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: Place: Chennai-7

(Dr. T. G. DEVANATHAN) Chairman APPROVED BY Chairman : Members

(Dr. T. G. DEVANATHAN)

: (Dr. K. KULASEKAR) (Dr. P. SRIDEVI)

(Dr. K. THILAK PON JAWAHAR)

Date:

(EXTERNAL EXAMINER)

CURRICULUM VITAE Name of the Candidate

:

M. KARUNAKARAN

Place of Birth

:

Gobichettipalayam

Date of Birth

:

21.03.1975

Major filed of specialization

:

Animal Reproduction,

Educational Status

:

Completed B.V.Sc., in 1998 at

Gynaecology and Obstetrics

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 Professional Experience

Marital Status

Permanent Address

:

:

:

Chennai – 600 007

Working as Scientist since 2003 at ICAR-RC- for Goa Old Goa Married

246, Asiriyar Nagar, Kullampalayam,

Gobichettipalayam, Publication made

Membership of Professional Society

:

:

Erode- 638 476.

Research articles

International : 14

National

:4

Life member of Indian Society for the Study 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. Joseph,

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

Chapter No.

CONTENTS Title LIST OF TABLES LIST OF PLATES

Page No.

INTRODUCTION

1

MOLECULAR INDICATORS OF FERTILITY

4

2.1.2

Seminal proteins and sperm function

5

2.2

BOVINE SEMINAL PLASMA AND SPERM MEMBRANE PROTEINS

8

2.2.2

Osteopontin

9

2.2.4

Fertility associated antigen

I

II

REVIEW OF LITERATURE

2.1.1

Seminal plasma and sperm membrane proteins

2.1

2.1.3

2.2.1 2.2.3

Electrophoretic profile of seminal plasma proteins

BSP proteins

Heparin binding proteins

4 4 7

8

11

12

2.2.5

Prostaglandin D- synthase

13

2.2.7

Spermadhesin Z13

15

2.2.9

Phospholipase A2

15

2.2.11

Acrosin

16

2.2.6 2.2.8 2.2.10 2.3

2.3.1 2.3.2 2.3.3 2.3.4 2.4

Type 2 Tissue inhibitor of metallo proteinases Clusterin

Heat shock protein

14 15

16

OXIDATIVE STRESS

16

Positive and negative effects of ROS

18

Origin of ROS in male reproductive system Lipid peroxidation

Cryopreservation and oxidative stress

EVALUATION OF IN VITRO SPERM CHARACTERS

17

18

19

20

Chapter No.

Title

2.4.2

Plasma membrane integrity

2.4.1

Sperm morphology

2.4.3

Functional membrane integrity

2.4.4 2.4.5 2.4.6 2.4.7 2.4.8

2.4.8.1 2.4.8.2

Mitochondrial membrane potential of sperm cells

Page No.

21

21

23

23

DNA integrity

24

Motility

27

Apoptosis

Capacitation status of sperm cells Acrosome integrity

Induction of capacitation

25

28

30

31

III

MATERIALS AND METHODS

33

3.2

Collection of semen

33

3.1 3.3 3.4 3.5 3.5.1 3.5.2 3.6 3.7

3.7.1 3.7.2 3.7.3 3.7.4 3.7.5 3.7.6

Experimental animals and source of semen

33

Extraction of seminal plasma and sperm membrane proteins

34

Characterization of seminal plasma and sperm membrane proteins by electrophoresis

35

Electrophoretic run

36

Isolation of heparin binding proteins

Sample preparation

34

35

Bull grouping

36

Sperm cell morphology

37

Simultaneous assessment of plasma membrane integrity and mitochondrial membrane potential

38

DNA integrity

39

Assessment of in vitro sperm characters Estimation of lipid peroxidation

Functional membrane integrity

Assessment of sperm cell apoptosis

36

37

38 39

Chapter No.

Title

3.8

Effect of fertility associated proteins on in vitro sperm characters

3.7.7

3.9

3.10

3.10.1 3.10.2 3.11 IV

4.1 4.1.1 4.1.2 4.1.3 4.1.4 4.2

4.2.1 4.2.2

4.2.2.1. 4.2.3.

4. 2. 3.1. 4. 2. 4.

4.2.4.1.

Sperm motility

Page No.

40

40

Induction of lipid peroxidation

41

Induction of capacitation with heparin

42

Assessment of capacitation status

Effect of fertility associated proteins on induction of capacitation

41

42

Data analysis

43

ISOLATION AND CHARACTERIZATION OF SEMINAL PLASMA AND SPERM MEMBRANE PROTEINS

45

RESULTS

45

Electrophoretic profile of seminal plasma proteins

45

Electrophoretic profile of heparin binding proteins of bovine seminal plasma

49

EVALUATION OF IN VITRO SPERM CHARACTERS

54

Electrophoretic profile of sperm membrane proteins

47

Electrophoretic profile of heparin binding proteins of bovine sperm membrane

51

Sperm morphology

54

Correlation between MDA levels and other in vitro sperm characters during the incubation at 37° C for 180 min

56

Correlation between plasma membrane integrity and other in vitro sperm characters during the incubation at 37° C for 180 min

60

Correlation between functional membrane integrity and other in vitro sperm characters during the incubation at 37°C for 180 min

63

Lipid peroxidation

Plasma membrane integrity

Functional membrane integrity

54

58

61

Chapter No.

Title

4.2.5.1.

Sperm cells with high mitochondrial membrane potency

Page No.

4.2.5.

Mitochondrial membrane potential of sperm cells

4.2.5.2.

Correlation between sperm cells HMMP and other in vitro sperm characters during the incubation at 37° C for 180 min

66

Correlation between sperm cells LWMMP and other in vitro sperm characters during the incubation at 37° C for 180 min

68

Correlation between sperm cells LTMMP and other in vitro sperm characters during the incubation at 37° C for 180 min

71

4.2.5.3. 4.2.5.4. 4.2.5.5. 4.2.5.6. 4.2.6.

4.2.6.1. 4.2.7.

4.2.7.1

4.2.7.2. 4.2.7.3. 4.2.7.4. 4.2.7.5. 4.2.7.6. 4.2.8.

4.2.8.1. 4.2.8.2.

Sperm cells with low mitochondrial membrane potency

Sperm cells with lost mitochondrial membrane potency

63

63

66

69

DNA integrity

72

Assessment of sperm cell apoptosis

75

Correlation between sperm cell DNA integrity and other in vitro sperm characters during the incubation at 37° C for 180 min

74

Viable sperm cells

75

Necrotic sperm cells

77

Correlation between viable sperm cells and other in vitro sperm characters during the incubation at 37° C for 180 min

77

Correlation between necrotic sperm cells and other in vitro sperm characters during the incubation at 37° C for 180 min

79

Correlation between apoptotic sperm cells and other in vitro sperm characters during the incubation at 37° C for 180 min

82

Apoptotic sperm cells

80

Motility and velocity parameters of sperm cells

83

Motility and velocity parameters of sperm cells- bull group II

86

Motility and velocity parameters of sperm cells- bull group I

83

Chapter No. 4.2.8.3. 4.2.8.4. 4.2.8.4.1. 4.2.8.4.2

4.2.8.4.3. 4.3.

4.3.1. 4.3.2. 4.3.3. 4.3.4. 4.3.5. 4.3.6. 4.4. V

5.1 5.1.1 5.1.2

5.1.3. 5.1.4. 5.2.

5.2.1.

Title Motility and velocity parameters of sperm cells- treatment with H2O2

Correlation between sperm cell motility parametersand other in vitro sperm characters during the incubation at 37° C for 180 min

Page No.

87 89

Progressive forward motility

89

Sperm velocity parameters

90

F pattern cells

91

Static sperm cells.

CAPACITATION STATUS OF SPERM CELLS

90 91

Correlation between F pattern sperm cells and other in vitro sperm characters during the incubation at 37° C for 180 min

93

Correlation between B pattern sperm cells and other in vitro sperm characters during the incubation at 37° C for 180 min

96

Correlation between AR pattern sperm cells and other in vitro sperm characters during the incubation at 37° C for 180 min

99

B pattern cells

AR pattern cells

94

97

RANKING OF BULL

100

CHARACTERIZATION OF SEMINAL PLASMA AND SPERM MEMBRANE PROTEINS

104

DISCUSSION

104

Electrophoretic profile of seminal plasma proteins

104

Heparin binding proteins of seminal plasma

107

Electrophoretic profile of sperm membrane proteins

106

Heparin binding proteins of sperm membrane

107

Sperm morphology

108

EVALUATION OF IN VITRO SPERM CHARACTERS

108

Chapter No.

Title

5.2.2.1.

Correlation between MDA levels and other in vitro sperm characters

112

Correlation between plasma membrane integrity and other in vitro sperm characters

115

Correlation between functional membrane integrity and other in vitro sperm characters

118

Correlation between mitochondrial membrane potential of sperm cellsand other in vitro sperm characters

120

5.2.2

5.2.3. 5.2.3.1. 5.2.4. 5.2.4.1. 5.2.5. 5.2.5.1. 5.2.6. 5.2.6.1. 5.2.7. 5.2.7.1. 5.2.8. 5.2.8.1. 5.3. 5.3.1. 5.4. VI

Lipid peroxidation

Plasma membrane integrity

Functional membrane integrity

Mitochondrial membrane potential of sperm cells

Page No.

109

113

116

118

DNA integrity

121

Assessment of sperm cell apoptosis

123

Correlation between sperm cell DNA integrity and other in vitro sperm characters Correlation between apoptotic sperm cells and other in vitro sperm characters

122

126

Motility and velocity parameters of sperm cells

126

CAPACITATION STATUS OF SPERM CELLS

131

Correlation between sperm cell motility parametersand other in vitro sperm characters Correlation between B pattern sperm cells and other in vitro sperm characters

130

134

RANKING OF BULL

134

REFERENCES

140

SUMMARY

136

Table No. 1 2 3 4 5 6 7

8

9

10

11

12

LIST OF TABLES Title Electrophoretic profile of bovine seminal plasma proteins assessed by SDS-PAGE

Page No.

46

Electrophoretic profile of bovine sperm membrane proteins assessed by SDS-PAGE

48

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

52

Electrophoretic profile of heparin binding proteins of bovine seminal plasma assessed by SDS-PAGE

50

MDA level (µ mol/ml) in frozen thawed bull semen samples

55

Correlation among in vitro sperm characters

57

treated with fertility associated protein and hydrogen peroxide Per cent of sperm cells with intact plasma membrane in frozen thawed bull semen samples treated with fertility associated

protein and hydrogen peroxide

Per cent of sperm cells with functional membrane integrity in frozen thawed bull semen samples treated with fertility

associated protein and hydrogen peroxide

Per cent of sperm cells with high mitochondrial membrane

potential in frozen thawed bull semen samples treated with fertility associated protein and hydrogen peroxide

Per cent of sperm cells with low mitochondrial membrane

potential in frozen thawed bull semen samples treated with fertility associated protein and hydrogen peroxide

Per cent of sperm cells with lost mitochondrial membrane

potential in frozen thawed bull semen samples treated with fertility associated protein and hydrogen peroxide

Per cent of sperm cells with intact DNA in frozen thawed bull

semen samples treated with fertility associated protein and hydrogen peroxide

59

62

65

67

70

72

Table No.

13

14

15

16 16a 17 18

19

20

21 22

Title Per cent of viable sperm cells in frozen thawed bull semen

samples treated with fertility associated protein and hydrogen peroxide

Page No.

76

Per cent of necrotic sperm cells in frozen thawed bull semen

78

Per cent of apoptotic sperm cells in frozen thawed bull semen

81

Motility and velocity parameters of frozen thawed bull semen

84

Velocity parameters of frozen thawed bull semen samples

85

Motility and velocity parameters of frozen thawed bull semen

88

Per cent of F pattern sperm cells in frozen thawed bull semen

92

samples treated with fertility associated protein and hydrogen peroxide samples treated with fertility associated protein and hydrogen peroxide samples treated with fertility associated protein treated with fertility associated protein samples treated with H2O2

samples treated with heparin and heparin with fertility associated protein

Per cent of B pattern sperm cells in frozen thawed bull semen

samples treated with heparin and heparin with fertility associated protein

Per cent AR pattern sperm cells in frozen thawed bull semen

samples treated with heparin and heparin with fertility associated protein

95

98

In vitro sperm characters after incubation for 180 min in

101

In vitro sperm characters after incubation for 180 min in

103

bull group I

bull group II

LIST OF PLATES Plate No.

Title

Between pages

1

SDS – PAGE apparatus

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

potential assessment

35-36

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

iii)

To rank the bulls included in the breeding programme based on the

peroxidation on the in vitro sperm characters. 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 coldshock-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

2.2.1.

BSP proteins

PROTEINS

PLASMA

AND

SPERM

MEMBRANE

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/ERKmediated 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 nonenzymatic 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, selfpropagating 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 (HeparinCL 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

elecrophoresis

sodium

dodecyl

sulphate

polyacrylamide

gel

(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) = 1.56 × 105 × test volume (1ml)

OD × 30 1.56

3.7.3

Simultaneous

assessment

of

plasma

mitochondrial membrane potential

membrane

integrityand

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 USA)

Assessment of sperm cell apoptosis Annexin-V-FITC apoptosis detection kit (Sigma – Aldrich, Saint Louis,

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 midpiece 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

4.1.1

Electrophoretic profile of seminal plasma proteins

PLASMA AND SPERM MEMBRANE PROTEINS

OF

SEMINAL

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 (BSPA1/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)

66

10 (100.00)

10 (100.00)

2 (100.00)

22 (100.00)

43

2 (20.00)

10 (100.00)

2 (100.00)

14 (63.64)

36

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)

97 55 41 28 25 17

7 (70.00) 6 (60.00) 7 (70.00)

10 (100.00) 9 (90.00) 4 (40.00)

7 (70.00) 6 (60.00) 1 (10.00)

10 (100.00) 6 (60.00) 8 (80.00)

15/14

10 (100.00)

10 (100.00)

3

1 (10.00)

3 (30.00)

6.5

2 (20.00)

Figures in parentheses indicate percentage to total

8 (80.00)

0 (0)

2 (100.00) 0 (0)

2 (100.00) 2 (100.00) 2 (100.00)

14 (63.64) 14 (63.64) 8 (36.36)

22 (100.00) 17 (77.27) 14 (63.64)

2 (100.00)

22 (100.00)

0 (0)

4 (18.18)

2 (100.00)

12 (54.55)

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 out of 22 bulls (36.36 %) were positive for 41 kDa protein.

25 kDa protein band was observed in 9 Jersey, 6 Jersey crossbred and 2

Holstein Friesian bulls. Over all 17 out of 22 bulls (77.27 %) were positive for 25

kDa protein. 17 kDa protein band was observed in 4 Jersey, 8 Jersey crossbred and 2 Holstein Friesian bulls. Over all 14 out of 22 bulls (63.64 %) were positive for 17 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 out of 22 bulls (18.18 %) were positive for 3 kDa protein. 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 (kDa)

Number of bulls positive for protein fractions Jersey (n = 10)

Jersey crossbred (n =10)

Holstein Friesian (n =2)

Total bulls (n = 22)

205

5 (50.00)

9 (90.00)

2 (100.00)

16 (72.73)

97

5 (50.00)

10 (100.00)

2 (100.00)

17 (77.27)

66

10 (100.00)

10 (100.00)

2 (100.00)

22 (100.00)

43

8 (80.00)

3 (30.00)

2 (100.00)

13 (59.09)

28

9(90.00)

10(100.00)

2(100.00)

21(95.45)

2(20.00)

3(30.00)

160 80 55 36 26 17

15/14 6.5

1 (10.00) 0 (0)

5(50.00)

4 (40.00) 6(60.00) 10(100.00) 6 (60.00)

3 0 (0) Figures in parentheses indicate percentage to total

8 (80.00)

2 (20.00) 4(40.00)

6 (60.00) 6(60.00)

2 (100.00) 0 (0)

2(100.00)

2 (100.00) 2(100.00) 0(0)

11 (50.00) 2 (9.09)

11(50.00)

12 (54.55) 14(63.64) 5(22.73)

10(100.00)

2(100.00)

22(100.00)

1 (10.00)

0 (0)

1 (4.55)

4 (40.00)

0 (0)

10 (45.45)

205 kDa protein band was observed in 5 Jersey, 9 Jersey crossbred and 2

Holstein Friesian bulls. Over all 16 out of 22 bulls (72.73 %) were positive for 205

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.

97 kDa protein band was observed in 5 Jersey, 10 Jersey crossbred and 2

Holstein Friesian bulls. Over all 17 out of 22 bulls (77.27 %) were positive for 97

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

43 kDa protein band was observed in 8 Jersey, 3 Jersey crossbred and 2

Holstein Friesian bulls. Over all 13 out of 22 bulls (59.09 %) were positive for 43 kDa protein.36 kDa protein band was observed in 4 Jersey, 6 Jersey crossbred and 2

Holstein Friesian bulls. Over all 12 out of 22 bulls (54.55 %) were positive for 36 kDa protein.

17 kDa protein band was observed in 2 Jersey and 3 Jersey crossbred

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) 205 160 66 43 36 28 15/14

Number of bulls positive for protein fractions Jersey (n = 10)

Jersey crossbred (n =10)

Holstein Friesian (n =2)

Total bulls (n = 22)

1 (10.00)

2 (20.00)

1 (50.00)

4 (18.18)

5 (50.00)

4 (40.00)

0 (0)

9 (40.91)

5 (50.00)

4 (40.00) 2 (20.00) 8 (80.00)

10 (100.00)

Figures in parentheses indicate percentage to total

9 (90.00)

2 (100.00)

5 (50.00)

1 (50.00)

8 (80.00)

2 (100.00)

1 (10.00)

10 (100.00)

0 (0)

2 (100.00)

16 (72.73)

10 (45.45) 3 (13.64) 18 (81.82)

22 (100.00)

Table 4: Electrophoretic profile of heparin binding proteins of bovine sperm membrane assessed by SDS-PAGE Protein molecular weight (kDa) 205 160 97 66 55 43 36 28 26/25 15/14

Number of bulls positive for protein fractions

Jersey (n = 10)

Jersey crossbred (n =10)

Holstein Friesian (n =2)

Total bulls (n = 22)

8 (80.00) 9 (90.00) 9 (90.00) 8 (80.00) 0 (0) 8 (80.00) 4 (40.00) 5 (50.00) 0 (0) 10 (100.00)

8 (80.00) 6 (60.00) 6 (60.00) 4 (40.00) 0 (0) 4 (40.00) 0 (0) 4 (40.00) 0 (0) 10 (100.00)

2 (100.00) 2 (100.00) 0 (0) 0 (0) 2 (100.00) 2 (100.00) 2 (100.00) 2 (100.00) 2 (100.00) 2 (100.00)

18 (81.82) 17 (77.27) 15 (68.18) 12 (54.55) 2 (9.09) 14 (63.64) 6 (27.27) 11 (50.00) 2 (9.09) 22 (100.00)

Figures in parentheses indicate percentage to total

4049; 8 Jersey crossbred bulls 2208, 5038, JS 149, 5011, 5049, 4072, 5055, JL 31 and 2 Holstein Friesian bulls HF 29, HF 30).

205 kDa protein band was observed in 1 Jersey, 2 Jersey crossbred and 1

Holstein Friesian bulls. Over all 4 out of 22 bulls (18.18 %) were positive for 205 kDa protein. 160 kDa protein band was observed in 5 Jersey, 9 Jersey crossbred and

2 Holstein Friesian bulls. Over all 16 out of 22 bulls (72.73 %) were positive for 160 kDa protein.

66 kDa protein band was observed in 5 Jersey and 4 Jersey crossbred

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.

205 kDa protein band was observed in 8 Jersey, 8 Jersey crossbred and 2

Holstein Friesian bulls. Over all 18 out of 22 bulls (81.82 %) were positive for 205 kDa protein. 160 kDa protein band was observed in 9 Jersey, 6 Jersey crossbred and

2 Holstein Friesian bulls. Over all 17 out of 22 bulls (77.27 %) were positive for 160 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 fertility associated protein and hydrogen peroxide Treatment Control Treatment with fertility associated protein Treatment with hydrogen peroxide

Bull group

Immediate

Post thaw MDA levels (µ mol/ml) 60 min

1.60± 0.15a

2.67± 0.19b p

1.60± 0.15a

1.86± 0.17ab q

Bull group

Immediate

10 min

Group II

1.73± 0.13a

Group I

Group II Group I

Group II Group I

1.73± 0.13a 1.73± 0.13a 1.60± 0.15a

120 min

2.79± 0.24 b p i

180 min

3.77± 0.41 c p i

3.04± 0.15b x

3.61± 0.28 b X ii

4.84± 0.46 c x ii

2.03± 0.12ab y

2.55± 0.13b Y

3.16± 0.14c y

1.79± 0.14a

2.85± 0.16b1

1.91± 0.14a

2.25± 0.19b q 30 min

3.26± 0.12b2

2.81± 0.26b q 60 min

3.04±0.161c

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 treated with fertility associated protein and hydrogen peroxide Treatment Control

Bull group Group I

Group II

Treatment with fertility Group I associated protein Group II Treatment with hydrogen peroxide

Bull group Group I

Group II

Sperm cells with intact plasma membrane during post thaw incubation (%) Immediate

60 min

120 min

58.37±2.79 a

43.30±2.80 b

28.22±3.10 c p

58.37±2.79a

47.13±2.39b

36.02±2.10c q

52.67±4.12 a 52.67±4.12a Immediate

58.37±2.79a 52.67±4.12a

38.98±3.48 b 44.24±3.38b 10 min

45.75±3.44 b i

31.79±3.34 b ii

180 min

15.27±2.11d p

25.96±3.32 c x

15.06±2.16 d x

32.50±3.03c y

23.36±2.17d y

30 min

26.03±2.53 c i

17.54±1.59 c ii

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.

26.69±2.06d q 60 min 0

0

4.2 4.2.1

EVALUATION OF IN VITRO SPERM CHARACTERS 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). 4.2.3.

Plasma membrane integrity 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 treated with fertility associated protein and hydrogen peroxide Treatment Control

Bull group Group I

Group II

Treatment with fertility Group I associated protein Group II Treatment with hydrogen peroxide

Bull group Group I

Group II

Sperm cells with functional membrane integrity during post thaw incubation (%) Immediate

55.61± 2.70a 49.12± 3.98a 55.61± 2.7a

60 min

120 min

40.69± 2.75b P

25.46± 2.95c p

11.59± 2.24d p

45.45± 2.59 b,1 Q

33.91± 2.31c,1 q

23.94± 2.08d q

36.29± 3.20 b x

22.13± 3.18c x

49.12± 3.98a

40.86± 2.30 b, 2 y

28.64± 3.03c,2 y

55.61± 2.7a

41.60± 3.06 b i

21.76± 2.27 c i

Immediate

49.12± 3.98a

180 min

10 min

28.63± 3.32

b ii

30 min

14.08± 1.46 c ii

11.52± 2.07d x 20.37± 2.12d y 60 min 0

0

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 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 Treatment Control Treatment with fertility associated protein Treatment with hydrogen peroxide

Bull group

Sperm cells with high mitochondrial membrane potential during post thaw incubation (%) Immediate

Group I

19.65±1.17 Aa

Group I

19.65±1.17a

60 min

12.32±1.04 b i p

17.51± 0.90i ab

14.54± 0.58i b q

10.54± 0.55ic q

10 min

30 min

60 min

12.71± 0.99 b ii

Group II

19.13± 0.99 a

13.57± 0.85 ii b

Group I

19.65±1.17a

15.30± 0.92b

Bull group Group II

Immediate

19.13± 0.99 a

180 min

16.72±1.02 B i

19.13± 0.99 a

Group II

120 min

8.39± 1.02 c ii

9.58± 0.76 ii c

14.86± 0.77b

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 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 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 .

0 0

8.31±0.78 c i p

5.19±1.00 d ii x 7.49±0.89 ii d y 0 0

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 Treatment Control Treatment with fertility associated protein Treatment with hydrogen peroxide

Bull group Group I

Sperm cells with low mitochondrial membrane potential during post thaw incubation (%)

Immediate

44.35± 3.95b

39.75± 5.07bc

52.96±2.50a

46.94± 3.64b

42.23± 4.73bc

Immediate

10 min

30 min

55.38± 1.73a

Group II

55.38± 1.73a

Group I

52.96±2.50a

Bull group Group II

120 min

52.96±2.50a

Group II Group I

60 min

55.38± 1.73a

45.68± 3.35b 47.01± 3.06b

16.03± 3.17b 13.36± 2.36b

Data shown all mean ± SEM (n = 11) Means with different superscripts (a, b, c, d) in a row differ significantly at P< 0.01.

180 min

32.03± 4.50c

35.34± 3.67c

33.58± 4.24c

39.84±3.58c

36.05± 4.11c

10.63± 1.43c

0

12.87± 1.33b

38.51± 4.01c 60 min 0

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 sperm characters during the incubation at 37° C for 180 min (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 capacitatedacrosome 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 Treatment Control Treatment with fertility associated protein Treatment with hydrogen peroxide

Bull group

Sperm cells with lost mitochondrial membrane potential during post thaw incubation (%)

Immediate

60 min

120 min

180 min

Group I

27.39± 2.35a

38.92± 3.65b

47.90± 5.05c

59.79± 4.45d

Group I

27.39± 2.35a

35.55± 3.38b

43.23± 4.53b

53.53± 3.78c

10 min

30 min

Group II Group II

Bull group Group I

Group II

25.49± 1.35a 25.49± 1.35a Immediate

27.39± 2.35a 25.49± 1.35a

41.62± 3.13b 39.42± 2.84b 68.69± 3.39b 71.78± 2.53b

Data shown all mean ± SEM (n = 11). Means with different superscripts (a, b, c, d) in a row differ significantly at P< 0.01.

56.32± 3.92c 51.14± 3.86c 89.37± 1.42c 87.13± 1.33c

61.13± 4.56d 56.46± 4.45c 60 min

100.00d 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 Treatment Control Treatment with fertility associated protein Treatment with hydrogen peroxide

Bull group Group I

Sperm cells with intact DNA during post thaw incubation (%)

Immediate

94.94± 0.61

60 min

94.66± 0.56

120 min

93.80±0.661

180 min

93.64± 0.661

Group II

93.55±0.70a

93.69±0.76ab

92.17±0.502b

91.99±0.432b

Group II

93.55±0.70

93.71±0.72

92.24±0.502

92.09± 0.452

94.80± 0.80

94.01±0.511

Group I

Bull group Group I

Group II

94.94± 0.61 Immediate

94.94± 0.61 93.55±0.70

94.72± 0.58 10 min

94.82± 0.76 93.30±0.82

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 1, 2 in a column differ significantly at P < 0.05.

93.95± 0.641 30 min

93.35±0.60

93.84± 0.691 60 min

92.26± 0.562

Table 13: Per cent of viable sperm cells in frozen thawed bull semen samples treated with fertility associated protein and hydrogen peroxide Treatment Control Treatment with fertility associated protein Treatment with hydrogen peroxide

Bull group

Per cent of viable sperm cells during post thaw incubation

Immediate

60 min

120 min

180 min

Group I

60.20± 3.93a

40.99± 4.66b P

25.34±5.29c p

13.39± 2.68d p

Group I

60.20± 3.93a

50.36± 4.20b Q

38.36± 4.72c q

30.24±2.621d q

Immediate

10 min

30 min

60 min

Group II Group II

Bull group Group I

Group II

50.51± 6.89a 50.51± 6.89a 60.20± 3.93a 50.51± 6.89a

35.78±6.08b

40.42± 4.75b

22.92± 4.27ib

11.54± 1.38iib

23.84± 5.84c 30.46± 5.20c 0

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 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

15.56±5.67d

20.68± 5.042d 0

0

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.

Mitochondrial membrane potential of sperm cells

4.2.5.1.

Sperm cells with high mitochondrial membrane potency

assessed

Sperm cells with high mitochondrial membrane potency (HMMP) 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 (treated with H2O2and withfertility associated protein) are presented in Table 10.

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 fertility associated protein and hydrogen peroxide Treatment Control Treatment with fertility associated protein Treatment with hydrogen peroxide

Bull group

Per cent of necrotic sperm cells during post thaw incubation

Immediate

60 min

120 min

180 min

Group I

32.30± 2.96a

50.15±4.17b P

64.56± 4.24c p

77.02± 2.32d p

Group I

32.30±2.96a

41.22±3.40b Q

50.60± 4.20c q

59.32± 4.24c q

Immediate

10 min

30 min

60 min

Group II Group II

Bull group Group I

Group II

39.37±5.27a 39.37±5.27a

32.30± 2.96a 39.37±5.27a

53.65±5.37b

48.80±4.62ab

66.55± 3.90ib

74.56± 1.55 ii b

64.83±6.18b

60.42±5.63bc 100

100

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 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

73.70±4.06c 69.75±5.21c 100

100

Table 15: Per cent of apoptotic sperm cells in frozen thawed bull semen samples treated with fertility associated protein and hydrogen peroxide Treatment Control Treatment with fertility associated protein Treatment with hydrogen peroxide

Bull group Group I

Per cent of apoptotic sperm cells during post thaw incubation

Immediate 7.50±1.07

60 min

8.86±1.23

120 min

10.10±1.37

180 min

9.58±1.21

Group II

10.12± 1.67

10.57±1.29

11.33±1.36

11.99±1.15

Group II

10.12±1.67

11.20±1.202

12.36±1.382

12.90±0.802

Bull group

Immediate

10 min

30 min

60 min

Group II

10.12± 1.67a

13.90±1.52iib

0

0

Group I

Group I

7.50±1.07

7.50±1.07a

8.00±1.041

10.53±1.35ib

Data shown all mean ± SEM (n = 11). 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

8.14±1.241

0

8.36±1.101

0

Table 18: Per cent of F pattern sperm cells in frozen thawed bull semen samples treated with heparin and heparin with fertility associated protein

Treatment Control Treatment with heparin Treatment with heparin and fertility associated protein

Bull group

Per cent of F pattern sperm cells during post thaw incubation

Immediate

60 min

120 min

180 min

Group I

51.73± 2.11a

43.19± 2.061b p

40.38± 2.011b p

37.48± 2.211b p

Group I

51.73± 2.11a

31.03±1.841b q

22.18±1.911c q

15.76±1.871d q

Group II Group II Group I

Group II

48.19± 2.39a 48.19± 2.39a 51.73± 2.11a 48.19± 2.39a

37.11± 2.112b x 37.43±2.162b x 30.19± 1.43b q 33.32±1.17b y

34.48± 2.142b x

31.44± 2.282b x

27.81±1.962c y

21.48± 1.592d y

24.07±1.99c z

17.60±1.54d z

21.87± 1.60c q

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 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, z in a column differ significantly at P< 0.01

15.63± 1.49d q

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 characters during the incubation at 37° C for 180 min (Table 6)

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 capacitatedacrosome intact sperm cells (- 0.488), AR pattern capacitated acrosome reacted

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 (treated with H2O2and withfertility associated protein) are presented in Table 11.

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.

When the semen samples were treated with fertility associated protein,

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 characters during the incubation at 37° C for 180 min (Table 6)

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 with heparin and heparin with fertility associated protein Treatment Control Treatment with heparin Treatment with heparin and fertility associated protein

Bull group

Per cent of B pattern sperm cells during post thaw incubation

Immediate

60 min

Group I

44.12± 1.86 a

50.86± 1.821 b p

Group I

44.12± 1.86a

62.37± 1.921b q

Group I

44.12± 1.86 a

63.24±1.55b q

Group II

46.30± 1.88 a

Group II Group II

46.30± 1.88 a 46.30± 1.88a

55.73±1.762 b x 54.68±1.652b x

59.61± 1.87 b y

120 min

53.10±1.761 b p

56.971.69 2 b x

69.79± 1.971c q

180 min

54.79± 1.701 b p

58.43±1.58 2 b x 74.48± 1.98ic q

62.30±2.662c y

66.28±2.16ii c y

66.57± 1.71c z

71.21± 1.23 d z

69.78± 1.41c q

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 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, z in a column differ significantly at P< 0.01

74.29± 1.33 c q

Table 20: Per cent AR pattern sperm cells in frozen thawed bull semen samples treated with heparin and heparin with fertility associated protein Treatment Control Treatment with heparin Treatment with heparin and fertility associated protein

Bull group Group I

Group II Group I

Group II Group I

Group II

Per cent of AR pattern sperm cells during post thaw incubation

Immediate

60 min

120 min

4.15±0.57a

6.09±0.78 b

6.51±0.87 bc

4.15±0.57a

6.60±0.81b

8.03±1.08 bc

PQ

4.15±0.57a

6.60±0.72 ab

8.54±0.77 b

Q

5.50±0.72 a

7.07±0.59 ab

5.50±0.72a 5.50±0.72a

7.14±0.82b 7.89±0.71b

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 of group I bulls with different superscripts P, Q in a column differ significantly at P< 0.05 Means of group II bulls with different superscripts x, y in a column differ significantly at P< 0.01

P

180 min

7.76±0.711 c

P

9.76±0.80 c

Q

8.55±0.90bc

10.14±0.732c x

9.90±0.73bc

12.32±0.94c y

9.37±0.45 b

10.08± 0.60 c

Q

11.15± 0.63c

xy

(- 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). 4.2.6.

DNA integrity Sperm cells with intact DNA assessed by acridine orange stainingin

frozen thawed semen samples of group I and II bulls with different treatments (treated with H2O2and withfertility associated protein) are presented in Table 12.

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 sperm characters during the incubation at 37° C for 180 min (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 protein) are presented in Table 13.

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 characters during the incubation at 37° C for 180 min (Table 6)

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 protein) are presented in Table 14.

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 characters during the incubation at 37° C for 180 min (Table 6)

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 presented in Table 15.

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 characters during the incubation at 37° C for 180 min (Table 6)

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 Parameter Progressive forward motility %

Non progressive motility

Total motility Static cells Curvilinear Velocity

VCL (µm/s)

Straight-line Velocity

VSL (µm/s)

Bull

groups

Immediate

60 min

Post thaw

Control

Group I

62.88±2.55

47.61±4.50

Group II

63.55±2.08p w

48.53±3.93a q

Group I

27.60±1.92 w

31.01±1.58 a

Group II Group I

Group II Group I

pw

28.02±2.38 w

90.48±2.19

pw

91.56±2.26 p w 9.53±2.19 p w

qA

120 min

FAP

treatment

39.23± 3.57

21.28±2.89 Bz

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

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

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

16.61±3.25 x

32.71±7.66 q r

35.51±3.38 y

43.46±7.39 r

77.42 ± 1.91x

18.69±5.11 pq

24.63±2.08 x

30.80±2.32

34.45±2.41 y

68.40±6.18 q r

59.63± 2.00 y

30.81±6.16 q r

40.37±1.99 y

Group II

10.78±4.39 p w 72.11±4.29 p w

65.61±3.87Apq

57.49±3.45 B x

Group II

73.49±6.15 p w

69.07±4.46Apq

57.31±1.41 B x

54.61±3.76 q

Group I

30.40±2.42 p w

26.19±1.45 pq

22.43±1.54 x

Group II

30.39±2.77 p w

26.87±1.93 pq

21.63±1.58 x

Group I

FAP treatment

29.49± 5.03A s

80.81±5.27 q 21.36± 5.21q

treatment

Control

31.32±3.06 B y

40.35±2.52 B x

39.30± 5.53

FAP

Ar

Bx

32.28±2.51A

78.62 ± 5.22 q

Control

180 min

60.99±5.59 q r

51.76±2.48 x

24.55±3.80 A

36.12±2.59 By

53.15±8.68 r

51.96± 1.99 z

43.68±8.80 r

48.05±1.98 z

42.22±3.48 z

51.75±6.29 r

45.52±2.77 y

56.04±2.34 x

53.39±4.64 q

50.68±3.42 x

22.09±2.16 q r

21.99±1.14 x

18.12±2.00 r

18.55±0.85 y

20.20±0.97 q

23.42±2.12 x

19.74±1.20 q

18.79±2.06 y

Data shown all mean ± SEM (n = 11) 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 Parameter Average Path Velocity VAP (µm/s) Linearity %

Straightness % Wobble % Amplitude of lateral head displacement ALH (µm) Beat / Cross Frequency BCF (Hz)

Bull

groups

Immediate

60 min

FAP

120 min

FAP

180 min

FAP treatment

Post thaw

Control

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

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

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 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 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

2.91±0.11

3.18±0.18

Group II Group II Group II Group I

41.71±1.90

69.92±2.14 w 59.42±1.23 3.36±0.16

39.19±1.86 65.97±1.89 59.12±1.20 3.29±0.15

treatment

40.06±2.99

64.21±2.38 w x 58.55±1.21

Control

37.71±1.47

treatment

41.31±1.84

65.03±1.75

66.49±2.94 w x

57.84±1.00 a

62.14±0.35 b 2.82±0.13

Control

38.08±1.82 65.45±2.40 58.08±1.35 2.82±0.21

38.66±1.72

62.79±2.50 x 58.52±0.89 2.57±0.12

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

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

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

Data shown all mean ± SEM (n = 11) 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.

min (16.61 ± 3.25 vs 21.36 ± 5.21), at 120 min (35.51 ± 3.38 vs 32.71 ± 7.66) and at 180 min of incubation (42.22 ± 3.48 vs 43.46 ± 7.39).

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).

There was no significant difference between fertility associated protein

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).

There was no significant difference between fertility associated protein

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 min (37.07 ± 1.99 vs 48.53 ± 3.93), at 120 min (25.18 ± 2.12 vs 37.60 ± 4.45) and at 180 min of incubation (15.85 ± 2.66 vs 28.61 ± 5.29).

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).

There was no significant difference between fertility associated protein

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).

There was no significant difference between fertility associated protein

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 presented in Table 17.

``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 capacitatedacrosome intact sperm cells (- 0.503), AR pattern capacitated acrosome reacted

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 characters during the incubation at 37° C for 180 min (Table 6)

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 characters during the incubation at 37° C for 180 min (Table 6)

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 characters during the incubation at 37° C for 180 min (Table 6)

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 Progressive forward motility (%) Non progressive forward motility (%) Static cells (%) Plasma membrane Integrity (%) Functional membrane integrity (%) F pattern cells (%) B pattern cells (%) AR pattern (%) MDA (µ mol/ml) Mitochondrial membrane Potential (%) Lost mitochondrial membrane potential (%) Curvilinear velocity (µ m/s) Amplitude of lateral head displacement (µm) Linearity (%) Straightness (%) DNA integrity (%) Data shown all mean ± SEM (n = 88).

Rank 1 (n= 5)

Rank 2 (n=5)

Rank 3 (n=1)

30.20±2.30

27.49±2.83

24.15

42.65±3.10

31.64±2.33

27.79

39.35±3.14

29.18±2.03

23.84

66.04±2.43

46.17±0.97

41.35

33.96±2.45

53.91±1.59

58.43

3.51±0.12

2.86±0.10

2.98

58.02±2.65

12.00±4.25

48.13±1.95 46.81±1.96 5.07±0.35 3.60±0.33

74.61±3.02

37.54±1.23 28.38±1.47 95.41±1.03

35.24±1.30

37.28±4.09

39.58±2.72 53.93±2.71 6.56±1.24 3.13±0.29

53.45±2.94

40.28±2.28 21.41±1.072 92.98±0.47

27.80

48.08

36.60 54.20 9.20 2.71

48.45

35.60 17.28 94.91

Table 22: In vitro sperm characters after incubation for 180 min in bull group II In vitro characters Progressive forward motility (%) Non progressive forward motility (%) Static cells (%) Plasma membrane Integrity (%) Functional membrane integrity (%) F pattern cells (%) B pattern cells (%) AR pattern (%) MDA (µ mol/ml) Mitochondrial membrane Potential (%) Lost mitochondrial membrane potential (%) Curvilinear velocity (µ m/s) Amplitude of lateral head displacement (µm) Linearity (%) Straightness (%) DNA integrity (%)

Data shown all mean ± SEM (n = 88).

Rank 1(n=4)

Rank 2 (n=4)

Rank 3 (n=3)

31.76±3.49

29.11±3.47

24.85±1.16

43.33±3.21

27.81±4.20

26.77±1.02

39.45±3.35

24.83±3.83

50.98±2.05

17.26±2.11

48.84±2.05 45.63±1.95 5.52±0.38 3.09±0.48

45.24±1.59 25.41±2.65

34.80±0.34 38.42±2.40 23.43±1.35

61.07±2.03

32.11±5.05 59.78±5.32 8.11±0.37 2.64±0.27

56.66±2.00

30.69±1.21 58.76±1.27 10.55±1.09 2.28±0.29

38.98±5.15

43.36±5.00

52.97±0.33

3.48±0.14

3.06±0.18

2.70±0.08

72.59±3.10

36.49±2.43 26.71±2.43 93.91±1.32

60.21±6.49

39.27±2.20 23.26±2.35 92.24±0.82

46.93±0.94 52.61±0.75 42.63±1.29 22.48±0.51 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

5.1.1

Electrophoretic profile of seminal plasma proteins

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). 5.2.3.

Plasma membrane integrity 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.

When semen samples were treated with H2O2 to induce oxidative stress,

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

in vitro sperm characters

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. 5.2.5.

Mitochondrial membrane potential of sperm cells 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).

When semen samples were treated with H2O2 to induce oxidative stress,

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. 5.2.6.

DNA integrity 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 (FuentesMascorro 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 uPAdependentMMP-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.

When the semen samples were treated with fertility associated protein,

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 vitro sperm characters

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 SDSPAGE 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



Exogenous addition of fertility associated proteins preserved the

damages to sperm plasma membrane and organelles.

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) 2)

All the bulls which have passed the breeding soundness examination

should be evaluated for the presence of fertility associated antigen.

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, Trishydroxyl 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

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

Sample buffer 5X

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

Dist water to make volume

:

100 ml

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

Dist water to make volume

:

100 ml

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

Separating gel 8 %

Volume 8 ml

5 µl

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 buffer

2 ml

Stacking buffer

0.37 ml

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

Ingredients

Stacking gel

Quantity

Ingredients

5%

Volume

Volume

1.32 ml

1.76 ml

3 ml

4 ml

Dist Water

2.3 ml

Dist Water

30 % Acrylamide

0.48 ml

0.64 ml

1.5 M separating buffer

1.75 ml

Stacking

0.6 ml

0.8 ml

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

30 % Acrylamide

2.8 ml

Separating gel 15 % ml Ingredients

buffer

Volume 7 Quantity

Stacking gel Ingredients

Dist Water

1.92

Dist Water

1.5 M separating buffer

2 ml

SDS 10 %

70 µl

TEMED

5 µl

30 % Acrylamide

APS 10 %

4 ml

80 µl

30 % Acrylamide

Volume 3 ml

1.32 ml

5% Volume 4 ml

1.76 ml

0.48 ml

0.64 ml

Stacking

0.6 ml

0.8 ml

SDS 10 %

24 µl

32 µl

TEMED

5 µl

5 µl

buffer

APS 10 %

10 µl

10 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

Ingredients

Quantity

Methanol

100 ml

Glacial acetic acid

20 ml

Dist water

80 ml Destaining solution II

Ingredients

Quantity

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

l

-

Microgram/millilitre

-

microlitre

-

Bovine Serum Albumin

-

Calcium ions

g/ml BSA º

C

Ca++

-

DMSO

-

DPBS

-

DNA

EDTA h

mM

Deoxyribonucleic acid

-

Ethylenediamine tetra acetic acid

-

Milli Molar

-

-

s

-

vs.

-

kDa

-

TALP V

Dimethyl Sulfoxide

-

min

ng/ml

Celsius Degree

Dulbeccos Phosphate Buffered Saline Hour

Minutes

-

Nanogram/milliliter

-

Tyrodes Albumin Lactate Pyruvate

seconds Versus

-

Volt

mOSM

-

Milli osmol

m

-

nm

mm

-

-

Kilo Dalton Nano meter

Micro meter

Milli meter