Effects of ascorbic acid supplementation on the post-thaw quality of epididymal bull spermatozoa

Cryopreservation of bovine semen supports artificial insemination by allowing the long-term storage and dissemination of genetic material; however, it also causes oxidative stress and structural damage to spermatozoa, decreasing motility and fertilising capacity. This study evaluated the effects of vitamin C, an antioxidant, on the post-thaw quality of bull epididymal spermatozoa. Sperm samples from 14 Holstein-Friesian bulls were cryopreserved in a Tris-based extender supplemented with vitamin C at 0, 25, 50, 100, and 200 µg/mL. After thawing, sperm motility parameters were assessed using computer-assisted sperm analysis to determine the optimal vitamin C concentration. Moderate supplementation levels (50–100 µg/mL) significantly improved total and progressive motility compared with the control, whereas 25 µg/mL did not and 200 µg/mL significantly reduced sperm motility. Average path velocity values were significantly higher at 50 and 100 µg/mL than at the other concentrations tested. In addition, while more sperm were motile at 50 μg/mL, they moved with greater speed and directionality at 100 μg/mL. At all concentrations (particularly 100 μg/mL), the curvilinear velocity was higher than the straight-line velocity, indicating a natural swimming pattern that is highly oscillatory rather than linear. A pronounced decline in all evaluated parameters was observed at 200 μg/mL, particularly in straight-line velocity, which was ±50% lower than that of the control group. This may suggest that excessive vitamin C concentrations impair progressive sperm movement, leading to more erratic or circular motility rather than effective forward progression. These findings indicate that vitamin C can enhance bovine epididymal spermatozoa cryo-survival at optimal concentrations, while excessive supplementation may be detrimental. (Submitted 21 April 2026; Accepted 05 October 2026; Published 03 November 2026)