Effect of temperature and incubation time on the evaluation of frozen ram semen membrane integrity

Abstract

The integrity of the sperm plasma membrane is an essential requirement for spermatozoa function and especially for fertilisation. Acrosome and plasma membrane of ram spermatozoa are the structures more damaged by freeze-thaw process. The aim of this study was to determine the importance of time and temperature of incubation, in the evaluation of membrane damage with the fluorophore probes Calcein AM, a membrane-permeant cytoplasmatic esterase marker and Ethidium homodimer, a membrane-impermeant DNA marker (CAM/EthD-1). This tecnique has the advantage of enabling estimation of the proportion of spermatozoa with intact plasma lemmae as well as the proportion exhibiting motility. The results obtained show that high temperature increased spermatozoa damage meanwhile the longer the incubation time as greater the lost of motility and plasma membrane damage.