An integrated microbiome- and metabolome-genome-wide association study reveals the role of heritable ruminal microbial carbohydrate metabolism in lactation performance in Holstein dairy cows

Abstract

Background: Despite the growing number of studies investigating the connection between host genetics and the rumen microbiota, there remains a dearth of systematic research exploring the composition, function, and metabolic traits of highly heritable rumen microbiota influenced by host genetics. Furthermore, the impact of these highly heritable subsets on lactation performance in cows remains unknown. To address this gap, we collected and analyzed whole-genome resequencing data, rumen metagenomes, rumen metabolomes and short-chain fatty acids (SCFAs) content, and lactation performance phenotypes from a cohort of 304 dairy cows.

Results: The results indicated that the proportions of highly heritable subsets (h² ≥ 0.2) of the rumen microbial composition (55%), function (39% KEGG and 28% CAZy), and metabolites (18%) decreased sequentially. Moreover, the highly heritable microbes can increase energy-corrected milk (ECM) production by reducing the rumen acetate/propionate ratio, according to the structural equation model (SEM) analysis (CFI = 0.898). Furthermore, the highly heritable enzymes involved in the SCFA synthesis metabolic pathway can promote the synthesis of propionate and inhibit the acetate synthesis. Next, the same significant SNP variants were used to integrate information from genome-wide association studies (GWASs), microbiome-GWASs, metabolome-GWASs, and microbiome-wide association studies (mWASs). The identified single nucleotide polymorphisms (SNPs) of rs43470227 and rs43472732 on SLC30A9 (Zn²⁺ transport) (P < 0.05/nSNPs) can affect the abundance of rumen microbes such as Prevotella_sp., Prevotella_sp._E15-22, Prevotella_sp._E13-27, which have the oligosaccharide-degradation enzymes genes, including the GH10, GH13, GH43, GH95, and GH115 families. The identified SNPs of chr25:11,177 on 5s_rRNA (small ribosomal RNA) (P < 0.05/nSNPs) were linked to ECM, the abundance alteration of Pseudobutyrivibrio_sp. (a genus that was also showed to be linked to the ECM production via the mWASs analysis), GH24 (lysozyme), and 9,10,13-TriHOME (linoleic acid metabolism). Moreover, ECM, and the abundances of Pseudobutyrivibrio sp., GH24, and 9,10,13-TRIHOME were significantly greater in the GG genotype than in the AG genotype at chr25:11,177 (P < 0.05). By further the SEM analysis, GH24 was positively correlated with Pseudobutyrivibrio sp., which was positively correlated with 9,10,13-triHOME and subsequently positively correlated with ECM (CFI = 0.942).

Conclusion: Our comprehensive study revealed the distinct heritability patterns of rumen microbial composition, function, and metabolism. Additionally, we shed light on the influence of host SNP variants on the rumen microbes with carbohydrate metabolism and their subsequent effects on lactation performance. Collectively, these findings offer compelling evidence for the host-microbe interactions, wherein cows actively modulate their rumen microbiota through SNP variants to regulate their own lactation performance. Video Abstract.

Keywords: Dairy cow; GWAS; Heritability; Host genetics; Lactation performance; Ruminal metabolome; Ruminal metagenome.

Fig. 1

Relationships between the rumen microbiota and the phenotype of dairy cows. A The relationship between the microbial diversity matrix and phenotype matrix was determined based on Mantel's test. The α diversity indices included ACE, Chao1, and Shannon indices. The β diversity indices included PC1, PC2, and PC3 from the PCoA. MY: milk yield, MF: milk fat, MP: milk protein, ML: milk lactose, ECM: energy-corrected milk, A:P: acetate/propionate, TA: total acid. B Differences in the rumen microbiome at the domain, KEGG level 1, and CAZy family levels among the low (31.8 kg/d), medium (42.6 kg/d), and high (52.4 kg/d) ECM groups. C The relationship between the phenotype matrix and the top 50 microbial matrices at the species level was determined based on Mantel's test. Lactation included milk yield, milk fat, milk protein, and milk lactose. The ruSCFAs included acetate, propionate, butyrate, A:P, and total acid. D The co-occurrence networks of the microbes at the species level with relative abundances greater than 0.01%

Fig. 2

The heritability and significant variants of the phenotype of dairy cows. A The heritability of lactation performance and rumen SCFAs. MY: milk yield, MF: milk fat, MP: milk protein, ML: milk lactose, ECM: energy-corrected milk, A:P: acetate/propionate, TA: total acid. B The Q‒Q plot of lactation performance. C Q‒Q plot of rumen SCFAs. D Manhattan plot of lactation performance. The significance threshold was 1/nSNP = 4.28E-07. The extremely significant threshold was 0.05/nSNP = 2.14E-08. E Manhattan plot of rumen SCFAs. The significance threshold was 1/nSNP = 4.28E-07. The extremely significant threshold was 0.05/nSNP = 2.14E-08

Fig. 3

The heritability and significant variants of the rumen microbiota of dairy cows. A The proportion of highly heritable microbes at the species level at the domain level, the proportion of highly heritable KEGG pathways at level 3 at level 1, and the proportion of highly heritable CAZy modules at the family level at the class level were calculated. B The linear relationship between node attributes (degree, closeness, and betweenness) and heritability. P < 0.05 was considered to indicate a linear relationship. C Manhattan plot of highly heritable rumen fat subsets from the top 100 microbes at the species level. The significance threshold was 1/nSNP = 4.28E-07. The extremely significant threshold was 0.05/nSNP = 2.14E-08

Fig. 4

The characteristics of highly heritable rumen microbes of dairy cows. A The relationship between rumen microbes (with high and low heritability) and KEGG pathway enrichment at level 3 in "metabolism". The 20 highly heritable microbes and 30 with lowly heritable microbes within the top 50 with relative abundance were selected at the species level. The microbes were connected to metabolism pathways based on relative contribution (%). The color of the lines was determined based on relative contribution and heritability. Gray: contribution < 1%, blue: low heritable microbes had the highest contribution for pathways, red: high heritable microbes had the highest contribution for pathways. B The effect of highly heritable and weakly heritable microbes on the ECM determined by A:P via SEM. The 5 microbes with the highest and lowest heritability were selected based on heritability at the species level. Highly heritable microbes are integrated into highly heritable latent variables. Lowly heritable microbes are integrated into a lowly heritable latent variable. Red arrows represent positive paths, and green arrows represent negative paths

Fig. 5

The characteristics of highly heritable rumen enzymes and metabolites in dairy cows. A The heritability of CAZy modules at the class level. B The proportion of highly heritable metabolites in the rumen. C The relationship between highly heritable CAZy modules from the top 100 modules at the family level and highly heritable metabolites from the top 50 metabolites, SCFAs. D The relationships between high-heritability enzymes involved in "butanoate metabolism", "glycolysis/gluconeogenesis", "pentose phosphate pathway", "propionate metabolism", "pyruvate metabolism" and the rumen A:P ratio

Fig. 6

Relationships among lactation performance, rumen microbial composition and function, and metabolites based on the mWAS. A Manhattan plot showing the microbes related to the ECM and the rumen A:P ratio based on microbiota-wide association studies (mWASs). B Venn diagram showing the proportion and quantity of microbes associated with the ECM and the rumen A:P ratio. C A density plot was generated to show the heritability of microbes associated with the ECM and the rumen A:P ratio. D A circular Manhattan plot showed that the same variant (chr25:11177 on 5 s_rRNA) of Pseudobutyrivibrio sp. (species-GWAS), GH24 (CAZy-GWAS), metab_11663 (9,10,13-TRIHOME) (metabolite-GWAS), and lactation performance (phenotype-GWAS)