This study assessed the optimum dietary vitamin B12 requirement of Pacific white shrimp, Penaeus vannamei, for growth, feed efficiency, hemocyte counts, innate immunity, and ammonia stress resistance. Semi-purified experimental diets were prepared by adding vitamin B12 at 0.0, 0.5, 1.0, 2.0, and 4.0 mg/kg (Con, CB0.5, CB1.0, CB2.0, and CB4.0) and a negative control (NCon) diet was prepared with 0.3% tetracycline hydrochloride. Four replicate groups of shrimp (0.17 ± 0.01 g, 32 shrimp per tank) were fed one of the diets at 3–6% of body Weight for 8 weeks. CB2.0 diet significantly increased weight gain and feed efficiency of the shrimp compared to Con diet. The expressions of insulin-like growth factor (IGF)-1, IGF-binding protein, regulatory-associated protein of mammalian target of rapamycin-like, crustin, methylmalonyl-CoA mutase, and methionine synthase-like genes in hepatopancreas were significantly upregulated with increasing dietary vitamin B12 levels. Total hemocyte count, nitroblue tetrazolium, lysozyme, and antiprotease activities were significantly increased at higher vitamin B12 inclusion levels above 1.00 mg/kg. Phenoloxidase, superoxide dismutase, glutathione peroxidase activities, and survival during the ammonia challenge were significantly higher in C2.0 group than in Con group. After the challenge test, C4.0 and C1.0 groups exhibited significantly higher nitroblue tetrazolium and phenoloxidase activities, respectively. Increasing dietary vitamin B12 levels linearly increased hemolymph, hepatopancreas, and fecal vitamin B12 levels. The polynomial regression analysis of final body weight indicated that the optimum dietary vitamin B12 requirement for juvenile Pacific white shrimp is 2.02 mg/kg.
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