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.

Context While conventional semen analysis is a simple, time-saving, and economical means to evaluate sperm quality, it leaves biochemical and metabolic characteristics of spermatozoa aside. To address this issue, the use of fluorescent probes assessing functional sperm parameters, such as JC-1, DiOC6(3) and MitoTracker, has increased over the last decades. Apparently contradictory observations have nevertheless fostered an ongoing debate on their sensitivity and ability to evaluate the mitochondrial membrane potential (MMP) of sperm cells, thus warranting a re-examination of these probes. Aims The present study aims to elucidate the suitability and sensitivity of each probe to evaluate the MMP of bovine spermatozoa by flow cytometry. Methods Cryopreserved spermatozoa from ten bulls were thawed, stained with JC-1/SYTOXRed, DiOC6(3)/propidium iodide (PI) or MitoTracker Deep Red (MTDR)/PI, and evaluated with flow cytometry and fluorescence microscopy. Key results DiOC6(3), JC-1 and MTDR can be simultaneously co-stained with a viability marker. The results of the present study support the ability of DiOC6(3)/PI and JC-1/SYTOXRed, but not that of MTDR/PI, to monitor the MMP of spermatozoa. Conclusions JC-1/SYTOXRed assessed by flow cytometry was found to be the most sensitive and robust fluorescent probe to assess MMP. Moreover, DiOC6(3)/PI could be a suitable alternative when the flow cytometer is not equipped with a red laser and/or an adequate optical filter. Implications Both DiOC6(3) and JC-1, but not MTDR, could be used as probes to assess the mitochondrial membrane potential of bovine spermatozoa.

This work aimed to investigate how stimulation of donkey sperm with red LED light affects mitochondrial function. For this purpose, freshly diluted donkey semen was stimulated with red light for 1, 5, and 10 min, in the presence or absence of oligomycin A (Omy A), a specific inhibitor of mitochondrial ATP synthase, or FCCP, a specific disruptor of mitochondrial electron chain. The results obtained in the present study indicated that the effects of red LED light on fresh donkey sperm function are related to changes in mitochondria function. In effect, irradiation of donkey sperm resulted in an increase in mitochondrial membrane potential (MMP), the activity of cytochrome C oxidase and the rate of oxygen consumption. In addition, in the absence of oligomycin A and FCCP, light-stimulation augmented the average path velocity (VAP) and modified the structure of motile sperm subpopulations, increasing the fastest and most linear subpopulation. In contrast, the presence of either Omy A or FCCP abolished the aforementioned effects. Interestingly, our results also showed that the effects of red light depend on the exposure time applied, as indicated by the observed differences between irradiation protocols. In conclusion, our results suggest that exposing fresh donkey sperm to red light modulates the function of their mitochondria through affecting the activity of the electron chain. However, the extent of this effect depends on the irradiation pattern and does not exclude the existence of other mechanisms, such as those related to thermotaxis.

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A survey of in vivo fertility data from 31 pig farms distributed worldwide was conducted to determine whether stimulating boar semen with LED‐based red light increases its reproductive performance following artificial insemination (AI). Red‐light stimulation with MaXipig® was found to increase farrowing rates (mean ± SEM, control: 87.2% ± 0.4% vs. light stimulation 90.3% ± 0.5%) and the number of both total and live newborn piglets. Red‐light stimulation increased farrowing rates in 27 farms, with an increase ranging from 0.2% to 9.1%. Similar results were observed in litter sizes. Suboptimal management after AI was suggested in those farms with no response to red‐light stimulation. Our results indicate that a routine use of red‐light stimulation of boar semen can have a positive effect on the reproductive performance. However, the effectiveness of this system appears to highly rely upon proper management of pig farms.