Freezability prediction of boar ejaculates assessed by functional sperm parameters and sperm proteins

The objective of this work was to look for useful predictive indicators of the potentially “good” or “poor” ability of a boar ejaculate to sustain cryopreservation by assessing both the conventional sperm quality parameters (Study 1) and the immunolabeling of three proteins involved in the physiology of the sperm cell: GLUT3, HSP90AA1 and Cu/ZnSOD (Study 2). Study 1 was carried out in three different steps during the cryopreservation process of the sperm-rich fraction of 29 Piétrain boar ejaculates (17 °C, 5 °C, and 240 min postthaw). These ejaculates were clustered based on sperm quality parameters analyzed at 240 min postthaw, obtaining 16 good freezability ejaculates (GFEs) and 13 poor freezability ejaculates (PFEs). The sperm linearity (LIN) and the straightforward (STR) indexes at 5 °C showed higher hyperactivated movement in the PFEs than in the GFEs, which suggests that analyzing these sperm kinematic parameters could be a useful tool for predicting the potential freezability of an ejaculate. This statement was demonstrated by grouping the 29 ejaculates into two clusters (A and B) based on LIN and STR values assessed after 30 min at 5 °C, which resulted in around 72% of coincidence with the GFE and PFE groups. Study 2, performed at 17 °C and 240 min postthaw, revealed no differences between GFEs and PFEs in the immunolabeling of the three proteins within a same step, in terms of location and reactivity, although reactivity was generally weaker at 240 min postthaw in both groups. Additional studies on Western blot are currently being carried out with the objective to quantify the expression of the three proteins in GFEs and PFEs in the three steps of the cryopreservation process.