ANTIOXIDANT FORTIFICATION OF SEMEN EXTENDER TO …

ANTIOXIDANT FORTIFICATION OF SEMEN EXTENDER TO

IMPROVE FREEZABILITY AND FERTILITY OF BUFFALO BULL

SPERMATOZOA

Muhammad Sajjad Ansari

(03-arid-781)

Department of Zoology

Faculty of Sciences Pir Mehr Ali Shah

Arid Agriculture University Rawalpindi Pakistan

2011

ii

ANTIOXIDANT FORTIFICATION OF SEMEN EXTENDER TO

IMPROVE FREEZABILITY AND FERTILITY OF BUFFALO BULL

SPERMATOZOA

By


(03-arid-781)

A thesis submitted in partial fulfillment of the

requirement of the degree of

Doctor of Philosophy

in

Zoology

Department of Zoology

Faculty of Sciences Pir Mehr Ali Shah

Arid Agriculture University Rawalpindi Pakistan

2011

iii

CERTIFICATION

I hereby undertake that this research is an original and no part of this thesis falls

under plagiarism. If found otherwise, at any stage, I will be responsible for the

consequences

Student’s Name: Muhammad Sajjad Ansari Signature: _________________

Registration No.: 03-arid-781 Date: _________________

Certified that the contents and the form of thesis entitled “Antioxidant

fortification of semen extender to improve freezability and fertility of buffalo bull

spermatozoa” submitted by Muhammad Sajjad Ansari have been found satisfactory

for the requirement of the degree.

Supervisor: ________________________________ (Dr. Shamim Akhter)

Member: __________________________________ (Dr. Afsar Mian)

Member: __________________________________

(Dr. Nemat Ullah)

Chairperson, Department of Zoology: ______________________

Dean, Faculty of Sciences: ______________________

Director, Advanced Studies: ______________________

iv

Dedicated to

Dr. Shamim Akhter

&

Dr. Nemat Ullah

v

CONTENTS

Page

ACKNOWLEDGEMENTS xiii

LIST OF ABBREVIATIONS xii

LIST OF TABLES ix

LIST OF FIGURES x

1. INTRODUCTION 1

2. REVIEW OF LITERATURE 8

2.1. DAIRY INDUSTRY IN PAKISTAN 8

2.2. MILK YIELD OF BUFFALO 9

2.3. AI IN BUFFALO 10

2.4. FREEZE-THAWING MEDIATED DAMAGE 11

2.5. OXIDATIVE DAMAGE TO SEMEN 12

2.5.1. Sperm Motility 13

2.5.2. Sperm Plasmalemma Integrity 14

2.5.3. Sperm Acrosomal Integrity 15

2.5.4. Sperm DNA Integrity 16

2.5.5. Sperm Lipid Peroxidation 18

2.5.6. Capacitation Status 19

vi

2.5.7. Antioxidant Activity 19

2.6. ANTIOXIDANT MEDIATED PROTECTION 20

2.7. THIOLS IN SEMEN STORAGE 23

2.8. GLUTATHIONE 24

2.8.1. Role of Glutathione in Bovine Semen 27

2.8.2. Role of Glutathione in Caprine Semen 28

2.8.3. Role of Glutathione in Ovine Semen 30

2.8.4. Role of Glutathione in Swine Semen 31

2.8.5. Role of Glutathione in Buffalo Semen 31

2.9. CYSTEINE 33

2.9.1. Role of Cysteine in Bovine Semen 33

2.9.2. Role of Cysteine in Canine and Feline Semen 34

2.9.3. Role of cysteine in Ovine Semen 34

2.9.4. Role of Cysteine in Swine Semen 35

2.9.5. Role of Cysteine in Buffalo Semen 36

2.10. THIOGLYCOL 36

2.10.1. Thioglycol in Protection of Gametes/embryos 36

3. MATERIALS AND METHODS 38

3.1. PREPARATION OF EXTENDERS 38

3.2. BUFFALO BULL SEMEN COLLECTION 38

3.3. PROCESSING OF BUFFALO SEMEN 40

vii

3.4. SEMEN QUALITY ASSESSMENT TECHNIQUES 40

3.4.1. Motility of Buffalo Spermatozoa 40

3.4.2. Buffalo Sperm Plasmalemma Integrity 41

3.4.3. Sperm Viability 41


3.4.5. In Vivo Fertility Rate 43

3.5. STATISTICAL ANALYSIS 43

4. RESULTS AND DISCUSSION 45

4.1. EFFECT OF GLUTATHIONE IN SEMEN EXTENDER 45

ON POST-THAW QUALITY OF BUFFALO BULL

SPERMATOZOA

4.1.1. Progressive Motility 45




4.2.1. EFFECT OF CYSTEINE IN SEMEN EXTENDER 51

ON POST-THAW QUALITY OF

BUFFALO BULL SPERMATOZOA





4.3. EFFECT OF THIOGLYCOL IN SEMEN EXTENDER 62

viii

ON POST-THAW QUALITY OF

BUFFALO BULL SPERMATOZOA





4.4. FERTILITY RATE OF BUFFALO SEMEN 68

CRYOPRESERVED IN BEST

EVOLVED EXTENDERS

4.5. GENERAL DISCUSSION 73

SUMMARY 80

LITERATURE CITED 82

APPENDIX I 107

ix

LIST OF TABLES

Table No.

Page

3.1. Composition of experimental extenders. 39

4.1. Overview of the studies on the role glutathione in semen storage. 77

4.2. Overview of the studies on the role of cysteine in semen storage. 78

4.3. Overview of the studies on the role of thioglycol in gamete/embryo

protection.

81

\

x

LIST OF FIGURES

Fig. No.

Page

2.1. Glutathione redox-cycle, glutathione peroxide (GPx) reduces H2O2

and hydrogen peroxide (ROOH) using reduced glutathione (GSH).

The oxidized form of glutathione (GSSG) is regenerated by the

glutathione reductase (GR) using NADPH. The energy is usually

drawn from the hexose monophosphate shunt-system.

26

4.1. Effect of glutathione addition in semen extender on the progressive

motility of buffalo bull spermatozoa at 0, 2 and 4 hours after

thawing. Bars with different letters differed significantly (P < 0.05)

at a given time.

47

4.2. Effect of glutathione addition in semen extender on viability of

buffalo bull spermatozoa at 0, 2 and 4 hours after thawing. Bars with

different letters differed significantly (P < 0.05) at a given time.

52

4.3. Effect of glutathione addition in semen extender on plasma

membrane integrity of buffalo bull spermatozoa at 0, 2 and 4 hours

after thawing. Bars with different letters differed significantly (P <

0.05) at a given time.

53

4.4. Effect of glutathione addition in semen extender on DNA integrity

of buffalo bull spermatozoa at 0, 2 and 4 hours after thawing. Bars

with different letters show significant (P < 0.05) differences at a

given time.

54

4.5. Effect of cysteine addition in semen extender on the progressive



at a given time.

57

4.6. Effect of cysteine addition in semen extender on viability of buffalo 59

xi

bull spermatozoa at 0, 2 and 4 hours after thawing. Bars with


4.7. Effect of cysteine addition in semen extender on the plasma

membrane integrity of buffalo bull spermatozoa at 0, 2 and 4 hours

after thawing. Bars with different letters differed significantly (P <

0.05) at a given time.

60

4.8. Effect of cysteine addition in semen extender on the DNA integrity

of buffalo bull spermatozoa at 0, 2 and 4 hours after thawing. Bars

with different letters differed significantly (P < 0.05) at a given time.

61

4.9. Effect of thioglycol addition in semen extender on progressive



at a given time.

65

4.10. Effect of thioglycol addition in semen extender on viability of



66

4.11. Effect of thioglycol addition in semen extender on plasma membrane

integrity of buffalo bull spermatozoa at 0, 2 and 4 hours after


at a given time.

69

4.12. Effect of thioglycol addition in semen extender on DNA integrity of



70

4.13. Effect of glutathione, cysteine and thioglycol on fertility rate (%) of

buffalo bull spermatozoa. Bars with different letters differed

significantly (P < 0.05).

72

xii

LIST OF ABBREVIATION

AI Artificial insemination

ANOVA Analysis of variance

GSH Glutathione

HOS Hypo-osmotic swelling

ROS Reactive oxygen species

LPO Lipid peroxidation

xiii

ACKNOWLEDGEMENTS

I would like to express my sincere thanks to my supervisor Dr. Shamim Akhter,

Associate Professor, Department of Zoology, Pir Mehr Ali Shah Arid Agriculture

University, Rawalpindi, for her kind supervision and sympathetic attitude throughout

my research in M. Sc., M. Phil. and now in PhD.

I am highly privileged in taking the opportunity to thank Prof. Dr. Nemat Ullah, Dean,

Faculty of Veterinary and Animal Sciences, Pir Mehr Ali Shah Arid Agriculture

University, Rawalpindi; Prof. Dr. Afsar Mian, Department of Zoology, Pir Mehr Ali

Shah Arid Agriculture University, Rawalpindi. I would express my appreciation to Prof.

Dr. Mazhar Qayyum, Chairman, Department of Zoology, Pir Mehr Ali Shah Arid

Agriculture University Rawalpindi and Prof. Dr. Mirza Azhar Beg, Department of Zoology,

Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi for their moral and technical

support. I appreciate and thank Higher Education Commission for providing financial

assistance under “Indigenous 5000 PhD Fellowship Program”.

I express my profound gratitude, sincere thanks and sense of obligations to Dr. S. M. H.

Andrabi, Principal Scientific Officer, National Agriculture Research Centre, Islamabad

and Ms. Bushra A. R., Department of Wildlife Management, Pir Mehr Ali Shah Arid

Agriculture University, Rawalpindi for their keen interest, valuable suggestions, kind

behaviour, timely help, generous support and skilful guidance during the entire

xiv

program. I also offer my thanks to Prof. Dr. W. V. Holt, Institute of Zoology,

Zoological Society of London, for his valuable and expert inputs in this project.

It is my pleasure to thank my friends Shahid Ali Khan, and Dr. Abdul Waheed and my

roommates, Dr. Adnan Umair, Dr. Ali Muhammad, Dr. Ayaz Mehmood, and Dr. Kashif

Bashir for their kind sincere and loving attitude during the course. I also thank to Ms. Saima

Qadeer and Ms. Rabea Ejaz, Research Associates of the Animal Physiology Laboratory,

Department of Zoology, Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi and Mr.

Iftikhar Mehdi for help during the entire tenure.

A feeling of pleasure is paid to my parents, brothers (Muhammad Ijaz Ansari,

Muhammad Ayyaz Ansari, Muhammad Sarfraz Ansari, Muhammad Shahbaz Ansari)

and sisters (Sajida Perveen, Abida Perveen) and grand parents for their love and support

that they gave to me. I love you all and appreciate the encouragement.

May Allah almightily bless them all.


80

SUMMARY

Freeze-thawing of spermatozoa is an oxidative processes which accelerates

the levels of reactive oxygen species (ROS) molecules due to plasma membrane lipid

peroxidation. Moreover, overproduction of ROS molecules increases the damages to

functional and structural integrity of buffalo spermatozoa during cryopreservation.

The experiments were conducted to explore the role of glutathione (GSH), cysteine

and thioglycol supplementation in semen extender on freezability and fertility of

buffalo (Bubalus bubalis) bull semen. Semen from three Nili-Ravi buffalo bulls was

cryopreserved with experimental tris-citric acid extenders containing 0.0 mM, 0.5

mM, 1.0 mM, 1.5 mM, 2.0 mM and 3.0mM of GSH or cysteine and thioglycol.

Sperm motility (%), sperm plasmalemma integrity (%), sperm viability (%) and

sperm DNA integrity was recorded at 0, 2 and 4 hours after thawing and incubation

at 37°C. Highest (P<0.05) sperm motility, plasmalemma integrity (structural and

functional) and viability of buffalo semen were recorded in extender containing

2.0mM of GSH as compared to control at 0, 2 and 4 hours after thawing. Motility,

plasmalemma integrity (structural and functional) and viability (live sperm with

intact acrosome) of buffalo semen were better in extender containing 1.0 mM and

1.5mM of cysteine as compared to control at 0, 2 and 4 hours post-thaw. Sperm

motility, plasmalemma integrity and viability of buffalo bull spermatozoa were

higher in extenders containing 1.0 mM, 1.5 mM, 2.0 mM and 3.0 mM of thioglycol

compared to control. Sperm DNA integrity was observed higher (P < 0.05) in all

extenders containing GSH, cysteine and thioglycol compared to control. Semen from

two Nili-Ravi buffalo bulls of known freezability and fertility was cryopreserved (as

described in earlier section) in extenders containing GSH (2.0mM), cysteine

81

(1.0mM), thioglycol (1.0mM) & control. The same semen was used for artificial

insemination in buffaloes. In vivo fertility rate was higher (P<0.05) in buffaloes when

using semen cryopreserved in extenders containing GSH and cysteine compared to

control under field conditions. In conclusion, supplementation of GSH (2.0mM) and

cysteine (1.0mM) in semen extender improved the post-thaw quality and in vivo

fertility rate of buffalo semen.

1

Chapter 1

INTRODUCTION

Milk and milk products are an integral part of the dining table and a preferred

part of the diet of people in Pakistan. In spite of this, per capita consumption of milk

and milk products in Pakistan remains considerably lower as compared to that in the

developed countries (Garcia et al., 2003). This low consumption of milk is mainly due

to its unavailability at a price affordable by the public. One of the main factors

responsible for the high price of milk and/or its products is their higher cost of

production. Cost of milk production largely depends upon the milk yield per animal

per lactation. Unfortunately the average milk production by our dairy buffalo is no

more than 3000 liters per lactation and it is this low genetic potential that makes our

milk production very costly (Ullah et al., 2010).

The population figures represent that buffalo is the main animal that

contributed 70% of the total milk yield in Pakistan (Garcia et al., 2003). From 1996 to

2006, the buffalo population was increased by 34.8% and milk yield increased by

20% (Livestock Census, 2006). The increase in milk production of the country was

mainly because of increase in number of dairy animals. Unfortunately, milk yield of

the dairy buffalo is too low, and the milk production of 7 to 10 buffalo is equal to one

dairy cow in the developed countries (Garcia et al., 2003). This difference in milk

yield between our buffalo and the dairy cow of developed countries is mainly due to a

difference in genetic potentials. Different assisted reproductive biotechnologies have

been available to improve the genetic potential of the livestock species. Artificial

insemination has been extensively used by developed countries for rapid genetic

2

improvement through exploiting the germplasm of the male. The benefits of artificial

insemination technique can be fully achieved by successful freezing of semen without

compromising its fertility, and so far, this has met with a little success in buffalo

(Anzar et al., 2003; Andrabi, 2009).

Cryo-damage during freeze-thawing process to buffalo semen was higher than

cattle spermatozoa due to unique physiology of the buffalo spermatozoa and higher

polyunsaturated phospholipids levels in the plasma membrane (Nair et al., 2006;

Sreejith et al., 2006; Andrabi, 2009). The fertility with frozen semen in buffaloes

under field conditions is very poor and has been considered as 30% (Abhi, 1982;

Chohan et al., 1992; Anzar et al., 2003; Andrabi, 2009). It is surprising that fertility

rate of 50% after AI with frozen semen in buffaloes is considered as good while the

same in cow is considered as poor (Andrabi, 2009). Cryopreservation processes

deteriorate the fertility of buffalo bull semen probably by affecting the sperm motility,

viability, plasma membrane, acrosomal and DNA integrity (Andrabi, 2009). The

reason for poor fertility rate in buffaloes under field conditions is poor post-thaw

characteristics of buffalo semen. This may be the reason that only 7% buffaloes are

bred through artificial insemination technique (Livestock Census, 2006). As a matter

of fact, the unique physiology of buffalo sperm requires buffalo specific semen

extender to reduce the cryo-damages. Improving the semen extender for the

cryopreservation that ensures no or little damage to sperm motility, plasmalemma,

acrosomal and chromatin integrity might enable us to achieve a comparatively higher

fertility rate with artificial insemination in buffalo under field conditions.

3

It is well documented that motility, plasma membrane, acrosome and DNA

integrity of buffalo bull spermatozoa significantly decreased after the process of

cryopreservation (Rasul et al., 2000; 2001; Kadirvel et al., 2009; Anzar et al., 2010;

Kumar et al., 2011). It was also observed that the process of cryopreservation induced

apoptosis in cryopreserved buffalo bull spermatozoa along with reduction in semen

quality parameters viz; motility and plasmalemma integrity (Khan et al., 2009).

Cryopreservation of buffalo semen increased the levels of reactive oxygen species

molecules that caused the lipid peroxidation of the bio-membrane system by reducing

the antioxidant potential of the cryopreserved semen (Kadirvel et al., 2009; Kumar et

al., 2011). The sources of the reactive oxygen species molecules were considered

impaired mitochondrial system and dead/damaged spermatozoa in cryopreserved

buffalo semen (Kadirvel et al., 2009). Motility, plasmalemma integrity and fertility of

bull semen negatively correlated with lipid peroxidation levels (Kasimanickam et al.,

2007).

The viability of the buffalo bull spermatozoa was reduced up to 50% during

the process of dilution, equilibration, freezing and thawing (Kumar et al., 2011; Rasul

et al., 2001; Anzar et al., 2010; Kadirvel et al., 2009). Oxidative stress during the

process of cryopreservation was considered one of the reason for reduced post-thawed

buffalo semen quality because of higher sensitivity of the buffalo sperm (Kumaresan

et al., 2005; 2006; Nair et al., 2006) which might be due to presence of higher

contents of polyunsaturated phospholipids present in sperm membrane (Sansone et

al., 2000). Overproduction of reactive oxygen species molecules increased the

damages to functional and structural integrity of the buffalo sperm during freeze-

thawing process (Kumaresan et al., 2005; 2006; Garg et al., 2009). Naturally, semen

4

is equipped with defensive system comprising enzymatic (catalase, glutathione

peroxidase, superoxide dismutase) and non-enzymatic (vitamin C, vitamin E,

glutathione, cysteine) antioxidants which protected the sperm from reactive oxygen

species mediated injuries (Bilodeau et al., 2000; 2001; 2002; Garg et al., 2009;

Andrabi, 2009). The indigenous antioxidant system is reported insufficient (Bilodeau

et al., 2000; Baumber et al., 2005; Nichi et al., 2006) to protect the sperm motility,

plasma membrane, acrosome, viability and DNA integrity from oxidative stress

during dilution, equilibration, freezing and thawing which causes higher lipid

peroxidation in buffalo semen (Kadirvel et al., 2009; Nair et al., 2006; Kumar et al.,

20011). In bovine semen activity of catalase enzyme decreased by 50% in frozen-

thawed semen compared to fresh ejaculate. Bilodeau et al. (2000) reported that the

level of glutathione and superoxide dismutase decreased by 78% and 50% in the

process of dilution, equilibration, freezing and thawing of bovine semen. In equine

semen, after dilution motility and plasmalemma integrity was highly positively

correlated with glutathione peroxidase and catalase activity in semen-extender (Pagl

et al., 2006). The freeze-thawing cycle also reduced the level of indigenous

antioxidants in bull semen (Alvarez and Storey, 1992; Beconi et al., 1993; Bilodeau et

al., 2000; Stradaioli et al., 2007). During freeze-thawing of buffalo bull spermatozoa

extra antioxidant supplements are required to protect the sperm functional and

structural intactness (Andrabi et al., 2008; Kumaresan et al., 2005, 2006).

The integrity of the sperm DNA is crucial for the development of viable

embryos following the completion of fertilization process (Andrabi, 2008). Chromatin

stability of the spermatozoa strongly correlated with poor/sub fertility of the bovine

semen (Ballachey et al., 1987; 1988). During the process of in vitro fertilization DNA

5

fragmentation of the spermatozoa strongly affected the fertility rate (Sun et al., 1997).

It is well recognized that reduction in sperm DNA integrity of mammalian

spermatozoa was highly related with higher membrane lipid peroxidation levels (Potts

et al., 2000) and oxidative stress (Hughes et al., 1996). Increase in reactive oxygen

species molecules have been observed and found associated with DNA fragmentation

after the cryopreservation process (Baumber et al., 2003). In bovine, semen lipid

peroxidation of sperm plasma membrane highly correlated with DNA fragmentation

index (Kasimanickam et al., 2007). The DNA integrity of buffalo semen in fresh

semen varied from 10 to 12% and 35% in frozen-thawed semen (Kumar et al., 2011).

Glutathione, cysteine and thioglycol are thiols which protected the sperm cell

from oxidative stress during cryopreservation artificially induced by hydrogen

peroxide (Ansari et al., 2010; 2011a,b; Bilodeau et al., 2001). Cysteine and thioglycol

can directly neutralize free radicals and/or act through glutathione mediated pathway

in the cell. Cryopreservation resulted in decreased thiols level in bovine semen

(Bilodeau et al., 2000; Stradaioli et al., 2007). Moreover, supplementation of

glutathione and cysteine in semen extender resulted in improved quality of semen

after thawing (Bilodeau et al., 2001; Tuncer et al., 2010; Ansari et al., 2010; 2011b).

In an another study, glutathione and cysteine addition in bovine semen extender

resulted in a decrease of the lipid peroxidation levels during freezing and ultimately

improved fertility rates with semen cryopreserved in extender containing glutathione

(Perumal et al., 2010). In some studies on chilled and frozen-thawed buffalo semen, it

was demonstrated that sperm motility, normal apical ridge and tail-plasmalemma

integrity was significantly better with the addition of glutathione and cysteine in

extender (Ansari et al., 2010; 2011a,b).

6

Several reports described that thioglycol protected the oocytes/embryo

viability by direct interaction with oxidized radicals and/or increasing intercellular

glutathione levels in oocytes (Takahashi et al., 2002). It was also reported that

thioglycol can influence the ATP metabolism of the oocyte and improved the number

of cumulus cells (Tsuzuki et al., 2005). Thioglycol showed the ability to prevent the

apoptosis of cloned embryos by antioxidant activity (Park et al., 2004a,b). A high

sperm oocyte penetration and late embryonic development was observed in the

presence of thioglycol in culture media (Funahashi, 2005). Thioglycol protected the

motility of bovine when spermatozoa in artificially induced oxidative stress was by

hydrogen peroxide in vitro (Bilodeau et al., 2001). Information regarding the role of

glutathione, cysteine and thioglycol at different concentrations in extender on

functional and structural plasmalemma intactness, viability, DNA integrity and in vivo

fertility of cryopreserved buffalo bull spermatozoa was lacking in published literature.

Therefore, it was hypothesized that glutathione, cysteine and thioglycol

supplementation in the freezing extender may improve the antioxidant potential of the

buffalo semen, which may be helpful in maintaining the quality and in vivo fertility of

buffalo semen in frozen state. Therefore, the study was planned to identify the impact

of various levels of glutathione, cysteine and thioglycol in semen extender on quality

and in vivo fertility of the Nili-Ravi buffalo bull spermatozoa.

The study was planned with following objectives:

1. To identify the impact of various levels of glutathione in semen extender on

post-thaw quality [motility, viability (live sperm with intact acrosome),

7

plasmalemma (structural and functional) and DNA integrity] of the Nili-Ravi

buffalo bull spermatozoa

2. To identify the impact of various levels of cysteine in semen extender on post-

thaw quality [motility, viability (live sperm with intact acrosome),



3. To identify the impact of various levels of thioglycol in semen extender on

post-thaw quality [motility, viability (live sperm with intact acrosome),



4. To determine the fertility rate of the Nili-Ravi buffalo bull spermatozoa frozen

in best evolved extenders

8

Chapter 2

REVIEW OF LITERATURE

2.1. DAIRY INDUSTRY IN PAKISTAN

In practical terms production of dairy items depends on buffalo and cattle in

Pakistan. The total population of buffalo and cattle in Pakistan are 27.33 and 29.36

million, respectively (Livestock Census, 2006). Although in our dairy industry cattle

population is higher than buffalo 70% of the total milk yield of the country is

contributed by buffalo (Garcia et al., 2003). These figures represent that the buffalo is

our main dairy animal in terms of milk production and has a higher production

potential than local cattle. In Pakistan, most of the cattle breeds are having lower milk

production potential.

According to Livestock Census (2006), the population of buffalo and cattle

increased by 34.8% and 44.7% from 1996 to 2006. However, the milk yield was

increased by 20% in buffalo and 11% in cattle with an overall increase of 17%. The

increase in milk production of the country was mainly are to the increase in the

number of dairy animals in Pakistan. The reason for poor milk yield of dairy animals

is the lack of effective genetic improvement program at national level. It is surprising

that to improve the genetic potential of the dairy animals an artificial insemination

program existed in the country from many decades but it did not contribute

effectively. The reason for the failure of the genetic improvement program in Pakistan

is poor acceptability of the artificial breeding among the farmers through frozen

9

semen because with frozen semen fertility rates are poor which resulted in financial

losses (Andrabi, 2009).

2.2. MILK YIELD OF BUFFALO

A careful analysis revealed that the increase in milk production in the buffalo

has mainly been due to an increase in the population of buffalo rather than any

increase in milk yield/animal. Ideally, any increase in milk production would have

been achieved by increasing the yield per animal as maintaining/feeding additional

animals means extra pressure on existing land resources which has now started to

affect our cereal crops production. Unfortunately, milk yield of the dairy buffalo is so

low that the production of 7 to 10 buffalo is equal to one dairy cow in the developed

countries (Garcia et al., 2003).

This huge difference in productivity between our buffalo and the dairy cow of

the West is mainly due to a difference in their genetic potentials (Garcia et al., 2003).

It is pertinent to mention that milk production of the dairy cow of the western

countries was just similar to the present milk production of our dairy buffalo. They

improved the milk yield of their dairy animals by improving the genetic potential of

the dairy animals adopting different assisted reproductive biotechnologies. We can

also improve the milk production of our dairy buffalo by improving their genetic

potential through development and successful application of suitable reproductive

techniques like artificial insemination (AI).

10

2.3. AI IN BUFFALO

AI is a technique that has been exploited by the western countries to improve

the genetic potential of their livestock breeds by exploiting the germplasm of superior

sires. To achieve the maximum benefits of the AI, successful freezing of the semen is

required along with an acceptable fertility rate; so far this has been met with little

success (Anzar et al., 2003).

Cryodamage during freeze-thawing of buffalo semen is higher than cattle

spermatozoa due to the unique physiology of the buffalo spermatozoa and higher

polyunsaturated phospholipids levels in the plasma membrane (Andrabi, 2009). The

fertility with frozen semen in buffalo under field conditions is very poor and has been

recorded as 45.6% (Shabbir et al., 1982), 31.7% (Abhi, 1982), 41.8% (Sharma and

Sahni, 1988), 33.22% (Chohan et al., 1992) and 19.9% (Anzar et al., 2003). The

reason for poor fertility rates in buffaloes under field conditions is poor post-thaw

quality of frozen semen. This is the reason that only 7% buffalo are bred through

artificial insemination technique (Livestock Census, 2006). The unique physiology of

the buffalo spermatozoa requires to adopt suitable approaches that ensure little

damage to buffalo spermatozoa thus resulting in acceptable fertility rates under field

conditions. By improving the fertility rate we can increase the acceptability of

artificial insemination technique among the farmers that will enhance the production

potential of the dairy buffalo (Andrabi, 2009).

11

2.4. FREEZE-THAWING MEDIATED DAMAGE

It is believed that sperm viability is reduced upto 50% after the freeze-thawing

process (Watson, 1995; Rasul et al., 2001; Kumar et al., 2011; Shannon and

Vishwanath, 1995; Hammerstedt et al., 1990). Freezing and thawing stages are

mainly responsible during cryopreservation for the reduction in semen quality

(Andrabi, 2009). In a recent study, motility, plasma membrane, acrosome and DNA

integrity of buffalo semen significantly decreased after cryopreservation (Kumar et

al., 2011). It was observed that cryopreservation process induced the apoptosis in

cryopreserved buffalo semen along with reduction in basic semen quality parameters

like motility and plasmalemma integrity (Khan et al., 2009).

The acrosome is a large Golgi/endoplasmic reticulum derived acidic secretory

organelle. It is filled with hydrolytic enzymes that are organized in a kind of enzyme

matrix and most enzymes are heavily glycosylated. The presence of normal acrosome

on a spermatozoon is essential for the acrosomal reaction that is being required at the

proper time to facilitate fertilization. The change in acrosomal cap is mainly due to

sperm aging or cryoinjury, which can be effectively determined by fixing the

specimen using phase contrast microcsopy. A high correlation between the percentage

of intact acrosome and fertility of frozen bovine spermatozoa was observed after 2

and 4 h of post-thaw incubation. Reduction in sperm motility and normal apical ridge

(O’Connor et al., 1981), extracellular release of enzymes viz; glutamic oxacloacetic

transaminase and acrosin (Graham et al., 1972; Palencia et al., 1996) and reduced

conception rate (Shannon and Vishwanath, 1995) are evidences of damage in bovine

semen.

12

2.5. OXIDATIVE DAMAGE TO SEMEN

The major limiting factor in mammalian semen preservation which

deteriorated the semen quality (Baumber et al., 2003; Sikka, 2004; El-Sisy et al.,

2007) and fertility (Maxwell and Salamon, 1993; Vishwanath and Shannon, 1997)

was oxidative stress. This resulted in the production of reactive oxygen species

molecules (ROS; superoxide, hydroxyl, hydrogen peroxide, nitric oxide,

peroxynitrile) (Baumber et al., 2005). The ROS increased the lipid peroxidation

(LPO) levels of unsaturated fatty acids in the membrane (El-Sisy et al., 2007;

Kadirvel et al., 2009).

Sperm respiration for energy production and the presence of dead/damaged

spermatozoa in semen were major sources of ROS production (Vishwanath and

Shannon, 1997). It is well documented that during preservation of buffalo semen,

oxidative stress caused the over production of ROS, resulting in higher LPO levels in

the sperm cell membrane (Nair et al., 2006; El-Sisy et al., 2007; Kadirvel et al.,

2009). The resulting oxidative stress is likely to cause mitochondrial dysfunction and

deterioration of sperm motility, viability, plasmalemma integrity and sperm

morphology (Nair et al., 2006; El-Sisy et al., 2007; Garg et al., 2009; Kadirvel et al.,

2009). Moreover, neither protection nor repair systems for sperm integrity are

available and, therefore, addition of exogenous protectants is required to reduce this

ROS-mediated damage (Vishwanath and Shannon, 1997). Additionally, oxidative can

stress cause DNA damage in spermatozoa (Donnelly et al., 1999; Andrabi, 2008).

13

It was reported that damaged/abnormal spermatozoa especially bovine

spermatozoa with abnormal mid piece and residual cytoplasm caused the production

of hydrogen peroxide in semen (Gadea et al., 2007). Free radical production during

cryopreservation was responsible for the deterioration of frozen-thawed semen quality

in bovine (Gadea et al., 2007) that cause cryo-injuries to spermatozoa (Wang et al.,

1997; Bilodeau 2002). It was observed that hydrogen peroxide production was the

main ROS molecule that caused damages to bovine spermatozoa during

cryopreservation in tris-egg yolk based extenders (Bilodeau et al., 2002) rather than

other free radicals.

2.5.1. Sperm Motility

It is pertinent to mention that subjective motility assessed post-thaw was

significantly correlated (r = 0.6772; P = 0.033) with in vivo fertility of bovine frozen

semen (Gillan et al., 2008).

Cryopreservation of mammalian semen reduced the motility and accelerated

the production of ROS molecules through lipid peroxidation levels (Rasul et al., 2001;

Chattergee and Gagnon, 2001; Kadirvel et al., 2009; Anzar et al., 2010) beyond the

physiological levels (Bailey et al., 2000). Sperm motility decreased after

cryopreservation along with the total antioxidant potential of the semen (Kumar et al.,

2011). It has been observed that levels of ROS in mammalian semen negatively

correlated with the motility (Kadirvel et al., 2009). The ROS molecules like H2O2

reduced the motility of buffalo and bull spermatozoa in vitro (Bilodeau et al., 2001;

Garg et al., 2009). It was noted that the motility improve recent in Bioxcell after

14

cryopreservation was due to the presence of glutathione at higher concentrations

(Stradaioli et al., 2007). In bovine semen addition of exogenous hydrogen peroxide

and/or production by enzymatic oxidation of phenylalanine reduced the motility of the

spermatozoa (Krzyzosiak et al., 2000). Motility was negatively correlated with lipid

peroxidation of bovine semen (Kasimanickam et al., 2007). In a another study, Khan

et al. (2009) reported reduction in motility of buffalo spermatozoa after the freeze

thawing process from 64.0 ± 2.1 to 49.4 ± 1.3%.

2.5.2. Sperm Plasmalemma Integrity

Cryopreservation of mammalian semen increased the levels of ROS molecules

that caused the lipid peroxidation of the membrane by reducing the antioxidant

potential of the cryopreserved semen (Kadirvel et al., 2009). Higher lipid peroxidation

levels beyond the physiological level caused higher fluidity of the cell membrane that

deteriorated the plasmalemma integrity of spermatozoa (Storey, 1997). The sources of

the ROS molecules were impaired mitochondrial system and dead/damage

spermatozoa in semen (Kadirvel et al., 2009). In vitro studies revealed that hydrogen

peroxide, a major naturally occurring ROS molecule in the semen (Kadirvel et al.,

2009) decreased the plasmalemma integrity of sperm (Garg et al., 2009). In bovine

semen addition of hydrogen peroxide and/or production by enzymatic oxidation of

phenylalanine reduced the plasmalemma integrity of spermatozoa (Krzyzosiak et al.,

2000). In frozen-thawed buffalo semen, damaged plasmalemma integrity as

determined by hypo-osmotic swelling technique was recorded 79.6 ± 0.5%, compared

to fresh semen that was observed 38.7 ± 0.3%. Plasmalemma integrity of the bovine

15

spermatozoa had negative association with lipid peroxidation of the bio-membrane

system (Kasimanickam et al., 2007).

2.5.3. Sperm Acrosomal Integrity

The assay used for the assessment of viability of buffalo spermatozoa was

Trypan-blue Giemsa that describes the live/dead status of the sperm and acrosomal

integrity simultaneously in the same semen sample. Sperm viability assessed through

this technique highly correlated (R2 = 0.62, P = 0.02) with in vitro fertility

(Tartaglione and Ritta, 2004). The viability of the frozen-thawed bovine semen was

also highly correlated with in vivo fertility under field conditions (r = 0.636, P =

0.048).

The freeze-thawing process decreased the population of sperm with intact

acrosomes (Rasul et al., 2001; Anzar et al., 2010; Kumar et al., 2011). The freeze-

thawing processes decreased the antioxidant potential of the semen during

cryopreservation and increased the lipid peroxidation levels and ROS molecules

(Kumar et al., 2011; Kadirvel et al., 2009). In vitro studies have already demonstrated

that hydrogen peroxide could reduce the percentage of sperm with intact acrosomes

(Garg et al., 2009). Higher percentage of acrosomal integrity of swine spermatozoa is

associated with higher levels of naturally occurring antioxidant semen (Gadea et al.,

2004). Naturally occurring antioxidants in semen protect the acrosomal integrity of

the spermatozoa by reducing levels of ROS molecules and lipid peroxidation of cell

membrane (Corton et al., 1989). Naturally occurring antioxidant added to semen

16

extender improved the sperm population with intact acrosomes in caprine and buffalo

semen (Sinha et al., 1996; Ansari et al., 2010; 2011a; 2011b).

2.5.4. Sperm DNA Integrity

The integrity of the sperm DNA integrity is critical for the development of

viable embryos following the completion of fertilization process (Andrabi, 2008). It

was observed that higher sperm DNA damage reduced the embryo development

(Benchaib et al., 2003; Yildiz et al., 2007). Severe damage to the spermatozoa DNA

integrity reduced its fertilization potential. It is interesting that sperm with apparently

normal motility, motion characteristics, intact plasma membrane and organelles could

fertilize the oocytes and produced embryos successfully. But development of the

embryos was impaired at four to eight cell stage and apoptosis initiated, if sperm

DNA fragmentation had occurred due to any reason (Seli et al., 2004; Yildiz et al.,

2007). Chromatin stability of the spermatozoa strongly correlated with poor/sub

fertility of bovine semen (Ballachey et al., 1987; 1988). During in vitro fertilization,

sperm DNA fragmentation of spermatozoa affected fertility rate (Sun et al., 1997).

The principal causes of the sperm DNA fragmentation were the oxidative

stress and the ROS molecules produced at the time of cryopreservation which resulted

in chromatin damage in the mammalian spermatozoa (Hammadeh et al., 2001; Linfor

and Meyers, 2002; Fraser and Strzezek, 2004; Preis et al., 2004). Chohan et al.,

(2004) reported that normal chromatin packaging was affected after cryopreservation

in the human spermatozoa. It is well recognized that reduction in sperm DNA

integrity of mammalian spermatozoa is related with membrane LPO levels (Chen et

17

al., 1997; Twigg et al., 1998a; Potts et al., 2000) and oxidative stress (Hughes et al.,

1996; Mckelvey-Martin et al., 1997; Lopes et al., 1998; Twigg at el., 1998b;

Donnelly et al., 1999). It was noted that hydrogen peroxide, a major ROS molecule in

the semen ((Baumber et al., 2003) reduced the chromatin stability and increased the

DNA sensitivity to in situ acid denaturation (Krzyzosiak et al., 2000). Increase in

ROS molecules were observed associated with DNA fragmentation during xanthine-

xanthine oxidase and caused oxidative stress in in vitro conditions (Baumber et al.,

2003). In bovine, semen lipid peroxidation of sperm plasma membrane positively

correlated with DNA fragmentation index (Kasimanickam et al., 2007). Moreover,

lipid peroxidation of the spermatozoa negatively correlated with plasmalemma

integrity and progressive motility.

It is now established that a higher rate of lipid peroxidation due to ROS

molecules produced during cryopreservation (Storey, 1997; Kadirvel et al., 2009)

were responsible for the formation of chromatin cross-linkages, alterations and DNA

breakages (Hughes et al., 1996; Kodama et al., 1997; Twigg et al., 1998b). Although

antioxidants were naturally present in the semen for protection against ROS

molecules their levels were insufficient (Bilodeau et al., 2000; Kumar et al., 2011).

Dilution and cryopreservation further caused reduction in antioxidants levels (Slaweta

et al., 1998). Increase in lipid peroxidation level of sperm membrane along with DNA

damage was observed when bull spermatozoa were given mercury-induced oxidative

stress in vitro (Arabi, 2005). It is demonstrated that cryopreservation of buffalo semen

increased the population of sperm with apoptotic like changes in frozen-thawed

semen (Khan et al., 2009). Moreover, it is suggested that difference in buffalo bull

fertility may be explained by the presence of apoptotic sperm in semen sample.

18

The DNA integrity of buffalo semen assessed through neutral comet assay

revealed that sperm with fragmented DNA in fresh semen varied from 10 to 12%.

However, after cryopreservation percentage of sperm with fragmented DNA was 35%

i.e. significantly higher compared to fresh semen. Sperm scoring in neutral comet

assay ranged from 1-2 in fresh buffalo semen but in cryopreserved semen its scoring

mostly fell in the range of 3-4 (Kumar et al., 2011). The DNA damage in buffalo

semen was unacceptability high, as it definitely reduces the fertility of cryopreserved

semen in AI programs. Therefore, studies are required for reducing the sperm DNA

fragmentation which may result in better fertility of cryopreserved buffalo semen.

2.5.5. Sperm Lipid Peroxidation

During cryopreservation excessive generation of ROS molecules caused the

higher lipid peroxidation of bovine spermatozoa (Chattergee and Gagnon, 2001). It

was observed that radical oxygen molecules levels increased during cooling of semen

to 4°C while level of nitric oxide did not changed after cooling (Chattergee and

Gagnon, 2001). It is interesting to know that the level of radical oxygen molecules

kept rising during deep storage. Sperm lipid peroxidation level of frozen thawed

bovine semen assessed through thiobarbituric acid reactive substance was observed

higher compared to fresh semen (16.4 ± 2.5 and 8 ± 2.6 nmole/µg, respectively). The

lipid peroxidation level after cooling remained higher than fresh semen and was

recorded 6.57 ± 2.9 nmole/µg. It was demonstrated that sperm lipid peroxidation

levels negatively correlates with the bull competitive index while DNA fragmentation

positively correlates with lipid peroxidation (Kasimanickam et al., 2007).

19

2.5.6. Capacitation Status

Spermatozoa have to undergo the process of capacitation in the female

reproductive tact to achieve fertilization. Capacitation causes hyper activation of the

sperm and increased fluidity of the membrane to achieve fertilizing ability of the

spermatozoa (Visconti et al., 1995). ROS molecules were responsible for the

premature initiation of sperm capacitation present in female reproductive tract (Bailey

et al., 2000).

Excessive ROS molecules produced during cryopreservation also caused

higher population of spermatozoa with premature capacitation in the frozen-thawed

bovine semen (Cormier et a., 1997). The presence of non-capacitated viable

spermatozoa in the semen sample was highly correlated with the fertilizing ability of

the bovine spermatozoa (Gadea et al., 2007).

2.5.7. Antioxidant Activity

Glutathione, glutathione peroxidase, reduced glutathione, catalase, superoxide

dismutase, vitamin C and E are the major antioxidants naturally present in

mammalian semen against ROS to protect the sperm from lipid peroxidation and to

maintain its integrity (Bilodeau et al., 2001; Gadea et al., 2004; Bucak et al., 2008;

Andrabi, 2009; Akhter et al., 2011). The levels of antioxidant decreased during the

freeze-thawing process by dilution of semen with extender and excessive generation

of ROS molecules (Andrabi, 2009; Kumar et al., 2011).

20

In bovine semen activity of catalase enzyme decreased by 50% in frozen-

thawed semen compared to the fresh ejaculate. Bilodeau et al. (2000) determined the

level of glutathione before and after cryopreservation of bull semen and recorded 78%

decrease in glutathione in frozen thawed semen. It was also observed that superoxide

dismutase levels reduced up to 50% following cryopreservation in bovine frozen-

thawed semen (Bilodeau et al., 2000). In equine semen, after dilution motility and

plasmalemma integrity was highly positively correlated with glutathione peroxidase

and catalase activity (Pagl et al., 2006).

2.6. ANTIOXIDANT MEDIATED PROTECTION

The use of antioxidant in extender is recommended to reduce the cryodamage

to spermatozoa (Sansone et al., 2000; Andrabi, 2009). It is known that all the

extenders belonging to third generation like AndroMed (Minitube, Germany),

Triladyl (Minitube, Germany), Bioxcell (IMV, France and are supplemented with

antioxidant. It is known that antioxidant potential of the mammalian semen is not

enough to protect the spermatozoa during cryopreservation against oxidative stress.

Therefore, in extender for improving the quality of bovine, caprine, canine,

equine, human, swine and buffalo spermatozoa in frozen and liquid state different

antioxidants were used viz; vitamin C (Andrabi et al., 2008; Akhter et al., 2011; Ball

et al., 2001; Michael et al., 2007), vitamin E (Upreti et al., 1997; Andrabi et al., 2008;

Akhter et al., 2011; Ball et al., 2001; Michael et al., 2007), catalase (Upreti et al.,

1997; El-Sisy et al., 2008; Michael et al., 2007), superoxide dismutase (El-Sisy et al.,

2008), glutathione (Munsi et al., 2007; Ansari et al., 2010; 2011a; Sinha et al., 1996;

21

Gadea et al., 2007; Uysal and Bucak, 2007; Buck and Tekin, 2007; Foote et al.,

2002), butylated hydroxyanisole (Upreti et al., 1997), n-propyl gallate (Upreti et al.,

1997), deferoxamine mesylate (Upreti et al., 1997), glutamine (Bucak et al., 2009),

hyaluronan (Buck et al., 2009; Bucak et al., 2007), hypotaurine (Funahashi and Sano,

2005) butylated hydroxytoluene (Ball et al., 2001; Ansari et al., 2011c), bovine serum

albumin (Ball et al., 2001; Uysal and Bucak, 2007), lycopene (Uysal and Bucak,

2007), cysteine (Buck and Tekin, 2007; Uysal and Bucak, 2007), methionine (Bucak

et al., 2010), inositol (Bucak et al., 2010), carnitine (Bucak et al., 2010), taurine

(Michael et al., 2007; Bucak et al., 2007), cysteamine (Bucak et al., 2007).

Carnitine and inositol addition in semen extender increased the subjective

motility but motility assessed through computer assisted sperm analyzer did not differ

in any extender containing methionine, inositol carnitine and control (Bucak et al.,

2010). The LPO and GSH level did not differ in all extenders containing antioxidants

compared with control. It is interesting that all the aforementioned antioxidants

improved the DNA integrity of the froze-thawed bovine semen (Bucak et al., 2010).

Glutamine supplementation at 2.5 mM and 5 mM concentration in extender

improved the progressive motility and plasmalemma integrity of the ram spermatozoa

(Buck et al., 2009). However, when hyaluronan was added to extender 500 and

1000µl/ml for cryopreservation of ram spermatozoa only 500 µl/ml was found

effective for improving the plasmalemma integrity of ram semen. These antioxidants

were found non-beneficial for acrosomal integrity, sperm abnormalities and catalase

activity in the cryopreserved semen (Buck et al., 2009). Bucak et al. (2007) observed

higher motility of the ram spermatozoa after thawing in extender containing trehalose,

22

taurine and cysteamine compared to control. However, morphology, acrosome and

plasmalemma integrity of ram spermatozoa did not differ in extender containing

trehalose, taurine and cysteamine and control. It was noted that ROS molecules,

glutathione and glutathione peroxidase activity did not differ in extender containing

aforementioned antioxidants. However, catalase activity was higher in extender

containing taurine compared to extender containing trehalose, cysteamine and control

and all the extenders showed higher levels of vitamin E (Bucak et al., 2007).

Vitamin C and E were tested in tris-citric acid extender to improve the

freezability of buffalo bull spermatozoa in liquid and frozen state (Akhter et al., 2011;

Andrabi et al., 2008). Sperm motility, plasma membrane and acrosomal integrity was

assessed in these studies. Andrabi et al. (2008) reported the beneficial effects of

vitamin E in the cryopreservation of buffalo bull spermatozoa by assessing frozen-

thawed semen. However, sperm progressive motility, plasmalemma and acrosome

integrity remained similar after supplementing vitamin E and C in extender during

liquid storage for five days at ambient temperature 4-5 °C (Akhter et al., 2011). This

difference in response of quality of the buffalo bull spermatozoa might have been due

to difference in extender type. Akhter et al. (2011) used extender based on skim milk

and it has been noted that supplementation of antioxidants in milk based semen

extender did not result in superior semen quality. It is suggested that presence of

casein in milk that also have antioxidant property reduced the requirement of the extra

antioxidant supplementation (Foote et al., 2002). Use of superoxide dismutase and

catalase in extender improved the progressive motility, viability (live/dead ratio),

morphology and plasmalemma integrity of buffalo bull spermatozoa (El-Sisy et al.,

2008). It is known that superoxide dismutase has the ability to convert radical oxygen

23

molecules into hydrogen peroxide while catalase detoxify the hydrogen peroxide and

form water and oxygen molecules (Aitken, 1984). Both the enzymes are naturally

present in bovine semen and were reduced up to 50% after cryopreservation in frozen-

thawed bovine semen (Bilodeau et al., 2000). Funahashi and Sano (2005) reported

that hypotaurine was non-beneficial for any semen quality parameter viz;

plasmalemma and acrosome integrity of swine spermatozoa in liquid state.

It was noted that butylated hydroxytoluene, vitamin C & E and bovine serum

albumin did not improve the motility and acrosome intactness of equine spermatozoa

stored at 5°C (Ball et al., 2001). The extender used for liquid storage was milk based

that already had casein as antioxidant (Foote et al., 2002).

Sperm motility was observed higher in extender containing catalase in frozen-

thawed canine semen (Michael et al., 2007). Similarly, viability of the canine

spermatozoa increased by adding catalase, taurine, N-acetyl-L-cysteine and vitamin E

in extender pre-freezing (Michael et al., 2007). It was observed that for canine

spermatozoa catalase was a more effective antioxidant that protected the motility and

percentage of live spermatozoa in frozen-thawed semen.

2.7. THIOLS IN SEMEN STORAGE

Thiols, a low molecular weight group of antioxidants, have an essential role in

the protective mechanism of sperm during oxidative stress (Bilodeau et al., 2001;

Gadea et al., 2004; Ansari et al., 2010; Ansari et al., 2011a,b). During

cryopreservation, naturally occurring thiols decreased and prove insufficient for the

24

protection of the spermatozoa (Bilodeau et al., 2000). This is the reason antioxidant

are continuously being tested in semen to identifying their protective effects for

spermatozoa.

Bilodeau et al. (2001) demonstrated that glutathione, oxidized glutathione,

cysteine, N-acetyl-L-cysteine and thioglycol could protect the sperm from oxidative

stress induced by hydrogen peroxide in vitro. In many studies, thiols have been tested

for improving the semen quality in bovine (Gadea et al., 2007; Ansari et al., 2011;

Gadea et al., 2007; Tuncer et al., 2010; Sariözkan et al., 2009), caprine (Sinha et al.,

1996), canine (Michel et al., 2007), ovine (Uysal and Bucak, 2007; Buck and Tekin,

2007), swine (Funahashi and Sano, 2005) and buffalo (Ansari et al., 2010; 2011a,b;

Perumal et al., 2010) in frozen and liquid state.

2.8. GLUTATHIONE

Glutathione is a tripeptide (gamma-glutamylcysteinyl-glycine) has a major

function as antioxidant in eliminating ROS molecules and maintaining the

intracellular redox state of the cell. Glutathione is a very important naturally occurring

antioxidant in mammalian semen. Which has a low molecular weight, and can easily

mobilize in the cell system for the removal of peroxides molecules by chain reactions

that result in the generation of oxidized glutathione (Meister and Anderson, 1983)

(Figure 2.1).

It is evident that glutathione present in mM concentrations in cell, is a very

rapid antioxidant system which has the capacity for the prevention of reactive oxygen

25

species molecules mediated damage to mammalian semen. After reaction with

peroxides molecules the product of glutathione oxidation, oxidized glutathione, is

toxic and is rapidly converted back to glutathione by the enzyme glutathione

reductase through a chain reaction.

It has been observed that glutathione can stimulate the mammalian

spermatozoa for fertilization that which inhibited by the ROS molecules from the

damaged spermatozoa in cyopreserved semen (Bath et al., 2010). Glutathione can

protect the semen quality when exposed to artificial oxidative stress induced by

hydrogen peroxide in frozen thawed semen (Bilodeau et al., 2001).

26

Figure 2.1. Glutathione redox-cycle, glutathione peroxide (GPx) reduces H2O2 and

hydrogen peroxide (ROOH) using reduced glutathione (GSH). The oxidized form of

glutathione (GSSG) is regenerated by the glutathione reductase (GR) using NADPH.

The energy is usually drawn from the hexose monophosphate shunt-system. (Bilodeau

et al., 2001)

27

2.8.1. Role of Glutathione in Bovine Semen

In a study on bull semen in liquid state using egg yolk citrate extender

glutathione was tested at concentrations of 0.5, 1.0, 2.0 and 3.0mM (Munsi et al.,

2007). In this study sperm motility, normal acrosome + mid piece + tail and head

abnormalities were assessed before and after freezing and on consecutive days of

storage up to the 5th day. Sperm motility was recorded higher with glutathione at 0.5,

1.0 and 2.0mM while 3mM of glutathione was found non-beneficial. Sperm

acrosomal integrity was conserved by 0.5mM of glutathione for five days. Therefore,

0.5mM of glutathione was recommended for use in extender for storage of bull semen

in egg yolk-citrate extender in liquid state. The limitations of the study were no

information regarding the live and dead status of the spermatozoa evaluated for

acrosomal integrity and the missing fertility test that is required before routine use in

an artificial insemination program. It was already observed that improvement in

motility and acrosomal integrity by glutathione supplementation in extender did not

ensure improvement in fertility rate (Sinha et al., 1996).

Gadea et al. (2007) tested glutathione supplementation in thawing medium for

improving the quality of bovine spermatozoa and studied the mechanism involved in

the improvement. Motion characteristics, membrane lipid packaging disorder,

capacitation status with live/dead distinction, free radical production, chromatin

condensation and DNA damage played a role as well as in vitro penetrability of

oocyte along with embryo production (Harrison et al., 1996; Gadea et al., 2005;

Evenson et al., 2002). Sperm DNA was assessed by two techniques using terminal

deoxynucleotidyl-transferase-mediated dUTP nicked labeling and acridine orange

28

staining through flow cytometry. Glutathione supplementation in thawing medium

was non-beneficial. The spermatozoa with lipid disorders decreased in thawing

medium containing glutathione compared to control. Free radical decreased in

thawing medium containing glutathione compared to control. Percentage of live

sperm with non-capacitated status improved in medium having glutathione.

Chromatin condensation was better in thawing medium following the addition of

glutathione. Sperm DNA fragmentation assessed through both techniques were better

compared to control. Pentratability of spermatozoa during in vitro fertilization was

higher from medium containing glutathione. Subsequently, embryo production was

better with semen from thawing medium containing glutathione. It is suggested that

glutathione protected the quality and fertility of the spermatozoa by improving all

aforementioned parameters (Gadea et al., 2007). Freeze-thawing cycle reduced the

glutathione level in bovine frozen-thawed semen (Bilodeau et al., 2000). So, addition

of glutathione in thawing medium might compensate the glutathione decrease during

cryopreservation and conserve the semen quality by reducing ROS levels in frozen

semen.

2.8.2. Role of Glutathione in Caprine Semen

Glutathione was tested in extender (2.0 mM and 5.0 mM) for the

cryopreservation of caprine semen in tris-based extender of goat breeds Beetal, Black

Bengal and Crossbred (Sinha et al., 1996). The semen quality testes used in the study

were motility and acrosomal integrity through Giemsa staining technique. This

technique does not provide the information regarding the live and dead status of the

spermatozoa (Hancock, 1952). After cryopreservation biochemical tests were also

29

preformed for aspartate aminotransferase, alanine transferase and lactate

dehydrogenase (Reitman and Frankel, 1957; Cabaud et al., 1958) to determine the

leakage of these enzymes from the cell in the surrounding medium. The in vivo

fertility test was also performed in this study with the same glutathione doses in

extender.

Sperm motility was higher after thawing in extender containing 5mM of

glutathione in Beetal, Black Bengal, crossbred and overall. Acrosomal integrity was

higher in extender containing 2mM of glutathione in Black Bengal, crossbred and

overall while in extender with glutathione 5mM acrosomal integrity was higher in

Beetal, Black Bengal and crossbred. It showed that in different goat breeds different

results of semen quality parameters were observed using glutathione in extender at

same concentrations. Aspartate aminotransferase, alanine transferase and lactate

dehydrogenase leakage in the surrounding medium was lower in the extender

containing 2mM and 5mM of glutathione. It represented that glutathione had the

ability to protect the acrosomal membrane and stop its contents from leakages.

The fertility rate of extenders containing 2 mM and 5 mM of glutathione was

determined by inseminating with frozen-thawed semen and performing pregnancy

diagnosis by abdominal palpation of the ewe 4 months post-insemination. The fertility

rates were recorded as 51.6%, 59.6% and 49.2% in extenders containing 2 mM and

5mM of glutathione compared to control. In this study, improvement in fertility was

observed with glutathione supplementation but it did not get statistical significance.

The reason of non-significant results of fertility might have been due to the lower

number of inseminations which were 98, 60 and 65 with extenders containing 2 mM

30

and 5 mM of glutathione and control. A minimum of 100 inseminations per extender

were recommended to get reliable fertility results (Akhter et al., 2007).

2.8.3. Role of Glutathione in Ovine Semen

Sperm progressive motility, viability, abnormalities, acrosome and

plasmalemma intactness were assessed of frozen-thawed semen cryopreserved in tris-

based extender containing 5.0mM of glutathione and oxidized glutathione (Buck et

al., 2008). Sperm quality was assessed through eosin-nigrocin, hypo-osmotic swelling

test and phase contrast microscopy, respectively. After thawing, biochemical testes

were performed to determine the levels of malondialdehyde (indicator of lipid

peroxidation), reduced glutathione, glutathione peroxidase, catalase and vitamin E in

extenders.

All the aforementioned semen quality parameters did not differ in extenders

containing 5.0mM of glutathione and oxidized glutathione compared with control.

Although activity of malondialdehyde and catalase remained similar, levels of

glutathione, glutathione peroxidase and vitamin E were higher in extenders containing

glutathione and oxidized glutathione (Bucak et al., 2008). In liquid state glutathione

(5-10mM) improved the viability of ram spermatozoa for up to 6 hours of storage

(Bucak and Tekin, 2007). However, other semen quality parameters like

plasmalemma integrity and sperm morphology did not differ in extenders containing

glutathione and control. Uysal and Bucak (2007) reported improvement in motility,

plasma membrane as well acrosome integrity and morphology of ram spermatozoa by

31

adding oxidized glutathione in extender that worked through the glutathione-mediated

protection system.

2.8.4. Role of Glutathione in Swine Semen

Glutathione (5.0mM) was tested in extender for liquid storage of boar semen

at 10°C to improve the viability and acrosomal integrity (Funahashi and Sano, 2005).

Sperm viability was assessed through SYBR 14 and propidium iodide while sperm

acrosomal integrity in terms of functional status was assessed through

chlortetracycline (Funahashi et al., 2000; Funahashi and Nagai, 2001). Sperm

viability increased by adding glutathione in extender. Sperm acrosomal integrity was

highest in extender containing glutathione compared to control (Funahashi and Sano,

2005).

2.8.5. Role of Glutathione in Buffalo Semen

For improving the quality of buffalo semen during liquid storage glutathione

was tested in extender at concentration of 0.5, 1.0, 2.0 and 3 mM (Ansari et al.,

2011a). Sperm progressive motility, plasmalemma and acrosomal integrity were

assessed for five days of storage at ambient temperature. It was noted that all the

aforementioned parameters improved in extender containing 0.5-1.0mM of

glutathione for five days. Similarly, the same concentrations of glutathione were

tested for cryopreservation of buffalo semen and after thawing the same parameters

were observed at 0, 3 and 6 hours of incubation at 37 °C (Ansari et al., 2010). In this

study, semen quality of frozen-thawed buffalo semen improved in extender containing

32

glutathione in concentrations of up to 2.0mM. The results showed that for frozen

semen the requirement of glutathione is higher compared to liquid semen. It was

suggested that buffalo bull spermatozoa have to face higher oxidative stress in freeze-

thawing process compared to liquid state. It was seen that the freeze-thawing process

induced more damage to spermatozoa compared to liquid storage (Andrabi, 2009).

The limitations of these studies were the use of basic semen quality tests with

poor prognostic value for the prediction of fertility. In these studies sperm plasma

membrane and acrosomal intactness was determined by hypo-osmotic swelling test

and phase contrast microscopy after fixing the semen sample in formal-citrate. The

hypo-osmotic swelling test basically provides information about the intactness of the

plasma membrane (Jeyendran et al., 1984). Direct assessment of wet samples under

phase contrast microscope for acrosomal integrity did not give information regarding

the live/dead status of the spermatozoa.

In these studies, no advanced test like DNA integrity was performed that has a

high positive correlation with fertility (Andrabi, 2008). Therefore, it is suggested that

a comprehensive study must be performed by using semen quality assays with more

prognostic value. After in vitro study in vivo fertility test must be performed to get

sufficient data for the recommendation of any extender in routine use under field

conditions.

33

2.9. CYSTEINE

Cysteine is an amino acid having the ability to penetrate the cell and protect

the cell bio-membrane system from the deleterious effects of free radicals by

scavenging them directly (Bilodeau et al., 2001). Along its direct action to cope the

ROS molecules it also enhances the intracellular level of glutathione that protect the

cell during oxidative stress indirectly (Buck et al., 2008). Cysteine has the ability to

protect the sperm motility in the presence of exogenous hydrogen peroxide in frozen-

thawed bull semen (Bilodeau et al., 2001). Sperm motility was recorded higher in

extender containing 5mM of cysteine compared to control post-thaw along with

higher catalase activity in ram semen after thawing (Buck et al., 2008). Funahashi and

Sano (2005) observed higher oocyte penetrability of swine spermatozoa during in

vitro fertilization by semen stored in extender containing cysteine.

2.9.1. Role of Cysteine in Bovine Semen

In a recent study, l-cysteine was used in extender at concentrations of 0.5, 1.0

and 2.0mM for the cryopreservation of Sahiwal bull spermatozoa (Ansari et al.,

2011d). Sperm progressive motility, plasmalemma integrity and viability were

assessed post-thaw and improvement in all aforementioned parameters was observed

in extender containing 1.0-2.0mM of cysteine. Sahiwal is a zebu breeds that has more

antioxidant potential compared to taurine breed (Nichi et al., 2006).

34

2.9.2. Role of Cysteine in Canine and Feline Semen

Sperm quality of canine semen was assessed after the supplementation of N-

acetyl-L-cysteine in semen extender (Tejada et al., 1984). It was noted that N-acetyl-

L-cysteine improved the sperm motility and DNA integrity of the canine spermatozoa

assessed after 2 and 4 hours post-thaw. N-acetyl-L-cysteine has the ability to protect

the sperm during artificially induced oxidative stress caused by adding hydrogen

peroxide (Bilodeau et al., 2001). Thuwanut et al. (2008) tested the 5mM of cysteine in

extender in the cryopreservation of cat epididymal spermatozoa. In this study,

motility, membrane and acrosome integrity was assessed of frozen-thawed semen

(Tejada et al., 1984). Sperm acrosome integrity did not differ between extender

containing cysteine and control. However, motility and DNA integrity of the cat

epididymal spermatozoa were higher in extender containing cysteine compared to

control.

2.9.3. Role of Cysteine in Ovine Semen

Sperm quality was assessed by frozen-thawed semen cryopreserved in tris-

citric acid based extender containing 5.0mM of cysteine (Bucak et al., 2008). Sperm

viability, plasmalemma integrity and morphology were assessed through eosin-

nigrocin, hypo-osmotic swelling test and phase contrast microscopy, respectively.

After thawing, biochemical testes were performed to determine the levels of

malondialdehyde, reduced glutathione, glutathione peroxidase, catalase and vitamin E

in extenders containing cysteine and control.

35

Sperm motility improved in extender containing 5mM of cysteine compared to

control post-thaw (Bucak et al., 2008). It is interesting to note that catalase activity

increased in extender containing 5.0mM of cysteine compared to control (Bucak et

al., 2008). It was thought that cysteine protected the ram spermatozoa by itself and by

increasing catalase activity in the frozen-thawed ram semen. It was demonstrated that

catalase activity decreased during cryopreservation of buffalo spermatozoa (El-Sisy et

al., 2007).

Uysal and Bucak (2007) identified the role of cysteine addition in tris-citric

acid based extender at concentrations of 5.0, 10 and 20mM. Sperm quality was

assessed post-thaw. Addition of cysteine addition in extender before freezing

improved the progressive motility, plasmalemma and acrosome integrity, viability and

reduced the abnormalities of the ram spermatozoa (Uysal and Bucak, 2007).

2.9.4. Role of Cysteine in Swine Semen

Funahashi and Sano (2005) tested 5.0mM of cysteine in extender for liquid

storage of boar semen at 10°C to improve the viability and acrosomal integrity. Sperm

viability and functional acrosomal integrity was assessed through SYBR 14 +

propidium iodide and chlortetracycline (Funahashi et al., 2000; Funahashi and Nagai,

2001). Sperm viability increased by adding 5.0mM of cysteine to the extender. Sperm

acrosomal integrity was highest in extender containing cysteine compared to control

(Funahashi and Sano, 2005). It was observed that the addition of 1.27mM of cysteine

hydrochloride improved the chromatin structure after during freezing (Szczesniak-

Fabianzyk et al., 2003).

36

2.9.5. Role of Cysteine in Buffalo Semen

Sperm quality was assessed after adding cysteine in semen extender before

cryopreservation of frozen-thawed semen (Ansari et al., 2011b). Higher motility,

plasma membrane and percentage of sperm with normal apical ridge were observed in

extender containing 1.0mM of cysteine.

2.10. THIOGLYCOL

2.10.1. Thioglycol in Protection of Gametes/embryos

Chilling of the buffalo spermatozoa resulted in decreased motility associated

with reduced antioxidant activity and higher ROS molecule levels (El-Sisy et al.,

2007). It was observed that motility of buffalo semen and total antioxidant potential

decreased during the cryopreservation process (Anzar et al., 2010; Kumar et al.,

2011). It was demonstrated that thioglycol had the ability to protect the motility of

bovine spermatozoa from deleterious effects of hydrogen peroxide in vitro, a major

ROS molecule naturally produced in the semen (Bilodeau et al., 2001). The results of

another study on buffalo semen also described hydrogen peroxide as blocker of

motility (Garg et al., 2009). The following studies described the protective properties

for the gametes/embryos.

Supplementation of thioglycol in IVF medium protected the bovine embryo

from oxidative stress and improved blastocyst quality (Feugang et al., 2004). It was

believed that thioglycol protected the oocytes/embryo viability by directly interacting

with oxidized radicals and/or increasing intercellular glutathione levels in bovine,

37

oocytes (Cornell and Crivaro, 1972; Wardman and Sonntag, 1995; Bannaї, 1992;

Takahashi et al., 2002; Takahashi et al., 1993). Thioglycol can stop the apoptosis in

cloned embryos by its antioxidant activity in swine (Park et al., 2004a,b). It was

demonstrated bovine that thioglycol increased the number of cumulus cells attached

to oocytes and cell numbers of blastocyst by influencing ATP metabolism (Tsuzuki et

al., 2005). Beneficial effects of thioglycol were evident for sperm penetration and

early embryonic development (Funahashi, 2005). Kim et al. (2004) described that

thioglycol presence in maturation media improved the maturation and M II stage of

canine oocytes in vitro. These all studies suggested that thioglycol should be tested in

extender for improving the quality of buffalo semen during cryopreservation.

38

Chapter 3

MATERIALS AND METHODS

3.1. PREPARATION OF EXTENDERS

Experimental extenders were prepared using Tris-citric egg yolk extender (pH

7.0; osmotic pressure 320 mOsmol Kg-1) that consisted of 1.56% citric acid (Fisher

Scientific, Loughborough, Leicestershire, UK) and 3.0% tris–(hydroxymethyl)-

aminomethane (Research Organics, Cleveland, OH, USA) were dissolved in distilled

water. Fructose (Scharlau, Barcelona, Spain) 0.2%; glycerol 7% (Merck, Darmstadt,

Germany); egg yolk 20% were added to the extender. To determine the role of

glutathione, cysteine and thioglycol, extenders were prepared by adding Glutathione

(Merck, Darmstadt, Germany), l-cysteine (Sigma, NY, USA) and thioglycol (Merck,

Darmstadt, Germany) at the rate of 0.0 mM, 0.5 mM, 1.0 mM, 1.5 mM, 2.0 mM or

3.0 mM in the extenders, respectively (Table 3.1).

3.2. BUFFALO BULL SEMEN COLLECTION

Three adult Nili-Ravi buffalo bulls of same cohort were used for this study.

Semen was collected from each bull in a graduated tube with an artificial vagina

(42°C) for three weeks for each experiment (two ejaculates/bull). The semen samples

were transferred to the laboratory within minutes of collection. Motility of buffalo

spermatozoa was determined microscopically (at 200x). Concentration of sperm in

semen was determined by bovine photometer ACCUCELL (IMV, France) at 539nm

wave length.

39

Table 3.1. Composition of experimental extenders.

Experiment Glutathione

(mM)

TRIS

(mM)

Citric Acid

(mM)

Fructose

(mM)

Glycerol

(%)

Egg yolk

(%)

I

0.0 274.6 74.4 11.1 7 20

0.5 274.6 74.4 11.1 7 20

1.0 274.6 74.4 11.1 7 20

1.5 274.6 74.4 11.1 7 20

2.0 274.6 74.4 11.1 7 20

3.0 274.6 74.4 11.1 7 20

Experiment Cysteine

II

0.0 274.6 74.4 11.1 7 20

0.5 274.6 74.4 11.1 7 20

1.0 274.6 74.4 11.1 7 20

1.5 274.6 74.4 11.1 7 20

2.0 274.6 74.4 11.1 7 20

3.0 274.6 74.4 11.1 7 20

Experiment Thioglycol

III

0.0 274.6 74.4 11.1 7 20

0.5 274.6 74.4 11.1 7 20

1.0 274.6 74.4 11.1 7 20

1.5 274.6 74.4 11.1 7 20

2.0 274.6 74.4 11.1 7 20

3.0 274.6 74.4 11.1 7 20

40

The qualifying ejaculates having motility >60%, volume >1 mL and concentration

>0.5 billion/ml from three bulls (Ansari et al., 2011e) were aliquoted into six parts

and sustained for 15 minutes in the water bath (37°C) prior dilution with six different

experimental extenders for each experiment on glutathione, cysteine and thioglycol.

3.3. PROCESSING OF BUFFALO SEMEN

Semen aliquots were diluted in a single step with the experimental extenders

for each experiment to a concentration of 50 × 106 spermatozoa mL-1 at 37°C. Then

semen after dilution was cooled to 4C in two hours. After cooling of semen,

equilibration was performed for 4 hours at the same temperature (4C). After the

completion of equilibration, semen was filled in medium size French straws (0.5 mL)

at 4C. The straws were kept on the liquid nitrogen vapours for 10 minutes. After

freezing, the straws were plunged into LN2 (-196C) for storage before post-thaw

analysis. Thawing of the experimental frozen straws was performed for 30 seconds at

37C. For each experimental extender, semen from the three straws were pooled and

incubated at 37C for the assessment of post-thaw semen quality after thawing at 0,

2, 4 hours.

3.4. SEMEN QUALITY ASSESSMENT TECHNIQUES

3.4.1. Motility of Buffalo Spermatozoa

Motility of buffalo spermatozoa was assessed as described in earlier section

3.2.

41

3.4.2. Buffalo Sperm Plasmalemma Integrity

An aliquot (5 μl) of semen after incubation in hypo-osmotic solution [0.735g

sodium citrate (Merck, Darmstadt, Germany) and 1.351g fructose (Scharlau,

Barcelona, Spain) in 100 ml distilled water; osmotic pressure ~ 190 mOsm Kg-1] was

placed on a slide. A droplet (5 μl) of eosin (Scharlau, Barcelona, Spain) (0.5%;

sodium citrate 2.92%) was mixed in for 10 seconds. A cover slip was placed on the

mixture and semen of each slide was evaluated under phase contrast microscopy

(400x; LABOMED LX400). A total of 100 spermatozoa per preparation were

observed in at least five different fields (Tartaglione and Ritta, 2004). The isotonic

medium of the hypo-osmotic solution caused coiling of the spermatozoa due to

osmotic difference. Unstained heads and tails, and swollen tails indicated intact,

biochemically active membranes, while pink heads and tails, and un-swollen tails

indicated disrupted, inactive membranes (Tartaglione and Ritta, 2004).

3.4.3. Sperm Viability

The dual staining procedure with Trypan blue-Giemsa stain was performed

following protocol described by Kovacs and Foote (1992). In this technique, Trypan-

blue distinguished live and dead spermatozoa and Giemsa evaluated the acrosomal

integrity at the same time (live sperm with intact acrosome). For this purpose, equal

volume of Trypan-blue (MP Biomedicals, Eschwege, Germany) solution (0.2%) and a

drop of semen was put on a microscope slide at room temperature and mixed with

edge of the cover slip. The prepared smears were then air-dried, and fixed in

formaldehyde-neutral red solution [86 ml 1M HCl + 14 ml 37% formaldehyde

42

(Merck, Darmstadt, Germany) + 0.2 g Neutral red (MP Biomedicals, Eschwege,

Germany)] for 5 minutes. After rinsing with running distilled water, Giemsa stain

(MP Biomedicals, Eschwege, Germany) (7.5%) was applied for 4 hours. Then after

staining the Giemsa slides were air dried and cover slip was placed on it before

mounting with Balsam of Canada (Merck, Darmstadt, Germany). A total of 100

hundred spermatozoa were evaluated from at least five different fields in each smear

with phase contrast microscope at 1000x (LABOMED LX400). As Trypan-blue

penetrates non-viable, dead spermatozoa with disrupted membrane, therefore, they

appeared stained in blue, while live, intact spermatozoa remained unstained. Whereas,

Giemsa accumulates in the spermatozoa with an intact acrosome, staining the

acrosome region in purple (Tartaglione and Ritta, 2004).


Three smears was prepared on glass slides from each semen sample and air-

dried. The smears were fixed by placing them in Carnoy’s solution [methanol (Merck,

Darmstadt, Germany) and glacial acetic acid (Merck, Darmstadt, Germany) in 3:1

ratio] over night. After that, slides were dried in air and placed in tampon solution (80

mM citric acid (Merck, Darmstadt, Germany) and 15 mM Sodium phosphate (Sigma,

NY, USA), pH 2.5) at 75°C for 5 minutes in water bath. Subsequently, processed

microscope slides were then stained with acridine orange (Sigma, NY, USA) (0.2

mg/ml) after incubation in tampon solution. The slides were then covered with cover

slip while they were still wet, and studied under fluorescent microscope (LABOMED

LX400). Acridine orange is a nucleic acid selective fluorescent cationic dye, cell-

permeable, and interacts with DNA with single and/or double strand

43

by intercalation or electrostatic attractions, respectively. One hundred sperm in each

prepared slide were evaluated. Sperms with intact DNA presented green whereas

sperm with damaged DNA presented yellow-green to red fluorescence in spectrum.

3.4.5. In Vivo Fertility Rate

The best evolved extenders containing antioxidants on the basis of the

experiments with glutathione cysteine and thioglycol were evaluated for in vivo

fertility rate of buffalo bull semen. Semen from two buffalo bulls was collected,

evaluated and cryopreserved in extenders containing antioxidants and without

antioxidant (control) as described earlier. After thawing, the inseminations were done

with experimental semen of two buffalo bulls under field conditions to get record

(pregnancies) of at least 200 inseminations performed per extender (total 800). The

experimental inseminations were done during peak breeding season (October to

December, 2010). The buffaloes were inseminated with experimental semen

approximately 24 hours after the onset of the standing heat (with at least one normal

pervious parturition record). After at least 90 days of insemination, buffalos were

checked for pregnancy through rectal palpation under field conditions by trained

technician.

3.5. STATISTICAL ANALYSIS

Results of the study were presented as means ± SEM. Effect of experimental

extenders on motility, viability, plasmalemma and DNA integrity were analyzed by

the analysis of variance. When the F–ratio was found significant (P<0.05), LSD test

44

was used, to separate the treatment means of each experiment. The data on in vivo

fertility trials were analyzed using chi-square test (MINITAB® Release 12.22, 1998).

45

Chapter 4

RESULTS AND DISCUSSION

4.1. EFFECT OF GLUTATHIONE IN SEMEN EXTENDER ON POST-THAW

QUALITY OF BUFFALO BULL SPERMATOZOA

4.1.1. Progressive Motility

The response to glutathione (GSH) addition in semen extender regarding

motility of buffalo bull spermatozoa is presented in Figure 4.1. Higher (P < 0.05)

sperm motility was observed in extender containing 2.0 mM of GSH as compared to

other experimental extenders and control at 0 hours post-thaw. At 2 hours after

thawing, motility remind higher (P < 0.05) in extender containing 1.0 mM, 1.5 mM

and 2.0 mM of GSH as compared to control, whereas, the sperm motility in extender

containing 0.5 mM of GSH remained similar (P > 0.05) as compared to control.

Likewise, best motility results were achieved (P < 0.05) with extender containing 1.0

mM, 1.5 mM and 2.0 mM of GSH as compared to control at 4 hours post-thaw.

However, sperm motility in extender containing 0.5mM of GSH did not vary (P >

0.05) as compared to control. Sperm motility in extender containing 3.0 mM of GSH

did not vary (P > 0.05) to the sperm motility in extenders containing 0.5 mM, 1.0 mM

and 1.5 mM of GSH at 0, 2 and 4 hours post-thaw but differed (P < 0.05) as compared

to control.

It is pertinent to describe that subjective motility assessed post-thaw was

significantly correlated (r = 0.672, P = 0.033) with in vivo fertility in bovine (Gillan et

al., 2008). Cryopreservation of buffalo semen reduced sperm motility (Rasul et al.,

46

2001) and accelerated the production of ROS molecules through higher lipid

peroxidation levels (Kadirvel et al., 2009) beyond the physiological levels. Kumar et

al. (2011) reported reduction in sperm motility along total antioxidant potential in

cryopreserved buffalo semen. Levels of ROS molecules were negatively correlated

with sperm motility of buffalo semen (Kadirvel et al., 2009).

An important ROS molecule, H2O2 reduced the sperm motility of buffalo

(Garg et al., 2009) and bovine semen in vitro (Bilodeau et al., 2001) while glutathione

protected the sperm from H2O2 mediated motility reduction. In some studies it was

reported that glutathione supplementation in semen extender resulted in high sperm

motility in bovine (Bilodeau et al., 2001; Munsi et al., 2007) and buffalo semen

(Ansari et al., 2010; Ansari et al., 2011a). Glutathione levels also decreased after

cryopreservation of bovine (Stradaioli et al., 2007) and swine spermatozoa (Gadea et

al., 2004). The above observations in various studies corroborated the findings of

current study where post-thaw sperm motility was increased due to the addition of

glutathione in the extender.


The response to glutathione addition in semen extender concerning viability of

buffalo bull spermatozoa is presented in Figure 4.2. Sperm viability was higher (P <

0.05) in extender containing 2.0 mM of GSH as compared to control at 0 hours after

thawing. At 2 hours post-thaw, sperm viability increased (P < 0.05) in extender

containing 1.5 mM and 2.0 mM of GSH as compared to control.

47

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

0 2 4

Post thaw hour

Sp

erm

mot

ility

(%

)

Control 0.5 mM 1.0 mM 1.5 mM 2.0 mM 3.0 mM

d

cb b

a

bc

c

bc

ab ab

a

b

d

cd

abcab

a

bc

Figure 4.1. Effect of glutathione addition in semen extender on the progressive

motility of buffalo bull spermatozoa at 0, 2 and 4 hours after thawing. Bars with


48

Moreover, sperm viability in extenders containing 0.5 mM and 1.5 mM of GSH did

not vary (P > 0.05) from each other but differed (P > 0.05) from the control. At 4

hours post-thaw, viability in extender containing 3.0 mM of GSH remained similar (P

> 0.05) to extenders containing 0.5 mM of GSH but differed (P < 0.05) compared to

control. Sperm viability in extender containing 3.0mM of GSH remained similar (P >

0.05) to extenders containing GSH 0.5 mM, 1.0 mM and 1.5 mM at 0, 2 and 4 hours

post-thaw but differed (P < 0.05) as compared to control.

Sperm viability was assessed by Trypan-blue Giemsa assay as described by

Tartaglione and Ritta (2004). Previous study (Sinha et al., 1996) reported improvement

in acrosomal integrity and decreased release of aspartate aminotransferase, alanine

aminotransferase and lactate dehydrogenase enzymes from the sperm in the surrounding

medium due to glutathione supplementation in caprine semen. An in vitro study on

bovine semen revealed that ROS reduced the sperm viability (Gillan et al., 2008) and

caused acrosomal reaction that resulted in poor sperm-oocyte fusion (Mammoto et al.,

1996). Freeze-thawing process decreased the population of sperm with intact

acrosomes (Rasul et al., 2001; Kumar et al., 2011) along with total antioxidant

potential and increased the lipid peroxidation levels in buffalo semen (Kadirvel et al.,

2009). Hydrogen peroxide reduced the percentage of sperm with intact acrosomes in

buffalo semen in vitro (Garg et al., 2009).

Gadea et al. (2004) observed a highly significant relationship between sperm

viability and glutathione contents of spermatozoa in swine. It was believed that

glutathione protected the sperm viability through scavenging ROS molecules

produced due to oxidative stress during cryopreservation and subsequently lipid

49

peroxidation of the spermatozoa (Cotran et al., 1989). In another study, a higher

percentage of sperm with intact acrosomes was reported in buffalo semen

cryopreserved in extender containing glutathione (Ansari et al., 2010). However, this

study did not provide information regarding the live/dead status of the spermatozoa. It

was inferred from the previous and current studies that glutathione in extender

improved the viability of buffalo semen by reducing oxidative stress.


The response to glutathione addition in semen extender in view of the

plasmalemma integrity of buffalo bull spermatozoa is presented in Figure 4.3.

Plasmalemma integrity was higher (P < 0.05) in extender containing 2.0 mM of GSH

compared to control at 0 hours post-thaw. At 2 hours after thawing, plasmalemma

integrity was observed higher (P < 0.05) in extender containing 1.0 mM, 1.5 mM and

2.0 mM GSH compared to control. However, sperm plasmalemma integrity in

extender containing 0.5mM of GSH remained similar (P < 0.05) compared to extender

without glutathione. At 4 hours after thawing, sperm plasmalemma integrity was

superior (P < 0.05) in extender containing 1.5 mM and 2.0 mM of GSH as compared

to control, whereas, the plasma membrane integrity in extender containing 0.5 mM of

GSH and control did not differ significantly (P > 0.05). Sperm plasma membrane

integrity in extender containing 3.0 mM of GSH remained similar (P > 0.05) to

extenders containing 0.5mM, 1.0mMm and 1.5 mM of GSH at 0 and 4 hours post-

thaw but differed (P < 0.05) compared to control. At 4 hours after thawing, sperm

plasmalemma integrity was higher in (P < 0.05) in extender containing 1.0 mM, 1.5

mM and 2.0 mM of GSH in comparison with control. Moreover, sperm plasmalemma

50

integrity differed (P < 0.05) in extender containing 0.5 mM of GSH compared with

control.

Sperm plasmalemma integrity was assessed through hypo-osmotic swelling test

in combination with eosin stain according to method of Tartaglione and Ritta (2004).

Buffalo sperm were observed more susceptible to ROS attack compared to cattle bull

spermatozoa because of higher levels of polyunsaturated fatty acids in plasma

membrane (Nair et al., 2006). It was believed that the lipid peroxidation cascade,

caused a loss of fluidity and integrity of the plasma membrane that was essential for

successful fertilization (Storey 1997). Cryopreservation of buffalo spermatozoa

resulted in higher levels of lipid peroxidation (Kadirvel et al., 2009) and reduction in

total antioxidant potential (Kumar et al., 2011) that damaged the sperm plasmalemma

integrity (Rasul et al., 2001; Kumar et al., 2011). The main sources of ROS were an

impaired mitochondrial system and dead or damaged spermatozoa in semen (Kadirvel

et al., 2009). The higher levels of ROS decreased the sperm plasmalemma integrity in

buffalo (Garg et al., 2009). Therefore, it was thought that higher sperm plasmalemma

integrity of buffalo spermatozoa observed after the addition of glutathione in extender

during the present study might have been due to lower levels of oxidative stress.


The data on the effect of glutathione addition to semen extender on DNA

fragmentation of buffalo bull spermatozoa is presented in Figure 4.4. In the present

study, DNA integrity of cryopreserved buffalo spermatozoa was higher in extender

containing GSH as compared to control. It was demonstrated that freeze-thawing of

51

buffalo semen causes DNA fragmentation in cryopreserved semen (Kumar et al.,

2011) along with reduction in total antioxidant capacity.

sperm with DNA fragmentation were recoded higher in cryopreserved compared to

fresh semen. This suggested the critical role of antioxidant in reducing DNA

fragmentation during cryopreservation of semen (Bucak et al., 2010). It was

demonstrated in vitro that ROS molecules were mainly responsible for causing sperm

DNA damage (Baumber et al., 2003). The results of present study also showed a

lower percentage of sperm with fragmented DNA in extender containing glutathione

supplementation which could be attributed to glutathione protection against ROS

molecules (Figure 4.4).

4.2. EFFECT OF CYSTEINE IN SEMEN EXTENDER ON POST-THAW



The data on the response to cysteine addition in semen extender with regard to

motility of buffalo bull spermatozoa is presented in Figure 4.5. Sperm motility (%)

was higher in extenders containing 1.0 mM and 1.5 mM of cysteine as compared to

control at 0, 2 and 4 hours after thawing. Sperm motility in extender containing 2.0

mM of cysteine did not vary (P > 0.05) compared to extender containing GSH 0.5

mM while the sperm motility percentage in extender containing 3.0 mM of GSH did

not vary (P > 0.05) to extender containing 0.5 mM of cysteine and control at 0 and 2

hours post-thaw.

52

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

0 2 4

Post thaw hour

Sp

erm

via

bili

ty (

%)


d

cbc b

a

bc

c

bb ab

a

b

c

b

ab ab

a

b

Figure 4.2. Effect of glutathione addition in semen extender on viability of buffalo

bull spermatozoa at 0, 2 and 4 hours after thawing. Bars with different letters differed

significantly (P < 0.05) at a given time.

53

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

0 2 4

Post thaw hour

Sp

erm

pla

smal

emm

a in

tigr

ity

(%)


d

cb b

a

bc

c

bcab

ab

a

b

d

cd

bab

a

c

Figure 4.3. Effect of glutathione addition in semen extender on plasma membrane

integrity of buffalo bull spermatozoa at 0, 2 and 4 hours after thawing. Bars with


54

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

0 2 4

Post thaw hour

Sp

erm

DN

A in

tigr

ity

(%)

Control

0.5 mM

1.0 mM

1.5 mM

2.0 mM

3.0 mM

b

aa a

aa

b

a a a a a

b

a a a a a

Figure 4.4. Effect of glutathione addition in semen extender on DNA integrity of

buffalo bull spermatozoa at 0, 2 and 4 hours after thawing. Bars with different letters

differed significantly (P < 0.05) at a given time.

55

At 4 hours after thawing, motility did not vary (P > 0.05) in extender containing 2.0

mM and 3.0 mM of cysteine as compared to the sperm motility in extender containing

0.5 mM of cysteine and control. Moreover, sperm motility was similar (P > 0.05) in

extender containing 2.0 mM of cysteine as compared to the extender containing 1.5

mM of cysteine at 4 hour post-thaw. The results are inline with studies on bovine

semen which had reported higher motility in semen cryopreserved in extender

containing cysteine to reduce oxidative stress (Johnson et al., 1954; Bilodeau et al.,

2001). In the present study, post-thaw sperm motility improved in the extender

containing cysteine which might be due to neutralization of ROS levels in the semen-

extender complex.


The data on the effect of cysteine addition to semen extender on viability of

buffalo bull spermatozoa is presented in Figure 4.6. Percentage of viable sperm was

observed to be higher (P < 0.05) in the experimental extenders supplemented with 1.0

mM of cysteine as compared to control at 0, 2 and 4 hours post-thaw. Sperm viability

in extender containing 2 mM of cysteine was similar (P > 0.05) as compared to the

experimental semen extender having 0.5 mM of cysteine, while the sperm viability in

extender containing 3.0 mM of cysteine did not differ (P > 0.05) as compared to the

sperm viability in semen extender containing 0.5 mM of cysteine and control at 0 and

2 hours post-thaw. At 4 hours after thawing, sperm viability was similar (P > 0.05) in

extender containing 0.5 mM, 1.5 mM, 2.0 mM and 3.0 mM of cysteine as compared

to control.

56

In a recent study, higher intact acrosome of cryopreserved buffalo bull

spermatozoa assessed through phase contrast microscopy was recorded in extender

containing cysteine however; no marker was used to separate the live/dead

spermatozoa (Ansari et al., 2010).

Supplementation of cysteine in extender improved the percentage of viable

spermatozoa along with increase in catalase activity (Bucak et al., 2008) in feline

(Thuwanut et al., 2008) swine (Funahashi and Sano, 2005) and ovine semen (Uysal

and Bucak, 2007; Bucak et al., 2008). It is shown in figure 4.6 that buffalo bull sperm

viability was conserved during the current study. It might had been due to the

presence of cysteine that had increased the intracellular activity of antioxidants.


The data on the effect of cysteine addition to semen extender on plasmalemma

integrity of buffalo bull spermatozoa is presented in Figure 4.7. Sperm plasmalemma

integrity was recorded superior (P < 0.05) in extenders containing cysteine 1.0 mM as

compared to control at 0, 2 and 4 hours post-thaw. At 0 hour post-thaw, plasmalemma

integrity did not vary (P > 0.05) in extender, containing 1.5 mM and 2.0 mM of

cysteine but was better than the control, while the plasmalemma integrity of

spermatozoa in extender containing 3.0 mM of cysteine was similar (P >0.05) as

compared to the experimental semen extender having 0.5 mM of cysteine and control.

At 2 hours, post-thaw.

57

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

0 2 4

Post thaw hour

Sp

erm

mot

ility

(%

)


c

bc

a a

b

c

c bc

a

a

b

c

bc bc

a

ab

bc

c

Figure 4.5. Effect of cysteine addition in semen extender on the progressive motility

of buffalo bull spermatozoa at 0, 2 and 4 hours after thawing. Bars with different

letters differed significantly (P < 0.05) at a given time.

58

The sperm plasmalemma integrity was similar (P >0.05) in extender

containing 2.0 mM and 0.5 mM of cysteine while the plasmalemma integrity in

extender containing 3.0 mM of cysteine was similar (P > 0.05) as compared to

experimental semen extender containing 0.5 mM of cysteine and control. At 4 hours

post-thaw plasmalemma integrity was similar in extenders containing 0.5 mM, 1.5

mM, 2.0 mM and 3.0 mM of cysteine and control.) A significantly higher percentage

of sperm with a functional plasma membrane after the addition of cysteine in extender

in bovine and ovine semen (Sariözkan et al., 2009; Uysal and Bucak, 2007).

Atessahin et al. (2008) reported a non significant increase in plasmalemma integrity

of the buck semen after the addition of cysteine. El-Sheshtawy et al. (2008) reported

significantly higher plasmalemma integrity after the addition of cysteine in Egyptian

buffalo semen. In the present study, it was observed that cysteine supplementation to

semen extender protected the membrane integrity which might be due to scavenging

the ROS molecules directly and/or indirectly in the semen-extender complex (Figure

4.7).


The data on the effect of cysteine addition to semen extender on DNA fragmentation

of buffalo spermatozoa is presented in Figure 4.8. It was observed that buffalo sperm

DNA integrity was higher in extender containing all experimental levels of cysteine

compared to control. It is known that the freeze-thawing process induced DNA

fragmentation in buffalo semen (Kumar et al., 2011) and result in reduced total

antioxidant capacity.

59

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

0 2 4

Post thaw hour

Sp

erm

via

bili

ty (

%)


c bc

aa

b

c

c bc

a

a

b

c

b b

a

abb

b

Figure 4.6. Effect of cysteine addition in semen extender on viability of buffalo bull

spermatozoa at 0, 2 and 4 hours after thawing. Bars with different letters differed


60

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

0 2 4

Post thaw hour

Sp

erm

pla

smal

emm

a in

tigr

ity

(%)


a

d

cd

ab

bc

d

cbc

a

a

b

c

bb

a

b

b

ab

Figure 4.7. Effect of cysteine addition in semen extender on the plasma membrane



61

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

0 2 4

Incubation hours

Sp

erm

DN

A in

tigr

ity

(%)

Control

0.5 mM

1.0 mM

1.5 mM

2.0 mM

3.0 mM

a a a a a

bb

aa a a a

b

a aa

a a

Figure 4.8. Effect of cysteine addition in semen extender on the DNA integrity of



62

It is believed that antioxidants have a protective role for DNA damage in the process

of semen freezing and thawing (Bucak et al., 2010). Glutathione, of which cysteine is

a precursor molecule, lowered the percentage of sperm with fragmented DNA by

protection against ROS molecules (Gadea et al., 2007; Tuncer et al., 2010). The

higher occurrence of DNA integrity of sperm in the current study might be due to the

reduction of oxidative stress in the presence of higher levels of cysteine in the

extender (Figure 4.8).

4.3. EFFECT OF THIOGLYCOL IN SEMEN EXTENDER ON POST-THAW



The data on the response to thioglycol addition to semen extender on the

percentage of motility of buffalo bull spermatozoa is presented in Figure 4.9. At 0, 2

and 4 hours after thawing, sperm progressive motility was higher in extenders

containing 1.0 mM, 1.5 mM, 2.0 mM and 3.0 mM of thioglycol compared to

extenders containing 0.5 mM of thioglycol and control.

Sperm motility is an indicator of semen quality routinely used to determine the

effect of experimental procedures and before artificial insemination. Sperm motility is

affected by physical and biochemical properties of the diluents (Akhter et al., 2008).

Chilling of buffalo spermatozoa resulted in decreased motility associated with

reduced antioxidant activity and higher ROS molecules levels (El-Sisy et al., 2007).

The results of one study (Garg et al., 2009) on buffalo spermatozoa also suggested

63

that H2O2 caused reduction of motility in vitro. Recently, it was observed that motility

of buffalo spermatozoa and total antioxidant potential decreased during the

cryopreservation process (Anzar et al., 2010; Kumar et al., 2011). Thioglycol had the

ability to protect the motility of bovine spermatozoa from deleterious effects of H2O2

in vitro, a major ROS molecule naturally produced in mammalian semen (Bilodeau et

al., 2001).

It was believed that thioglycol protected the motility of buffalo bull semen

from deleterious effects of oxidative stress produced during the freeze-thawing

process by inhibiting the ROS-mediated damage. Moreover, it was observed that

thioglycol in maturation media enhanced the ATP metabolism in the oocyte (Tsuzuki

et al., 2005). In light of the results of this study and above discussion, it was thought

that the improvement in sperm motility in extender containing thioglycol might be

associated with the reduction in oxidative stress and enhancement of ATP metabolism

in spermatozoa (Figure 4.9).


The data on the effect of thioglycol addition to semen extender on the

percentage of viable (live sperm with intact acrosome) buffalo bull spermatozoa are

presented in Figure 4.10. At 0, 2 and 4 hours after thawing, sperm viability was higher

in extenders containing 1.0 mM, 1.5 mM 2.0 mM and 3.0 mM of thioglycol as

compared to extenders containing 0.5 mM of thioglycol and control.

64

Sperm viability assessment with a dual staining procedure could be an

effective technique for indirect prediction of fertilizing ability of spermatozoa

(Tartaglione and Ritta, 2004). There was a positive relation between the percentage of

sperm with intact acrosomes and fertility in frozen bovine semen (Saacke and White,

1972). It was generally observed that the freeze-thawing process reduced the

acrosomal integrity of the buffalo spermatozoa (Rasul et al., 2001; Anzar et al., 2010;

Kumar et al., 2011). Thioglycol protected the bovine embryo against oxidative stress

and improved the blastocyst quality (Feugang et al., 2004). Thioglycol protected the

oocyte/embryo viability by directly interacting with oxidized radicals and/or

increasing intracellular glutathione levels in oocytes (Cornell and Crivaro, 1972;

Bannaï, 1992; Takahashi et al., 1993; Wardman and Sonntag, 1995; Matos and

Furnus, 2000; Takahashi et al., 2002; Matos et al., 2002). It was believed that

thioglycol levels in extenders of present study might have reduced the acrosomal

damage as was observed in another study (Sinha et al., 1996).


The data on the effect of glutathione addition to semen extender on the

percentage of buffalo bull spermatozoa with intact plasma membrane is presented in

Figure 4.11. At 0, 2 and 4 hours after thawing, sperm plasmalemma was observed

higher in extenders containing 1.0 mM, 1.5 mM, 2.0 mM and 3.0 mM of thioglycol as

compared to extenders containing 0.5 mM of thioglycol and control. Buffalo sperm

plasmalemma integrity decreased during cryopreservation (Rasul et al., 2001; Anzar

et al., 2010) due to poor levels of antioxidant potential of the semen (Kumar et al.,

2011).

65

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

0 2 4

Post thaw hour

Sp

erm

mot

ility

(%

)


c

b

a

a

aa

c

b

a a a

a

c

b

a a ab a

Figure 4.9. Effect of thioglycol addition in semen extender on progressive motility of


show significant (P < 0.05) differences at a given time.

66

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

0 2 4

Post thaw hour

Sp

erm

via

bili

ty (

%)


c

b

a a a a

c

b

a a a a

c

b

aa a a

Figure 4.10. Effect of thioglycol addition in semen extender on viability of buffalo

bull spermatozoa at 0, 2 and 4 hours after thawing. Bars with different letters differed


67

LPO has recorded higher in frozen-thawed spermatozoa compared to fresh buffalo

spermatozoa (Kadirvel et al., 2009). ROS molecules produced during

cryopreservation could caused damage to the sperm membrane by the initiation of the

lipid peroxidation cascade (Sharma and Agarwal, 1996). Sperm cells were susceptible

to oxidative stress because of higher levels of polyunsaturated phospholipids in the

membrane, which might decrease the fertilizing ability of the spermatozoa (Storey,

1997). Thioglycol had antioxidant activity that protected the sperm bio-membrane

system by reducing ROS mediated damage to bovine spermatozoa in vitro (Bilodeau

et al., 2001). The sperm plasmalemma integrity was recorded higher during this study

in extender containing thioglycol which might have protected the plasma membrane

by reducing lipid peroxidation levels (Figure 4.11).


The data on the effect of thioglycol addition to semen extender on DNA

integrity of buffalo bull spermatozoa is presented in Figure 4.12. In the present study,

DNA integrity of buffalo spermatozoa was higher in all experimental extenders

containing thioglycol compared to control. It was observed that oxidative stress

during cryopreservation could damage the DNA integrity of mammalian spermatozoa

(Aitken et al., 1998; Andrabi, 2009; Bucak et al., 2010) and resulted in reduced

fertilizing ability of the spermatozoa. It was reported that cryopreservation reduced

the DNA intactness of buffalo spermatozoa and was associated with reduction in total

antioxidant potential of semen (Kumar et al., 2011). A significant correlation was

recorded between DNA fragmentation and levels of ROS molecules in buffalo semen

(Kadirvel et al., 2009). Thioglycol improved the development of cloned embryo by

68

inhibiting the apoptosis process (Park et al., 2004a,b) by its antioxidant activity. It is

concluded that thioglycol reduced the DNA fragmentation of buffalo bull

spermatozoa due to its protective effects for gamete DNA.

4.4. FERTILITY RATE OF BUFFALO SEMEN CRYOPRESERVED IN BEST

EVOLVED EXTENDERS

The data on the fertility rate of buffalo bull spermatozoa cryopreserved in the

best evolved extenders containing glutathione (2 mM), cysteine (1.0 mM) and

thioglycol (1.0 mM) is given in Figure 4.13-4.15.

The fertility rate of cryopreserved buffalo bull spermatozoa in terms of

positive pregnancy at 2 month post-insemination was recorded higher (P < 0.05) in

extender containing GSH and cysteine as compared to control. Recently, in vivo

fertility rate of semen from crossbred Jersey breed cryopreserved in extender

containing GSH was observed significantly higher compared to extender with out

GSH, 68% vs 49%, respectively (Perumal et al., 2010). Higher in vivo fertility rates of

caprine semen cryopreserved in extender containing GSH was recorded (Sinha et al.,

1996). It was observed that addition of GSH to the fertilization medium significantly

increased the fertilizing ability of mouse spermatozoa by reducing ROS molecules

(Bath et al., 2010). Addition of GSH to the dilution medium after thawing improved

the fertilizing ability of bovine spermatozoa (Gadea et al., 2007). However, it was

also observed that bovine semen cryopreserved in GSH supplemented milk based

extender Laiciphose® did not improve the fertility rate (Tuncer et al., 2010).

69

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

0 2 4

Post thaw hour

Sp

erm

pla

smal

emm

a in

tigr

ity

(%)


c

b

aa a a

c

b

aa a a

c

b

aa a a

Figure 4.11. Effect of thioglycol addition in semen extender on plasma membrane



70

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

0 2 4

Post thaw hour

Sp

erm

DN

A in

tigr

ity

(%)

Control

0.5 mM

1.0 mM

1.5 mM

2.0 mM

3.0 mM

b

a a a a a

b

a a a a a

b

a a a a a

Figure 4.12. Effect of thioglycol addition in semen extender on DNA integrity of



71

Similarly, fertility of semen cryopreserved in milk extender containing GSH and

superoxide dismutase did not result in any improvement (Foote et al., 2002). It was

concluded that the presence of naturally occurring antioxidant (casein) in milk based

extender reduced the requirement of extra antioxidant supplementation.

The higher percentage of fertility based on 59-day non-returns was observed

(74.54% vs 57.145) in animals inseminated with extender containing cysteine and

control in bovine (Sariözkan et al., 2009). Tuncer et al. (2010) observed that

supplementation of cysteine in Laiciphose® extender did not improve the conception

rate in bovine. It was observed in a previous study that presence of naturally occurring

antioxidant (casein) in milk extender reduced the requirement of extra antioxidant

supplementation (Foote et al., 2002).

In the present study overall in vivo fertility rate after using cryopreserved

buffalo bull semen remained similar (P>0.05) in extender containing thioglycol and

control. It was observed that thioglycol supplementation in in vitro maturation media

improved the quality of buffalo embryos (Shang et al., 2007). Thioglycol added in the

in vitro culture media protected the bovine embryos from oxidative stress and

improved the quality of blastocysts by reducing apoptosis induction (Feugang et al.,

2004). In an in vitro study it was observed that the addition of thioglycol increased the

number of cumulus cells attached to oocytes and cell numbers of blastocysts by

influencing ATP metabolism (Tsuzuki et al., 2005). Funahashi (2005) reported

beneficial effect of thioglycol for sperm penetration and early embryonic

development. Thioglycol presence in maturation media also improved the maturation

and M II stage of canine oocytes in vitro (Kim et al., 2004).

72

0

10

20

30

40

50

60

70

Control GSH Cysteine Thioglycol

Extnders

Fer

tilit

y (%

)

a

a ab

b

Figure 4.13. Effect of glutathione, cysteine and thioglycol on fertility rate (%) of

buffalo bull spermatozoa. Bars with different letters differed significantly (P < 0.05).

73

4.5. GENERAL DISCUSSION

Freeze-thawing of mammalian semen is associated with oxidative stress that

causes overproduction of reactive oxygen species resulting in lipid peroxidation of

plasma membrane (Aitken et al., 1998). Buffalo sperm is composed of a plasma

membrane which has a higher content of polyunsaturated fatty acids compared to

cattle bull sperm (Parks et al., 1987; Cheshmedjieva and Dimov 1994). This makes

the buffalo sperm more susceptible to oxidative stress during the process of

cryopreservation (Nair et al., 2006). Consequently, there is a reduction in motility

and integrity of plasma membrane, acrosome and chromatin of buffalo spermatozoa

(Rasul et al., 2001; Anzar et al., 2010; Kumar et al., 2011). Likewise, there is also a

decrease in fertility of buffalo spermatozoa compared to cattle bull spermatozoa

(Andrabi, 2009).

Buffalo semen has enzymatic and non-enzymatic antioxidants to protect the

spermatozoa from oxidative stress (Sansone et al., 2000; Andrabi, 2009). However, it

is believed that the indigenous defense system of buffalo semen against the oxidative

stress during the process of freezing and thawing is insufficient. This weakness is

mainly due to low concentrations of naturally occurring antioxidants in buffalo

semen, which is further decreased during the extension and freezing process (El-Sisy

et al., 2007; Andrabi, 2009; Kumar et al., 2011).

Glutathione, cysteine and thioglycol are antioxidants that protected the sperm

cell from oxidative stress (Ansari et al., 2010; 2011a,b; Bilodeau et al., 2001).

Cysteine and thioglycol can directly neutralized free radicals and/or act through

74

glutathione mediated cellular pathway. Cryopreservation resulted in a decrease of

thiols levels in bovine semen (Bilodeau et al., 2000). Moreover, supplementation of

glutathione and cysteine in extender resulted in better post-thaw semen quality

(Bilodeau et al., 2001; Tuncer et al., 2010; Ansari et al., 2011a,b). In an another

study, glutathione and cysteine addition to bovine semen extender resulted in a

decrease of the lipid peroxidation levels during freezing and ultimately improved

fertility rates with semen cryopreserved in extender containing glutathione (Perumal

et al., 2010). In other studies on chilled and frozen thawed buffalo semen, it was

demonstrated that motility, normal apical ridge and plasmalemma integrity was

significantly better with the addition of glutathione and cysteine in extender (Ansari et

al., 2010; 2011a,b).

In the present study, glutathione, cysteine and thioglycol were tested at the

concentration of 0.5, 1.0, 1.5, 2.0 and 3.0mM in extender for cryopreservation of

buffalo semen. Sperm motility, plasmalemma integrity (structural and functional) and

viability (live sperm with intact acrosome) was recorded higher in extenders

containing glutathione 2.0mM, cysteine 1.0mM and thioglycol 1.0mM. Sperm DNA

integrity was higher in extender containing glutathione, cysteine and thioglycol at any

concentration during the study. Semen containing antioxidants in a concentration

exhibiting higher semen quality (glutathione 2.0mM, cysteine 1.0mM and thioglycol

1.0mM) were tested for in vivo fertility rate under field conditions. It was observed

that glutathione and cysteine was the best antioxidant for improving fertility rate

under field conditions during the present study. The findings of the present study are

inline with the results of previous studies performed on buffalo semen in which

75

improvements in on post-thaw semen quality parameters were observed by adding

glutathione and cysteine to extender (Ansari et al., 2010; 2011a,b).

In an in vitro study, it was demonstrated that glutathione, cysteine and

thioglycol had the ability to protect the motility of frozen-thawed bovine spermatozoa

at 0.5mM concentration during in vitro incubation and artificially induced oxidative

stress generated by adding hydrogen peroxide in diluting media (Bilodeau et al.,

2001). In the present study, tris-citric egg yolk extender was used for cryopreservation

of buffalo semen. It was evident that hydrogen peroxide was the most prominent

reactive oxygen species molecule in egg yolk based extender (Bilodeau et al., 2002).

This might be the reason that glutathione, cysteine and thioglycol protected the

quality of buffalo semen efficiently by interacting with hydrogen peroxide. Using

different antioxidants beneficial to non-beneficial effects have been reported for

cryopreservation of semen but more consistent beneficial effects have been reported

using glutathione in extender.

It is relevant to mention that the addition of glutathione and cysteine at the rate

of 2.0 and 3.0 mM to extender, proved to be ineffective for cryopreservation of

buffalo spermatozoa (Ansari et al., 2011a,b). Addition of cysteine protected the

spermatozoa by synthesis of glutathione. However, selenium is also essential for the

formation of phospholipid hydroperoxide glutathione peroxidase, which becomes a

structural protein in the mid-piece of spermatozoa. When either substance is deficient

or in other words if glutathione levels are higher than the selenium levels, it can lead

to instability of the mid-piece of the spermatozoa, resulting in reduced sperm quality

(Slaweta et al., 1988; Meseguer et al., 2004).

76

The highest fertility rate was observed in extender containing glutathione and

cysteine. Gadea at el. (2007) reported higher oocyte penetrability of the bovine

spermatozoa with glutathione in thawing extender along with higher embryo

production in vitro. Supplementation of cysteine in Laiciphose® extender did not

improve the conception rate in bovine (Tuncer et al., 2010). Laiciphose® is a milk

based extender and has an antioxidant, namely casein, which might decrease the extra

antioxidant supplementation requirement (Foote et al., 2002). In an in vitro study it

was observed that the addition of thioglycol increased the number of cumulus cells

attached to oocytes and cell numbers of blastocysts by influencing ATP metabolism

(Tsuzuki et al., 2005). Funahashi (2005) reported beneficial effect of thioglycol for

sperm penetration and early embryonic development. Keeping in view the results of

previous studies it was thought that higher fertility rate observed in the present study

with semen cryopreserved in extender containing antioxidants might have been due to

the ability of these substances to decrease the oxidative stress.

77

Table 4.1. Overview of the studies on the role glutathione in semen storage.

Reference Specie Extender State Doses (mM) SM SPMI SV SAI SDI SF

Ansari et al. (2011a) Buffalo Tris-citric acid-fructose-yolk Liquid 0.5, 1, 2, 3 ↑ ↑ -- ↑ -- --

Ansari et al. (2010) Buffalo Tris-citric acid-fructose-yolk-glycerol Frozen 0.5, 1, 2, 3 ↑ ↑ -- ↑ -- --

Perumal et al. (2010) Cattle Tris-citric acid-fructose-yolk-glycerol Frozen 5 -- ↑ -- ↑ -- ↑

Bucak et al. (2008) Sheep Tris-citric acid-fructose-yolk-glycerol Frozen 5 -- -- -- -- -- --

Bucak and Tekin (2007) Sheep Tris-citric acid-fructose-yolk Liquid 5, 10 -- -- ↑ -- -- --

Gadea et al. (2007) Cattle Thawing medium Liquid 1, 5 -- -- -- ↑ ↑ ↑

Munsi et al. (2007) Cattle Egg yolk-citrate Liquid 0.5, 1, 2, 3 ↑ -- -- ↑ -- --

Sinha et al. (1996) Goat Tris-citric acid-fructose-yolk-glycerol Frozen 2, 5 ↑ -- -- ↑ -- NS

In present study Buffalo Tris-citric acid-fructose-yolk-glycerol Frozen 0.5, 1, 1.5, 2, 3 ↑ ↑ ↑ ↑ ↑ ↑

↑ = Improvement -- = Not performed NS = Non-significant SM = Sperm motility SPMI = Sperm plasmalemma integrity SAI= Sperm acrosomal integrity SV = Sperm viability SDI = Sperm DNA integrity SF = Semen fertility

78

Table 4.2. Overview of the studies on the role of cysteine in semen storage.

Reference Specie/breed Extender State Doses (mM) SM SPMI SV SAI SDI SF

Ansari et al. (2011b) Nili-Ravi buffalo Tris-citric acid- fructose-yolk-glycerol Frozen 0.5, 1, 2, 3 ↑ ↑ -- ↑ -- --

Ansari et al. (2011d) Sahiwal bull Tris-citric acid- fructose-yolk-glycerol Frozen 0.5, 1, 2 ↑ ↑ -- ↑ -- --

Bucak et al. (2008) Sheep Tris-citric acid- fructose-yolk-glycerol Frozen 5 ↑ -- -- -- -- --

Perumal et al. (2010) Cattle Tris-citric acid- fructose-yolk-glycerol Frozen 5 NS -- -- ↑ -- NS

Uysal and Bucak (2007) Sheep Tris-citric acid- fructose-yolk-glycerol Frozen 5, 10, 20 ↑ ↑ ↑ ↑ -- --

Funahashi and Sano (2005) Boar Modified Modena solution Liquid 5 -- -- ↑ ↑ -- --

In present study Buffalo Tris-citric acid- fructose-yolk-glycerol Frozen 0.5, 1, 1.5, 2, 3 ↑ ↑ ↑ ↑ ↑ ↑

↑ = Improvement -- = Not performed NS = Non-significant SM = Sperm motility SPMI = Sperm plasmalemma integrity SV = Sperm viability SAI= Sperm acrosomal integrity SDI = Sperm DNA integrity SF = Semen fertility

79

Table 4.3. Overview of the studies on the role of thioglycol in gamete/embryo protection.

Reference Protection by thioglycol

Bilodeau et al. (2001) Protected the motility of bovine spermatozoa in vitro during induced oxidative stress by hydrogen peroxide

Tsuzuki et al. (2005) Increased the ATP metabolism of the oocyte and number of cumulus cells

Takahashi et al. (2002)

de Matos and Furnus (2000)

Improved the viability of oocyte/embryo by reducing oxidative stress directly and/or by increasing

intracellular glutathione synthesis

Feugang et al. (2004) Prevented the apoptosis in cloned embryo

Shang et al. (2007) Improved the quality of buffalo embryos

Funahashi (2005) Improved the sperm penetration of oocyte and embryonic development

In present study Improved the motility, plasma membrane, acrosome and DNA integrity of buffalo spermatozoa

82

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Aitken, R. 1984. A free radicals theory of male infertility. Reprod. Fertil. Develop., 6:

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Aitken, R. J., E. Gordon., D. Harkiss., J. P. Twigg., P. Milne., Z. Jenning and D. S.

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Akhter, S., M. S. Ansari., S. M. H. Andrabi., N. Ullah and M. Qayyum. 2008. Effect

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Akhter, S., B. A. Rakha., M. S. Ansari., S. M. H. Andrabi and N. Ullah. 2011. Storage

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cryodamage to human sperm during cryopreservation. J. Andrology, 13: 232–

241.

Andrabi, S. M. H. 2008. Mammalian sperm chromatin structure and assessment of

DNA fragmentation. J. Assist. Reprod. Genet., 24: 561-569.

Andrabi, S. M. H. 2009. Factors affecting the quality of cryopreserved buffalo

(Bubalus bubalis) bull spermatozoa. Reprod. Domest. Anim., 44: 552-569.

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APPENDIX I

Appendix 1. Analysis of variance of sperm motility for glutathione at 0 hour post-

thaw.

Source of variation SS df MS F-value p-value

Treatments 679.17 5 135.833 54.33 0.00

Blocks 58.33 2 29.167 11.67 0.00

Error 25.00 10 2.500

Total 762.50 17

SS = Sum of square df = Degree of freedom MS = Mean sum of square

Appendix 2. Analysis of variance of sperm motility for glutathione at 2 hours post-

thaw.


Treatments 1,244.44 5 248.889 16.91 0.00

Blocks 352.78 2 176.389 11.98 0.00

Error 147.22 10 14.722

Total 1,744.44 17


108

Appendix 3. Analysis of variance of sperm motility for glutathione at 4 hours post-

thaw.


Treatments 1,244.44 5 248.889 16.91 0.00

Blocks 352.78 2 176.389 11.98 0.00

Error 147.22 10 14.722

Total 1,744.44 17


Appendix 4. Analysis of variance of sperm viability for glutathione at 0 hour post-

thaw.


Treatments 1,244.44 5 248.889 16.91 0.00

Blocks 352.78 2 176.389 11.98 0.00

Error 147.22 10 14.722

Total 1,744.44 17


109

Appendix 5. Analysis of variance of sperm viability for glutathione at 2 hours post-

thaw.


Treatments 1,639.83 5 327.967 27.25 0.00

Blocks 324.33 2 162.167 13.48 0.00

Error 120.33 10 12.033

Total 2,084.50 17


Appendix 6. Analysis of variance of sperm viability for glutathione at 4 hour post-

thaw.


Treatments 1,903.61 5 380.722 18.26 0.00

Blocks 293.44 2 146.722 7.04 0.012

Error 208.56 10 20.856

Total 2,405.61 17


110

Appendix 7. Analysis of variance of sperm plasma membrane integrity for

glutathione at 0 hour post-thaw.


Treatments 948.44 5 189.689 79.40 0.00

Blocks 37.44 2 18.722 7.84 0.01

Error 23.89 10 2.389

Total 1,009.78 17



glutathione at 2 hours post-thaw.


Treatments 1,677.11 5 335.422 19.59 0.00

Blocks 294.78 2 147.389 8.61 0.01

Error 171.22 10 17.122

Total 2,143.11 17


111


glutathione at 4 hours post-thaw.


Treatments 1,739.11 5 347.822 33.06 0.00

Blocks 244.11 2 122.056 11.60 0.00

Error 105.22 10 10.522

Total 2,088.44 17


Appendix 10. Analysis of variance of sperm DNA integrity for glutathione at 0 hour

post-thaw.


Treatments 235.33 5 47.067 48.69 1.07E-06

Blocks 1.00 2 0.500 0.52 .6113

Error 9.67 10 0.967

Total 246.00 17


112

Appendix 11. Analysis of variance of sperm DNA integrity for glutathione at 2 hours

post-thaw.


Treatments 294.67 5 58.933 65.48 0.00

Blocks 0.33 2 0.167 0.19 0.83

Error 9.00 10 0.900

Total 304.00 17


Appendix 12. Analysis of variance of sperm DNA integrity for glutathione at 4 hours

post-thaw.


Treatments 294.67 5 58.933 65.48 0.00

Blocks 0.33 2 0.167 0.19 0.83

Error 9.00 10 0.900

Total 304.00 17


113

Appendix 13. Analysis of variance of sperm motility for cysteine at 0 hour post-thaw.


Treatments 990.28 5 198.056 41.94 0.00

Blocks 2.78 2 1.389 0.29 0.75

Error 47.22 10 4.722

Total 1,040.28 17


Appendix 14. Analysis of variance of sperm motility for cysteine at 2 hours post-

thaw.


Treatments 990.28 5 198.056 41.94 0.00

Blocks 2.78 2 1.389 0.29 0.75

Error 47.22 10 4.722

Total 1,040.28 17


114

Appendix 15. Analysis of variance of sperm motility for cysteine at 4 hours post-

thaw.


Treatments 683.33 5 136.667 12.62 0.00

Blocks 158.33 2 79.167 7.31 0.01

Error 108.33 10 10.833

Total 950.00 17


Appendix 16. Analysis of variance of sperm viability for cysteine at 0 hour post-

thaw.


Treatments 1,225.11 5 245.022 65.24 0.00

Blocks 5.78 2 2.889 0.77 0.49

Error 37.56 10 3.756

Total 1,268.44 17


115

Appendix 17. Analysis of variance of sperm viability for cysteine at 2 hours post-

thaw.


Treatments 1,865.83 5 373.167 97.35 0.00

Blocks 80.33 2 40.167 10.48 0.0035

Error 38.33 10 3.833

Total 1,984.50 17


Appendix 18. Analysis of variance of sperm viability for cysteine at 4 hours post-

thaw.


Treatments 926.94 5 185.389 9.59 0.0014

Blocks 105.44 2 52.722 2.73 0.1133

Error 193.22 10 19.322

Total 1,225.61 17


116

Appendix 19. Analysis of variance of sperm plasma membrane integrity for cysteine

at 0 hour post-thaw.


Treatments 1,046.28 5 209.256 40.59 0.00

Blocks 5.78 2 2.889 0.56 0.58

Error 51.56 10 5.156

Total 1,103.61 17



at 2 hours post-thaw.


Treatments 1,717.33 5 343.467 81.13 0.00

Blocks 64.33 2 32.167 7.60 0.01

Error 42.33 10 4.233

Total 1,824.00 17


117


at 4 hours post-thaw.


Treatments 643.33 5 128.667 9.37 0.0016

Blocks 121.33 2 60.667 4.42 .0422

Error 137.33 10 13.733

Total 902.00 17


Appendix 22. Analysis of variance of sperm DNA integrity for cysteine at 0 hour

post-thaw.


Treatments 643.33 5 128.667 9.37 0.0016

Blocks 121.33 2 60.667 4.42 0.0422

Error 137.33 10 13.733

Total 902.00 17


118

Appendix 23. Analysis of variance of sperm DNA integrity for cysteine at 2 hours

post-thaw.


Treatments 643.33 5 128.667 9.37 0.0016

Blocks 121.33 2 60.667 4.42 0.0422

Error 137.33 10 13.733

Total 902.00 17


Appendix 24. Analysis of variance of sperm DNA integrity for cysteine at 4 hours

post-thaw.


Treatments 232.28 5 46.456 39.07 0.00

Blocks 15.44 2 7.722 6.50 0.02

Error 11.89 10 1.189

Total 259.61 17


119

Appendix 25. Analysis of variance of sperm motility for thioglycol at 0 hour post-

thaw.


Treatments 1,066.67 5 213.333 85.33 0.00

Blocks 58.33 2 29.167 11.67 0.0024

Error 25.00 10 2.500

Total 1,150.00 17


Appendix 26. Analysis of variance of sperm motility for thioglycol at 2 hours post-

thaw.


Treatments 1,066.67 5 213.333 85.33 0.00

Blocks 58.33 2 29.167 11.67 0.0024

Error 25.00 10 2.500

Total 1,150.00 17


120

Appendix 27. Analysis of variance of sperm motility for thioglycol at 4 hours post-

thaw.


Treatments 1,066.67 5 213.333 85.33 0.00

Blocks 58.33 2 29.167 11.67 0.0024

Error 25.00 10 2.500

Total 1,150.00 17


Appendix 28. Analysis of variance of sperm viability for thioglycol at 0 hour post-

thaw.


Treatments 1,214.28 5 242.856 133.27 0.00

Blocks 5.78 2 2.889 1.59 0.25

Error 18.22 10 1.822

Total 1,238.28 17


121

Appendix 29. Analysis of variance of sperm viability for thioglycol at 2 hours post-

thaw.


Treatments 1,215.17 5 243.033 54.82 0.00

Blocks 139.00 2 69.500 15.68 0.0008

Error 44.33 10 4.433

Total 1,398.50 17


Appendix 30. Analysis of variance of sperm viability for thioglycol at 4 hours post-

thaw.

Source of variation SS Df MS F-value p-value

Treatments 1,215.17 5 243.033 54.82 0.00

Blocks 139.00 2 69.500 15.68 0.001

Error 44.33 10 4.433

Total 1,398.50 17


122


thioglycol at 0 hour post-thaw.


Treatments 1,286.28 5 257.256 50.22 0.00

Blocks 68.78 2 34.389 6.71 0.0142

Error 51.22 10 5.122

Total 1,406.28 17



thioglycol at 2 hours post-thaw.


Treatments 1,197.11 5 239.422 93.69 0.00

Blocks 172.44 2 86.222 33.74 0.00

Error 25.56 10 2.556

Total 1,395.11 17


123


thioglycol at 4 hours post-thaw.


Treatments 1,255.11 5 251.022 34.28 0.00

Blocks 201.44 2 100.722 13.76 0.0013

Error 73.22 10 7.322

Total 1,529.78 17


Appendix 34. Analysis of variance of sperm DNA integrity for thioglycol at 0 hours

post-thaw.


Treatments 356.94 5 71.389 98.85 0.00

Blocks 7.44 2 3.722 5.15 0.0290

Error 7.22 10 0.722

Total 371.61 17


124


post-thaw.

Source f variation SS df MS F-value p-value

Treatments 357.61 5 71.522 94.66 0.00

Blocks 0.44 2 0.222 0.29 0.7514

Error 7.56 10 0.756

Total 365.61 17



post-thaw.


Treatments 357.61 5 71.522 94.66 0.00

Blocks 0.44 2 0.222 0.29 0.75

Error 7.56 10 0.756

Total 365.61 17


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