Importance of cooling rate and animal variability for boar sperm cryopreservation: Insights from the cryomicroscope

Abstract

A series of experiments was set up to investigate the effect of different cooling rates on boar sperm cryosurvival using cryomicroscopy. The cooling protocols were split into two stages: (i) from +5°C to -5°C and (ii) from -5°C to -50°C. Fluorescent probes (SYBR14 and propidium iodide) were used to monitor plasma membrane integrity during the entire process. Cooling rates in the range 3°C min-1 to 12°C min-1 did not cause significant damage to the sperm plasma membrane between +5°C and -5°C; however, spermatozoa cooled at 24°C min-1 to -5°C were slightly damaged. Motility was not particularly sensitive to variations in cooling rate. Cooling rates in the range 15°C min-1 to 60°C min-1 did not produce differences in sperm cryosurvival during freezing between -5°C and -50°C, or after thawing. In addition, cooling rates in the range 3°C min-1 to 80°C min-1 did not produce significant differences in sperm cryosurvival. However, slow freezing (3°C min-1) induced a slight increase in the percentage of plasma membrane-damaged spermatozoa (propidium iodide-positive) at -50°C. Inter-ejaculate and inter-boar differences in sperm cryosurvival were manisfested independently of cooling rate. The sperm plasma membrane remained intact (SYBR14-positive) during cooling and freezing, but upon rewarming, the plasma membrane of a high proportion of spermatozoa was damaged (propidium iodide-positive), indicating that rewarming is a critical step of the freezing-thawing process.