Bovine serum albumin and skim-milk improve boar sperm motility by enhancing energy metabolism and protein modifications during liquid storage at 17 °C

•BSA and skim-milk have protective effects on boar sperm protein phosphorylation, acylation and ubiquitination.•We explored the protective roles of BSA and skim-milk from the perspective of energy metabolism and protein modification.•This study provides the feasibility of using skim-milk instead of BSA as a boar semen extender supplement.

Both bovine serum albumin (BSA) and skim-milk have been reported to improve sperm quality, primarily by enhancing sperm motility, but the underlying molecular mechanism remains unknown. In this study, boar semen samples were collected and diluted with Androstar® Plus extender containing different concentrations (0, 2, 4 g/l) of BSA and skim-milk. On days 0, 3, 5 and 7, the sperm motility parameters were determined using computer-assisted sperm analysis (CASA), and the ATP concentrations, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity and mitochondrial membrane potential were evaluated using commercial kits. The levels of protein phosphorylation, acylation and ubiquitination were analyzed by western blot. The results showed that supplementation with BSA and skim-milk provided higher sperm motility parameters, ATP levels, GAPDH activity and mitochondrial membrane potential than the control group (P < 0.05). Interestingly, we found that the levels of protein phosphorylation, acetylation and succinylation of the spermatozoa in the treated groups were dramatically higher than those in the control group (P < 0.05). Though the protein ubiquitination level had a decreasing trend, the change in ubiquitination modification was not significantly different between the control group and treated groups. Moreover, the changes in protein modifications between the BSA treated group and skim-milk treated group were not distinctly dissimilar. Taken together, these results suggest that BSA and skim-milk had a positive role in the regulation of boar sperm motility by influencing sperm protein modifications changes as well as increasing the GAPDH activity, mitochondrial membrane potential, and intracellular ATP content. This research provides novel insights into the molecular mechanisms underlying BSA and skim-milk protective effects on boar sperm in the male reproductive system and suggests the feasibility of using skim-milk instead of BSA as a boar semen extender supplement.