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

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 +5degreesC to -5degreesC and (ii) from -5degreesC to -50degreesC. Fluorescent probes (SYBR14 and propidium iodide) were used to monitor plasma membrane integrity during the entire process. Cooling rates in the range 3degreesC min(-1) to 12degreesC min(-1) did not cause significant damage to the sperm plasma membrane between +5degreesC and -5degreesC; however, spermatozoa cooled at 24degreesC min(-1) to -5degreesC were slightly damaged. Motility was not particularly sensitive to variations in cooling rate. Cooling rates in the range 15degreesC min(-1) to 60degreesC min(-1) did not produce differences in sperm cryosurvival during freezing between -5degreesC and -50degreesC, or after thawing. In addition, cooling rates in the range 3degreesC min(-1) to 80degreesC min(-1) did not produce significant differences in sperm cryosurvival. However, slow freezing 3degreesC min(-1)) induced a slight increase in the percentage of plasma membrane-damaged spermatozoa (propidium iodide-positive) at -50degreesC. Inter-ejaculate and inter-boar differences in sperm cryosurvival were manifested 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.