113 Effect of antioxidants on motility and fertility of liquid-stored bovine sperm

Abstract

It is difficult to maintain sperm in liquid storage for a long time, compared with permanent frozen storage in liquid nitrogen. Antioxidants have been reported to improve the quality and fertility of liquid-stored semen. In this study, we investigated whether antioxidants can extend the motility and fertility of frozen-thawed sperm in liquid storage. Frozen-thawed semen from one Japanese black bull (one ejaculate) was diluted in Tris-citrate-fructose (TCF) diluent with 10% (v/v) egg yolk to a sperm concentration of 1 × 10⁷ sperm mL⁻¹. The antioxidants β-mercaptoethanol (βMe) and glutathione (GSH) were added independently, at various concentrations (0.1, 0.5, 1, and 5 mM) to sperm suspensions, and these preparations were compared with Control (no added antioxidant). Sperm suspensions were packaged in centrifuge tubes and placed at 17°C in air and monitored daily until sperm motility had stopped (up to 14 days). Sperm motility was analysed by the Sperm Motility Analysis System (SMAS; Ditect Co. Ltd), and the percentage of progressively motile sperm (straight-line velocity (VSL) of >25 μm s⁻¹; Grade A classified by WHO manual), compared with that recorded on Day 0 (100%), was determined each day. For evaluation of fertilizing ability, after incubation in liquid storage for 0, 3, 5, and 7 days, sperm were used for IVF with in vitro-matured oocytes (30 oocytes per treatment, three replicates). Embryo development was recorded as the proportion of embryos that reached blastocyst by 8 days after IVF. Data for motility were analysed using one-way ANOVA with Tukey test, and embryo development using chi-squared test. A P-value < 0.05 was considered statistically significant. At 7 days, the percentage of progressively motile sperm was significantly higher for 0.5, 1, and 5 mM βMe than for Control (30.8%, 48.1%, and 50.3%, vs. 0%, respectively). Treatments with 1 and 5 mM βMe maintained some sperm progressive motility for 14 days (9.5% and 14.5%). Treatment with GSH showed the same trend at 7 days (32.2%, 36.3%, and 13.7% for 0.5, 1, and 5 mM, vs. 0% for Control); 1 and 5 mM GSH maintained sperm progressive motility over 10 days (24.8% and 4.4%). In both antioxidant treatments, embryo development was achieved with sperm stored for up to 5 days (Day 0 vs. Day 5 for 0.1 mM βMe: 17.6% vs. 13.8%; for 1.0 mM GSH: 26.0% vs. 6.7%; for Control: 17.6% vs. 0%). In this study, antioxidants extended both motility and fertility of frozen-thawed bovine sperm in liquid storage. This result suggests the possibility of application to AI using liquid-stored bovine semen.