Post-Thaw Parameters of Buck Semen Quality after Soy Lecithin Extender Supplementation with Fumaric Acid

SIMPLE SUMMARY: The cryopreservation of spermatozoa causes biochemical and ultrastructural changes that compromise their structural and functional integrity, thereby affecting their fertilizing ability. These alterations are influenced and/or mediated by oxidative stress. Since goat seminal plasma interacts with egg yolk and milk constituents, soy-lecithin-based extenders have been used for the cryopreservation of goat spermatozoa for over a decade. Fumaric acid, a substance that enhances antioxidant enzyme activity, has been extensively used in therapeutics due to its antioxidant, anti-inflammatory and immunoregulating properties. We tested the addition of fumaric acid (0 mM, 2.15 mM, 10 mM, 30 mM) in a soy lecithin extender on post-thaw buck sperm quality and function variables (sperm motility and kinetics (CASA); viability (eosin–nigrosin); acrosome integrity (SpermBlue(®)); membrane functional integrity (hypo-osmotic swelling test; HOST) and mitochondrial function (MF; Rhodamine 123/SYBR-14/PI)). Our results indicated a beneficial effect of 2.15 mM fumaric acid supplementation on frozen–thawed buck sperm viability, acrosome integrity, plasma membrane functional integrity and mitochondrial function. Further research related to sperm-fertilizing capacity under in vivo conditions is currently in progress. ABSTRACT: The supplementation of cryopreservation media with antioxidants improves the post-thaw quality and fertilizing ability of spermatozoa. To maximize the fertility of frozen–thawed buck spermatozoa, further research is required to overcome obstacles that have yielded controversial results and standardize protocols. In the present work, the effect of adding fumaric acid (a well-described antioxidant) to a soy lecithin semen extender on certain quality parameters of spermatozoa following freezing and thawing was examined for the first time. Five sexually mature Skopelos bucks were used, and ejaculates were collected with an artificial vagina. The semen samples (98 samples, five replicates) were diluted (400 × 10(6) spermatozoa/mL) with OviXcell(®), supplemented with fumaric acid (0 mM, 2.15 mM, 10 mM or 30 mM), equilibrated (5 °C; 3 h), packed (0.5 mL straws), frozen and stored (−196 °C) until further processing. After thawing, the spermatozoa total and progressive motility (CASA), viability (eosin–nigrosin), membrane functional integrity (HOST), acrosome integrity (SpermBlue(®)) and mitochondrial function (Rhodamine-123/SYBR-14/PI) were evaluated. Statistical analysis was performed with one-way ANOVA, followed by Duncan’s test; significance was set at 0.05. The addition of 2.15 mM fumaric acid improved (p < 0.05) spermatozoa viability, membrane functional integrity, acrosome integrity and mitochondrial function compared to all other concentrations. The addition of 30 mM fumaric acid decreased (p < 0.05) spermatozoa viability and mitochondrial function compared to all other concentrations. These results indicate a beneficial effect of a 2.15 mM fumaric acid addition to a soy lecithin extender on post-thaw buck spermatozoa quality. Further research is required to evaluate the in vivo fertility of frozen–thawed buck spermatozoa treated with fumaric acid, as well as to elucidate the mechanism of action of fumaric acid in spermatozoa.