Single-cell transcriptomics across 2,534 microbial species reveals functional heterogeneity in the rumen microbiome.

Deciphering the activity of individual microbes within complex communities and environments remains a challenge. Here we describe the development of microbiome single-cell transcriptomics using droplet-based single-cell RNA sequencing and pangenome-based computational analysis to characterize the functional heterogeneity of the rumen microbiome. We generated a microbial genome database (the Bovine Gastro Microbial Genome Map) as a functional reference map for the construction of a single-cell transcriptomic atlas of the rumen microbiome. The atlas includes 174,531 microbial cells and 2,534 species, of which 172 are core active species grouped into 12 functional clusters. We detected single-cell-level functional roles, including a key role for Basfia succiniciproducens in the carbohydrate metabolic niche of the rumen microbiome. Furthermore, we explored functional heterogeneity and reveal metabolic niche trajectories driven by biofilm formation pathway genes within B. succiniciproducens. Our results provide a resource for studying the rumen microbiome and illustrate the diverse functions of individual microbial cells that drive their ecological niche stability or adaptation within the ecosystem.

- Invited Review - Understanding the functionality of the rumen microbiota: searching for better opportunities for rumen microbial manipulation.

Rumen microbiota play a central role in the digestive process of ruminants. Their remarkable ability to break down complex plant fibers and proteins, converting them into essential organic compounds that provide animals with energy and nutrition. Research on rumen microbiota not only contributes to improving animal production performance and enhancing feed utilization efficiency but also holds the potential to reduce methane emissions and environmental impact. Nevertheless, studies on rumen microbiota face numerous challenges, including complexity, difficulties in cultivation, and obstacles in functional analysis. This review provides an overview of microbial species involved in the degradation of macromolecules, the fermentation processes, and methane production in the rumen, all based on cultivation methods. Additionally, the review introduces the applications, advantages, and limitations of emerging omics technologies such as metagenomics, metatranscriptomics, metaproteomics, and metabolomics, in investigating the functionality of rumen microbiota. Finally, the article offers a forward-looking perspective on the new horizons and technologies in the field of rumen microbiota functional research. These emerging technologies, with continuous refinement and mutual complementation, have deepened our understanding of rumen microbiota functionality, thereby enabling effective manipulation of the rumen microbial community.

Dynamic fecal microenvironment properties enable predictions and understanding of peripartum blood oxidative status and nonesterified fatty acids in dairy cows.

The transition period in dairy cows is a critical stage and peripartum oxidative status, negative energy balance (NEB), and inflammation are highly prevalent. Fecal microbial metabolism is closely associated with blood oxidative status and nonesterified fatty acids (NEFA) levels. Here, we investigated dynamic changes in total oxidative status markers and NEFA in blood, fecal microbiome, and metabolome of 30 dairy cows during transition (-21, -7, +7, +21 d relative to calving). Then the Bayesian network and 9 machine-learning algorithms were applied to dismantle their relationship. Our results show that the oxidative status indicator (OSI) of -21, -7, +7 d was higher than +21 d. The plasma concentration of NEFA peaked on +7 d. For fecal microenvironment, a decline in bacterial α diversity was observed at postpartum and in bacterial interactions at +7 d. Conversely, microbial metabolites involved in carbohydrate, lipid, and energy metabolism increased on +7 d. A correlation analysis revealed that 11 and 10 microbial metabolites contributed to OSI and NEFA variations, respectively (arc strength >0.5). The support vector machine (SVM) radial model showed the highest average predictive accuracy (100% and 88.9% in the test and external data sets) for OSI using 1 metabolite and 3 microbiota. The SVM radial model also showed the highest average diagnostic accuracy (100% and 91% in the test and external data sets) for NEFA with 2 metabolites and 3 microbiota. Our results reveal a relationship between variation in the fecal microenvironment and indicators of oxidative status, NEB, and inflammation, which provide a theoretical basis for the prevention and precise regulation of peripartum oxidative status and NEB.

Age- and Microbiota-Dependent Cell Stemness Plasticity Revealed by Cattle Cell Landscape.

Newborn ruminants are considered functionally monogastric animals. The poor understanding of cellular differences between newborn and mature ruminants prevents the improvement of health and performance of domestic ruminants. Here, we performed the single-cell RNA sequencing on the rumen, reticulum, omasum, abomasum, duodenum, jejunum, ileum, cecum, colon, rectum, liver, salivary gland, and mammary gland from newborn and adult cattle. A comprehensive single-cell transcriptomic atlas covering 235,941 high-quality single cells and 78 cell types was deciphered. A Cattle Cell Landscape database (http://cattlecelllandscape.zju.edu.cn) was established to elaborately display the data and facilitate effective annotation of cattle cell types and subtypes for the broad research community. By measuring stemness states of epithelial cells in each tissue type, we revealed that the epithelial cells from newborn forestomach (rumen, reticulum, and omasum) were more transcriptionally indistinct and stochastic compared with the adult stage, which was in contrast to those of abomasum and intestinal tissues. The rapid forestomach development during the early life of calves was driven by epithelial progenitor-like cells with high DNA repair activities and methylation. Moreover, in the forestomach tissues of newborn calves, the Megasphaera genus was involved in regulating the transcriptional plasticity of the epithelial progenitor-like cells by DNA methylation regulation. A novel cell type, the STOML3+ cell, was found to be newborn-specific. It apparently plays a crucial role in stemness maintenance of its own and cholangiocytes in the hepatic microenvironment. Our results reveal that the age- and microbiota-dependent cell stemness plasticity drives the postnatal functional maturity of ruminants.

The hindgut microbiome contributes to host oxidative stress in postpartum dairy cows by affecting glutathione synthesis process.

BACKGROUND: Dairy cows are susceptible to postpartum systemic oxidative stress (OS), which leads to significant production loss and metabolic disorders. The gut microbiota has been linked to host health and stress levels. However, to what extent the gut microbiota is associated with postpartum OS remains unknown. In this study, the contribution of the fecal microbiota to postpartum systemic OS and its underlying mechanisms were investigated by integrating 16S rRNA gene sequencing, metagenomics, and metabolomics in postpartum dairy cattle and by transplanting fecal microbiota from cattle to mice. RESULTS: A strong link was found between fecal microbial composition and postpartum OS, with an explainability of 43.1%. A total of 17 significantly differential bacterial genera and 19 species were identified between cows with high (HOS) and low OS (LOS). Among them, 9 genera and 16 species showed significant negative correlations with OS, and Marasmitruncus and Ruminococcus_sp._CAG:724 had the strongest correlations. The microbial functional analysis showed that the fecal microbial metabolism of glutamine, glutamate, glycine, and cysteine involved in glutathione synthesis was lower in HOS cows. Moreover, 58 significantly different metabolites were identified between HOS and LOS cows, and of these metabolites, 19 were produced from microbiota or cometabolism of microbiota and host. Furthermore, these microbial metabolites were enriched in the metabolism of glutamine, glutamate, glycine, and cysteine. The mice gavaged with HOS fecal microbiota had significantly higher OS and lower plasma glutathione peroxidase and glutathione content than those orally administered saline or LOS fecal microbiota. CONCLUSIONS: Integrated results suggest that the fecal microbiota is responsible for OS and that lower glutathione production plays a causative role in HOS. These findings provide novel insights into the mechanisms of postpartum OS and potential regulatory strategies to alleviate OS in dairy cows. Video Abstract.

Gut microbiome is linked to functions of peripheral immune cells in transition cows during excessive lipolysis.

BACKGROUND: Postpartum dairy cows experiencing excessive lipolysis are prone to severe immunosuppression. Despite the extensive understanding of the gut microbial regulation of host immunity and metabolism, its role during excessive lipolysis in cows is largely unknown. Herein, we investigated the potential links between the gut microbiome and postpartum immunosuppression in periparturient dairy cows with excessive lipolysis using single immune cell transcriptome, 16S amplicon sequencing, metagenomics, and targeted metabolomics. RESULTS: The use of single-cell RNA sequencing identified 26 clusters that were annotated to 10 different immune cell types. Enrichment of functions of these clusters revealed a downregulation of functions in immune cells isolated from a cow with excessive lipolysis compared to a cow with low/normal lipolysis. The results of metagenomic sequencing and targeted metabolome analysis together revealed that secondary bile acid (SBA) biosynthesis was significantly activated in the cows with excessive lipolysis. Moreover, the relative abundance of gut Bacteroides sp. OF04 - 15BH, Paraprevotella clara, Paraprevotella xylaniphila, and Treponema sp. JC4 was mainly associated with SBA synthesis. The use of an integrated analysis showed that the reduction of plasma glycolithocholic acid and taurolithocholic acid could contribute to the immunosuppression of monocytes (CD14+MON) during excessive lipolysis by decreasing the expression of GPBAR1. CONCLUSIONS: Our results suggest that alterations in the gut microbiota and their functions related to SBA synthesis suppressed the functions of monocytes during excessive lipolysis in transition dairy cows. Therefore, we concluded that altered microbial SBA synthesis during excessive lipolysis could lead to postpartum immunosuppression in transition cows. Video Abstract.

Microbiota-host crosstalk in the newborn and adult rumen at single-cell resolution.

BACKGROUND: The rumen is the hallmark organ of ruminants, playing a vital role in their nutrition and providing products for humans. In newborn suckling ruminants milk bypasses the rumen, while in adults this first chamber of the forestomach has developed to become the principal site of microbial fermentation of plant fibers. With the advent of single-cell transcriptomics, it is now possible to study the underlying cell composition of rumen tissues and investigate how this relates the development of mutualistic symbiosis between the rumen and its epithelium-attached microbes. RESULTS: We constructed a comprehensive cell landscape of the rumen epithelium, based on single-cell RNA sequencing of 49,689 high-quality single cells from newborn and adult rumen tissues. Our single-cell analysis identified six immune cell subtypes and seventeen non-immune cell subtypes of the rumen. On performing cross-species analysis of orthologous genes expressed in epithelial cells of cattle rumen and the human stomach and skin, we observed that the species difference overrides any cross-species cell-type similarity. Comparing adult with newborn cattle samples, we found fewer epithelial cell subtypes and more abundant immune cells, dominated by T helper type 17 cells in the rumen tissue of adult cattle. In newborns, there were more fibroblasts and myofibroblasts, an IGFBP3+ epithelial cell subtype not seen in adults, while dendritic cells were the most prevalent immune cell subtype. Metabolism-related functions and the oxidation-reduction process were significantly upregulated in adult rumen epithelial cells. Using 16S rDNA sequencing, fluorescence in situ hybridization, and absolute quantitative real-time PCR, we found that epithelial Desulfovibrio was significantly enriched in the adult cattle. Integrating the microbiome and metabolome analysis of rumen tissues revealed a high co-occurrence probability of Desulfovibrio with pyridoxal in the adult cattle compared with newborn ones while the scRNA-seq data indicated a stronger ability of pyroxidal binding in the adult rumen epithelial cell subtypes. These findings indicate that Desulfovibrio and pyridoxal likely play important roles in maintaining redox balance in the adult rumen. CONCLUSIONS: Our integrated multi-omics analysis provides novel insights into rumen development and function and may facilitate the future precision improvement of rumen function and milk/meat production in cattle.

Heritable and Nonheritable Rumen Bacteria Are Associated with Different Characters of Lactation Performance of Dairy Cows.

Recent studies have reported that some rumen microbes are heritable. However, it is necessary to clarify the functions and specific contributions of the heritable rumen microbes to cattle phenotypes (microbiability) in comparison with those that are nonheritable. This study aimed to identify the distribution and predicted functions of heritable and nonheritable bacterial taxa at species level in the rumen of dairy cows and their respective contributions to energy-corrected milk yield, protein content and yield, and fat content and yield in milk. Thirty-two heritable and 674 nonheritable bacterial taxa were identified at species level, and the functional analysis revealed that predicted microbial functions for both groups were mainly enriched for energy, amino acid, and ribonucleotide metabolism. The mean microbiability (to reflect a single taxon's contribution) of heritable bacteria was found to range from 0.16% to 0.33% for the different milk traits, whereas the range for nonheritable bacteria was 0.03% to 0.06%. These findings suggest a strong contribution by host genetics in shaping the rumen microbiota, which contribute significantly to milk production traits. Therefore, there is an opportunity to further improve milk production traits through attention to host genetics and the interaction with the rumen microbiota. IMPORTANCE Rumen bacteria produce volatile fatty acids which exert a far-reaching influence on hepatic metabolism, mammary gland metabolism, and animal production. In the current study, 32 heritable and 674 nonheritable bacterial taxa at species level were identified, and shown to have different microbiability (overall community contribution) and mean microbiability (the average of a single taxon's contribution) for lactation performance. The predicted functions of heritable and nonheritable bacterial taxa also differed, suggesting that targeted nutritional and genetic breeding approaches could be used to manipulate them to improve dairy cow performance.

An Age Effect of Rumen Microbiome in Dairy Buffaloes Revealed by Metagenomics.

Age is an important factor in shaping the gut microbiome. However, the age effect on the rumen microbial community for dairy buffaloes remains less explored. Using metagenomics, we examined the microbial composition and functions of rumen microbiota in dairy Murrah buffaloes of different ages: Y (1 year old), M (3−5 years old), E (6−8 years old), and O (>9 years old). We found that Bacteroidetes and Firmicutes were the predominant phyla, with Prevotella accounting for the highest abundance at the genus level. The proportion of Bacteroides and Methanobrevibacter significantly increased with age, while the abundance of genus Lactobacillus significantly decreased with age (LDA > 3, p < 0.05). Most differed COG and KEGG pathways were enriched in Y with carbohydrate metabolism, while older buffaloes enriched more functions of protein metabolism and the processing of replication and repair (LDA > 2, p < 0.05). Additionally, the functional contribution analysis revealed that the genera Prevotella and Lactobacillus of Y with more functions of CAZymes encoded genes of glycoside hydrolases and carbohydrate esterases for their roles of capable of metabolizing starch and sucrose-associated oligosaccharide enzyme, hemicellulase, and cellulase activities than the other three groups (LDA > 2, p < 0.05), thus affecting the 1-year-old dairy buffalo rumen carbohydrate metabolism. This study provides comprehensive dairy buffalo rumen metagenome data and assists in manipulating the rumen microbiome for improved dairy buffalo production.

Formation of Blood Neutrophil Extracellular Traps Increases the Mastitis Risk of Dairy Cows During the Transition Period.

The current study was conducted to analyze the functions of blood neutrophils in transition cows and their association with postpartum mastitis risk as indicated by somatic cell counts (SCCs) in milk. Seventy-six healthy Holstein dairy cows were monitored from Week 4 prepartum to Week 4 postpartum. Five dairy cows with low SCCs (38 ± 6.0 × 103/mL) and five with high SCCs (3,753 ± 570.0 × 103/mL) were selected based on milk SCCs during the first three weeks of lactation. At Week 1 pre- and postpartum, serum samples were obtained from each cow to measure neutrophil extracellular trap (NET)-related variables, and blood neutrophils were collected for transcriptome analysis by RNA sequencing. The serum concentration of NETs was significantly higher (P < 0.05) in cows with high SCCs than in cows with low SCCs (36.5 ± 2.92 vs. 18.4 ± 1.73 ng/mL). The transcriptomic analysis revealed that the transcriptome differences in neutrophils between high- and low-SCC cows were mainly in cell cycle-related pathways (42.6%), including the cell cycle, DNA damage, and chromosomal conformation, at Week 1 prepartum. The hub genes of these pathways were mainly involved in both the cell cycle and NETosis. These results indicated that the formation of NETs in the blood of transition dairy cows was different between cows with low and high SCCs, which may be used as a potential indicator for the prognosis of postpartum mastitis risk and management strategies of perinatal dairy cows.

Feedomics provides bidirectional omics strategies between genetics and nutrition for improved production in cattle.

Increasing the efficiency and sustainability of cattle production is an effective way to produce valuable animal proteins for a growing human population. Genetics and nutrition are the 2 major research topics in selecting cattle with beneficial phenotypes and developing genetic potentials for improved performance. There is an inextricable link between genetics and nutrition, which urgently requires researchers to uncover the underlying molecular mechanisms to optimize cattle production. Feedomics integrates a range of omic techniques to reveal the mechanisms at different molecular levels related to animal production and health, which can provide novel insights into the relationships of genes and nutrition/nutrients. In this review, we summarized the applications of feedomics techniques to reveal the effect of genetic elements on the response to nutrition and investigate how nutrients affect the functional genome of cattle from the perspective of both nutrigenetics and nutrigenomics. We highlighted the roles of rumen microbiome in the interactions between host genes and nutrition. Herein, we discuss the importance of feedomics in cattle nutrition research, with a view to ensure that cattle exhibit the best production traits for human consumption from both genetic and nutritional aspects.

Cross-tissue single-cell transcriptomic landscape reveals the key cell subtypes and their potential roles in the nutrient absorption and metabolism in dairy cattle.

Introduction: Dairy cattle are a vitally important ruminant in meeting the demands for high-quality animal protein production worldwide. The complicated biological process of converting human indigestible biomass into highly digestible and nutritious milk is orchestrated by various tissues. However, poorly understanding of the cellular composition and function of the key metabolic tissues hinders the improvement of health and performance of domestic ruminants. Objectives: The cellular heterogeneity, metabolic features, interactions across ten tissue types of lactating dairy cattle were studied at single-cell resolution in the current study. Methods: Unbiased single-cell RNA-sequencing and analysis were performed on the rumen, reticulum, omasum, abomasum, ileum, rectum, liver, salivary gland, mammary gland, and peripheral blood of lactating dairy cattle. Immunofluorescences and fluorescence in situ hybridization were performed to verify cell identity. Results: In this study, we constructed a single-cell landscape covering 88,013 high-quality (500 < genes < 4,000, UMI < 50, 000, and mitochondrial gene ratio < 40% or 15%) single cells and identified 55 major cell types in lactating dairy cattle. Our systematic survey of the gene expression profiles and metabolic features of epithelial cells related to nutrient transport revealed cell subtypes that have preferential absorption of different nutrients. Importantly, we found that T helper type 17 (Th17) cells (highly expressing CD4 and IL17A) were specifically enriched in the forestomach tissues and predominantly interacted with the epithelial cell subtypes with high potential uptake capacities of short-chain fatty acids through IL-17 signaling. Furthermore, the comparison between IL17RAhighIL17RChigh cells (epithelial cells with IL17RA and IL17RC expression levels both greater than 0.25) and other cells explained the importance of Th17 cells in regulating the epithelial cellular transcriptional response to nutrient transport in the forestomach. Conclusion: The findings enhance our understanding of the cellular biology of ruminants and open new avenues for improved animal production of dairy cattle.

Breed dependent regulatory mechanisms of beneficial and non-beneficial fatty acid profiles in subcutaneous adipose tissue in cattle with divergent feed efficiency.

The current study aimed to determine whether breed and feed efficiency affect the molecular mechanisms regulating beneficial and non-beneficial fatty acid profiles in subcutaneous adipose tissue of beef steers. Fatty acid profiling and RNA-Seq based transcriptome analysis were performed on subcutaneous adipose tissues collected from beef steers with three divergent breeds (Angus, ANG, n = 47; Charolais, CHAR, n = 48; Kinsella Composite, KC, n = 48) and different residual feed intake (RFI, a measure of feed efficiency). The comparison of fatty acid profiles showed that KC had higher beneficial FAs compared to the other two breeds. Distinct FA profiles between H-RFIfat and L-RFIfat steers was more obvious for KC steers, where H-RFIfat steers tended to have higher proportion of healthy FAs and lower proportion of the unhealthy FAs. A higher number of differentially expressed (DE) genes were observed for KC steers, whereas ANG and CHAR steers had a lower number of DE genes between H- and L-RFIfat steers. The association analyses of the gene expressions and FA profiles showed that 10 FA metabolism-associated genes together with the one upstream regulator (SREBF1) were associated with the proportion of C18:2n-6, total n-6, PUFA and PUFA/SFA for KC steers but not the other two breeds. Subcutaneous adipose tissue FA profiles and healthy FA index differed in cattle with divergent feed efficiency and such variation was unique for the three examined cattle breeds. Key FA metabolism-associated genes together with SREBF1 which is the upstream regulator of a set of genes involved in lipid metabolism may be of importance for genetic selection of meat with higher healthy FA index in beef cattle.

Integrated meta-omics reveals new ruminal microbial features associated with feed efficiency in dairy cattle.

BACKGROUND: As the global population continues to grow, competition for resources between humans and livestock has been intensifying. Increasing milk protein production and improving feed efficiency are becoming increasingly important to meet the demand for high-quality dairy protein. In a previous study, we found that milk protein yield in dairy cows was associated with the rumen microbiome. The objective of this study was to elucidate the potential microbial features that underpins feed efficiency in dairy cows using metagenomics, metatranscriptomics, and metabolomics. RESULTS: Comparison of metagenomic and metatranscriptomic data revealed that the latter was a better approach to uncover the associations between rumen microbial functions and host performance. Co-occurrence network analysis of the rumen microbiome revealed differential microbial interaction patterns between the animals with different feed efficiency, with high-efficiency animals having more and stronger associations than low-efficiency animals. In the rumen of high-efficiency animals, Selenomonas and members of the Succinivibrionaceae family positively interacted with each other, functioning as keystone members due to their essential ecological functions and active carbohydrate metabolic functions. At the metabolic level, analysis using random forest machine learning suggested that six ruminal metabolites (all derived from carbohydrates) could be used as metabolic markers that can potentially differentiate efficient and inefficient microbiomes, with an accuracy of prediction of 95.06%. CONCLUSIONS: The results of the current study provided new insights into the new ruminal microbial features associated with feed efficiency in dairy cows, which may improve the ability to select animals for better performance in the dairy industry. The fundamental knowledge will also inform future interventions to improve feed efficiency in dairy cows. Video Abstract.

Investigation of fiber utilization in the rumen of dairy cows based on metagenome-assembled genomes and single-cell RNA sequencing.

BACKGROUND: Dairy cows utilize human-inedible, low-value plant biomass to produce milk, a low-cost product with rich nutrients and high proteins. This process largely relies on rumen microbes that ferment lignocellulose and cellulose to produce volatile fatty acids (VFAs). The VFAs are absorbed and partly metabolized by the stratified squamous rumen epithelium, which is mediated by diverse cell types. Here, we applied a metagenomic binning approach to explore the individual microbes involved in fiber digestion and performed single-cell RNA sequencing on rumen epithelial cells to investigate the cell subtypes contributing to VFA absorption and metabolism. RESULTS: The 52 mid-lactating dairy cows in our study (parity = 2.62 ± 0.91) had milk yield of 33.10 ± 6.72 kg. We determined the fiber digestion and fermentation capacities of 186 bacterial genomes using metagenomic binning and identified specific bacterial genomes with strong cellulose/xylan/pectin degradation capabilities that were highly associated with the biosynthesis of VFAs. Furthermore, we constructed a rumen epithelial single-cell map consisting of 18 rumen epithelial cell subtypes based on the transcriptome of 20,728 individual epithelial cells. A systematic survey of the expression profiles of genes encoding candidates for VFA transporters revealed that IGFBP5+ cg-like spinous cells uniquely highly expressed SLC16A1 and SLC4A9, suggesting that this cell type may play important roles in VFA absorption. Potential cross-talk between the microbiome and host cells and their roles in modulating the expression of key genes in the key rumen epithelial cell subtypes were also identified. CONCLUSIONS: We discovered the key individual microbial genomes and epithelial cell subtypes involved in fiber digestion, VFA uptake and metabolism, respectively, in the rumen. The integration of these data enables us to link microbial genomes and epithelial single cells to the trophic system. Video abstract.

Multi-omics revealed the effects of rumen-protected methionine on the nutrient profile of milk in dairy cows.

Cow's milk is a highly-nutritious dairy product part of human diet worldwide. Rumen-protected methionine (RPM) is widely used to improve lactation performance of dairy cows, but understanding of the effects of RPM on milk nutrients composition are still limited. In this study, twenty mid-lactating dairy cows were supplemented with 20 gm/day RPM for 8 weeks to investigate the responses of milk nutritional composition to RPM. Metabolomics was applied for analyzing milk metabolites and 16S rRNA gene sequencing was used for analysis of rumen microbial composition. Milk fat content and yield were significantly increased after RPM supplementation. Totally 443 compounds belonging to 15 classes were identified, among which 15 metabolites were significantly changed. The functional nutrient α-ketoglutaric acid were significantly increased in the milk after RPM supplementation. We found 48 significantly differing bacterial genera in the rumen after supplementing RPM. Multi-omics integrated analysis revealed the higher abundance of Acetobacter, unclassified_f_Lachnospiraceae and Saccharofermentan contributed to the improved milk fat. In addition, the enriched abundance of Thermoactinomyces, Asteroleplasma, and Saccharofermentan showed positive correlations with higher α-ketoglutaric acid of milk. Our results uncover the metabolomic fingerprint and the key functional metabolites in the milk after supplementing RPM in dairy cows, as well as the key rumen bacteria associated with them. These findings provide novel insights into the development of functional dairy products that enriched the functional nutrient α-ketoglutaric acid or high milk fat.

Metagenomics analysis revealed the distinctive ruminal microbiome and resistive profiles in dairy buffaloes.

BACKGROUND: Antimicrobial resistance poses super challenges in both human health and livestock production. Rumen microbiota is a large reservoir of antibiotic resistance genes (ARGs), which show significant varations in different host species and lifestyles. To compare the microbiome and resistome between dairy cows and dairy buffaloes, the microbial composition, functions and harbored ARGs of rumen microbiota were explored between 16 dairy cows (3.93 ± 1.34 years old) and 15 dairy buffaloes (4.80 ± 3.49 years old) using metagenomics. RESULTS: Dairy buffaloes showed significantly different bacterial species (LDA > 3.5 & P < 0.01), enriched KEGG pathways and CAZymes encoded genes (FDR < 0.01 & Fold Change > 2) in the rumen compared with dairy cows. Distinct resistive profiles were identified between dairy cows and dairy buffaloes. Among the total 505 ARGs discovered in the resistome of dairy cows and dairy buffaloes, 18 ARGs conferring resistance to 16 antibiotic classes were uniquely detected in dairy buffaloes. Gene tcmA (resistance to tetracenomycin C) presented high prevalence and age effect in dairy buffaloes, and was also highly positively correlated with 93 co-expressed ARGs in the rumen (R = 0.98 & P = 5E-11). In addition, 44 bacterial species under Lactobacillus genus were found to be associated with tcmA (R > 0.95 & P < 0.001). L. amylovorus and L. acidophilus showed greatest potential of harboring tcmA based on co-occurrence analysis and tcmA-containing contigs taxonomic alignment. CONCLUSIONS: The current study revealed distinctive microbiome and unique ARGs in dairy buffaloes compared to dairy cattle. Our results provide novel understanding on the microbiome and resistome of dairy buffaloes, the unique ARGs and associated bacteria will help develop strategies to prevent the transmission of ARGs.

Ruminal resistome of dairy cattle is individualized and the resistotypes are associated with milking traits.

BACKGROUND: Antimicrobial resistance is one of the most urgent threat to global public health, as it can lead to high morbidity, mortality, and medical costs for humans and livestock animals. In ruminants, the rumen microbiome carries a large number of antimicrobial resistance genes (ARGs), which could disseminate to the environment through saliva, or through the flow of rumen microbial biomass to the hindgut and released through feces. The occurrence and distribution of ARGs in rumen microbes has been reported, revealing the effects of external stimuli (e.g., antimicrobial administrations and diet ingredients) on the antimicrobial resistance in the rumen. However, the host effect on the ruminal resistome and their interactions remain largely unknown. Here, we investigated the ruminal resistome and its relationship with host feed intake and milk protein yield using metagenomic sequencing. RESULTS: The ruminal resistome conferred resistance to 26 classes of antimicrobials, with genes encoding resistance to tetracycline being the most predominant. The ARG-containing contigs were assigned to bacterial taxonomy, and the majority of highly abundant bacterial genera were resistant to at least one antimicrobial, while the abundances of ARG-containing bacterial genera showed distinct variations. Although the ruminal resistome is not co-varied with host feed intake, it could be potentially linked to milk protein yield in dairy cows. Results showed that host feed intake did not affect the alpha or beta diversity of the ruminal resistome or the abundances of ARGs, while the Shannon index (R2 = 0.63, P < 0.01) and richness (R2 = 0.67, P < 0.01) of the ruminal resistome were highly correlated with milk protein yield. A total of 128 significantly different ARGs (FDR < 0.05) were identified in the high- and low-milk protein yield dairy cows. We found four ruminal resistotypes that are driven by specific ARGs and associated with milk protein yield. Particularly, cows with low milk protein yield are classified into the same ruminal resistotype and featured by high-abundance ARGs, including mfd and sav1866. CONCLUSIONS: The current study uncovered the prevalence of ARGs in the rumen of a cohort of lactating dairy cows. The ruminal resistome is not co-varied with host feed intake, while it could be potentially linked to milk protein yield in dairy cows. Our results provide fundamental knowledge on the prevalence, mechanisms and impact factors of antimicrobial resistance in dairy cattle and are important for both the dairy industry and other food animal antimicrobial resistance control strategies.

Predicting Daily Dry Matter Intake Using Feed Intake of First Two Hours after Feeding in Mid and Late Lactation Dairy Cows with Fed Ration Three Times per Day.

The objective of this study was to evaluate the feasibility of using the dry matter intake of first 2 h after feeding (DMI-2h), body weight (BW), and milk yield to estimate daily DMI in mid and late lactating dairy cows with fed ration three times per day. Our dataset included 2840 individual observations from 76 cows enrolled in two studies, of which 2259 observations served as development dataset (DDS) from 54 cows and 581 observations acted as the validation dataset (VDS) from 22 cows. The descriptive statistics of these variables were 26.0 ± 2.77 kg/day (mean ± standard deviation) of DMI, 14.9 ± 3.68 kg/day of DMI-2h, 35.0 ± 5.48 kg/day of milk yield, and 636 ± 82.6 kg/day of BW in DDS and 23.2 ± 4.72 kg/day of DMI, 12.6 ± 4.08 kg/day of DMI-2h, 30.4 ± 5.85 kg/day of milk yield, and 597 ± 63.7 kg/day of BW in VDS, respectively. A multiple regression analysis was conducted using the REG procedure of SAS to develop the forecasting models for DMI. The proposed prediction equation was: DMI (kg/day) = 8.499 + 0.2725 × DMI-2h (kg/day) + 0.2132 × Milk yield (kg/day) + 0.0095 × BW (kg/day) (R2 = 0.46, mean bias = 0 kg/day, RMSPE = 1.26 kg/day). Moreover, when compared with the prediction equation for DMI in Nutrient Requirements of Dairy Cattle (2001) using the independent dataset (VDS), our proposed model shows higher R2 (0.22 vs. 0.07) and smaller mean bias (-0.10 vs. 1.52 kg/day) and RMSPE (1.77 vs. 2.34 kg/day). Overall, we constructed a feasible forecasting model with better precision and accuracy in predicting daily DMI of dairy cows in mid and late lactation when fed ration three times per day.

Gene co-expression and alternative splicing analysis of key metabolic tissues to unravel the regulatory signatures of fatty acid composition in cattle.

Increasing the healthy/unhealthy fatty acid (FA) ratio in meat is one of the urgent tasks required to address consumer concerns. However, the regulatory mechanisms ultimately resulting in FA profiles vary among animals and remain largely unknown. In this study, using ~1.2 Tb high-quality RNA-Seq-based transcriptomic data of 188 samples from four key metabolic tissues (rumen, liver, muscle, and backfat) together with the contents of 49 FAs in backfat, the molecular regulatory mechanisms of these tissues contributing to FA formation in cattle were explored. Using this large dataset, the alternative splicing (AS) events, one of the transcriptional regulatory mechanisms in four tissues were identified. The highly conserved and absent AS events were detected in rumen tissue, which may contribute to its functional differences compared with the other three tissues. In addition, the healthy/unhealthy FA ratio related AS events, differential expressed (DE) genes, co-expressed genes, and their functions in four tissues were analysed. Eight key genes were identified from the integrated analysis of DE, co-expressed, and AS genes between animals with high and low healthy/unhealthy FA ratios. This study provides an applicable pipeline for AS events based on comprehensive RNA-Seq analysis and improves our understanding of the regulatory mechanism of FAs in beef cattle.