Cold-induced damage in boar sperm cooled to 5°C in Lactose Egg Yolk (LEY) medium with different Glycerol concentrations

Boar sperm is highly susceptible to cold damage. When temperature drops to 5 degrees C, the plasmatic membrane is destabilized. The freezing process causes a reduction of the fertility window because frozen/thawed boar sperm has less survivability. The aim of this work was to analyze the effect on sperm characteristics and response to capacitation stimuli of cooling to 5 degrees C using a controlled protocol. Also, we evaluated if the addition of Glycerol 2% or 3% at 5 degrees C was able to modify these parameters. For this purpose, we assessed motility, plasmatic membrane integrity and acrosomal membrane status. Capacitation was induced using Tyrode<acute accent>s capacitating medium (TCM) and assessed by chlortetracycline stain and induction of acrosomal reaction with Progesterone. Motility patterns were analyzed using a CASA system. These tests were performed at three different points of the freezing curve: 37 degrees C; 17 degrees C and 5 degrees C. Response to TCM vs TBM was only significant at 37 degrees C. While at 37 degrees C and 17 degrees C capacitated sperm was below 20%, at 5 degrees C reached 50% both in the TBM and TCM. CASA analysis showed that spermatozoa exposed to TCM had higher LIN and WOB than those in TBM. All parameters were similar in the Glycerol concentrations studied. These results suggest that the chilling process may be causing an effect similar to cryocapacitation along the cooling curve, starting subtle at 17 degrees C and reaching 50% of the sperm population at 5 degrees C, being independent of Glycerol concentration.