Effects of cooling time and thaw rate on membrane integrity and motility of frozen-thawed canine spermatozoa using commercial semen extenders.

Abstract

Cryopreservation of bovine spermatozoa has been successful for many years. More recently, cryopreservation of canine spermatozoa has been requested. Protocols using different extenders, different freeze rates and different thaw rates have been empirically derived over the years. The objective of this study was to compare the current laboratory protocol with two other protocols, one using different cooling times and a different extender and one using different thaw temperatures and a different extender [1]. A single ejaculate was collected from 11 mature dogs of different breeds. Each ejaculate was prepared for cryopreservation with two different commercial semen extenders, a human extender (Irvine Scientific, Santa Ana, CA, 92705-5588)(HEYE) or a canine extender (3560 Pine Grove, Unit 227, Port Huron, MI 48060)(CEYE). For each extender, two different cooling times were used, 30 (30m) and 60 (60m) minutes, before adding the corresponding extender containing 12% glycol (resulting in a final glycerol concentration of 6%). Each of the four aliquots were loaded into 0.5 mL straws, placed on a boat 4 cm over liquid nitrogen for 10 minutes and then plunged. For each aliquot, two different thawing protocols were used, one 50oC for 10 sec (50o) and one 37oC for 30 sec (37o). Five minutes after thawing, each sample was assessed for 13 different motility parameters using a computer assisted sperm analyzer (SpermVision, Minitüb, 419 Venture Court, Verona, WI 53593, USA) and for membrane integrity using SYBR-14/PI (LIVE/DEAD® Sperm Viability Kit, by Invitrogen™, 5791 Van Allen Way Carlsbad, CA 92008 USA). Only membrane integrity, progressive and total motility data are presented. Results showed that changing the extender, the cool down protocol or the thawing protocol offered no advantage over the current employed laboratory protocol.