| The Chinese Holstein dairy cow is the largest dairy breed in China and has a high economic value.Milk fat content is an important indicator for evaluating the production performance of cows and an important factor affecting the economic efficiency of the dairy industry.The high fluctuation of milk fat rate in dairy cows has a serious impact on milk quality.There are a surprising number of microorganisms in the rumen of dairy cows,which have an important impact on growth and digestion,energy supply,improvement of body health and feed efficiency.Furthermore,rumen microorganisms and their metabolites have an important impact on milk fat percentage.In order to investigate the differences in rumen microorganisms and their metabolites between Chinese Holstein dairy cows with different milk fat rates,cows with high(n=5,H group)and low(n=5,L group)milk fat rates were selected based on DHI data from a large farm in Jiangsu Province in 2020.There was no difference in parity,lactation days and milk yield between two groups(P>0.05).Milk,blood and ruminal fluid samples were collected on Jan 16th 2021.The milk composition,blood indexes,rumen fluid digestive enzyme activities and volatile fatty acids were analyzed.Runinal fluid was subsequently used for 16S rDNA high-throughput sequencing and untargeted liquid chromatography-mass spectrometry(LC-MS)metabolomic analysis.Finally,lauric acid,a key differential metabolite affecting milk fat rate,was selected based on the metabolomic result and its function was validated at the cellular level.The results were as follows:1.The average milk fat rate throughout the year was highly significantly higher in H group than in L group(P<0.01).The milk fat rate and whole milk solids content were significantly higher in H group than in L group(P<0.05)on Jan 16th 2021.Compared to L group,the acetic acid/propionic acid ratio and xylanase activity in ruminal fluid were significantly higher in H group(P<0.05),while lipase and protease activities were significantly lower in H group(P<0.05).The β-hydroxybutyric acid concentration in the plasma of H group was higher than L group(P<0.05),while the rest of indexes in plasma did not differ between the two groups(P>0.05).2.16S rDNA analysis revealed that the rumen microbial composition of the two groups was relatively similar at the phylum level.Firmicutes,Bacteroidetes and Proteobacteria accounted for more than 93%of the total bacterial flora.Compared to L group,the abundance of Kiritimatiellaeota was lower in H group(P<0.01).At the genus level,Prevotella 1 had the highest relative abundance,followed by Succiniclasticum and Succinibrionaceae UCG-001.Compared to L group,H group showed a significant decrease in the abundance of Christensenellaceae R-7,Eubacterium coprostanoligenes,Ruminococcaceae UCG-010,Butyrivibrio 2,Methanobrevibacter,p-1088-a5 gut,Acetitomaculum,Lachnoclostridium 10 and Lachnospiraceae ND3007 group,while the abundance of Eubacterium xylanophilum was increased(P<0.05).Metabolomic analysis revealed that four differential metabolites(FC≥1.5 and P<0.05)were identified in negative ion mode(NEG).The concentrations of lauric acid,succinic acid,ethyl laurate and 12,13-dihydroxyoctadecenoic acid were higher in the ruminal fluid of H group compared to L group.These metabolites mainly are lipids and organic acids,enriched in pathways such as fatty acid synthesis and amino acid metabolism.3.Bovine mammary epithelial cells(BMEC)were used to validate the function of lauric acid in regulating milk lipid metabolism.The survival rate of BMEC treated with different concentrations of lauric acid(50,100,200,300 and 400 μM)for 2 d was assessed by the CCK-8 assay and found that 50 μM lauric acid significantly stimulated BMEC proliferation(P<0.05).Meanwhile,EdU incorporation assay revealed that the percentage of EdU positive cell was significantl decreased in 300 and 400 μM lauric acid-treated group compared to NC(P<0.05).Flow cytometry showed that the apoptosis rate of BMEC treated with 400 μM was significantly higher than NC(P<0.05).Oil red O staining and intracellular triglyceride analysis showed that the number of red-stained lipid droplets was higher in the 200 mM lauric acid-treated group compared to NC,and the intracellular triglyceride content was also significantly higher(P<0.05).The expression of genes related to milk fat synthesis was detected by fluorescence quantitative PCR.The relative expression level of Diacylglycerol acyltransferase 1(DGAT1),Sterol reglatory element binding proteins(SREBP1)and Glycerol-3-phosphate acyltransferase(GPAM)was significantly higher in the 200 μM lauric acid-treated group compared to NC(P<0.01),and the expression of DGAT2 and Fatty acid translocase(CD36)were increased(P<0.05).While the expression of Peroxisome proliferator-activated receptor gamma(PPARγ)and Fatty acid synthase(FASN)were significantly decreased(P<0.05).Western Blot showed that the protein expression leverl of SREBP1,CD36 and DGAT1 were increased in the lauric acid-treated group compared to NC.In summary,hemicellulose-degrading bacteria were enriched in the rumen of cows with high milk fat rate,whereas cellulose-degrading and acid-producing bacteria were enriched in the rumen of cows with low milk fat rate,and the differential metabolites in rumen between the two groups were mainly lipids and organic acids.Lauric acid could promote the expression of genes and proteins involved in fatty acid transport(CD36)and triglyceride synthesis(DGAT1,DGAT2,GPAM)in BMEC cells.This study provides a theoretical basis for clarifying the regulatory mechanisms of rumen microbes and their metabolites on milk lipid metabolism. |
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