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

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.