Background

: Establishing an efficient, simple and inexpensive method for freezing ram epididymal sperm so that the quality and fertility of sperm 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 sperm 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 sperm 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 hours. The motility characteristics and functional membrane integrity (FMI) of sperm were evaluated after thawing, after dilution (t0), and after incubation (t4). The in vitro fertility of the sperm was assessed at t0 and t4.

Results

: For both CPAs, the highest motility parameters and FMI was found for sperm frozen at 3 cm above LN2 and thawed at 50 and 65°C (P<0.05). In comparison to the sperm of GLY group, sperm of EG group had higher total and progressive motility at t0, as well as higher FMI, total and progressive motility, and linearity at t4(P<0.05). Fertility of frozen-thawed sperm was higher than that of fresh sperm at t0(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 EG group.

Conclusion

: Based on the findings, EG can be a more suitable CPA for freezing ram epididymal sperm.

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