Effect of In Vitro Addition of Melatonin and Glutathione on Seminal Parameters of Rams in Diluted Semen and after Thawing

SIMPLE SUMMARY: The cryopreservation of semen has numerous advantages but decreases sperm quality, compromising its viability and functionality. Oxidative stress is considered the main cause of this decrease, so using antioxidants such as glutathione (GSH) and melatonin (MLT) may be a way to overcome this problem. We aimed to evaluate whether adding 1 mM MLT, 5 mM GSH, or both improves ram semen quality after thawing. We observed that, in fresh semen, the separate addition of MLT and GSH had a negative effect on sperm motility parameters. In thawed semen, MLT increased progressive sperm motility and combined with MLT, attenuated the negative effects of GSH. This study provides valuable information for selecting antioxidants or possible associations to be added to ram freezing-semen extenders. ABSTRACT: The present study aimed to evaluate the effects of melatonin (MLT), glutathione (GSH), and their combination on ram semen quality after thawing. During eight weekly sessions, semen from three Merino rams was pooled, diluted with an egg-yolk-based semen extender, and divided into four groups: control, 1 mM MLT, 5 mM GSH, and 1 mM MLT + 5 mM GSH. Diluted semen was evaluated before and after the freezing process. The supplementation of diluted semen with GSH at 5 mM had a deleterious effect on total motility progressive (TPM), curvilinear velocity (VCL), straight-line velocity (VSL), average-path velocity (VAP), linearity (LIN), and straightness (STR) and increased slow spermatozoa (%). MLT at 1 mM also had a negative effect on TPM, VSL, and STR in diluted semen. In thawed semen, 1 mM MLT increased the TPM compared with the control group. VSL was lower in the 5 mM GSH group than in the 1 mM MLT group. Additionally, the combination of both antioxidants attenuated the negative effect of 5 mM GSH on TPM, VSL, and BCF. These results indicate that 5 mM GSH impairs or does not improve sperm kinetic parameters in either diluted or thawed semen. They also suggest that MLT combined with GSH plays a protective role against these effects.