Substituting fish meal with castor meal in diets of hybrid grouper (Epinephelus fuscoguttatus♀ × E. lanceolatus♂): Effects on growth performance, immune response, antioxidant and digestive enzyme activities, gut morphology, and inflammatory-related gene expression.

The present study was conducted to investigate the effects of replacing fishmeal (FM) with castormeal (CM) on the growth performance, immune response, antioxidant and digestive enzyme activities, intestinal morphology, and expression of inflammatory-related genes in juvenile hybrid grouper (Epinephelus fuscoguttatus♀ ×E. lanceolatus♂). Six iso-nitrogenous (50% crude protein) and iso-lipidic (10% crude lipid) diets were formulated; namely, a reference diet (FM) containing 50% FM and five experimental diets (4% (CM4), 8% (CM8), 12% (CM12), 16% (CM16), and 20% (CM20)) in which FM protein was substituted with CM at varying levels to feed fish (initial weight: 9.12 ± 0.01 g) for 8 weeks. The results showed that the final weight, weight gain rate, and specific growth rate were highest in the FM, CM4, and CM8 groups, whereas the feed conversion ratio, hepatosomatic and viscerosomatic indexes were significantly enhanced in the CM4 group in comparison to the others. The CM4 and CM12 groups were observed to show the highest intestinal length index values compared to the other groups, with the CM20 revealing the worst growth performance. The serum total protein content first increased (P < 0.05) in the CM4 group and decreased (P < 0.05) afterward. Nonetheless, a decreasing significant (P < 0.05) cholesterol and triglyceride contents were witnessed with the increasing replacement of FM with CM. Compared to the control group, a significant increase (P < 0.05) in the activities of serum and liver immunoglobulin-M, superoxide dismutase, glutathione peroxidase, total antioxidant capacity, and complement-3 (except serum activity for CM12 group); liver lysozyme; intestinal amylase, and lipase, was witnessed in the CM groups. However, the serum lysozyme activity was highest (P < 0.05) in the CM4 group and lowest in the CM20 group. While the least serum malondialdehyde contents were observed in the CM4 group, that of the liver malondialdehyde was least witnessed in the FM, CM4, CM8, CM12, and CM16 groups as compared to the CM20. The intestinal histological examination revealed a significantly decreasing trend for villi height and villi width with increasing replacement levels. However, the muscle thickness, crypt depth, and type II mucus cells first increased upto 4% replacement level and later decreased. The increasing of dietary replacement levels significantly up-regulated pro-inflammatory (il-1ß, tnf-α, myd88, ifn-γ, tlr-22, and il-12p40) and down-regulated anti-inflammatory (il-10, tgf-ß, mhc-iiß) and anti-bacterial peptide (epinecidin and hepcidin) mRNA levels in the intestine. The mRNA levels of il-6 was up-regulated firstly upto 4 and 8% replacement levels, and later down-regulated with increasing replacement. These results suggested that, although higher dietary CM replacement enhances the immune, antioxidant and digestive enzymes, it aggravates intestinal inflammation. Replacing 4 and 8% of FM with CM could enhance the growth performance of fish.

Ultra-Performance Liquid Chromatography-Mass Spectrometry-Based Untargeted Metabolomics Reveals the Key Potential Biomarkers for Castor Meal-Induced Enteritis in Juvenile Hybrid Grouper (Epinephelus fuscoguttatus♀ × E. lanceolatus♂).

The intensification of aquaculture to help kerb global food security issues has led to the quest for more economical new protein-rich ingredients for the feed-based aquaculture since fishmeal (FM, the ingredient with the finest protein and lipid profile) is losing its acceptability due to high cost and demand. Although very high in protein, castor meal (CM), a by-product after oil-extraction, is disposed-off due to the high presence of toxins. Concurrently, the agro-industrial wastes' consistent production and disposal are of utmost concern; however, having better nutritional profiles of these wastes can lead to their adoption. This study was conducted to identify potential biomarkers of CM-induced enteritis in juvenile hybrid-grouper (Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂) using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) alongside their growth and distal intestinal (DI) health evaluation. A total of 360 fish (initial weight = 9.13 ± 0.01g) were randomly assigned into three groups, namely, fish-meal (FM) (control), 4% CM (CM4), and 20% CM (CM20). After the 56-days feeding-trial, the DI tissues of FM, CM4, and CM20 groups were collected for metabolomics analysis. Principal components analysis and partial least-squares discriminant-analysis (PLS-DA, used to differentiate the CM20 and CM4, from the FM group with satisfactory explanation and predictive ability) were used to analyze the UPLC-MS data. The results revealed a significant improvement in the growth, DI immune responses and digestive enzyme activities, and DI histological examinations in the CM4 group than the others. Nonetheless, CM20 replacement caused DI physiological damage and enteritis in grouper as shown by AB-PAS staining and scanning electron microscopy examinations, respectively. The most influential metabolites in DI contents identified as the potential biomarkers in the positive and negative modes using the metabolomics UPLC-MS profiles were 28 which included five organoheterocyclic compounds, seven lipids, and lipid-like molecules, seven organic oxygen compounds, two benzenoids, five organic acids and derivatives, one phenylpropanoids and polyketides, and one from nucleosides, nucleotides, and analogues superclass. The present study identified a broad array of DI tissue metabolites that differed between FM and CM diets, which provides a valuable reference for further managing fish intestinal health issues. A replacement level of 4% is recommended based on the growth and immunity of fish.