This work analyzes the effects of red LED light on mammalian sperm mitochondrial function, using the pig as an animal model. Liquid-stored pig semen was stimulated with red-light for 1, 5 and 10 min in the presence or absence of oligomycin A, a specific inhibitor of mitochondrial ATP synthase, or carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), a specific disruptor of mitochondrial electron chain. Whereas exposure for 1 and 5 min significantly (p < 0.05) decreased total motility and intracellular ATP levels, irradiation for 10 min induced the opposite effect. Oligomycin A abolished the light-effects on intracellular ATP levels, O₂ consumption and mitochondrial membrane potential, whereas compared to non-irradiated samples, FCCP significantly (p < 0.05) increased O₂ consumption when sperm were irradiated for 1 min. Both oligomycin A and FCCP significantly (p < 0.05) decreased total motility. Red-light increased cytochrome c oxidase activity with a maximal effect after 5 min of irradiation, which was abolished by both oligomycin A and FCCP. In conclusion, red-light modulates sperm mitochondrial function via electron chain activity in an exposition, time-dependent manner.

This work sought to address whether the presence of exogenous bicarbonate is required for pig sperm to elicit in vitro capacitation and further progesterone-induced acrosome exocytosis. For this purpose, sperm were either incubated in a standard in vitro capacitation medium or a similar medium with different concentrations of bicarbonate (either 0 mM, 5 mM, 15 mM or 38 mM) and BSA (either 0 mg/mL or 5 mg/mL). The achievement of in vitro capacitation and progesterone-induced acrosomal exocytosis was tested through the analysis of sperm motility, plasma membrane integrity and lipid disorder, acrosome exocytosis, intracellular calcium levels, mitochondria membrane potential, O₂ consumption rate and the activities of both glycogen synthase kinase 3 alpha (GSK3α) and protein kinase A (PKA). While sperm incubated in media without BSA or BSA/bicarbonate, they did not achieve in vitro capacitation; those incubated in media with BSA achieved the capacitated status under any bicarbonate concentration, even when bicarbonate was absent. Moreover, there were differences related to the concentration of bicarbonate, since sperm incubated in media with BSA and with no bicarbonate or 5 mM bicarbonate showed lower overall efficiency in achieving in vitro capacitation than those incubated in the presence of BSA and 15 mM or 38 mM bicarbonate. Additionally, at the end of the experiment, sperm incubated in the presence of BSA and 38 mM bicarbonate showed significantly (p < 0.05) lower values of motility and plasma membrane integrity than those incubated in media with BSA and lower concentrations of bicarbonate. In conclusion, BSA is instrumental for pig sperm to elicit in vitro capacitation and trigger the subsequent progesterone-induced acrosome exocytosis. Furthermore, while exogenous bicarbonate does not seem to be essential to launch sperm capacitation, it does modulate its efficiency.

This work sought to test the positive effect of red LED light on frozen bovine sperm resistance to thawing in suboptimal conditions. Moreover, a preliminary study explores whether this improvement could have any repercussions on in vivo fertilizing ability. Thus, frozen bull semen straws (n = 16) were thawed (a) with irradiation for 5 min at 20 °C (2 min light, 1 min darkness, 2 min light; PHOTO); (b) without irradiation for 5 min at 20 °C (ET); or (c) through immersion into a water bath at 38 °C for 40 s as standard control (CONTROL). Sperm quality and preliminary, purely descriptive AI trials were performed. The PHOTO samples demonstrated significantly (p < 0.05) improved percentages of intact acrosomes, progressive motility, DNA condensation and fragmentation, and viable sperm with high ROS/superoxides. The viability of PHOTO samples decreased significantly (p < 0.05) when compared with the ET ones. Overall results of both the PHOTO and ET samples were poorer than those of the CONTROL. Otherwise, the PHOTO straws yielded greater pregnancy rates (64.0% vs. 49.4% in CONTROL) when evaluated at two different farms. The results suggest that irradiating cryopreserved bovine sperm during thawing in suboptimal conditions could improve AI pregnancy rates, although more in vivo studies are needed to support this conclusion.