Introduction
P. vannamei is one of the most important aquaculture species worldwide, however, intensive culture conditions frequently expose shrimp to environmental stressors including salinity fluctuations and ammonia accumulation, which can compromise physiological homeostasis and reduce productivity. Nutritional strategies using functional feed additives have been extensively explored to mitigate these challenges. Astaxanthin (Ax), a lipophilic carotenoid, is known to improve growth performance, antioxidant capacity, immune responses, and stress tolerance. However, its efficacy may be constrained by limited bioavailability, potentially due to inefficient lipid digestion and emulsification in shrimp. Dietary emulsifiers (Em) have been suggested to enhance the absorption of lipophilic compounds and improve their utilization efficiency. Therefore, co-supplementation of Ax with an emulsifier may enhance its functional efficacy through improved bioavailability and potential synergistic interactions. Nevertheless, the combined effects of Ax and Em have not been systematically evaluated in P. vannamei. This study aimed to evaluate the interactive effects of dietary Ax and Em on physiological performance, immune response, antioxidant capacity, intestinal microbiota, and stress resistance under salinity and ammonia challenges in P. vannamei.
Materials and Methods
A basal diet (Con) was formulated without Ax and Em and four additional diets were prepared: 50 mg/kg Ax (A50), 25 mg/kg Ax + 350 mg/kg Em (A25E350), 50 mg/kg Ax + 350 mg/kg Em (A50E350) and 50 mg/kg Ax + 700 mg/kg Em (A50E700). Shrimp (initial body weight: 1.42 ± 0.01 g) were stocked into 25 tanks with 30 shrimp per tank in five replicates per dietary treatment and fed for 10 weeks. At the end of the feeding trial, growth performance and feed utilization were evaluated. Hemolymph, hepatopancreas, intestine and muscle samples were collected for analysis of immune responses (lysozyme, antiprotease, nitro blue tetrazolium and phenoloxidase), antioxidant capacity (superoxide dismutase, glutathione peroxidase, catalase and malondialdehyde), digestive enzyme activities (amylase, lipase, trypsin and chymotrypsin), gene expression related to antioxidant defense, pro-inflammatory and lipid metabolism, histological examination, Ax retention and meat quality (cooking loss, texture properties and color indices). Chromium oxide (Cr2O3, 1%) was incorporated into the diets as an inert marker to determine the apparent digestibility coefficients (ADCs) of protein, lipid, fatty acids and energy. Intestinal microbiota was evaluated by quantifying total viable bacteria (TVB), lactic acid bacteria (LAB), Vibrio spp., and Gram-negative bacteria using selective culture media. For the salinity stress test, shrimp (18 shrimp per tank, triplicate) were exposed to low salinity (5 psu) for 2 h, followed by recovery to normal salinity condition (25 psu). Hemolymph was collected at 2, 24 and 48 h post-exposure for flow cytometry analysis. Flow cytometry was conducted to assess hemocyte count, cell mortality, nitric oxide and reactive oxygen species production, and phagocytic activity. For the ammonia challenge, shrimp (10 shrimp per tank, triplicate) were exposed to 2.8 mg/L ammonia, and survival was monitored for 60 h.
Results
Dietary supplementation of Ax significantly improved growth performance, feed efficiency, immune response and antioxidant capacity in shrimp, with additional improvements observed when combined with an Em. Co-supplementation (A50E350 and A50E700) resulted in improved growth and feed utilization efficiency, along with elevated high-density lipoprotein levels and reduced low-density lipoprotein levels. Co-supplementation of Ax and Em enhanced phenoloxidase, lysozyme, antiprotease and antioxidant enzymes activities, while reducing malondialdehyde levels. Em supplementation groups showed significantly improved lipase activity and ADCs of protein, lipid, energy and fatty acids. Gene expression analysis revealed the upregulation of genes related to antioxidant defense (catalase, glutathione peroxidase, superoxide dismutase) and lipid metabolism along with the downregulation of pro-inflammatory (I��B kinase ��, Tumor necrosis factor-α, Interleukin-16) and stress-related genes (heat shock protein 70) in co-supplemented groups. Ax concentration in muscle was significantly increased and further enhanced by Em, accompanied by improved meat quality, including increased hardness and enhanced coloration. Under salinity stress conditions, the A25E350 group showed significantly higher cumulative survival compared to the Con group. Under ammonia stress conditions, the A50, A25E350, A50E350, and A50E700 groups exhibited significantly higher cumulative survival than the Con group. Flow cytometry analysis showed that Em supplementation increased hemocyte counts and immune cell function activities while reducing hemocyte mortality, nitric oxide production and reactive oxygen species levels. Em supplementation favorably modulated intestinal microbiota as indicated by increased lactic acid bacteria and reduced Vibrio spp.
Conclusions
Co-supplementation of Ax and an Em effectively enhanced growth performance, physiological responses and stress resistance in P. vannamei. The improved outcomes were associated with enhanced immune function, antioxidant capacity, and nutrient utilization, along with increased Ax accumulation in muscle and improved meat quality. Notably, Em supplementation enhanced the functional efficacy of Ax, resulting in favorable modulation of intestinal microbiota, as well as improved stress tolerance and immune stability under salinity and ammonia challenges. Overall, these findings indicate that dietary Em may serve as an effective nutritional strategy to enhance the bioefficacy of Ax, providing a practical nutritional approach to improve productivity and robustness in shrimp culture.