Analyses Of Mechanism Of Brisket Disease In Holstein Heifers Based On Multi-omics Technology

The present study focused on Holstein heifers transported to high-altitude environment suffered from brisket disease(BD).Multi-omics of transcriptome,metabolome and microbiome analyses were used,in which differentially expressed genes,metabolites and microbes were identified.Through this study,we wish to provide strategies and theoretic support for the prevention of BD caused by pulmonary hypertension.In this study,2000 Holstein heifers that were 13–15 months old were transported from a low altitude of 1,027 m(Xian,Shaanxi,China)to a high altitude of 3,658 m(Lhasa,Tibet,China).After 3-month of adaption,ten of the Holstein heifers(16–18 months old,non-pregnant,495 ± 15 kg)from the BD(n = 5;pulmonary arterial pressure ? 49 mm Hg)and healthy heifers(HH;n = 5;pulmonary arterial pressure < 49 mm Hg)groups were selected for the multi-omics analyses and physiological indicators testing.Trial I: The jugular vein blood and tail root artery blood of two groups were used for physiological,blood biochemical,and blood gas tests.The result showed that the average pulmonary hypertension and hypertension of BD were significantly higher than that of HH(P<0.05);Indicators of the respiratory frequency,blood oxygen saturation,red blood cell count,average hemoglobin amount,hemoglobin concentration,hematocrit,sodium,chloride,iron,magnesium,and glucose were significantly lower in BD group than that in HH(P<0.05).For HH exposed to hypoxic environment,the above indicators have undergone an adaptive change in hypoxic acclimatization.Trial II: The liver tissues were collected from five cows in each group after slaughter.A total of 1,146 differentially expressed genes were identified in BD vs.HH,in which most of them were down-regulated.The apolipoprotein gene Apoe participates in the Bmp2(bone morphogenetic protein 2)/Ppar?(peroxisome proliferator-activated receptor)/Apoe axis as a transcription target downstream of Bmp2 and Ppar? signals.The transmission of pulmonary artery plays a role by Bmp2 and Ppar? agonists in the remodeling of the pulmonary artery and participates in the occurrence and development of BD.The Rbp4 gene is significantly down-regulated in a high-pressure and hypoxic environment,and it is related to glucose metabolism,leading to insufficient energy supply in the cattle,which may be one of the reasons for the occurrence of BD.The lipid metabolism-related genes of Lcat,Apoc4,Rbp4,Apoc3 and Apoe(down-regulated)were involved in LXR/RXR and FXR/RXR pathways,which mainly inhibits inflammation,inhibiting anti-inflammatory effect.Cytochrome P450 pathway,with Cyp1a2 involved,is involved in remodeling of pathological vascular,inhibition of inflammation and BD.Trial III: The liver tissues were also used in metabolomes analysis.Two modes were used for identification of differential metabolites,electrospray positive ionization mode and negative ionization mode.A total of 1,325 and 1,442 differential metabolites were identified in BD compared with HH in positive and negative modes,respectively;The main metabolites with significant differences were: alanine,aspartic acid and glutamate metabolism;D-glutamine and glutamate metabolism;butyrate metabolism;folic acid;pantothenic acid and Co A biosynthesis.The levels of sugar(such as D-ribose,etc.)and amino acids(such as alanine-glycine,histidine-isoleucine,p-chlorophenylalanine,glycyl-L-Leucine,etc.)were significantly decreased in BD than HH.The combined analysis of transcriptomics and metabolomics found that the expression level of carbohydrate-related genes and metabolites were significantly reduced in high altitude sickness dairy cows,and D-ribose,?-D-fructose,1,4-?-D-glucose glycans,sucrose,glucose6-phosphate,and ethyl acetyltyrosine are negatively related to pulmonary artery pressure.The reduction of these sugars and lipids leads to insufficient energy supply,which may be one of the reasons for the occurrence of altitude sickness.Therefore,timely and adequate sugar supplementation may play a key role in the prevention of altitude sickness.More importantly,adequate supplementation of carbohydrates,lipids and amino acids may be particularly important for the prevention of altitude sickness.Trial IV: The contents of the cecum and colon taken from each group(n=5)were used for 16 S r RNA sequencing analysis.The result of cecum and colon showed that the phylum Firmicutes significantly decreased and Bacteroides significantly increased in BD.The occurrence of BD might decrease the ratio of Firmicutes/Bacteroidetes(F/B)and enhance inflammation.The genus of Blautia,Coprobacillus,Dorea,Oscillospira,Ruminococcaceae?UCG?009,Ruminococcaceae?UCG?013,Ruminococcaceae?UCG?014,and Ruminococcus?2 was significantly decreased in heifers in BD group and were positively correlated with sugars(?-D-fructose,D-ribose,sucrose,glucose-6-phosphate and 1,4-?-D-glucan),lipids(acetyl?tyrosine?ethyl?ester),and amino acids(L-asparagine,Ala-Gly,His-Ile,Glycyl-L-leucine).Trial V: The contents of rumen taken from each group(n=5)were used for 16 S r RNA sequencing analysis.The results showed that genus of Spirochaetes,Spirochaetales,Spirochaetaceae,Treponema?2,Bacteroidales?RF16?group,Ruminococcus?2,and Butyrivibrio are essential for healthy heifers.The correlation analysis revealed that Ruminococcus,Treponema,Blautia positively correlated with total volatile fatty acids and negatively correlated with pulmonary artery pressure.Campylobacter is significantly increased in BD groups and increased with the concentration of volatile fatty acids in rumen.Ruminococcus?2,Blautia,and Eubacterium?brachy?group were all significantly decreased in samples of cecum,colon and rumen,which closely correlated with the occurrence of BD.In summary,we hypothesized that genes down-regulated in BD were mainly related with vascular remodeling(Apoe and Cyp1a2),anti-inflammatory response,glucose and lipid metabolism.Moreover,the genus of Ruminococcus?2,Eubacterium?brachy?group as well as F/B ratio in intestinal tract were reduced,Campylobacter and Anaerobic Clostridium in rumen were increased,which caused abnormal dysregulation of sugars and lipids(1,4-?-D-glucan,sucrose,glucose-6-phosphate and acetyl?tyrosine?ethyl?ester)in liver,insufficient energy supply,and strengthened inflammation.The pulmonary artery pressure might be increased for the above reasons,which might also be the main cause of occurrence and development of BD in heifers under high-altitude environment.We will identify the functional mechanism of key genes,metabolites,and genus on BD in our future work.