Effects of complex protein sources on the growth and intestinal microflora of hybrid grouper

Abstract: Methanotroph bacteria meal (MBM) is one type of single-cell protein produced by Methylococcus capsulatus. The greenhouse gas methane can be taken as the sola carbon source, similar to the protein content and amino acid composition of the fish meal. Cottonseed meal (CSM) is a reliable plant protein source with more than 60% protein content by removing some fiber from cottonseed. According to the amount of MBM in the previous experiment, this study aims to improve intestinal health and the safe application range of CSM in the fishmeal replacement for fish. A complex protein (MC) mixed with the 4% MBM and 12% CSM was formulated and then used in the hybrid grouper's feed. An investigation was made to clarify the effects of MC in the fishmeal-reducing diet on the growth performance, body composition, serum biochemical indices, liver and intestinal histomorphology, and the intestinal microflora of Epinephelus fuscoguttatus ♀×E. lanceolatus ♂. An 8-week feeding trial was conducted with the 180 healthy juvenile grouper in six tanks (30 fish in each tank, and 3 tanks in each group) at an average weight of (28.85 ± 0.04) g. Two iso-nitrogenous and lipid diets were formulated to contain a control group (FM group) with 40% fishmeal as the basic protein source, and an experimental group (MC group) that reduced the fishmeal content to 30% and added the MC (consisting of 4% MBM and 12% CSM) to balance the protein content. The results showed that there was no significant difference in the weight gain rate, specific growth rate, survival rate, feed conversion ratio, feed intake rate, fatness, viscera/body index, and liver/body index in the two groups. The moisture of whole fish in the FM group was higher than that in the MC group. The contents of alanine, histidine and arginine in muscle were significantly lower than those in the MC group, whereas, the content of proline in muscle was significantly higher than that in the MC group. The contents of serum triglyceride, total cholesterol, aspartate aminotransferase activity, and liver malondialdehyde in the MC group were significantly lower than those in the FM group. The total intestinal antioxidant capacity in the MC group was significantly higher than that in the FM group, whereas, the length of intestinal villi was significantly longer, and the intestinal wall thickness was significantly lower than that in the FM group. 16s rDNA sequencing results showed that the Chao1 and ACE indexes of alpha diversity of intestinal microbiota in the MC group were lower than those in the FM group, while the Shannon and Simpson indexes were higher than those in the FM group. There was a variation in the structure of intestinal microflora in the two groups. In the MC group, the relative abundance of Proteobacteria decreased at the phylum level, and the relative abundance of Alpharoteobacteria and Deltaroteobacteria increased, whereas, the relative abundance of Gammaroteobacteria decreased at the class level, while the total abundance of Vibrio, Photobacterium and Aeromonas decreased at the genus level. The microbial functional taxa predicted that the potential pathogen taxa decreased in the MC group. The KEGG pathways enrichment analysis of intestinal microflora genes showed that the MC group presented a much higher enrichment in the metabolism and organic system than the FM group, but a lower enrichment in the cell process and environmental information processing. To sum up, there was no negative effect on the juvenile grouper's survival, the growth and feed utilization feeding MC added in the fish meal-reducing diet. There was a significant increase in the dry matter of whole fish, in order to improve the antioxidant capacity of the liver and intestine. The intestinal morphological development was stimulated to enhance the intestinal microbial richness, and then to reduce the pathogenic bacteria abundance for a stable intestinal microflora. Anyway, the optimal strategy can be the complex protein mixed with the MBM and CSM used in a fishmeal-reducing diet for the juvenile hybrid grouper. As such, the utilization efficiency of protein sources can be improved by the optimal combination.