Aim: This study aimed to study the electrophoretic properties of seminal plasma and sperm proteins of Black Bengal buck semen and their correlation with in vitro sperm characters and freezability.

Materials and Methods: Semen ejaculates from nine Black Bengal bucks were collected by artificial vagina (n=20/buck). Ejaculates were evaluated for in vitro sperm characters and electrophoretic profile of seminal protein. In vitro sperm characters were evaluated immediately after collection, after completion of equilibration period, and after freeze-thawing. For seminal protein studies, seminal plasma proteins were precipitated by ice-cold ethanol method, and sperm proteins were extracted by Triton X detergent extraction method. Discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to assess the molecular weight of seminal proteins. Correlation between in vitro sperm characters and protein bands was determined by Pearson's correlation coefficient, and two-way ANOVA was applied to find the individual buck differences.

Results: Significant difference (p<0.01) among the bucks was noticed in the in vitro sperm characters evaluated at all the three stages of semen evaluation such as immediately after collection, after completion of equilibration period, and post-freeze thawing. Progressive loss of sperm motility, membrane integrity, and other in vitro sperm characters were noticed during cryopreservation. A total of ten protein bands in the molecular weight ranging from 17 to 180 kDa were found in the SDS-PAGE of seminal plasma proteins, while nine bands of 17-134 kDa were observed in sperm proteins. Seminal plasma proteins of molecular weight 75, 62-49, 20, and 17 kDa and sperm proteins of 75, 20, and 17 kDa were present in all the nine bucks (100%) screened, and variation among the bucks was noticed for the presence of other proteins. Seminal plasma protein of 180-134 kDa showed a negative correlation with individual motility (−0.716) and functional membrane integrity of sperm cells (−0.724) in post-freeze-thaw analysis and 48 kDa protein had a positive correlation with individual motility (0.649) and functional membrane integrity of sperm cells (0.664) in post-thaw analysis. Sperm proteins of 63 kDa had a negative correlation (−0.616) with sperm concentration in neat semen.

Conclusion: Variation among the bucks was noticed in the in vitro sperm characters and semen freezability. Correlation between seminal proteins and in vitro sperm characters and semen freezability had been found which might be useful as a tool to select breeding bucks.

Keywords: buck, in vitro characters, semen, seminal proteins.

1. Chowdhury, S.A., Bhuiyan, M.S.A. and Faruk, S. (2002) Rearing black Bengal goat under semi-intensive management. Asian Australas. J. Anim. Sci., 15(1): 477-484. [Crossref]

2. Nandi, D., Roy, S., Bera, S., Kesh, S.S. and Samanta, A.K. (2011) The rearing system of black Bengal goat and their farmers in West Bengal, India. Vet. World, 4(6): 254-257. [Crossref]

3. Khandoker, M.A.M., Apu, A.S., Husain, S.S. and Notter, D.R. (2011) A baseline survey on the availability of black Bengal breeding bucks in different districts of Bangladesh. J. Bangladesh Agric. Univ., 9(1): 91-96. [Crossref]

4. Konyak, P., Mandal, A., Mondal, M., Bhakat, C., Das, S.K., Saroj, R., Ghosh, M.K. and Karunakaran, M. (2018) Preservation of black Bengal buck semen in soybean lecithin based chemically defined extender. Indian J. Anim. Res., 52(8): 1151-1154. [Crossref]

5. Karunakaran, M., Konyak, P., Mandal, A., Mondal, M., Bhakat, C., Mandal, D.K., Das, S.K. and Ghosh, M.K. (2017) Chemically well-defined extender for preservation of black Bengal buck semen. Res. Rev. J. Dairy Sci. Technol., 6(2): 7-10.

6. Karunakaran, M., Konyak, P., Mandal, A., Mondal, M., Bhakat, C., Saroj, R., Ghosh, M.K. and Behera, R. (2019) Effect of trehalose-an impermeant cryoprotectant on cryopreservation of black Bengal buck semen. Indian J. Anim. Res., 53(1): 37-40. [Crossref]

7. Larson, J.L. and Miller, D.J. (2000) Can relative spermatozoal galactosyltransferase activity be predictive of dairy bull fertility? J. Dairy Sci., 83(11): 2473-2479. [Crossref]

8. Karunakaran, M., Devanathan, T.G., Jawahar, T.P., Manimaran, K., Chitra, A., Dhali, A. and Selvaraju, S. (2012) Electrophoretic profile of bull sperm membrane proteins as a tool for selection of breeding bulls. Indian J. Anim. Sci., 82(11): 1303-1305.

9. Karunakaran, M., Devanathan, T.G., Kulasekar, K., Sridevi, P., Jawahar, T.P., Loganathasamy, K., Dhali, A. and Selvaraju, S. (2012) Effect of heparin-binding protein and hydrogen peroxide on lipid peroxidation status of bovine sperm cells. Indian J. Anim. Sci., 82(9): 976-978.

10. Karunakaran, M., Devanathan, T.G., Kulasekar, K., Sridevi, P., Jawahar, T.P., Loganatahsamy, K., Dhali, A. and Selvaraju, S. (2012) Effect of fertility associated protein on oxidative stress of bovine sperm cells. Indian J. Anim. Reprod., 33(1): 43-46.

11. Krishnan, G., Thangvel, A., Loganathasamy, K., Veerapandian, C., Kumarasamy, P. and Karunakaran, M. (2015) Effect of fertility associated proteins on lipid peroxidation production in Holstein Friesian semen. Indian J. Anim. Sci., 85(11): 1176-1180.

12. Desnoyers, L. and Manjunath, P. (1992) Major proteins of bovine seminal plasma exhibit novel interactions with phospholipid. J. Biol. Chem., 267(14): 10149-10155.

13. Yanagimachi, R. (1994) Fertility of mammalian spermatozoa: Its development and relativity. Zygote, 2(4): 371-372. [Crossref]

14. Russell, L.D., Montag, B., Hunt, W. and Peterson, R.N. (1985) Properties of boar sperm plasma-membranes (pm)-proteins released by washing and differential solubility in salts, detergents, and sensitivity to surface radiolabeling. Gamete Res., 11: 237-252. [Crossref]

15. Caballero, I., Vazquez, J.M., Garcia, E.M., Roca, J., Martinez, E.A., Calvete, J.J., Sanz, L., Ekwall, H. and Rodriguez-Martinez, H. (2006) Immunolocalization and possible functional role of pspi/psp-ii heterodimer in highly extended boar spermatozoa. J. Androl., 27(6): 766-773. [Crossref] [PubMed]

16. Moura, A.A., Koc, H., Chapman, D.A. and Killian, G.J. (2006) Identification of proteins in the accessory sex gland fluid associated with fertility indexes of dairy bulls: A proteomic approach. J. Androl., 27(2): 201-211. [Crossref] [PubMed]

17. Karunakaran, M. and Devanathan T.G. (2016) Evaluation of bull semen for fertility-associated protein, in vitro characters and fertility. J. Appl. Anim. Res., 45(1): 136-144. [Crossref]

18. Krishnan, G., Thangvel, A., Loganathasamy, K., Veerapandian, C., Kumarasamy, P. and Karunakaran, M. (2016) The presence of heparin-binding proteins and their impact on semen quality of Holstein Friesian bulls. Indian J. Anim. Sci., 86(4): 392-396.

19. Prien, S. and Iacovides, S. (2016) Cryoprotectants and cryopreservation of equine semen: A review of industry cryoprotectants and the effects of cryopreservation on equine semen membranes. J. Dairy Vet. Anim. Res., 3(1): 1-8. [Crossref]

20. Gurler, H., Malama, E., Heppelmann, M., Calisici, O., Leiding, C., Kastelic, J.P. and Bollwein, H. (2016) Effects of cryopreservation on sperm viability, synthesis of reactive oxygen species and DNA damage of bovine sperm. Theriogenology, 86(2): 562-571. [Crossref] [PubMed]

21. Thurston, L.M., Siggins, K., Mileham, A.J., Watson, P.F. and Holt, W.V. (2002) Identification of amplified restriction fragment length polymorphism markers linked to genes controlling boar sperm viability following cryopreservation. Biol. Reprod., 66(3): 545-554. [Crossref]

22. Asadpour, R., Alavi-Shoushtari, S.M., Rezaii, S.A. and Ansari, M.K. (2007) SDS-polyacrylamide gel electrophoresis of buffalo bulls seminal plasma proteins and their relation with semen freezability. Anim. Reprod. Sci., 102(3-4): 308-313. [Crossref] [PubMed]

23. Nass, S.J., Miller, D.J., Winner, M.A. and Ax, R.L. (1990) Male accessory sex glands produce heparin-binding proteins that bind to cauda epididymal spermatozoa and are testosterone dependent. Mol. Reprod. Dev., 25(3): 237-246. [Crossref] [PubMed]

24. Laemmli, V.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259): 660-685. [Crossref]

25. Purdy, P. (2006) A review on goat sperm cryopreservation. Small Rumin. Res., 63(3): 215-225. [Crossref]

26. Afroz, S., Islam, M., Khandoker, M. and Akter, Q. (2008) Cryopreservation of black Bengal buck semen: Effects of diluents and freezing on sperm motility and morphology. Anim. Sci. J., 79(5): 550-553. [Crossref]

27. Apu, A.S., Khandoker, M.A.M., Husain, S.S., Rahman, S.M.H. and Notter, R.D. (2008) Investigation of seminal attributes and fertility of black Bengal goat. Bangladesh J. Anim. Sci., 37(2): 17-24. [Crossref]

28. 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(4): 871-891. [Crossref]

29. Teixeira, D., Cavada, B., Sampaio, A., Havt, A., Bloch, C Jr., Prates, M., Moreno, F., Santos, E., Gadelha, C., Gadelha, T., Crisostomo, F. and Freitas, V. (2002) Isolation and partial characterisation of a protein from buck seminal plasma (Capra hircus), homologous to spermadhesins. Protein Pept. Lett., 9(4): 331-335. [Crossref]

30. Yue, W., Shi, L., Bai, Z., Ren, Y. and Zhoa, Y. (2009) Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of ram seminal plasma proteins and their correlation with semen characteristics. Anim. Reprod. Sci., 116(3-4): 386-391. [Crossref] [PubMed]

31. Deori, S., Deka, B., Biswas, R., Nahardeka, N., Arangasamy, A., Bhuyan, D., Kalita, D., Borah, R. and Phookan, A. (2018) Characteristics and freezability of Assam Hill goat semen. Indian J. Anim. Res., 52(1): 25-28. [Crossref]

32. Karunakaran, M., Ratnakaran, U., Naik, P.K. and Chakurkar, E.B. (2016) Electrophoretic profile of boar seminal proteins. J. Appl. Anim. Res., 44(1): 403-405. [Crossref]

33. Barrios, B., Fernandez, J.M., Muino, B.T. and Cebrian, P.J.A. (2005) Immuno-cytochemical localization and biochemical characterization of two seminal plasma proteins that protect ram spermatozoa against cold shock. J. Androl., 26(4): 539-549. [Crossref] [PubMed]

34. Villemure, M., Lazure, C. and Manjunath, P. (2003) Isolation and characterization of gelatin-binding proteins from goat seminal plasma. Reprod. Biol. Endocrinol., 1: 39-45. [Crossref] [PubMed] [PMC]

35. Killian, G.J., Chapman, D.A. and Rogowski, L.A. (1993) Fertility associated proteins in Holstein bull seminal plasma. Biol. Reprod., 49(6): 1202-1207. [Crossref]

36. Cancel, A.M., Chapman, D.A. and Killian, G.J. (1997) Osteopontin is the 55-kilodalton fertility-associated protein in Holstein bull seminal plasma. Biol. Reprod., 57(6): 1293-1301. [Crossref]

37. Gerena, R.L., Irikura, D., Urade, Y., Eguchi, N., Chapman, D.A. and Killian, G.J. (1998) Identification of a fertility-associated protein in bull seminal plasma as lipocalin-type prostaglandin D synthase. Biol. Reprod., 58(3): 826-833. [Crossref]

38. Bellin, M.E., Oyarzo, J.N., Hawkins, H.E., Zhang, H., Smith, R.G., Forrest, D.W., Sprott, L.R. and Ax, R.L. (1998) Fertility-associated antigen on bull sperm indicates fertility potential. J. Anim. Sci., 76(8): 2032-2039. [Crossref]

39. Sprott, L.R., Harris, M.D., Forrest, D.W., Young, J.H., Zhang, M., Oyarzo, J.N., Bellin, M.E. and Ax, R.L. (2000) Artificial insemination outcomes in beef females using bovine sperm with a detectable fertility-associated antigen. J. Anim. Sci., 78(4): 795-798. [Crossref]