New insights in the study of boar semen cryopreservation

Résumé

The PhD Thesis aimed to improve the current knowledge of boar sperm cryopreservation. To achieve it, three specific objectives were proposed: (1) to address the role of antioxidants of seminal plasma (SP) in protecting boar sperm against cryopreservation stress (exp. 1); (2) to evaluate whether SP from post-sperm rich ejaculate fraction (post-SRF) is the only reason why sperm from the entire ejaculate cryopreserve worse than those from the SRF (exp. 2); and (3) to evaluate whether cryostorage time of boar frozen semen doses in liquid nitrogen (LN2) tanks affects the further post-thaw sperm quality (exp. 3). In exp. 1, ten ejaculates were manually collected in fractions: the first 10 mL of SRF (P1), the rest of the SRF (P2) and the post-SRF. A portion of each fraction was used for sperm cryopreservation (using a standard protocol for 0.5 mL straws) while the rest was used to collect SP for antioxidant assessment. Frozen-thawed (FT) sperm from the SRF (P1 and P2) showed higher motility and viability than those from post-SRF (P<0.01). Viable FT-sperm from SRF generated less intracellular reactive oxygen species and experienced less lipid peroxidation (LPO) than those from the post-SRF (P<0.01). Regression analyses indicated that some SP-antioxidants influenced sperm freezability. Specifically, superoxide dismutase (SOD), trolox-equivalent antioxidant capacity (TEAC) and ferric-reducing ability showed good predictive value for post-thaw recovery rates of total motility (R2 = 54.8%, P < 0.001), whereas SOD, paraoxonase 1, glutathione peroxidase 5 and TEAC for post-thaw recovery rates of viability (R2 = 56.1%, P < 0.001). These results demonstrated that SP-antioxidant are directly involved in boar sperm freezability. In experiment 2, twelve ejaculates were manually collected in fractions: P1, P2 and post-SRF. Samples were centrifuged to separate sperm and SP. Sperm from P1 and P2 were re-diluted with either its own SP or SP from post-SRF, then overnight storage at 17 ºC and frozen the next day. After overnight storage, sperm motility was impaired (P < 0.05) in both P1 and P2 when diluted with SP from post-SRF. In contrast, sperm viability was higher (P < 0.05) in P1 than in P2, regardless of the SP used during overnight storage. After thawing, sperm quality and functionality were affected by sperm origin, regardless of the SP used during pre-freezing storage. Sperm from P1 showed highest (P < 0.05) motility and viability and lowest (P<0.05) plasma membrane fluidity and LPO. Protein profile of sperm from P1 and P2 was analyzed by 2D-PAGE, showing qualitatively differences among them. These results highlighted that SP from post-SRF is not detrimental for boar sperm freezability and also suggested that differences in sperm proteome could explain the differences between ejaculate fractions in sperm freezability. In experiment 3, post-thaw sperm motility, including kinetics, and viability was evaluated in 58 semen samples cryostored in LN2 for up to 8 yr. Post-thaw sperm quality was evaluated after 2, 4 or 8 yr of cryostorage, and values were compared to those obtained in straws of the same semen samples thawed few days after freezing (15 d). Sperm viability was not affected by cryostorage length, but total and progressive sperm motility were lower (P < 0.01) in semen samples cryostored for 4 or 8 yr than in those thawed 15 d after freezing. Cryostorage time also affected sperm kinetics, with greater intensity in the samples cryostored for 4 yr (p < 0.001) than in those for 2 yr (P < 0.01). These results demonstrated that more than to 2 yrs of cryostorage time negatively influences the quality of FT-boar sperm samples.