Objective: The frozen semen has the significant advantages of long-term storage and long-range transportation. However, due to the low cryotolerance of boar sperm, the global utilization of frozen boar semen in artificial insemination was less than 1% until the year 2000.Methods: In this study, the effects of five cryoprotectants at different concentrations on the cryotolerance of boar semen were evaluated when they were added separately, and the optimal concentrations for each cryoprotectant were determined, then their combined additive effects were further assessed.Results: At a glycerol (GLY) concentration of 5%, the quality of frozen-thawed sperm reached its maximum value, which was significantly higher than the 4% GLY group (p<0.05) and 0% GLY group (p<0.01). The straight-line velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), sperm plasma membrane integrity (SPMI), sperm acrosome integrity (SAI) and sperm mitochondrial activity (SMA) of the frozen-thawed sperm in treated egg yolk group exhibited significant improvements compared to the untreated egg yolk group (p<0.05). The total motility (TM), progressive motility (PM), SPMI, SAI, and SMA of 2% Equex STM paste group were significantly higher than the rest groups (p<0.05). The TM, PM, VSL, VCL, and VAP of frozen-thawed sperm in the 250 nM and 300 nM Mitoquinone mesylate groups showed significant improvements compared to the other groups (p<0.05), and the reactive oxygen species levels in sperm cells were also significantly lower (p<0.05). The quality of frozen-thawed boar sperm in 0.6 mM L-ergothioneine group reached its peak value and was significantly higher than the rest groups (p<0.05). When these five cryoprotectants were used in combination, the quality of frozen-thawed boar sperm exhibited a significant improvement compared to when they were used individually (p0.05).Conclusion: The optimized boar semen cryopreservation system can substantially enhance the quality of frozen-thawed boar sperm, making it suitable for artificial insemination in pig farm.

This study systematically compared the effects of dietary supplementation with glutamine (Gln) and its precursors, including glutamic acid (GA) and α-ketoglutarate (AKG), on growth performance, serum antioxidant and immune parameters, and multi-region gastrointestinal microbiota in suckling lambs. Forty healthy suckling Hu lambs with similar body weight (7.37 ± 1.18 kg) and age (7 ± 0.8 d) were selected and randomly allocated into four groups (n = 10 per group): a control group (CON, without additive), and three treatment groups (GA, AKG, and Gln), each receiving 2 g per animal per day of the corresponding additive. The experimental period lasted for 42 d. All three additives showed a tendency to increase the final body weight (p = 0.056) and significantly increased the average daily gain (ADG) of lambs (p < 0.05). GA supplementation increased the dry matter intake throughout the entire trial (p < 0.05), whereas the addition of AKG and Gln increased the dry matter intake only during the later period (d 21–42) (p < 0.05). The feed-to-gain ratios did not differ among all groups (p > 0.05). Compared with the CON group, all three treatment groups showed elevated serum activities of catalase, glutathione peroxidase, and total antioxidant capacity, as well as increased IgA and IgG contents (p < 0.05). In addition, malondialdehyde concentration was decreased in all three treatment groups (p < 0.05). Moreover, GA supplementation reduced the ruminal alpha diversity while increasing the abundance of butyrate-producing bacteria (Ruminococcaceae UCG-014) (p < 0.05). All three interventions consistently decreased the abundance of the intestinal pathogen Escherichia-Shigella in the ileum (p < 0.05). Correlation analyses showed that ruminal Treponema 2 abundance was negatively correlated with ADG, whereas jejunal Methylobacterium and ileal [Eubacterium] coprostanoligenes group were positively correlated with final body weight or ADG. In conclusion, glutamine and its precursors play an important role in modulating gastrointestinal bacterial diversity and composition, enhancing antioxidant and immune functions, and improving the growth performance of suckling lambs.