Alterations in the gut microbiota and its metabolites contribute to metabolic maladaptation in dairy cows during the development of hyperketonemia

Abstract

Metabolic maladaptation in dairy cows after calving can lead to long-term elevation of ketones, such as β-hydroxybutyrate (BHB), representing the condition known as hyperketonemia, which greatly influences the health and production performance of cows during the lactation period. Although the gut microbiota is known to alter in dairy cows with hyperketonemia, the association of microbial metabolites with development of hyperketonemia remains unknown. In this study, we performed a multi-omics analysis to investigate the associations between fecal microbial community, fecal/plasma metabolites, and serum markers in hyperketonemic dairy cows during the transition period. Dynamic changes in the abundance of the phyla Verrucomicrobiota and Proteobacteria were detected in the gut microbiota of dairy cows, representing an adaptation to enhanced lipolysis and abnormal glucose metabolism after calving. Random forest and univariate analyses indicated that Frisingicoccus is a key bacterial genus in the gut of cows during the development of hyperketonemia, and its abundance was positively correlated with circulating branched-chain amino acid levels and the ketogenesis pathway. Taurodeoxycholic acid, belonging to the microbial metabolite, was strongly correlated with an increase in blood BHB level, and the levels of other secondary bile acid in the feces and plasma were altered in dairy cows prior to the diagnosis of hyperketonemia, which link the gut microbiota and hyperketonemia. Our results suggest that alterations in the gut microbiota and its metabolites contribute to excessive lipolysis and insulin insensitivity during the development of hyperketonemia, providing fundamental knowledge about manipulation of gut microbiome to improve metabolic adaptability in transition dairy cows.IMPORTANCEAccumulating evidence is pointing to an important association between gut microbiota-derived metabolites and metabolic disorders in humans and animals; however, this association in dairy cows from late gestation to early lactation is poorly understood. To address this gap, we integrated longitudinal gut microbial (feces) and metabolic (feces and plasma) profiles to characterize the phenotypic differences between healthy and hyperketonemic dairy cows from late gestation to early lactation. Our results demonstrate that cows underwent excessive lipid mobilization and insulin insensitivity before hyperketonemia was evident. The bile acids are functional readouts that link gut microbiota and host phenotypes in the development of hyperketonemia. Thus, this work provides new insight into the mechanisms involved in metabolic adaptation during the transition period to adjust to the high energy and metabolic demands after calving and during lactation, which can offer new strategies for livestock management involving intervention of the gut microbiome to facilitate metabolic adaptation.

Keywords: dairy cows; gut microbiota; hyperketonemia; metabolic maladaptation; microbial metabolites; multi-omics.

Fig 1

Establishment of two cohorts of dairy cows. (A) Overview of the study design. (B) Dynamic changes of serum variables in HE and HYK dairy cows from late gestation (d −7) to early lactation (d +14). TCHO, total cholesterol; LC-MS/MS, liquid chromatography-tandem mass spectrometry.

Fig 2

Longitudinal changes of the fecal microbial community in HE and HYK dairy cows. (A) Alpha-diversity of the fecal microbiota, including Chao1 and Shannon indices, at different time points. (B) Principal coordinates analysis (PCoA) plot of beta-diversity based on the Bray-Curtis distance. (C) Associations between blood BHB level and microbial diversity index according to Spearman’s rank correlation coefficient. (D) Percentage accumulation chart displaying the relative abundance of microbial taxa at the phylum level. (E) Ratio of Firmicutes to Bacteroidota (F/B) within and between the HE and HYK cows at each time point. Data are presented as means ± SEM. (F) Dynamic changes of relative abundance of the microbial phyla Verrucomicrobiota, Proteobacteria, and Actinobacteriota. *P < 0.05. **P < 0.01.

Fig 3

Co-occurrence network analysis of gut microbiota. (A) The co-occurrence networks of genera in HE and HYK dairy cows during d −7 to d +14. Only genera with a correlation coefficient >0.5 or <−0.5 and significance of P < 0.05 are shown. The node size indicates the proportion of mean abundance in the genus and the node color represents the relative abundance in different phases. (B) Venn diagram indicating the edge numbers in each network and their overlap. (C and D) Histograms displaying the edge numbers for the top 15 microbial taxa of HE (blue) and HYK (pink) cows, respectively, at the genus level.

Fig 4

Links between gut microbiota and ketogenesis in dairy cows. (A) Volcano plot indicating the differences in the abundances of gut bacteria at the genus level between the HE and HYK cows at each time point. (B) Comparisons of Lactobacillus and Frisingicoccus abundances between the HE and HYK cows over time. Data are presented as means ± SEM. (C) Associations between the abundance of Lactobacillus or Frisingicoccus and circulating BHB levels in dairy cows based on Spearman’s correlation analysis. (D) The heatmap displays the associations between serum marker level and the abundance of Lactobacillus or Frisingicoccus. TKA, total ketogenic amino acids; TGA, total glucogenic amino acids; TGKA, total both glucogenic and ketogenic amino acids; TAA, total amino acids; TCHO, total cholesterol. (E) Dynamic change of circulating individual amino acids involved in the glucogenic and ketogenic pathways. The circle color indicates the fold change between HE and HYK groups at each time point. The line color indicates the metabolic pathway of amino acids. PEP, phosphoenolpyruvate; TCA, tricarboxylic acid. (F) Metabolism pathway of branched-chain amino acids (BCAAs), involving key Kyoto Encyclopedia of Genes and Genomes orthologs (KO) of microbial taxa related to glucogenesis and ketogenesis. The line chart indicates the dynamic changes of KO abundance between the HE and HYK groups from d −7 to d +14. k00632, fadA/fadI (acetyl-CoA acyltransferase); k01825, fadB (3-hydroxyacyl-CoA dehydrogenase); k01965, pccA (propionyl-CoA carboxylase alpha chain); k11532, glpX-SEBP (fructose-1,6-bisphosphatase II/sedoheptulose-1,7-bisphosphatase); k01623, ALDO (fructose-bisphosphate aldolase, class I). 0.01 < *P < 0.05, **P < 0.01.

Fig 5

Alterations in fecal metabolites between HE and HYK dairy cows. (A) Scatter plot showing differential metabolites between the HYK and HE groups at each time point based on the variance in projection (VIP) value of OPLS-DA. (B and C) Change and origin of shared differential metabolites in the non-hyperketonemia and hyperketonemia periods, respectively. (D) Associations of blood BHB level with the abundance of fecal TDCA. (E) Differential analysis of secondary bile acid (SBA) biosynthesis between HE and HYK cows based on functional prediction of gut microbiota. (F) Change of SBA biosynthesis correlated with an increase in the circulating BHB level of dairy cows from late gestation to early lactation. (G) Lollipop plot displaying the differences in the levels of individual bile acids in feces of cows in the HE and HYK groups at each time point. 0.01 < *P < 0.05, **P < 0.01.

Fig 6

Plasma bile acid (BA) profiles in the HE and HYK dairy cows. (A) TBA. (B) Ratio of SBA to TBA. (C) Ratio of 12-OH to non-12-OH BAs (log₁₀-transformed data). (D) Associations of 12-OH/non-12-OH BAs with SBA/TBA based on Spearman’s correlation analysis. (E) The classification of BA differences in the HE and HYK groups at different time points. Cholic acid (CA) species comprise CA, glycolic acid (GCA), and taurocholic acid (TCA). DCA species comprise DCA, glycodeoxycholic acid (GDCA), TDCA, isoDCA, and norDCA. Chenodeoxycholic acid (CDCA) species comprise CDCA, glycochenodeoxycholic acid (GCDCA), and taurochenodeoxycholic acid (TCDCA). Lithocholic acid (LCA) species comprise LCA, glycolithocholic acid (GLCA), taurolithocholic acid (TLCA), isoLCA, 7-ketoLCA, and 12-ketoLCA. Other BA species include glycoursodeoxycholic acid (GUDCA), 3-dehydrocholic acid (DHCA), and 7-DHCA. (F) Comparison of plasma levels of individual BAs between the HE and HYK groups at each time point. The node size indicates the P-value after −log₁₀ transformation. The node color indicates the fold change (FC) of individual BA levels between the HE and HYK cows; a log₂(FC) greater than 0 indicates relatively higher concentration in the HYK cows, whereas a log₂(FC) less than 0 indicates a lower concentration in HYK cows than in HE cows. 0.01 < *P < 0.05, **P < 0.01.

Fig 7

Links between gut microbial community and host phenotypes in non-hyperketonemia and hyperketonemia periods. The heatmap displays the relationships among serum markers, plasma bile acids, and plasma amino acids composition based on Spearman’s correlation analysis. The line indicates the relationship of the gut microbiota matrix with the blood variables matrix based on the Mantel test. The line color indicates the P-value in the Mantel test and the thickness of the line indicates the correlation coefficient. GLCA, glycollithocholic acid; TCA, taurocholic acid; GCA, glycolic acid; GDCA, glycodeoxycholic acid; S/T, secondary bile acids/total bile acids ratio; CHO, cholesterol; TKA, total ketogenic amino acids; TGA, total glucogenic amino acids; TGKA, total glucogenic and ketogenic amino acids.