Irradiating frozen-thawed stallion sperm with red-light increases their resilience to withstand post-thaw incubation at 38 °C

The aim of this study was to evaluate whether red-light stimulation increases the longevity and resilience of cryopreserved stallion sperm to withstand post-thaw incubation for 120 min. Sixteen frozen straws of 0.5 mL from eight stallions were used. Samples were cryopreserved, thawed through incubation at 38 °C for 30 s and divided into the control and samples exposed to red-light using a triple LED photo-activation system (wavelength: 620-630 nm). Three irradiation protocols consisting of different light-dark-light intervals (1-1-1, 2-2-2 and 3-3-3 min) were tested. Sperm quality parameters were analyzed immediately after light-stimulation (0 min) and after 120 min of incubation at 38 °C. Sperm motility was evaluated using a Computerized Semen Analysis System (CASA), and flow cytometry and different fluorochromes were used to evaluate the integrity and lipid disorder of plasma membrane, mitochondrial membrane potential and intracellular levels of peroxides and superoxides. Irradiation significantly increased the percentages of spermatozoa with high mitochondrial membrane potential (1-1-1 pattern) and the intracellular levels of peroxides (2-2-2 pattern) at 0 min. In addition, sperm kinematic parameters (2-2-2 and 3-3-3 patterns) and percentages of viable spermatozoa with low membrane lipid disorder (3-3-3 pattern) were significantly higher in irradiated samples than in the control at 120 min. Our results indicate that red-light stimulation could help increase the resilience of frozen-thawed stallion sperm to withstand post-thaw incubation at 38 °C for 120 min and that these effects rely on the irradiation pattern. Further research should evaluate whether light-stimulation could also have a positive on fertility rates after artificial insemination.