Phosphorus supplementation mitigates high Clostridium autoethanogenum protein-induced growth retardation and metabolic dysregulation in Procambarus clarkii

A 10-week feeding experiment was conducted to investigate the effects of dietary phosphorus supplementation on the fishmeal replacement with Clostridium autoethanogenum protein (CAP) in the diet of red swamp crayfish ( Procambarus clarkii ). Six isonitrogenous and isolipidic diets were formulated: the FM diet contains 10 % fishmeal; the CAP, CAPSP1, CAPSP2 and CAPSP3 diets were that fishmeal in the FM diet was replaced with CAP and supplemented with 0, 2.5 %, 3 % and 3.5 % Ca(H 2 PO 4 ) 2 respectively (equivalent to dietary phosphorus level of 0.66 %, 1.27 %, 1.40 % and 1.52 %); the FMSP1 diet was supplemented 2.5 % Ca(H 2 PO 4 ) 2 (equivalent to dietary phosphorus level of 1.41 %). A total of 720 crayfish with an initial mean weight of 5.01 g were selected and randomly assigned to 18 cages for feeding and sampled at the end of the experiment. The results showed that the weight gain rate and the activities of antioxidant enzymes in the hemolymph of crayfish in the CAPSP2 and CAPSP3 groups were significantly elevated compared with those in the CAP group. The crayfish in the CAPSP2 group presented the highest activity of hemolymph alkaline phosphatase (AKP) among all groups, while the CAP group had the highest malondialdehyde (MDA) content and the lowest lysozyme (LZM) activity. It was also found that supplementation of dietary phosphorus levels to 1.40 %-1.52 % significantly increased the expression of antioxidant-related genes, in contrast to the oxidative stress exhibited by the CAP group. In addition, crayfish in the CAP group showed fusion and shortening of intestinal villi, as well as increased abundance of pathogenic bacteria Shewanella . In contrast, crayfish in the CAPSP2 and CAPSP3 groups showed significant improvements in villus height and muscle layer thickness, as well as increased intestinal protease activity and abundance of probiotic bacteria Bacillus . Overall, supplementation of dietary phosphorus levels to 1.40 %-1.52 % was effective in mitigating the adverse effects of growth retardation, oxidative stress, and intestinal damage induced by high levels of CAP-replaced fishmeal. It is recommended to ensure a phosphorus level higher than 1.40 % when using CAP as a substitute for fishmeal in crayfish diets. • Dietary phosphorus alleviates growth inhibition of crayfish caused by high- Clostridium autoethanogenum protein diet • Phosphorus regulates immunity and antioxidation to improve the physiological state of crayfish • Phosphorus optimizes the intestinal microbiota and improves crayfish digestion an absorption