Influences of fishmeal substitution with protein blends on growth performance, protein digestibility, intestinal health, and gut microbiota of gibel carp (Carassius gibelio var. CAS Ⅴ) under thermal stress

Temperature is a crucial environmental factor for poikilothermic aquatic species and can affect their protein utilization. This study examined the impacts of temperature (optimal 27 °C vs. high 33 °C) and protein blends (formulated at 1: 1: 8: 2, 1: 1: 6: 4, and 1: 1: 4: 6 ratios using Chlorella meal, Tenebrio molitor meal, Clostridium autoethanogenum protein, and cottonseed protein concentrate as fishmeal substitutes) on growth performance, health status, and gut microbiota of gibel carp. Healthy fish (3. 45 ± 0. 03 g) were randomly distributed among 24 tanks (30 fish/tank), with each temperature condition assigned four experimental diets and each treatment replicated in three tanks for an 8-week feeding trial. High temperature (33 °C) enhanced feed intake and final body weight, but reduced feed efficiency (FE) and the apparent digestibility coefficients of crude protein and total essential amino acid. It also decreased intestinal superoxide dismutase, glutathione, and glutathione peroxidase activities, increased malondialdehyde content and elevated heat shock protein 70 (HSP70) and HSP90 levels. Moreover, it down-regulated il10 and tgfβ transcription and reduced villus height with lymphocyte infiltration. Furthermore, there was an enrichment in phyla Proteobacteria and Actinobacteriota and genera Mycobacterium, while beneficial Cetobacterium abundance was decreased. Compared to fishmeal, the protein blend in a 1: 1: 6: 4 ratio improved FE and protein retention efficiency at 33 °C. Concurrently, this blend augmented the abundance of phyla Firmicutes and genera Clostridiumₛensuₛtricto₁2. In summary, although elevated temperature enhanced growth via hyperphagia, it substantially compromised intestinal health and nutrient utilization. This adverse effect was mitigated through nutritional intervention with the optimized 1: 1: 6: 4 protein blend formulation.