Gossypol is the main limiting factor in the application of cottonseed meal in grass carp feed production: Involvement of growth, intestinal physical and immune barrier, and intestinal microbiota

Abstract

The objective of this study is to investigate the effects of free gossypol in cottonseed meal (CM) on the growth and intestinal health of grass carp, as well as to determine whether free gossypol is the primary factor restricting high-level inclusion of cottonseed meal. This study was designed with five groups: the control group (32% soybean concentrate protein), the CM group (44% CM), the cottonseed protein concentrate (CPC) group (33% CPC), and the CPC and control groups with 400 and 600 ​mg/kg gossypol added, designated as CON, CM, CPC, CPC ​+ ​GP, and CON ​+ ​GP, respectively. The experiment lasted eight weeks, and each group had three replicates. This experiment employed one-way ANOVA and Duncan's multiple comparisons of the means. The findings revealed that in comparison to the CON group, the growth of fish in the CM, CPC ​+ ​GP, and CON ​+ ​GP groups significantly decreased. Intestinal inflammation damage was observed. This was indicated by a significant upregulation of pro-inflammatory factors, including tnf-α, nf-κb, il-6, il-8, il-12β, and il-1β, along with significant downregulation of anti-inflammatory factors, including tgf-β1, il-15, and il-10. Additionally, significant downregulation of antioxidant enzyme-related genes, including nrf2, cat, CuZnsod, gpx4, and gpx1, was observed and the intestinal physical barrier function was compromised. In addition, the intestinal microbiota composition was affected, with a significant reduction in Bacillus and Cetobacterium abundances and a remarkable increase in the abundance of Aeromonas, resulting in dysregulation of the intestinal microbiota function. However, in comparison to the CM group, the growth rate of fish in the CPC group exhibited a marked increase; nonetheless, it persisted at a level lower than that observed in the CON group and the degree of intestinal damage was significantly improved. Additionally, the intestinal microbiota structure was found to be similar to that of the CON group. In conclusion, excessive CM negatively affects grass carp growth and intestinal health, whereas replacing CM with CPC mitigates these effects. Adding gossypol equivalent to CM group in CON and CPC diets reduces growth performance and impairs intestinal function, indicating gossypol as a primary limiting factor for high CM inclusion in diets.