Ram epididymal sperm frozen in an extender containing ethylene glycol have higher post‐thaw longevity and in vitro fertility

Abstract

Background: Establishing an efficient, simple and inexpensive method for freezing ram epididymal sperm so that the quality and fertility of spermatozoa could be maintained for a longer period after thawing is of great practical value. Objectives:To optimize freezing and thawing protocol for ram epididymal sperm using either ethylene glycol (EG) or glycerol (GLY) as cryoprotectants (CPAs). Then, to evaluate the post-thaw longevity and in vitro fertility of spermatozoa that were frozen and thawed according to the optimized protocol. Materials and methods:At first, an optimum protocol for freezing and thawing sperm using EG or GLY were investigated, and the next experiments were performed using the spermatozoa that had been frozen and thawed according to the optimized protocol for each CPA. In the next experiments, frozen-thawed and fresh sperm were diluted in an isotonic culture medium and subsequently incubated at 39 • C for 4 h. The motility characteristics and functional membrane integrity (FMI) of spermatozoa were evaluated after thawing, after dilution (t 0 ), and after incubation (t 4 ). The in vitro fertility of the spermatozoa was assessed at t 0 and t 4 .Results: For both CPAs, the highest motility parameters and FMI was found for spermatozoa frozen at 3 cm above LN2 and thawed at 50 and 65 • C (P < 0.05). In comparison to the spermatozoa of GLY group, the spermatozoa of the EG group had higher total and progressive motility at t 0 , as well as higher FMI, total and progressive motility, and linearity at t 4 (P < 0.05). Fertility of frozen-thawed sperm was higher than that of fresh sperm at t 0 (P < 0.05). Incubation treatment increased the fertility of fresh sperm while decreased the fertility of frozen-thawed sperm, and this decline was more severe in GLY than in the EG group. Conclusion:Based on the findings, EG can be a more suitable CPA for freezing ram epididymal sperm.