EFFECT OF EQUILIBRATION PROCESS ON CANINE SPERMATOZOA AFTER VITRIFICATION USING COCONUT WATER EXTENDER WITH ADDITION OF SOY LECITHIN AND SUCROSE AS NONPERMEABLE CRYOPROTECTANTS

Abstract

The aim of the present study was to evaluate the effect of equilibration process on canine sperms after vitrification using coconut water extender with addition of soy lecithin and sucrose as nonpermeable cryoprotectants. Twelve ejaculates were collected separately by digital manipulation from 12 adult dogs. Only the second fraction of the ejaculate was used in this study, which was evaluated about volume, concetration, viability, total and progressive motility, kinetic parameters and morphology. After evaluation, semen was diluted with a coconut water extender (50% coconut water (v/v), 25% (v/v) distilled water and 25% (v/v) 5% anhydrous monosodium citrate solution) with addition of soy lecithin and fructose at 1% (v/v) and 0.25 M sucrose until final concentration of 100x106 spermatozoa/ml. Samples were divided into three aliquots and each of them was processed at different regimens: without equilibration, 5ºC for 30 minutes and 5ºC for 60 minutes and then vitrified by dropping 30 μl of sperm suspension directly into liquid nitrogen. Sperm pellets were devitrified at least one week later as three of them were dropped into 0.3 mL of CaniPlus AI (Minitüb, Germany), which was previously warmed in a water bath at 37ºC for 2 minutes. Sperm concentration and motility parameters were assayed using a computer-aided sperm analysis (CASA) system, viability-by supravital staining technique and morphology parameters were evaluated in Haemacolor® stained semen samples. In conclusion, our results demonstrate that when vitrification and coconut water extender with addition of 1% soy lecithin and 0.25 M sucrose as cryoprotectants were used, presence of equilibration time of 60 minutes returned the best canine sperm quality results.