The preservation of sperm functional parameters and fertility post-cryopreservation largely varies in the porcine, a species with a fractionated ejaculate. Although intrinsic individual differences have primarily been linked to this variation, differences in protein abundance among frozen-thawed (FT)-spermatozoa are far more relevant. This study, performed in two experiments, looked for proteomic quantitative differences between FT-sperm samples differing in post-thaw viability, motility, apoptosis, membrane lipid peroxidation and nuclear DNA fragmentation. The spermatozoa were either derived from the sperm-rich ejaculate fraction (SRF) or the entire ejaculate (Experiment 1) or from the first 10 mL of the SRF, the remaining SRF and the post-SRF (Experiment 2). Quantitative sperm proteomic differences were analysed using a LC-ESI-MS/MS-based SWATH approach. In Experiment 1, FT-spermatozoa from the SRF showed better preservation parameters than those from the entire ejaculate, with 26 Sus scrofa proteins with functional sperm relevance showing relative quantitative differences (FC ≥ 1.5) between sperm sources. In Experiment 2, FT-spermatozoa from the first 10 mL of the SRF and the remaining SRF were qualitatively better than those from the post-SRF, and 187 proteins showed relative quantitative differences among the three ejaculate sources. The results indicate that quantitative proteome differences are linked to sperm cryosurvival.
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