Abstract
This study aimed to evaluate the nutritional efficacy of various cottonseed meal processing methods to enable cottonseed meal to be used as an alternative to soybean meal in aquatic feed for grass carp (Ctenopharyngodon idella). A total of 720 juvenile fish (50.02 ± 0.23 g) were randomly assigned to six diets; one with 44% soybean meal (SBM) and five where SBM was fully replaced by unprocessed (CM), hydrolyzed (HCM), concentrated (CPC), roasted (RCM), or fermented cottonseed meal (FCM) for an eight-week feeding trial with three replicates of 40 fish each. Fish fed CM diets showed significantly impaired (P < 0.05) growth, muscle quality, antioxidant capacity, intestinal morphology, and microbiota structure. Reprocessed CM diets mitigated these adverse effects to varying extents, with HCM yielding the most notable improvements. Specifically, the HCM, RCM, and CPC groups exhibited improved growth performance, enhanced digestive enzyme activities, elevated muscle free amino acid levels, and significant upregulation of intestinal antioxidant genes (nrf 2, cat, cuznsod, gpx 1, and gpx 4), along with reduced expression of inflammatory markers (tnf-α, il-1β, il-6, il-8, il-12β, and nf-κb) (P < 0.05). Notably, the HCM group outperformed all other reprocessed CM groups in these metrics, whereas the FCM group showed no significant (P < 0.05) improvement compared to the unprocessed CM group. These effects were primarily mediated by modulation of the nuclear factor erythroid 2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) and nuclear factor kappa-B (NF-κB) signaling pathways. Moreover, the HCM diet reversed the CM-induced elevation of the potential pathogen Aeromonas (P < 0.001) and the reduction of beneficial genera such as Akkermansia (P < 0.001) and Fusibacter (P < 0.001), and further increased the abundance of Streptococcus (P = 0.001). These findings underscore that hydrolysis markedly improved the nutritional and functional properties of cottonseed meal, supporting its potential as a viable and sustainable protein source for aquaculture feeds.