Effects Of Fermented Pellet Feeds On Growth,Intestinal Flora And Metabolomic Analysis Of Procambarus Clarkii

In this experiment,Procambarus clarkii were treated with microbial and fermentation enhancers to compare their effects on growth performance,intestinal digestive enzyme activity,serum biochemical parameters,hepatopancreas antioxidant capacity,muscle nutrient content,intestinal tissue morphology and intestinal flora,and metabolomic analysis and intestinal flora association analysis on the intestine(with contents)of crayfish.The aim was to investigate the effects of fermented pellet feed on the growth,digestive physiology,antioxidant capacity and intestinal flora of crayfish.A total of 360 juvenile crayfish of body mass(5.00±0.01 g)were randomly divided into 3 groups with 4 replicates each.The control group(Group Ⅰ)was fed a commercial complete formla pellet feed,and the control group was supplemented with microbial bacterium(Group Ⅱ)and microbial bacterium+fermentation enhancer(1:1)to produce a biofermented feed(Group Ⅲ)for a 60-day growth trial.1.The effects of fermented pellet feed on growth,intestinal digestive enzyme activity,serum biochemical indexes,hepatopancreas antioxidant capacity and intestinal histology of crayfish were investigated by measuring the growth indexes and intestinal,serum and hepatopancreas enzyme activity indexes of crayfish in each experimental group,combined with the histological observation of shrimp intestine.The results showed that the intestinal lipase activity of shrimp in the fermented pellet feed groups(Group Ⅱ and Group Ⅲ)increased by 56.6%and 84.2%,respectively,compared with the control group(P<0.05);the trypsin activity was highest in Group Ⅱ and decreased by 33.0%and 36.8%in Group Ⅲcompared with Groups Ⅰ and Ⅱ,respectively(P<0.05).The serum glutamic transaminase of shrimp in group Ⅱ was significantly higher than that of the control group(P<0.05)and the serum glutamic alanine transaminase of shrimp in group Ⅲ was significantly higher than that of the control group(P<0.05).Glutathione peroxidase(GSH-Px),total superoxide dismutase(T-SOD)and total antioxidant capacity(T-AOC)in the hepatopancreas of shrimp in group Ⅲ were significantly lower than those in the control group(P<0.05);the highest catalase(CAT)activity and the lowest malondialdehyde(MDA)content in the hepatopancreas of shrimp in group Ⅱ were significantly different from those in groups Ⅰ andⅢ(P<0.05).The height of intestinal villi in group Ⅱ shrimp was significantly greater than that in the control group(P<0.05),and the width of folds in group Ⅲ was significantly smaller than that in groups Ⅰ and Ⅱ(P<0.05).Compared with the control group,the overall contour of the intestine of shrimp in the two fermented pellet feed groups was clearer,and the number of longitudinal ridges formed by intestinal mucosal folds increased;the connective tissue in the intestinal wall was tighter,and the muscular layer was more obvious and complete.2.The effect of fermented pellet feed on the nutrient composition and content of the muscles of crayfish was investigated by measuring the conventional nutrients and amino acid composition and content in the muscles of crayfish in each test group.The results showed that the protein content of shrimp muscle in group Ⅲ was 6.0%lower and the moisture content was 1.6%higher than that in group Ⅰ(P<0.05).Glycine content was significantly reduced by 5.6%in group Ⅲ compared to groups Ⅰ and Ⅱ(P<0.05);16 amino acids,7 essential,2 semi-essential and 7 non-essential,were detected in the muscles of crayfish in all three test groups.3.Bacterial flora analysis of the test feed and the gut(with contents)of crayfish was performed by 16S rDNA high-throughput sequencing analysis to compare the composition of the fermented pellet feed with that of the control feed and its effect on the intestinal flora of crayfish.The results showed that:The total number of OTUs generated from 18 samples of the test feeds in Groups Ⅰ,Ⅱand Ⅲ was 1149,of which 149 OTUs were unique to Group Ⅰ,67 OTUs to Group Ⅱ and 509 OTUs to Group Ⅲ.At the phylum level,the relative abundance of the phylum Bacteroidetes in Group Ⅰ was 29.25%,the relative abundance of the phylum Thick-walled was 1.67%and the relative abundance of the phylum Cyanobacteria was After fermentation,the relative abundance of Bacillus phylum increased to 25.07%and 26.32%,the relative abundance of Bacillus phylum decreased to 17.00%and 19.31%,and the relative abundance of Bacillus phylum decreased to 0.50%and 1.18%in Group Ⅱ and Group Ⅲ respectively.The relative abundance of Acidobacteria(2.51%)and Green Curvilinear Phylum(1.45%)increased in Group Ⅲ compared with Group Ⅰ(0.22%and 0.08%).At the genus level,the relative abundance of Streptococcus lactis,Mycobacterium spp.,Chloroplast_norank spp.and Vibrio spp.decreased in Group Ⅱ and Ⅲ test diets compared to Group Ⅰ.The relative abundance of Lactobacillus spp.,Clostridium sensu stricto 1,Burkholderia spp.and Bacillus spp.increased in Group Ⅱ and Ⅲ test diets compared to Group Ⅰ.The relative abundance of Clostridium sensu stricto 1,Burkholderia spp.The Simpson index of test feeds in group Ⅲwas significantly smaller than those in groups Ⅰ and Ⅱ(P<0.05),and the Chao1 index,Richness and ACE index of test feeds in group Ⅱ were smaller than those in groups Ⅰ and Ⅲ,but there was no significant difference(P>0.05);the sequencing depth ranged from 99.87%to 99.97%,indicating that the sequencing data were valid and reliable,the microbial species were basically covered,and the sequencing results could Beta diversity analysis showed that the microbial composition of fermented pellet feeds in Group Ⅱ and Group Ⅲ changed significantly compared with the control group,and LEFSE analysis showed that the significantly different groups of fermented pellet feeds in Group Ⅱ were mainly Lactobacillus and in Group Ⅲ were mainly Bacillus,indicating that the addition of microbicides,microbicides+fermentation enhancers had a significant impact on the microbial composition of Group Ⅱ and Group Ⅲ.The addition of microbicides and microbicides+fermentation enhancers produced significant differences in the microbial composition of the fermented pellet feeds in Group Ⅱ and Group Ⅲ.A total of 2069 OTUs were generated from 18 samples from the intestinal tract of shrimp in groups Ⅰ,Ⅱ and Ⅲ,of which 119 OTUs were unique to group Ⅰ,145 OTUs to group Ⅱ and 226 OTUs to group Ⅲ.At the phylum level,the composition of the dominant phylum in the intestinal tract of shrimp in groups Ⅰ,Ⅱ and Ⅲ was similar,with the highest relative abundance of Proteobacteria,followed by Firmicutes and Bacteroidota in descending order.These three dominant phyla accounted for more than 75%of the intestinal flora,and the sum of the relative abundance of the top seven phyla accounted for more than 96.50%.The relative abundance of the thick-walled Bacteroides:Bacteroides(abbreviated as F:B,the same below)was 1.64 for group Ⅰ,2.22 for group Ⅱ and 5.77 for group Ⅲ.At the genus level,the dominant genus(>5%)and relative abundance of the shrimp intestinal flora differed somewhat between groups.The dominant genera in group Ⅰ were Vibrionimonas,Bradyrhizobium and RsaHf231_norank;the dominant genera in group Ⅱ were RsaHf231_norank and[Anaerorhabdus]furcosa group;the dominant genera in group Ⅲwere The dominant genera in Group Ⅲ are RsaHf231_norank,Citrobacter and ZOR0006.The relative abundance of the genera RsaHf231_norank,ZOR0006 and[Anaerorhabdus]furcosa group was higher than that of group Ⅰ.The relative abundance of Vibrionimonas and Bradyrhizobium was lower than that of group Ⅰ.The relative abundance of the genera ZOR0006 and[Anaerorhabdus]furcosa group was higher than that of group Ⅰ.The relative abundance of Vibrionimonas and Bradyrhizobium was lower than that of group Ⅰ.The relative abundance of ZOR0006 and Citrobacter was the highest in all groups.The relative abundance of ZOR0006 spp.and Citrobacter spp.in group Ⅲ was the highest among all groups.The Chao1 index,Richness,Shannon index and ACE index in group Ⅱ were greater than those in the control group,and those in group Ⅲ were greater than those in group Ⅱ,but there was no significant difference between the groups(P>0.05).Beta diversity analysis showed that the composition of gut microbial species of shrimps in groups Ⅱ and Ⅲ changed compared with that in group Ⅰ.4.A non-targeted metabolomic study of the gut(with contents)of each test group of crayfish based on liquid-liquid mass spectrometry(LC-MS)technology and an intestinal flora-metabolite association analysis were done to compare the effects of fermented pellet feed and control feed on the intestinal metabolism of crayfish.The results showed that compared to the control group,there were 4 differential metabolites that were highly significantly up-regulated(P<0.01),11 differential metabolites that were significantly up-regulated(P<0.05)and 3 differential metabolites that were significantly down-regulated(P<0.05)in group Ⅱ;7 differential metabolites that were highly significantly up-regulated(P<0.01),29 differential metabolites that were significantly up-regulated(P<0.05)and 29 differential metabolites that were significantly down-regulated(P<0.05)in group Ⅲ.There were 4 differential metabolites that were highly significantly down-regulated(P<0.01)and 16 differential metabolites that were significantly down-regulated(P<0.05).In group Ⅲcompared to group Ⅱ,there were 2 differential metabolites that were highly significantly upregulated(P<0.01),7 differential metabolites that were significantly upregulated(P<0.05),2 differential metabolites that were highly significantly downregulated(P<0.01)and 16 differential metabolites that were significantly downregulated(P<0.05)in group Ⅲ.A total of 18 metabolic pathways were enriched by the differential metabolites in group Ⅱ and Group Ⅰ,with no significant enrichment(P>0.05);a total of 60 metabolic pathways were enriched by the differential metabolites in group Ⅲ and Group Ⅰ,with one significant enrichment(P<0.05)for fatty acid biosynthesis,and three differential metabolites were enriched The former was up-regulated and the latter two were down-regulated;a total of 40 metabolic pathways were enriched for differential metabolites in Group Ⅲ and Group Ⅱ,of which 2 were significantly enriched(P<0.05),namely taurine and hypotaurine metabolism and primary bile acid biosynthesis.bile acid biosynthesis),and three of the enriched differential metabolites,namely taurine,taurine,and taurocholic acid,were downregulated.Analysis of the association of intestinal flora and metabolites between group Ⅰ and group Ⅱshowed that Clostridium sensu stricto 5 was associated with MGMG(18:2),Leucine-enkephalin,2’-Deoxyadenosine 5’-monophosphate(dAMP)was highly significantly positively correlated,[Eubacterium]brachy group was highly significantly positively correlated with Estradiol Benzoate,Blastopirellula was highly significantly positively correlated with Epoxomicin,PC(18:4e/20:5)and Muribaculaceae_norank was highly significantly positively correlated with Protopine.In summary,under the present experimental conditions,compared with unfermented pellet diets,feeding fermented pellet diets increased lipase activity in the intestine,serum glutamic transaminase and glutamic aminotransferase activity and intestinal villus height of crayfish,improved the morphology of shrimp intestinal tissues and thus promoted shrimp growth,but had some effect on the antioxidant capacity of shrimp hepatopancreas,The use of microbial agents and fermentation enhancers increased the relative abundance of beneficial bacteria and reduced the relative abundance of harmful bacteria in the fermented pellet feed,thus improving the structure of the intestinal flora of crayfish.The use of microbial agents and fermentation enhancers increased the relative abundance of beneficial bacterial genera in the fermented pellet feeds,but the strains in the test feeds may not colonise the gut of crayfish;there was a significant effect on metabolites such as lipids and fatty acid anabolic pathways in the gut of crayfish and a link between genera and metabolites in the gut of crayfish.