Dietary Arginine Supplementation Mitigates Heat Stress-Induced Testicular Dysfunction Through Arginine-Related Metabolic Remodeling and Autophagy-Associated Stress Adaptation in a Rongchang Boar Model

Heat stress compromises male reproductive function in livestock and other mammals. Arginine is a conditionally essential amino acid involved in metabolic regulation; however, its protective effects on the male reproductive system under heat stress remain unclear. Using a heat-stressed Rongchang boar model, this study aimed to explore the possible mechanism of dietary arginine supplementation on sperm quality and testicular function. Twenty-four Rongchang boars were assigned to three treatments: thermoneutral control (CON), heat-stressed control (HS), or heat-stressed treatment supplemented with 0.8% dietary arginine (HA). Results showed that heat stress reduced feed intake, body weight, sperm viability, sperm concentration, and sperm motion parameters (p < 0.05), accompanied by testicular histological damage. However, arginine supplementation improved sperm viability and straight-line velocity (p < 0.05), with partial preservation of testicular morphology. Furthermore, single-cell RNA sequencing revealed heat stress-associated changes in germ-cell composition and enrichment of autophagy-related pathways. Transmission electron microscopy and qRT-PCR showed endoplasmic reticulum swelling, autophagic vesicle accumulation, and increased expression of GABARAPL1, ULK1, BECN1, and CALM3 in heat-stressed testes (p < 0.05); these alterations were attenuated by arginine supplementation, as indicated by reduced ULK1 and BECN1 expression (p < 0.05). Metabolomic and targeted analyses indicated improved circulating arginine availability and reshaped testicular arginine-related amino acid/polyamine metabolism after arginine supplementation (p < 0.05). Overall, dietary arginine supplementation alleviated heat stress-induced testicular dysfunction, which was accompanied by alterations in arginine-related metabolic regulation and autophagy-associated cellular stress responses.