BACKGROUND
This study evaluated whether dietary alpha-lipoic acid (α-LA), a widely recognized antioxidant, could improve growth, biochemical health markers, liver histology, antioxidant status, and hepatic gene expression in juvenile hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus polyphekadion ♂). The rationale was that aquaculture intensification increases environmental and disease stress, and antioxidant supplementation may help preserve physiological homeostasis. Although α-LA has been studied in several aquatic species, the authors noted that there were no prior reports of α-LA supplementation in grouper diets.
METHODS
Four isonitrogenous diets were prepared containing 0 (SL0), 0.4 (L1), 0.6 (L2), and 1.2 (L3) g/kg α-LA. A total of 360 fish with an initial body weight of 24.06 ± 0.15 g were randomly assigned to 12 fiberglass tanks, with 3 replicates per diet group, and fed for 56 days in an indoor flowing-water system. Water temperature was maintained at 28.5 ± 2.0 °C, pH at 7.6–8.2, dissolved oxygen above 6 mg/L, and total nitrite and ammonia below 0.04 mg/L. Fish were fed twice daily at 5–8% of body weight. Outcomes included growth performance, feed utilization, serum biochemical indexes, liver histomorphology, and liver antioxidant parameters. Serum markers included triglyceride (TG), total cholesterol (TCHO), total protein (TP), albumin (ALB), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), aspartate aminotransferase (AST), and alanine aminotransferase (ALT). Liver antioxidant markers included glutathione peroxidase (GSH-Px), catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA). Transcriptome sequencing was performed on liver tissue from SL0 and L3 using DESeq2, with differentially expressed genes defined as FDR < 0.05 and |log2(Fold Change)| > 1.
KEY RESULTS
Growth performance was negatively affected at the lower α-LA doses. Weight gain rate (WGR) was 333.26 ± 30.76% in SL0, 312.25 ± 3.37% in L1, 296.33 ± 17.75% in L2, and 338.19 ± 19.59% in L3. The authors reported that WGR was significantly lower in L1 and L2 than in SL0 and L3 (p < 0.05). Feed conversion ratio (FCR) was 0.82 ± 0.06 in SL0, 0.83 ± 0.05 in L1, 0.88 ± 0.06 in L2, and 0.81 ± 0.04 in L3, with L2 significantly higher than the other groups (p < 0.05). No significant differences were found in other growth or morphology indices.
Serum biochemical findings suggested improved protein-related measures and lower lipid and liver injury markers at the highest dose. TG decreased from 1.19 ± 0.22 mmol/L in SL0 to 0.80 ± 0.06 mmol/L in L3, and TCHO decreased from 1.60 ± 0.14 mmol/L in SL0 to 1.21 ± 0.15 mmol/L in L3; L3 was significantly lower than SL0 for both, and lower than L1 for TG (p < 0.05). TP increased from 19.32 ± 0.58 g/L in SL0 to 24.42 ± 1.57 g/L in L1, 28.48 ± 2.85 g/L in L2, and 29.44 ± 3.52 g/L in L3; SL0 was significantly lower than the other three groups (p < 0.05). ALB was 4.45 ± 0.32 g/L in SL0, 4.03 ± 0.51 g/L in L1, 5.26 ± 0.70 g/L in L2, and 5.99 ± 0.35 g/L in L3, with L3 significantly higher than SL0 and L1 (p < 0.05). LDL-C was unexpectedly higher in L2 at 0.80 ± 0.02 mmol/L and in L3 at 0.42 ± 0.06 mmol/L compared with SL0 at 0.34 ± 0.05 mmol/L and L1 at 0.29 ± 0.01 mmol/L (p < 0.05). AST decreased from 41.89 ± 3.77 U/L in SL0 to 33.40 ± 2.12 U/L in L3, and ALT decreased from 581.11 ± 18.34 U/L in SL0 to 390.53 ± 19.07 U/L in L3; the authors reported significantly lower AST in L3 than the other groups and significantly higher ALT in SL0 than the other groups (p < 0.05).
Liver histology showed qualitative improvement in all α-LA groups. The control group showed serious cell vacuolation, swelling, disordered arrangement, and nuclear migration, whereas L1, L2, and L3 showed reduced vacuolation and nuclear migration with more regular cell morphology.
Antioxidant capacity improved mainly at the 0.6 and 1.2 g/kg doses. GSH-Px rose from 312.35 ± 12.26 U/mgprot in SL0 to 346.87 ± 6.48 in L1, 350.37 ± 6.88 in L2, and 371.72 ± 7.18 in L3; L3 was significantly higher than all other groups, and L1 and L2 were significantly higher than SL0 (p < 0.05). SOD was 51.64 ± 1.61 U/mgprot in SL0, 44.02 ± 3.86 in L1, 62.01 ± 4.33 in L2, and 61.03 ± 5.33 in L3; L2 and L3 were significantly higher than SL0 and L1 (p < 0.05). CAT and MDA showed no significant differences (p > 0.05).
Transcriptome sequencing generated 37760784900 bp of RawData and 37,229,629,218 bp of CleanData. GC content ranged from 49.58–50.10%, Q20 exceeded 98.13%, and Q30 exceeded 94.62%. Comparing SL0 with L3, 42 differentially expressed genes were identified, including 31 up-regulated and 11 down-regulated genes. Notable genes included ifnk (log2(FC) +10.632, p-Value 0.000, FDR 0.039), prl3b1 (+12.004, 0.000, 0.000), prl4a1 (+11.728, 0.000, 0.000), ctsl (+7.349, 0.000, 0.000), gapdh (−6.806, 0.000, 0.000), and eno1 (+1.640, 0.000, 0.010). KEGG analysis found 12 significantly enriched pathways, including the JAK/STAT signaling pathway, prolactin signaling pathway, antigen processing and presentation, and glycolysis/gluconeogenesis.
CLINICAL IMPLICATIONS
This is an aquaculture nutrition study rather than a human clinical investigation, but its applied significance is clear for fish health management. The data suggest that α-LA does not support growth at 0.4 or 0.6 g/kg and therefore may impair production efficiency at these doses. In contrast, 1.2 g/kg α-LA did not significantly improve growth, but it reduced TG, TCHO, AST, and ALT, improved liver histologic appearance, increased GSH-Px and SOD activity, and altered hepatic immune and glucose homeostasis pathways. Overall, the study supports 1.2 g/kg α-LA as a potentially useful feed additive for improving liver health and antioxidant defense in juvenile hybrid grouper, while indicating that the optimal dose for growth remains unresolved.