Research About Ketosis Of Dairy Cattle Based On Fecal Microbes Sequence And Feeding Procyanidins

Ketosis is one of the high incidence nutritional metabolic diseases in early lactation of high producing dairy cows,it has a major impact on the health of cows.In order to understand the occurrence mechanism of ketosis,this research studied the difference of gut microecology between ketosis cows and healthy cows by means of gut microbiome.In addition,grape seed proanthocyanidin extract(GSPE)was administered to early lactating cows to understand its effect on related blood plasma parameters of ketosis,and the parameters of oxidant and antioxidant etc.Specific research contents are as follows:1.The difference analysis of gut microflora between healthy dairy cows and cows with ketosisAccording to the principle of pairing design Ketosis group(KET)and control group(CON group)were divided into 22 cows from the early milk lactation in a dairy farm.The KET group was composed of clinical ketosis(CK group,n = 3)and subclinical ketosis(SK group,n = 19).On the day of detection of ketosis,the rectal intestinal feces of cows of KET and CON group were collected,and the genomic DNA of all its microorganisms was extracted.It was sequenced by Illumina HiSeq sequencing platform using 16S rDNA amplicons.Results:(1)Analysis and species annotation of operational classification unit(OTUs):75724 OTUs,effective data 3739252Tags.According to the GraPhlAn map,the dominant microorganisms of CK group were firmicutes,proteobacteria,bacteroides and actinomycetes,and a small number of Euryarchaeota.SK group were firmicutes,proteobacteria and Bacteroidetes,but not Euryarchaeota.CON group were firmicutes,actinomycetes and bacteroides,and a small amount of Spirochaetes and Euryarchaeota.The columnar accumulation figure showed that the three groups of species with the highest abundance were similar at the phylum level,in which the sum of firmicutes and bacteroides was more than 80%,while the species with the highest abundance at the genus level were significantly different.Streptococcus and lachnoclostridium were only in the top ten in the CK group,and buchenella was only in the top ten in the SK group.(2)OTUs sharing analysis between groups:Wayne diagram of three groups of sample OTUs shows that the CK group shares 1967 OTUs with the CON group,696 OTUs in the CON group and 35 OTUs in the CK group.SK group Shared 2,544 OTUs with CON group,SK group Shared 191 OTUs and CON group Shared 119 OTUs.CK group shares 1967 OTUs with SK group,CK only has 35 OTUs and SK only has 768 OTUs.(3)Inter-group comparison analysis:the principal coordinate analysis showed that the Unweighted algorithm showed that the gut microcommunity of cows in KET group and CON group was significantly different,and the Weighted algorithm showed that the composition structure of three groups of samples was similar.Principal component analysis(PCA)showed that the composition of three groups of samples was similar.Non-metric multidimensional calibration(NMDS)showed that the gut microorganism of cow was different between KET and CON.Anosim test showed that the differences between the groups were significant and greater than the differences within the group,the differences between CK and CON,between SK and CON were significant,while the differences between CK and SK were not significant.The analysis of displacement multi-factor variance(ADONIS)showed that the differences between KET and SK were significant,while the differences between CK and CON,between CK and SK were not significant.Ketosis had a significant influence on the intestinal microorganisms of cows.T-test showed that different microbes at the phylum level were Saccharibacteria in CK and CON;Euryarchaeota in SK and CON,KET and CON,but not in CK and SK.At the genus level the different microbes of the CK and the CON were Candidatus_Saccharimonas,erysipelotrichaceae_ugg-008.In SK and CON were streptococcus(with very little content in SK group),Methanobrevibacter,Prevotellaceae_UGG-001,Erysipelotrichaceae_UGG-009,Lachnospiraceae_UGG-010,Parabacteroides and Atopobium,while in KET and CON were Ruminococcaceae,Methanobrevibacter,Erysipelotrichaceae_UGG-009,Lachnospiraceae_UGG-010 and Atopobium.No difference in microorganism was found between CK group and SK group.The different microbes at the species level between SK and CON were Clostridium_butyricum,there were no different microbe between CK and CON,CK and SK,KET and CON.LefSe analysis showed that the core microfloras of SK group were proteobacteria,Buchnere and Enterobacteriaceae.The core microfloras of CK group were Gamma-proteobacteria and Enterobacteriaceae.The abundance of Ruminococcaceae_gnavus in CK group was higher than that in SK group,Clostridia and Christensenellaceae in SK were higher than that in CK,but the differences were not significant.2.Effects of GSPE feeding on plasma level of ketosis related indicators and antioxidant indicators of dairy cowsThe 48 early lactating Holstein cows that met the inclusion criteria were divided into four groups according to the completely random design principle:Control group(GO)and three experimental groups(G20,G40 and G80),each group of 12 cows.The cows in each group were given 0,20,40 and 80 mg GSPE/kg BW/day respectively,and the milk yield(MY)was calculated on day 0(before perfusion),10,20,30,40 and 50d.Milk samples and blood samples were collected on day 0(before perfusion),7,14,21,28,35 and 42d.the following indexes were determined,in cluding ketone bodies(KB),free fatty acids(NEFA),blood glucose(GLU),total antioxidant capacity(TAC),superoxide dismutase(SOD),glutathione peroxidase(GPX),hydrogen peroxide(H2O2),malondialdehyde(MDA),Alanine Aminotransferase(ALT);Aspartate Aminotransferase(AST);Gamma Glutamyl Transferase(GGT);Albumin(ALB);Total Protein(TP);Globulin(GLOB);Blood Urea Nitrogen(BUN);Uric Acid(UA),cholesterol,Milk protein(MP),non-fat milk solids(SNF),milk fat(MF),somatic cell count(SCC).The results showed that there was no significant difference between the plasma KB,NEFA and GLU levels in the three test groups and the CON group,but there was a significant difference in KB’s time,and there was a significant difference in KB’s interaction with DIM(P = 0.0003).GPX,SOD,TAC and MDA were all significantly correlated with test time(P<0.0001).MDA content in G20 significantly increased compared with GO at 14d(P =0.0371).The levels of ALT,AST,ALB,GLOB,TP,BUN and UA,as well as GPX,SOD,TAC and H2O2 in the experimental group were not significantly different from those in the control group.3.Safety study on dairy cows fed by GSPEAccording to the completely random design principle,16 early lactation Holstein cows that met the inclusion criteria were divided into four groups:control group(g0 group)and three experimental groups(g20,g40 and g80).Each group was given 0,20,40 and 80 mg GSPE per kg of body weight(BW)per day(mg GSPE/kg BW/day),and were given 1 time every 24 hours for 168h.At 0(before perfusion),3,6,12,24,48,72,96,72,96 and 168 h blood samples were collected and the following indicators were determined,including ALT,AST,GGT,TP,ALB,GLOB,BUN and UA.The results showed that the ALB(P=0.0361)and BUN(P=0.0478)of the g20 group were significantly higher than those of the control group at 120h,while the BUN of the g40 and g80 groups were not significantly different from those of the control group.There were no significant differences in ALT,AST,GGT,GLOB,TP and UA between the g20,g40 and g80 groups and the control group.Conclusion:(1)ketosis has a significant impact on the microbial flora in the posterior intestinal tract.The difference between CK and CON at the phylum level is Euryarchaeota,and at the genus level are Ruminococcaceae,Methanobrevibacter,Erysipelotrichaceae_UGG-009,Lachnospiraceae and Atopobium.(2)Compared with CON,the gut Euryarchaeota of dairy cows with subclinical ketosis were significantly reduced,especially the Methanobrevibacter.And Streptococcus,Methanobrevibacter,Prevotellaceae,Erysipelotrichaceae_ugg-009,Parabacteroides and Atopobium at the genus level in the posterior intestinal tract were significantly reduced,while the significantly increased Lachnospiraceae at the genus level and Clostridium_butyricum at the species level were the characteristics of subclinical ketosis in cows.(3)there was no significant difference in the plasma KB,NEFA and GLU levels in the early lactation cows between fed by GSPE and the CON group,but there were significant differences in between KB and the time and DIM(P = 0.0003).(4)When feeding GSPE to cows,GPX,SOD,TAC and MDA were significantly correlated with the time(P<0.0001),while MDA showed significant differences in the interaction between treatment group and the time(P = 0.0463).As it is not clear that GSPE has a significant positive effect on ketosis treatment,further study is needed to decide whether to recommend this product.