EFFECT OF DIFFERENT CRYOPROTECTIVE AGENTS ON SKIM MILK AND DIMITROPOULUS EXTENDER FOR STALLION SEMEN CRYOPRESERVATION

Abstract

Cryoprotective agents (CPAs) protect sperm during cryopreservation. The objective of this study was to assess different CPAs on stallion semen cryopreservation. Skim milk (SM) and Dimitropoulos (DV) were the extenders used in this study; each was added by glycerol (Gly), combination of ethylene glycol-glycerol (EG+Gly) or dimethilformamide (DMF). Each semen sample was evaluated and divided equally into six tubes; semen in the three tubes was diluted 1:1 with (SM), while in the remaining tubes the semen was diluted 1:1 by DV. After being diluted, all tubes were centrifuged at 1006xg for 10 minutes. The supernatan discarded, the pellet was rediluted by SM trehalosa or DV trehalose, and added by G, EG+Gly, or DMF to reach the final sperm concentration of 200x106/ml. The extended semen was individually packed in 0.3 ml minitube, equilibrated at 4oC for 2 hours, frozen in liquid nitrogen vapor for 10 minutes, and then was stored in liquid nitrogen container at -196 oC. After 24 hours, the semen was thawed at 37 oC for 30 second. There were no significantly different (p>0.05) on the percentages of motile and viable sperm in SMT (21.7% and 43.4%, respectively) compared with those extended with DV T extender (26.9% and 50.8%, respectively). DMF demonstrated better results as CPA compared to the others; and DVTDMF combination had the best protection during cryopreservation in this study. Keywords: cryopreservation, stallion sperm, sugar, cryoprotective agents