1Department Veterinary Gynaecology, Directorate of Veterinary and A.H. Services, Manipur, Sanjenthong, India
2Department of Veterinary Gynaecology and Obstetrics, Faculty of Veterinary and Animal Science, WBUAFS, 37, K.B. Sarani, Kolkata, 700037, West Bengal, India
Receive Date: 11 April 2015,
Revise Date: 03 June 2015,
Accept Date: 15 June 2015
This study was conducted to assess the physical and morphological characteristics of fresh, pre-freeze and post-thaw black Bengal buck (Capra hircus) semen processed in Tris-egg yolk-citrate-fructose-glycerol (TEYCFG) extender containing 4 different concentrations (2.5, 5, 7.5 and 10%) of egg-yolk. Ejaculates were collected once a week for eight weeks from six mature bucks (48 ejaculates) using an artificial vagina. It was found that a general decrease in values of pre-freeze and post-thaw semen parameters such as progressive sperm motility (%), live sperm (%), acrosomal integrity (%) and positive sperm (%) in the hypo-osmotic swelling (HOS) test, but an over-all increase in abnormal spermatozoa compared to fresh semen for extenders containing different concentrations of egg-yolk. Best values for all the semen parameters were obtained using extender with 2.5% egg-yolk. Of all egg-yolk concentrations, the 10% egg-yolk supplement caused the highest percentage of abnormal sperm, which was significant (P<0.05), compared to 2.5% and 5% egg-yolk containing extenders, but non-significantly (P>0.05) different compared with 7.5% egg-yolk containing extender. Only progressive motility was significantly different (P<0.05) within extenders containing 2.5%, 5%, 7.5% and 10% egg-yolk. We conclude that Black Bengal buck semen can be cryopreserved effectively with tris-egg yolk-citrate-fructose-glycerol extender containing 2.5% egg yolk (V/V).
Buck are used either in natural service or as contributors of ejaculate for use in artificial insemination (AI). In natural service, each ejaculate are used to breed a single doe but in case of AI a large number of doses of semen could be prepared from single ejaculate and utilized for insemination of several females. This is one of the major advantages of artificial insemination (AI) over natural service. However, the fertility or reproductive ability of the individual buck is an important factor to determine the reproductive performance of does. Buck with good breeding soundness and proven fertility are essential for breeding the doe to obtain the large number of offspring. The commonly used components of semen extenders for cryopreservation include egg-yolk, buffer, sugars, glycerol and antibiotics, each providing a complex and important role in protecting the spermatozoa (Purdy, 2006). Egg yolk used in semen extenders plays a major role during the freezing steps of buck sperm cryopreservation (Aboagla and Terada, 2004) and provide sperm cells with nutrients like protein (Ritar et al. 1990). On the other hand, egg yolk might have a negative effect on frozen buck semen due to the presence of phospholipase, an enzyme present in the seminal plasma which catalyzes the hydrolysis of lecithin in egg yolk to fatty acids and lysolecithins, which proved toxic to spermatozoa and cause coagulation of the storage medium (Iritani and Nishikawa, 1972). The cryopreserved sperm cells can be stored for a long period of time before being used. Cryopreservation of buck semen also extends the reproductive life of a buck after his own life (Rahman et al. 2008). Here buck semen has been cryopreserved, using tris-based extender, which contains a wide margin of egg yolk. Hence, the present work was carried out with the objective to assess the spermatic parameters of the Black Bengal buck semen cryopreserved in extender containing different levels of egg-yolk.
MATERIALS AND METHODS
The experiment was conducted at the Department of Veterinary Gynaecology and Obstetrics, West Bengal University of Animal and Fishery Sciences, Belgachia, Kolkata-37. The experiment was carried out from February to July, 2012. Six sexually matured black Bengal bucks aged 21/2 to 4 years were used for semen collection. All experimental animals used in this experiment were maintained by the Department of Livestock Production and Management, WBUAFS, under appropriate and uniform conditions of feeding and management. Before selection, all the bucks were thoroughly examined for general and reproductive health conditions. Proper deworming and vaccination were carried out. Experimental animals were grazing on natural pasture from 7 a.m. to 3 p.m. every day. Water was provided ad libitum throughout the study period.
Semen collection and evaluation
Semen was collected once a week for 8 weeks by artificial vagina (AV) method. A total number of 48 ejaculates were used in this study. Semen samples collected from each buck were processed individually and evaluated after i) collection, ii) before freezing and iii) after thawing for progressive motility, live and dead sperm percentage, sperm abnormalities, acrosome intactness and functional membrane integrity. The ejaculates were assessed and accepted for experiments if the following criterion was met: volume between 0.5-2 mL; minimum sperm concentration ≥ 2.5 × 109 sperm/mL; motility ≥ 80%. Progressive motility as an indicator of semen quality was assessed using a phase contrast microscope (Olympus, India), fitted with a warm stage at 37 ˚C. The viability was evaluated by eosin-nigrosin staining according the method described by Rao (1957). The percentage of sperm with abnormal morphology was determined according to Hancock (1951). Acrosomal integrity was assessed according to the method of Hancock (1952), modified by Sarma (1995). The hypo-osmotic swelling test (HOST) was used to evaluate the functional integrity of the sperm membrane, based on curled and swollen tails. The test was done according to Jeyendran et al. (1984).
Semen processing and evaluation
Tris-citrate-egg-yolk based cryodiluent was used as an experimental extender in this study and it was composed of 2.422 g Tris-hydroxymethyl-aminomethane, 1.36 g citrate, 1.00 g fructose, 90.5- 83 mL triple distilled water, 2.5%, 5%, 7.5% and 10% (V/V) egg yolk, 7% (V/V) glycerol. Antibiotics viz., penicillin (100000 IU), streptomycin sulphate 100 mg/100 mL were added to the extender.
Extension of semen
Immediately after evaluation, the ejaculate was divided into 4 equal aliquots and diluted in one step at room temperature, to a concentration of 100 × 106 sperm/mL, with this extender. The pH of the diluents was adjusted to 6.8 with an appropriate amount of citrate.
The extended semen samples from each buck were kept in a cold handling cabinet at +5 ˚C for 3 h of equilibration period, and subsequently, the different parameters of semen were evaluated.
Freezing, thawing and evaluation
Straws (0.25 mL, French) were filled with extended semen using the comb and hand pump (IMV, India) immediately after equilibration. Then the straws were sealed by polyvinyl alcohol (PVA). The straws were then racked before and exposed to LN2 vapour by placing those 5 cm above the LN2 in a thermo box for 10 min and then plunged the straws into LN2. Thawing of the straws was carried out individually after 24 h of freezing at 37 ˚C for 30 s in water bath for post-thaw sperm evaluation.
Data were analysed using one-way analysis of variance (ANOVA), followed by the Duncan’s multiple range test to determine significant difference in all parameters between groups using the SPSS software system (SPSS, 2011). Differences with values of P < 0.05 were considered to be statistically significant.
RESULTS AND DISCUSSION
In the fresh semen, the average values of progressive motility (%), live (%), abnormal spermatozoa (%), intact acrosome (%) and HOS positive sperm (%) was depicted in Table 1. There was a general decrease trend in values of pre-freeze and post thawed semen parameters such as progressive motility, live sperm, intact acrosome and HOS positive sperm, but mainly, increase in percentage of abnormal sperm in the semen diluted in extenders containing different concentrations of egg-yolk in comparison to fresh semen. The percentage of progressive motility, live sperm, intact acrosome and HOS positive sperm were significantly (P<0.05) higher, when 2.5% egg-yolk was added to the extender. Conversely, the lower percentage of abnormal sperm (P<0.05) was obtained in the same group. The 10% egg-yolk containing extender showed all parameters presented lower results than other treatments, except sperm abnormality (Table 1 and 2). Cryopreservation as a technique for storage of buck semen has advantages but the freezing and thawing processes induce detrimental effects on the sperm ultra-structure, and the biochemical and functional integrity (Watson, 2000). Eggyolk, is a crude lipid and is a common component for sperm refrigeration, the role of which is protecting sperm plasma membrane against cold shock, and interacts with sperm plasma membrane (Watson, 1975; Watson, 1976; Foulkes, 1977). The present study showed that 2.5% egg-yolk in TEYCFG extender provided better pre-freeze sperm protection than all other egg-yolk concentrations tested. The findings of pre-freeze seminal parameters are in close agreement with values of a study undertaken by Priyadharsini et al. (2011). On the contrary, a study conducted by Yimer et al. (2014), showed that progressive motility (%) at 10% omega-3 egg-yolk in citrate extender was better than 2.5% omega-3 egg-yolk in citrate extender during cryopreservation of buck semen. Current study revealed that progressive sperm motility (%) after thawing remains above 50% only when using the 2.5% egg-yolk concentration. Similar trend of changes in respect to sperm motility have been reported by Shamsuddin et al. (2000). The decline in sperm motility after thawing may be due to formation of peroxides from free radicals. This oxidative stress causes damage to biomolecules and cellular components (Halliwell, 1991). The sperm membrane contains a high number of unsaturated fatty acids, which are predisposed to damage due to peroxidation, which destroys the structural integrity of plasma membrane leading to loss in motility (Aitken et al. 1989; Salamon and Maxwell, 2000). The toxicity of cryoprotective agent glycerol in semen diluents causes a reduction in sperm motility and to alter the acrosome integrity by interfering with the permeability of the sperm membrane (Maxwell and Salamon, 1993; Maxwell and Watson, 1996). The percentage of post-thaw motility of spermatozoa was decreased significantly (P<0.05) between 2.5% to 10% egg-yolk extender in this study. Similar trend of changes in sperm motility have been reported by Ritar et al. (1990). These findings may signify that when egg yolk level increased in the extended semen, hydrolysis of lecithin led to further deterioration of sperm motility. The current results came in line with those obtained by El-Maghraby (2007) and Ashmawy et al. (2010) who found an optimal level of 2.5% egg yolk with tris-based extender for dilution of buck semen. Bispo et al. (2011) has also observed that low level inclusion of egg yolk significantly improved post-thaw physic-morphological seminal parameters compared to inclusion of 20% egg yolk in the extender. This result coincide with those Cabrera et al. (2005) has included 1.5, 6 and 12% egg yolk for freezing canary buck semen. The finding with respect to the quality of post-thawed spermatozoa in the present study was in disagreement with the findings of Cabrera et al. (2005). Ranjan et al. (2009) investigated the effect of 2.5, 5, 7.5 and 10% egg yolk level and concluded that 10% showed higher post-thaw motility and liveability in sirohi buck semen held at refrigeration temperature for 72 h. On the other hand, our study exhibited that progressive motility and viability at 2.5% egg-yolk level in TEYCFG extender was highest compared to other extender, which was in discrepancy with the result of Ranjan et al. (2009). The comparative study of extender containing egg-yolk at elevated concentration hampered the seminal parameters suggesting that egg yolk could contain some deleterious components which are potent to reduce sperm motility at higher concentrations. The ingredients of egg-yolk defend the spermatozoa from deleterious effect of cryopreservation but not all of its components are favourable. Addition of more amount of egg-yolk increases the concentration of these deleterious components and that might be a cause for the excess damage caused by 10% egg-yolk level in this trial. Moreover, egg yolk increases the risk of microbial contamination and thereby releases excess endotoxin which may reduce the fertilizing capacity of spermatozoa (Aires et al. 2003; Bousseau et al. 1998). On evaluation of effect of freezing, it was observed that, the motility of spermatozoa were significantly influenced by freezing and thawing, which reiterates earlier findings in Boer cross and Barbari bucks (Sundararaman and Edwin, 2005). All the sperm parameters were significantly reduced in the post-thawing semen. Changes in the osmotic pressure during semen processing for cryopreservation critically affect the spermatozoa. This may be the most important deterrent to sperm survival during cryopreservation (Watson, 1995).
Table 1 Mean ± SEM of fresh and pre-freeze semen quality parameters (%) extended with different concentrations of egg-yolk in the extender
HOST: hypo-osmotic swelling test.
SEM: standard error of the means.
The means within the same row with at least one common letter, do not have significant difference (P>0.05).
Table 2 Mean ± SEM of fresh and post-thaw buck semen quality parameters (%) extended with different concentrations of egg-yolk in the extender
HOST: hypo-osmotic swelling test.
SEM: standard error of the means.
The means within the same row with at least one common letter, do not have significant difference (P>0.05).
Furthermore, membrane destabilization can occur when the sperm plasma membrane undergoes a phase transition from the liquid crystalline phase to the gel phase due to a decrease in temperature (Barrea- Compean et al. 2005). The irreversible changes in the sperm membrane induced by lipid phase transitions during cooling warming may possibly affect the movement characteristics of spermatozoa during semen processing for cryopreservation (Deleeuw et al. 1990). In addition, frozen-thawed sperm are more vulnerable to oxidative stress due to peroxidation than sperm in freshly diluted semen (Neild et al. 2005). As semen is diluted many fold in the extender it reduces the total antioxidant concentration in the medium and cells (Kumar and Das, 2005). Many sperm are killed during cryopreservation. Thus, it is likely that cryopreserved sperm cells are posed to more reactive oxygen species concentration and therefore many of the surviving cells post-thaw exhibit as if they are capacitated or acrosome reacted (Bailey et al. 2000). The overall effects of these events may adversely affect quality of post-thaw semen. It is most likely that the cryo damages are due to the irreversible destruction of individual components of the structural organization of sperm cells. Sperm are subjected to major changes in osmotic pressure during freezing and thawing. The stress on sperm membranes is dependent upon the basic extender used and the concentration of cryo-protectant as they interact with the freezing and thawing rates (Curry and Watson, 1994; Curry et al. 2000). The post-thaw viability was highest in 2.5% egg-yolk followed by 5 %, 7.5 % and 10% egg-yolk and lowest in 10% egg-yolk diluents. There was highly significant difference of sperm liveability between 2.5 %, 5 %, 7.5 % and 10% egg-yolk. The live sperm percentages of post-thaw spermatozoa of the present study were in disagreement with the findings of Priyadharsini et al. (2011), who reported that the post-thaw liveability of Jakhrana buck semen in TEYCFG extender diluted with 10% and 20% egg-yolk levels were 53.4 ± 0.80 and 53.2 ± 1.12, respectively. The proportion of abnormality of post-thaw spermatozoa was lowest at the 2.5% egg-yolk, increased with 5%, 7.5% and 10% egg-yolk extender. There was a highly significant difference (P<0.05) in morphological abnormality of spermatozoa of post-thaw semen between 2.5% and 5% egg-yolk and non-significant difference between 7.5% and 10% egg-yolk. The present findings of sperm abnormality of post-thaw semen were in agreement with the observation by Apu et al. (2012) and disparate with the findings of Batista et al. (2009). The most probable reason for this morphological abnormality seems to be the physical and chemical environments to which a spermatozoon is exposed during the preservation. On the other hand, Medeiros et al. (2002) and Ozkavukcu et al. (2008) noted that the sperm cell water exchange during the early stages of the preservation causes swellings and shrinkages which may be intolerable for the majority of organelles and might predispose to spermatozoan morphological abnormality. It was noteworthy that number of abnormal spermatozoa were significantly increased (P<0.01) as the number of motile spermatozoa decreased following preservation. The values of post-thaw acrosomal integrity were highest at 2.5% egg-yolk followed by 5%, 7.5% and 10% egg-yolk diluents in the present study. There was highly significant difference (P<0.05) of acrosomal integrity between 2.5% and 5% egg-yolk diluents but there was non-significant (P>0.05) difference of acrosomal integrity between 7.5% and 10% egg-yolk extender. The present observations of acrosomal integrity were in close agreement with the findings of Batista et al. (2009) and Nally et al. (2010). The values of HOS positive sperm of post-thaw were highest in 2.5% egg-yolk and followed by 5% and 7.5% egg-yolk and lowest in 10% egg-yolk. There was highly significant difference of HOS positive sperm between 2.5% and 5% egg-yolk. There was no significant difference of HOS positive sperm between 7.5 and 10% egg-yolk. Pramanik and Raina (2001) studied hypo-osmotic swelling response of buffalo spermatozoa in various extenders. The value of HOS positive at post-freezing stage in skim milk extender (SEM) and Tris were 60.25 ± 1.44% and 57.75 ± 2.66% respectively and showed significantly (P<0.01) better performance over egg-yolk-citrate (46.75±2.87%). Our findings of HOS positive sperm was in close agreement with the findings of Priyadharsini et al. (2011), who reported that post-thaw HOS positive of Jakhrana buck semen in TEYCFG extender diluted with 10% and 20% egg-yolk levels were 60.56 ± 1.31 and 52.7 ± 1.87 respectively.
On the basis of the present result, it is concluded that 2.5% egg-yolk level used in tris-egg yolk-citrate-fructose-glycerol (TEYCFG) extender was best for cryopreservation of black Bengal buck semen.
I acknowledge with gratitude, the support of the Department of Livestock Production and Management, WBUAFS, for its assistance in providing the animals for the study.
Aboagla E.M.E. and Terada T. (2004). Effects of egg yolk during the freezing step of cryopreservation on the viability of goat spermatozoa. Theriogenology. 62, 1160.
Aires V.A., Hinsch K.D., Schloesser F.M., Bogner K., Schloesser S.M. and Hinsch E. (2003). In vitro and in vivo comparison of egg yolk based and soya bean extenders for cryopreservation of bovine semen. Theriogenol. 60, 269-279.
Aitken R.J., Clarkson J.S. and Fishel S. (1989). Generation of reactive oxygen species, lipid peroxidation, and human sperm function. Biol. Reprod. 41, 183-197.
Apu A.S., Khandoker M.A.M.Y., Hussain S.S., Fakruzzaman M. and Notter D.R. (2012). A comparative study of fresh and frozen-thawed quality in relation to fertility of black Bengal goats. Iranian J. Appl. Anim. Sci. 2(2), 157-161.
Ashmawy T.A.M., Sallam A.A., El-Khalek A.E.A., El-Saidy B.E. and Gabr M.G. (2010). Recovery and fertilization rates of goat spermatozoa as affected by different levels of egg yolk, dilution rates, freezing method and months of the year. Egypt J. Sheep. Goat. Sci.5(1), 283-293.
Bailey J.L., Bilaodeau J.F. and Cormier N. (2000). Semen cryopreservation in domestic animals: a damaging capacitating phenomenon. J. Androl. 20, 1-7.
Barrea-Compean M.H., Purdy P.H., Dzakurna J.M., Newton G.R. and Nuti L.C. (2005). Cholesterol-loaded cyctodextrin improves post-thaw goat sperm motility. J. Anim. Sci. 83(1), 153-153.
Batista M., Nino T., Alamo D., Castro N., Santana M., Gonzalez F., Cabrera F. and Gracia A. (2009). Successful artificial insemination using semen frozen and stored by an ultrafreezer in the Majorera goat breed. Theriogenology. 71, 1307-1315.
Bispo C.A.S., Pugliesi G., Galvao P., Rodrigues M.T., Ker P.G., Filgueiras B. and Carvalho G.R. (2011). Effect of low and high egg yolk concentrations in the semen extender for goat semen cryopreservation. Small Rumin. Res. 100, 54-58.
Bousseau S., Brillard J.P., Guienne M., Guerin B., Camus A. and Lechat M. (1998). Comparison of bacteriological qualities of various egg yolk sources and then in vitro fertilizing potential of bovine semen frozen in egg yolk or lecithin based diluents. Theriogenology. 50, 669-706.
Cabrera F., Gonzalez F., Batista M., Calero P., Medrano A. and Gracia A. (2005). The effect of removal of seminal plasma, egg yolk level and season on sperm freeze ability of canary buck (Capra hircus). Reprod. Domest. Anim. 40, 191-195.
Curry M.R. and Watson P.F. (1994). Osmotic effects on ram and human sperm membranes in relation to thawing injury. Cryobiology. 31, 39-46.
Curry M.R., Kleinhans F.W. and Watson P.F. (2000). Measurement of the water permeability of the membranes of boar, ram, and rabbit spermatozoa using concentration-dependent self-quenching of an entrapped fluorophore. Cryobiology. 41, 167-173.
Deleeuw F.E., Chen H.C., Colenbrander B. and Verkleij A.J. (1990). Cold-induced ultrastructural changes in bull and boar sperm plasma membranes. Cryobiology. 27, 171-183.
EI-Maghraby M.M. (2007). Physiological studies on reproduction in goats. Ph D. Thesis. MonsouraUniv., Monsoura, Egypt.
Foulkes J.A. (1977). The separation of lipoprotein from egg yolk and their effect on the motility and integrity of bovine spermatozoa. J. Reprod. Fertil. 49, 277-284.
Halliwell B. (1991). Reactive oxygen species in living systems: source, biochemistry and role in human disease. Am. J. Med. 91, 14-22.
Hancock J.L. (1951). A staining technique for the study of temperature-shock in semen. Nature. 167, 323-324.
Hancock L. (1952). The morphology of bull spermatozoa. Br. J. Exp. Biol. 29, 445-453.
Iritani A. and Nishikawa Y. (1972). Studies on the egg yolk coagulation enzyme (Phopholipase) in goat semen. IX. Enzyme concentration in the semen collected from the Cowper’s gland removed goat. Mem. Coll. Agric. Kyoto Univ. 10, 44.
Jeyendran R.S., Vander Ven H.H., Perez-Pelaez M., Crabo B.G. and Zaneveld L.J.D. (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.
Kumar S. and Das G.K. (2005). Frozen sperm quality with reference to reactive oxygen species: a review. Indian J. Anim. Sci. 75, 874-884.
Maxwell W.M.C. and Salamon S. (1993). Liquid storage of ram semen. Reprod. Fertil. Dev. 5, 613-638.
Maxwell W.M.C. and Watson P.F. (1996). Recent progress in the preservation of ram semen. Anim. Reprod. Sci. 42, 55-65.
Medeiros C.M., Forell F., Oliveira A.T. and Rodrigues J.L. (2002). Current status of sperm cryopreservation: why isn't it better? Theriogenology.57, 327-344.
Nally W.M.M., Handarani R., Yusuf T.L., Purwantra P. and Semiadi G. (2010). The effect of glycerol concentration in tris glucose egg yolk extender on the quality of timor deer frozen semen. J. Indonesian Trop. Anim. Agric. 36(2), 91-96.
Neild D.M., Brouwers J.P., Colenbrander B., Aguero A. and Gadella B.M. (2005). Lipid peroxide formation in relation to membrane stability of fresh and frozen-thawed stallion spermatozoa. Mol. Reprod. Dev. 72, 230-238.
Ozkavukcu S., Erdemli E., Isik A., Oztuna D. and Karahuseyinoglu S. (2008). Effects of cryopreservation on sperm parameters and ultrastructural morphology of human spermatozoa. J. Assist. Reprod. Genet. 25, 403-411.
Pramanik P.S. and Raina V.S. (2001). HOS response and post-thaw incubation motility of buffalo spermatozoas in various extenders. Indian Vet. Med. J. 25, 129-131.
Priyadharsini R., Jindal S.K., Sharma D., Ramachandran N., Karche S.D. and Goel A.K. (2011). Effect of different egg yolk level on the cryopreservation capability of Jakhrana goat semen. J. Anim. Sci. Adv. 1(1), 28-37.
Purdy P.H. (2006). A review on goat sperm cryopreservation. Small Rumin. Res. 63, 215-225.
Rahman A.N.M.A., Abdullah R.B. and Khadijah W.E.W. (2008). A review of reproductive biotechnologies and their application in goat. Biotechnology. 7(2), 371-384.
Ranjan R., Ramchandran N., Jindal S.K., Goel A.K. and KharcheS.D. (2009). Effect of egg yolk levels on keeping quality of Marwari buck semen at refrigeration temperature. Indian J. Anim. Sci. 79(7), 662-664.
Rao C.K. (1957). Different staining of live and dead sperm. Indian Vet. J. 29, 307-309.
Ritar A.J., Ball P.D. and O'May P.J. (1990). Examination of methods for the deep freezing of goat semen. Reprod. Fertil. Dev. 2, 27-34.
Salamon S. and Maxwell W.M.C. (2000). Storage of ram semen. Anim. Reprod. Sci. 62, 71-111.
Sarma P.V. (1995). A simplified staining technique for evaluation of acrosomal status of sperm cells. J. Anim. Sci.16, 127-127.
Shamsuddin M., Amiri Y. and Bhuiyan M.M.U. (2000). Characteristic of buck semen with regards to ejaculate numbers, collection intervals, dilution and preservation periods. Reprod. Domest. Anim.35, 53-57.
SPSS Inc. (2011). Statistical Package for Social Sciences Study. SPSS for Windows, Version 20. Chicago SPSS Inc.
Stanger J.D., Long V., Yovich J.L. and Almahbobi G. (2010). Hypo osmotic swelling test identifies individual spermatozoa with minimal DNA fragmentation. Reprod. Biomed. Online. 21, 474-484.
Sundararaman M.N. and Edwin M.J. (2005). Evaluation of sperm motion characteristics of pre-freeze and post-thaw goat spermatozoa on computer assisted semen analysis (CASA). Indian J. Small Rumin. 11, 92-95.
Watson P.F. (1975). The interaction of egg yolk and ram spermatozoa studied with fluorescent probe. J. Reprod. Fertil. 42, 105-111.
Watson P.F. (1976). The protection of ram and bull spermatozoa by the low density lipoprotein fraction of egg yolk during storage at 5 ˚C and deep-freezing. J. Thermal. Biol. 1, 137-141.
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.
Watson P.F. (2000). The causes of reduced fertility with cryopreserved semen. Anim. Reprod. Sci. 60, 481-492.
Yimer N., Noraisyah A.H., Rosnina Y., Wahid H., Sarsaifi K. and Hafizal A.M. (2014). Comparison of cryopreservative effect of different levels of omega-3 egg-yolk in citrate extender on the quality of goat spermatozoa. Pakistan Vet. J. 34(3), 347-350.