Exogenous protease supplementation in high- and low-fishmeal diets for Pacific white shrimp (Penaeus vannamei): Comparative effect on growth, immunity, nutrient digestibility and gut health

The present study evaluated the effects of exogenous protease supplementation in low- and high-fishmeal (FM) diets on growth performance, feed utilization, innate immunity, digestive enzyme activity, nutrient digestibility, gut microbiota and intestinal morphology of Pacific white shrimp (<i>Penaeus vannamei</i>). A 2 × 3 factorial experiment was designed with two dietary FM levels (200 g/kg, positive control [PC]; and 100 g/kg, negative control [NC]) and three protease levels (0, 400 and 800 mg/kg) to obtain six experimental diets designated as PC, PC400, PC800, NC, NC400, and NC800. Six replicate groups of 30 shrimp (initial weight 0.30 ± 0.01 g) were fed the diets for 8 wk. Protease supplementation significantly improved (<i>P</i> < 0.001) growth performance and feed utilization efficiency in both high- and low-FM diets. Innate immunity and antioxidant enzyme activities were significantly enhanced (<i>P</i> < 0.001) with increasing FM and protease levels. Furthermore, the inclusion of protease in low-FM diets significantly increased (<i>P</i> < 0.001) total hemocyte count and phagocytic, phenoloxidase, lysozyme and superoxide dismutase activities, reaching levels comparable to the PC group. Increasing FM and protease levels significantly upregulated (<i>P</i> < 0.001) the expression of <i>proPO</i>, crustin, <i>TGF-β</i>, <i>Lv</i> <i>IKK-β</i> and <i>TLR3</i> genes, while downregulating <i>TNF-α</i>. The inclusion of protease in the low FM diet significantly increased (<i>P</i> < 0.05) digestive enzyme activities, intestinal villi length, whole-body amino acid composition and nutrient digestibility to the levels comparable to the PC group. The relative abundance of heterotrophic marine bacteria (<i>P</i> < 0.001), Gram-positive bacteria (<i>P</i> = 0.034) and <i>Lactobacilli</i> spp. (<i>P</i> < 0.001) in the gut significantly increased (<i>P</i> < 0.05) with increasing protease levels, while an inverse relationship was observed for <i>Vibrio</i> spp. (<i>P</i> < 0.001). These results demonstrated that protease supplementation in either the high- or low-FM diets could improve shrimp growth, feed utilization efficiency, immunity, nutrient digestibility, intestinal morphology and gut microbiome. Notably, supplementing the low-FM diet with 800 mg/kg protease improved shrimp performance, reaching levels comparable to those obtained with the PC diet.