Abstract: In order to investigate the influence of adding glutathione （GSH） in frozen diluent on cryopreservation effect of canine semen, the seminal samples were collected from 5 hybrid dogs by massage method. After centrifugation to remove the seminal plasma, 0.5, 1.0, 1.5, 2.0 and 2.5 mmol/L of GSH was supplemented in the frozen diluent, respectively. All of the above samples with volume of 0.25 mL containing different concentrations of GSH were prepared for cryopreservation. The seminal samples without addition of GSH was used as a control group. After thawing, the samples were incubated for 10 hours in air containing 5% CO₂ at 37 ℃ with relative saturated humidity. The sperm motility of the samples was determined at 0, 2, 4, 6, 8, 10 h after incubation, respectively. The results showed that at 0 h after freezing and thawing, the sperm motility of the 0.5 and 1.0 mmol/L GSH treatment groups was 0.36 and 0.38, respectively, which were both significantly （P<0.05） higher than those of the control group and 2.0 and 2.5 mmol/L GSH treatment groups, and no significant difference （P>0.05） were observed between them; the highest （85.10%） sperm acrosome integrity rate and the lowest （23.00%） sperm deformity rate were simultaneously observed in the 1.0 mmol/L GSH treatment group, and they were significantly （P<0.05） higher and significantly （P<0.05） lower than those of the control group, respectively. After incubation for 2 and 4 hours after freezing and thawing, the sperm motility of the 0.5 and 1.0 mmol/L GSH treatment groups was relatively higher, and that of the 0.5 mmol/L GSH treatment group after in vitro incubation for 4 hours still reached up to 0.30; after in vitro incubation for 6 hours, the highest sperm motility was observed in the 1.0 mmol/L GSH treatment group, which was significantly （P<0.05） higher than those of the control group and 2.0 and 2.5 mmol/L GSH treatment groups; after in vitro incubation for 8 hours, the sperm motility of all GSH treatment groups was significantly （P<0.05） higher than that of the control group; after in vitro incubation for 10 hours, the sperm motility of each GSH treatment group was all decreased compared with other incubation time points, and there was no sperm with linear motion in the control group. In summary, the addition of 0.5-1.0 mmol/L GSH in freezing solution of canine semen can significantly improve the sperm quality and in vitro survival time after freezing and thawing.

Key words: glutathione, dog, cryopreserved semen, sperm motility