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 10 mM raffinose, 5 mM hypotaurine, 5 mM raffinose +2.5 mM hypotaurine (H + R) and no antioxidant (control), were cooled to 5 degrees C and frozen in 0.25 ml French straws and stored in liquid nitrogen. Frozen straws were then thawed individually at 37 degrees C for 25 s 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, 5 mM 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. Crown Copyright (C) 2013 Published by Elsevier Inc. All rights reserved.