Effect of pasteurized chicken egg yolk diluent on the cryopreservation of boar semen

In the context of semen cryopreservation, egg yolk is frequently utilized as a protective agent; however, it poses a potential risk of contamination by pathogenic microorganisms. Pasteurization of egg yolk can effectively eliminate pathogenic bacteria while retaining its active components. This study established two groups for comparison: (1) the experimental group (PS), which utilized a diluent containing pasteurized chicken egg yolk, and (2) the control group (Control), which employed a diluent with chicken egg yolk mixed at ambient temperature. The diluents from both groups were subjected to microscopic structural observation, bacterial isolation and culture, as well as Gram staining. Boar semen was cryopreserved using two different diluents following standard methods. Following thawing, sperm motility parameters, acrosome and plasma membrane integrity, antioxidant indicators, reactive oxygen species (ROS) levels, and the expression of genes related to oxidative stress and apoptosis were assessed. Microscopic observation revealed no significant differences in yolk granule morphology between PS and the control. Bacterial isolation and Gram staining results indicated the presence of small, spherical Gram-positive bacteria in the control, whereas no bacterial growth was observed in the PS. Post-thaw sperm analysis showed that, compared to the control, the PS exhibited significantly lower sperm survival rate and motility (P < 0.05), but significantly higher values for straight line velocit (VSL), curvilinear velocity (VCL), and average path velocity (VAP) (P < 0.05). Although the integrity of the acrosome and plasma membrane in sperm from the PS group showed improvement compared to the control, this difference did not reach statistical significance (P > 0.05). The levels of malondialdehyde (MDA) and total antioxidant capacity (T-AOC) in the PS group were significantly lower (P < 0.05), while the levels of superoxide dismutase (SOD) and catalase (CAT) showed a decreasing trend, but the difference was not significant (P > 0.05).The content of reactive oxygen species (ROS) in the PS group was significantly lower than that in the control group (P < 0.05). Gene expression analysis related to cellular oxidative stress and apoptosis showed that the mRNA levels of CAT, SOD, P53, and Bax were lower in the PS than in the control, with CAT expression significantly diminished (P < 0.05), and Bcl-2 expression slightly elevated (P > 0.05). In conclusion, pasteurized chicken egg yolk diluent can effectively eliminate pathogenic bacteria, reduce oxidative damage to sperm, and lower sperm apoptosis rate. Nonetheless, it is important to note that in the PS group, the post-thaw survival rate and motility of porcine sperm were markedly diminished, accompanied by an upregulation of certain apoptosis-related genes.