during
different point of incubation- 60 min (11.20 ± 1.20 vs 10.57 ± 1.29), 120 min (12.36
± 1.38 vs 11.33 ± 1.36) and 180 min (12.90 ± 0.80 vs 11.99 ± 1.15).
4.2.7.6. Correlation between apoptotic sperm cells and other in vitro sperm
Apoptotic sperm cells had highly significant (P <0.01) negative

correlation with progressive forward motility (- 0.332), sperm cells with low
mitochondrial membrane potency (- 0.338), curvilinear velocity of sperm cells (-
0.275), straight line velocity of sperm cells (- 0.316), average path velocity of sperm
cells (- 0.286), DNA integrity (- 0.365) and sperm cell viability (- 0.555). Apoptotic
sperm cells had significant (P <0.05) negative correlation with amplitude of lateral
head displacement (- 0.221).
Apoptotic sperm cells had non significant (P <0.05) negative correlation

with MDA (- 0.017), plasma membrane integrity (- 0.140), functional membrane
integrity (- 0.151), sperm cells with high mitochondrial membrane potency (- 0.204),
non progressive forward motility (- 0.030), linearity (- 0.098), straightness of sperm
cell motility (- 0.105), wobble (- 0.046) and AR pattern capacitated acrosome
reacted sperm cells (- 0.066).
Apoptotic sperm cells had highly significant (P <0.01) positive

correlation with necrotic sperm cells (0.403) and sperm cells with lost mitochondrial
membrane potency (0.343). Apoptotic sperm cells had significant (P <0.05) positive
correlation with static sperm cells (0.256). Apoptotic sperm cells had non significant
positive correlation with beat cross frequency (0.061) and AR pattern capacitated
acrosome reacted sperm cells (0.195).
4.2.8. Motility and velocity parameters of sperm cells
Motility and velocity parameters of sperm cells assessed by CASA in

frozen thawed semen samples of group I and II bulls during different periods of
incubationin control and treated with fertility associated protein is presented in
Table16 and 16a.
Significant reductions in progressive forward motility, total motility,

straight line velocity and average path velocity were observed during incubation in
control as well as in fertility associated protein treated samples. Treatment with
fertility associated protein caused significant reductions in progressive forward
motility, total motility, straight line velocity and average path velocity of the sperm
cells during incubation when compared to the untreated control. Bulls in group I
and II did not differ each other significantly in the motility and velocity parameters
during different points of incubation in control and in fertility associated protein
treatment.
4.2.8.1. Motility and velocity parameters of sperm cells in group Ibulls
The per cent of sperm cells with progressive forward motility was
significantly lower in fertility associated protein treated group than the control at 60
min (39.23 ± 3.57 vs 47.61 ± 4.50), at 120 min (31.32 ± 3.06 vs 39.30 ± 5.53) and at
180 min of incubation (21.28 ± 2.89 vs 29.49 ± 5.03).
Per cent of sperm cells with non progressive motility significantly

increased in fertility associated protein treated group than the control at 60 min
(42.16 ± 1.81 vs 31.01 ± 1.58), at 120 min (34.97 ± 2.80 vs 28.23 ± 2.68) and at 180
min of incubation (36.51 ± 2.15 vs 26.83 ± 3.08).
There was no significant difference between fertility associated protein

treated group and control group in total motility at 60 min (81.39 ± 3.25 vs 78.62 ±
5.22), at 120 min (66.29 ± 2.78 vs 67.53 ± 7.60) and at 180 min of incubation (57.79
± 3.48 vs 56.32 ± 7.46). Similarly there was no significant difference between
fertility associated protein treated group and control in per cent of static cells at 60
Table 16: Motility and velocity parameters of frozen thawed bull semen samples treated with fertility associated protein
60 min 120 min 180 min

Bull Immediate
Parameter FAP FAP FAP
groups Post thaw Control Control Control
treatment treatment treatment
Progressive Group I 62.88±2.55 pw
47.61±4.50 qA
39.23± 3.57 Bx
39.30± 5.53 Ar
31.32±3.06 B y 29.49± 5.03A s 21.28±2.89 Bz
forward
Group II 63.55±2.08p w 48.53±3.93a q 37.07±1.99 b x 37.60±4.45A r 25.18±2.12 B y 28.61±5.29 A s 15.85±2.66 Bz
motility %
Non progressive Group I 27.60±1.92 w 31.01±1.58 a 42.16±1.81b x 28.23±2.68A 34.97±2.80 B y 26.83±3.08 A 36.51±2.15 By
motility Group II 28.02±2.38 w 32.28±2.51A 40.35±2.52 B x 30.80±2.32 34.45±2.41 y 24.55±3.80 A 36.12±2.59 By
Total motility Group I 90.48±2.19 pw
78.62 ± 5.22 q 81.39±3.25 x 67.53 ± 7.60 r 66.29±2.78 y 56.32 ± 7.46 s 57.79±3.48 z
Group II 91.56±2.26 p w 80.81±5.27 q 77.42 ± 1.91x 68.40±6.18 q r 59.63± 2.00 y 53.15±8.68 r 51.96± 1.99 z
Static cells Group I 9.53±2.19 p w 21.36± 5.21q 16.61±3.25 x 32.71±7.66 q r 35.51±3.38 y 43.46±7.39 r 42.22±3.48 z
Group II 10.78±4.39 p w 18.69±5.11 pq 24.63±2.08 x 30.81±6.16 q r 40.37±1.99 y 43.68±8.80 r 48.05±1.98 z
Curvilinear Group I 72.11±4.29 p w 65.61±3.87Apq 57.49±3.45 B x 60.99±5.59 q r 51.76±2.48 x 51.75±6.29 r 45.52±2.77 y
Velocity
Group II 73.49±6.15 p w 69.07±4.46Apq 57.31±1.41 B x 54.61±3.76 q 56.04±2.34 x 53.39±4.64 q 50.68±3.42 x
VCL (µm/s)
Straight-line Group I 30.40±2.42 p w 26.19±1.45 pq 22.43±1.54 x 22.09±2.16 q r 21.99±1.14 x 18.12±2.00 r 18.55±0.85 y
Velocity
Group II 30.39±2.77 p w 26.87±1.93 pq 21.63±1.58 x 20.20±0.97 q 23.42±2.12 x 19.74±1.20 q 18.79±2.06 y
VSL (µm/s)

Means in a row with different superscripts p, q, r, s and w, x, y, z differ significantly at P <0.01
Means in a row, for a particular incubation period with different superscripts a, b and A, B differ significantly at P < 0.01 and P < 0.05, respectively.
Table 16a: Velocity parameters of frozen thawed bull semen samples treated with fertility associated protein
60 min 120 min 180 min

Bull Immediate
Parameter FAP FAP FAP
groups Post thaw Control Control Control
treatment treatment treatment
Average Path Group I 42.89±3.05 p w 39.25±1.92 pq 35.45±2.45 x 34.51±3.15 q r 32.95±1.99 x 29.95±3.62 r 26.09±1.77 y
Velocity VAP
Group II 46.99±2.69 p w 40.69±2.60 p 35.92±2.27 x 31.37±1.90 q 34.99±1.39 x 30.86±2.59 q 27.93±2.98 y
(µm/s)
Linearity % Group I 41.80 ±1.32 40.30±1.48 41.34±2.28 36.44±1.61 A 42.24±1.56 B 35.88±1.82 39.23±2.18
Group II 41.71±1.90 39.19±1.86 40.06±2.99 37.71±1.47 41.31±1.84 38.08±1.82 38.66±1.72
Straightness % Group I 70.53±1.38 p w 66.71±1.39 pq 66.59±1.87 w x 64.14±2.01 q 67.59±1.46 x 61.81±2.70 q 65.07±2.74 x
Group II 69.92±2.14 w 65.97±1.89 64.21±2.38 w x 65.03±1.75 66.49±2.94 w x 65.45±2.40 62.79±2.50 x
Wobble % Group I 59.17±0.96 60.28±1.23 60.07±1.28 56.62±1.00 a 60.31±1.28 b 57.93±0.94 58.02±1.39
Group II 59.42±1.23 59.12±1.20 58.55±1.21 57.84±1.00 a 62.14±0.35 b 58.08±1.35 58.52±0.89
Amplitude of lateral Group I 3.36±0.16 3.29±0.15 2.91±0.11 3.18±0.18 2.82±0.13 2.82±0.21 2.57±0.12
head displacement
Group II 3.33±0.25 3.34±0.14 A 2.98±0.05B 2.91±0.14 2.67±0.08 2.87±0.18 2.50±0.14
ALH (µm)
Beat / Cross Group I 9.45±0.45 10.11±0.31 10.75±0.49 9.22±0.61 10.88±0.28 8.42±0.76 9.91±0.61
Frequency BCF
Group II 9.15±0.73 9.69±0.50 10.64±0.59 9.61±0.27 a 11.50 ±0.33 b 9.35±0.39 10.31 ± 0.63
(Hz)

Means in a row with different superscripts p, q, r, s and w, x, y, z differ significantly at P <0.01
Means in a row, for a particular incubation period with different superscripts a, b and A, B differ significantly at P < 0.01 and P < 0.05, respectively.
Treatment with fertility associated protein significantly (P< 0.05)

inhibited curvilinear velocity of sperm cells in treatment group than the control at 60
min of incubation (57.49 ± 3.45 vs 65.61 ± 3.87). But there was no significant
difference between treatment groups at 120 min (51.76 ± 2.48 vs 60.99 ± 5.59) and
at 180 min incubation (45.52 ± 2.77 vs 51.75 ± 6.29).

treated group and control in straight-line velocity of sperm cells at 60 min (22.43 ±
1.54 vs 26.19 ± 1.45), at 120 min (21.99 ± 1.14 vs 22.09 ± 2.16) and at 180 min of
incubation (18.55 ± 0.85 vs 18.12 ± 2.00).

treated group and control in average path velocity of sperm cells at 60 min (35.45 ±
2.45 vs 39.25 ± 1.92 ), at 120 min (32.95 ± 1.99 vs 34.51 ± 3.15) and at 180 min of
incubation (26.09 ± 1.77 vs 29.95± 3.62). Treatment with fertility associated protein
significantly increased the per cent of linearity (42.24 ± 1.56 vs 36.44 ± 1.61),
wobble (61.31 ± 1.28 vs 56.62 ± 1.00) and beat cross frequency (10.88 ± 0.28 vs
9.22± 0.61) than the control at 120 min of incubation.
4.2.8.2. Motility and velocity parameters of sperm cells in group IIbulls
The per cent of sperm cells with progressive forward motility was
significantly lower in fertility associated protein treated group than the control at 60
Per cent of sperm cells with non progressive motility significantly

increased in fertility associated protein treated group than the control at 60 min
(40.35 ± 2.52 vs 32.28 ± 2.51) and at 180 min of incubation (36.12 ± 2.59 vs 24.55 ±
3.80).
treated group and control in total motility at 60 min (77.42 ± 1.91 vs 80.81 ± 5.27),
at 120 min (59.63 ±2.00 vs 68.40 ± 6.18) and at 180 min of incubation (51.96± 1.99
vs 53.15 ± 8.68). Similarly there was no significant difference between fertility
associated protein treated group and control in per cent of static cells at 60 min
(24.63 ± 2.08 vs 18.69 ± 5.11), at 120 min (40.37 ± 1.99 vs 30.81± 6.16) and at 180
min of incubation (48.05 ± 1.98 vs 43.68 ± 8.80).
Treatment with fertility associated protein significantly (P< 0.05)

inhibited curvilinear velocity of sperm cells in treatment group than the control at 60
min of incubation (57.31 ± 1.41 vs 69.07 ± 4.46). But there was no significant
difference between treatment groups at 120 min (56.04 ± 2.34 vs 54.61 ± 3.76) and
at 180 min incubation (50.68 ± 3.42 vs 53.39 ± 4.64).

treated group and control in straight-line velocity of sperm cells at 60 min (21.63 ±
1.58 vs 26.87 ± 1.93), at 120 min (23.42 ± 2.12 vs 20.20 ± 0.97) and at 180 min of
incubation (18.79 ± 2.06 vs 19.74 ± 1.20). Similarly, there was no significant
difference between fertility associated protein treated group and control in average
path velocity of sperm cells at 60 min (35.92 ± 2.27 vs 40.69± 2.60), at 120 min
(34.99 ± 1.39 vs 31.37 ± 1.90) and at 180 min of incubation (27.93± 2.98 vs 30.86 ±
2.59).
There was no significant difference between treatment groups in per cent

of linearity and straightness of sperm cell motility during different periods of
incubation. Fertility associated protein treated group had significantly higher per
cent of wobble (62.14 ± 0.35 vs 57.84 ± 1.00) and beat cross frequency (11.50 ±
0.33 vs 9.61 ± 0.27) than the control at 120 min of incubation.
4.2.8.3. Motility and velocity parameters of sperm cells- treatment with H2O2
Motility and velocity parameters of sperm cells in group I and II bulls

during incubation of frozen thawed bull semen samples treated with H2O2 is
``Significant reductions in progressive forward motility, total motility, curvilinear
velocity, straight line velocity and average path velocity were observed in both the
bull groups during incubation of semen samples withH2O2. But there was no
significant difference between group I and II bulls observed in motility and velocity
parameters during different periods of incubation.
There was no significant difference between bulls in group I and II after

10 min of incubation with H2O2 in progressive forward motility 22.92 ± 3.09 vs
19.66 ± 2.11; non progressive motility 10.86 ± 1.82 vs 12.03 ± 3.08; static cells
66.23 ± 3.29 vs 62.39 ± 6.26; curvilinear velocity 61.75 ± 4.94 vs 57.03 ± 5.46;
straight line velocity 28.26 ± 3.37 vs 23.85 ± 2.48; average path velocity 37.03 ±
3.13 vs 32.95 ± 2.89; linearity 44.83 ± 2.79 vs 41.93± 2.21; straightness 74.09 ±
3.11 vs 71.42 ± 2.55; wobbliness 60.10 ± 1.98 vs 58.51 ± 1.91; amplitude of lateral
head displacement 2.98 ± 0.19 vs 2.84 ± 0.19 and beat cross frequency 9.93 ± 0.76
vs 9.45 ± 0.65.Progressive forward motility of the sperm cells was lost within 30
min of incubation with H2O2and all the cells became static.
4.2.8.4. Correlation between sperm cell motility parametersand other in

vitro sperm characters during the incubation at 37° C for 180 min
(Table 6)
4.2.8.4.1. Progressive forward motility
Sperm cells with progressive forward motility had highly significant (P

<0.01) positive correlation with sperm cell viability (0.596), plasma membrane
integrity (0.697), functional membrane integrity (0.702), sperm cells with high
mitochondrial membrane potency (0.592), sperm cells with low mitochondrial
membrane potency (0.946), non progressive forward motility (0.354), curvilinear
velocity of sperm cells (0.708), straight line velocity of sperm cells (0.743), average
path velocity of sperm cells (0.797), amplitude of lateral head displacement (0.651),
F pattern uncapacitated- acrosome intact sperm cells (0.603) and DNA integrity
(0.495). Sperm cells with progressive forward motility had non significant (P <0.01)
positive correlation with linearity (0.045), straightness of motility (0.076) and beat
cross frequency (0.026).
Sperm cells with progressive forward motility had highly significant (P

<0.01) negative correlation with MDA (- 0.342), sperm cells with lost mitochondrial
membrane potency (- 0.971), static sperm cells (- 0.898), B pattern capacitated-
sperm cells (- 0.603), apoptotic sperm cells (-0.332) and necrotic sperm cells (-
0.594).
4.2.8.4.2. Static sperm cells
Static sperm cells had highly significant (P <0.01) positive correlation

with MDA (0.285), sperm cells with lost mitochondrial membrane potency (0.887),
B pattern capacitated- acrosome intact sperm cells (0.358), AR pattern capacitated
acrosome reacted sperm cells (0.563) and necrotic sperm cells (0.520). Static sperm
cells had significant (P <0.05) positive correlation with apoptotic sperm cells
(0.256). Static sperm cells had non significant positive correlation with linearity
(0.192), straightness (0.181), wobble (0.175) and beat cross frequency (0.088).
Static sperm cells had highly significant (P <0.01) negative correlation

with sperm cell viability (- 0.502), plasma membrane integrity (- 0.548), functional
membrane integrity (- 0.551), sperm cells with high mitochondrial membrane
potency (- 0.554), sperm cells with low mitochondrial membrane potency (- 0.840),
progressive forward motility (- 0.898), non progressive forward motility (- 0.678),
curvilinear velocity of sperm cells (- 0.647), straight line velocity of sperm cells (-
0.564), average path velocity of sperm cells (-0.688), amplitude of lateral head
displacement (- 0.628), F pattern uncapacitated- acrosome intact sperm cells (-
0.469) and DNA integrity (- 0.356).
4.2.8.4.3. Sperm velocity parameters
Sperm cell velocity parameters such as curvilinear velocity, straight line

velocity, average path velocity, linearity, straightness of sperm cell motility and
amplitude of lateral head displacement had positive correlation among themselves
and with other in vitro sperm characters such as plasma membrane integrity,
functional membrane integrity, progressive forward motility, F pattern
(uncapacitated- acrosome intact) sperm cells, DNA integrity, viable sperm cells and
sperm cells high mitochondrial membrane potential. Sperm cell velocity parameters
had negative correlations with sperm static cells, cells with lost mitochondrial
membrane potential, B pattern (capacitated- acrosome intact) sperm cells, AR
pattern (capacitated – acrosome reacted) sperm cells, necrotic sperm cells and MDA.
4.3. CAPACITATION STATUS OF SPERM CELLS
4.3.1. F pattern cells
Capacitation status of sperm cells – F pattern (uncapacitated acrosome

intact) cells assessed by chlortetracycline staining in frozen thawed semen samples
of bulls in group I and II with different treatments (treated with heparin and heparin
with fertility associated proteins) are presented in Table 18.
In untreated control, the per cent of F pattern sperm reduced significantly

from immediate post thaw level to 60 min of incubation in both the bull groups.
After 60 min of incubation the rate of reduction in F pattern cells were not
statistically significant. Though it was not significant, bulls in group I had more
number of F pattern sperm cells than bull group II (51.73 ± 2.11 vs 48.19 ± 2.39) at
immediate post thaw. But bulls in group I had significantly (P < 0.05) more number
of F pattern cells at 60 min (43.19 ± 2.06 vs 37.11 ± 2.11), at 120 min (40.38 ± 2.01
vs 34.48 ± 2.14) and at 180 min post thaw incubation (37.48 ± 2.21 vs 31.44 ± 2.28)
than group II.
When semen samples were treated with heparin, per cent of F pattern
cells reduced significantly during different points of incubation in both the bull
group I and II. Bulls in group I had significantly (P < 0.05) low number of F pattern
cells at 60 min (31.03 ± 1.84 vs 37.43 ± 2.16), at 120 min (22.18 ± 1.91 vs 27.81 ±
1.96) and at 180 min post thaw incubation (15.76 ± 1.87 vs 21.48 ± 1.59) than the
group II. Similarly, when the semen samples were treated with heparin along with
fertility associated protein, per cent of F pattern cells reduced significantly during
different points of incubation in both the group I and II bulls. The per cent of sperm
cells with F pattern in group I and II bulls did not differ significantly at different
periods of incubation (30.19 ± 1.43 vs 33.32 ± 1.17 at 60 min; 21.87 ± 1.60 vs 24.07
± 1.99 at 120 min; 15.63 ± 1.49 vs 17.60 ± 1.54 at 180 min).
In group Ibulls, the per cent of F pattern cells in heparin treated group
and heparin with fertility associated proteins treated group did not differ
significantly at any point of incubation. But F pattern cells in heparin and heparin
with fertility associated proteins treated groups were significantly (P < 0.01) lower
than the control group at 60 min (31.03 ± 1.84, 30.19 ± 1.43 and 43.19 ± 2.06), at
120 min (22.18 ± 1.91, 21.87 ± 1.60 and 40.38 ± 2.01) and at 180 min incubation
(15.76 ± 1.87, 15.63 ± 1.49 and 37.48 ± 2.21).
In group IIbulls, the per cent of sperm cells with F pattern in heparin with
fertility associated proteins treated group (33.32 ± 1.17) was significantly (P < 0.01)
lower than the control (37.11 ± 2.11) and with the heparin treated group (37.43 ±
2.16) at 60 min post thaw incubation. The per cent of sperm cells with F pattern in
control, heparin treated and heparin with fertility associated proteins treated groups
differed significantly (P< 0.01) each other at 120 min (34.48 ± 2.14 vs 27.81 ± 1.96
vs 24.07 ± 1.99) and at 180 min post thaw (31.44 ± 2.28 vs 21.48 ± 1.59 vs 17.60 ±
1.54).
4.3.2. Correlation between F pattern sperm cellsand other in vitro sperm

F pattern uncapacitated- acrosome intact sperm cellshad highly

significant (P <0.01) positive correlation with sperm cell viability (0.397),plasma
membrane integrity (0.838), functional membrane integrity (0.822),sperm cells with
high mitochondrial membrane potency (0.306),sperm cells with low mitochondrial
membrane potency (0.584),progressive forward motility (0.603),curvilinear velocity
of sperm cells (0.432),straight line velocity of sperm cells (0.542),average path
velocity of sperm cells (0.521),amplitude of lateral head displacement (0.390)and
DNA integrity (0.368). F pattern uncapacitated- acrosome intact sperm cellswere
having non significant positive correlation with non progressive forward motility
(0.108), linearity (0.124), straightness (0.162), wobble (0.016) and beat cross
frequency (0.146).
F pattern uncapacitated- acrosome intact sperm cellshad highly significant (P
<0.01) negative correlation with MDA (- 0.274),sperm cells with lost mitochondrial
membrane potency (- 0.566),static sperm cells (- 0.469),B pattern capacitated-
acrosome intact sperm cells (- 0.962),AR pattern capacitated acrosome reacted
sperm cells (- 0.674)and necrotic cells (- 0.432).
4.3.3. B pattern cells
Capacitation status of sperm cells – B pattern (capacitated acrosome

intact) cells assessed by chlortetracycline staining in frozen thawed semen samples
of group I and II bulls with different treatments (treated with heparin and heparin
with fertility associated proteins) are presented in Table19.
In untreated control, the per cent of B pattern sperm increased

significantly from immediate post thaw level to 60 min of incubation in both the bull
groups. After 60 min of incubation the rate of increase in B pattern cells was not
statistically significant. At immediate post thaw though it was not significant, bulls
in group I had low number B pattern sperm cells than group II (44.12 ± 1.86 vs
46.30 ± 1.88). But bulls in group I had significantly low (P < 0.05) number of sperm
cells with B pattern at 60 min (50.86 ± 1.82 vs 55.73 ± 1.76), at 120 min (53.10 ±
1.76 vs 56.97 ± 1.69) and at 180 min post thaw incubation (54.79 ± 1.70 vs 58.43 ±
1.58) than the group II.
When semen samples were treated with heparin, per cent of B pattern
cells increased significantly during different periods of incubation in both the group
I and IIbulls. Bulls in group I had significantly (P < 0.05) more number of sperm
cells with B pattern at 60 min (62.37 ± 1.92 vs 54.68 ± 1.65), at 120 min (69.79 ±
1.97 vs 62.30 ± 2.66) than the group II. At 180 min post thaw incubation the
difference was highly significant (74.48 ± 1.98 vs 66.28 ± 2.16).
Similarly, when the semen samples were treated with heparin along with
fertility associated protein, per cent of B pattern cells increased significantly during
different points of incubation in both the group I and II. Per cent of sperm cells with
B pattern in group I and II bulls did not differ significantly at different periods of
incubation (63.24 ± 1.55 vs 59.61 ± 1.87 at 60 min; 69.78 ± 1.41 vs 66.57 ± 1.71 at
120 min; 74.29 ± 1.33 vs 71.21 ± 1.23 at 180 min) when semen samples treated with
heparin and fertility associated proteins.
In group Ibulls, the per cent of sperm cells with B pattern in heparin and
heparin with fertility associated proteins treated groups were significantly (P < 0.01)
higher than the control group at 60 min (62.37 ± 1.92, 63.24 ± 1.55 and 50.86 ±
1.82), at 120 min (69.79 ± 1.97, 69.78 ± 1.41 and 53.10 ± 1.76) and at 180 min post
thaw incubation (74.48 ± 1.98, 74.29 ± 1.33 and 54.79 ± 2.08). But B pattern cells in
heparin treated group and heparin with fertility associated proteins treated group did
not differ significantly at any point of incubation.
In group IIbulls, the per cent of sperm cells with B pattern in heparin
with fertility associated proteins treated group (59.61 ± 1.87) was significantly (P <
0.01) higher than the control (55.73 ± 1.76) and heparin treated group (54.68 ± 1.65)
at 60 min post thaw incubation. The per cent of sperm cells with B pattern in
control, heparin treated and heparin with fertility associated proteins treated groups
differed significantly (P < 0.01) with each other at 120 min (56.97 ± 1.69 vs 62.30 ±
1.66 vs 66.57 ± 1.71) and at 180 min post thaw (58.43 ± 3.09 vs 66.28 ± 2.16 vs
71.21 vs 1.23).
4.3.4. Correlation between B pattern sperm cellsand other in vitro sperm

B pattern capacitated- acrosome intact sperm cells had highly significant

(P <0.01) positive correlation with sperm cells with lost mitochondrial membrane
potency (0.457), static sperm cells (0.358), AR pattern capacitated acrosome reacted
sperm cells (0.457) and necrotic cells (0.352). B pattern capacitated- acrosome intact
sperm cells were having significant (P <0.05) positive correlation with MDA
(0.251).
B pattern capacitated- acrosome intact sperm cells had highly significant

(P <0.01) negative correlation with sperm cell viability (- 0.315), plasma membrane
integrity (- 0.764), functional membrane integrity (- 0.743), sperm cells with low
mitochondrial membrane potency (- 0.488), progressive forward motility (- 0.503),
curvilinear velocity of sperm cells (- 0.362), straight line velocity of sperm cells (-
0.470), average path velocity of sperm cells (- 0.441), amplitude of lateral head
displacement (- 0.299), F pattern uncapacitated- acrosome intact sperm cells (-
0.962).and DNA integrity (- 0.346).
B pattern capacitated- acrosome intact sperm cells had significant (P

<0.05) negative correlation with sperm cells with high mitochondrial membrane
potency (- 0.226) and non significant negative correlation with non progressive
forward motility (- 0.025), linearity (-0.163), straightness (- 0.185), wobble (-
0.071), beat cross frequency (- 0.175) and apoptotic sperm cells (- 0.066).
4.3.5. AR pattern cells
Capacitation status of sperm cells – AR pattern (capacitated acrosome

reacted) cells assessed by chlortetracycline staining in frozen thawed semen samples
of group I and II bulls with different treatments (treated with heparin and heparin
with fertility associated proteins) are presented in Table 20.
The per cent of AR pattern sperm cells increased significantly during

different points of incubation in both the groups. In untreated control, though it was
not significant group I bulls had low number of AR pattern sperm cells at immediate
post thaw (4.15 ± 0.57 vs 5.50 ± 0.72), at 60 min (6.09 ± 0.78 vs 7.14± 0.82) and at
120 min (6.51 ± 0.87 vs 8.55 ± 0.90) than group II. But at 180 min post thaw
incubation group I bulls had significantly (P < 0.05) low number of AR pattern cells
(7.76 ± 0.71 vs 10.14 ± 0.73) than group II.
When semen samples were treated with heparin, the per cent of sperm
cells with AR pattern did not differ significantly between bull groups throughout the
incubation period (6.60 ± 0.81 vs 7.89 ± 0.71 at 60 min; 8.03 ± 1.08 vs 9.90 ± 0.73
at 120 min; 9.76 ± 0.80 vs 12.32 ± 0.94 at 180 min). Similarly, the per cent of sperm
cells with AR pattern in group I and II bulls did not differ significantly at different
periods of incubation 6.60 ± 0.72 vs 7.07 ± 0.59 at 60 min; 8.54 ± 0.77 vs 9.37 ±
0.45 at 120 min; 10.08 ± 0.60 vs 11.15 ± 0.63 at 180 min when semen samples were
treated with heparin and fertility associated proteins.
In group Ibulls, the per cent of sperm cells with AR pattern in heparin with
fertility associated proteins treated group was significantly higher (P < 0.05) than the
control group at 120 min incubation (8.54 ± 0.77 vs 6.51± 0.87) and significantly
higher (P < 0.01) at 180 min post thaw incubation (10.08 ± 0.60 vs 7.76± 0.71). AR
pattern cells in heparin treated group and heparin with fertility associated proteins
treated group did not differ significantly at any point of incubation.
In group IIbulls, the per cent of sperm cells with AR pattern in heparin
treated group was significantly higher (P < 0.01) than the control group at 180 min
incubation (12.32 ± 0.94 vs 10.14 ± 0.73). AR pattern cells in heparin treated group
and heparin with fertility associated proteins treated group did not differ
significantly at any point of incubation.
4.3.6. Correlation between AR pattern sperm cells and other in vitro sperm
AR pattern capacitated acrosome reacted sperm cells had highly

significant (P <0.01) negative correlation with sperm cell viability (- 0.440), plasma
membrane integrity (- 0.638), functional membrane integrity (- 0.643), sperm cells
with high mitochondrial membrane potency (- 0.374), sperm cells with low
mitochondrial membrane potency (- 0.581), progressive forward motility (- 0.603),
non progressive forward motility (- 0.293), curvilinear velocity of sperm cells (-
0.420), straight line velocity of sperm cells (- 0.466), average path velocity of sperm
cells (- 0.489), amplitude of lateral head displacement (- 0.441), F pattern
uncapacitated- acrosome intact sperm cells (- 0.674) and DNA integrity (- 0.300).
AR pattern capacitated acrosome reacted sperm cells were having non significant
negative correlation with straightness (- 0.018).
AR pattern capacitated acrosome reacted sperm cells had highly

significant (P <0.01) positive correlation with sperm cells with lost mitochondrial
membrane potency (0.602), static sperm cells (0.563), B pattern capacitated-
acrosome intact sperm cells (0.457) and necrotic cells (0.455).AR pattern
capacitated acrosome reacted sperm cells had non significant positive correlation
with MDA (0.177), linearity (0.040), wobble (0.133), beat cross frequency (0.023)
and apoptotic sperm cells (0.195).
4.4. RANKING OF BULL
In present study, when semen samples from the 22 bulls were screened
for the presence of fertility associated proteins, 11 bulls were positive for 28 kDa
heparin binding protein (FAA) in the sperm membrane. Based on the presence of 28
kDa heparin binding protein in sperm membrane bulls were categorized into two
bull groups. Bulls in group I were positive for the protein fraction and bulls in group
II were negative for the protein fraction.The bulls under group I were 5 Jersey bulls
(bull No. 621, 627, 662, 670, 4049); 4 Jersey crossbred bulls (bull No. 4072, 5011,
JS 149, 2208) and 2 Holstein Friesian bulls (bull No. HF 29, HF 30) and the bulls
under the group II were 5 Jersey bulls (612, 624, 626, 657, 658) and 6 Jersey
crossbred bulls (JL 31, 4021, 5038, 5049, 5052, 5055).
To rank the bulls included in the study, the in vitro sperm characters in
untreated control group during incubation (immediate post thaw to 180 min) were
analyzed by Duncan Multiple Range Test (DMRT). Following the DMRT test bulls
were categorized under different subsets for each in vitro character and suitable rank
was given to the bulls that were categorized under same subsets. In group I, a total
of 5 bulls such as NJ 670, NJ 627, 4072, 4049, JS 149 were categorized under rank 1
and another 5 bulls such as NJ 621, 5011, HF 29, HF 30, 2208 were categorized
under rank 2 and one bull NJ 662 was categorized under rank 3. The respective
values of the in vitro characters in the bulls ranked as 1, 2 and 3 (Table 21) for
progressive forward motility were 58.02±2.65 vs 35.24±1.30 vs 27.80, non
progressive motility were 30.20±2.30 vs 27.49±2.83 vs 24.15, per cent of static cells
were 12.00±4.25 vs 37.28±4.09 vs 48.08, per cent of sperm cells with intact plasma
membrane were 42.65±3.10 vs 31.64±2.33 vs 27.79, per cent of sperm cells with
functional membrane integrity were 39.35 ± 3.14 vs 29.18 ± 2.03 vs 23,84, per cent
of sperm cells with F pattern were 48.13±1.95 vs 39.58±2.72 vs 36.60, per cent of
sperm cells with B pattern were 46.81±1.96 vs 53.93±2.71 vs 54.20, per cent of
sperm cells with AR pattern were 5.07±0.35 vs 6.56±1.24 vs 9.20, concentration of
malondioldehyde µ mol/ml were 3.60 ± 0.33 vs 3.13 ± 0.29 vs 2.71, per cent of
sperm cells with mitochondrial membrane potency were 66.04±2.43 vs 46.17±0.97
vs 41.35, per cent of sperm cells with lost mitochondrial membrane potency were
33.96±2.45 vs 53.91±1.59 vs 58.43, curvilinear velocity of sperm cells were
Table 21: In vitro sperm characters after incubation for 180 min in bull group I
In vitro characters Rank 1 (n= 5) Rank 2 (n=5) Rank 3 (n=1)
Progressive forward
58.02±2.65 35.24±1.30 27.80
motility (%)
Non progressive
30.20±2.30 27.49±2.83 24.15
forward motility (%)
Static cells (%) 12.00±4.25 37.28±4.09 48.08
Plasma membrane
42.65±3.10 31.64±2.33 27.79
Integrity (%)
Functional membrane
39.35±3.14 29.18±2.03 23.84
integrity (%)
F pattern cells (%) 48.13±1.95 39.58±2.72 36.60
B pattern cells (%) 46.81±1.96 53.93±2.71 54.20
AR pattern (%) 5.07±0.35 6.56±1.24 9.20
MDA (µ mol/ml) 3.60±0.33 3.13±0.29 2.71
Mitochondrial membrane
66.04±2.43 46.17±0.97 41.35
Potential (%)
Lost mitochondrial
33.96±2.45 53.91±1.59 58.43
membrane potential (%)
Curvilinear velocity (µ m/s) 74.61±3.02 53.45±2.94 48.45
Amplitude of lateral head
3.51±0.12 2.86±0.10 2.98
displacement (µm)
Linearity (%) 37.54±1.23 40.28±2.28 35.60
Straightness (%) 28.38±1.47 21.41±1.072 17.28
DNA integrity (%) 95.41±1.03 92.98±0.47 94.91
Table 22: In vitro sperm characters after incubation for 180 min in bull group II
In vitro characters Rank 1(n=4) Rank 2 (n=4) Rank 3 (n=3)

Progressive forward
50.98±2.05 45.24±1.59 34.80±0.34
motility (%)
Non progressive
31.76±3.49 29.11±3.47 24.85±1.16
forward motility (%)
Static cells (%) 17.26±2.11 25.41±2.65 38.42±2.40
Plasma membrane
43.33±3.21 27.81±4.20 26.77±1.02
Integrity (%)
Functional membrane
39.45±3.35 24.83±3.83 23.43±1.35
integrity (%)
F pattern cells (%) 48.84±2.05 32.11±5.05 30.69±1.21
B pattern cells (%) 45.63±1.95 59.78±5.32 58.76±1.27
AR pattern (%) 5.52±0.38 8.11±0.37 10.55±1.09
MDA (µ mol/ml) 3.09±0.48 2.64±0.27 2.28±0.29
Mitochondrial membrane
61.07±2.03 56.66±2.00 46.93±0.94
Potential (%)
Lost mitochondrial
38.98±5.15 43.36±5.00 52.97±0.33
membrane potential (%)
Curvilinear velocity (µ m/s) 72.59±3.10 60.21±6.49 52.61±0.75
Amplitude of lateral head
3.48±0.14 3.06±0.18 2.70±0.08
displacement (µm)
Linearity (%) 36.49±2.43 39.27±2.20 42.63±1.29
Straightness (%) 26.71±2.43 23.26±2.35 22.48±0.51
DNA integrity (%) 93.91±1.32 92.24±0.82 92.26±0.46

74.61±3.02 vs 53.45±2.94 vs 48.45 and amplitude of lateral head displacement of
sperm cells were 3.51±0.12 vs 2.86±0.10 vs 2.98, linearity of sperm cell movement
were 37.54±1.23 vs 40.28±2.28 vs 35.60, straightness of sperm cell motility were
28.38±1.47 vs 21.41±1.072 vs 17.28 and DNA integrity were 95.413±1.03 vs
92.98±0.47 vs 94.91.
In group II, a total 4 bulls such as NJ 658, JL 31, 5052, NJ 612 were
categorized under rank 1 and another 4 bulls such as NJ 626, NJ 657, NJ 624, 4021
were categorized under rank 2 and 3 bulls such as 5038, 5049, 5055 were
categorized under rank 3. The respective values of the in vitro characters in the bulls
ranked as 1, 2 and 3 (Table 22) for progressive forward motility were 50.98±2.05 vs
45.24±1.59 vs 34.80±0.34, per cent of non progressive motile cells were 31.76±3.49
vs 29.11±3.47 vs 24.85±1.16, per cent of static cells were 17.26±2.11 vs 25.41±2.65
vs 38.42±2.40, per cent of sperm cells with intact plasma membrane were
43.33±3.21 vs 27.81±4.20 vs 26.77±1.02, per cent of sperm cells with functional
membrane integrity were 39.45±3.35 vs 24.83±3.83 vs 23.43±1.35, per cent of
sperm cells with F pattern were 48.84±2.05 vs 32.11±5.05 vs 30.69±1.21, per cent
of sperm cells with B pattern were 45.63±1.95 vs 59.78±5.32 vs 58.76±1.27, per
cent of sperm cells with AR pattern were 5.52±0.38 vs 8.11±0.37 vs 10.55±1.09,
concentration of malondioldehyde µ mol/ml were 3.09±0.48 vs 2.64±0.27 vs
2.28±0.29, per cent of sperm cells with mitochondrial membrane potency were
61.07±2.03 vs 56.66±2.00 vs 46.93±0.94, per cent of sperm cells with lost
mitochondrial membrane potency were 38.98±5.15 vs 43.36±5.00 vs 52.97±0.33,
curvilinear velocity of sperm cells were 72.59±3.10 vs 60.21±6.49 vs 52.61±0.75,
amplitude of lateral head displacement of sperm cells were 3.48±0.14 vs 3.06±0.18
vs 2.70±0.08, linearity of sperm cell movement were 36.49±2.43 vs 39.27±2.20 vs
42.63±1.29, straightness of sperm cell motility were 26.71±2.43 vs 23.26±2.35 vs
22.48±0.51 and DNA integrity were 93.91±1.32 vs 92.24±0.82 vs 92.26±0.46.
CHAPTER V
DISCUSSION
There were many proteins fractions seen in seminal plasma as well as in

sperm membrane. Some of the protein fractions found in seminal plasma were not
detectable in sperm membrane and vice versa. Attempts have been made to analyze
the roles of different protein fractions on the fertility of semen samples (Yue et al.,
2009). Proteins such as 55 kDa molecular weight osteopontin(Moura et al., 2006),
26 kDa lipocallin type prostaglandin D synthase (Miller et al., 1990), 15 – 17 kDa
BSP A1/ A2, BSP A3, 28 - 30 kDa molecular weight BSP 30, 15/14 kDa and 28 – 30
kDa heparin binding proteins were found highly correlated with fertility of the bulls
(Miller et al., 1990; Bellin et al, 1998; Sprott et al., 2000).
5.1 CHARACTERIZATION OF SEMINAL PLASMA AND SPERM

MEMBRANE PROTEINS
Studies have been carried out to characterize the proteins present in the
seminal plasma and sperm membrane by chromatography, SDS-PAGE (Arangasamy
et al., 2005), polymerase chain reactions, Western blotting (Bellin et al., 1998) and
amino acid sequencing (Yue et al., 2009). In the present study, the SDS- PAGE of
seminal plasma protein revealed a total of 15 protein bands with the molecular
weight ranging from 3 to 205 kDa. Protein bands of varying intensities were
observed in the gel with dense bands at 15/14 and 28 kDa. Proteins related with bull
fertility such as 15/14 kDa and 28 kDawere present in all the 22 bulls (100 %), while
26 kDa protein was present in 18 bulls (81.82 %) and 55 kDa in 14 bulls (63.63 %).
Seshagiri and Pattabiraman (1991) observed 8 protein fractions in the

seminal plasma of Jersey and Sindhi bulls, and 7 protein fractions in crossbred bulls.
Kulkarni et al. (1996) observed protein fractions with molecular weight ranging
from 11 to 92 kDa in cattle seminal plasma. Arangasamy et al. (2005) and Harshan
et al. (2006) reported a total of 18 and 19 protein bands respectively, in buffalo
seminal plasma with molecular weight ranging from 3 to 205 kDa. Rafiq et al.
(2009) reported a total of 18 protein bands with molecular weight ranging from 11 to
201 kDa in Tharparkar bull seminal plasma. In the present study, dense bands were
observed at 28 kDa and 15/14 kDa, while Arangasamy et al. (2005) and Harshan et
al. (2006) reported dense proteins at lesser than 25 kDa in buffalo seminal plasma.
The more number of bands observed in the present study than those
reported by Seshagiri and Pattabiraman (1991) might be due to the 12 per cent
resolving and 5 per cent stacking gel buffer used in the SDS-PAGE, which was
responsible for better running of protein fractions of less than 250 kDa molecular
weight. The reason for the difference in the number of protein bands in the present
study when compared with the reports of Arangasamy et al. (2005), Harshan et al.
(2006) and Rafiq et al. (2009) might be the species/breed differences and method of
protein isolation. In the present study, the seminal plasma proteins were precipitated
by ice cold ethanol method while Arangasamy et al. (2005) and Harshan et al.
(2006) used the seminal plasma directly for SDS- PAGE.
Moura et al. (2006) reported that 15/14 kDa proteins (BSP A1/ A2, BSP
A3) and 28 kDa proteins (BSP 30) of the seminal plasma were the secretory products
of seminal vesicle and ampulla. All these BSP proteins bound to sperm at
ejaculation via their interaction with phospholipids of sperm membrane (Desnoyers
and Manjunath,1992) and appeared to be immunologically ubiquitous in mammalian
species (Calvette et al., 1994). Gwathmey et al. (2006) suggested that BSP proteins
mediated sperm binding to the oviductal epithelium, helping to preserve sperm
viability and motility while in the oviductal reservoir.
Rodriguez et al.(2000) established that the 55 kDa protein OPN present

in bull seminal plasma originated from the ampulla and seminal vesicles, with the
majority of the protein being synthesized by the epithelial cells of the
ampulla.Killian et al. (1993) and Cancel et al.(1997) reported a high correlation (r =
0.89) between presence ofOPN proteins and actual fertility data indicating that OPN
was more prevalent in the seminal plasma of bulls with higher fertility.
5.1.2 Electrophoretic profile of sperm membrane proteins

SDS- PAGE of the sperm membrane protein revealed a total of 14
protein bands with molecular weight ranging from 3 to 205 kDa. The protein band at
25 kDa which was present in seminal plasma protein was absent in sperm membrane
protein. Wolfe et al. (1993) used whole sperm for electrophoretic study and they
reported 15- 30 bands in beef bull sperm protein. In the present study, proteins
present in the sperm membrane were extracted by using Triton X 100 detergent
which was probably responsible for less number of protein bands observed. Proteins
related with bull fertility such as 15/14 kDa (BSP A1/ A2, BSP A3) was observed in
all the 22 bulls (100 %), 28 kDa protein (BSP 30) in 21 bulls (95.45 %), 26 kDa
protein (lipocallin type prostaglandin D synthase) in 14 bulls (63.64 %) and 55 kDa
protein (osteopontin) in 11 bulls (50.00 %). Nauc and Manjunath (2000) reported 3
protein fractions of BSP- A1/ A2, BSP A3 and BSP 30 in the sperm membrane by
SDS- PAGE and radio immuno-assay.They also reported a reduction in the
concentration of these membrane proteins during cryopreservation.
Souza et al. (2006) reported that BSP proteins were observed in the
acrosome and mid piece of the spermatozoa. It was suggested that localization of
BSP proteins in the midpiece closeto the mitochondria was indicative of the fact that
BSP proteins controlled sperm motility. BSP A1/A2was shown to stimulate
membrane-bound calcium ATPase activity in bull sperm (Sanchez-Luengo et al.,
2004). Thefunctional connection between binding of BSPproteins to the midpiece
and stimulation of mitochondrialactivity and sperm motility suggested multifaceted
role for BSPproteins as sperms were prepared for fertilization. Moura (2005) and
Moura et al. (2006) suggested that the 55 kDa osteopontin mediated sperm–oocyte
interaction and fertilization. Souza et al. (2008) reported that osteopontin was
localized on the post-equatorial segment and acrosomal cap ofejaculated sperm.
OPN had known activities inanti-apoptosis and cell survival (Wai and Kuo, 2004;
Rangaswami et al.,2006; Chakraborty et al., 2006; Khodavirdi et al., 2006; Lee et
al., 2007).
5.1.3. Heparin binding proteins of seminal plasma

A total of 7 heparin binding proteins with the molecular weight range of
15 kDa to 205 kDawere observed in the present study. Fertility related heparin
binding proteinwith molecular weight 15/14 kDa was present in all the 22 bulls
(100.00 %) and 28 kDa protein was present in 18 out of 22 bulls (81.82 %). Miller et
al. (1990) reported 3 heparin binding proteins in the seminal plasma. Arangasamy et
al. (2005) found 8 bands of heparin binding proteins in buffalo seminal plasma.
Similarly, Rafiq et al. (2009) reported 8 bands in Tharparkar bull semen. The
variations in the number of bands might be due to aggregation of products of low
molecular weight proteins or degradation product of high molecular weight or due to
species/ breed variations (Arangasamy et al., 2005).
Heparin binding proteins were secreted from the prostate, seminal

vesicles and bulbourethral glands into seminal fluid and bind to sperm at ejaculation
(Miller et al., 1990; Nass et al., 1990). Bulls with increased fertility produced sperm
with greater affinity to bind heparin like complex sugars that were commonly found
in the reproductive tract of females (Marks and Ax, 1985). Heparin binding proteins
promote capacitation of sperm cells by increasing the number of heparin binding
sites on the sperm surface (Therien et al., 1995) and stimulating cholesterol release
from the membrane (Therein et al., 1998). In female reproductive tract, heparin
binding proteins bound sperm interacted with oviductal components like high
density lipoproteins which stimulated a second cholesterol efflux resulting in
capacitation (Therien et al., 1998). Thus, a positive effect of heparin binding
proteins on fertility could be linked to its ability to mediate these events which are
crucial for successful fertilization.
5.1.4. Heparin binding proteins of sperm membrane
A total of 10 heparin binding proteins with the molecular weight range of

15 kDa to 205 kDawere observed in the present study. Fertility related heparin
binding protein with molecular weight 15/14 kDa was present in all the 22 bulls
(100.00 %) and 28 kDa protein was present in 11 out of 22 bulls (50.00 %).
Bulls which were positive for 28 kDa heparin binding proteins in sperm
membrane were also positive for the other fertility associated proteins in seminal
plasma and sperm membrane such as 55 kDa, 28 kDa, 26 kDa, 15/14 kDa proteins
as well as 15/14 kDa, 28 kDa heparin binding proteins in seminal plasma and 15/14
kDa heparin binding proteins in sperm membrane.
The 28 – 30 kDa heparin binding protein in sperm membrane has been

designated as Fertility Associated Antigen (FAA) and it is a heritable trait (Ax,
2004). Bull semen with the presence of FAA in sperm membranes had increased
fertility by 9 to 40 per centunder natural service and artificial breeding in beef cattle
(Bellin et al., 1996; Sprott et al., 2000).
In the present study, 11 out of 22 bulls (50.00 %) were positive for 28

kDa heparin binding protein in sperm membrane. Bellin et al. (1998) reported that
the percentage of bulls that were FAA negative among 44 herds ranged from zero to
50 % (average, 12 %; n = 2,191 bulls). Given this wide range, the chance of having
FAA negative bulls may be relatively high in some herds. Thus screening semen
samples for FAA after a breeding soundness examination for all bulls is prudent
whether natural mating or AI is to be employed. FAA negative bulls should be
eliminated regardless of whether they are to be used in natural or artificial breeding
programme (Sprott et al., 2000). Hence, based on the presence of 28 kDa heparin
binding protein in sperm membrane, bulls in the present study were categorized into
two groups. Group I bulls were positive for the protein fraction and Group II bulls
were negative for the protein fraction.
5.2. EVALUATION OF IN VITRO SPERM CHARACTERS
5.2.1. Sperm morphology
The role of morphologically normal sperm in fertility has been widely

accepted. Normal sperm morphology may be an indicator of the fertility potential of
a given male (Padrick and Jaakma, 2002; Esteso et al., 2006). The association of
increased morphological abnormalities of spermatozoa with reduced reproductive
efficiency has been reported in bulls (Walters et al., 2005). In bulls, higher
proportion of abnormal spermatozoa could be of genetically heritable character
(Hafez, 1987). Morphometric analysis of sperm heads has been shown to be an
indicator of in vitro fertility (Kruger et al., 1993).
In this study more than 80 per cent of spermatozoa were morphologically

normal in both the group I and II bulls. These findings were obviously due to the
fact that the bulls involved in the experiment were selected and reared as artificial
insemination sires and were in use for frozen semen production. In fertile bulls the
incidence of abnormal morphology of spermatozoa was found to be 10 to 18 per
cent (Rao et al., 1980). The 17.70 ± 2.50 per cent sperm cell abnormalities in group I
bulls and 15.90 ± 1.25 per cent in group IIbulls in the present studywere in
accordance with the findings of Hallap et al. (2004) but lower than the report of
Sundararaman et al. (2007). The incidence of tail abnormality was most frequent in
this experiment and did show similarity in all the bulls.The proportion of sperm
abnormalities in this study was within the threshold value of 20 per cent.
5.2.2 Lipid peroxidation
Reactive oxygen species (ROS) generated by spermatozoa play an

important role in normal physiological processes such as, sperm capacitation,
acrosome reaction and maintenance of fertilizing ability (Desai et al., 2010). When
the ROS were generated in excess, they caused oxidative stress to sperm cells
(Bansal and Bilaspuri, 2007). Mature spermatozoa have little capacity for repairing
oxidative damage because their cytoplasm contains low concentrations of
scavenging enzymes (Alvarez and Storey, 1989). Seminal plasma is endowed with
enzymatic and non-enzymatic antioxidant capacity (Jimenez et al., 1990) and should
be capable of scavenging ROS and protect spermatozoa against oxidative stress.
Semen dilution with extenders reduces the potential protective capacity

of plasma (Maxwell and Stojanov, 1996). During cryopreservation, sperm cells are
exposed to cold shock and atmospheric oxygen, which in turn increases the
production of ROS and decreases antioxidant level (Bucak et al., 2010). The
freezing and thawing procedures also causes severe damages and/or death to certain
per cent of sperm cells, which generate ROS in excess via an aromatic amino acid
oxidase catalyzed reaction (Sariozkhan et al., 2009).
The mechanism of ROS-induced damage to spermatozoa includes an
oxidative attack on the sperm membrane lipids leading to initiation of lipid
peroxidation (LPO) cascade (Sharma and Agarwal, 1996). The susceptibility of
ruminant spermatozoa to oxidative stress is a consequence of the abundance of poly
unsaturated fatty acids (PUFAs) in sperm plasma membrane and the presence of
double bonds in these molecules makes them vulnerable to free radicals attack and
the initiation of LPO cascade. This results in a subsequent loss in membrane and
morphological integrity, impaired cell functions, along with impaired sperm motility
and induction of sperm apoptosis (Bucak et al., 2010). A simple tool to evaluate the
level of lipid peroxidation in the spermatozoan is the assay of sperm
malondialdehyde (MDA), a thiobarbituric- acid- reactive substance (TBARS) that is
a stable lipid peroxidation product (Kasimanickam et al., 2006).
In the present study, in untreated control the MDA level increased

significantly (P < 0.01) during different periods of incubation from the immediate
post thaw level in bothGroup I and II. Elevation of MDA level during different
periods of incubation suggests that the resumption of metabolic activity of the sperm
cells after thawing leads to generation of excessive ROS (Agarwal et al., 2005).
Activation of an aromatic amino acid oxidase enzyme from dead sperm cells also
might be an additional source of ROS in frozen thawed semen samples (Upretti et
al., 1998; Slaweta et al., 1988). The MDA values obtained for cattle bull sperms in
the present study were in accordance with the reported values of Beorlegui et al.
(1997) who reported values ranging between 0.34 ± 0.18 and 4.95 ± 0.31µM/ ml in
frozen thawed bovine semen sample and lesser than the reported values of Selvaraju
et al. (2008), who observed 8.00 ± 0.31 µM/ml at immediate post thaw and 9.36 ±
0.36 µM/ml at 120 min post thaw incubation in buffalo frozen semen. The elevated
level of MDA concentration in buffalo sperm in comparison with bull sperm might
be due to the fact that the sperm membrane of buffalo is rich in poly unsaturated
fatty acids (Nair et al., 2006).
When the semen samples were treated with 0.1 mM H2O2, the MDA
levels increased significantly (P < 0.01) from immediate post thaw in both the
groups during incubation. The MDA level in H2O2treated group was significantly
higher than their controls at 60 min of incubation. Garg et al. (2009) reported a two
fold increase in MDA level when the buffalo semen samples were treated with 50
µM H2O2 for 1 hour. They further observed that H2O2 induced lipid peroxidation of
sperm lipids in a dose and time dependent manner and cryopreserved sperm samples
were more susceptible to lipid peroxidation than fresh semen. Alvarez et al. (1987)
indicated that H2O2 was the main reactive oxygen species responsible for the
oxidative damage to sperm cells. H2O2 reacts with transition metals (Fe, Cu) to form
more reactive radicals like hydroxyl radicals that were believed to damage proteins
and lipids (Halliwell and Gutterige, 1984) and start lipid peroxidation.
Among the group I and II bulls, group I bulls had low level MDA than
the group II at different points of incubation in control as well as when treated with
H2O2. This clearly indicates that the bulls positive for fertility associated protein had
significantly better protection against oxidative stress and were able to control the
generation of lipid peroxides during incubation as well as when challenged with
ROS like H2O2.
To assess whether supplementation of fertility associated protein would

control the elevation of MDA level, 25 µg of heparin binding protein was added to
the frozen thawed semen samples and MDA level were analyzed at 60 min, 120 min
and 180 min post thaw incubation. Though the MDA level showed an increase
during incubation, it was significantly (P < 0.01) lower than the untreated control
samples at different points of incubation in both groups I and II. But the MDA level
in group I and II did not show statistically significant difference during different
points of incubation. The results indicated that fertility associated proteins helped in
combating oxidative stress of sperm cells in bulls of both groups. The response of
group I bulls to exogenous addition of protein in controlling MDA level may be
because some of the native proteins of sperm might have reduced during
cryopreservation as reported by Nauc and Manjunath (2000) or the fertility
associated proteins might counteract the lipid peroxidation on dose dependent
manner as reported by Schoneck et al. (1996). Marti et al. (2008) reported that
seminal proteins added alone or withother compounds showed a protective effect
and accounted foran increase in the sperm enzyme activitylevels not only in the
fresh sample but also after coolingand freezing/thawing.
5.2.2.1. Correlation between MDA levels and other in vitro sperm characters
In the present study, MDA had negative correlation with sperm cell
viability, plasma membrane integrity, functional membrane integrity, sperm cells
with high mitochondrial membrane potency, F pattern (uncapacitated- acrosome
intact) sperm cells, progressive forward motility, straight line velocity of sperm
cells, curvilinear velocity, wobble, amplitude of lateral head displacement and beat
cross frequency. MDA had positive correlation with static sperm cells, sperm cells
with lost mitochondrial membrane potency and necrotic sperm cells. The
observations in the present study were in accordance with Nair et al. (2006) and
Kasimanickam et al. (2007), who reported negative correlation of sperm lipid
peroxidation with plasma membrane integrity (- 0.93), progressive motility (- 0.90)
and positive correlation with DNA fragmentation index. Kasimanickam et al. (2007)
inferred that the deleterious effect of sperm lipid peroxidation imposed on the
plasma membrane integrity and sperm DNA resulted in loss of bull fertilization
potential. Selvaraju et al. (2009) and Garg et al. (2009) also reported a negative
correlation with progressive forward motility and functional membrane integrity and
viability.
In accordance with the present study, Garg et al. (2009) reported negative
correlation between MDA level and acrosome integrity and Baumber et al. (2000)
reported significant decline in per cent of total motility, curvilinear velocity, straight
line velocity, amplitude of lateral head displacement with rise in LPO. They
proposed that sperm immobilization was due to a decreased phosphorylation of
axonemal proteins required for sperm movement, inhibition of oxidative
phosphorylation, glycolysis, or both, thus limiting ATP generation by sperm cell.
Alternatively, enzyme inhibition could be induced indirectly by products of lipid
peroxidation, especially malondialdehyde and 4- hydroxynonenol. Low
concentrations of these substances have been shown to inhibit a large number of
cellular enzymes and functions, anaerobic glycolysis, DNA, RNA, and protein
synthesis (Comporti, 1989). In the present study, MDA had negative correlation
with mitochondrial membrane potency of sperm cells (- 0.689) which is in the
accordance with Martnez –Poastor et al. (2009). ROS may adversely affect sperm
motility via alterations in mitochondrial function (Cummings et al., 1994).
Poly unsaturated fatty acids (PUFA) in the sperm membrane is required

to give the plasma membrane the fluidity it needs for sperm motility and
participation in the membrane fusion events associated with fertilization, as well as
the structural integrity required for viability. The abundance of PUFA in sperm
plasma membrane and the presence of double bonds in these molecules make them
vulnerable to free radicals attack and the initiation of lipid peroxidation cascade.
This results in loss of membrane and morphological integrity (Aitken et al., 1989).
The sperm plasma membrane is the primary site where lesions occur during
freezing-thawing of semen (Hammerstedt et al., 1990). Hence, the assessment of
plasma membrane integrity is considered to be useful for predicting the fertilizing
ability of sperm (Brito et al., 2003). Plasma membrane integrity in this study was
assessed by using fluorogenic stain carboxy fluoresin diacetate (CFDA) and
propidium iodide (PI).
In the present study, the per cent of sperm cells with intact plasma
membrane in the control group decreased significantly (P < 0.01) from the
immediate post thaw level during different periods of incubation in both group I and
II bulls. The observations are well in accordance with Selvaraju et al. (2009) who
reported 59.65 ± 1.60, 29.61 ± 2.20 and 26.66 ± 4.40 per cent of sperm cells with
intact plasma membrane at immediate post thaw, 60 min and 120 min of incubation,
respectively. Sperm plasma membranes suffered cryoinjury when exposed to
tremendous stress during freezing and thawing (Watson, 1981). As a result of
cryoinjury, lipids and proteins were released leading to morphological damages to
sperm plasma membrane and acrosome. Accumulation of metabolic end products
and subsequent development of oxidative stress may further reduce the sperm cell
viability (Aitken et al., 1989).
When semen samples were treated with H2O2 to induce oxidative stress,
the per cent of sperm cells with intact plasma membrane were decreased
significantly (P < 0.01) from immediate post thaw level in both group I and II bulls.
The significant reduction in per cent of sperm cells with intact plasma membrane
when treated with H2O2 is in accordance with the observations of Garg et al. (2009)
and they reported a dose and time dependent reduction in sperm cell viability when
sperm cells were treated with H2O2. When the lipid peroxidation cascade is
stimulated, 60 per cent of the fatty acid is lost from the membrane, resulting in loss
of membrane integrity (Jones et al., 1979). de Lamirande and Gagnon (1992)
reported that reactive oxygen species like H2O2 act on membrane lipids leading to
membrane damage and reduction in number of sperm cells with intact acrosome.
Bilodeau et al. (2001) suggested that H2O2 impaired plasma membrane by producing
sulphydryl group oxidation or by lowering the redox potential.
The semen samples when treated with fertility associated protein showed
a decrease in the per cent of sperm cells with intact plasma membrane during
incubation. However, the per cent of intact cells were significantly (P < 0.01) higher
than the untreated control samples at 120 minand at 180 min of incubation in group I
and II bulls. The observations of the present study are in accordance with the
findings of Harshan et al. (2006) when they treated epididymal sperm with fertility
associated protein PDC (BSP A1/ A2and BSP A3) at the rate of 40µg/ml. They
reported that protein treated sperm cells responded to osmotic resistance test
significantly higher than the untreated control. Fertility associated protein PDC –
109 (BSP A1/ A2, BSP A3) has been reported to stabilize the sperm membrane in a
first step (Greube et al., 2001). Barrios et al. (2000) reported that plasma membrane
damages in ram membrane were controlled by the addition of seminal proteins P14
and P 20. The adsorption of proteins by sperm cells reverted the damage as
confirmed by the results of electron microscopy and chemical analysis, possibly by
occupying sites on the plasma membrane surface. The results obtained with
increasing amounts of proteins also supported this suggestion. Barrios et al. (2000)
also suggested that the protein adsorption would repair damage caused by the cold
shock, restoring the impermeability of the plasma membrane to propidium iodide.
The beneficial effect of the inclusion of seminal proteins into the cold-shock
medium is well established, since the viability of control samples with only bovine
serum albumin as a supplement was strongly decreased as a consequence of the
cold-shock treatment.
In control as well as in fertility associated protein treatment, though it

was not statistically significant, bulls of group I had more per cent of sperm cells
with intact plasma membrane than the group II during incubation. In hydrogen
peroxide treatment, bulls of group I had significantly (P < 0.01) more per cent of
sperm cells with intact plasma membrane than the group II both at 10 min and at 30
min of incubation. From the results it was evident that the presence of fertility
associated proteins in the sperm membrane might have played a role in the
protection of membrane integrity. Desnoyers and Manjunath (1992) suggested that
the proteins adsorbed onto the sperm membrane maintain the stability of membrane
up to the process of capacitation. Barrios et al. (2005) reported that the quantity of
sperm membrane proteins P14 and P20 on the surface of ram spermatozoa could be
related to their membrane stabilizing effect. They also hypothesized that P14 takes
part in the protein structure surrounding the spermatozoa in a way similar to that of
fibronectin, stabilizing membrane phospholipids, and the cytoskeleton. Comparative
sequence analysis P 14 showed the highest homology with bovine sperm membrane
protein PDC-109.
5.2.3.1. Correlation between plasma membrane integrity and other in vitro

sperm characters
In the present study, plasma membrane integrity of the sperm cells

assessed by fluorogenic staining was having highly significant (P <0.01) positive
correlation with sperm cells with high mitochondrial membrane potency,
progressive forward motility, functional membrane integrity, F pattern
(uncapacitated- acrosome intact) sperm cells and DNA integrity. This is in
accordance with the findings of Bollwein et al. (2008), Selvaraju et al. (2008) and
Selvaraju et al. (2009).
5.2.4. Functional membrane integrity

Vital staining of sperm evaluates the structural integrity of the plasma
membrane which indicates whether the sperm cell is viable or dead. But in the viable
cells the functional integrity of plasma membrane has to be evaluated since sperm
require an active membrane during fertilization and will fail to fertilize ovum if
plasma membrane is functionally inactive. The ability of spermatozoa to swell in the
presence of hypo-osmotic medium reflects water transport across the sperm
membrane, which is a sign of membrane functional integrity (Jeyendran, et al.,
1984). Brito et al. (2003) concluded that HOST was the only plasmalemma
functional evaluation method that significantly contributed in predicting fertilization
rate.
In the present study, in the untreated control group the per cent of sperm
cells with functional membrane integrity decreased significantly (P < 0.01) during
different periods of incubation from the immediate post thaw level in both group I
and II bulls.Similar to the findings of the present study, Selvaraju et al. (2009) also
reported a significant reduction in per cent of cells with functional membrane
integrity during different periods of incubation in buffalo semen. In the present study
though the per cent of sperm cells with functional membrane integrity cells assessed
by hypo-osmotic swelling was lesser than the per cent of structurally intact cells
assessed by fluorogenic staining, it was not statistically significant. But Selvaraju et
al. (2009) reported a significant difference between the per cent of structural intact
cells and functional intact cells. This might be because of that, they used eosin-
nigrosine staining to estimate the structural integrity of the sperm cells. Woelders
(1991) reported that for light microscopic evaluation of membrane integrity, a
relatively high concentration of the dye is required, at these concentrations eosin and
many other dyes are toxic, which can lead to under estimation of the proportion of
live cells.
the per cent of sperm cells with functional membrane integrity were decreased
significantly (P < 0.01) from immediate post thaw level in both the bull group I and
II. As discussed earlier, when the lipid peroxidation cascade was stimulated the fatty
acid was lost from the membrane resulting in loss of membrane integrity (Jones et
al., 1979). Baumber et al. (2000) reported that lipid peroxides cause loss of
membrane integrity that lead to increase in membrane permeability and loss in
capacity to regulate the movement of intracellular components.
In the fertility associated protein treated group, though the per cent of
sperm cells with functional membrane integrity showed a decrease during
incubation, the per cent of functional membrane intact cells were significantly
(P < 0.01) higher than the untreated control samples at 120 min and at 180 min of
incubation in group I bullsand at 180 min of incubation in group II bulls. The
observations in the preset study are in accordance with that of Arangasamy et al.
(2005) in buffalo epididymal sperm. They reported that heparin binding proteins as
well as gelatin binding proteins at the concentration of 20 and 40µg/ml gave better
protection to functional integrity of sperm cells when compared to the untreated
controls. Harshan et al. (2006) suggested that, the influence of BSP proteins BSP
A1/ A2, BSP A3 and BSP 30 on sperm hypo-osmotic swelling response could be due
to the removal of cholesterol by these proteins.
Group I bulls had non significantly more number of sperm cells with
intact functional membrane in untreated control and significantly more number of
sperm cells when treated with H2O2and with fertility associated protein than the
group II bulls. From the results it was evident that the presence of fertility associated
proteins in the sperm membrane played a role in the protection of membrane
integrity. FN- 2 domain present in the fertility associated protein (BSP/ PDC- 109)
binds to extra cellular matrix and cytoskeleton components and stabilizes the
membrane up to the process of capacitation (Greube et al., 2001; Barrios et al.,
2005; Juan et al., 2006).
5.2.4.1. Correlation between functional membrane integrity and other

Functional membrane integrity of the sperm cells assessed by hypo

osmotic swelling test had highly significant (P <0.01) positive correlation with
plasma membrane integrity, sperm cells with high mitochondrial membrane potency,
F pattern (uncapacitated- acrosome intact) sperm cells, progressive forward motility
and DNA integrity. The observations are in accordance with the findings of
Bollwein et al. (2008), Selvaraju et al.(2008) and Selvaraju et al. (2009). El-Sisy et
al. (2010) also obtained significant positive correlation of HOST positive cells with
progressive forward motility (0.918), DNA integrity (0.378) and uncapacitated
sperm cells (0.305). Henry et al. (1993) suggested that plasma membranes and
mitochondria in sperm were similarly affected by cryopreservation. Moskovtsev et
al. (2005) reported that patients with abnormal HOST results had a higher likelihood
of DNA fragmentation and concluded that a common sub lethal insult might be
responsible for the damages to functional membrane integrity as well as DNA
integrity.
Sperm mitochondria are located in the mid piece and enrolled over the
principal part of the flagellum. Mitochondria produce ATP by oxidative
phosphorylation and provide accessible energy to the tail filaments, thus facilitating
efficient propulsion for the sperm both to reach the oocyte and to penetrate through
zona pellucida (O’ Connell et al., 2002). Mitochondria also provided the required
ATP for Na+/ K+ gradient over the plasma membrane. The Na+/ K+ ATPase regulate
the chemical and electrical gradient of the plasma membrane. Thus, the functional
integrity of mitochondria was important for the sperm survival in the female genital
tract. Mitochondrial membrane potential was assessed by using JC-1 (5, 5’, 6, 6’-
tetrachloro-1, 1’, 3, 3’-tetraethyl benzimidazole carbocyanine iodide) and carboxy
fluoresin diacetate (CFDA) in fluorescent microscope (Salvioli et al., 1997).
In the present study, in untreated control group, the per cent of sperm
cells with high and low mitochondrial membrane potentialdecreased significantly (P
< 0.01), while the sperm cells with lost mitochondrial membrane potential increased
significantly (P < 0.01) from the immediate post thaw level during incubation in
both the group I and II bulls. The results obtained in the present study were higher
than the value of 6.70 per cent of sperm cells with high mitochondrial membrane
potential reported by Selvaraju et al. (2008) in buffalo semen. Kadirvel et al. (2009)
reported 68.60 ± 2.90 per cent of sperm with high mitochondrial membrane potential
in fresh semen and it was reduced to 25.40 ± 2.10 per cent after freezing and
thawing. Martin et al. (2004) and Kadirvel et al. (2009) reported a 2.5 fold reduction
in mitochondrial membrane potential in buffalo sperm cells, after freezing and
thawing and suggested that impairment of mitochondrial function might be due to
the physical and chemical stress induced during freezing and thawing. During cell
death, the release of apoptotic factors located between the outer and inner membrane
of mitochondria was another cause for the loss of its integrity (Ravagnan et al.,
2002).
the per cent of sperm cells with high and low mitochondrial membrane
potentialdecreased significantly (P < 0.01), while the sperm cells with lost
mitochondrial membrane potential increased significantly (P < 0.01)from immediate
post thaw level in both the group I and II bulls. Armstrong et al. (1999) and
Martinez- Poster et al. (2009) also reported a similar reduction in the mitochondrial
membrane potential when sperm cells were treated with different concentrations of
H2O2. The coupling of electron transport to oxidative phosphorylation maintains the
mitochondrial membrane potential and this electron transport process could be
disrupted by ROS, resulting in a decrease in the sperm mitochondrial membrane
potential (de Lamirande and Gagnon, 1992).
In fertility associated protein treated group, though the per cent of sperm
cells with high mitochondrial membrane potential showed a decrease during
incubation, the per cent of sperm cells with high mitochondrial membrane potential
in fertility associated protein treated group were significantly (P < 0.01) higher than
the untreated control samples at 120 min and at 180 min of incubation in group
Ibulls, at 180 min of incubation in group II bulls. Though it was not statistically
significant, the fertility associated protein treated group had more number of sperm
cells with low mitochondrial membrane potential and less number of sperm cells
with lost mitochondrial potential than the control, in both the group I and II bulls.
Among the group I and II, group I bullshad significantly (P<0.01) more per cent of
sperm cells with high mitochondrial membrane potential than the group II bulls
during incubation in control as well as in fertility associated protein treatment. From
the results, it was evident that the fertility associated proteins played a role in the
protection of mitochondrial integrity. Centurion et al. (2003) reported that addition
of the fertility associated protein spermadhesin PSP-I /PSP-II heterodimer to a final
concentration of 1.5 mg/ml in the incubation medium preserved mitochondrial
activity of spermatozoa up to 5 h of incubation, while most of the sperm cells lost
their mitochondrial activity in the untreated control group during the incubation
period. Caballero et al. (2006) confirmed the protective effect role of PSP proteins
by electron microscopic study and reported that these proteins were localized on the
acrosomal and post acrosomal regions of the sperm head and were distributed to
other parts during incubation.
5.2.5.1. Correlation between mitochondrial membrane potential of sperm

cellsand other in vitro sperm characters
Sperm cell mitochondrial membrane potency had highly significant (P

<0.01) positive correlation with plasma membrane integrity, functional membrane
integrity, progressive forward motility, straight line velocity of sperm cells, average
path velocity of sperm cells and F pattern (uncapacitated- acrosome intact) sperm
cells. Selvaraju et al. (2008) also reported a similar correlation of mitochondrial
potential with progressive motility (0.64), plasma membrane integrity (0.67),
functional membrane integrity (0.74), and acrosomal integrity (0.06). They also
reported the correlation with zona binding (0.90) and cleavage rate (0.70) in their in
vitro fertilization studies. Marchetti et al. (2002) reported that high mitochondrial
membrane potential had positive correlation with fertility.
Sperm DNA integrity was vital for successful pregnancy and

transmission of genetic material to the offspring. The sperm DNA is organized in a
specific way that keeps the chromatin compact and stable in the nucleus (Fuentes-
Mascorro et al., 2000). Sperm DNA fragmentation may result from aberrant
chromatin packaging during spermatogenesis (Gorczyca et al., 1993; Manicardi et
al., 1995; Sailer et al., 1995), defective apoptosis before ejaculation (Sakkas et al.,
1999; Sakkas et al., 2002), or excessive production of reactive oxygen species in the
ejaculate (Kodama et al., 1997; Aitken and Krausz, 2001; Moustafa et al., 2004).
Sperm cells with fragmented DNA reduced the fertility of bulls (Kasimanickam et
al., 2006). The acridine orange staining is a simple microscopic technique to
evaluate the DNA integrity (Chogan et al., 2004).
In the present study the loss of DNA integrity during incubation in untreated control
was very low in the group I bulls. The per cent of sperm cells with intact DNA in the
group at immediate post thaw, 60 min, 120 min and 180 min of incubation were
94.94±0.61, 94.66±0.56, 93.80±0.66 and 93.64±0.66, respectively. The per cent of
cells with intact DNA in group II bulls reduced significantly from the immediate
post thaw level of 93.55±0.70 to 92.17±0.50 at 120 min and to 91.99±0.43 at 180
min of incubation. The per cent of intact DNA cells at immediate post thaw in the
present study was in accordance with the reports of Waterhouse et al. (2010). They
reported that the proportions of spermatozoa with fragmented DNA increased from
4.80 per cent in fresh semen to 8.90 per cent after freezing and thawing of bull
semen. The destabilizing effect of cryopreservation on sperm chromatin may stem
from the high ionic strength in frozen nuclei (Courtens et al., 1989) and excessive
intracellular influx of free calcium ions (Zhao and Buhr, 1995) leading to activation
of nucleoprotein-degrading enzymes such as acrosin (Zirkin et al., 1980),
endonucleases (Maione et al., 1997; Krzyzosiak et al., 2000) and phospholipases
with their toxic metabolites, lysolecithins (Upreti et al., 1999). Evenson et al. (1999)
suggested that loss of DNA integrity ≥ 20 per cent might announce lower fertility.
Loss of DNA integrity during incubation in the present study was lesser than the
reports of Bollwein et al. (2008) who reported the per cent of DNA fragmentation
index increased from 5.70 ± 1.50 at immediate post thaw to 10.90 ± 4.40 at 3 h of
incubation.
When semen samples were treated with H2O2 to induce oxidative stress, there was a
decrease in the per cent of sperm cells with intact DNA in both the group I and II
bulls. However, the rate decrease was not statistically significant. Several authors
reported that ROS induced many kinds of DNA damage, including DNA base
modifications, chromatin cross linkage and breakage of DNA strand (Kodama et al.,
1997; Twigg et al., 1998; Evenson and Wixon, 2006). Aitken (2006) further
suggested that peroxidative damage of membranes and destabilization of
deoxyribonucleoprotein (DNP) complex lead to the loss of sperm viability, motility
and chromatin stability.
In the fertility associated protein treated group, the DNA integrity of the sperm cells
were maintained without significant loss during 180 min of incubation in both
groups. In untreated control, group II bulls had a significant loss in DNA integrity at
120 min and 180 min of incubation, but when samples were treated with fertility
associated protein the DNA integrity of the sperm cells were sustained without
significant loss during the incubation period. Dilution of semen to low cell numbers
can reduce proteins and natural antioxidants along with other components in seminal
plasma necessary for preservation of sperm chromatin stability (Bedford et al., 1973;
Maxwell and Johnson, 1999). Exogenous addition of fertility associated proteins
protected the sperm cells against the oxidative stress, which is the cause for damage
to DNA integrity. In the present study group I bulls that were positive for fertility
associated protein had significantly more number of DNA intact cells than the group
II bulls during incubation in control, treatment with fertility associated proteins and
with H2O2.The results clearly indicated that fertility associated proteins preserved
the DNA integrity of the sperm cells.
5.2.6.1. Correlation between sperm cell DNAintegrity and other in vitro

sperm characters
In the present study, sperm cells with intact DNA had highly significant
(P <0.01) positive correlation with plasma membrane integrity, functional
membrane integrity, progressive forward motilityand F pattern (uncapacitated-
acrosome intact) sperm cells. Observations were similar to Selvaraju et al. (2008)
who reported a correlation of DNA integrity with progressive forward motility
(0.28), plasma membrane integrity (0.55), functional membrane integrity (0.57),
acrosomal integrity (0.37) and mitochondrial membrane potential (0.39).
Kasimanickam et al. (2007) reported a correlation of DNA fragmentation index with
MDA level (0.86), progressive forward motility (- 0. 67), plasma membrane
integrity (- 0.76) and competitive indices of dairy bulls (- 0.87). Kadirvel et al.
(2009) reported ROS had a correlation with DNA damage (0.32) in frozen thawed
semen samples.
5.2.7. Assessment of sperm cell apoptosis
Cell death in general can occur through two distinct ways, necrosis and
apoptosis. Necrosis is a passive process that results from injury while apoptosis is an
active reaction that follows a sequence of controlled steps leading to locally and
temporally defined self-destruction without causing an inflammatory reaction
(Lockshin, 1964; Kerr et al., 1972; Wyllie et al., 1980). Apoptosis was a complex
phenomenon that can be divided into three phases: induction, execution, and
degradation.After induction of apoptosis, mitochondrial pores are opened,
characterized by decreased mitochondrial membrane potential. Opening of
mitochondrial pores leads to the release of pro-apoptotic factors from the
mitochondria (Ravagnanet al., 2002). Phosphatidylserine, ordinarily sequestered in
the plasma membrane inner leaflet, appears in the outer leaflet, where it triggers
noninflammatory phagocytic recognition of the apoptotic cell (Bratton et al., 1997).
The externalization of phosphatidylserine was one of the hallmarks of apoptosis.
The disturbance of membrane function was detectable as an increase in the cell
ability to bind the calcium-dependent binding of annexin-V to the outwardly
translocated phosphatidylserine (Andree et al., 1990) and it could be used as a
marker for measuring apoptosis in human (Duru et al., 2001) and bovine (Anzar et
al., 2002) spermatozoa.
In the present study,the per cent of viable sperm cells decreased

significantly (P < 0.01) during incubation while the per cent of necrotic cells
increased in bulls of both the group I and II. The per cent of apoptotic cells also
showed a minor increase during incubation. The observation in the present study is
in accordance with findings of Januskauskas et al. (2003) in frozen thawed bull
semen samples. Feitosa et al. (2008) reported a significant increase in the per cent of
apoptotic cells during incubation over a period of 2 h in frozen thawed bovine semen
samples. Lachaud et al. (2004) suggested that the presence of apoptotic markers in
ejaculated spermatozoa was a consequence of processes started before ejaculation.
They further reported that ejaculated spermatozoa lack the capacity of initiating the
apoptotic path way of cell death and the death of healthy ejaculated spermatozoa
occurs only by necrosis rather than by apoptosis. In contrast, Martin et al. (2007)
observed that cryopreservation induces the occurrence of some apoptotic features in
bovine spermatozoa and they also observed that these apoptotic features appeared as
ordered events during the cryopreservation process such as decrease of the
mitochondrial membrane potential, caspase activation and changes in membrane
permeability. The decrease in mitochondrial membrane potential might be facilitated
by the fact that bovine spermatozoa contain the pro-apoptotic factor Bax,
cytochrome c and flavoprotein apoptosis inducing factor. The consequence of
increased permeability of spermatozoa membrane could lead to early cell death. In
accordance with Martin et al. (2007), a significant decrease in membrane integrity
and significant loss in mitochondrial membrane potential were observed during
incubationin the present study. Paasch et al. (2004) also confirmed the activation of
apoptotic factors such as caspase-3, 8, and 9 as well as disruption of mitochondrial
membrane potential during cryopreservation of ejaculated spermatozoa.
Januskauskas et al. (2003) reported that cryopreservation causes changes in lipid
composition and organization of membrane bilayer and expression of
phosphatidylserine on the sperm plasma membrane surface. These changes indicate
that the cells were not completely competent, since redistribution of phospholipids
affects membrane function and charge and even lead to severe membrane
dysfunction. Januskauskas et al. (2003) further suggested that annexin V positive
cells represent a transitory step between cell viability and necrosis. The incidence of
these transitory cells is dependent on incubation condition and might represent the
rate at which sperm cells undergo necrosis in vitro.
In the present study, when the semen samples were treated with H2O2,
the per cent of viable cells decreased significantly while the per cent of necrotic and
apoptotic cells increased significantly after 30 min of incubation in both the group I
and II bulls. In accordance to the present study, Martinez Pastor et al. (2009)
reported that H2O2 induced some elements of apoptotic pathways, in a reduced
subpopulation of spermatozoa, in response to oxidative stress. Kumaresan et al.
(2009) reported a positive correlation between the lipid peroxidation and appearance
of apoptotic features in boar sperm cells. It was suggested that high ROS levels in
semen might be associated with the presence of apoptotic markers (Wang et al.,
2003, Moustafa et al., 2004).
There was no significant difference observed between fertility associated

protein treated samples and untreated control samples in the per cent of apoptotic
cells during incubation period. However, the per cent of viable cells were
significantly (P < 0.01) higher in the semen samples treated with fertility associated
protein, than the untreated control samples at 120 and180 min of incubation in group
I bulls while the group II bulls had a non significantly higher per cent of viable cells
in comparison to control. The per cent of necrotic cells in group I and II bulls were
lower when the semen samples were treated with fertility associated protein when
compared to untreated control during incubation. The results clearly indicated that
treatment with fertility associated protein helped the sperm cells in maintaining their
viability and this might be through protection of their membrane integrity and
mitochondrial membrane potential. Barrios et al. (2000) reported that plasma
membrane damages in ram sperm were controlled by the addition of seminal
proteins P14 and P 20, possibly by occupying sites on the plasma membrane surface.
They also suggested that the protein adsorption would repair the damage and
restorethe plasma membrane. Centurion et al. (2003) reported that addition of the
fertility associated protein spermadhesin PSP-I /PSP-II in the incubation medium
preserved mitochondrial activity of spermatozoa up to 5 h of incubation, while most
of the sperm cells lost their mitochondrial activity in the untreated control group
during the incubation period.
Between groups I and II, group I bulls had significantly more per cent of
viable cells, low per cent of apoptotic and necrotic cells than the group II during
incubation with fertility associated protein as well as with H2O2. Fertility associated
protein, osteopontin has known activities inanti-apoptosis and cell survival through
activation of integrins and CD44 membrane receptorsand signal transduction
mechanisms, including activation of Map kinases, phosphoinositide (PI)3-
kinase/Akt-dependent NFkB, IKK/ERK-mediated pathways, which stimulates uPA-
dependentMMP-9 activation, PLC-g/PKC/PI 3-kinase pathways (Wai and Kuo,
2004; Rangaswami et al.,2006; Chakraborty et al., 2006; Khodavirdi et al., 2006;
Lee et al., 2007). Fertility associated protein PDC – 109 (BSP A1/ A2, BSP A3) was
reported to stabilize the sperm membrane (Greube et al., 2001).
5.2.7.1. Correlation between apoptotic sperm cells and other in vitro sperm
characters
In the present study, apoptotic sperm cells had negative correlation with
progressive forward motility, MDA, plasma membrane integrity, functional
membrane integrity and sperm cells with high mitochondrial membrane potency. In
accordance with the present observations, Janukauskas et al. (2003) also reported a
correlation between apoptotic cells and motile spermatozoa (- 0.06), linearity of
sperm cell movement (0.17) and per cent viable cells (- 0.10).
5.2.8. Motility and velocity parameters of sperm cells
Though motility and kinetic parameters of sperm cells could not be

considered as a reliable marker for the fertilizing ability of a given ejaculate, it was
reasonable to presume that the sperm cells with progressive forward motility had
higher chance of reaching the site of fertilization (Muinoet al., 2008). Since,
subjective estimation of motility was affected by numerous factors (Verstegenet al.,
2002; Rijsselaere et al., 2003),about 30 to 60 per cent of variations in subjective
evaluation of motility characters was reported by Amann, 1989 and Auger et al.
1993. However, the computer-assisted semen analysis (CASA), had a high degree of
accuracy, repeatability and ability to find subtle difference between bulls or
treatments (Verstegenet al., 2002). It gave extensive information about kinetic
properties of ejaculate based on measurements of the individual sperm cells and was
able to minimize the errors in the evaluation of motility characters.
In the present study, in control the per cent of sperm cells with
progressive forward motility decreased significantly (P<0.01) from more than 60 per
cent at immediate post thaw to less than 30 per cent at 180 min of incubation in both
the group I and II bulls. Similarly, the per cent of total motile cells decreased
significantly from more than 90 per cent to around 50 per cent during the incubation.
The per cent of static sperm cells increased significantly from 10 per cent to 40 per
cent during the incubation period. There was no significant difference observed in
the per cent of sperm cells with non progressive motility during the incubation
period.In accordance with the present study, Bag et al. (2004) reported a significant
reduction in motile sperm cells during post-thaw incubation of ram spermatozoa.
The decrease in motility during incubation might be due to a gradual declinein the
ability of spermatozoa to generate ATP through mitochondrial respiration as
aconsequence of mitochondrial aging (Viswanathet al., 1997) or the toxic effect of
membrane-boundaromatic amino acid oxidase (AAAO) enzyme released from dead
spermatozoa duringlong storage in ambient temperature (Shannon and Curson,
1972; 1982). Within an aerobic or even a partiallyaerobic system, the production of
reactive oxygen species(ROS) were suggested as the principal cause ofdecrease in
sperm survivability and motility (Viswanathet al., 1997).
The curvilinear velocity of the sperm cells in both group I and II bulls
decreased significantly from the initial value of more than 70 µm/s to around 50
µm/s during the 180 min of incubation periodin the untreated control semen
samples. The straight line velocity of the sperm cells were reduced significantly
from more than 30 µm/s to less than 20 during the period, while average path
velocity was reduced significantly from 40 µm/s to 30 µm/s. Linearity of the sperm
cell motility varied between 40 and 35 per cent, the straightness of the movement
varied between 70 and 65 per cent with the per cent of wobble around 60. Amplitude
of lateral head displacement of sperm cells was around 3 µm and the beat cross
frequency was 9 Hz, during the incubation period. Though Krzyzosiak et al. (2000)
and Bag et al. (2004)reported that velocity parameters did not change during
incubation, the reduction in the velocity parameters observed in the present study
was well in accordance with the observations of Gil et al. (2000) and Moce et al.
(2008). Variations in the reports on sperm velocity parameters might be due to
differences in the settings used in the CASA such as framerate and frames per field,
chamber and time of analysis, sample preparations including thawing temperature,
sperm sample concentration and media used for dilution (Contri et al., 2010).
When the semen samples were treated with H2O2, the progressive
forward motility of the sperm cells in bulls of both group I and II had reduced
significantly (P<0.01) from the level of more than 60 per cent at immediate post
thaw to about 20 per cent within 10 min of incubation and further reduced to 6 – 7
per cent at 30 min of incubation. There was a significant reduction in the per cent of
non progressive motile and total motile sperm cells with significant increase in static
sperm cell population within 10 min of incubation. Reductions in sperm velocity
parameters such as curvilinear velocity, straight line velocity, average path velocity,
amplitude of lateral head displacement and beat cross frequency were also observed
during the incubation period. In accordance to the present study, Garg et al. (2009)
reported a dose and time dependent reduction in sperm motility. They also observed
a significant reduction in sperm motility within 15 – 30 min of incubation with 50
µM H2O2 and no motility observed after 60 min of incubation. Baumber et al.
(2000) reported a significant decline in per cent of total motility, curvilinear
velocity, straight line velocity, amplitude of lateral head displacement, linearity and
average path velocity when the semen samples were exposed to reactive oxygen
species. Similar reports were also given by de Lamirande and Gagnon (1992) in
human sperm cells. Paul et al. (1986) proposed that H2O2 causes perturbations in
important biochemical functions, including increased formation of oxidized
intracellular sulfydryls, rapid decrease in ATP levels and a consequent depression of
glycolytic flux. de Lamirande and Gagnon (1992) confirmed that the inhibition of
sperm motility after incubation with ROS was caused by depletion of ATP. ROS
inhibited one or more enzymes of oxidative phosphorylation, glycolysis or both,
thus limiting ATP generation by the sperm cells. Alternatively, enzyme inhibition
could be induced indirectly by products of lipid peroxidation, especially
malondioldehyde. Low concentrations of these substances had been shown to inhibit
a larger number of cellular enzymes and functions, anaerobic glycolysis, DNA,
RNA and protein synthesis (Comporti, 1989). Martinez Pastor et al. (2009) also
confirmed that high doses of H2O2 caused an immediate inhibition of motility and
suggested that loss of mitochondrial membrane potential would be a cause for loss in
sperm motility.
the progressive forward motility was significantly reduced during incubation in
comparison to untreated control. However, there was no significant difference in the
per cent of total motile sperm cells and static cells between the control and treatment
groups. Also there was no significant difference between control and treatment with
fertility associated protein in the velocity parameters such as curvilinear velocity,
straight line velocity, average path velocity, linearity, straightness, wobble and
amplitude of lateral head displacement. The beat cross frequency in the fertility
associated protein treatment was significantly higher than the untreated control
during incubation. The results indicated that the addition of fertility associated
protein reduced the progressive forward motility of the sperm cells but it did not
cause a total loss of sperm motility. The reductions in the progressive motility upon
addition of proteins were explained by Kumar et al. (2005) and Harshan et al.
(2006) that the addition of fertility associated proteins such as BSP, heparin binding
protein and PDC 109 influenced the motility of sperm cells on dose dependent
manner and at higher concentrations, they inhibited the motility in the sperm cells
harvested from epididymis. Einspanier et al. (1971) and Schoneck et al. (1996)
observed that higher concentration of acidic seminal proteins present in the ampulla
inhibited the motility of sperm cells to conserve their energy. Dostalova et al. (1994)
suggested that removal of adhered proteins from sperm membrane in the female
reproductive tract restores the motility of the sperm cell. It was further proved by
Centurion et al. (2003) that, upon high dilution PDC homologue protein PSP I/II
enhanced motility, mitochondrial activity and viability of the sperm cells. Sanchez-
Luengo et al. (2004) found that addition of PDC – 109 protein at lower
concentrations of 2 µM in the extender improved the straight line velocity, average
path velocity and curvilinear velocity of the sperm cells.In the present study, the
retention motility potential in the sperm cells treated with fertility associated
proteins was clearly indicated by maintenance of high mitochondrial membrane
potential.
In the present study, group I bulls showed significantly more per cent of
sperm cells with high mitochondrial membrane potential than the group II bulls
during the treatments which indicated that group I bulls had sufficient energy
reserve to maintain the sperm motility. Souza et al. (2006) reported that BSP
proteins were observed in the acrosome and mid piece of the spermatozoa. It was
suggested that localization of BSP proteins in the midpiece closeto the mitochondria
was indicative of the fact that BSP proteins controlled sperm motility. BSP
A1/A2was shown to stimulate membrane-bound calcium ATPase activity in bull
sperm (Sanchez-Luengo et al., 2004). Thefunctional connection between binding of
BSPproteins to the midpiece and stimulation of mitochondrialactivity and sperm
motility suggested multifaceted role for BSPproteins as sperms were prepared for
fertilization.
5.2.8.1. Correlation between sperm cell motility parametersand other

In the present study, sperm cells with progressive forward motility had
highly significant (P <0.01) positive correlation with plasma membrane integrity,
functional membrane integrity,sperm cells with high mitochondrial membrane
potency,F pattern uncapacitated- acrosome intact sperm cells and DNA integrity. In
accordance with the present study, Selvaraju et al. (2008) also reported that the
progressive forward motility had correlation with plasma membrane integrity (0.73),
functional membrane integrity (0.89), acrosomal integrity (0.61), DNA integrity
(0.28), mitochondrial membrane potential (0.93), sperm- zona binding and cleavage
rate (0.56). Gillan et al (2008) reported that F-pattern (uncapacitated) spermatozoa
have high amplitude of lateral head displacement, low straightness and linearity.
Spermatozoa displaying the B pattern (capacitated) were present in samples of low
amplitude of lateral head displacement and linearity.
5.3. Capacitation status of sperm cells
Freshly ejaculated mammalian spermatozoa must undergo a maturation

process termed ‘capacitation’ before fertilizing an oocyte (Austin, 1952).
Capacitation is a continuous biochemical change associated with the functional and
structural changes in the sperm. Removal of cholesterol from sperm membrane
increases membrane fluidity (Langlais et al., 1988) resulting in increase in calcium
influx (Singh et al., 1978), cAMP level (White and Aitken, 1989) and changes in
enzymatic activities such as protein kinase C (Furuya et al., 1993). Capacitation
process ended with the acrosome reaction, an essential stage for oocyte fertilization
(O’Flaherty et al., 1999). The physiologic mammalian acrosome reaction was
experienced only by sperm that have been previously capacitated.The destabilization
of sperm membranes could be evaluated by tracking the distribution of Ca2+ in
spermatozoa. Antibiotic chlortetracycline (CTC), which accumulates in organelles
containing high concentrations of Ca2+ was used to evaluate the capacitation status
in sperm cells.
In the present study per cent of sperm cells with F pattern (uncapacitated-
acrosome intact) decreased significantly (P < 0.01) at 60 min of incubation from
immediate post thaw level in both group I and II. Though there was a decrease in the
values during further incubation up to 180 min, it was not statistically significant.
The per cent of sperm cells with B pattern (capacitated- acrosome intact) were
increased significantly (P < 0.01) at 60 min of incubation from immediate post thaw
level in both the group I and IIbulls. Though there was an increase in the values
during further incubation up to 180 min, it was not statistically significant. The per
cent of sperm cells with AR pattern (capacitated – acrosome reacted) increased
significantly during incubation in both the bull groups. In contrast to the present
study, Gil et al. (2000) reported slightly more number of F pattern cells (65.60 ±
2.40), less number of B pattern cells (28.60 ± 2.10) and similar number of AR
pattern cells (5.80 ± 0.70) at immediate post thaw in bovine semen samples.
Kadirvel et al. (2009) reported slightly less per cent of F pattern cells (36.75± 1.37),
similar per cent of B pattern cells (42.21 ± 2.23) and more per cent of AR pattern
acrosome reacted cells (23.34± 1.31) at immediate post thaw in buffalo semen
samples. All these findings confirmed the presence of a sperm population already
capacitated in post thaw semen, probably induced by the freezing procedures as
observed by Watson (1995). Maxwell and Johnson (1997) reported similarities
between the changes associated with capacitation and cryoinjury, such as plasma
membrane reorganization, fluidization, calcium influx and ability to undergo the
acrosomal reaction. This cryo-capacitation is thought to be partly responsible for the
reduced fertility of frozen thawed bull semen (Cormier and Bailey, 2003).
When the semen samples were treated with heparin to induce
capacitation, the per cent of F pattern (uncapacitated) cells were reduced
significantly while, the per cent of B pattern (capacitated) sperm cells were
increased significantly during different points of incubation in both the groups I and
II bulls when compared to the untreated control. AR pattern (acrosome reacted) cells
also showed an increasing trend during incubation with heparin when compared to
control. Observations in the present study are in accordance with Berquist et al.
(2007). They compared various glycosaminoglycons (GAGs) in stimulating
capacitation in frozen thawed bull semen. GAGs have been ascribed among a
variety of substances within the oviductal fluid as causing sperm capacitation (Lee et
al., 1986, Tienthai et al., 2004). Heparin stimulates capacitation by binding to and
removing seminal proteins associated with sperm membrane (Miller et al., 1990)
and calcium uptake (Parrish et al., 1999). Mehmood et al. (2007) reported that
heparin induces capacitation in dose and time dependent mannerin frozen buffalo
semen.
When semen samples were treated with heparin along with fertility
associated protein to induce capacitation, the per cent of F pattern cells decreased
significantly while the per cent of B pattern cells increased significantly and the AR
pattern cells increased non significantly during incubation in bulls of both group I
and II.
In group Ibulls, the per cent of F pattern cells were significantly lower in
the group treated with heparin along with fertility associated protein than the control,
but there was no significant difference between the two treatments, treatment with
heparin and treatment with heparin along with fertility associated protein. Whereas,
in group IIbulls, the per cent of F pattern cells in the group treated with heparin
along with fertility associated protein were significantly lower than the control and
treatment with heparin alone.
In group Ibulls, the per cent of B pattern cells were significantly higher
than the control group at 60 min, 120 min and 180 min of incubation, but there was
no significant difference between the treatment with heparin and treatment with
heparin and fertility associated protein in the per cent of B pattern cells. But in group
II bulls, the percentage of B pattern cells in heparin along with fertility associated
protein treatment were significantly higher than the control and treatment with
heparin at 60 min, 120 min and 180 min of incubation.
In the present study, the group II bulls that were negative for fertility
associated proteins in sperm membrane had significant improvement in the
induction capacitation when supplemented with fertility associated protein. There
was a significant difference in the percentage of B pattern (capacitated) sperm cells
between heparin along with fertility associated protein treatment vs control and
treatment with heparin alone. These findings are in agreement with Arangasamy et
al. (2005) and Harshan et al. (2006). They reported a time and dose dependent
increase in capacitation and acrosomal reaction in response to treatment with heparin
binding proteins in buffalo epididymal spermatozoa. Fiol de Cuneo et al. (2004)
observed an increase in the induction of acrosome reaction when ejaculated bovine
spermatozoa were incubated with PDC- 109. Similar results were reported by
Manjunath et al. (1994) who proposed that PDC 109 binds preferentially to the
sperm head and could be a physiological acrosome reaction inducer. They reported
that this protein could bind to other substances like apoliprotein A-I, fibrinogen,
calmodulin and phospholipase A2. There was an increase in the acrosome reacted
spermatozoa with incubation which was in agreement with the findings of Therien et
al. (1995), Arangasamy et al. (2005) and Harshan et al. (2006).
5.3.1. Correlation between B pattern sperm cellsand other in vitro sperm

characters
B pattern (capacitated- acrosome intact sperm) cells were having highly

significant (P <0.01) positive correlation with sperm cells with lost mitochondrial
membrane potency, static sperm cells, AR pattern capacitated acrosome reacted
sperm cells and necrotic cells. B pattern capacitated- acrosome intact sperm cells
were having significant (P <0.05) positive correlation with MDA.
B pattern capacitated- acrosome intact sperm cells were having highly

significant (P <0.01) negative correlation with sperm cell viability, plasma
membrane integrity, functional membrane integrity, sperm cells with low
mitochondrial membrane potency, progressive forward motility, curvilinear velocity
of sperm cells, straight line velocity of sperm cells, average path velocity of sperm
cells, amplitude of lateral head displacement,F pattern (uncapacitated- acrosome
intact) sperm cells and DNA integrity. Kadirvel et al. (2009) reported that B pattern
(capacitated) cells had potential association with cholesterol level, membranefluidity
and intracellular calcium. They further observed that B pattern cells had positive
correlation with proportion of sperm with high intracellular calcium (r = 0.81) and
high membrane fluidity (r = 0.65), and negativelycorrelated with cholesterol level (r
=−0.56) in frozen-thawed semen.
5.4. RANKING OF BULL
In the present study, when semen samples from 22 bulls were screened
for the presence of fertility associated proteins, 11 bulls were positive for 28 kDa
heparin binding protein (FAA) in the sperm membrane. These 11 bulls were also
positive for the other fertility associated proteins such as 55 kDa (osteopontin), 28
kDa (BSP 30), 26 kDa (lipocallin type prostaglandin D synthase) and 15 kDa (BSP
A1/ A2, BSP A3), heparin binding proteins such as 15/14 kDa and 28 kDa in seminal
plasma as well as sperm membrane. Based on the presence of 28 kDa heparin
binding protein in sperm membrane bulls in the present study were categorized into
group I positive for 28 kDa heparin binding protein (FAA) and group II negative for
the protein fraction.
Saacke and White (1972) and Bollwein et al. (2008) opined that sperm
characteristics assessed during 2 – 4 h of thawing were more closely related to bull
fertility than those measured only at immediately post thaw, since the sperm cells
has to remain in the female reproductive tract for some period of time to acquire
fertilizing capacity and to reach the site of fertilization. Amann et al., (1999)
suggested that the most reliable approach to predict the potential fertility of a semen
sample was to evaluate different attributes in an individual spermatozoan. Variation
in fertility and semen quality traits usually was greater among the males than it was
from ejaculate to ejaculate within males (Januskauskas et al. 2003; Den Dass et al.,
1988). Hence, in the present study, the in vitro sperm characters during incubation
from immediate post thaw to 180 min in the frozen thawed semen samples of both
bull group I and II were analyzed by Duncan Multiple Range Test (DMRT).
Following the DMRT test, bulls in the either group were categorized under different
subsets for each in vitro character and suitable rank was given to the bulls that were
categorized under same subsets. Bulls categorized under the rank 1 had better values
than the rank 2 and 3 bulls in the following in vitro sperm characters namely,
progressive forward motility, per cent of static cells, per cent of sperm cells with
intact plasma membrane, per cent of sperm cells with intact functional membrane,
per cent of sperm cells with F pattern, per cent of sperm cells with AR pattern, per
cent of sperm cells with mitochondrial membrane potency, per cent of sperm cells
with lost mitochondrial membrane potency, curvilinear velocity and straightness of
sperm cell motility.
In bull group I, a total of 5 bulls such as NJ 670, NJ 627, 4072, 4049, JS

149 were categorized under rank 1 and another 5 bulls such as NJ 621, 5011, HF 29,
HF 30, 2208 were categorized under rank 2 and one bull (NJ 662) was categorized
under rank 3. In bull group II, a total 4 bulls (NJ 658, JL 31, 5052, NJ 612) were
categorized under rank 1 and another 4 bulls (NJ 626, NJ 657, NJ 624, 4021) were
categorized under rank 2 and 3 bulls (5038, 5049, 5055) were categorized under
rank 3. From the results it was observed that the group positive for FAA had more
number of bulls falling under rank 1 and 2 when compared to group II which did not
had FAA. This infers that the FAA positive group had a better in vitro sperm
characters than the group II. Hence it is considered that the evaluating for the
presence of FAA which is a heritable trait will give a better value to the breeding
bulls.
CHAPTER VI
SUMMARY
At present bulls are included in semen collection programme based on

the breeding soundness examination which includes evaluation for physical
conformation, normal development of reproductive organs and analysis of semen for
its volume, sperm cell concentration, motility, viability, morphology etc. Since the
subjective estimation of sperm characters was affected by numerous factors, the
significance of these parameters in assessing the fertilizing capacity of an ejaculate
or a bull had its own limitations. Hence, it has become imperative that to ensure the
fertility of a bull, a reliable marker has to be identified which will remain as a
constant factor throughout the semen collection period of the bull. Under these
circumstances the Fertility Associated Antigen (FAA) identified in the sperm
membrane which was considered as heritable factor was projected as a marker for
measuring the fertilizing potential of the bull. It has been well established that the
bulls positive for this protein had 9 to 40 per cent more conception rate. It has also
been established that 0 – 50 per cent of bulls in different herds were lacking this
FAA protein. Hence, in the present study an attempt was made to establish the
presence of FAA in breeding bulls maintained at bull stations
Fresh semen samples were collected from 22 numbers of breeding bulls

maintained at Nucleus Jersey and Stud Farm, Udhagamandalam and Semen Bank,
Department of Animal Genetics and Breeding, Madras Veterinary College, Chennai.
Seminal plasma and sperm cells were separated immediately after collection.
Seminal plasma proteins were isolated by ice cold ethanol and the sperm membrane
proteins were separated by detergent extraction method. Heparin binding proteins of
the seminal plasma and sperm membrane proteins were isolated by using Heparin
CL agarose column. Molecular weights of the proteins were determined by SDS-
PAGE analysis.
Seminal plasma proteins of the bulls revealed 15 numbers of protein

bands. The fertility related proteins such as 15/14 kDa and 28 kDa were present in
all the 22 bulls (100 %), while 26 kDa protein was present in 18 bulls (81.82 %) and
55 kDa protein was present in 14 bulls (63.3 %). SDS- PAGE of sperm membrane
protein revealed a total of 14 protein bands. Proteins related with bull fertility such
as 15/14 kDa protein was observed in all the 22 bulls (100 %), 28 kDa protein was
present in 21 bulls (95.45 %), 26 kDa protein was present in 14 bulls (63.64 %) and
55 kDa protein was present in only 11 bulls (50.00 %).
Heparin binding proteins of the seminal plasma revealed 7 protein bands,

the fertility related proteins such as 15/14 kDa protein was present in all the 22 bulls
(100 %) and 28 kDa protein was present in 18 bulls (81.82 %). 10 protein bands
were observed in the heparin binding proteins of the sperm membrane. 15/14 kDa
fertility related protein was present in all the bulls, while 28 kDa protein was present
in 11 bulls (50.00 %). Bulls positive for 28 kDa heparin binding protein in sperm
membrane (FAA) were also positive for the other fertility related proteins such as 55
kDa, 28 kDa, 26 kDa and 15/14 kDa proteins in seminal plasma and sperm
membrane, 15/14 kDa and 28 kDa heparin binding proteins in seminal plasma and
15/14 kDa heparin binding protein in sperm membrane. Based on the presence of 28
kDa heparin binding protein in sperm membrane, bulls in the present study were
categorized into group I bulls which were positive for the protein and group II bulls
negative for the protein.
Significant reductions in structural and functional integrity of sperm

plasma membrane, mitochondrial membrane potential, sperm cell viability and
progressive forward motility were observed during incubation of frozen thawed
semen at 37° C for 180 min in both the groups I and II. Significant elevation in the
level of lipid peroxide compound malondioldehyde during the incubation was also
observed.
Addition of fertility associated protein (25 µg) preserved sperm cell

integrity and the fertility associated protein treated group had significantly more per
cent of sperm cells with structural and functional intact plasma membrane, more per
cent of sperm cells with high mitochondrial membrane potential, more per cent of
viable cells than the control, during incubation. Addition of fertility associated
protein also helped in controlling the oxidative stress as indicated by significantly
low level of malondioldehyde than the control during incubation.
Freezing and thawing procedure of semen causes death and damages to

certain population of sperm cells. Aromatic amino acid oxidase from the affected
sperm cells release excessive quantities of reactive oxygen species (ROS), which
leads to oxidative stress. In the present study, the excessive level of ROS, a cause for
deterioration of sperm quality was mimicked by the addition of 0.1 mM H2O2. The
H2O2 caused rapid and significant reductions in plasma membrane integrity,
mitochondrial membrane potential, sperm cell viability with significant elevations in
the lipid peroxide product malondioldehyde within a short period of incubation.
Semen samples from the group I bulls positive for fertility associated
antigen showed better in vitro sperm characters such as more per cent of sperm cells
with plasma membrane integrity, functional membrane integrity, more per cent of
sperm cells with high mitochondrial membrane potential, intact DNA and more per
cent of viable cells than group II bulls, during incubation in control and following
treatment with H2O2and with fertility associated protein. Group I bulls shown
significantly low level of malondioldehyde than the group II.
Group I bulls had less per cent of cryo-capacitated (B pattern) sperm

cells and more per cent of uncapacitated (F pattern) sperm cells in the frozen thawed
semen samples. When capacitation was induced with heparin, group I bulls positive
for 28- 30 kDa heparin binding protein readily responded and had significantly more
per cent of capacitated (B pattern) sperm cells than the group II. Addition of fertility
associated proteins promoted the capacitation induction by heparin in the group II
bulls which were negative for the protein.
When the bulls were ranked by analyzing the in vitro sperm characters
during incubation from immediate post thaw to 180 min in the frozen thawed semen
samples by Duncan Multiple Range Test, bulls in the group I had 5 bulls each in
rank 1 and rank 2, while only one bull was categorized under rank 3. Group II bulls
had 4 bulls each in rank 1 and 2 and 3 bulls in rank 3. It was inferred from the
present study that FAA was present only in 50 per cent of the breeding bulls. The
bulls which did not have FAA were also being used as semen donors for AI.
Inclusion of such bulls in breeding programme would result in failure to achieve the
desirable 1.5 services per conception and tend to increase the calving interval in the
breedable population. Hence, it is imperative to maintain only the bulls positive for
FAA in the bull station and the rest of the bulls may be removed from breeding
programme.
CONCLUSION
From the present study, it could be concluded that,
 Variations in the electrophoretic profile of seminal plasma and sperm

membrane proteins were observed among the breeding bulls. Fertility
associated antigen was present only in 50 per cent of the breeding bulls
screened.
 Reactive oxygen species when generated in excess caused severe

damages to sperm plasma membrane and organelles.
 Exogenous addition of fertility associated proteins preserved the

structural and functional integrity of plasma membrane and organelles
and protected the sperm cells from oxidative stress. Fertility associated
proteins also enhanced the induction of capacitation in the sperm cells
negative for fertility associated proteins.
 Sperm cells positive for fertility associated antigen had better in vitro
sperm characters, better protection against oxidative stress and they
readily underwent capacitation induction by heparin than the negative
cells.
Based on the above findings, the following recommendations are given,
1) All the bulls which have passed the breeding soundness examination
should be evaluated for the presence of fertility associated antigen.
2) Only those positive for the protein should be included in the breeding
programme.
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APPENDIX
Chemicals
Ethylene diamine tetra acetic acid (EDTA), glycerol, glacial acetic acid,
bromophenol blue, hydrochloric acid (HCl), methanol, ethanol, isopropanol, Tris-
hydroxyl methyl amino methane, fructose, citric acid, all were in GR-grade,
procured from Merck India. Glycine, urea, acrylamide, bisacrylamide, ammonium
persulphate, agarose, sodium dodecyl sulphate (SDS), tetramethylethylenediamine
(TEMED) and coomassie brilliant blue R-250, all were molecular biology grade,
procured from SRL, India. Protein molecular markers were procured from
Bangalore Genei, India. All other chemicals used in the experiments were procured
from Sigma Aldrich, USA.
TC Buffer
Tris – hydroxyl methyl amino methane : 4840 mg
CaCl2 : 290 mg
Sodium azide : 100 mg
Distilled water : to make 1000 ml
Sample buffer 5X
SDS 10 % : 0.2 g
1 % mercaptoethanol : 0.1 ml
Tris – HCl pH 6.8 : 2.5 ml
0.1 % Bromophenol blue : 0.01 g
200 mM EDTA : 0.25 ml
Dist water to make up to : 10 ml

Bis – Acrylamide 30%
Acrylamide : 29.2 g
N” methylene bisacrylamide : 0.8 g
Dist water to make upto : 100 ml
(The solution is to be mixed well. If not mixed properly water bath can be used. To
be filtered through Whatman filter paper and stored in brown coloured container and
store at 4 C)
5X Running Buffer
Tris base : 15.0 g
Glycine : 72.0 g
SDS : 5.0 g
Dist water to make volume : 1000 ml
pH 8.3
(To make 1 X running buffer take 1 ml of 5X buffer add 4 ml of distilled water.

Tank or electrode buffer)
Resolving or separating gel buffer
Tris : 18.17g
1.5 M Tris-HCl (pH 8.8)
First dissolve in 70 ml, adjust pH and make up to 100ml
Stacking gel buffer 0.5 M Tris-HCl (pH 6.8)
Tris : 6.057 g

Composition of separating / resolving gel and stacking gel
Separating gel 10 % Volume 8 ml Stacking gel 4 % Volume 3 ml
Ingredients Quantity Ingredients Quantity
Dist Water 2.8 ml Dist Water 2.04 ml
30 % Acrylamide 2.3 ml 30 % Acrylamide 0.57 ml
1.5 M separating buffer 1.75 ml Stacking buffer 0.37 ml
SDS 10 % 70 µl SDS 10 % 35 µl
APS 10 % 35µl APS 10 % 30 µl
TEMED 5 µl 5 µl
Separating gel 8 % Volume 8 ml Stacking gel 4 % Volume 3 ml
Ingredients Quantity Ingredients Quantity
Dist Water 3.7 ml Dist Water 2.04 ml
30 % Acrylamide 2.13 ml 30 % Acrylamide 0.57 ml
1.5 M separating 2 ml Stacking buffer 0.37 ml

buffer
SDS 10 % 70 µl SDS 10 % 35 µl
APS 10 % 80 µl APS 10 % 30 µl
TEMED 5 µl TEMED 5 µl
Separating gel 12 % Volume 7 ml Stacking gel 5%
Volume Volume
Ingredients Quantity Ingredients
3 ml 4 ml
Dist Water 2.3 ml Dist Water 1.32 ml 1.76 ml
30 % Acrylamide 2.8 ml 30 % Acrylamide 0.48 ml 0.64 ml
1.5 M separating buffer 1.75 ml Stacking 0.6 ml 0.8 ml

buffer
SDS 10 % 70 µl SDS 10 % 24 µl 32 µl
APS 10 % 35 µl APS 10 % 10 µl 10 µl
TEMED 5 µl TEMED 5 µl 5 µl
Separating gel 15 % Volume 7 Stacking gel 5%

ml
Ingredients Quantity Ingredients Volume Volume

3 ml 4 ml
Dist Water 1.92 Dist Water 1.32 ml 1.76 ml
30 % Acrylamide 4 ml 30 % 0.48 ml 0.64 ml

Acrylamide
1.5 M separating 2 ml Stacking 0.6 ml 0.8 ml

buffer buffer
SDS 10 % 70 µl SDS 10 % 24 µl 32 µl
APS 10 % 80 µl APS 10 % 10 µl 10 µl
TEMED 5 µl TEMED 5 µl 5 µl
Commassie Brilliant Blue stain
Ingredients Quantity
Commassie brilliant blue R- 250 0.2 g
Methanol 40 ml
Acetic acid 10 ml
Dist water 50 ml
Destaining solution I
Methanol 100 ml
Glacial acetic acid 20 ml
Dist water 80 ml
Destaining solution II
Methanol 25 ml
Glacial acetic acid 50 ml
Dist water 425 ml
Keep the gel in Commassie brilliant blue stain for over night. Remove from
Commassie brilliant blue stain after over night; wash with distilled water two times.
Modified Tyrode’s solution (mTALP)
(Florman and First 1988)
NaCl 100mM
KCl 3.1mM
MgCl2 1.5mM
CaCl2 2.1 mM
KH2PO4 0.29mM
NaHCO3 25mM
Na-Hepes 10mM
Na-lactate 21.6mM
Phenol red 10 μg/ml
NaOH / HCl titrated to pH 7.4
Millipore filtered (0.22 μm) and stored at 4° C.
Washing medium was prepared daily with mTALP supplemented with fraction V
bovine serum albumin (BSA 6 mg/ml) and pyruvate (1 mM)
ABBREVIATIONS
g - Micro gram
g/ml - Microgram/millilitre
l - microlitre
BSA - Bovine Serum Albumin

º
C - Celsius Degree
Ca++ - Calcium ions
DMSO - Dimethyl Sulfoxide
DNA - Deoxyribonucleic acid
DPBS - Dulbeccos Phosphate Buffered Saline
EDTA - Ethylenediamine tetra acetic acid
h - Hour
mM - Milli Molar
min - Minutes
ng/ml - Nanogram/milliliter
s - seconds
TALP - Tyrodes Albumin Lactate Pyruvate
vs. - Versus
V - Volt
kDa - Kilo Dalton
mOSM - Milli osmol
nm - Nano meter
m - Micro meter
mm - Milli meter

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EVALUATION OF BULL SEMEN QUALITY IN RELATION TO

FERTILITY ASSOCIATED PROTEINS, LIPID PEROXIDATION

DEPARTMENT OF ANIMAL REPRODUCTION,GYNAECOLOGY

Thesis submitted in partial fulfilment of

Tamilnadu Veterinary and AnimalSciencesUniversity

DEPARTMENT OF ANIMAL REPRODUCTION,

Department of Animal Reproduction,

Date: (Dr. T. G. DEVANATHAN)

Chairman : (Dr. T. G. DEVANATHAN)

Members : (Dr. K. KULASEKAR)

(Dr. K. THILAK PON JAWAHAR)

Date: (EXTERNAL EXAMINER)

Name of the Candidate : M. KARUNAKARAN

Shri Maragathavalli Nayaki Sametha

With sincere gratitude and indebtedness, I express my deep sense of

I express my heartfelt gratitude to members of advisory committee

I express my profound and deep sense of gratitude and personal indebtedness

No words can ever express my sincere gratitude to Dr. S. Selvaraju,

I express my sincere gratitude to Dr. K. Manimaran, Assistant Professor,

I am much obliged to Dr. A. Subramanian, Professor (Retd), Dr. Cecilia

My special thanks are due to Dr. K. Loganathasamy, Assistant Professor,

I thank Dr. S. A. Asokan, Professor and Head and Dr.R.C. Rajasundaram,

I register my profound gratitude to Dr. M. Selvaraju, Associate Professor,

I avail this opportunity to express my affectionate to Dr. Kulanthaivel,

I am indebted to all who provided me with information, guidance and

Title : Evaluation of bull semen quality in relation

Name and Degree : M. KARUNAKARAN

Name of the Chairman : Dr. T.G. DEVANATHAN, Ph.D.,

University : Tamilnadu Veterinary and Animal Sciences

Bulls in group I when treated with fertility associated proteinhad

Significant reductions in motility and velocity parameters were observed

Plate Title Between

2 SDS – PAGE power pack assembly 35-36

3 Electrophoretic pattern of seminal plasma proteins 36-37

4 Electrophoretic pattern of sperm membrane proteins 36-37

5 Sperm cell morphology assessment 37-38

6 Plasma membrane integrity and mitochondrial membrane 37-38

7 Functional membrane integrity assessment 38-39

8 DNA integrity assessment 38-39

9 Apoptosis assay 40-41

10 CASA- sperm motility patterns 40-41

11 CASA- sperm velocity parameters 40-41

12 Capacitation status – F pattern cells 42-43

13 Capacitation status – B pattern cells 42-43

14 Capacitation status – AR pattern cells 42-43

Artificial insemination (AI) is the reproductive biotechnology that has

Currently breeding soundness examination (BSE) is carried out before

Reactive oxygen species (ROS) generated by spermatozoa play an

i) To study the presence of fertility associated proteins, status of lipid

ii) To determine role of fertility associated proteins and lipid

2.1. MOLECULAR INDICATORS OF FERTILITY

Fertility associated proteins such as osteopontin, prostaglandin D

2.1.1. Seminal plasma and sperm membrane proteins

2.1.2 Seminal proteins and sperm function

The role of seminal plasma proteins in the regulation of sperm function

Immunocytochemical analysis showed that membrane alterations induced

Seminal plasma proteins protected sperm from peroxidative damage

Blanco (2008) reported that the addition of seminal plasma proteins to

Henault and Killian (1996) reported that the fertility of sub-fertile

2.1.3 Electrophoretic profile of seminal plasma proteins

Seminal plasma proteins partly originated from the blood plasma by

Arora and Jain (1995) observed 6 protein components in seminal plasma