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Cryo-survival, cryo-capacitation and oxidative stress assessment of buffalo spermatozoa cryopreserved in new soya milk extender
Livestock Science 160 (2014) 214–218
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Livestock Science journal homepage: www.elsevier.com/locate/livsci
Cryo-survival, cryo-capacitation and oxidative stress assessment of buffalo spermatozoa cryopreserved in new soya milk extender$ V.K. Singh, R. Kumar, S.K. Atreja n Reproductive Biochemistry Laboratory, Animal Biochemistry Division, National Dairy Research Institute, Karnal 132001, Haryana, India
a r t i c l e in f o
abstract
Article history: Received 3 January 2013 Received in revised form 27 November 2013 Accepted 10 December 2013
Egg yolk is most commonly used in semen extenders for cryopreservation but, wide variability in composition and potential risk of xenobiotic contamination has raised questions upon the use of egg yolk. Therefore, the present study was designed to develop a soya milk based phytoextender for buffalo semen cryopreservation. Soya milk was prepared from fresh soya beans in laboratory. Soya milk (25% v/v) and glycerol (6.4% v/v) was added in Tris citrate buffer to prepare Soya Milk Tris (SMT) extender. To improve postthaw sperm quality, cryo-protectants like trehalose (100 mM) or taurine (50 mM) was supplemented in SMT extender before semen cryopreservation. Post-thaw quality parameters, degree of cryocapacitation and oxidative stress (lipid peroxidation) of spermatozoa cryopreserved in SMT with or without additives were assessed and compared with Egg Yolk Tris (EYT) extender. SMT extender showed no significant (P 40.05) differences in sperm motility, viability, membrane integrity and acrosome integrity as compared to EYT extender. The degree of cryocapacitation and oxidative damage were found significantly (P o 0.05) lower in spermatozoa cryopreserved in SMT extender as compared to EYT extender. Supplementation of additives (trehalose or taurine) in SMT extender significantly (P o 0.05) reduced oxidative damage. Thus, soya milk extender (25% soya milk v/v; 6.4% Glycerol in tris citrate buffer) with additives (trehalose or taurine) may substitute conventional egg yolk extender (20% egg yolk v/v; 7% Glycerol in tris citrate buffer) for better cryopreservation of buffalo semen. & 2013 Elsevier B.V. All rights reserved.
Keywords: Murrah Buffalo Soya milk extender Spermatozoa Cryopreservation Cryocapacitation
1. Introduction Artificial Insemination (AI) extensively depends on frozen semen. However, the process of cryopreservation damages plasma membrane, cytoskeleton, motility apparatus, and nucleus of spermatozoa (Ragoonanan et al.,
Abbreviations: EYT, egg yolk tris; SMT, soya milk tris; HOST, hypo-osmotic swelling test ☆ This study was partly funded by World Bank funded National Agricultural Innovation Project number-C4/C30014 (Code-41420102), ICAR, New Delhi. n Corresponding author. E-mail address: [email protected] (S.K. Atreja). 1871-1413/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.livsci.2013.12.013
2010). The process of cryopreservation also induces premature capacitation (Reddy et al., 2010), osmotic stress and oxidative damage (Thuwanut et al., 2008) that reduce functional life of spermatozoa. Therefore, quality evaluation of spermatozoa based on motility is not sufficient to judge true fertilizing capacity of spermatozoa. Currently, egg yolk-based extenders are globally used for cryopreservation of semen from bull and many other species. However, wide variability, risk of xenobiotic contamination (Aires et al., 2003) and presence of steroid hormones (Lipar et al., 1999) have raised questions upon the use of egg yolk in freezing extender. Furthermore, egg yolk contains substances (e.g. high-density lipoproteins and minerals) that inhibit cellular respiration and affect
V.K. Singh et al. / Livestock Science 160 (2014) 214–218
metabolic activities (Pace and Graham, 1974). Hence, to circumvent these problems a non animal origin based semen extender with comparable cryoprotective effect was desirable. Recently, many commercial extenders like AndroMeds (Aires et al., 2003) and Bioxcells (Akhter et al., 2010) have been evaluated for preservation of bovine and buffalo semen and found to maintain better semen quality than egg yolk based extenders. But, high cost and limited availability of these commercial extenders make them unobtainable for emerging and small-scale semen banks especially in developing countries. We have developed an economic, pathogen free and non animal origin based soya milk semen extender to replace egg yolk based extender and reported successful liquid preservation of buffalo semen upto 72 h (A.K. Singh et al., 2012). A further study was required to establish this extender for cryopreservation of buffalo semen. Recently, cryoprotectants like taurine or trehalose, have been supplemented in freezing extenders of bull (Sariozkan et al., 2009) and buffalo (Kumar and Atreja, 2012, Kumar et al., 2013) semen prior to cryopreservation, to improve the post thaw sperm quality. Hence, the present study was designed first; to cryopreserve buffalo semen in soya milk tris (SMT) extender, second; to evaluate cryo-survival, cryo-capacitation and oxidative stress of sperm cryopreserved in SMT extender in comparison to conventional EYT extender and third; to further improve the post thaw sperm quality by supplementing cryoprotectants like trehalose or taurine in SMT extender.
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2. Results 2.1. Optimization of glycerol for cryopreservation Post thaw motility of buffalo spermatozoa cryopreserved in SMT extender with gradient of glycerol ranging from 6.0–7.0% with a difference of 0.2% revealed that motility of spermatozoa cryopreserved in SMT extender with 6.4% of glycerol was significantly higher (P o0.05) than other concentrations (Fig. 1). 2.2. Post-thaw motility, viability, membrane integrity and acrosome integrity The standard semen quality parameters such as percent motility, viability, membrane integrity and acrosomal integrity in fresh semen ejaculates and post thawed semen are shown in Table 1. Cryopreservation of buffalo spermatozoa resulted in a significant (Po0.05) decrease in all the standard semen quality parameters as compared to fresh. Buffalo spermatozoa cryopreserved in SMT extender did not show significant (P 40.05) differences in any of the standard sperm quality parameters as compared to spermatozoa cryopreserved in EYT extender. Supplementation of trehalose (100 mM) or taurine (50 mM) to freezing extender showed a significantly (Po0.05) higher postthaw sperm motility, viability, percent membrane integrity and acrosome integrity as compared to sperm cryopreserved in their absence (Table 1). 2.3. Extent of cryocapacitation of spermatozoa
60 c
Motility (%)
45
a
b d
d
d
30
6.0 % Glycerol 6.2 % Glycerol 6.4 % Glycerol 6.6 % Glycerol 6.8 % Glycerol 7.0 % Glycerol
15
0 6.0 % G6.2 % G6.4 % G6.6 % G6.8 % G7.0 % G
Fig. 1. Post thaw sperm motility of buffalo spermatozoa cryopreserved in soya milk extender with different glycerol concentration for optimization of glycerol percentage. Values are the mean 7 S.E.M. of nine experiments. Means with different letters are significantly different (Po 0.05).
Sperm cryocapacitation was assessed by lysophosphatidyl choline (LPC)-induced acrosome reaction followed by dual staining of spermatozoa. Cryopreservation of buffalo spermatozoa in EYT extender resulted in a significant (P o0.05) increase of capacitation as compared to freshly ejaculated buffalo semen. On the other hand, extent of cryocapacitation was found significantly (P o0.05) lower in buffalo spermatozoa cryopreserved in SMT extender than EYT extender. Moreover, Supplementation of 100 mM trehalose or 50 mM taurine to the freezing medium prior to cryopreservation significantly (P o0.05) decreased the extent of cryocapacitation in buffalo spermatozoa as compared to control (Table 1).
Table 1 Post thaw parameters of freshly ejaculated and cryopreserved Buffalo spermatozoa in extended in egg yolk tris extender (EYT), soya milk tris extender (SMT), soya milk tris extender supplemented trehalose (SMTþ Trehalose) and soya milk tris extender supplimented with taurine (SMTþ Taurine) respectively. Values are Mean 7 Standard Error Mean (S.E.M.) of nine experiments. Mean 7 S.E.M. with different letters (a,b,c,d) in row are significantly different (P o 0.05).
Motility (%) Viability (%) Membrane integrity (%) Acrosome integrity (%) Sperm capacitation (%) Lipid peroxidation (nmol MDA/108cells)
Fresh
EYT
SMT
SMTþTrehalose (100 mM)
SMTþTaurine (50 mM)
81.0 7 1.87a 89.0 7 1.83a 74.6 7 3.04a 96.2 7 1.48a 5.6 7 0.67a 1.137 0.31a
47.0 7 1.22b 61.4 7 1.12b 38.2 7 1.31b 84.4 7 3.40b 17.2 7 0.73b 3.26 7 0.10b
46.0 7 1.87b 62.2 7 1.58b 39.2 7 1.52b 86.4 7 2.20bc 12. 7 0.83c 2.487 0.10c
54.0 71.87c 69.2 71.11c 42.4 71.93b 90.6 72.28cd 9.8 70.58d 1.48 70.02d
57.0 7 1.22c 70.07 1.26c 42.2 7 2.03b 91.4 7 2.16d 9.0 7 0.44d 1.477 0.16d
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2.4. Lipid peroxidation assay The extent of lipid peroxidation (LPO; production of MDA) by freshly ejaculated and cryopreserved buffalo spermatozoa are shown in Table 1. The values for lipid peroxidation in freshly ejaculated spermatozoa was found to be 1.1370.31 nmol MDA/108cells and it increased significantly (Po0.05) upon cryopreservation. There was a significant (Po0.05) difference in the value of lipid peroxidation of cryopreserved spermatozoa in EYT extender as compared to SMT extender. Lipid peroxidation was significantly (Po0.05) reduced upon supplementation of SMT with trehalose or taurine than control. However, there was no significant (P40.05) difference in lipid peroxidation of spermatozoa cryopreserved in these two additives. 3. Discussion Egg yolk is rich in lipids like lecithin that interacts with sperm membrane and show a stabilizing effect during cold shock (Pettitt and Buhr, 1998). Soya bean also have high content of lecithin. Therefore, soya milk prepared from soya beans possesses cryoprotective potential similar to egg yolk. In an earlier study, we have reported that soya milk (25% v/v) can replace egg yolk (20% v/v) in tris based semen extender for liquid preservation of buffalo semen up to 72 h with better keeping quality (A.K. Singh et al., 2012). Glycerol, a permeating cryoprotectant, is incorporated in freezing extender for semen cryopreservation. Abbas and Andrabi (2002) studied the effects of different concentrations of glycerol on post-thaw sperm quality and found that 7% glycerol is significantly (Po0.05) better than other concentrations of glycerol. In another study, Kumar et al. (1992) reported that the best level of glycerol is 6% for Tris- and milk-based diluents, and 9% for the sodium citrate diluents. However, in this study 6.4% glycerol was found significantly (Po0.05) better than other concentrations of glycerol in SMT extender for buffalo semen cryopreservation. Cryopreservation process causes damage to plasma membrane, cytoskeleton, motility apparatus, and the nucleus of spermatozoa (Ragoonanan et al., 2010) resulting into decreased motility, membrane integrity and increased premature capacitation (Akhter et al., 2010). In consistent to these reports, we also found significantly (P o0.05) decreased motility, viability, membrane integrity, acrosome integrity and an increased degree of cryocapacitation in spermatozoa cryopreserved in EYT or SMT extenders as compared to freshly ejaculated spermatozoa. There were no significant (P40.05) difference in post thaw semen quality parameters between two extenders but degree of cryocapacitation was significantly (Po0.05) lesser in spermatozoa cryopreserved in SMT extender than EYT extender. This suggests that keeping quality of SMT extender is comparable with EYT extender but SMT extender reduces premature capacitation during cryopreservation. Damage that occurs during cryopreservation is due to extracellular ice crystals formation. Furthermore, the membrane stretching during freezing and thawing results in decreased lateral packing of lipids (Lehtonen and Kinnunen, 1995) and reorganization of the actin cytoskeleton (Correa et al., 2007) leaving the cell membrane vulnerable to reactive oxygen species (ROS) attack. Thus, cryopreservation not only
induces an osmotic stress to cells, it also makes spermatozoa susceptible to oxidative stress (Thuwanut et al., 2008) and causes DNA damage. Recent studies indicate that low levels of ROS play important role in controlling various sperm functions like capacitation, acrosome reaction, hyperactivation and sperm-oocyte fusion (deLamirande and Lamothe, 2009) but excessive generation of ROS have a detrimental effect on spermatozoa. Though, it was reported that ROS is produced by defective spermatozoa and contaminating leukocytes (Aitken et al., 1995), it is now evident that that process of cryopreservation also increases the level of ROS in spermatozoa (Thuwanut et al., 2008). It is interesting to note that, sperm membrane is rich in unsaturated fatty acids and they have limited ability to store antioxidant enzymes so they are very susceptible to oxidative stress and peroxidative attack by ROS (Baker and Aitken, 2005). Our results showed that spermatozoa cryopreserved in SMT extender showed significantly (Po0.05) lesser lipid peroxidation as compared to EYT extender. Recently, several reports have suggested that supplementation of additives like trehalose or taurine to the freezing extender improves semen quality in many species (Kumar and Atreja, 2012; Kumar et al., 2013; Reddy et al., 2010 ; Sariozkan et al., 2009). In close agreement to these reports, we also found significant (Po0.05) improvement in post thaw sperm quality and reduced degree of premature capacitation and oxidative damage upon supplementation of trehalose (100 mM) or taurine (50 mM) to SMT extender as compared to control. Trehalose, a non-reducing disaccharide, causes cellular osmotic dehydration before freezing (Molinia et al., 1994). On the other hand, Taurine, a permeating sulfonic amino acid analog, inhibit lipid peroxidation, protects the cells against accumulation of ROS (Chen et al., 1993), modulate Ca2 þ uptake (V.K. Singh et al., 2012) and inhibit protein tyrosine phosphorylation (Kumar and Atreja, 2012). This showed that soya milk might have some antioxidant property. On other hand oxidative damage can be further mitigated through supplementation of cryoprotectants like trehalose or taurine in freezing extender. 4. Conclusion Cryopreservation of Buffalo sperm in newly developed SMT extender showed comparable post thaw semen quality parameters and significantly (Po0.05) lesser oxidative damage and cryocapacitation as compared to spermatozoa cryopreserved in conventional EYT extender. Moreover, supplementation of trehalose (100 mM) or taurine (50 mM) in SMT extender significantly (Po0.05) improved post thaw sperm quality and decreased oxidative damage and cryocapacitation. Hence, soya milk (25% v/v; 6.4% glycerol v/v) with additives (trehalose or taurine) can be used to substitute egg yolk (20% v/v; 7% glycerol v/v) in tris buffer based extender for better cryopreservation of buffalo semen. 5. Materials and methods 5.1. Semen extenders Egg yolk tris (EYT) semen extender (20% Egg yolk v/v diluted in TCG buffer; 274 mM Tris, 87 mM Citric acid,
V.K. Singh et al. / Livestock Science 160 (2014) 214–218
43 mM Glucose, 1,000,000 IU Benzyl penicillin per litre, 750,000 IU Streptomycin per litre) was prepared as described by Ahmed and Foote (1986). Whereas, Soya milk used in the semen extender was prepared as described by A.K. Singh et al. (2012). SMT extender was prepared by adding soya milk (25% v/v) in TCG buffer. 5.2. Semen collection and cryopreservation Murrah buffalo bulls, 3–5 years of age, housed at Artificial Breeding Research Centre, National Dairy Research Institute, Karnal, India, under uniform nutritional conditions were used for semen collection. A total of nine ejaculates were collected using artificial vagina (IMV, L0 Aigle Cedex, France) from nine randomly chosen bulls. Ejaculated semen having milky white color, score of þ3 or more mass activity and more than 80% progressive motility was used in all the experiments. Each ejaculate was split into five aliquots and one aliquot was used for fresh semen analysis whereas other four were diluted with the EYT extender, SMT extender and SMT extender containing taurine (50 mM) or trehalose (100 mM) respectively. Sperm concentrations in semen samples were determined by hemocytometer and a final concentration of 80 106 sperms ml 1 was achieved. Diluted semen was filled and sealed into French straws (IMV, L0 Aigle cedex, France) and kept for 4 h in cold room at 4 1C for equilibration. Equilibrated semen straws were then kept five centimeter above the liquid nitrogen for 10 min and plunged in liquid nitrogen for storage at 196 1C for 3–4 weeks. Frozen straws were thawed at 37 1C for 45 s in a water bath and used for assessment of various semen quality parameters. 5.3. Optimization of glycerol for cryopreservation In EYT extender glycerol was added at the rate of 7.0% (Ahmed and Foote, 1986). To optimize glycerol concentration in SMT extender, gradient of glycerol ranging from 6.0% to 7.0% with a difference of 0.2% (v/v) was used in the cryopreservation of buffalo bull semen. Optimum percentage of glycerol in SMT extender was decided on the basis of post thaw motility of spermatozoa. 5.4. Semen quality assessment For all quality assessment a minimum of 200 cells were counted per slide from at least five randomly chosen microscopic fields and two slides were evaluated per sample. Mean of observations was taken as the final score.
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The eosin–nigrosin stained sperm was smeared on a greasefree slide and allowed to dry for 10–15 min. Viable (white) and non-viable (red) sperms were counted under microscope (BX-51, Olympus, Japan) at 400 magnification. 5.4.3. Membrane integrity The hypo-osmotic swelling test (HOST) was used to evaluate functional membrane integrity of spermatozoa. In brief, 100 ml of diluted semen was added to 900 ml of 100 mOsM/Kg hypo-osmotic solution (49.95 mM fructose, 16.66 mM trisodium citrate) and incubated for 1 h at 37 1C. After incubation, a small drop of sperm suspensions was placed on clean, dry and grease-free glass slides and covered with cover-slip. The slide was examined under 400 magnification using phase-contrast microscope for different type of swelling patterns. Similarly, spermatozoa were counted in 300 mOsml/Kg isotonic solution (99.96 mM fructose, 29.97 mM trisodium citrate) and treated as control.
5.4.4. Acrosome integrity Spermatozoal percent acrosome integrity was assessed according to the method of Hancock (1952). In brief, a thin smear of diluted semen was smeared in a glass slide, airdried and fixed at room temperature before transferring into freshly prepared Giemsa solution for 90 min. Acrosomal integrity of spermatozoa were recorded under 1000 magnification.
5.5. Assessment of sperm cryocapacitation Immediately after thawing, semen samples were collected separately into 15 ml polypropylene tubes and washed as described by Reddy et al. (2010). Washed spermatozoa (100 106 cells/ml) were then incubated with 100 mg/ml Lyso-Phosphatidyl Choline (LPC, a known inducer of acrosome reaction in capacitated cells only) for 15 min at 38.5 1C in a cell culture incubator with 5% CO2 and 85% relative humidity in air. The samples were then subjected to a double staining procedure using trypan blue and giemsa stains to differentiate between acrosome-reacted (live/dead) and nonreacted (live/dead) cells (Sidhu et al., 1992). The percentage of live spermatozoa undergoing acrosome reaction was counted under bright field microscope (BX-51, Olympus, Japan) at 400 magnification. 5.6. Lipid peroxidation of spermatozoa
5.4.1. Individual motility Individual motility of spermatozoa was assessed using Phase contrast microscope under 200 magnifications. In brief, 5 ml of diluted semen was transferred on a greasefree glass slide maintained over 37 1C heating plate and covered with cover slip and flagellar movement of spermatozoa were observed (Reddy et al., 2010). 5.4.2. Viability For viability evaluation, 10 ml of diluted semen was mixed with 10 ml of eosin–nigrosin dye (5% eosin B, 10% nigrosin) on a microscopic slide and incubated for 2 min at 37 1C.
Lipid peroxidation of freshly ejaculated and cryopreserved spermatozoa was measured by following protocol of Alvarez and Story (1982). Production of malonaldehyde (MDA) was used as an index of spontaneous lipid peroxidation in spermatozoa using a modified thiobarbituric acid (TBA) assay. Malonaldehyde, obtained by acid hydrolysis of the 1,1,3,3-tetraethoxypropane in the TCA solution followed by TBA reaction were used as standard. A graph of absorbance vs. MDA concentration of standards was plotted on Log paper and concentration of MDA production in samples were determined by interpolation.
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5.7. Statistical analysis All the experiments were done in nine replicates i.e. a total of nine ejaculates from nine randomly chosen bulls. One way analysis of variance (ANOVA) was used to analyze the data in SigmaPlot 11s software package (Systat software Inc., USA). The level of significance was considered at (Po0.05). Post hoc comparisons (Tukey test) were evaluated between sample treatments. For motility percentage Arc Sine transformation was carried out to get normalized data before analysis. Results were expressed as the means7S.E.M. Author contribution Authors V. K. Singh, Raj Kumar and S. K. Atreja were involved in plan of work, execution of work, analysis of data and manuscript preparation. Conflict of interest statement None of the authors have any conflict of interest to declare. Acknowledgment We sincerely thank the Director, National Dairy Research Institute, Karnal, India, for providing financial assistance and necessary facilities during the course of study. This study was supported by World Bank funded National Agricultural Innovation Project number-C4/C30014 (Code-41420102). References Abbas, A., Andrabi, S.M.H., 2002. Effect of different glycerol concentrations on motility before and after freezing, recovery rate, longevity and plasma membrane integrity of Nili-Ravi buffalo bull spermatozoa. Pak. Vet. J. 22, 1–4. Ahmed, K., Foote, R.H., 1986. Post thaw survival of frozen bull spermatozoa treated with antibiotics and detergent. J. Dairy Sci. 69, 535–541. Aires, V.A., Hinsch, K.D., Mueller-Schloesser, F., Bogner, K., MuellerSchloesser, S., Hinsch, E., 2003. In vitro and in vivo comparison of egg yolk-based and soybean lecithin-based extenders for cryopreservation of bovine semen. Theriogenology 60, 269–279. Aitken, R.J., Buckingham, D.W., Brindle, J., Gomez, E., Baker, H.W., Irvine, D.S., 1995. Analysis of sperm movement in relation to the oxidative stress created by leukocytes in washed sperm preparations and seminal plasma. Hum. Reprod. 10, 2061–2071. Akhter, S., Ansari, M.S., Rakha, B.A., Andrabi, S.M.H., Iqbal, S., Ullah, N., 2010. Cryopreservation of buffalo (Bubalus bubalis) semen in Bioxcells extender. Theriogenology 74 (6), 951–955. Alvarez, J.G., Story, B.T., 1982. Spontaneous lipid peroxidation in rabbit epidydimal spermatozoa: its effect on sperm motility. Biol. Reprod. 27, 1102–1110. Baker, M.A., Aitken, R.J., 2005. Reactive oxygen species in spermatozoa: methods for monitoring and significance for the origins of genetic disease and infertility. Reprod. Biol. Endocrinol. 3, 67.
Chen, Y., Foote, R.H., Brockett, C.C., 1993. Effect of sucrose, trehalose, hypotaurine, taurine and blood serum on survival of frozen bull Sperm. Cryobiology 30, 423–431. Correa, L.M., Thomas, A., Meyers, S.A., 2007. The macaque sperm actin cytoskeleton reorganizes in response to osmotic stress and contributes to morphological defects and decreased motility. Biol. Reprod. 77, 942–953. de Lamirande, E., Lamothe, G., 2009. Reactive oxygen-induced reactive oxygen formation during human sperm capacitation. Free Radic. Biol. Med. 46, 502–510. Hancock, J.L., 1952. The morphology of bull spermatozoa. J. Exp. Biol. 29, 445–453. Kumar, R., Atreja, S.K., 2012. Effect of incorporation of additives in trisbased egg yolk extender on buffalo (Bubalus bubalis) sperm tyrosine phosphorylation during cryopreservation 47 (3), 485–490Reprod. Domest. Anim. 47 (3), 485–490. Kumar, R., Singh, V.K., Chhillar, S., Atreja, S.K., 2013. Effect of supplementation of taurine or trehalose in extender on immunolocalization of tyrosine phosphoproteins in buffalo and cattle (Karan Fries) cryopreserved spermatozoa. Reprod. Domest. Anim. 48 (3), 407–415. Kumar, S., Sahni, K.L., Mohan, G., 1992. Effect of different levels of glycerol and yolk on freezing and storage of buffalo semen in milk, Tris and sodium citrate buffers. Buffalo J. 8, 151–156. Lehtonen, J.Y., Kinnunen, P.K., 1995. Phospholipase A2 as a mechanosensor. Biophys. J. 68, 1888–1894. Lipar, J.L., Ketterson, E.D., Nolan Jr., V., Casto, J.M., 1999. Egg yolk layers vary in the concentration of steroid hormones in two avian species. Gen. Comp. Endocrinol. 115 (2), 220–227. Molinia, F.C., Evans, G., Casares, P.I., Maxwell, W.M.C., 1994. Effect of monosaccharides and disaccharides in Tris-based diluents on motility, acrosome integrity and fertility of pellet frozen ram spermatozoa. Anim. Reprod. Sci. 36, 113–122. Pace, M.M., Graham, E.F., 1974. Components in egg yolk which protect bovine spermatozoa during freezing. J. Anim. Sci. 39, 1144–1149. Pettitt, M.J., Buhr, M.M., 1998. Extender components and surfactants affect boar sperm function and membrane behavior during cryopreservation. J. Androl. 19, 736–746. Ragoonanan, V., Hubel, A., Aksan, A., 2010. Response of the cell membrane-cytoskeleton complex to osmotic and freeze/thaw stresses. Cryobiology 61 (3), 335–344. Reddy, N.S.S., Gali, J.M., Atreja, S.K., 2010. Effects of adding taurine and trehalose to a tris-based egg yolk extender on buffalo (Bubalus bubalis) sperm quality following cryopreservation. Anim. Reprod. Sci. 119 (3–4), 183–190. Sariozkan, S., Bucak, M.N., Tuncer, P.B., Ulutas, P.A., Bilgen, A., 2009. The influence of cysteine and taurine on microscopic-oxidative stress parameters and fertilizing ability of bull semen following cryopreservation. Cryobiology 58, 134–138. Sidhu, K.S., Dhindsa, J.S., Guraya, S.S., 1992. A simple staining procedure for detecting the true acrosome reaction in buffalo (Bubalus bubalis) spermatozoa. Biotech. Histochem. 67 (1), 35–39. Singh, A.K., Singh, V.K., Narwade, B.M., Mohanty, T.K., Atreja, S.K., 2012. Comparative quality assessment of Buffalo (Bubalus bubalis) semen chilled (5 1C) in egg yolk- and soya milk-based extenders. Reprod. Domest. Anim. 47 (4), 596–600. Singh, V.K., Atreja, S.K., Kumar, R., Chhillar, S., Singh, A.K., 2012. Assessment of intracellular Ca2 þ , cAMP and 1, 2-diacylglycerol in cryopreserved buffalo (bubalus bubalis) spermatozoa on supplementation of taurine and trehalose in the extender. Reprod. Domest. Anim. 47 (4), 584–590. Thuwanut, P., Chatdarong, K., Techakumphu, M., Axner, E., 2008. The effect of antioxidants on motility, viability, acrosome integrity and DNA integrity of frozen-thawed epididymal cat spermatozoa. Theriogenology 70, 233–240.
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Livestock Science journal homepage: www.elsevier.com/locate/livsci
Cryo-survival, cryo-capacitation and oxidative stress assessment of buffalo spermatozoa cryopreserved in new soya milk extender$ V.K. Singh, R. Kumar, S.K. Atreja n Reproductive Biochemistry Laboratory, Animal Biochemistry Division, National Dairy Research Institute, Karnal 132001, Haryana, India
a r t i c l e in f o
abstract
Article history: Received 3 January 2013 Received in revised form 27 November 2013 Accepted 10 December 2013
Egg yolk is most commonly used in semen extenders for cryopreservation but, wide variability in composition and potential risk of xenobiotic contamination has raised questions upon the use of egg yolk. Therefore, the present study was designed to develop a soya milk based phytoextender for buffalo semen cryopreservation. Soya milk was prepared from fresh soya beans in laboratory. Soya milk (25% v/v) and glycerol (6.4% v/v) was added in Tris citrate buffer to prepare Soya Milk Tris (SMT) extender. To improve postthaw sperm quality, cryo-protectants like trehalose (100 mM) or taurine (50 mM) was supplemented in SMT extender before semen cryopreservation. Post-thaw quality parameters, degree of cryocapacitation and oxidative stress (lipid peroxidation) of spermatozoa cryopreserved in SMT with or without additives were assessed and compared with Egg Yolk Tris (EYT) extender. SMT extender showed no significant (P 40.05) differences in sperm motility, viability, membrane integrity and acrosome integrity as compared to EYT extender. The degree of cryocapacitation and oxidative damage were found significantly (P o 0.05) lower in spermatozoa cryopreserved in SMT extender as compared to EYT extender. Supplementation of additives (trehalose or taurine) in SMT extender significantly (P o 0.05) reduced oxidative damage. Thus, soya milk extender (25% soya milk v/v; 6.4% Glycerol in tris citrate buffer) with additives (trehalose or taurine) may substitute conventional egg yolk extender (20% egg yolk v/v; 7% Glycerol in tris citrate buffer) for better cryopreservation of buffalo semen. & 2013 Elsevier B.V. All rights reserved.
Keywords: Murrah Buffalo Soya milk extender Spermatozoa Cryopreservation Cryocapacitation
1. Introduction Artificial Insemination (AI) extensively depends on frozen semen. However, the process of cryopreservation damages plasma membrane, cytoskeleton, motility apparatus, and nucleus of spermatozoa (Ragoonanan et al.,
Abbreviations: EYT, egg yolk tris; SMT, soya milk tris; HOST, hypo-osmotic swelling test ☆ This study was partly funded by World Bank funded National Agricultural Innovation Project number-C4/C30014 (Code-41420102), ICAR, New Delhi. n Corresponding author. E-mail address: [email protected] (S.K. Atreja). 1871-1413/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.livsci.2013.12.013
2010). The process of cryopreservation also induces premature capacitation (Reddy et al., 2010), osmotic stress and oxidative damage (Thuwanut et al., 2008) that reduce functional life of spermatozoa. Therefore, quality evaluation of spermatozoa based on motility is not sufficient to judge true fertilizing capacity of spermatozoa. Currently, egg yolk-based extenders are globally used for cryopreservation of semen from bull and many other species. However, wide variability, risk of xenobiotic contamination (Aires et al., 2003) and presence of steroid hormones (Lipar et al., 1999) have raised questions upon the use of egg yolk in freezing extender. Furthermore, egg yolk contains substances (e.g. high-density lipoproteins and minerals) that inhibit cellular respiration and affect
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metabolic activities (Pace and Graham, 1974). Hence, to circumvent these problems a non animal origin based semen extender with comparable cryoprotective effect was desirable. Recently, many commercial extenders like AndroMeds (Aires et al., 2003) and Bioxcells (Akhter et al., 2010) have been evaluated for preservation of bovine and buffalo semen and found to maintain better semen quality than egg yolk based extenders. But, high cost and limited availability of these commercial extenders make them unobtainable for emerging and small-scale semen banks especially in developing countries. We have developed an economic, pathogen free and non animal origin based soya milk semen extender to replace egg yolk based extender and reported successful liquid preservation of buffalo semen upto 72 h (A.K. Singh et al., 2012). A further study was required to establish this extender for cryopreservation of buffalo semen. Recently, cryoprotectants like taurine or trehalose, have been supplemented in freezing extenders of bull (Sariozkan et al., 2009) and buffalo (Kumar and Atreja, 2012, Kumar et al., 2013) semen prior to cryopreservation, to improve the post thaw sperm quality. Hence, the present study was designed first; to cryopreserve buffalo semen in soya milk tris (SMT) extender, second; to evaluate cryo-survival, cryo-capacitation and oxidative stress of sperm cryopreserved in SMT extender in comparison to conventional EYT extender and third; to further improve the post thaw sperm quality by supplementing cryoprotectants like trehalose or taurine in SMT extender.
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2. Results 2.1. Optimization of glycerol for cryopreservation Post thaw motility of buffalo spermatozoa cryopreserved in SMT extender with gradient of glycerol ranging from 6.0–7.0% with a difference of 0.2% revealed that motility of spermatozoa cryopreserved in SMT extender with 6.4% of glycerol was significantly higher (P o0.05) than other concentrations (Fig. 1). 2.2. Post-thaw motility, viability, membrane integrity and acrosome integrity The standard semen quality parameters such as percent motility, viability, membrane integrity and acrosomal integrity in fresh semen ejaculates and post thawed semen are shown in Table 1. Cryopreservation of buffalo spermatozoa resulted in a significant (Po0.05) decrease in all the standard semen quality parameters as compared to fresh. Buffalo spermatozoa cryopreserved in SMT extender did not show significant (P 40.05) differences in any of the standard sperm quality parameters as compared to spermatozoa cryopreserved in EYT extender. Supplementation of trehalose (100 mM) or taurine (50 mM) to freezing extender showed a significantly (Po0.05) higher postthaw sperm motility, viability, percent membrane integrity and acrosome integrity as compared to sperm cryopreserved in their absence (Table 1). 2.3. Extent of cryocapacitation of spermatozoa
60 c
Motility (%)
45
a
b d
d
d
30
6.0 % Glycerol 6.2 % Glycerol 6.4 % Glycerol 6.6 % Glycerol 6.8 % Glycerol 7.0 % Glycerol
15
0 6.0 % G6.2 % G6.4 % G6.6 % G6.8 % G7.0 % G
Fig. 1. Post thaw sperm motility of buffalo spermatozoa cryopreserved in soya milk extender with different glycerol concentration for optimization of glycerol percentage. Values are the mean 7 S.E.M. of nine experiments. Means with different letters are significantly different (Po 0.05).
Sperm cryocapacitation was assessed by lysophosphatidyl choline (LPC)-induced acrosome reaction followed by dual staining of spermatozoa. Cryopreservation of buffalo spermatozoa in EYT extender resulted in a significant (P o0.05) increase of capacitation as compared to freshly ejaculated buffalo semen. On the other hand, extent of cryocapacitation was found significantly (P o0.05) lower in buffalo spermatozoa cryopreserved in SMT extender than EYT extender. Moreover, Supplementation of 100 mM trehalose or 50 mM taurine to the freezing medium prior to cryopreservation significantly (P o0.05) decreased the extent of cryocapacitation in buffalo spermatozoa as compared to control (Table 1).
Table 1 Post thaw parameters of freshly ejaculated and cryopreserved Buffalo spermatozoa in extended in egg yolk tris extender (EYT), soya milk tris extender (SMT), soya milk tris extender supplemented trehalose (SMTþ Trehalose) and soya milk tris extender supplimented with taurine (SMTþ Taurine) respectively. Values are Mean 7 Standard Error Mean (S.E.M.) of nine experiments. Mean 7 S.E.M. with different letters (a,b,c,d) in row are significantly different (P o 0.05).
Motility (%) Viability (%) Membrane integrity (%) Acrosome integrity (%) Sperm capacitation (%) Lipid peroxidation (nmol MDA/108cells)
Fresh
EYT
SMT
SMTþTrehalose (100 mM)
SMTþTaurine (50 mM)
81.0 7 1.87a 89.0 7 1.83a 74.6 7 3.04a 96.2 7 1.48a 5.6 7 0.67a 1.137 0.31a
47.0 7 1.22b 61.4 7 1.12b 38.2 7 1.31b 84.4 7 3.40b 17.2 7 0.73b 3.26 7 0.10b
46.0 7 1.87b 62.2 7 1.58b 39.2 7 1.52b 86.4 7 2.20bc 12. 7 0.83c 2.487 0.10c
54.0 71.87c 69.2 71.11c 42.4 71.93b 90.6 72.28cd 9.8 70.58d 1.48 70.02d
57.0 7 1.22c 70.07 1.26c 42.2 7 2.03b 91.4 7 2.16d 9.0 7 0.44d 1.477 0.16d
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2.4. Lipid peroxidation assay The extent of lipid peroxidation (LPO; production of MDA) by freshly ejaculated and cryopreserved buffalo spermatozoa are shown in Table 1. The values for lipid peroxidation in freshly ejaculated spermatozoa was found to be 1.1370.31 nmol MDA/108cells and it increased significantly (Po0.05) upon cryopreservation. There was a significant (Po0.05) difference in the value of lipid peroxidation of cryopreserved spermatozoa in EYT extender as compared to SMT extender. Lipid peroxidation was significantly (Po0.05) reduced upon supplementation of SMT with trehalose or taurine than control. However, there was no significant (P40.05) difference in lipid peroxidation of spermatozoa cryopreserved in these two additives. 3. Discussion Egg yolk is rich in lipids like lecithin that interacts with sperm membrane and show a stabilizing effect during cold shock (Pettitt and Buhr, 1998). Soya bean also have high content of lecithin. Therefore, soya milk prepared from soya beans possesses cryoprotective potential similar to egg yolk. In an earlier study, we have reported that soya milk (25% v/v) can replace egg yolk (20% v/v) in tris based semen extender for liquid preservation of buffalo semen up to 72 h with better keeping quality (A.K. Singh et al., 2012). Glycerol, a permeating cryoprotectant, is incorporated in freezing extender for semen cryopreservation. Abbas and Andrabi (2002) studied the effects of different concentrations of glycerol on post-thaw sperm quality and found that 7% glycerol is significantly (Po0.05) better than other concentrations of glycerol. In another study, Kumar et al. (1992) reported that the best level of glycerol is 6% for Tris- and milk-based diluents, and 9% for the sodium citrate diluents. However, in this study 6.4% glycerol was found significantly (Po0.05) better than other concentrations of glycerol in SMT extender for buffalo semen cryopreservation. Cryopreservation process causes damage to plasma membrane, cytoskeleton, motility apparatus, and the nucleus of spermatozoa (Ragoonanan et al., 2010) resulting into decreased motility, membrane integrity and increased premature capacitation (Akhter et al., 2010). In consistent to these reports, we also found significantly (P o0.05) decreased motility, viability, membrane integrity, acrosome integrity and an increased degree of cryocapacitation in spermatozoa cryopreserved in EYT or SMT extenders as compared to freshly ejaculated spermatozoa. There were no significant (P40.05) difference in post thaw semen quality parameters between two extenders but degree of cryocapacitation was significantly (Po0.05) lesser in spermatozoa cryopreserved in SMT extender than EYT extender. This suggests that keeping quality of SMT extender is comparable with EYT extender but SMT extender reduces premature capacitation during cryopreservation. Damage that occurs during cryopreservation is due to extracellular ice crystals formation. Furthermore, the membrane stretching during freezing and thawing results in decreased lateral packing of lipids (Lehtonen and Kinnunen, 1995) and reorganization of the actin cytoskeleton (Correa et al., 2007) leaving the cell membrane vulnerable to reactive oxygen species (ROS) attack. Thus, cryopreservation not only
induces an osmotic stress to cells, it also makes spermatozoa susceptible to oxidative stress (Thuwanut et al., 2008) and causes DNA damage. Recent studies indicate that low levels of ROS play important role in controlling various sperm functions like capacitation, acrosome reaction, hyperactivation and sperm-oocyte fusion (deLamirande and Lamothe, 2009) but excessive generation of ROS have a detrimental effect on spermatozoa. Though, it was reported that ROS is produced by defective spermatozoa and contaminating leukocytes (Aitken et al., 1995), it is now evident that that process of cryopreservation also increases the level of ROS in spermatozoa (Thuwanut et al., 2008). It is interesting to note that, sperm membrane is rich in unsaturated fatty acids and they have limited ability to store antioxidant enzymes so they are very susceptible to oxidative stress and peroxidative attack by ROS (Baker and Aitken, 2005). Our results showed that spermatozoa cryopreserved in SMT extender showed significantly (Po0.05) lesser lipid peroxidation as compared to EYT extender. Recently, several reports have suggested that supplementation of additives like trehalose or taurine to the freezing extender improves semen quality in many species (Kumar and Atreja, 2012; Kumar et al., 2013; Reddy et al., 2010 ; Sariozkan et al., 2009). In close agreement to these reports, we also found significant (Po0.05) improvement in post thaw sperm quality and reduced degree of premature capacitation and oxidative damage upon supplementation of trehalose (100 mM) or taurine (50 mM) to SMT extender as compared to control. Trehalose, a non-reducing disaccharide, causes cellular osmotic dehydration before freezing (Molinia et al., 1994). On the other hand, Taurine, a permeating sulfonic amino acid analog, inhibit lipid peroxidation, protects the cells against accumulation of ROS (Chen et al., 1993), modulate Ca2 þ uptake (V.K. Singh et al., 2012) and inhibit protein tyrosine phosphorylation (Kumar and Atreja, 2012). This showed that soya milk might have some antioxidant property. On other hand oxidative damage can be further mitigated through supplementation of cryoprotectants like trehalose or taurine in freezing extender. 4. Conclusion Cryopreservation of Buffalo sperm in newly developed SMT extender showed comparable post thaw semen quality parameters and significantly (Po0.05) lesser oxidative damage and cryocapacitation as compared to spermatozoa cryopreserved in conventional EYT extender. Moreover, supplementation of trehalose (100 mM) or taurine (50 mM) in SMT extender significantly (Po0.05) improved post thaw sperm quality and decreased oxidative damage and cryocapacitation. Hence, soya milk (25% v/v; 6.4% glycerol v/v) with additives (trehalose or taurine) can be used to substitute egg yolk (20% v/v; 7% glycerol v/v) in tris buffer based extender for better cryopreservation of buffalo semen. 5. Materials and methods 5.1. Semen extenders Egg yolk tris (EYT) semen extender (20% Egg yolk v/v diluted in TCG buffer; 274 mM Tris, 87 mM Citric acid,
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43 mM Glucose, 1,000,000 IU Benzyl penicillin per litre, 750,000 IU Streptomycin per litre) was prepared as described by Ahmed and Foote (1986). Whereas, Soya milk used in the semen extender was prepared as described by A.K. Singh et al. (2012). SMT extender was prepared by adding soya milk (25% v/v) in TCG buffer. 5.2. Semen collection and cryopreservation Murrah buffalo bulls, 3–5 years of age, housed at Artificial Breeding Research Centre, National Dairy Research Institute, Karnal, India, under uniform nutritional conditions were used for semen collection. A total of nine ejaculates were collected using artificial vagina (IMV, L0 Aigle Cedex, France) from nine randomly chosen bulls. Ejaculated semen having milky white color, score of þ3 or more mass activity and more than 80% progressive motility was used in all the experiments. Each ejaculate was split into five aliquots and one aliquot was used for fresh semen analysis whereas other four were diluted with the EYT extender, SMT extender and SMT extender containing taurine (50 mM) or trehalose (100 mM) respectively. Sperm concentrations in semen samples were determined by hemocytometer and a final concentration of 80 106 sperms ml 1 was achieved. Diluted semen was filled and sealed into French straws (IMV, L0 Aigle cedex, France) and kept for 4 h in cold room at 4 1C for equilibration. Equilibrated semen straws were then kept five centimeter above the liquid nitrogen for 10 min and plunged in liquid nitrogen for storage at 196 1C for 3–4 weeks. Frozen straws were thawed at 37 1C for 45 s in a water bath and used for assessment of various semen quality parameters. 5.3. Optimization of glycerol for cryopreservation In EYT extender glycerol was added at the rate of 7.0% (Ahmed and Foote, 1986). To optimize glycerol concentration in SMT extender, gradient of glycerol ranging from 6.0% to 7.0% with a difference of 0.2% (v/v) was used in the cryopreservation of buffalo bull semen. Optimum percentage of glycerol in SMT extender was decided on the basis of post thaw motility of spermatozoa. 5.4. Semen quality assessment For all quality assessment a minimum of 200 cells were counted per slide from at least five randomly chosen microscopic fields and two slides were evaluated per sample. Mean of observations was taken as the final score.
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The eosin–nigrosin stained sperm was smeared on a greasefree slide and allowed to dry for 10–15 min. Viable (white) and non-viable (red) sperms were counted under microscope (BX-51, Olympus, Japan) at 400 magnification. 5.4.3. Membrane integrity The hypo-osmotic swelling test (HOST) was used to evaluate functional membrane integrity of spermatozoa. In brief, 100 ml of diluted semen was added to 900 ml of 100 mOsM/Kg hypo-osmotic solution (49.95 mM fructose, 16.66 mM trisodium citrate) and incubated for 1 h at 37 1C. After incubation, a small drop of sperm suspensions was placed on clean, dry and grease-free glass slides and covered with cover-slip. The slide was examined under 400 magnification using phase-contrast microscope for different type of swelling patterns. Similarly, spermatozoa were counted in 300 mOsml/Kg isotonic solution (99.96 mM fructose, 29.97 mM trisodium citrate) and treated as control.
5.4.4. Acrosome integrity Spermatozoal percent acrosome integrity was assessed according to the method of Hancock (1952). In brief, a thin smear of diluted semen was smeared in a glass slide, airdried and fixed at room temperature before transferring into freshly prepared Giemsa solution for 90 min. Acrosomal integrity of spermatozoa were recorded under 1000 magnification.
5.5. Assessment of sperm cryocapacitation Immediately after thawing, semen samples were collected separately into 15 ml polypropylene tubes and washed as described by Reddy et al. (2010). Washed spermatozoa (100 106 cells/ml) were then incubated with 100 mg/ml Lyso-Phosphatidyl Choline (LPC, a known inducer of acrosome reaction in capacitated cells only) for 15 min at 38.5 1C in a cell culture incubator with 5% CO2 and 85% relative humidity in air. The samples were then subjected to a double staining procedure using trypan blue and giemsa stains to differentiate between acrosome-reacted (live/dead) and nonreacted (live/dead) cells (Sidhu et al., 1992). The percentage of live spermatozoa undergoing acrosome reaction was counted under bright field microscope (BX-51, Olympus, Japan) at 400 magnification. 5.6. Lipid peroxidation of spermatozoa
5.4.1. Individual motility Individual motility of spermatozoa was assessed using Phase contrast microscope under 200 magnifications. In brief, 5 ml of diluted semen was transferred on a greasefree glass slide maintained over 37 1C heating plate and covered with cover slip and flagellar movement of spermatozoa were observed (Reddy et al., 2010). 5.4.2. Viability For viability evaluation, 10 ml of diluted semen was mixed with 10 ml of eosin–nigrosin dye (5% eosin B, 10% nigrosin) on a microscopic slide and incubated for 2 min at 37 1C.
Lipid peroxidation of freshly ejaculated and cryopreserved spermatozoa was measured by following protocol of Alvarez and Story (1982). Production of malonaldehyde (MDA) was used as an index of spontaneous lipid peroxidation in spermatozoa using a modified thiobarbituric acid (TBA) assay. Malonaldehyde, obtained by acid hydrolysis of the 1,1,3,3-tetraethoxypropane in the TCA solution followed by TBA reaction were used as standard. A graph of absorbance vs. MDA concentration of standards was plotted on Log paper and concentration of MDA production in samples were determined by interpolation.
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5.7. Statistical analysis All the experiments were done in nine replicates i.e. a total of nine ejaculates from nine randomly chosen bulls. One way analysis of variance (ANOVA) was used to analyze the data in SigmaPlot 11s software package (Systat software Inc., USA). The level of significance was considered at (Po0.05). Post hoc comparisons (Tukey test) were evaluated between sample treatments. For motility percentage Arc Sine transformation was carried out to get normalized data before analysis. Results were expressed as the means7S.E.M. Author contribution Authors V. K. Singh, Raj Kumar and S. K. Atreja were involved in plan of work, execution of work, analysis of data and manuscript preparation. Conflict of interest statement None of the authors have any conflict of interest to declare. Acknowledgment We sincerely thank the Director, National Dairy Research Institute, Karnal, India, for providing financial assistance and necessary facilities during the course of study. This study was supported by World Bank funded National Agricultural Innovation Project number-C4/C30014 (Code-41420102). References Abbas, A., Andrabi, S.M.H., 2002. Effect of different glycerol concentrations on motility before and after freezing, recovery rate, longevity and plasma membrane integrity of Nili-Ravi buffalo bull spermatozoa. Pak. Vet. J. 22, 1–4. Ahmed, K., Foote, R.H., 1986. Post thaw survival of frozen bull spermatozoa treated with antibiotics and detergent. J. Dairy Sci. 69, 535–541. Aires, V.A., Hinsch, K.D., Mueller-Schloesser, F., Bogner, K., MuellerSchloesser, S., Hinsch, E., 2003. In vitro and in vivo comparison of egg yolk-based and soybean lecithin-based extenders for cryopreservation of bovine semen. Theriogenology 60, 269–279. Aitken, R.J., Buckingham, D.W., Brindle, J., Gomez, E., Baker, H.W., Irvine, D.S., 1995. Analysis of sperm movement in relation to the oxidative stress created by leukocytes in washed sperm preparations and seminal plasma. Hum. Reprod. 10, 2061–2071. Akhter, S., Ansari, M.S., Rakha, B.A., Andrabi, S.M.H., Iqbal, S., Ullah, N., 2010. Cryopreservation of buffalo (Bubalus bubalis) semen in Bioxcells extender. Theriogenology 74 (6), 951–955. Alvarez, J.G., Story, B.T., 1982. Spontaneous lipid peroxidation in rabbit epidydimal spermatozoa: its effect on sperm motility. Biol. Reprod. 27, 1102–1110. Baker, M.A., Aitken, R.J., 2005. Reactive oxygen species in spermatozoa: methods for monitoring and significance for the origins of genetic disease and infertility. Reprod. Biol. Endocrinol. 3, 67.
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