Cryopreservation of Boar Semen Impact of the Use of Specific Ejaculate Portions, Concentrated Packaging, and Simplified Freezing Procedures on Sperm Cryosurvival and Potential Fertilising Capacity

Generally, spermatozoa from most boars survive poorly the customary process of cryopreservation. Post-thaw (PT) survivors often show unstable membranes, short life-span, and low fertilising capacity when artificial insemination (AI) is used. This thesis aimed at improving this situation by (i) freezing high sperm numbers in novel packages (MiniFlatPacksTM, MFP) and exploring their degree of PT-survival in vitro and fertility in vivo using deep intrauterine AI (DIU-AI). Moreover, it attempted to find out (ii) why particular portions of the ejaculate better resist cryopreservation and (iii) whether the process could be made attractive for routine use. Spermatozoa from the sperm-rich fraction of the ejaculate (SRF) were densely (2×10 sperm/mL) packed into MFP, frozen and studied post-thaw, alongside controls handled in plastic straws. Cells handled in MFP survived better in vitro than controls. In vivo, single DIU-AIs of these spermatozoa led, despite the very low volume inseminated, to pregnancies, fertilisation rates being higher in MFP than controls, when the interval between DIU-AI and ovulation was -8 to -4 h. Two specific portions of the ejaculate, namely the first 10 mL from the SRF (P1) and (P2) i.e. the rest of the SRF and the post-sperm fraction, were further tested for their resilience to extension, cooling and freezing-thawing in vitro, using a battery of tests for kinematics, membrane integrity/stability and chromatin intactness. Most often, P1spermatozoa best sustained all handling procedures, revealing that capacitation-like sperm destabilisation did not occur during controlled cooling. Such in vitro resilience was not intrinsically sperm-related, but differently influenced by the seminal plasma (SP) of the portions (SP-P1, SP-P2). Pooled SP-P1 (with fewer and different proteins, as well as less bicarbonate, than SP-P2) was able to increase PT motility of cleansed P2-spermatozoa to P1 levels. Using all previous findings, a final study was designed to simplify cryopreservation, by freezing P1 in MFP with a shorter (from 8 to 3.5 h) freezing protocol (SF), excluding primary extension and removal of SP by centrifugation. The P1-SF-processed spermatozoa in MFP survived the process equally as well as SRF controls, but without variation among males or ejaculates, implying that this simpler freezing protocol could be advantageously applied to build genetic banks or commercialise frozen semen primarily for DIU-AI using P1. The P2 could still be used for production of conventional AI doses, allowing the AI enterprise to maintain routine management of commercially relevant stud boars.