Alpha-Lipoic Acid Alleviates Lead-Induced Testicular Damage in Roosters by Reducing Oxidative Stress and Modulating Key Pathways

Abstract

(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 (CH₃OO)₂Pb (LPB); CON + 300 mg/kg (CH₃OO)₂Pb (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 (p < 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 (p < 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.

Keywords: Pb; alpha-lipoic acid; oxidative stress; rooster; testicular toxicity.

Figure 1

The effects of ALA supplementation on serum testosterone levels and the testicular Pb content in Pb-exposed roosters. (A) A statistical analysis of serum testosterone levels. (B) A statistical analysis of the testicular Pb content. CON, roosters fed a basal diet; LPB, roosters fed a basal diet supplemented with 150 mg/kg (CH₃OO)₂Pb; LPB, roosters fed a basal diet supplemented with 300 mg/kg (CH₃OO)₂Pb; ALA, roosters fed a basal diet supplemented with 300 mg/kg ALA; ALP, roosters fed a basal diet supplemented with 300 mg/kg ALA and 150 mg/kg (CH₃OO)₂Pb; AHP, roosters fed a basal diet supplemented with 300 mg/kg ALA and 300 mg/kg (CH₃OO)₂Pb. The data are presented as means ± SEM (n = 8). Bars with different letters indicate significant differences (p < 0.05).

Figure 2

The effects of ALA supplementation on testicular histomorphology in Pb-exposed roosters. (A–F) HE images of the testis. (G) A statistical analysis of the seminiferous epithelium height. (H) A statistical analysis of the seminiferous tubule diameter. (I) A statistical analysis of the testis scores. CON, basal diet; LPB, basal diet supplemented with 150 mg/kg (CH₃OO)₂Pb; LPB, basal diet supplemented with 300 mg/kg (CH₃OO)₂Pb; ALA, basal diet supplemented with 300 mg/kg ALA; ALP, basal diet supplemented with 300 mg/kg ALA and 150 mg/kg (CH₃OO)₂Pb; AHP, basal diet supplemented with 300 mg/kg ALA and 300 mg/kg (CH₃OO)₂Pb. The triangle indicates spermatogenic cell shedding, and the arrow indicates vacuolation of the spermatogenic epithelium. Scale bar = 20 μm. The data are presented as means ± SEM (n = 8). Bars with different letters denote significant differences (p < 0.05).

Figure 3

Effects of ALA supplementation on testicular oxidative status in Pb-exposed roosters. (A) Total antioxidant capacity of the testis. (B) Superoxide dismutase activity of the testis. (C) Testicular malondialdehyde content. (D) Testicular catalase activity. (E) Glutathione peroxidase activity of the testis. T-AOC, total antioxidant capacity; SOD, superoxide dismutase; MDA, malondialdehyde; CAT, catalase; GPx, glutathione peroxidase. Groups: CON, basal diet; LPB, basal diet supplemented with 150 mg/kg (CH₃OO)₂Pb; LPB, basal diet supplemented with 300 mg/kg (CH₃OO)₂Pb; ALA, basal diet supplemented with 300 mg/kg ALA; ALP, basal diet supplemented with 300 mg/kg ALA and 150 mg/kg (CH₃OO)₂Pb; AHP, basal diet supplemented with 300 mg/kg ALA and 300 mg/kg (CH₃OO)₂Pb. Data are presented as means ± SEM (n = 8). Bars with different letters indicate significant differences (p < 0.05).

Figure 4

Effects of ALA supplementation on DEG expression in Pb-exposed roosters. (A) PCA plot of sample distribution. (B) Venn diagram of DEGs. (C,D) Volcano plot of significant DEGs detected from data sets. (E,F) Heatmaps showing distribution of DEGs in data sets. Groups: CON, basal diet; HPB, basal diet supplemented with 300 mg/kg (CH₃OO)₂Pb; AHP, basal diet supplemented with 300 mg/kg ALA and 300 mg/kg (CH₃OO)₂Pb. SC, subcluster.

Figure 5

A KEGG pathway enrichment analysis of DEGs in two comparison groups of rooster testes. (A) HPB vs. CON. (B) AHP vs. HPB. Groups: CON, basal diet; HPB, basal diet supplemented with 300 mg/kg (CH₃OO)₂Pb; AHP, basal diet supplemented with 300 mg/kg ALA and 300 mg/kg (CH₃OO)₂Pb. The X-axis represents the rich factor, which is the ratio of the number of genes enriched in this pathway to the number of annotated genes. A larger rich factor indicates a higher degree of enrichment. The Y-axis represents the pathway. Number: The size of the dots represents the number of genes in this pathway. Padjust: the p-value corrected using the Benjamini–Hochberg method. Padjust < 0.05 was considered to be statistically significant. The colors of the dots correspond to different Padjust ranges. The top 20 enrichment results are displayed under the premise that Padjust < 1.

Figure 6

The relative mRNA expression of DEGs in rooster testes. Groups: CON, roosters fed a basal diet; HPB, roosters fed a basal diet supplemented with 300 mg/kg (CH₃OO)₂Pb; AHP, roosters fed a basal diet supplemented with 300 mg/kg ALA and 300 mg/kg (CH₃OO)₂Pb. The data are presented as means ± SEM (n = 3). Bars with different letters indicate significant differences (p < 0.05).

Figure 7

Hypothetical molecular model of how ALA alleviates Pb-induced testicular damage and semen quality by reducing oxidative stress and regulating key signal pathways. Dotted arrows represent promotion.