| Soybean meal has become a common alternative protein source for fish meal in aquatic feed due to its advantages of high protein content and relatively balanced amino acid composition.However,the anti-nutritional factors present in plant raw materials are one of the important factors that limit the application of plant proteins in aquatic compound feed.Glycinin and ?-conglycinin are anti-nutritional factors with high content and strong thermal stability in soybean meal,but their effects on the intestinal health and growth performance of crustaceans are rarely studied.This study investigated the effects of glycinin and ?-conglycinin on the growth and intestinal health of juvenile Chinese mitten crab(Eriocheir sinensis)by molecular biology and histomorphology.Based on the different effects of two antigen proteins on the intestinal tract of crab,we explored the improvement of N-acetylcysteine and sodium butyrate on growth inhibition and intestinal damage induced by ?-conglycinin and glycinin,respectively.The effect of sodium butyrate on the intestinal microbiota structure of crab analyzed by using 16 S RNA sequencing method.In vitro screening of probiotics can utilize glycinin and improve intestinal immunity,and then evaluate the beneficial effects of the screened probiotics on Chinese mitten crab by in vivo feeding experiments.Results in the study not only filled the gap of the nutritional regulation of the intestinal health in E.sinensis,but also can provide us a new insight to the efficient use of plant protein sources in diet of aquatic animal.1.Effects of glycinin and ?-conglycinin on growth performance and intestinal health in juvenile Chinese mitten crabsThis study investigates the effects of two soybean antigens(glycinin and ?-conglycinin)as an antinutritional substance in the diet on the growth,digestive ability,intestinal health and microbiota of juvenile Chinese mitten crabs.The isonitrogenous and isolipidic diets contained two soybean antigens at two levels each(7% and 14% ?-conglycinin,8% and 16% glycinin)and a control diet without ?-conglycinin or glycinin supplementation,and were used respectively to feed juvenile E.sinensis for seven weeks.Dietary inclusion of either glycinin or ?-conglycinin significantly reduced crab survival and weight gain.The crabs fed diets containing soybean antigens had higher malondialdehyde concentrations and lower catalase activities in the intestine than those in the control.The activities of trypsin and amylase in the intestine were suppressed by dietary ?-conglycinin and glycinin.Dietary glycinin or ?-conglycinin impaired the immunity and morphological structure of intestine,especially the peritrophic membrane.The m RNA expression of constitutive and inducible immune responsive genes(lipopolysaccharide-induced TNF-? factor and interleukin-2 enhancer-binding factor 2)increased while the m RNA expression of the main genes related to the structural integrity peritrophic membrane(peritrophin-like gene and peritrophic 2)significantly decreased in the groups with soybean antigen addition.Soybean antigen could also change the intestinal microbial community.The abundance of pathogenic bacteria(Ochrobactrum,Burkholderia and Pseudomonas)increased significantly in both soybean antigen groups.Although pathogenic bacteria Vibrio were up-regulated in the glycinin group,the abundance of Dysgonomonas that degraded lignocellulose and ameliorated the gut environment decreased in the glycinin group.This study indicates that existence of soybean antigens(glycinin or ?-conglycinin)could induce gut inflammation,reshape the community of gut microbiota,and cause digestive dysfunction,ultimately leading to impaired growth in crabs.2.Improvement of dietary N-acetylcysteine on growth inhibition and intestinal damage induced by two soybean antigens in juvenile Chinese mitten crabsIn order to investigate the mitigative effects of N-acetylcysteine(NAC)on growth inhibition and intestinal damage induced by ?-conglycinin or glycinin in juvenile Chinese mitten crabs,two experiments were designed.In experiment 1,all three experimental diets contained 7% ?-conglycinin,but with three levels of NAC in eachdiet(0%,0.05% and 0.1% respectively).There is no ?-conglycinin nor NAC in the control diet.In experiment 2,all three experimental diets contained 8% glycinin,but with three levels of NAC in each diet(0%,0.05% and 0.1% respectively).There is no glycinin nor NAC in the control diet.Juvenile crabs(3.06 ± 0.03g)were respectively fed with these diets for eight weeks.The results showed that in experiment 1,0.1% NAC significantly improved the survival and weight gain of the crab compared with those in the 0% NAC group,and showed no difference with the control group.The crabs fed diets containing ?-conglycinin had lower glutathione and catalase activities but higher malondialdehyde in the intestine than those in the control group.Moreover,dietary ?-conglycinin improved the level of histamine in the intestine and increased the activity of diamine oxidase in serum.The activities of lipase in hepatopancreas and trypsin in both hepatopancreas and intestine were restrained by ?-conglycinin,while addition of NAC reversed these negative effects.The addition of NAC restored the impaired immunity and morphological structure of the intestine.Moreover,dietary NAC downregulated m RNA levels of pro-inflammatory factor genes(lipopolysaccharide-induced TNF-? factor,Es Relish and interleukin-2 enhancerbinding factor 2)while upregulated the structural integrity peritrophic membrane related gene expression levels(peritrophin-like gene,peritrophic 1 and 2).The results showed that in experiment 2,glycinin significantly reduced the survival and weight gain of crab.0.1% NAC could only significantly improve the survival of crab,and it was still significantly lower than that of the control group.The crabs fed diets containing glycinin had lower glutathione and catalase activities but higher malondialdehyde in the intestine than those in the control group.The activities of trypsin in both hepatopancreas and intestine were restrained by glycinin,while addition of NAC reversed these negative effects.In addition,NAC alleviated the intestinal peritrophic membrane damage and down-regulation of peritrophin m RNA caused by glycinin,but it did not return to the control group.Dietary glycinin improved the levelof histamine in the intestine and increased the activity of diamine oxidase in serum,while reducing the gene expression of anti-lipopolysaccharide factors(ALF1,ALF2,and ALF3).In addition to increasing the expression of ALF1,NAC has no effect on other indicators.This study indicates that NAC is a useful feed additive to ameliorate the growth inhibition,intestinal oxidative damage and inflammation induced by ?-conglycinin in crab diets.NAC can alleviate the intestinal oxidative stress,but it has not significantly improved the intestinal peritrophic membrane damage and decreased intestinal immunity caused by glycinin.3.Sodium butyrate can improve intestinal integrity and immunity in juvenile Chinese mitten crab fed glycininSince NAC only improved the intestinal oxidative stress of juvenile E.sinensis caused by glycinin,this study investigates the mitigative effects of sodium butyrate(Na B)on growth inhibition and intestinal damage induced by glycinin in juvenile E.sinensis from the perspective of reshaping intestinal microbiota.In this experiment,based on the addition of 1 g/kg NAC in all treatment groups,all four experimental diets containing 8% glycinin were formulated with 0%,1%,2% and 4% Na B respectively.There was no glycinin or Na B in the control diet.Juvenile crabs(0.33 ± 0.01g)were respectively fed with these five diets for eight weeks.The diets with 1% and 2% Na B significantly improved the survival and weight gain of the crabs compared with those in the 0% Na B group,and showed no difference with the control group.The crabs fed diets containing glycinin without Na B had lower glutathione and glutathione peroxidase activities but higher malondialdehyde in the intestine than those in the control group.Moreover,dietary glycinin decreased the lysozyme and phenoloxidase activities and improved the level of histamine in the intestine compared with the control group,while the supplementation of Na B counteracted these negative effects.The addition of Na B could also restore the impaired immunity and morphological structureof the intestine.Dietary Na B could increase the m RNA expression of antimicrobial peptides genes(anti-lipopolysaccharide factor 1 and 2)and decrease the content of proinflammatory factor TNF-?.The Na B could restore the intestinal microbial community disorganized by glycinin.The abundance of pathogenic bacteria(Aeromonas,Vibrio and Pseudomonas)decreased significantly and the potential probiotic bacteria(Bacillus,Lactobacillus,Chitinibacter and Dysgonomonas)increased significantly in the 1% Na B group.This study suggests that sodium butyrate supplementation can mitigate the negative effects induced by glycinin such as growth inhibition,intestinal inflammation and reduction of beneficial flora in the gut.4.Screening and evaluation of probiotics that promote efficient utilization of glycininSince the addition of sodium butyrate in the diet can significantly improve the growth inhibition and intestinal injury of Chinese mitten crab caused by glycinin,this experiment selected the intestinal samples of crabs fed on 1% sodium butyrate diet.In vitro screening of probiotics can utilize glycinin and improve intestinal immunity,and then evaluate the beneficial effects of the screened probiotics on Chinese mitten crab by in vivo feeding experiments.Pre-screening of 12 candidate probiotics based on criteria such as utilization of glycinin,extracellular enzyme production,haemolytic activity.According to criteria for the ability to resist pathogenic bacteria,tolerance to gastrointestinal stress,cell surface hydrophobicity,and auto-agglutination,three potential probiotic strains were finally identified: Z9,Z34 and Z98,which showed good probiotic characteristics on all criteria above.Biochemical and 16 S r RNA sequencing analysis confirmed Z9,Z34 and Z98 as Shewanella putrefaciens,Bacillus subtilis and Bacillus pumilus,respectively.Five isonitrogenous and isolipidic diets were designed,with fish meal and casein as the main protein source for the positive control(C)and the addition of 8% glycinin as the negative control(11S).On the basis of 11 S diet,109 cfu/ g of Z9,Z34 and Z98 diets were added respectively,which were recorded as SP,BS and BP.Juvenile crabs with an initial average weight of(0.28 ± 0.01g)were selected for an 8-week breeding experiment.The results showed that the survival of SP and BP groups were significantly higher than that of 11 S group,and showed no difference with the control group.The weight gain and specific growth rate of the BP and BS groups were significantly higher than those of the 11 S group,and showed no difference with the control group.All three strains of probiotics can significantly increase the tryptase in hepatopancreas and intestine of crabs,reduce the reactive oxygen species level in hemolymph and the activity of serum diamine oxidase.The BP and SP groups can restore the intestinal peritrophic membrane and improve intestinal immunity of crabs.The intestinal microbiota results showed that the BP group can increase the diversity of the flora,and the three probiotics can increase the abundance of probiotics and reduce the abundance of pathogenic bacteria.Finally,the intestinal microbial metabolic activity of crab was compared and analyzed by using Biolog-ECO method in the later period of feeding.The average color change rate of the experimental group fed with probiotics increased significantly,especially in the BP group,indicating that probiotics enhanced the intestinal microorganism activity.This experiment demonstrated that three probiotics can improve the antioxidant and intestinal capacity and digestive ability of crab,ameliorate the intestinal inflammation caused by glycinin,and ultimately improve the growth performance of crab.Based on the indicators of survival rate and weight gain,it was concluded that Bacillus pumilus Z98 can be used as the best probiotic to improve the utilization of glycinin by crabs. |
PDF Full Text Request