The objective of this study was to investigate the effects of trehalose and different doses of melatonin and lipid mixtures on the quality parameters of post-thaw bull sperm during cryopreservation. The ejaculates of three mature bulls were pooled and divided into ten equal aliquots. These aliquots were diluted with a Tris-based extender, which was supplemented with either 5% glycerol (G5) or 3% glycerol combined with 60 mM trehalose (G3T), alongside different doses of melatonin and lipid mixtures. Ten experimental groups were established as follows: G5, G5+0.25 mM melatonin (G5M0.25), G5+0.75 mM melatonin (G5M0.75), G5+ 1.25 μl/ml lipid mixtures (G5L1.25), G5+3.75 μl/ml lipid mixtures (G5L3.75), G3T, G3T+0.25 mM melatonin (G3TM0.25), G3T+0.75 mM melatonin (G3TM0.75), G3T+1.25 μl/ml lipid mixtures (G3TL1.25), and G3T+3.75 μl/ml lipid mixtures (G3TL3.75). No significant interaction was detected between any of the groups containing the G5 and G3T extenders, and all groups were found to be similar for sperm motility and flow cytometry analysis results (p > 0.05). The G5M0.25 and G5L1.25 groups had a higher recovery of post-thaw motility compared to the other groups containing 5% glycerol (p = 0.0004). In terms of motility rates, groups G3TM0.25 and G3TL1.25 displayed a higher level of protection compared to the other groups, and this protection was significantly greater than that determined in groups G3TM0.75 and G3TL3.75 (p = 0.001). The expression of the GFPT1, PFKP, FBF2, HK1, and ALDH2 genes was found to be significantly increased in the G5 and G3T groups containing both doses of lipid mixtures (L1.25 and L3.75) compared to the groups without additives (G5 and G3T) (p < 0.001-0.0001). However, the expression of the SORD gene was found to be increased only in the G3TL1.25 and G3TL3.75 groups compared to group G3T (p < 0.01-0.001). GFPT1 and FBF2 gene expressions were significantly increased in the G5M0.75 group compared to group G5 (p < 0.05). The G3T groups containing both doses of melatonin were found to display increased GFPT1 (p < 0.01) and PFKP (p < 0.05-0.01) expression levels compared to group G3T. It was determined that the addition of lipid mixtures at both doses to both G5 and G3T resulted in a significant transcriptional increase in all of the genes studied (except for SORD gene expression in the G5L1.25 and G5L3.75 groups), compared to the lipid mixtures-free groups (p < 0.05-0.001). Based on the evaluation of all results, G3T can be used as a substitute for G5 to reduce glycerol toxicity.
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