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
Muhammad Sajjad Ansari
(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
3.4.4. Sperm DNA Integrity 42
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
4.1.2. Sperm Viability 46
4.1.3. Sperm Plasmalemma Integrity 49
4.1.4. Sperm DNA Integrity 50
4.2.1. EFFECT OF CYSTEINE IN SEMEN EXTENDER 51
ON POST-THAW QUALITY OF
BUFFALO BULL SPERMATOZOA
4.2.1. Progressive Motility 51
4.2.2. Sperm Viability 55
4.1.3. Sperm Plasmalemma Integrity 56
4.1.4. Sperm DNA Integrity 58
4.3. EFFECT OF THIOGLYCOL IN SEMEN EXTENDER 62
viii
ON POST-THAW QUALITY OF
BUFFALO BULL SPERMATOZOA
4.1.1. Progressive Motility 62
4.1.2. Sperm Viability 63
4.1.3. Sperm Plasmalemma Integrity 64
4.1.4. Sperm DNA Integrity 67
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
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.
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
different letters differed significantly (P < 0.05) at a given time.
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
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.
65
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 significantly (P < 0.05) at a given time.
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
thawing. Bars with different letters differed significantly (P < 0.05)
at a given time.
69
4.12. Effect of thioglycol 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.
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.
Muhammad Sajjad Ansari
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),
plasmalemma (structural and functional) and DNA integrity] of the Nili-Ravi
buffalo bull spermatozoa
3. To identify the impact of various levels of thioglycol in semen extender on
post-thaw quality [motility, viability (live sperm with intact acrosome),
plasmalemma (structural and functional) and DNA integrity] of the Nili-Ravi
buffalo bull spermatozoa
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).
3.4.4. Sperm DNA Integrity
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.
4.1.2. Sperm Viability
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
different letters differed significantly (P < 0.05) at a given time.
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.
4.1.3. Sperm Plasmalemma Integrity
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.
4.1.4. Sperm DNA Integrity
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
QUALITY OF BUFFALO BULL SPERMATOZOA
4.2.1. Progressive Motility
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 (
%)
Control 0.5 mM 1.0 mM 1.5 mM 2.0 mM 3.0 mM
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
(%)
Control 0.5 mM 1.0 mM 1.5 mM 2.0 mM 3.0 mM
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
different letters differed significantly (P < 0.05) at a given time.
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.
4.2.2. Sperm Viability
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.
4.2.3. Sperm Plasmalemma Integrity
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
(%
)
Control 0.5 mM 1.0 mM 1.5 mM 2.0 mM 3.0 mM
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).
4.2.4. Sperm DNA Integrity
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 (
%)
Control 0.5 mM 1.0 mM 1.5 mM 2.0 mM 3.0 mM
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
significantly (P < 0.05) at a given time.
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
(%)
Control 0.5 mM 1.0 mM 1.5 mM 2.0 mM 3.0 mM
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
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
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
buffalo bull spermatozoa at 0, 2 and 4 hours after thawing. Bars with different letters
differed significantly (P < 0.05) at a given time.
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
QUALITY OF BUFFALO BULL SPERMATOZOA
4.3.1. Progressive Motility
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).
4.3.2. Sperm Viability
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).
4.3.3. Sperm Plasmalemma Integrity
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
(%
)
Control 0.5 mM 1.0 mM 1.5 mM 2.0 mM 3.0 mM
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
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.
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 (
%)
Control 0.5 mM 1.0 mM 1.5 mM 2.0 mM 3.0 mM
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
significantly (P < 0.05) at a given time.
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).
4.3.4. Sperm DNA Integrity
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
(%)
Control 0.5 mM 1.0 mM 1.5 mM 2.0 mM 3.0 mM
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
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.
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
buffalo bull spermatozoa at 0, 2 and 4 hours after thawing. Bars with different letters
differed significantly (P < 0.05) at a given time.
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
LITERATURE CITED
Abhi, H. L. 1982. Note on the freezing of buffalo semen by Landshut method and fertility
of frozen semen in comparison to liquid semen under field conditions. Ind. J.
Anim. Sci., 52: 809-811.
Aitken, R. 1984. A free radicals theory of male infertility. Reprod. Fertil. Develop., 6:
19-24.
Aitken, R. J., E. Gordon., D. Harkiss., J. P. Twigg., P. Milne., Z. Jenning and D. S.
Irvine. 1998. Relative impact of oxidative stress on the functional competence
and genomic integrity of human spermatozoa. Biol. Reprod., 59: 1037-1046.
Akhter, S., M. Sajjad., S. M. H. Andrabi., N. Ullah and M. Qayyum. 2007. Effect of
antibiotics in extender on fertility of liquid buffalo bull semen. Pak. Vet. J.,
27: 13-16.
Akhter, S., M. S. Ansari., S. M. H. Andrabi., N. Ullah and M. Qayyum. 2008. Effect
of antibiotics in extender on bacterial control and spermatozoal quality of
cooled buffalo (Bubalus bubalis) bull semen. Reprod. Dom. Anim., 43: 272-
278.
83
Akhter, S., B. A. Rakha., M. S. Ansari., S. M. H. Andrabi and N. Ullah. 2011. Storage
of Nili-Ravi buffalo (Bubalus bubalis) semen in skim milk extender
supplemented with ascorbic acid and α-tocopherol. Pak. J. Zool., 43: 273-277.
Alvarez, J. G. and B. T. Storey. 1992. Evidence for increased lipid peroxidation
damage and loss of superoxide dismutase activity as a mode of sublethal
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.
Andrabi, S. M. H., M. S. Ansari., N. Ullah and M. Afzal. 2008. Effect of non-
enzymatic antioxidants in extender on post-thaw quality of buffalo (Bubalus
bubalis) bull spermatozoa. Pak. Vet. J., 28: 159-162.
Ansari, M. S., B. A. Rakha., N. Ullah., S. M. H. Andrabi and S. Akhter. 2011a.
Glutathione addition in tris-citric egg yolk extender improves the quality of
cooled buffalo (Bubalus bubalis) bull semen. Pak. J. Zool., 43: 46-55.
Ansari, M. S., B. A. Rakha., N. Ullah., S. M. H. Andrabi., M. Khalid and S. Akhter.
2011b. Effect of L-cysteine in tris-citric egg yolk extender on post thaw
84
quality of Nili-Ravi buffalo (Bubalus bubalis) bull spermatozoa. Pak. J. Zool.,
43: 41-47.
Ansari, M. S., B. A. Rakha., N. Ullah., S. M. H. Andrabi., S. Iqbal., M. Khalid and S.
Akhter. 2010. Effect of exogenous glutathione in extender on the freezability
of Nili-Ravi buffalo (Bubalus bubalis) bull spermatozoa. Anim. Sci. Pap.
Rep., 28: 235-244.
Ansari, M. S., B. A. Rakha and S. Akhter. 2011c. Effect of butylated hydroxytoluene
in extender on motility, plasmalemma and viability of Sahiwal bull
spermatozoa. Pak. J. Zool., 43: 311-314.
Ansari, M. S., B. A. Rakha and S. Akhter. 2011d. Effect of L-cysteine in extender on
post-thaw quality of Sahiwal bull semen. Anim. Sci. Pap. Rep., 29: 197-203.
Ansari, M. S., B. A. Rakha., S. M. H. Andrabi and S. Akhter. 2011e. Effect of straw
size and thawing time on quality of cryopreserved buffalo (Bubalus bubalis)
semen. Reprod. Biol., 11: 49-55.
Anzar, M., U. Farooq., M. A. Mirza., M. Shahab and N. Ahmed. 2003. Factors
affecting the efficiency of artificial insemination in cattle and buffalo in
Punjab, Pakistan. Pak. Vet. J., 23: 106-113.
85
Anzar, M., Z. Rasul., T. A. Ahmed and N. Ahmad. 2010. Response of buffalo
spermatozoa to low temperatures during cryopreservation. Reprod. Fertil.
Dev., 22: 871-880.
Arabi, M. 2005. Bull spermatozoa under mercury stress. Reprod. Domest. Anim., 40:
454-459.
Atessahin, A., M. N. Bucak., P. B. Tuncer and M. Kizil. 2008. Effects of antioxidant
additives on microscopic and oxidative parameters of Angora goat semen
following the freeze–thawing process. Small Rum. Res., 77: 38-44.
Bailey, J. L., J. F. Bilodeau and N. Cormier. 2000. Semen cryopreservation in
domestic animals: a damaging and capacitating phenomena. J. Androl., 22:
508-515.
Ball, B. A., V. Medina., C. G. Gravance and J. Baumber. 2001. Effect of antioxidants
on preservation of motility, viability and acrosomal integrity of Equine
spermatozoa during storage at 5oC. Theriogenology, 56: 577-589.
Ballachey, B. E., W. D. Honenboken and D. Evenson. 1987. Heterogeneity of sperm
nuclear chromatin structure and its relationship to bull fertility. Biol. Reprod.,
36: 915-925.
86
Ballachey, B. E., D. P. Evenson and R. G. Saacke. 1988. The sperm chromatin
structure assay: relationship with alternate tests of semen quality and
heterospermic performance of bulls. J. Androl., 9: 109-115.
Bannaï, S. 1992. Use of 2-mercaptoethanol in the cell culture. Human Cell, 5: 292-
297.
Bath, M. L. 2010. Inhibition of in vitro fertilizing capacity of cryopreserved mouse
sperm by factors released by damaged sperm, and stimulation by glutathione.
PLoS One, 24: e9387.
Baumber, J., B. A. Ball and J. J. Linfor. 2005. Assessment of the cryopreservation of
equine spermatozoa in the presence of enzyme scavengers and antioxidants.
Am. J. Vet. Res., 66: 772-779.
Baumber, J., B. A. Ball., C. G. Gravance., V. Medina and M. C. G. Davies-Morel.
2000. The effect of reactive oxygen species on equine sperm motility,
viability, acrosomal integrity, mitochondrial membrane potential, and
membrane lipid peroxidation. J. Androl., 21: 895-902.
Baumber, J., B. A. Ball., J. J. Linfor and S. A. Meyers. 2003. Reactive oxygen species
and cryopreservation promote DNA fragmentation in equine spermatozoa. J.
Androl., 24: 621-628.
Benchaib, M., V. Braun., J. Lornage., S. Hadj., B. Salle., H. Lejeune and J. F.
87
Guerin. 2003. Sperm DNA fragmentation decreases the pregnancy rate in an
assisted reproductive technique. Hum. Reprod., 18: 1023–1028.
Bilodeau, J. F., S. Blanchette., C. Gagnon and M. A. Siarrd. 2001. Thiols prevent
H2O2- mediated loss of sperm motility in cryopreserved bull semen.
Theriogenology, 56: 275-286.
Bilodeau, J. F., S. Blanchette., C. Gagnonm and M. A. Sirard. 2000. Levels of
antioxidant defenses are decreased in bovine spermatozoa after a cycle of
freezing and thawing. Mol. Reprod. Develop., 55: 282-288.
Bilodeau, J. J., S. Blanchette., N. Cormier and M. A. Sirard. 2002. Reactive oxygen
species-mediated loss of bovine sperm motility in egg yolk tris extender:
protection by pyruvate, metal chelators and bovine liver or oviductal fluid
catalase. Theriogenology, 57: 1105-1122.
Bucak, M. N., S. Sarıözkan., P. B. Tuncer., P. A. Ulutaş and H. I. Akçadağ. 2009.
Effect of antioxidants on microscopic semen parameters, lipid peroxidation
and antioxidant activities in Angora goat semen following cryopreservation.
Small Rum. Res., 81: 90-95.
Bucak, M. N., A. Atessahin and A. Yuce. 2008. Effect of anti-oxidants and
oxidative stress parameters on ram semen after the freeze-thawing process.
Small Rum. Res., 75: 128-134.
88
Bucak, M. N., A. Ateşşahin., Ömer Varışlı., A. Yüce., N. Tekin and A. Akçay. 2007.
The influence of trehalose, taurine, cysteamine and hyaluronan on ram semen
microscopic and oxidative stress parameters after freeze-thawing process.
Theriogenology, 67: 1060-1067.
Bucak, M. N. and N. Tekin. Protective effect of taurine, glutathione and trehalose on
the liquid storage of ram semen. Small Rum. Res., 73: 103-108.
Bucak, M. N., P. B. Tuncer., S. Sarıözkan., N. Başpınar., M. Taşpınar., K. Coyan., A.
Bilgili., P. P. Akalın., S. Büyükleblebici., S. Aydos., S. Ilgaz., A. Sunguroğlu
and D. Oztuna. 2010. Effects of antioxidants on post-thawed bovine sperm and
oxidative stress parameters: antioxidants protect DNA integrity against
cryodamage. Cryobiology, 61: 248-253.
Cabaud, P. G., F. Wreblewski and V. Rauggiero. 1958. Colorimeteric measurement of
LDH acitivity of body fluids. Am. J. Clin. Pathol., 30: 234.
Chen, C. S., H. T. Chao., R. L. Pan and Y. H. Wei. 1997. Hydroxyl radical-induced
decline in motility and increase in lipid peroxidation and DNA modification in
human sperm. Biochem. Mol. Biol. Int., 43: 291–303.
Cheshmedjieva, S. B. and V. N. Dimov. 1994. Effect of freezing on phospholipid
distribution of buffalo spermatozoa plasma membranes. In: Vale, W. G..,
V. H. Barnabe., J. C. A. de Mattos. Proceedings of 4th World Buffalo
89
Cong., Sao Paulo, Brazil. International Buffalo Federation, Roma, pp. 519-
521.
Chohan, A. R., J. Iqbal., A. A. Asghar and M. A. Chaudhry. 1992. Fertility of liquid and
frozen semen in Nili Ravi buffaloes. Pak. Vet. J., 12: 4-5.
Chohan, K. R., J. T. Griffin and D. T. Carrell. 2004. Evaluation of chromatin integrity
in human sperm using acridine orange staining with different fixatives and
after cryopreservation. Andrologia, 36: 321–326.
Chtterjee, S., and Gagnon. 2001. Production of reactive oxygen species by
spermataozoa undergoing cooling, freezing, and thawing. Mole. Reprod.
Develop., 59: 451-458.
Cormier, N., M. Sirad and J. L. Bailey. 1997. Premature capaacitation of bovine
spermatozoa is initiated by cryopreservation. J. Androl., 18: 461-468.
Cornell, N. W. and K. E. Crivaro. 1972. Stability constant for zinc–dithiothreitol
complex. Anal. Biochem., 47: 203–208.
Cotran, R. S., V. Kumar and S. L. Robins. 1989. Pathologic basis of diseases, 4th edn,
W. B. Saunders Co. Philadelphia, 9-16.
90
Donnelly, E. T., N. McClure and S. E. Lewis. 1999. The effect of ascorbate and alpha-
tocopherol supplementation in vitro on DNA integrity and hydrogen peroxide-
induced DNA damage in human spermatozoa. Mutagenesis, 14: 505–512.
El-Sisy, G.A., W. S. El-Nattat and R. I. El-Sheshtawy. 2007. Buffalo semen quality,
antioxidants and peroxidation during chilling and cryopreservation. J. Vet.
Res., 11: 55-61.
El-Sisy, G. A., W. S., El-Nattat and R. I. El-Sheshtawy. 2008. Effect of superoxide
dismutatse and catalase on viability of cryopreserved buffalo spermatozoa.
Global Vet., 2: 56-61.
El-Sheshtawy, R. I., G. A. EL-SISY and W.S. El-Nattat. 2008. Use of selected
amino acids to improves buffalo bull semen cryopreservation. Global
Vetrinaria, 2: 146-150.
Evenson, D. P., K. Larson and L. K. Jost. 2002. Sperm chromatin structure assay: its
clinical use for detecting sperm DNA fragmentation in male infertility and
comparisons with other techniques. J. Androl., 23: 25-43.
Feugang, J. M., R. de Roover., A. Moens., S. Léonard., F. Dessy and I. Donnay. 2004.
Addition of beta-mercaptoethanol or Trolox at the morula/blastocyst stage
improves the quality of bovine blastocysts and prevents induction of apoptosis
and degeneration by prooxidant agents. Theriogenology, 61: 71-90.
91
Foote RH, Brockett CC, Kaproth MT. 2002. Motility and fertility of bull sperm in
whole milk extender containing antioxidants. Anim. Reprod. Sci., 71: 13-23.
Fraser, L. and J. Strzezek. 2004. The use of comet assay to assess DNA integrity of
boar spermatozoa following liquid preservation at 5 and 16ºC. Folia
Histochemica et Cytobiologica, 42: 49–55.
Funahashi, H., 2005. Effect of beta-mercaptoethanol during in vitro fertilization
procedures on sperm penetration into porcine oocytes and the early
development in vitro. Reproduction, 130: 889-898.
Funahashi, H., T. Fujiwara., T. Nagai. 2000. Modulation of the function of boar
spermatozoa via adenosine and fertilization promoting peptide receptors
reduce the incidence of polyspermic penetration into porcine oocytes. Biol.
Reprod., 63: 1157-1163.
Funahashi, H. and T. Nagai. 2001. Regulation of in vitro penetration of frozen-thawed
boar spermatozoa by caffeine and adenosine. Mol. Reprod. Dev., 58: 424-431.
Funahashi, H. and T. Sano. 2005. Select antioxidants improve the function of
extended boar semen stored at 10 °C. Theriogenology, 63: 1605-1616.
Gadea, J., D. Gumbao., S. C. Novas., F. A. Z. Zquez., L. A. Grullo and G. C. Gardo.
2007. Supplementation of the dilution medium after thawing with reduced
glutathione improves function and the in vitro fertilizing ability of frozen-
thawed bull spermatozoa. Andrology, 7: 1-10.
92
Gadea, J., E. Selles., M. A. Marco., P. Copy., C. Matas., R. Romar and S. Ruiz. 2004.
Decrease in glutathione content in boar sperm cryopreservation. Effect of the
addition of reduced glutathione to the freezing and thawing extenders.
Theriogenology, 62: 690-701.
Gadea, J., D. Gumbao., C. Matas and R. Romar. 2005. Supplementation of the
thawing media with reduced glutathione improves function and the in vitro
fertilization ability of boar sperm after cryopreservation. J. Androl., 26: 749-
756.
Garg, A., A. Kumaresan and M. R. Ansari. 2009. Effect of hydrogen peroxide (H2O2)
on fresh and cryopreserved buffalo sperm functions during incubation at 37oC
in vitro. Reprod. Domestic Anim., 44: 907-912.
Gillan, L., T. Kroetsch., W. M. Maxwell., G. Evans. 2008. Assessment of in vitro
sperm characteristics in relation to fertility in dairy bulls. Anim Reprod Sci.,
103: 201-14.
Garcia, O., K. Mahmood and T. Hemme. 2003. A review of milk production in
Pakistan with particular emphasis on small scale producers. International Farm
Comparison Network (IFCN).
Graham, E. F., B. G. Crabo and K. I. Brown. 1972. Effect of some zwitter ion buffers
on the freezing and storage of spermatozoa. I. Bull. J. Dairy Sci., 55: 372–378.
93
Hancock, J. L. 1952. The morphology of bull spermatozoa. J. Exp. Biol., 29: 445-453.
Hammadeh, M. E., S. Greiner., P. Rosenbaum and W. Schmidt. 2001. Comparison
between human sperm preservation medium and TEST-yolk buffer on
protecting chromatin and morphology integrity of human spermatozoa in fertile
and subfertile men after freeze-thawing procedure. J. Androl., 22: 1012–1018.
Hammerstedt, R. H., J. K. Graham and J. P. Nolan. 1990. Cryopreservation of
mammalian sperm: what we ask them to survive. J. Androl., 11: 73-88.
Harrison, R. A., P. J. Ashworth and N. G. Miller. 1996. Bicarbonate/Co2, an effector
of capacitation, induces a rapid and reversible change in the lipid architecture
of boar sperm plasma membranes. Mol. Reprod. Dev., 45: 378-391.
Hughes, C. M., S. E. Lewis., V. J. McKelvey-Martin and W. Thompson. 1996. A
comparison of baseline and induced DNA damage in human spermatozoa
from fertile and infertile men, using a modified comet assay. Mol. Human
Reprod., 2: 613–619.
Johnson, P. E., R. J. Flipse and J. O. Almquist. 1954. The effect of cysteine
hydrochloride on the livability of bull spermatozoa in unheated skim milk.
Pennsylvania Agri. Exp. Station, 53-57.
94
Jeyendran, R. S., H. H. Van Der Ven., M. Perez-Pelaez., B. G., Crabo., L. J. D.
Zaneveld. 1984. Development of an assay to assess the functional integrity of
the human sperm membrane and its relationship to other semen characteristics.
J. Reprod. Fertil., 70: 219-228.
Kadirvel, G., K. Satish and A. Kumaresan. 2009. Lipid peroxidation, mitochondrial
membrane potential and DNA integrity of spermatozoa in relation to
intracellular reactive oxygen species in liquid and frozen-thawed buffalo
semen. Anim. Reprod. Sci., 114: 125-134.
Kasimanickam, R., V. Kasimanickam., C. D. Thatcher., R. L. Nebel and B. G.
Cassell. 2007. Relationships among lipid peroxidation, glutathione peroxidase,
superoxide dismutase, sperm paramters, and competitive index in dairy bulls.
Theriogenology, 67: 1004-1012.
Kim, M. K., Y. H. Fibrianto., J. O. Hyun., G. Jang., H. J. Kim., K. S. Lee., S. K.
Kang., B. C. Lee and W. S. Hwang. 2004. Effect of β-mercaptoethanol or
epidermal growth factor supplementation on in vitro maturation of canine
oocytes collected from dogs with different stages of the estrus cycle. J. Vet.
Sci., 5: 253-258.
Khan, D. R., N. Ahmad., M. Anzar and A. A. Channa. 2009. Apoptosis in fresh and
crryopreserved buffalo sperm. Theriogenology, 71: 872-876.
95
Kodama, H., R. Yamaguchi., J. Fukuda., H. Kasai and T. Tanaka. 1997. Increased
oxidative deoxyribonucleic acid damage in the spermatozoa of infertile male
patients. Ferti. Steri. 68: 519–524.
Kovacs, A. and R. H. Foote. 1992.Viability and acrosome staining of bull, boar and
rabbit spermatozoa. Biotech. Histochem., 67:119–24.
Krzyzosiak, J., D. Evenson., C. Pitt., L. Jost., P. Molan and R. Vishwanath. 2000.
Changes in susceptibility of bovine sperm to in situ DNA denaturation during
prolonged incubation at ambient temperature under conditions of exposure to
reactive oxygen species and nuclease inhibitor. Reprod. Fertil., 12: 251-261.
Kumar, R., G. Jagan Mohanarao., Arvind and S. K. Atreja. 2011. Freeze-thaw
induced genotoxicity in buffalo (Bubalus bubalis) spermatozoa in relation to
total antioxidant status. Mol. Boil. Rep., 38: 1499-1506.
Kumaresan, A., M. R. Ansari and A. Garg. 2005. Modulation of post thaw sperm
functions with oviductal proteins in buffaloes. Anim. Reprod. Sci., 90: 73-84.
Kumaresan, A., M. R. Ansari., A. Garg and M. Kataria. 2006. Effect of oviductal
proteins on sperm functions and lipid peroxidation levels during
cryopreservation in buffaloes. Anim. Reprod. Sci., 93: 264-257.
96
Linfor, J. J. and S. A. Mayers. 2002. Detection of DNA damage in response to cooling
injury in equine spermatozoa using single-cell gel electrophoresis. J. Androl.
23: 107–113.
Livestock Census. 2006. Pakistan Livestock Census. Agriculture Census
Organization.
Lopes, S., A. Jurisicova., J. G. Sun and R. F. Casper. 1998. Reactive oxygen species:
potential cause for DNA fragmentation in human spermatozoa. Human
Reprod., 13: 896–900.
Mammoto, A., N. Masumoto., M. Tahara., Y. Ikebuchi., M. Ohmichi., K. Tasaka and
A. Miyake. 1996. Reactive oxygen species block sperm-egg fusion via
oxidation of sperm sulfhydryl proteins in mice. Biol. Reprod., 55:1063-1068.
Matos, D. G. and C. C. Furnus. 2000. The importance of having high glutathione
(GSH) level after bovine in vitro maturation on embryo development: Effect
of β-mercaptoethanol, cysteine and cystine. Theriogenology, 53: 761-771.
Matos, D. G., B. Gasparrini., S. R. Pasqualini and J. G. Thompson. 2002. Effect of
glutathione synthesis stimulation during in vitro maturation of ovine oocyte on
embryo development and intracellular peroxide content. Theriogenology 57:
1443-1445.
97
Maxwell, W.M. and S. Salamon. 1993. Liquid storage of ram semen: a review.
Reprod. Fertil. Dev., 5: 613-638.
McKelvey-Martin, V. J., N. Melia., I. K. Walsh., S. R. Johnston., C. M. Hughes., S. E.
Lewis and W. Thompson. 1997. Two potential clinical applications of the
alkaline single-cell gel electrophoresis assay: (1). Human bladder washings
and transitional cell carcinoma of the bladder; and (2). human sperm and male
infertility. Mut. Res., 375: 93–104.
Meseguer, M., N. Garrido., C. Simón., A. Pellicer and J. Remohí. 2004.
Concentration of glutathione and expression of glutathione peroxidases 1 and
4 in fresh sperm provide a forecast of the outcome of cryopreservation of
human spermatozoa. J. Androl., 25: 273-80.
Meister, A. and M. E. Anderson. 1983. Glutathione. Annual Rev. Biochem., 52: 711-
760.
Michael, A., C. Alexopoulos., E. Pontiki., D. Hadjipavlou-Litina., P. Saratsis and C.
Boscos. 2007. Effect of antioxidant supplementation on semen quality and
reactive oxygen species of frozen-thawed canine spermatozoa.
Theriogenology, 68: 204-212.
Munsi, M. N., M. M. U. Bhuiyan., S. Majumder and M. G. S. Alam. 2007. Effects of
exogenous glutathione on the quality of chilled bull semen. Reprod. Domest.
Anim., 42: 358–362.
98
Nair, S. J., A. S. Brar., C. S. Ahuja., S. P. Sangha, , K. C. Chaudhary. 2006. A
comparative study on lipid peroxidation, activities of antioxidant enzymes and
viability of cattle and buffalo bull spermatozoa during storage at refrigeration
temperature. Anim. Reprod. Sci., 96: 21-29.
Nichi, M., P. E. J. Bols., R. M. Zuge., V. H. Barnabe., I. G. F. Goovaerts, R. C.
Barnabe., C. N. M. Cortada. 2006. Seasonal variation in semen quality in Bos
indicus and Bos taurus bulls raised under tropical conditions. Theriogenology.
66: 822-828.
O’Connor, M. T., R. P. Amann and R. G. Saacke. 1981. Comparisons of computer
evaluations of spermatozoal motility with standard laboratory tests and their
use for predicting fertility. J. Anim. Sci., 53: 1368–1376.
Pagl, R., C. Aurich and M. Kankofer. 2006. Anti-oxidative status and semen quality
during cooled storage in stallions. J. Vet. Med. A., 53: 486-489.
Palencia, D. D., D. L. Garner and D. Hudig. 1996. Determination of activable
proacrosin/acrosin in bovine sperm using an irreversible isocoumarin serine
protease inhibitor. Biol. Reprod., 55: 536–542.
Parks, J. E., J. A. Arion and R. H. Foote. 1987. Lipids of plasma membrane and
outer acrosomal membrane from bovine spermatozoa. Biol. Reprod., 37:
1249-1258.
99
Park, E. S., W. S. Hwang., G. Jang., J. K. Cho., S. k. Kang., B. C. Lee., J. Y. Han and
J. M. Lim. 2004a. Incidence of apoptosis in clone embryos and improved
development by the treatment of donors somatic cells with putative apoptosis
inhibitors. Mol. Reprod. Dev., 68: 65-71.
Park, E. S., W. S. Hwang., S. k. Kang., B. C. Lee., J. Y. Han and J. M. Lim. 2004b.
Improved embryos development with decreased apoptosis Incidence of
apoptosis in clone embryos and improved development by the treatment of
donors somatic cells with putative apoptosis inhibitors. Mol. Reprod. Dev., 68:
65-71.
Peris, S. I., A. Morrier., M. Dufour and J. L. Bailey. 2004. Cryopreservation of ram
semen facilitates sperm DNA damage: relationship between sperm
andrological parameters and the sperm chromatin structure assay. J. Androl.,
25: 224–233.
Perumal, P., S. Selvaraju., S. Selvakumar., A. Barik., D. Mohanty., S. Das., R. Das
and P. Mishra. 2010. Effect of pre-freeze addition of cysteine hydrochloride
and reduced glutathione in semen of crossbred Jersey bulls on sperm
parameters and conception rates. Reprod. Domest. Anim., DOI:
10.1111/j.1439-0531.2010.01719.x
100
Potts, R. J., L. J. Notarianni and T. M. Jefferies. 2000. Seminal plasma reduces
exogenous oxidative damage to human sperm, determined by the measurement
of DNA strand breaks and lipid peroxidation. Mut. Res., 447: 249–256.
Rasul, Z., M, Anzar., S. Jalali and N. Ahmad. 2000. Effect of buffering system on
post-thaw motion characteristics, plasma membrane integrity and acrosome
morphology of buffalo spermatozoa. Anim. Reprod. Sci., 59: 31-41.
Rasul, Z., N. Ahmad and M. Anzar. 2001. Changes in motion characteristics, plasma
membrane integrity, and acrosome morphology during cryopreservation of
buffalo spermatozoa. J. Androl., 22: 278-83.
Reitman, S. and S. Frankel. 1957. A colorometric method for the determination of
serum glutamic oxaloacetic and glutamic pyruvic transaminases. Am. J. Clin.
Pathol., 28: 56.
Saack, R. G. and J. M. White. 1972. Semen quality tests and their relationship to
fertility. Proc. Fourth Tech. Conf. Artif. Insem, and Reprod. p 22.
Sariözkan, S., M. n. Bucak., P. B. Tuncer., P. A. Ulutaş., and A. Bilgen. 2009. The
influence of cysteine and taurine on microscopic-oxidative stress parameters
and fertilizing ability of bull semen following cryopreservation. Cryobiology,
58: 134-1338.
101
Sansone, G., M. J. F. Nastri and A. Fabbrocini. 2000. Storage of buffalo (Bubalus
bubalis) semen. Anim. Reprod. Sci., 62: 55-76.
Seli, E., D. K. Gardner., W. B. Schoolcraft., O. Moffatt and D. Sakkas. 2004. Extent
of nuclear DNA damage in ejaculated spermatozoa impacts on blastocyst
development after in vitro fertilization. Fertil. Steril., 82: 378–383.
Shabbir, S. G., C. S. Ali., H. A. Samad and S. H. Hanjra. 1982. Comparative study of
fertility of frozen and fluid semen of buffalo. Pak. Vet. J., 2: 179-180.
Shang, J. H., Y. J. Huang., X. F. Zhang., F. X. Hung and J. Qin. 2007. Effect of β-
mercaptoethanol and buffalo follicular fluid on fertilization and subsequent
embryonic development of water buffalo (Bubalus bubalis) oocytes derived
from in vitro maturation. Ital. J. Anim. Sci., 6: 751-754.
Shannon, P. and R. Vishwanath. 1995. The effect of optimal and suboptimal
concentrations of sperm on the fertility of fresh and frozen bovine semen and a
theoretical model to explain the fertility differences. Anim. Reprod. Sci., 39:
1–10.
Sharma, R. K. and A. Agarwal. 1996. Role of reactive oxygen species in male
infertility. Urology, 48: 835 -850.
Sharma, N. C. and K. L. Sahni. 1988. Fertility of frozen and liquid semen in
buffaloes. 2nd World Buffalo Congress New Delhi, India, 100.
102
Sikka, S. C. 2004. Role of oxidative stress and antioxidants in andrology and assisted
reproductive technology. J. Andrology. 24: 5-18.
Sinha, M. P., A. K. Sinha., B. K. Singh and P. L. Prasad. 1996. The effect of
glutathione on the motility, enzyme leakage and fertility of frozen goat
semen. Theriogenology, 41: 237–243.
Sławeta, R., T. Laskowska and E. Szymańska. 1988. Seasonal changes in total and
selenium-dependent glutathione peroxidase activity in bovine semen in
relation to lipid peroxide. Anim. Reprod. Sci., 17: 303-307.
Slaweta, R., T. Laskowska and E. Szymanska. 1998. Lipid peroxides, spermatozoa
quality and activity of glutathione peroxidase in bull semen. Acta Physiol.
Pol., 39: 207–214.
Sreejith, J. N., A. S. Brar., C. S. Ahuja., S. P. S. Sangha and K. C. Chaudhary. 2006.
A comparative study on lipid peroxidation, activities of antioxidant enzymes
and viability of cattle and buffalo bull spermatozoa during storage at
refrigeration temperature. Anim. Reprod. Sci., 96: 21-29.
Storey, B. T. 1997. Biochemistry of the induction and prevention of lipoperoxidative
damage in human spermatozoa. Mol. Human Reprod., 3: 203–213.
103
Stradaioli, G., T. Noro., L. Sylla and M. Monaci. 2007. Decrease in glutathione
(GSH) content in bovine sperm after cryopreservation: Comparison between
two extenders. Theriogenology, 67: 1249-1255.
Sun, J. G., A. Jurisicova and R. F. Casper. 1997. Detection of deoxyribonucleic acid
fragmentation in human sperm: correlation with fertilization in vitro. Biol.
Reprod., 56: 602-607.
Szczesniak-Fabianzyk, B., M. Bockenek., Z. Smorag and F. Ryszka. 2003. Effect of
antioxidants added to boar semen extender on the semen survival time and
sperm chromatin structure. Reprod. Biol., 3: 83-89.
Takahashi, Y., T. Nagai., N. Okamura., H. Takahashi and A. Okano. 2002. Promoting
effect of β-mercaptoethanol on in vitro development under oxidative stress and
cysteine uptake of bovine embryos. Biol. Reprod., 6: 562-567.
Takahashi, Y., T. Nagai., S. Hamano., M. Kuwayama., N. Okamura and A. Okano.
1993. Effect of thiols compounds on in vitro development and intracellular
glutathione content of bovine embryos. Biol. Reprod., 49: 228-232.
Tartaglionea, C. M. and M.N. Ritta. 2004. Prognostic value of spermatological
parameters as predictors of in vitro fertility of frozen-thawed bull semen.
Theriogenology, 62: 1245-1252.
104
Tejada, R. I., J. C. Mitchell., A. Norman., J. J. Marik and S. Friedman. 1984. A test
for the practical evaluation of male fertility by acridine orange (AO)
fluorescence. Fertil. Steril., 42: 87-91.
Thuwanut, P., K. Chatdarong., M. Techakumphu and E Axnér. 2008. The effect of
antioxidants on motility, viability, acrosome integrity and DNA integrity of
frozen-thawed pididymal cat spermatozoa. Theriogenology, 70: 233-40.
Tsuzuki, Y., Y. Saigoh and Koji Ashizawa. 2005. Effects of β-mercaptoethanol on
ATP contents in cumulus cell-enclosed bovine oocytes matured in vitro and
sequential development of resultant embryos from In vitro fertilization. J.
Mamm. Ova. Res., 22: 33-39.
Tuncer, P. B., M. N. Bucak., S. Büyükleblebici., S. Sarıözkan., D. Yeni., A. Eken., P.
P. Akalın., H. Kinet., F. Avdatek., A. F. Fidan and M. Gündoğan., 2010. The
effect of cysteine and glutathione on sperm and oxidative stress parameters of
post-thawed bull semen. Cryobiology, 61: 303-307.
Twigg, J., N. Fulton., E. Gomez., D. S. Irvine and R. J. Aitken. 1998a. Analysis of the
impact of intracellular reactive oxygen species generation on the structural and
functional integrity of human spermatozoa: lipid per-oxidation, DNA
fragmentation and effectiveness of antioxidants. Human Reprod., 13: 1429–
1436.
105
Twigg, J. P., D. S. Irvine and R. J. Aitken. 1998b. Oxidative damage to DNA in
human spermatozoa does not preclude pronucleus formation at intracytoplas-
mic sperm injection. Human Reprod., 13: 1864–1871.
Upreti, G. C., K. Jensen., J. E. Oliver., D. M. Duganzich., R. Munday and J. F. Smith.
1997. Motility of ram spermatozoa during storage in a chemically-defined
diluent containing antioxidants. Anim. Reprod. Sci., 48: 269-278.
Ullah, N., M. Anwar., S. M. H. Andrabi., M. S. Ansari., S. Murtaza., Q. Ali and M.
Asif. 2010. Effect of mineral supplementation on post partum ovarian activity
in Nili-Ravi buffaloes (Bubalus bubalis). Pak, J. Zool., 43: 195-2000.
Uysal, O. and M. N. Bucak. 2007. Effect of oxidized serum albumin, cysteine and
lycopene on the quality of frozen thawed ram semen. Acta Vet. Brno., 76:
383-390.
Visconti, P. E., J. L. Bailey., G. D. Moore., D. Pan., P. Olds-Clarke and S. Kopf.
1995. Capacitation of mouse spermatozoa I. Correlation between the
capacitation status and protein tyrosine phosphorylation. Develop., 121: 1129-
1137.
Vishwanath, R. and P. Shannon. 1997. Do sperm cells age? A review of the
physiological changes in sperm during storage at ambient temperature.
Reprod. Fertil. Dev., 9: 321-332.
106
Wardman, P. and C. Von Sonntag. 1995. Kinetic factors that control the fate of thiol
radicals in cells. Meth. Enz. 251: 31-45.
Watson, P. F. 1995. Recent developments and concepts in the cryopreservation of
spermatozoa and the assessment of their post-thawing function. Reprod. Fertil.
Dev., 7: 871–891.
Wang, A. W., H. Zhang., I. Ikemoto., D. J. Anderson and K. R. Loughlin. 1997.
Reactive oxygen species generation by seminal cells during
cryopreservation. Urology, 49: 921-925.
Yildiz, C. P., N. Ottaviani., R. Law., L. Ayearst., C. Liu and McKerlie. 2007.
Effects of cryopreservation on sperm quality, nuclear DNA integrity, in vitro
fertilization, and in vitro embryo development in the mouse. Reproduction,
133: 585-595.
107
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.
Source of variation SS df MS F-value p-value
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
SS = Sum of square df = Degree of freedom MS = Mean sum of square
108
Appendix 3. Analysis of variance of sperm motility for glutathione at 4 hours post-
thaw.
Source of variation SS df MS F-value p-value
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
SS = Sum of square df = Degree of freedom MS = Mean sum of square
Appendix 4. Analysis of variance of sperm viability for glutathione at 0 hour post-
thaw.
Source of variation SS df MS F-value p-value
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
SS = Sum of square df = Degree of freedom MS = Mean sum of square
109
Appendix 5. Analysis of variance of sperm viability for glutathione at 2 hours post-
thaw.
Source of variation SS df MS F-value p-value
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
SS = Sum of square df = Degree of freedom MS = Mean sum of square
Appendix 6. Analysis of variance of sperm viability for glutathione at 4 hour post-
thaw.
Source of variation SS df MS F-value p-value
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
SS = Sum of square df = Degree of freedom MS = Mean sum of square
110
Appendix 7. Analysis of variance of sperm plasma membrane integrity for
glutathione at 0 hour post-thaw.
Source of variation SS df MS F-value p-value
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
SS = Sum of square df = Degree of freedom MS = Mean sum of square
Appendix 8. Analysis of variance of sperm plasma membrane integrity for
glutathione at 2 hours post-thaw.
Source of variation SS df MS F-value p-value
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
SS = Sum of square df = Degree of freedom MS = Mean sum of square
111
Appendix 9. Analysis of variance of sperm plasma membrane integrity for
glutathione at 4 hours post-thaw.
Source of variation SS df MS F-value p-value
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
SS = Sum of square df = Degree of freedom MS = Mean sum of square
Appendix 10. Analysis of variance of sperm DNA integrity for glutathione at 0 hour
post-thaw.
Source of variation SS df MS F-value p-value
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
SS = Sum of square df = Degree of freedom MS = Mean sum of square
112
Appendix 11. Analysis of variance of sperm DNA integrity for glutathione at 2 hours
post-thaw.
Source of variation SS df MS F-value p-value
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
SS = Sum of square df = Degree of freedom MS = Mean sum of square
Appendix 12. Analysis of variance of sperm DNA integrity for glutathione at 4 hours
post-thaw.
Source of variation SS df MS F-value p-value
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
SS = Sum of square df = Degree of freedom MS = Mean sum of square
113
Appendix 13. Analysis of variance of sperm motility for cysteine at 0 hour post-thaw.
Source of variation SS df MS F-value p-value
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
SS = Sum of square df = Degree of freedom MS = Mean sum of square
Appendix 14. Analysis of variance of sperm motility for cysteine at 2 hours post-
thaw.
Source of variation SS df MS F-value p-value
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
SS = Sum of square df = Degree of freedom MS = Mean sum of square
114
Appendix 15. Analysis of variance of sperm motility for cysteine at 4 hours post-
thaw.
Source of variation SS df MS F-value p-value
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
SS = Sum of square df = Degree of freedom MS = Mean sum of square
Appendix 16. Analysis of variance of sperm viability for cysteine at 0 hour post-
thaw.
Source of variation SS df MS F-value p-value
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
SS = Sum of square df = Degree of freedom MS = Mean sum of square
115
Appendix 17. Analysis of variance of sperm viability for cysteine at 2 hours post-
thaw.
Source of variation SS df MS F-value p-value
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
SS = Sum of square df = Degree of freedom MS = Mean sum of square
Appendix 18. Analysis of variance of sperm viability for cysteine at 4 hours post-
thaw.
Source of variation SS df MS F-value p-value
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
SS = Sum of square df = Degree of freedom MS = Mean sum of square
116
Appendix 19. Analysis of variance of sperm plasma membrane integrity for cysteine
at 0 hour post-thaw.
Source of variation SS df MS F-value p-value
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
SS = Sum of square df = Degree of freedom MS = Mean sum of square
Appendix 20. Analysis of variance of sperm plasma membrane integrity for cysteine
at 2 hours post-thaw.
Source of variation SS df MS F-value p-value
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
SS = Sum of square df = Degree of freedom MS = Mean sum of square
117
Appendix 21. Analysis of variance of sperm plasma membrane integrity for cysteine
at 4 hours post-thaw.
Source of variation SS df MS F-value p-value
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
SS = Sum of square df = Degree of freedom MS = Mean sum of square
Appendix 22. Analysis of variance of sperm DNA integrity for cysteine at 0 hour
post-thaw.
Source of variation SS df MS F-value p-value
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
SS = Sum of square df = Degree of freedom MS = Mean sum of square
118
Appendix 23. Analysis of variance of sperm DNA integrity for cysteine at 2 hours
post-thaw.
Source of variation SS df MS F-value p-value
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
SS = Sum of square df = Degree of freedom MS = Mean sum of square
Appendix 24. Analysis of variance of sperm DNA integrity for cysteine at 4 hours
post-thaw.
Source of variation SS df MS F-value p-value
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
SS = Sum of square df = Degree of freedom MS = Mean sum of square
119
Appendix 25. Analysis of variance of sperm motility for thioglycol at 0 hour post-
thaw.
Source of variation SS df MS F-value p-value
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
SS = Sum of square df = Degree of freedom MS = Mean sum of square
Appendix 26. Analysis of variance of sperm motility for thioglycol at 2 hours post-
thaw.
Source of variation SS df MS F-value p-value
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
SS = Sum of square df = Degree of freedom MS = Mean sum of square
120
Appendix 27. Analysis of variance of sperm motility for thioglycol at 4 hours post-
thaw.
Source of variation SS df MS F-value p-value
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
SS = Sum of square df = Degree of freedom MS = Mean sum of square
Appendix 28. Analysis of variance of sperm viability for thioglycol at 0 hour post-
thaw.
Source of variation SS df MS F-value p-value
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
SS = Sum of square df = Degree of freedom MS = Mean sum of square
121
Appendix 29. Analysis of variance of sperm viability for thioglycol at 2 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 0.0008
Error 44.33 10 4.433
Total 1,398.50 17
SS = Sum of square df = Degree of freedom MS = Mean sum of square
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
SS = Sum of square df = Degree of freedom MS = Mean sum of square
122
Appendix 31. Analysis of variance of sperm plasma membrane integrity for
thioglycol at 0 hour post-thaw.
Source of variation SS df MS F-value p-value
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
SS = Sum of square df = Degree of freedom MS = Mean sum of square
Appendix 32. Analysis of variance of sperm plasma membrane integrity for
thioglycol at 2 hours post-thaw.
Source of variation SS df MS F-value p-value
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
SS = Sum of square df = Degree of freedom MS = Mean sum of square
123
Appendix 33. Analysis of variance of sperm plasma membrane integrity for
thioglycol at 4 hours post-thaw.
Source of variation SS df MS F-value p-value
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
SS = Sum of square df = Degree of freedom MS = Mean sum of square
Appendix 34. Analysis of variance of sperm DNA integrity for thioglycol at 0 hours
post-thaw.
Source of variation SS df MS F-value p-value
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
SS = Sum of square df = Degree of freedom MS = Mean sum of square
124
Appendix 35. Analysis of variance of sperm DNA integrity for thioglycol at 2 hours
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
SS = Sum of square df = Degree of freedom MS = Mean sum of square
Appendix 36. Analysis of variance of sperm DNA integrity for thioglycol at 4 hours
post-thaw.
Source of 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 0.75
Error 7.56 10 0.756
Total 365.61 17
SS = Sum of square df = Degree of freedom MS = Mean sum of square