100 A COMPARISON OF GLYCEROL AND EGTA FOR ULTRARAPID FREEZING OF BULL EPIDIDYMAL SPERM

Abstract

The ultrarapid freezing technique was developed as an alternative to slow conventional freezing to avoid formation of ice crystals (Tucker MJ and Liebermann J 2007, Vitrification in Assisted Reproduction, 87-92). The recent use of ethylene glycol tetraacetic acid (EGTA) instead of glycerol as a cryoprotectant for sperm is a result of efforts to reduce osmotic and cytotoxic effects. Accordingly, the objective of the present study was to compare the cryoprotective effects of EGTA v. glycerol on bovine sperm frozen using an ultrarapid method. A pool of epididymal sperm collected from 5 bulls was maintained in Botusui extender (Biotech, Botucatu, Brazil) containing 5% BSA, either EGTA (5%) or glycerol (5%), and 0 or 2 mg mL of polyvinyl alcohol (PVA). All samples were loaded into 0.25-mL French straws and either placed into a freezing machine (TK 4000 compacta, TK equipamentos para reproducao, Uberaba, Brazil) and cooled at a rate of 50°C/min, or exposed to liquid nitrogen (LN2) vapor for 5 to 15 min before plunging into LN2. After overnight storage in LN2, straws were thawed and examined using CASA (HTM IVOS 12, Hamilton Thorne Research, Beverly, MA, USA) for total motility (TM), progressive motility (PM), path velocity (VAP), progressive velocity (VSL), and track speed (VCL). Acridine Orange (AO) was used to check DNA integrity of sperm cells (Chirinea VH et al. 2006 Ciencia Animal Brasileira 7, 407-415). Our results indicate that, despite the potentially damaging effects of the extreme temperature changes, epididymal sperm showed 95% of DNA integrity and, therefore, should be able to participate in the fertilization process. Addition of PVA had a negative effect on sperm motility characteristics. CASA revealed that glycerol was a more suitable cryoprotectant for ultrarapid freezing of bull epididymal spermatozoa than was EGTA (see Table 1). Table 1. CAPES.