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The effect of collection temperature, cooling rate and warming rate on chilling injury and cryopreservation of mouse spermatozoa

The experiments presented here identify several factors that affect survival (motility) of cryopreserved mouse spermatozoa after freezing and thawing. Among these factors are: (i) the temperature at which spermatozoa are collected, (ii) the cooling rate to 0°C and (iii) the warming rate from –196°C to ambient. When excised epididymides were cooled to near 0° (1–4°C) and spermatozoa collected and mixed with cryoprotectant at that temperature, motilities after subsequent freezing and thawing were 8–10 times higher than when the spermatozoa were collected from the epididymides at 22°C. In addition, the survival rates of spermatozoa warmed at rates ranging from 150 to 2000°C min^–1 were about five times higher than those in suspensions warmed at about 7500°C min^–1. The combination of a low collection temperature and the lower warming rates resulted in approximately 50% motility relative to unfrozen controls. Motility was reduced to 6–8% when the collection temperature was 22°C, and to approximately 10% when frozen suspensions of spermatozoa collected in the cold were rapidly warmed from –196°C. When spermatozoa collected at 22°C were abruptly cooled to 0°C, 40–80% of the cells suffered an irreversible loss of motility after warming. In contrast, when spermatozoa were cooled to 0°C at 1°C min^–1 and warmed (either rapidly or slowly), motilities were similar to those of uncooled controls (75–90%). These findings indicate sensitivity to cold shock. Finally, the addition of raffinose or sucrose to the suspending medium did not affect the survival of the spermatozoa cooled slowly, but it did increase the survival of spermatozoa that were rapidly cooled to 0°C (55–60% versus 25–30%).

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