Global Proteomic Analysis of Sperm and Seminal Plasma From Tropically-Adapted Rams With Contrasting Parameters of Frozen-Thawed Semen.

The aim of this study was to characterize the proteome of sperm and seminal plasma (SP) from rams with high semen freezability (HF) and low semen freezability (LF). Eleven ejaculates were collected from 12 rams, at 3-day intervals, and cryopreserved (132 semen samples). For proteomic analysis, sperm and SP samples were pooled separately for each ram. Pools 1 and 2 included ejaculates obtained on odd-numbered and even-numbered collection days, respectively. This generated 24 sperm and 24 SP pools. Sperm and SP proteins were analyzed by mass spectrometry, and computational biology platforms, used for calculation of differentially abundant proteins (DAPs; p < 0.05) in HF (n = 5) and LF (n = 7) rams. Total sperm motility decreased from 91.87% ± 0.63% in fresh to 65.65% ± 2.58% in frozen-thawed semen of HF rams; and decreased from 89.47% ± 1.15% to 50.05% ± 2.33% in LF rams. Reduction in total sperm motility after cryopreservation was lower in HF (26.22% ± 2.39%) compared to LF rams (37.70% ± 2.23%; p = 0.006). Sperm progressive motility decreased less in HF rams (from 87.20% ± 0.50% to 58.12% ± 2.82%) than in LF rams (from 85.54% ± 1.41% to 44.00 ± 2.13; p = 0.006). Rams with the HF phenotype had better plasma membrane (39.00% ± 2.79% vs. 15.14% ± 0.92%) and acrosome integrity (37.54% ± 2.72% vs. 15.40% ± 1.92%; p < 0.001) than LF rams. Sperm motility was similar in fresh semen of HF and LF rams. There were 1599 and 1677 sperm proteins identified in HF and LF rams, with 73 and 76 DAPs, respectively. SP had 1127 proteins, with 25 and 45 DAPs in HF and LF rams. This is the greatest coverage ever published about the ram SP proteome. Sperm proteins with greater abundance in HF rams were extracellular and immune-related proteins, antiproteases, chaperones and structural regulators, contributing to cytoskeletal integrity, apoptotic resistance and maintenance of proteostasis. Sperm proteins with greater abundance in LF rams were mainly mitochondrial subunits, axonemal components, and bioenergetic enzymes, indicating greater reliance on oxidative processes. Major components of HF SP participate in sperm protection, stabilization of membrane lipids, calcium fluxes, epididymal maturation and ATP availability. The major LF SP proteome was associated with cellular stress, inflammatory responses, oxidative damage and ATP production. Thus, the nature of sperm and SP proteome atlas potentially defines ram sperm cryoresilience.