| Megalobrama amblycephala,commonly known as Wuchang bream,is a commercially important freshwater fish species that is native to China.At present,our laboratory has successively carried out studies on the construction of nutritional requirements database,screening of high-quality feed sources,immune function regulation and other related technologies for M.amblycephala.However,there are few reports on the response mechanism of M.amblycephala to deprivation of food.Therefore,this experiment took M.amblycephala as the research object,and focused on the compensatory growth effect after starvation and refeeding.And mainly through the analysis of intestinal morphology and pathological labeling,as well as the study of intestinal microbial community changes to clarify there may be irreversible point of compensatory growth after starvation.In addition,the effects of feeding regulation in fish body after starvation were studied from the molecular level,so as to provide some basis for the healthy breeding technology of M.amblycephala.The research results provide theoretical basis and data support for exploring the scientific feeding system,improving feed efficiency and saving production costs of M.amblycephala.1.Effects of starvation and compensatory growth on growth,blood biochemical parameters,digestive enzyme and antioxidant enzyme activity in juvenile blunt snout bream in summer,M,amblycephala:A feeding trial was conducted to investigate the effects of starvation and compensatory growth on growth,blood biochemical parameters,digestive enzyme and antioxidant enzyme activities in juvenile blunt snout bream in summer.Five groups were formulated respectively hungering 0,5,10,15,20 days and then refeeding 3 weeks,marked as S0,S5,S10,S15,S20.Two hundred seventy juvenile blunt snout breams with an average body weight of(22.03 ± 0.04)g were randomly divided into 5 groups.Each group has 3 replicates.The results showed as follows:(1)The final fish weight,weight gain,specific growth rate and feed efficiency ratio were highest in S10 group and the feeding rate and food conversion rate increased(P<0.05);(2)The contents of triglycerides in S5 and S10 groups were lower than other groups obviously(P<0.05);however,the activities of aspartate transaminase in S20 group were higher than other groups obviously(P<0.05)which indicated that long-term starvation and refeeding after may damage the liver.No differences were detected among the groups in glucose,total protein,total cholesterol concentrations and alanine aminotransferase activity(P>0.05).(3)The activities of lipase in S15 group(P<0.05)and the activities of protease in S20 group were both significantly higher than other groups except for the S5 group(P<0.05),which indicated that long-term starvation and refeeding after may improve protein digestion in vivo.(4)The activities of superoxide dismutase and catalase in starvation and refeeding groups were higher than those in the control group.The activity of glutathione peroxidase in S20 group was significantly lower than other test groups,which indicated that long-term starvation may repress the recovery of antioxidant capacity.It is concluded that in this experiment juvenile blunt snout breams after 10-day starvation and 3-week refeeding achieve the best compensatory growth effect in summer,the immune and antioxidant capacity also increase certainly.2.Effects of starvation on growth,blood biochemical parameters,intestinal digestion and health of juvenile blunt snout bream,M.amblycephala:This study aimed to investigate the effects of starvation on intestinal digestion and health of M.amblycephala.Six hundred and thirty juvenile blunt snout breams with an average body weight of(8.20±0.01 g)were randomly divided into 6 groups(S0,S1F1,S3F1,S7F1,S13F1 and F0)with 3 replicates in each group,which were re-fed for 1 day after starved for 0,1,3,7,13 and 56 days for 8 weeks respectively.The results showed as follows:(1)Final weight,weight gain,specific growth rate,hepatosomatic index and condition factor decreased significantly with the duration of starvation increasing(P<0.05).(2)Plasma glucose concentrations of the fish in S13F1 group were the highest(P<0.05).Total protein,total cholesterol and triglycerides concentrations decreased significantly with the duration of starvation increasing(P<0.05).(3)Highest lipase activities were found in S0 group(P<0.05)and protease activities of the fish in S7F1 and S13F1 groups were higher than those of the fish in S3F1 and F0 groups(P>0.05)but not significantly.(4)In the F0 group,the morphology of intestinal villi was irregular.Intestinal epithelial cells were necroticthe and leucocyte infiltration in lamina propria were slightly increased.(5)The 16S rRNA sequencing was carried out on the intestinal microflora showed that,Cyanobacteria,Proteobacteria,Firmicutes,Verrucomicrobia and Fusobacteria were the main bacteria in the blunt snout bream intestine.At genus level,Bacillus and Lactobacillus in the intestinal microorganism of fish in the S3F1 and S7F1 groups was in the majority.On the contrary,Aeroomonas and Vibrio were a large proportion in the intestinal microorganism of fish in the S13F1 and F0 groups.In conclusion,long-term starvation has a negative effect on the growth and physiology performance of juvenile blunt snout bream,resulting in the destruction of intestinal tissue structure and the changes in intestinal microflora diversity.3.Molecular cloning of AGRP and NPY and their expression analysis under fasting and refeeding in juvenile blunt snout bream,M.amblycephala:The purpose of this study was to investigate the expression patterns of AGRP and NPY in the regulation of fasting and refeeding in M.amblycephala.Four hundred and fifty-six juvenile blunt snout breams with an average body weight of(5.30±0.01)g were randomly divided into 4 groups with 3 replicates in each group.Respectively for the Control group(the daily feeding experiment diet at 3%of the weight),the F/Refed group(fasted from 1d to 14 d,refed from 15 d to 28 d and the daily feeding experiment diet at 3%of the weight,no residual feed within 30 minutes),the F/Excessive Refed group(fasted from 1d to 14 d,refed from 15 d to 28 d and the daily feeding experiment diet at 8%of the weight,remaining feed within 30 minutes),the Fasted group(fasted for 28 d)during the test.The results showed as follows:(1)The full length cDNA of M.amblycephala AGRP and NPY are respectively 770 bp and 557 bp,consisting of a 5’ untranslated region of 77 bp and 101 bp,a 3’ untranslated region of 315 bp and 120 bp,and an open reading frame of 378 bp and 336 bp,encoding a preproghrelin with 125 amino acid(aa)and 111 amino acid(aa).Based on amino similarity comparison,AGRP is relatively conservative and has the highest similarity to that of AGRP in Cyprinus carpio L.,and the phylogenetic tree analysis indicated that AGRP of M.amblycephala is associated with AGRP in other carp fish;NPY of M.amblycephala has close relationgship with Anabarilius grahami,Carassius auratus and Sinocyclocheilus graham.(2)Quantitative real-time PCR analysis showed that AGRP and NPY were expressed in all tested tissues.AGRP was expressed in M.amblycephala with highest expression in brain and then hindgut,and NPY was expressed in M.amblycephala with highest expression in brain and then liver and hindgut.(3)AGRP mRNA expression in both brain and intestine tissues was first down,then up and then down during starvation,while NPY mRNA expression in both brain and intestine tissues increased and then decreased during starvation.The expression of AGRP mRNA in the brain decreased and then increased in F/Refed group after refeeding,while the expression of NPY mRNA in the brain and intestine decreased significantly(P<0.05).The expression of AGRP and NPY mRNA in the brain was both significantly decreased in F/Excessive refed group after refeeding(P<0.05),while the expression of AGRP mRNA in the intestine was significantly increased in this group(P<0.05)and the expression of NPY mRNA in the intestine in this group increased and then decreased after re-feeding(P<0.05).In conclusion,this suggested that AGRP and NPY are involved in the regulation of food intake in M.amblycephala. |
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