Melatonin Supplementation to the Freezing Extender Improves the Quality of Canine Epididymal Spermatozoa

Abstract

The present study aimed to determine the cryoprotective effect of melatonin supplements on the extender on the quality of canine epididymal spermatozoa. Epididymis from 20 castrated dogs were minced and incubated in Tris buffer. Spermatozoa were diluted in tris-citric acid-fructose (TCF) extender with two doses of melatonin (0.002 and 0.0035 mol) as well as a control (0.0 mol). The samples were packed in 0.25 ml straws after 2 hr of equilibration at 5°C and kept in liquid nitrogen (−196°C). Sperm parameters such as viability, motility, integrity of acrosome, sperm membrane functioning, and DNA integrity were assessed after thawing (37°C for 30 s). In addition, lipid peroxidation was assessed using the determination of malondialdehyde (MDA) levels. Results revealed that adding 0.0035 mol melatonin to the cryopreservation medium enhanced ($P<0.05$) the measured parameters of spermatozoa compared to the control and 0.002 mol melatonin. Levels of MDA were obviously ($P<0.05$) lower when 0.0035 mol melatonin was added in comparison to 0.002 mol melatonin and the control. Eventually, adding 0.0035 mol melatonin to the TCF extender significantly improved the percentage of motility, viability, and integrity of both plasma membrane and acrosome integrity of dog epididymal spermatozoa. In addition, melatonin supplementation improved DNA integrity and reduced membrane lipid peroxidation.