Effect of lactose, skim milk and Tris diluents on frozen buffalo spermatozoa

Subject Areas : Veterinary Clinical Pathology

Ali  Rastegarnia ¹ (Department of Clinical Sciences, Faculty of Veterinary Medicine, Islamic Azad University of Urmia, Urmia, Iran)
وحید  Shafepour ² (Buffalo Breeding and Extension Training Center, Urmia, Urmia, Iran)

Received: 2007-04-23 Accepted : 2007-11-11 Published : 2007-08-23

Keywords: Buffalo, Spermatozoa, Extender,

Abstract :

The composition of the extender in which semen is diluted before freezing plays a major role in successful cryopreservation of spermatozoa. This study was carried out to identify the suitable buffer for cryopreservation of buffalo semen. Sixteen split pooled ejaculates from two buffalo bulls possessing more than 70% visual sperm motility, were diluted at 370c either in lactose, skim milk or Tris extenders. The diluted semen was cooled to 40c within 2 hours, equilibrated at 40c for 4-6 hours following the addition of glycerol, filled in 0.5 ml French straws and frozen in a programmable cell freezer before plunging into liquid nitrogen. Semen was thawed at 370c for 30 seconds after 48 hours of storage inside liquid nitrogen. Post thaw visual sperm motility, plasma membrane integrity and acrosome morphology of each semen sample were assessed by warm plate microscopy at 370c, hypo-osmotic swelling test (HOST) and giemsa staining, respectively. Analysis of variance revelated that percentage of post thaw visual sperm motility (Mean± standard deviation) tended to be higher in Tris (50±3.6) than skim milk (44.5±2.5) and lower in lactose (24.4±10.5) extenders (P<0.01). The percentage of spermatozoa with normal plasma membrane and intact acrosome averaged 41.4±1.2 , 32.6±3.8 , 24.0±9.4 and 54.0±3.1 ,50.4±4.1, 27.3±12.2 for Tris, skim milk and lactose diluents, respectively (P<0.01). In conclusion, the result of this study indicated that the use of Tris based buffering system is suitable and recommendable for cryopreservation of buffalo spermatozoa in comparison to skim milk or lactose diluents.  

References:

1. Azam, M., Anzar, M. and Arslan, M. (1988): Assessment of post-thaw semen quality of buffalo and sahiwal bulls using new semen assay. Pakistan Vet.J.18 (2), pp: 74-80.

1. Baruselli, P. S. (1994): Basic requirement for artificial insemination & embryo transfer in buffaloes. Buffalo. J. Suppl. 2, pp: 53-60.

3. Dhami, A.J., and Kodagali, S.B. (1990): Freezability, enzyme leakage and fertility of buffalo spermatozoa in relation to the quality of semen ejaculates and extenders. Theriogenology. 34 (5): 853-863.


4. Dhami, A., Mohan, G. and Sahni, K.L. (1993): Effect of extender and additives on preservability of cattle and buffalo semen at 5⁰C and -196⁰C. Indian Journal of Animal Sciences. 63(5): 492-498.


5. Dhami, A.J., Sahni, K.L., Mohan,G. and Jani,V.R. (1996): Effect of differences variables on frezeability, post-thaw longevity and fertility of buffalo spermatozoa in the tropics. Theriogenology. (10), pp: 109-120.


6. Gokhale, S.B., Joshi, P.H., Mushtaque, M. and  Mokashi, S.P. (2002): Studies on the effect of hydrogen ion concentration (PH) of extender of semen characters of surti buffalo bulls. Buffalo-bulletin. 21 (2): 29-34.


7. Jeyendran, R.S., Van-dan-ven, H.H., Perez-Pelaez, M., Crabo, B.G. and Zeneveld, L.J.D. (1984): Development an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J. Reprod.Fert. 70: 219-228.


8. Kaker, S.S. and Anand, S.R. (1981): Changes in adenosine 5´- triphosphate adeneylate energy changes and adenosine 3´5´- cyclic monophosphate during the freezing of buffalo semen. J.Reprod.Fertil. 62, 543-548.


9. Khan, I.Q., Ahmad, K., Akhtar, N. and Khan, A. (1988): Effect of cryopreservation on the post-thaw surviveability of buffalo bull spermatozoa without seminal plasma. Pkistan.Vet. J. 18 (4), pp: 177-179.


10. Kolev, S.V. and Dimov, K. (1999): Freezing ability of sperm from young buffalo and cattle bulls. Bulgarian- journal-of- Agriculture- Science. 5(3): 497-501.


11. Kumar, S., Sahni, K.L. and Mohan, G. (1993-a) Effect of different extender formulation on acrosomal maintenance of buffalo spermatozoa frozen in milk, Tris and sodium citrate dilutors. Indian Journal of Animal Sciences. 63 (12): 1233-1239.


12. Kumar, S., Sahni, K.L. and Mohan, G. (1993-b) Freezing of buffalo semen in milk. Tris and sodium citrate dilutors with different levels of yolk and glycerol in relation to PH of dilutors. Indian Journal of Animal Sciences. 63(5): 499-504.


13. Medeiros, C.M.O., Forell, F., Oliveira, A.T.D. and Rodrigues, J.L. (2002): Current status of sperm cryopreservation: Why isn’t it better. Theriogenology. 57: 327-344.


14. Pampapathi, J., Mohan, G., Kumar, S. and Sahni. K.L. (1997): Effect of dilution rate and yolk levels on preservability of Murrah buffalo semen. Indian-journal of dairy science. 50(5): 352-358.


15. Rasul, Z., Anzar, M., Jalali, S. and Ahmad, N. (2000): Effect of buffering systems on post-thaw motion characteristics, plasma membrane integrity, and acrosome morphology of buffalo spermatozoa. Animal reproduction science. 59, 31-41.


16. Rasul, Z., Ahmad, N. and Anzar, M. (2001): Changes in motion characteristics, plasma membrane integrity, and acrosome morphology during cryopreservation of buffalo spermatozoa. Journal of Andrology. 22(2):278-283.


17. Sukhato, P., Thongsodseang, S., Utha, A. and Songsasen, N. (2001): Effect of cooling and warming conditions on post-thawed motility and fertility of cryoprederved buffalo spermatozoa. Animal Reproduction Science. (67), pp: 69-77.


18. Watson, P.F. (1975): Use of giemsa stain to detect change in the acrosome of frozen ram spermatozoa. Veterinary Record. 97: 12-15.