| Summary: | (1) Background: This study aimed to detect whether alpha-lipoic acid (ALA) supplementation could reduce lead (Pb)-induced testicular toxicity in roosters. (2) Methods: A total of 48 roosters, aged 20 weeks, were selected and randomly allocated to six treatment groups: basic diet (CON); CON + 150 mg/kg (CH<sub>3</sub>OO)<sub>2</sub>Pb (LPB); CON + 300 mg/kg (CH<sub>3</sub>OO)<sub>2</sub>Pb (HPB); CON + 300 mg/kg ALA (ALA); LPB + 300 mg/kg ALA (ALP); and HPB + 300 mg/kg ALA (AHP). (3) Results: The testicular Pb content was obviously higher in the LPB and HPB groups than in the CON group, while ALA supplementation reduced the testicular Pb content (<i>p</i> < 0.05). Roosters showed a significant increase in serum testosterone, sperm viability, sperm concentration, and testicular score in the AHP group compared with the HPB group. Pb exposure caused a remarkable increase in sperm abnormality and testicular malondialdehyde level, which were down-regulated by ALA supplementation (<i>p</i> < 0.05). RNA sequencing identified 227 differentially expressed genes (DEGs) between the HPB and CON groups and 220 DEGs between the HPB and AHP groups. (4) Conclusions: ALA supplementation mitigated Pb-induced testicular damage, suggesting its potential as a therapeutic agent for Pb toxicity in birds and potentially other species.
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