| In this study,Rhynchocypris lagowskii with an initial mass of(10.00±0.22)g/tail was used as the research object.The concentration of Rhynchocypris lagowskii’s source Bacillus amyloliquefaciens LSG2-8 was added to the feed by spraying,and the breeding experiment was carried out for 8 weeks.After the culture experiment,a 14-day challenge experiment was carried out with Aeromonas hydrophila.This study provides theoretical basis and technical reference for the rational development of special probiotic feed and healthy and efficient breeding technology for Rhynchocypris lagowskii by exploring the effects of Bacillus amyloliquefaciens LSG2-8 on the growth,immunity and intestinal health of Rhynchocypris lagowskii.The experiment consists of three parts,and the results are as follows:1.Effects of Bacillus amyloliquefaciens LSG2-8 on growth,digestive enzyme activities and muscle nutrient composition of Rhynchocypris lagowskiiThe results showed that compared with the control group,Bacillus amyloliquefaciens LSG2-8 had no significant effects on growth performance,feed efficiency ratio,water content,ash content and crude fat content in muscle of Rhynchocypris lagowskii at the middle stage of culture.Crude protein content in muscle of D-6,D-7 and D-8 groups was significantly higher than that of control group(P<0.05).At the end of culture stage,the final body weight,weight gain rate,specific growth rate,feed efficiency ratio and crude protein content in muscle in D-6,D-7,D-8 and D-9 groups were significantly higher than those in control group(P<0.05),and the effect of D-8 group was the best.However,Bacillus amyloliquefaciens LSG2-8 diet had no significant effects on fatness,hepatobody ratio,viscerobody ratio,intestinal length index and intestinal body index,and the contents of moisture,ash and ether extract in muscle(P>0.05).The results of intestinal digestive enzyme activities showed that the trypsin and amylase activities in Rhynchocypris lagowskii treatment groups were significantly higher than those in control group(P<0.05),while lipase,Na~+/K~+-ATPase andγ-glutamyltransferase activities had no significant changes(P>0.05).At the end of culture,compared with the control group,the activities of trypsin,lipase,amylase,Na~+/K~+-ATPase andγ-glutamyltransferase in all treatment groups were significantly increased(P<0.05),and the effects of D-7 and D-8 groups were better.2.Effects of Bacillus amyloliquefaciens LSG2-8 on antioxidant capacity and immunity of Rhynchocypris lagowskiiBy the antioxidant enzyme activity detection result:in the middle of the aquaculture,the treatment group of Rhynchocypris lagowskii catalase activity were higher than control group(P<0.05),and the treatment group of Rhynchocypris lagowskii malondialdehyde content was significantly lower than control group(P<0.05),the total superoxide dismutase and glutathione peroxidase activity than the control group no significant change(P>0.05).At the end of culture,compared with the control group,the activities of total superoxide dismutase,catalase and glutathione peroxidase in Rhynchocypris lagowskii treatment groups were significantly increased(P<0.05),and the malondialdehyde content in Rhynchocypris lagowskii treatment groups was significantly decreased(P<0.05),among which the effects of D-7 and D-8 groups were better.The results of antioxidant gene expression showed that Keap-1 m RNA expression level and Nrf-2 m RNA expression level in Rhynchocypris lagowskii treatment groups were significantly higher than those in control group(P<0.05),and the effect of D-8 group was better.The results of immune-related enzyme activity test showed that Bacillus amyloliquefaciens LSG2-8 had significant effects on antioxidant capacity and immunity of Rhynchocypris lagowskii(P<0.05).At the middle stage of culture,lysozyme activity in Rhynchocypris lagowskii treatment groups was significantly higher than that in control group(P<0.05),while alkaline phosphatase and acid phosphatase activities had no significant changes compared with control group(P>0.05).At the end of culture,the activities of alkaline phosphatase,acid phosphatase and lysozyme in Rhynchocypris lagowskii treatment groups were significantly increased compared with the control group(P<0.05),and the effects of D-7 and D-8 groups were better.The expression levels of IL-1βm RNA and IL-8 m RNA in Rhynchocypris lagowskii treatment groups were significantly lower than those in control group(P<0.05),and the effect of D-8 group was better.The expression levels of IL-10 m RNA and TGF-βm RNA in Rhynchocypris lagowskii treatment groups were significantly higher than those in control group(P<0.05),and the effect of D-8 group was the best.The results of the disease resistance test showed that the mortality of Rhynchocypris lagowskii in D-6(60.7%),D-7(40.5%),D-8(36.5%)and D-9(50.2%)groups was significantly lower than that in the control group(70.6%)(P<0.05),D-8 group had the lowest mortality rate.The lysozyme activity and the contents of complement C3,complement C4 and immunoglobulin M of Rhynchocypris lagowskii in D-6,D-7,D-8 and D-9 groups were significantly higher than those in the control group(P<0.05).D-8 group had the most significant effect.3.Effects of Bacillus amyloliquefaciens LSG2-8 on intestinal health of Rhynchocypris lagowskiiThe results of intestinal paraffin sections showed that Bacillus amyloliquefaciens LSG2-8had a significant effect on the intestinal tissue structure of Rhynchocypris lagowskii(P<0.05).The fore gut,mid gut and hind gut of Rhynchocypris lagowskii in each treatment group had clear and complete tissue structure,well-developed intestinal villi,clear mucosa,and no shedding phenomenon.Among them,the fold height,lamina propria width and muscle layer thickness of Rhynchocypris lagowskii intestinal tract in D-8 group were significantly higher than those in control group(P>0.05).The results of the intestinal barrier gene detection test showed that,compared with the control group,the Claudin-3 m RNA expression and ZO-1 m RNA expression of Rhynchocypris lagowskii in each treatment group were significantly higher than those in the control group(P<0.05),D-8 group have better effect.From the detection results of intestinal flora diversity,it can be seen that the sequencing results of the intestinal flora diversity of each experimental group basically covered all the microorganisms in the intestinal tract of Rhynchocypris lagowskii,and the proportion of OTUs shared among the samples was relatively high.Proteobacteria,Firmicutes,Bacteroides and Actinobacteria were the dominant populations in the Rhynchocypris lagowskii gut.At the genus level,the major intestinal flora were Bacillus and Gemmobacter.And adding Bacillus amyloliquefaciens LSG2-8 to the feed can reduce the relative abundance of immune system disease microorganisms and increase the relative abundance of amino acids,carbohydrates,energy and vitamin metabolizing microorganisms in the intestinal tract of Rhynchocypris lagowskii.In conclusion,adding Bacillus amyloliquefaciens LSG2-8 to the feed at a concentration of1.0×10~7～1.0×10~8 CFU/g can improve the growth performance,immunity intestinal health of Rhynchocypris lagowskii. |
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