The present study was conducted to evaluate whether supplementation of semen extender with glutathione (GSH) can maintain the quality of frozen-thawed canine spermatozoa. Eighteen ejaculates were obtained from 5 dogs and placed in extender (20% egg yolk, Tris, citric acid, lactose, raffinose, antibiotics and 6.5% glycerol) containing 0 (control), 2.5, 5, 7.5 or 10 mM GSH. The samples were cooled to 4 C and then frozen in liquid nitrogen vapor. Motility parameters of the sperm were evaluated at 0, 1, 2, 3, 4, 12 and 24 h after thawing. Sperm motility was higher in the 5 mM GSH group than in the control or 2.5 and 10 mM GSH groups; this effect was observed at 1 to 24 h after thawing (P < 0.05). The 5 mM GSH group had a higher sperm viability index at 12 and 24 h after thawing compared with the other groups (P < 0.05). Acrosome integrity, evaluated at 4 h after thawing, was greater in two of the GSH-treated groups (5 and 10 mM) compared with the control. Lipid peroxidation (LP) levels immediately after thawing were lower in the 5 and 10 mM GSH groups compared with the control, while those at 12 h after thawing did not differ significantly. Frozen-thawed semen in the 5 mM GSH group was used for transcervical insemination of 4 bitches, resulting in delivery of 5 puppies from 2 bitches. These results indicate that supplementation of semen extender with 5 mM GSH was effective in improving motility, longevity and acrosomal integrity and inhibiting LP levels in post-thaw canine spermatozoa, without any adverse impacts on full-term development after transcervical insemination.

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Trans-cervical insemination (TCI) with cryopreserved semen offers a potentially effective approach for breeding canids with specific genetic traits, such as guide dogs for the blind. However, there are technical difficulties in canine sperm cryopreservation, such as the composition of semen extender. The aim of this study was to evaluate the effects of glutathione (GSH) as an antioxidant in the semen extender to improve the quality of frozen-thawed dog sperm. A Tris-egg yolk-citrate extender containing 15.7 mg mL–1 of TRIS, 8.8 mg mL–1 of citric acid, 14.1 mg mL–1 of lactose, 25.4 mg mL–1 of raffinose, 1% (vol/vol) antibiotics, and 20% (vol/vol) egg yolk in ultra-pure water was used as the base medium. Twelve ejaculates were collected from 7 dogs. Each ejaculate was divided into 2 to 5 aliquots and extended with base extender supplemented with 0, 2.5, 5, 7.5, and 10 mM GSH as first dilution. The extended semen was equilibrated for 3 h at 4°C. An equal volume of second extender was added to obtain a final concentration of 6.5% glycerol and sperm per milliliter. The sperm samples were loaded in straws and frozen at 6 cm above the surface of LN2 for 15 min in a styrene foam box and plunged into the LN2. The frozen semen was thawed for evaluation. The motility of sperm was estimated with a phase-contrast microscope and the motile patterns were classified into the following grades: progressively motile at a high speed (+++), progressively motile at a moderate and low speed (++), motile without progression (+), and immotile (–). Then, the sperm motility index (SMI) was determined from the following formula as described previously (Iritani et al., 1975), with some modifications: the percentage of (+++) sperm + the percentage of (++) sperm × 0.75 + the percentage of (+) sperm × 0.5. Sperm motility and the SMI were determined at 0, 1, 2, 3, 4, 12, and 24 h after thawing. Acrosome status was evaluated at 4 h after thawing. Lipid peroxidation (LP) levels at 0 and 12 h after thawing were used to examine the antioxidant ability of GSH. Trans-cervical insemination was carried out on 5 bitches to evaluate the fertility of GSH-treated sperm. The TCI were performed nonsurgically with a laparoscope and deposited 2 mL of semen through a catheter. Each bitch was inseminated 1 to 2 times during oestrus. Data were analysed using ANOVA with the Tukey-Kramer method. We found that the rate of (+++) sperm in the 5 mM GSH group was higher than that in the 0 mM group from 1 to 24 h after thawing (P < 0.05). The SMI was higher in the 5 and 7.5 mM GSH groups than in the 0 mM group (P < 0.05). There were no significant differences in the control and 2.5 and 10 mM GSH groups. Long-term survival was increased in the 5 mM GSH group. Acrosome integrity was higher in the GSH-treated group. The level of LP was lower in the GSH-treated groups at 0 h after thawing (P < 0.05). Trans-cervical insemination with the 5 mM GSH-treated semen resulted in the delivery of 5 pups from 2 bitches. These results indicate that the cryopreservation with 5 mM GSH can improve the motility, viability, and fertility of frozen-thawed canine sperm by its antioxidant effects on the sperm membrane.