| In modern dairy production,supplying cows with high-grain diets is a widely adopted feeding regimen in pursuit of ever-increasing milk yield,which leads conversely to a higher incidence of subacute ruminal acidosis(SARA),affecting cow health and farm profitability.Therefore,knowledge about the effects of SARA on rumen health and strategies on the effective prevention and regulation of SARA have become the main concerns for the dairy production.Numerous previous studies suggested that SARA challenge affected rumen fermentation parameters,rumen microbiota composition,and rumen epithelial structure and function(based on the quantification of a handful of genes).However,the effects of SARA on the function of rumen microbota and on the overall function changes of rumen epithelium are still unclear.Moreover,in recent years,it is found that rumen content transplantation(RCT)exhibited an effect on rumen digestion and metabolic regulation.Therefore,the present study comprehensively investigated the effects of the SARA on rumen homeostasis and the RCT on rumen homeostasis remodeling in lactating dairy cows by metagenomics sequencing,transcriptome sequencing,16 S r RNA gene sequencing,and liquid chromatography-mass spectrometry(LC-MS)metabolomics analyses using a cow model induced by a high-grain(HG)diet.The specific contents are as follows:1.Effects of SARA on rumen fermentation and structure and function of rumen microbial community in lactating dairy cowsThe objective of the present study was to investigate the fluctuations of rumen fermentation and the structure and function of rumen microbiota during SARA induced by a high-grain(HG)diet in lactating dairy cows.Eight multiparous lactating Holstein cows,all of which were ruminally cannulated,were selected for a 21 d experiment.The cows were randomly divided into 2 groups,fed either a conventional diet(CON;40% concentrate;dry matter basis)or a HG diet(60% concentrate;dry matter basis).Rumen p H was monitored at0 h before morning feeding and 2,4,6,8,and 12 h later on the third day and the last 2 d of each week.The rumen content samples were collected at 0 h before morning feeding and 4,8,12 h later on the third day and the last day of each week,for the determination of rumen VFA and lactic acid concentrations.Furthermore,the rumen content samples taken at 0 h before morning feeding on the last day of each week were also used for metagenomics sequencing.Compared with the CON diet,the HG diet reduced ruminal average daily p H,acetate concentration,acetate ratio,and the ratio of the concentrations of acetate to propionate(P < 0.05),but increased the concentrations of total volatile fatty acids,propionate,and valerate and the propionate ratio(P < 0.05).Taxonomic analysis indicated that the HG cows had a higher relative abundance of Ruminococcus,Eubacterium,Selenomonas,Ruminobacter,Succinimonas,Methanomicrobium,and Methanocaldococcus accompanied by a lower relative abundance of unclassified Firmicutes,unclassified Bacteroidetes,Bacteroides,Fibrobacter,Alistipes,Candidatus Methanoplasma,Methanomassiliicoccus,and Methanolobus(P < 0.05).Carbohydrate-active enzyme annotation suggested that there was an enriched relative abundance of glycosyltransferases(GT)2,glycoside hydrolase(GH)13,GH24,carbohydrate-binding module(CBM)26,GH73,GH25,CBM12,GH23,GT8,CBM50,and GT9 and reduced relative abundance of GH78,GH31,S-layer homology,GH109,carbohydrate esterase 1,GH3,carbohydrate esterase 10,and GH43 in the HG group(P < 0.05).Functional profiling revealed that the HG feeding mainly downregulated the cellulose degradation pathway,pentose phosphate pathway of carbohydrate catabolism,acetate metabolism,and methane metabolism,whereas it upregulated the starch degradation pathway,the Embden-Meyerhof-Parnas and Entner-Doudoroff pathways of glycolysis,and the citrate cycle.These results elucidated the changes in the structure of rumen microbiota and the function adjustment of rumen microbiota for carbohydrate metabolism during SARA induced by a HG diet in lactating dairy cows,which eventually altered the rumen fermentation,that led to a decrease in the acetate concentration and an increase in the concentrations of propionate,valerate,and total volatile fatty acids.2.Effects of SARA on rumen epithelial morphology,blood biochemical parameters,and rumen epithelium transcriptome in lactating dairy cowsThe objective of the present chapter was to investigate the effects of high-grain-induced SARA on rumen epithelial morphology,blood biochemical parameters,and rumen epithelium transcriptome in lactating dairy cows.The experiment design was the same as in Chapter 1.Blood samples were collected via the tail vein at 6 h after morning feeding on the last day of each week for the determination of blood biochemical parameters.Rumen epithelial papillae were biopsied from the ventral sac before evening feed delivery on the last day of each week for morphological observation and transcriptome sequencing.The results showed that the HG-feeding cows had a thickened rumen epithelial papilla with edge injury and a decreased BHBA concentration compared with the CON-feeding cows(P < 0.05).Transcriptome sequencing indicated that a total of 696 differentially expressed genes(DEGs)were identified between the CON and HG groups with a cutoff condition of an absolute fold change of >1.2 and a Benjamini-Hochberg-adjusted P-value(Q)of <0.05.Enrichment analysis showed that the DEGs were significantly enriched in KEGG pathways of signaling molecules and interaction,cellular processes,and the immune system(Q < 0.05),and DEGs corresponding to these pathways were basically all upregulated in the HG group.Through WGCNA analysis,we obtained a coexpressed gene module(M4 module)that showed the most significant correlation with rumen fermentation parameters,ruminal epithelium thickness,and blood biochemical parameters.And the markedly enriched functions of the M4 module were mainly concentrated on RNA metabolic processes,protein synthesis,the metabolism of some amino acids and organic acids,the tricarboxylic acid cycle,RNA degradation,the proteasome,and steroid biosynthesis,with almost all corresponding genes also being upregulated in the HG group.Preliminary analysis on the pathway of steroid biosynthesis showed that it was mainly enriched in the cholesterol synthesis pathway.Further analysis found that cholesterol synthesis-related genes in the rumen epithelium were all upregulated in the HG group.Generally,these results indicated that the high-grain-induced SARA promoted cell proliferation,caused an inflammatory event,and upregulated the expression of the genes for cholesterol biosynthesis in the ruminal epithelium.Besides,the high-grain-induced SARA further caused changes in host metabolism and reduced plasma BHBA concentration.3.Changes in rumen fermentation and bacterial community in lactating dairy cows with SARA following rumen content transplantationThe objective of the present chapter was to explore the changes in rumen fermentation and bacterial community in lactating dairy cows with SARA following rumen content transplantation(RCT).The entire experiment comprised 2 periods: the SARA induction period and the RCT period.Each period lasted 21 d.During the SARA induction period,12 ruminally cannulated lactating Holstein cows were selected and allocated into 2 groups at random,fed either a conventional diet [CON;n = 4;40% concentrate,dry matter(DM)basis]or a high-grain diet(HG;n = 8;60% concentrate,DM basis).Rumen p H was monitored at 0h before the morning feed delivery and 2,4,6,8,and 12 h later on the third day and the last2 d of each week.Rumen content samples were collected on the third day and the last day of each week before morning feeding and 4,8,and 12 h later for determination of VFA concentrations.After the SARA induction period,the RCT period began.The HG cows were randomly divided into 2 groups: the donor-recipient(DR)group and the self-recipient(SR)group,and their rumen contents were removed completely before the morning feeding.For the DR group,cows received 70% rumen content from the CON cows,paired based on comparable body weight;for the SR group,each cow received 70% rumen content,selfderived.After the transplantation,the diets for all 12 cows were changed to the CON diet.The day of transfer was considered time point 0 d.Rumen p H was monitored before the transfer and on d 1,2,4,5,6,12,13,19,and 20 after transfer.The rumen content samples were obtained before the transfer and on d 1,2,4,6,13,and 20 after transfer,for the determination of VFA concentrations and 16 S r RNA gene sequencing.The results showed that nearly all rumen fermentation parameters returned to the normal levels that the cows had before SARA induction after 6 d of transplantation,regardless of RCT.The concentrations of acetate,valerate,and total volatile fatty acids(VFA)were not recovered in the SR cows,whereas all of them were recovered in the DR cows.The 16 S r RNA gene ssequencing results indicated that both the SR and DR cows rebuild their rumen bacterial homeostasis quickly within 4 d after RCT,and the DR group showed a higher level of bacterial community diversity.At the genus level,the DR cows displayed an improved proportion of unclassified Ruminococcaceae and Saccharofermentans compared with the SR cows(P < 0.05).Correlation analysis between the rumen bacteria and rumen fermentation suggested some potential relationships between the predominant transplantation-sensitive operational taxonomic units and VFA.Co-occurrence network analysis revealed that RCT affected only those rumen bacterial taxa that showed weak interactions with other taxa and did not affect the pivotal rumen bacteria with high levels of co-occurrence.Our findings indicate that RCT contributes to the restoration of rumen bacterial homeostasis and rumen fermentation in cows suffering from SARA without affecting the core microbiome.4.Changes in rumen epithelial morphology and transcriptome,rumen metabolome,and blood biochemical parameters in lactating dairy cows with SARA following rumen content transplantationThe objective of the present chapter was to explore the changes in rumen epithelial morphology and transcriptome,rumen metabolome,and blood biochemical parameters in lactating dairy cows with SARA following rumen content transplantation(RCT).The experiment design was the same as in Chapter 3.Rumen epithelial papillae were biopsied from the ventral sac before evening feed delivery on the day before transfer and on d 1,2,4,6,8,13,16,and 20 after transfer for morphological observation.Moreover,rumen epithelial papillae biopsied on d 2,4,8,and 16 d were also used for transcriptome sequencing.Rumen content samples were obtained before morning feeding on d 1,2,4,6,13,and 20 after transfer for rumen metabolomics analysis.Blood samples were collected via the tail vein at 6 h after morning feeding on the day before transfer and on d 1,2,4,6,13,and 20 after transfer for the determination of blood biochemical parameters.Cross sections of the rumen papillae revealed that there were no significant differences(P > 0.05)in the thicknesses of the stratum corneum,granulosum,and spinosum/basale layers as well as the total depth of the epithelium between the SR and DR groups;and they were all obviously decreased and changed dynamically after the RCT.Transcriptome sequencing indicated that a total of 277 differentially expressed genes(DEGs)were identified between the two groups with a cutoff condition of an absolute fold change of >1.2 and a Benjamini-Hochberg-adjusted P-value(Q)of <0.05.Enrichment analysis showed that the DEGs were significantly enriched in GO biological processes of cellular process and metabolic process as well as the KEGG pathways of glycan biosynthesis and metabolism,amino acid metabolism,lipid metabolism,and the immune system(P < 0.05).Meanwhile,DEGs corresponding to almost each of these terms did not show complete group-dependent up-or down-regulation of expression.The changes in the SR group became stabilized basically after 13 or 16 d of transfer,whereas they became stabilized basically after 8 or 13 d of transfer in the DR group.The metabolomics analysis of rumen fluid samples by liquid chromatography-mass spectrometry indicated that a total of33 differential metabolites were detected between the SR and DR groups(VIP > 1 and P <0.05),with 19 of them showing upregulated levels and 14 of them showing downregulated levels in the DR group.Pathway analysis showed that the 33 metabolites were mainly enriched in five key differential metabolic pathways of plant polysaccharide and starch degradation,lipid metabolism,amino sugar and nucleotide sugar metabolism,purine metabolism,and the Krebs cycle.And levels of all the metabolites that enriched in plant polysaccharide and starch degradation,amino sugar and nucleotide sugar metabolism,and purine metabolism were observed upregulated in the DR group.The results of blood biochemical parameters showed that the triglyceride concentration of the DR cows was significantly higher than that of the SR cows,which was comparable to the level of the CON group during the SARA induction period;whereas the cholesterol concentration had a tendency to decrease in the DR group(P = 0.050).In general,these results indicated that the RCT could accelerate morphology recovery of rumen epithelium,but could not promote its function recovery.Furthermore,the RCT enhanced rumen plant polysaccharide and starch degradation,amino sugar and nucleotide sugar metabolism,and purine metabolism,and further promoted the recovery of the blood biochemical parameters in cows suffering from SARA.In summary,the high-grain-induced SARA disrupted rumen homeostasis,causing changes in the structure and function of rumen microbiota in lactating dairy cows,which altered the rumen fermentation,that led to a decrease in the acetate concentration and an increase in the concentrations of propionate,valerate,and total VFA.Alterations in rumen fermentation affected the structure and function of the rumen epithelium.The rumen epithelial papillae became darkened and thickened,with the appearance of edge injury,and the expression of cholesterol biosynthesis genes in the rumen epithelium was up-regulated,triggering epithelial inflammation.The disruption of rumen homeostasis further caused variations in host metabolism and reduced plasma BHBA concentration.The RCT contributed to the restoration of rumen bacterial homeostasis and rumen fermentation,and the morphology recovery of rumen epithelium,and enhanced rumen plant polysaccharide and starch degradation,amino sugar and nucleotide sugar metabolism,and purine metabolism in cows suffering from SARA.Moreover,the recovery of rumen fermentation parameters was 1 to 2 d slower than the recovery of the rumen bacterial community,and the recovery of rumen epithelial structure was about 1 week slower than the recovery of rumen fermentation parameters.However,the RCT could not promote the recovery of rumen epithelial function.In addition,RCT promoted the recovery of the blood biochemical parameters in cows suffering from SARA. |
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