84 EFFECT OF A STRESSOR ON CANINE SPERM DNA FRAGMENTATION USING THE SPERM CHROMATIN DISPERSION TEST

Recently, a new procedure for the analysis of sperm DNA fragmentation has been developed for human and different mammalian species (Sperm-Halomax®), based on the sperm chromatin dispersion test (SCDt); however, no studies has been performed specifically on canine frozen–thawed-stressed semen but is there for cooled semen. The aim of this work was to assess the effect of a stressor (24 h in an oven at 38°C) on canine frozen–thawed semen using the SCDt to resemble what happens in the female reproductive tract. For this purpose, ejaculates were collected by digital manipulation from 4 healthy beagle dogs and the sperm-rich fraction of the ejaculates from 3 different dogs was pooled each time. All the pooled semen samples (n = 4) used presented physiological values concerning to routine semen parameters (motility, morphology, and sperm concentration). After evaluation, semen samples were centrifuged and the sperm pellet resuspended to a final concentration of 100 × 106 sperm mL–1 in 2 steps with CaniPRO Freeze (Minitub, Tiefenbach, Germany). Sperm were slowly cooled to 5°C and then loaded into 0.5-mL plastic straws. After that, straws were frozen in liquid-nitrogen vapours for 10 min and stored into a nitrogen tank. Straws were thawed in a water bath (30 s/37°C) and incubated for 24 hours at 38°C before analysis. The sperm DNA fragmentation was assessed in fresh semen and frozen–thawed-stressed samples using the Sperm-Halomax® commercial kit specifically developed for canine semen (Halotech DNA SL, Madrid, Spain) following the manufacturer’s instructions. Slides were stained for green fluorescence staining and 500 sperm per slide were counted using fluorescence microscopy. The sperm DNA fragmentation index (%) was compared between fresh and frozen–thawed-stressed semen samples by ANOVA. Results were expressed as mean ± standard error of the mean. The results obtained showed that subjecting thawed semen to 24 h in an oven at 38°C significantly increased (P < 0.05) DNA fragmentation compared with fresh semen (2.7% ± 0.2 v. 1.4 ± 0.1%). The stress factor was performed to simulate the viability of canine thawed sperm (12–24 h) when a bitch is inseminated with frozen semen. It would be interesting to perform further studies to relate sperm DNA fragmentation and fertility of frozen–thawed canine semen. In conclusion, frozen–thawed-stressed semen samples increased the sperm DNA fragmentation index measured using a SCDt. Further studies are needed to relate sperm DNA fragmentation with fertility rates or cryopreservation success.