Synergistic Effects of Cysteine and Ascorbic Acid on Post-Thaw Rabbit Semen Quality and Fertility During the Hot Summer Season by Modulating Redox and Endoplasmic Reticulum Stress

The present work aims to determine whether supplementation of cysteine and ascorbic acid in rabbit semen extender can maintain post-thawing sperm physiochemical quality and farm fertility under ambient heat stress conditions, and to clarify the molecular mechanisms involved. Mature bucks (n = 15) were used to collect semen samples, which were pooled and divided into six portions, including the control (C0A0) that extended with Tris-Citric-Glucose (TCG) without any additives. The other extenders were prepared by supplementing TCG with 5 mM cysteine (C5A0); 10 mM cysteine (C10A0); 5 mM cysteine and 5 mM ascorbic (C5A5); 10 mM cysteine and 5 mM ascorbic (C10A5) and 5 mM ascorbic (C0A5). Physiochemical characteristics and transcript abundance of sperm viability and redox status genes were measured in post-thawed semen samples. Results revealed that antioxidant supplementation, especially C5A5 and C10A5, significantly improved post-thaw sperm quality, redox status, and transcript abundance compared to control. This enhanced biochemical profile reduced oxidative stress, subsequently increasing conception rates (ranging from 44.0% to 56.0% in treated groups vs. 40.0% in control) and litter sizes (5.58 - 5.92 in combined treatment groups vs. 4.44 in control). At the molecular regulation level, antioxidant transcripts (SOD1, CAT, PRDX6, TXN, and NRF2) were upregulated while endoplasmic reticulum stress genes (CHOP, XBP1 and ATF6) were downregulated in C5A5 and C10A5 compared with the control. Conclusively, supplementing rabbit semen extenders with cysteine (5 or 10 mM) combined with ascorbic acid (5 mM) synergistically enhance post-thaw sperm quality and in-vivo fertility under ambient heat stress conditions by maintaining sperm DNA and acrosome intactness through modulation of redox enzymes and gene networks regulating cellular death and defense against stress.