Effects of substitution of dietary fishmeal with protein blend and stocking density on growth performance, stress response, and protein utilization of gibel carp (Carassius gibelio, CAS V)

Stocking density and feed protein sources are critical in aquaculture. This study investigated their effects on growth, stress response, and protein utilization in gibel carp. Fishmeal served as the control diet (diet 1), while it was replaced by a blend of Tenebrio molitor meal, Chlorella meal, Clostridium autoethanogenum protein, and cottonseed protein concentrate in various ratios: 1:1:8:2 (diet 2), 1:1:6:4 (diet 3), and 1:1:4:6 (diet 4). Fish were raised at either a low stocking density (100 fish/cage, 20 fish/m 3 ) or a high density (400 fish/cage, 80 fish/m 3 ), with 6000 fish in total. The interaction of stocking density and diet affected growth and protein utilization. At a stocking density of 20 fish/m 3 , diet 2 effectively replaced fishmeal without compromising growth performance. At 80 fish/m 3 , only diet 3 achieved full fishmeal replacement without negative impacts on growth. High stocking density induced chronic stress, as seen by lower plasma glucose and higher muscle heat shock protein 70 and lactate levels. It also impaired intestinal function and muscle growth at the transcript level, reducing villus height and resulting in poorer growth performance. Diet 4 decreased trypsin and chymotrypsin activities, lowered villus height and the villus height/crypt depth ratio, and reduced plasma and muscle amino acid levels. It also downregulated genes involved in muscle protein synthesis, leading to reduced protein utilization. In conclusion, the optimal protein blend ratio was 1:1:8:2 ​at lower stocking densities and 1:1:6:4 ​at higher stocking densities, both of which achieved growth performance comparable to fishmeal. This density-specific protein blending strategy offers a scalable approach to formulating fishmeal-free feeds for intensive aquaculture systems without compromising productivity. • Stocking density influenced the utilization of blended proteins in gibel carp. •High stocking density induced stress responses. •High stocking density compromised intestinal function and inhibited muscle growth. •An optimal protein blend could fully replace fishmeal under high stocking density.