Effects of Starvation and Refeeding on Growth, Digestion, Nonspecific Immunity and Lipid-Metabolism-Related Genes in Onychostoma macrolepis

**Background:** Food restriction is a common stressor for fish, which often adapt by mobilizing energy reserves, particularly lipids. Onychostoma macrolepis is a national second-class protected animal in China with both conservation and aquaculture value. No prior studies had examined the regulation of lipid metabolism gene expression during short-term starvation and refeeding in this species. This study aimed to characterize the physiological and metabolic changes in O. macrolepis during starvation and subsequent refeeding.

**Methods:** 120 fish (initial weight 2.50 ± 0.14 g and 2.51 ± 0.17 g) were randomly assigned to two groups with six replicates each: a control group (continuous feeding for six weeks) and a starved–refed group (starvation for three weeks, then refeeding for three weeks). Fish were fed a commercial diet (crude protein ≥35%, crude fat ≥6%) to satiation three times daily. Samples were collected weekly. Body weight, condition factor (CF), visceral index (VSI), hepatopancreas index (HSI), and intraperitoneal fat index (IPFI) were measured. Serum triglycerides (TG), total cholesterol (T-CHOL), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) were analyzed. Intestinal digestive enzyme activities (amylase, lipase, protease) and immune enzyme activities (lysozyme [LZM], acid phosphatase [ACP], alkaline phosphatase [ALP], superoxide dismutase [SOD], glutathione peroxidase [GSH-PX], catalase [CAT]) in hepatopancreas and intestines were measured using commercial kits. TG content in hepatopancreas, muscle, and intraperitoneal fat was determined. Gene expression of hormone-sensitive lipase (HSL), carnitine palmitoyl transferase 1 isoform A (CPT-1A), sterol regulatory element binding protein 1 (SREBP1), and fatty acid synthase (FAS) was quantified by real-time qPCR. Statistical analysis used two-way ANOVA with p < 0.05 considered significant.

**Key Results:** After three weeks of starvation, the starved–refed group showed significantly lower BW (1.44 g vs. 5.72 g), CF (1.17% vs. 1.85%), VSI (3.96% vs. 6.35%), HSI (0.93% vs. 2.04%), and IPFI (0.70% vs. 1.92%) compared to controls (all p < 0.05). Serum TG (0.83 vs. 1.69 mmol/L), T-CHOL (1.15 vs. 1.86 mmol/L), HDL (1.13 vs. 1.62 mmol/L), and LDL (0.46 vs. 0.63 mmol/L) were significantly lower (p < 0.05). Intestinal amylase, lipase, and protease activities were significantly reduced (p < 0.05). Hepatic LZM, ACP, and ALP activities peaked at week 2 of starvation and were significantly higher than controls (p < 0.05). Intestinal immune enzyme activities also increased significantly. Antioxidant enzymes (SOD, GSH-PX, CAT) in both hepatopancreas and intestines were significantly elevated, while MDA levels were significantly reduced (p < 0.05). The lowest TG contents in hepatopancreas, muscle, and intraperitoneal fat were observed after starvation, with significantly upregulated expression of lipolysis genes (HSL, CPT-1A) and downregulated expression of lipogenesis genes (SREBP1, FAS) (p < 0.05). After three weeks of refeeding, BW recovered to 5.49 g, serum TG and T-CHOL showed no significant difference from controls, digestive enzyme activities (amylase, lipase) recovered to control levels, and hepatic ALP activity normalized. TG content in tissues increased, and SREBP1 and FAS expression in the hepatopancreas returned to control levels.

**Clinical Implications:** This study demonstrates that O. macrolepis can withstand three weeks of starvation by mobilizing lipid reserves through enhanced lipolysis and fatty acid oxidation while suppressing lipogenesis, and by upregulating antioxidant and immune defenses. The compensatory growth and recovery of lipid metabolism upon refeeding suggest that short-term fasting followed by refeeding is a viable strategy in aquaculture, potentially reducing feed costs without permanently impairing fish health. These findings provide a physiological and molecular reference for the large-scale culture, proliferation, and release of this protected species.