Puja GOEL, Anil Kumar GOEL, Ashok Kumar BHATIA, Suresh Dinkar KHARCHE

Effect of capacitating agents on sperm pretreatment during in vitrofertilization for blastocyst production in caprines

The present study aimed at comparing the relative efficacy of different capacitating agents used for sperm pretreatment during in vitro fertilization (IVF) and subsequent embryo development of caprine oocytes. In experiment 1, the effect of different concentrations of heparin (0, 20, and 50 μg/mL) on sperm pretreatment for different periods of time (30 and 60 min) was studied. In experiment 2, the dose-dependent effect of calcium ionophore (0, 0.1, 0.2, and 0.5 μM) on sperm pretreatment was assessed. Experiment 3 validated and compared the effects of both capacitating agents on sperm pretreatment for capacitation based on the results of the first two experiments. The treated sperms from each group were used for in vitro fertilization and subsequent embryo development. The results indicated that in experiment 1, capacitation with heparin at 20 μg/mL for 60 min had significantly higher (P < 0.05) cleavage and blastocyst production. In experiment 2, capacitation with calcium ionophore at 0.1 μM had significantly more morulae (P < 0.05) and numerically more blastocyst production. In experiment 3, capacitation with heparin at 20 μg/mL had a significantly higher (P < 0.05) blastocyst production rate as compared to calcium ionophore treatment. Based on this study, heparin can be used to enhance capacitation of freshly collected sperms during in vitro fertilization and subsequent embryo development.

PDF

___

1. Nandi P, Ghosh S, Jana K, Sen PC. Elucidation of the involvement of p14, a sperm protein during maturation, capacitation and acrosome reaction of caprine spermatozoa. PLoS ONE 2012;7: e30552.
2. Austin CR. Observation on the penetration of the sperm into the mammalian egg. Aust J Sci Res 1951; 4: 581-596.
3.Breitbart H. Intracellular calcium regulation in sperm capacitation and acrosomal reaction. Mol Cell Endocrinol 2002; 187: 139-144.
4. Lv L, Yue W, Liu W, Ren Y, Li F, Lee KB, Smith GW. Effects of sperm pretreatments and in vitro culture systems on development of in vitro fertilized embryos derived from prepubertal Boer goat oocytes in China. Asian-Aust J Anim Sci 2009; 22: 969-976.
5. Hanada A. In vitro fertilization in cattle with particular reference to sperm preparation with ionophore A 23187. Japanese J Anim Reprod 1985; 31: 56-61.
6. Parrish JL, Susko-Parrish JL, Winer MA, First NL. Capacitation of bovine sperm by heparin. Biol Reprod 1988; 38: 1171-1180.
7. Kharche SD, Goel AK, Jindal SK, Yadav P, Sinha R, Sinha NK. Effect of serum albumin supplementation on in-vitro capacitation and fertilization of caprine oocytes. Small Rumin Res 2009; 81: 85-89.
8. Kharche SD, Goel AK, Jindal SK, Goel P, Jha BK. Birth of twin kids following transfer of in-vitro produced goat embryos. Indian J Anim Sci 2011; 81: 1132-1134.
9. Yadav P, Kharche SD, Goel AK, Jindal SK, Goel P. Assessment of nuclear maturation and subsequent in vitro embryo development of caprine oocytes with different supplementations in maturation medium. Indian J Anim Sci 2013; 83: 1048-1052.
10. Kharche SD, Goel AK, Jindal SK, Sinha NK, Yadav P. Effect of somatic cells co-culture on cleavage and development of in-vitro fertilized caprine embryos. Indian J Anim Sci 2008; 78: 686-692.
11. Singh KP, Saxena A, Kharche SD, Singh P. In vitro fertilization and cleavage rate of in vitro matured prepubertal and pubertal goat oocytes matured in maturation media with different supplementation. Indian J Anim Sci 2008; 78: 1078-1081.
12. Snedecor GW, Cochran WG. Statistical Methods. 6th ed. Ames, IA, USA: Iowa State University Press; 1989.
13. Parrish JL, Susko-Parrish JL, Handrow RR, Sims MM, First NL. Capacitation of bovine spermatozoa by oviduct fluid. Biol Reprod 1989; 40: 1020-1025.
14. Lenz RW, Ball GD, Lohse JK, First NL, Ax RL. Chondroitin sulfate facilitates an acrosome reaction in bovine spermatozoa as evidenced by light microscopy, electron microscopy and in vitro fertilization. Biol Reprod 1983; 28: 683-690.
15. Ksiazkiewicz-Katska L, Rynska B, Gajda B, Smorag Z. Effect of donor stimulation, frozen semen and heparin treatment on the efficiency of in vitro embryo production in goats. Theriogenology 2004; 62: 576-586.
16. Lu KH, Seidel GE. Effects of heparin and sperm concentration on cleavage and blastocyst development rates of bovine oocytes inseminated with flow cytometrically-sorted sperm. Theriogenology 2004; 62: 819-830.
17. Izquierdo D, Villamediana P, Palomo MJ, Mogas T, Paramio MT. Effect of sperm capacitation and fertilization media on IVF and early embryo development of prepubertal goat oocytes. Theriogenology 1998; 49: 1501-1513.
18. Shi DS. Studies related to factors affecting the yield of bovine embryos produced by in vitro techniques. PhD, National University of Ireland, Dublin, Ireland, 1987.
19. First NL, Parrish JJ. Preparation of sperm for in vitro fertilization. In: Proceedings of the Symposium on Application of Egg and Embryo Technologies to Domestic Animals; 1991. pp. 33-35.
20. Wang B, Baldassarre H, Tao T, Gauthier M, Neveu N, Zhou JF. Transgenic goats produced by DNA pronuclear microinjection of in vitro derived zygotes. Mol Reprod Dev 2002; 63: 437-443.
21. Mendes JOB, Burns PD, De La Torre-Sanchez JF, Seidel GE Jr. Effect of heparin on cleavage rates and embryo production in four bovine sperm preparation protocols. Theriogenology 2003; 60: 331-340.
22. Park CK, Ohgoda O, Niwa K. Penetration of bovine follicular oocytes by frozen thawed spermatozoa in the presence of caffeine and heparin. J Reprod Fertil 1989; 86: 577-582.
23. Zhou JB, Wu YG, Liu LQ, Luo MJ, Chang ZL, Ta n X W, Liu N, Tan JH. Studies on in vitro capacitation of goat spermatozoa by heparin treatment. Sheng Wu Gong Cheng Xue Bao 2004; 20: 252-256 (in Chinese with English abstract).
24. Luconi M, Krausz C, Forti G, Baldi E. Extracellular calcium negatively modulates tyrosine phosphorylation and tyrosine kinase activity during capacitation of human spermatozoa. Biol Reprod 1996; 55: 207-216.
25. Tanphaichitr N, Hansen C. Production of motile acrosome reacted mouse sperm with nanomolar concentration of calcium ionophore A23187. Mol Reprod Dev 1994; 37: 326-334.
26. Wang S, Liu Y, Holyoak GR, Evans RC, Bunch TD. A protocol for in vitro maturation and fertilization of sheep oocytes. Small Ruminant Res 1998; 29: 83-88.
27. Pereira, RJ, Tuli RK, Wallenhorst S, Holtz W. The effect of heparin, caffeine and calcium ionophore A23187 on in vitro induction of the acrosome reaction in frozen-thawed bovine and caprine spermatozoa. Theriogenology 2000; 54: 185-192.
28. Ho HC, Suarez SS. An inositol 1,4,5-trisphosphatereceptor-gated intracellular Ca2+ store is involved in regulating sperm hyper activated motility. Biol Reprod 2001; 65: 1606-1615.
29. Watson PF, Plummer JM, Jones PS. The ionophore induced acrosome reaction differs structurally from the spontaneous acrosome reaction. J Exp Zoo1 1992; 264: 231-235.
30. Poulin N, Guler A, Pignon P, Cognie Y. In vitro production of goat embryos: heparin in IVF medium affect developmental ability. In: Proceedings of the 6th International Conference on Goats; 1996. pp. 838-840.