Raffinose and hypotaurine improve the post-thawed Merino ram sperm parameters

Abstract

The aim of this study was to determine the effects of raffinose and hypotaurine on sperm parameters after the freeze–thawing of Merino ram sperm. Totally 40 ejaculates of five Merino ram were used in the study. Semen samples, which were diluted with a Tris-based extender containing 10mM raffinose, 5mM hypotaurine, 5mM raffinose +2.5mM hypotaurine (H+R) and no antioxidant (control), were cooled to 5°C and frozen in 0.25ml French straws and stored in liquid nitrogen. Frozen straws were then thawed individually at 37°C for 25s in a water bath for evaluation.The addition of raffinose led to higher percentages of subjective and CASA motilities (47.5±12.2%, 46.3±13.6%) compared to controls (38.8±13.8%, 30.5±11.7%, P<0.05). For the CASA progressive motility, 5mM raffinose (20.12±8.82%) had increasing effect in comparison to control (10±7.94%, P<0.05) following the freeze–thawing process. Raffinose and hypotaurine led to higher viability (40.8±4.68%, 40.8±4.7%), high sperm mitochondrial activity (29.5±5.4%, 27.3±4.9%) and acrosome integrity (50.8±8.1, 50.7±4.4) percentages, compared to control groups (31.5±3.5%, 9.5±8.2%, 42.8±7.3%, P<0.05). H+R group only led to high sperm mitochondrial activity when compared to control group. In the comet test, raffinose and hypotaurine resulted in lower sperm with damaged DNA (6.2% and 3.9%) than that of control (9.1%), reducing the DNA damage. For TUNEL assay, The TUNEL-positive cell was distinguished by distinct nuclear staining. Raffinose and H+R groups resulted in lower sperm with TUNEL-positive cell (1.5±1.2% and 2.1±0.9%) than that of control (4.9±2.5%) (P<0.05). In conclusion, findings of this study showed that raffinose and hypotaurine supplementation in semen extenders provided a better protection of sperm parameters against cryopreservation injury, in comparison to the control groups.