43 Boosting boar fertility: the impact of glutathione on Kolbroek sperm parameters

As a long-term method of preserving an animal’s genetic potential for breeding, cryopreservation of semen helps conserve endangered species. However, lower temperatures cause an increase in osmotic pressure and result in oxidative stress, which in turn reduces sperm motility and the rate of fertilization. Owing to its strong antioxidant potential, which may protect sperm from oxidative stress, glutathione (GSH) has drawn attention. The aim of the study was to determine the effect of GSH supplementation of boar sperm freezing extender at various concentrations (0, 1, 5, and 10 mM) on sperm motility and velocity parameters and sperm viability of frozen-thawed Kolbroek sperm. Six ejaculates for each group were collected from three Kolbroek boars, pooled, and centrifuged at 800g for 10 min. Sperm pellets were re-extended with Fraction A extender: control (egg yolk 20% + Beltsville thawing solution [BTS] 80%), 1 mM (egg yolk 20% + BTS 78% + GSH 2%), 5 mM (egg yolk 20% + BTS 70% + GSH 10%), or 10 mM (egg yolk 20% + BTS 60% + GSH 20%). After cooling at 5°C for 60 min, Fraction B extender, control (egg yolk 20% + BTS 72% + glycerol 8%), 1 mM (egg yolk 20% + BTS 70% + glycerol 8% + GSH 2%), 5 mM (egg yolk 20% + BTS 62% + 8% glycerol + GSH 10%), or 10 mM (egg yolk 20% + BTS 52% + glycerol 8% + GSH 20%), was added into semen samples. All straws were cryopreserved in parallel using an automatic controlled-rate freezer (Custom BioGenic Systems CBS) at −5°C min⁻¹ from +4°C to −10°C; −40°C min⁻¹ from −10°C to −100°C; and −20°C min⁻¹ from −100°C to −140°C. After thawing (37°C), sperm motility and velocity traits were evaluated using a computer-aided sperm analyzer (Microptic). Sperm viability was determined microscopically with the use of eosine nigrosin. The data were analyzed using one-way ANOVA and are presented as mean ± SEM. Treatment means were compared using Least Significant Difference t-test. The results showed a difference in boar’s sperm progressive motility (%) on the control (51.4 ± 23.7) as compared with 1 mM (18.8 ± 14.2), 5 mM (6.2 ± 2.3), and 10 mM (35.1 ± 22.0). A difference was recorded in boar’s sperm total motility (%) 1 mM (38.5 ± 20.3) as compared with 0 mM (67.1 ± 20.0), 5 mM (57.4 ± 12.8), and 10 mM (53.8 ± 19.7). There was a significant difference in 1 mM (18.8 ± 14.2) on sperm progressive motility (%) compared with 5 mM (6.2 ± 2.3). The 0 mM (74.8 ± 22.9), 5 mM (80.8 ± 9.6), and 10 mM (78.5 ± 78) on boar’s sperm straightness (%) showed higher percentages compared with 1 mM (58.3 ± 34.2; P < 0.05). Boars sperm straight-line velocity (μm s⁻¹) was numerically lower in 1 mM (46.6 ± 30.1) as compared with other treatments (P > 0.05). The boar’s rapid sperm (%) in 5 mM (4.3 ± 2.8) was numerically lower as compared with 0 mM (9.1 ± 9.2), 1 mM (16.1 ± 11.1), and 10 mM (16.1 ± 15.2; P > 0.05). Results indicated that 10 mM (39.2 ± 6.4) in live boar’s sperm was numerically higher as compared with 0 mM (38.8 ± 3.8), 1 mM (28.6 ± 9.8), and 5 mM (27.6 ± 7.1; P > 0.05). A difference on 10 mM (5.7 ± 2.5) in live boar’s sperm tail (%) was observed as compared with the other treatments (P < 0.05). No difference was observed on head, midpiece, proximal droplets, and distal droplets in live sperm abnormalities. In conclusion, 10 mM was the optimum concentration of GSH to be added to the freezing extender for cryopreserving semen from Kolbroek boars.