Melatonin Supplementation in Sex-Sorted Nili-Ravi Buffalo Semen: Effect on Sperm Quality, Subsequent in Vitro Embryo Development, and Pregnancy Outcomes

Melatonin (MLT) is a potent antioxidant that reduces oxidative stress (OS)-induced sperm damage. However, few studies have explored its effects in the field investigated here. This study aimed to evaluate the effects of MLT supplementation in extenders used for staining, sorting, and freezing on the quality of sorted Nili-Ravi buffalo sperm, embryo development after in vitro fertilization (IVF), and pregnancy outcomes following artificial insemination (AI). Computer-aided sperm analyzer (CASA) showed that progressive motility (PM) and velocity parameters of sorted, frozen-thawed sexed sperm were higher in the MLT-treated groups (<i>p</i> < 0.05). Fluorescence microscopy demonstrated that MLT significantly improved sperm plasma membrane integrity after staining and increased the proportion of frozen-thawed sex-sorted sperm with high mitochondrial membrane potential (HMMP) (<i>p</i> < 0.05). Raman spectroscopy further identified several distinctive Raman bands at 936, 1300, and 1651-1652 cm^-1, which may serve as biomarkers for assessing sperm quality. Additionally, cleavage rate, blastocyst formation, and pregnancy rates following IVF and AI were higher in the MLT-treated group (<i>p</i> < 0.05). In conclusion, MLT can serve as a valuable additive during sperm sex-sorting procedures to enhance sperm quality, thereby improving embryo developmental competence and pregnancy outcomes.