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

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 × 10⁶ 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.

Keywords: buck; cryopreservation; fumaric acid; goat; semen; soy lecithin extender; spermatozoa.

Figure 1

Percentages (means ± SD) of total motile spermatozoa (CASA) in Skopelos buck semen cryopreserved in commercial soy-lecithin-based extender (OviΧcell^®) and supplemented with 0 mM (n = 25), 2.15 mM (n = 24), 10 mM (n = 25) and 30 mM (n = 24) fumaric acid. a, b: Bars with different letters differ significantly (p < 0.0005).

Figure 2

Percentages (means ± SD) of progressively motile spermatozoa (CASA) in Skopelos buck semen cryopreserved in commercial soy-lecithin-based extender (OviΧcell^®) and supplemented with 0 mM (n = 25), 2.15 mM (n = 24), 10 mM (n = 25) and 30 mM (n = 24) fumaric acid. a, b: Bars with different letters differ significantly (p < 0.0005).

Figure 3

Percentages (means ± SD) of live spermatozoa (eosin–nigrosin) in Skopelos buck semen cryopreserved in commercial soy-lecithin-based extender (OviΧcell^®) and supplemented with 0.0 mM (n = 25), 2.15 mM (n = 24), 10.0 mM (n = 25) and 30.0 mM (n = 24) fumaric acid. a, b, c: Bars with different letters differ significantly (p < 0.0005).

Figure 4

Percentages (means ± SD) of spermatozoa with functional cell membranes (HOST) in Skopelos buck semen cryopreserved in commercial soy-lecithin-based extender (OviΧcell^®) and supplemented with 0.0 mM (n = 25), 2.15 mM (n = 24), 10.0 mM (n = 25) and 30.0 mM (n = 24) fumaric acid. a, b: Bars with different letters differ significantly (p < 0.0005).

Figure 5

Percentages (means ± SD) of spermatozoa with intact acrosomal membranes (SpermBlue^®) in Skopelos buck semen cryopreserved in commercial soy-lecithin-based extender (OviΧcell^®) and supplemented with 0.0 mM (n = 25), 2.15 mM (n = 24), 10.0 mM (n = 25) and 30.0 mM (n = 24) fumaric acid. a, b: Bars with different letters differ significantly (p < 0.0005).

Figure 6

Percentages (means ± SD) of live spermatozoa with high mitochondrial membrane potential (SYBR-14/PI) in Skopelos buck semen cryopreserved in commercial soy-lecithin-based extender (OviΧcell^®) and supplemented with 0.0 mM (n = 25), 2.15 mM (n = 24), 10.0 mM (n = 25) and 30.0 mM (n = 24) fumaric acid. a, b c: Bars with different letters differ significantly (p < 0.0005).