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Research (Published online: 02-05-2016)

1. Minimization of apoptosis-like changes in cryopreserved buffalo bull sperm by supplementing extender with Bcl-2 protein - Jasmer Dalal, Ajeet Kumar, Mrigank Honparkhe, Dipak Deka and Narinder Singh

Veterinary World, 9(5): 432-436

 

 

   doi: 10.14202/vetworld.2016.432-436

 

 

Jasmer Dalal: Department of Veterinary Gynaecology and Obstetrics, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India; jasmer.sl.dalal@gmail.com

Ajeet Kumar: Department of Veterinary Gynaecology and Obstetrics, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India; ajeetvet@yahoo.com

Mrigank Honparkhe: Department of Veterinary Gynaecology and Obstetrics, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India; honparkhem@rediffmail.com

Dipak Deka: School of Animal Biotechnology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India; dekadipak@rediffmail.com

Narinder Singh: Department of Veterinary Gynaecology and Obstetrics, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India; narinder2002us@yahoo.com

 

Received: 28-01-2016, Accepted: 16-04-2016, Published online: 02-05-2016

 

Corresponding author: Ajeet Kumar, e-mail: ajeetvet@yahoo.com


Citation: Dalal J, Kumar A, Honparkhe M, Deka D, Singh N (2016) Minimization of apoptosis-like changes in cryopreserved buffalo bull sperm by supplementing extender with Bcl-2 protein, Veterinary World, 9(5): 432-436.



Aim: This study was aimed at evaluating the anti-apoptotic effects of Bcl-2 protein in cryopreserved buffalo bull sperm.

Materials and Methods: A total 10 ejaculates from two buffalo bulls (5 each) were collected using artificial vagina method, and semen was evaluated using a standard protocol. Semen was extended by Tris egg yolk extender supplemented with Bcl-2 protein at 5, 10, and 15 μM. Semen was cryopreserved at ultra-low temperature using traditional vapor freezing method. Prefreeze and post-thaw semen samples were evaluated for percent motility, viability, hypo-osmotic swelling test (HOST) reactive sperms; status of mitochondrial membrane activity and status of sperm phospholipase A1 and phospholipase A2 activity.

Results: There were no significant effects of Bcl-2 protein supplementation on pre-freeze sperm quality. Percent motility and active mitochondria in post-thaw Bcl-2 supplemented and control groups were also similar. However, viable sperms were significantly (p<0.05) higher (74.294.23%) in Bcl-2 supplemented group (5 μM) as compared to control (51.65.77%). The proportion of HOST reactive sperms was also higher (63.16.73%) in Bcl-2 supplemented (5 μM) group as compared to control (50.76.98%). The sperm with low PLA activity (non-apoptotic) was significantly (p<0.05) higher in all the supplemented doses of Bcl-2 protein, i.e., at 5 μM (73.425.79%), 10 μM (75.516.22%), and 15 μM (74.785.89%) as compared to control (60.234.45%). We found that Bcl-2 protein supplementation at 5 μM dose improved the postthaw semen quality indicated by higher viability, HOST reactive sperms, and sperm with low PLA activity (non-apoptotic sperms).

Conclusion: Bcl-2 protein supplementation exerts its protective effect on spermatozoa against apoptosis-like changes developed during cryopreservation.

Keywords: anti-apoptotic effects, Bcl-2 protein, buffalo bull, semen cryopreservation.



1. Chaveiro, A., Machado, L., Frijters, A., Engel, B. and Woelders, H. (2006) Improvement of parameters of freezing medium and freezing protocol for bull sperm using two osmotic supports. Theriogenology, 65(9): 1875-1890.
http://dx.doi.org/10.1016/j.theriogenology.2005.10.017
PMid:16310842
 
2. Aitken, R.J. and Baker, M.A. (2013) Causes and consequences of apoptosis in spermatozoa; Contributions to infertility and impacts on development. Int. J. Dev. Biol., 57(2-4): 265-272.
http://dx.doi.org/10.1387/ijdb.130146ja
PMid:23784837
 
3. Aitken, R.J. and Koppers, A.J. (2011) Apoptosis and DNA damage in human spermatozoa. Asian J. Androl., 13: 36-42.
http://dx.doi.org/10.1038/aja.2010.68
PMid:20802502 PMCid:PMC3739394
 
4. Martin, G., Cagnon, N. and Sabido, O. (2007) Kinetics of occurrence of some features of apoptosis during the cryopreservation process of bovine spermatozoa. Hum. Reprod., 22: 380-388.
http://dx.doi.org/10.1093/humrep/del399
PMid:17092986
 
5. Dogan, S., Mason, M.C., Govindaraju, A., Belser, L., Kaya, A., Stokes, J., Rowe, D. and Memili, E. (2013) Interrelationships between apoptosis and fertility in bull sperm. J. Reprod. Dev., 59: 18-26.
http://dx.doi.org/10.1262/jrd.2012-068
PMid:22986927 PMCid:PMC3943226
 
6. Korsmeyer, S.J., Knudson, C.M., Tung, K.S., Tourtellotte, W.G. and Brown, G.A. (1995) Bax-deficient micewith lymphoid hyperplasia and male germ cell death. Science, 270: 96-99.
http://dx.doi.org/10.1126/science.270.5233.96
PMid:7569956
 
7. Korsmeyer, S.J., MacGregor, G.R., Ross, A.J., Richardson, L.L., Mahar, P.L., Waymire, K.G., Knudson, C.M., Thompson, C.B., Handel, M.A. and Russell, L.D. (1999) Analysis of Bcl2 family function in spermatogenesis. In: Goldberg, E., editor. XVth Testis Workshop. Serono Symposia USA, Inc., Louisville, Kentucky. p55.
 
8. Nasimi, P. and Roohi, S. (2012) Cell Death in Animals and Plants; Apoptosis, Necrosis & Autophagy. Islamic Azad University, Masjed Soleyman Press, Masjed Soleyman.
 
9. Simon, L., Lutton, D., McManus, J. and Lewis, S.E. (2011) Sperm DNA damage measured by the alkaline Comet assay as an independent predictor of male infertility and in vitro fertilization success. Fertil. Steril., 95: 652-657.
http://dx.doi.org/10.1016/j.fertnstert.2010.08.019
PMid:20864101
 
10. Aitken, R.J., Findlay, J.K., Hutt, K.J. and Kerr, J.B. (2011) Apoptosis in the germ line. Reproduction, 141: 139-150.
http://dx.doi.org/10.1530/REP-10-0232
PMid:21148286
 
11. Anzar, M., Erickson, L. and Kroetsch, T. (2015) Relationship between sperm apoptosis and bull fertility: In vivo and in vitro studies. Reprod. Fertil. Dev., 2: 58.
 
12. Belka, C. and Budach, W. (2002) Anti-apoptotic Bcl-2 proteins: structure, function and relevance for radiation biology. Int. J. Radiat. Biol., 78(8): 643-658.
http://dx.doi.org/10.1080/09553000210137680
PMid:12194748
 
13. Kontos, C.K., Christodoulou, M.L. and Scorilas, A. (2013) Apoptosis-related BCL2-family members: Key players in chemotherapy. Anticancer Agents Med. Chem., 13: 1-22.
 
14. Blom, E. (1977) The evaluation of bull semen with special reference to its use in artificial insemination. Anim. Breed. Abst., 19: 648.
 
15. Jeyendran, R.S., Vander-Ven, H.H., Perez-Pelaez, M., Crabo, B.G. and Zanevld, L.J.D. (1984) Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characters. J. Reprod. Fertil., 70: 219-228.
http://dx.doi.org/10.1530/jrf.0.0700219
PMid:6694140
 
16. Oldereid, N.N., Angelis, P.D., Wiger, R. and Clausen, O.P. (2001) Expression of Bcl-2 family proteins and spontaneous apoptosis in normal human testis. Mol. Hum. Reprod., 7(5): 403-408.
http://dx.doi.org/10.1093/molehr/7.5.403
 
17. Deming, P.B., Schafer, Z.T., Tashker, J.S., Potts, M.B., Deshmukh, M. and Kornbluth, S. (2004) Bcr Abl-mediated protection from apoptosis downstream of mitochondrial cytochrome c release. Mol. Cell. Biol., 23: 10289-10299.
http://dx.doi.org/10.1128/MCB.24.23.10289-10299.2004
PMid:15542838 PMCid:PMC529043
 
18. Llambi, F., Moldoveanu, T., Tait, S.W., Bouchier-Hayes, L., Temirov, J., McCormick, L.L., Dillon, C.P. and Green, D.R. (2011) A unified model of mammalian BCL-2 protein family interactions at the mitochondria. Mol. Cell., 44(4): 517-531.
http://dx.doi.org/10.1016/j.molcel.2011.10.001
PMid:22036586 PMCid:PMC3221787
 
19. Reed, J.C. (1997) Double identity for proteins of the Bcl-2 family. Nature, 387: 773-776.
http://dx.doi.org/10.1038/42867
PMid:9194558
 
20. Strasser, A., Cory, S. and Adams, J.M. (2011) Deciphering the rules of programmed cell death to improve therapy of cancer and other diseases. EMBO J., 30(18): 3667-3683.
http://dx.doi.org/10.1038/emboj.2011.307
PMid:21863020 PMCid:PMC3173800
 
21. Garcia-Saez, A.J. (2012) The secrets of the Bcl-2 family. Cell Death Differ., 19(11): 1733-1740.
http://dx.doi.org/10.1038/cdd.2012.105
PMid:22935609 PMCid:PMC3469065
 
22. Vines, C.M. and Bill, C.A. (2015) Phospholipases. In: Encyclopedia of Life Sciences. Chichester: John Wiley & Sons Ltd., p1-9.
http://dx.doi.org/10.1002/9780470015902.a0001394.pub3
 
23. Robertson, L., Bailey, J.L. and Buhr, M.M. (1990) Effects of cold shock and phospholipase A2 upon intact boar spermatozoa and sperm head plasma membranes. Mol. Reprod. Dev., 26: 143-149.
http://dx.doi.org/10.1002/mrd.1080260208
PMid:2372397
 
24. Shier, W.T. (1979) Activation of high levels of endogenous phospholipase A2 in cultured cells. Proc. Natl. Acad. Sci. USA.,76: 195-199.
http://dx.doi.org/10.1073/pnas.76.1.195
 
25. Farkas, D.L., Febbroriello, W.P., Carson, J.H. and Loew, L.M. (1989) Simultaneous imaging of cell and mitochondrial membrane potentials. Biophys. J., 56: 1053-1069.
http://dx.doi.org/10.1016/S0006-3495(89)82754-7
 
26. Glander, H.J., Schiller, J., Suss, R., Paasch, U., Grunewald, S. and Arnhold, J. (2002) Deterioration of spermatozoal plasma membrane is associated with an increase of sperm lyso-phosphatidylcholines. Andrologia, 34(6): 360-366.
http://dx.doi.org/10.1046/j.1439-0272.2002.00508.x
 
27. Tavalaee, M., Deemeh, M.R., Arbabian, M., Kiyani, A. and Nasr-Esfahani, M.H. (2014) Relationship between fertilization rate and early apoptosis in sperm population of infertile individuals. Andrologia, 46: 36-41.
http://dx.doi.org/10.1111/and.12038
PMid:23134226