Semen cryopreservation is associated with cryo-damages that compromises sperm quality parameters post-thawing. Hence, the present study was planned to evaluate the influence of supplementation of puerarin (PUE) to Tris-based extender on post-thaw semen quality parameters of ram semen, with a view to determine the optimal puerarin concentration for the cryopreservation of Hu ram semen. For this purpose, 100 ejaculates collected twice weekly from five Hu rams were used. Ejaculates collected from 5 rams on each collection day were pooled to yield 20 pooled samples for further processing. These samples were diluted with a Tris-based extender supplemented with 0, 25, 50, 75 and 100µmol/L of PUE solution in DMSO, cryopreserved and stored in liquid nitrogen. Post-thaw results showed that the addition of 75µmol/L PUE significantly improved total sperm motility, progressive sperm motility, as well as some bio-kinetic characteristics, compared with the control and other PUE supplementation groups (P<0.05). Sperm bio-kinetic characteristics including amplitude of lateral head displacement, angular displacement and wobble in PUE 75 and 100µmol/L supplementations were also higher than those of the control group (P<0.05). Moreover, sperm acrosome integrity, plasma membrane integrity, along with semen total antioxidant content, activities of catalase and superoxide dismutase enzymes, and mitochondrial membrane potential were correspondingly higher in the PUE 75µmol/L group post-thaw (P<0.05). In addition, PUE 75µmol/L treatment significantly decreased semen ROS and malondialdehyde level (P<0.05). In-vitro fertilization rate of semen in the PUE 75µmol/L group (57.14%) was higher than 38.00% for the control group (P<0.05). In conclusion, supplementation of 75µmol/L PUE to a Tris-based extender was considered optimal, as it improved post-thaw sperm motility indexes, bio-kinetic characteristics, mitochondrial function, antioxidant enzymes activity, and fertilization potential of Hu ram semen.

To Cite This Article: Sohail T, Sun Y, Chen F, Sun X, Wei Z and Li Y, 2026. Puerarin supplementation into a tris-based extender improves sperm quality, antioxidant capacity, and in vitro fertility during cryopreservation of hu ram semen. Pak Vet J, 46(5): 1284-1292. http://dx.doi.org/10.29261/pakvetj/2026.115