| The hybrid grouper(Epinephelus lanceolatus ? × E.fuscoguttatus ?)with an initial weight of 9-12 g was selected as the research object,and an 8-week breeding experiment was conducted to evaluate the correlation of growth,serum biochemical indexes and intestinal function of hybrid grouper with enzymatic chicken mash,and explore the effect of enzymatic chicken mash on the healthy growth of hybrid grouper.To evaluate the soy protein concentration on the growth,feed utilization,liver antioxidant and intestinal immunity to meet the maximum use of soy protein concentration in the diet of hybrid grouper.The experimental results of this paper are as follows:(1)Effect of enzymatic chicken mash on the growth and serum biochemical indexes of hybrid grouperThe hybrid grouper were randomly divided into three groups with three replicates in each group,which were the control grouper(C),3.5 % enzymatic chicken mash 1 addition group(E1)and 3.5 % enzymatic chicken mash 2 addition group(E2).The results showed that the weight gain,specific growth rate and condition factor of group E2 were significantly higher than those of the groups C and E1(P < 0.05),while there was no significant difference between the groups C and E1(P > 0.05).The serum glucose level in the groups E1 and E2 were significantly reduced(P < 0.05).The total cholesterol level in group E1 was significantly increased(P < 0.05),while that of group E2 was higher than the group C with no significant difference(P > 0.05).The low-density lipoprotein cholesterin of group E2 was significantly decreased(P < 0.05),which was not significantly different from that of the group E1(P > 0.05).The cumulative mortality rate of group E2 was the highest and significantly higher that of group C(P < 0.05),and there was no significant difference between groups E1 and C(P > 0.05).Our studies have shown that E2 is better than E1 in improving the growth performance of hybrid grouper,and E1 and E2 can reduce the capacity of disease resistance of hybrid grouper.(2)Effect of enzymatic chicken mash on the intestinal function of hybrid grouperTo tissue section observation and microbial diversity analysis of the intestine of hybrid grouper in the previous chapter and the results showed that compared with group C,the proximal intestinal ZO-1,ZO-2,ZO-3,occluding and claudin-3 m RNA levels in the groups E1 and E2 were significantly increased(P < 0.05),while claudin-12 and claudin-15 had no significant change in m RNA levels(P > 0.05).The mid intestinal ZO-3 and claudin-3 m RNA levels in the group E1 were significantly reduced(P < 0.05)and claudin-12 was significantly increased(P < 0.05).In the group E2,ZO-1,ZO-2,occludin,claudin-12 and claudin-15 were significantly increased(P < 0.05).The gene expression of ZO-1,ZO-2,ZO-3,claudin-3,claudin-12 and claudin-15 in the distal intestine of groups E1 and E2 were significantly down-regulated(P < 0.05),and the gene expression of occludin in the group E1 was significantly up-regulated(P < 0.05),while the occludin in the group E2 down-regulated significantly(P < 0.05).Compared with the group C,the proximal intestinal villus width in the group E1 was significantly reduced(P < 0.05),and the villus height and the muscle thickness were not significantly changed(P > 0.05).The muscle thickness of mid intestine in the group E1 was significantly lower than that in the group E2(P < 0.05).The distal intestinal villus height in groups E1 and E2 were significantly decreased(P < 0.05),as well as the villus width in the group E1(P < 0.05).Compared with group C,the Shannon index and OTUs of the groups E1 and E2 were decreased.At the genus level,Bacillus and Acinetobacter constituted the core microbial genus.The groups E1 and E2 showed the higher abundance of Bacillus and lower abundance of Acinetobacter than those of the group C.Studies have shown that the enzymatic chicken mash in the feed can promote the development of the proximal intestine,cause damage to the mid and distal intestine,reduce the intestinal microbial abundance and increase the intestinal beneficial bacteria abundance.(3)Transcriptome sequencing analysis of the effect of enzymatic chicken mash on the intestinal gene expression of hybrid grouperTranscriptome analysis of the intestine of hybrid grouper of growth experiment.The results showed that a total of 111,196 Unigenes were obtained,of which 36,295 Unigenes were compared to the four major annotation libraries(Nr,Swissport,KOG and KEGG).There were 1936 differentially expressed genes between groups C and E1,of which 1355 were up-regulated and 571 were down-regulated.There were 550 differentially expressed genes between groups C and E2,of which 383 were up-regulated and 167 were downregulated.Among the differential genes in the groups C and E1,the GO annotation secondary classification can be divided into 47 subclasses,and more genes are annotated to the binding,cell process,and catalytic activity.Among the differential genes in the groups C and E2,the GO annotation secondary classification can be divided into 34 subclasses,and more genes are annotated to binding,single-organism process and cell process.KEGG enrichment shows that the differentially expressed genes between groups C and E1 are mostly concentrated in the phosphatidylinositol signal system and glycerol phospholipid metabolism while the differentially expressed genes between groups C and E2 are mostly concentrated in amino acid metabolism.Based on this,we speculate that the enzymatic hydrolysis of chicken pulp may change the expression of genes related to the amino acid metabolism and phosphatidylinositol signal system of the intestine to affect the growth performance and immunity of hybrid grouper,but the mechanism remains to be further studied.(4)Effect of replacement of fish meal by soy protein concentrate on the growth performance,liver antioxidant capacity and immunity of hybrid grouperAn 8-week feeding trial was conducted to investigate the effects of partial replacement of fish meal by soy protein concentrate(SPC)on the growth performance,immune responses,intestine morphology and relation gene expression of intestinal inflammation for juvenile hybrid grouper(Epinephelus fuscoguttatus ? × E.lanceolatus ?)(initial weight 12.5 ± 0.00g).Eight isonitrogenous and isolipidic diets(48.61 % protein and 11.17 % lipid)were formulated by replacing 0 %(the control),11 %,22 %,33 %,44 %,55 %,66 %,and 77 % of fish meal(FM)with SPC,respectively(the eight dietary be named FM,S11,S22,S33,S44,S55,S66,and S77,respectively).With the replacement level increased,the final body weight,weight gain ratio(WGR),specific growth rate(SGR),and survival rate of fish were significantly decreased(P < 0.05)compared with the group FM.By contrast,the feed conversion ratio(FCR)of fish was significantly increased(P < 0.05)when the replacement level up to 44%.Partial FM replacement by SPC(ranging from 11 % to 77 %)substantially reduced(P < 0.05)the serum total protein,albumin,and total cholesterol contents compared with the group FM.Liver total superoxide dismutase,glutathione peroxidase,catalase activities,and total antioxidant capacity showed the same trend of gradual increase first and then decrease.Their highest values were found in the replacement levels of 55 %,33 %,22 %,and 55 % and were significantly higher(P < 0.05)than the control group.The lowest malondialdehyde content was observed in group S77 and was significantly lower(P < 0.05)than that of the control group.The complements C3 and C4 contents of fish fed with experimental diets(replacement level ranged from 11 % to 66 %)were significantly higher(P < 0.05)than the group FM.The liver lysozyme activity of the control group was the lowest and was significantly lower than that of other dietary treatments(P < 0.05).Villus length and muscle thickness in the intestine of fish were significantly lower(P < 0.05)than other groups when the replacement level exceeded 44 %.(5)Effect of replacement of fish meal with soy protein concentrate on the intestinal function of hybrid grouperWith dietary replacement levels increased,the TLR22,My D88,p65,pro-inflammatory cytokines(IL-1?,TNF-?,IL-12P40 and IFN-?)and anti-inflammatory cytokines(TGF-?,IL-10,epinecidin,MHCII? and hepcidin)m RNA levels in the proximal intestine were significantly up-regulated(P < 0.05).The TLR22,My D88,p65,pro-inflammatory cytokines(IL-1?,TNF-?,IL-12P40 and IFN-?)and anti-inflammatory cytokines(TGF-?,IL-10,MHCII? and hepcidin)m RNA levels in the mid intestine were significantly upregulated(P < 0.05).The m RNA levels of TLR22,anti-inflammatory cytokines(IL-1?,TNF-?,IL-12P40,IFN-?)and anti-inflammatory cytokines(TGF-?,IL-10,epinecidin,MHCII? and hepcidin)in the distal intestine were significantly down-regulated(P < 0.05).The m RNA expression of My D88 and p65 m RNA were showed a tend increased first and then decreased,and the highest values were observed in group S33 and S55(P < 0.05),respectively.Based on the present work,the correlation between WGR and FM replacement level with SPC was described using the broken-line model,which estimated the optimum FM replacement to 37.23% for juvenile hybrid grouper dietary. |
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