Adjustments on the cryopreservation conditions reduce the incidence of boar ejaculates with poor sperm freezability

The objective of the present study was to evaluate the effectiveness of different cryopreservation conditions (CCs) for freezing and thawing boar ejaculates, focusing on those having sub-optimal sperm freezability. Using a split-ejaculate technique, single ejaculates from 53 boars were diluted in lactose-egg yolk extender, containing a final glycerol concentration (GLY) of 2 or 3%, packaged in 0.5 mL straws and were cooled at rates of -10, -40 or -60 °C/min (cooling rate: CR). Thereafter, the frozen sperm samples were thawed by warming them at rates of approximately 1200 or approximately 1800 °C/min (warming rate: WR). Frozen-thawed sperm samples were assessed for the sperm motility (CASA system) and flow cytometric analysis of plasma and acrosomal membranes integrity. Cooling rate had no influence (P > 0.05) on sperm quality parameters, however GLY and WR independently affected (P < 0.05) all assessed sperm parameters. Evaluating the combined effect of GLY and WR (four different CCs resulting of a 2 x 2 factorial design), the best post-thaw quality results were achieved for sperm samples frozen with 3% glycerol and thawed at 1800 °C/min (CC4). However, there was a significant interaction (P < 0.001) between CC and ejaculate for all post-thaw sperm quality assessments. Therefore, ejaculates were classified in three different populations according to the post-thaw sperm quality achieved using control CC (CC1: 2% of glycerol and 1200 °C/min of warming). The effectiveness of CCs was different (P < 0.05) in the three ejaculate populations. Spermatozoa from ejaculates considered as “good” freezers were relatively unaffected (P > 0.05) by the modifications in the CCs, whereas those from “moderate” and, mainly, “bad” freezers were very sensitive (P < 0.05). In conclusion, optimization of the CCs -- GLY and WR -- can improve the cryosurvival of spermatozoa in some ejaculates, particularly in those having poor sperm freezing ability.