Dilution of spermatozoa results in improved viability following a 24 h storage period but decreased acrosome integrity following cryopreservation

Abstract

This study was designed to investigate the physiological factors affecting the reduced viability of cryopreserved spermatozoa following dilution. Ninety-six ejaculates were collected from 13 bulls and diluted to 10 × 10 6 and 60 × 10 6 sperm/ml in a commercial long term extender (Eqcellsire ®; IMV) and in an egg yolk extender. Samples diluted in the egg yolk extender were frozen in 0.25 ml straws. Samples diluted in the Eqcellsire were stored at room temperature for 24 h and assessed for sperm cell viability using SYBR14 and PI (Molecular Probes) and osmotic resistance. Frozen samples were thawed and assessed for viability, osmotic resistance and acrosome intergrity. Acrosome integrity was measured using Mitotracker, PI and PNA-FITC (Molecular Probes). Spermatozoa diluted to 10 × 10 6 sperm/ml and stored at ambient temperature had a higher proportion of viable spermatozoa ( P < 0.01) and were less susceptible to osmotic stress ( P < 0.01) than sperm diluted to 60 × 10 6 sperm/ml. Following cryopreservation there was no concentration-related difference in the proportion of viable spermatozoa and their relative susceptibility to osmotic stress. Spermatozoa diluted to lower cell concentrations had a higher proportion of viable cells that were acrosome reacted ( P < 0.001). It is suggested that the higher proportion of acrosome reacted cells may result from an increased proportion of cells in a capacitated-like state in the spermatozoa diluted to lower concentrations. A Spearmans ranked correlation demonstrates a relationship between individual bull spermatozoa following dilution or cryopreservation for viability ( r 2 = 0.98; P < 0.001) or osmotic resistance ( r 2 = 0.87; P < 0.001) suggesting a variation in these characteristics between bulls.