The effect of dietary vitamin A supplementation on growth, gonadal development, molting, immunity, and ammonia nitrogen resistance in sub-adult male Chinese mitten crab, Eriocheir sinensis

Vitamin A (VA) is closely related to male gonadal development; however, whether it can promote the speed of gonadal maturity in male Chinese mitten crab, Eriocheir sinensis, is unknown. This study investigated the effects of dietary VA on growth, gonadal development, molting, immunity, and ammonia nitrogen resistance in sub-adult male E. sinensis to determine the appropriate dietary VA supplementation level. A total of 280 crabs (61.22 +/- 0.45 g) were divided into seven experimental groups with four replicates (n = 10 per treatment replicate). Crabs in the experimental treatments were fed a basal diet (1846 IU/kg VA; 38.11% crude protein, 7.65% crude lipid) supplemented with 0, 10,000, 20,000, 40,000, 80,000, 160,000, or 320,000 IU/kg VA for 8 weeks. Subsequently, a 48-h ammonia nitrogen stress test was conducted on a subset of crabs (total 105 crabs, seven treatments with three replicates, n = 5 per treatment replicate). Orthogonal polynomial contrasts were used to determine the associations between VA levels and outcome measures. In the feeding trial, dietary VA supplementation was significantly negatively associated with final body weight and significantly positively associated with feed conversion ratio (both P < 0.01 for linear model). Final total weight of crabs in a rearing unit, hepatopancreas index, and survival rate (SR) was significantly linearly and quadratically associated with VA supplementation, with the highest levels in crabs fed 10,000-20,000 IU/kg VA-supplemented diets (both P < 0.05). VA supplementation was significantly associated with gonadosomatic index (whole gonad weight / body weight) and testis index (testis weight / body weight; all P < 0.01 for linear and quadratic models), with crabs fed a 20,000 IU/kg dietary VA-supplemented diet having the highest levels that were 1.19-fold and 1.27-fold higher than those supplemented with 0 IU/kg VA, respectively; mRNA expression of genes related to testis development [DNA meiotic recombinase 1, nanos, and cullin 4] were in alignment with this pattern. Molting frequency was also highest in crabs receiving 20,000 IU/kg dietary VA (2.00 +/- 0.08) among all experimental diets, and lowest in those receiving the 320,000 IU/kg treatment (1.75 +/- 0.13; linear and quadratic models, both P < 0.01); mRNA expression of molting-related genes [crustacean hyperglycemic hormone, molt-inhibiting hormone, ecdysone receptor, and retinoid X receptor] exhibited analogous patterns. Evaluation of immunity-related biochemical indicators [lysozyme, acid phosphatase, and alkaline phosphatase] and gene expressions [toll-like receptor 2, relish, myeloid differentiation factor 88, crustin, anti-lipopolysaccharide factor, and prophenoloxidase] indicated that the 20,000 IU/kg VA treatment conferred the highest nonspecific immunity (quadratic associations, all P < 0.05). In the acute ammonia nitrogen stress test, crabs fed a 20,000 IU/kg VA-supplemented diet for 8 weeks had the highest SR of 60%, while no crab survived in VA320000 treatment (linear and quadratic models, both P < 0.001). Antioxidant-related biochemical indicators [superoxide dismutase (sod), glutathione peroxidase (gpx), malondialdehyde, and total antioxidant capacity] and gene expressions [sod, gpx, thioredoxin 1, and peroxiredoxin 6] were also significantly quadratically associated with VA levels (all P < 0.01). In conclusion, 20,000 IU/kg dietary VA supplementation proved advantageous for growth, gonadal development, molting, immunity, and antioxidant capacity in sub-adult male E. sinensis; however, dietary VA supplementation levels over 80,000 IU/kg appeared to be detrimental to crabs.