Effect of antioxidants on motility and morphology of chilled-stored canine semen

INTRODUCTION Generation of reactive oxygen species and lipid peroxidation of the sperm cell membranes during liquid storage or semen cryopreservation are associated with loss of motility and reduced viability (1). It has been shown that the addition of antioxidants to semen extenders can improve the survival of spermatozoa (2). Antioxidants are present in seminal plasma and inhibit damage induced by free radicals and hydrogen peroxide. In this study, the antioxidants ascorbic acid and catalase were added to dog semen diluted in different extenders. Motility and morphology of spermatozoa during 96h storage at 5°C were investigated. MATERIALS AND METHODS Three sexually mature cross-bred male dogs, aged 1 to 6 yrs, were used as the semen donor. Semen was collected by manual stimulation into prewarmed glass tubes. Only the first two fractions of the ejaculate were collected. The percentage of motile spermatozoa was estimated by the CASA (Computer Assisted Semen Analysis) system. Semen smears were stained with fast-green FCF and rose bengal for examination of sperm morphology. Only ejaculates containing a minimum of 70% progressively motile and 50% morphologically normal spermatozoa were included in the study. The three extenders used were egg-yolk-tris (EYT, 3), skim milk (SM, 4), caprogen (CAP, 3) supplemented with antibiotics, with or without antioxidants. The antioxidants were added in the following concentrations: ascorbic acid (0.9 g/L) or catalase (cat, 200,000 U/L). Semen was stored at 5°C for 96h, motility and morphology were evaluated after dilution (0h) and at 6, 24, 48, 72, 96 h of storage. Data were analyzed using general linear model procedure (SAS). A P value <0.05 was considered significant. Values given are least squares means (LSMEAN) + standard error of mean (SEM). RESULTS AND DISCUSSION The motility of sperm cells after dilution (0h) was strongly depressed and this was independent of the extender used. This dilution effect was transitory and it was not observed at the following time points. At 96h of storage, the results showed that the extender CAP maintains higher motility (57.6%+6.7) compared to EYT (40.1%+6.7) and SM (30.5%+6.7). The addition of ascorbic acid did not result in any improvement when added to EYT, while it significantly (P<0.05) decreased the motility in SM and CAP. The addition of catalase in CAP significantly increased (62%+6.7; P<0.005) the motility at the end of storage compared to the other extenders (EYT-cat 27.5%+6.7; SM-cat 31.3%+6.7). The beneficial effect of catalase on survival at 96h was only observed when added to the extender CAP (Fig. 1). The positive interaction between CAP and catalase was also clearly evident in the evaluation of the sperm morphology. In fact, this treatment maintains the highest percentage (96.2%+4.9) of morphologically normal cells at 96h. The present study demonstrated that the addition of catalase to CAP extender reduces the loss of motility of canine spermatozoa after 96h storage at 5°C. Although further research is needed, the presence of antioxidants, such as catalase, in the extender is advisable for the liquid storage of canine semen. REFERENCES (1) Alvarez J.G., Touchstone J.C., Blasco L. & Storey B.T. (1987) J. Androl. 8: 338-348. (2) Maxwell W.M.C. & Stojanov T. (1996) Reprod. Fertil. Dev. 8: 1013-1020. (3) Province C.A., Amann R.P., Pickett B.W. & Squires E.L. (1984) Theriogenology 22: 409-415. (4) Kenney R.M., Bergam R.V., Cooper W.L. & Morse G.W. (1975) Proc. 21st Ann. Conv. A.A.E.P.: 327-336.