Abstract
This study tested the hypothesis that sperm membrane changes during cooling contribute substantially to the membrane damage observed after cryopreservation of boar spermatozoa. Flow cytometry was used to assess viability (percentages of live and dead cells) of boar sperm cells after staining with SYBR-14 and propidium iodide (PI) and acrosome status after staining with FITC-pisum sativum agglutenin and PI. Incubation (38 °C, 4 h), cooling (to 15 or 5 °C) and freezing reduced the proportion of live spermatozoa compared with those in fresh semen. There were more membrane changes in spermatozoa cooled to 5 °C than to 15 °C. The proportion of live spermatozoa decreased during processing for cryopreservation and cooling to 5 °C, but was unaffected by freezing and thawing if held at 15 °C for 3.5 h during cooling. Spermatozoa not held during cooling exhibited further loss of viability after freezing and thawing. Holding the spermatozoa also increased the proportion of acrosome-intact spermatozoa at both 15 °C and 5 °C and at thawing compared with that of the unheld controls. The results of this study sugget that a substantial proportion of the membrane changes associated with cryopreservation of boar spermatozoa may be attributed to the cooling of the cells to 5 °C rather than to the freezing and thawing process, and that sperm membrane changes are reduced when semen is held at 15 °C during cooling.
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