Viruses contribute to microbial diversification in the rumen ecosystem and are associated with certain animal production traits.

BACKGROUND: The rumen microbiome enables ruminants to digest otherwise indigestible feedstuffs, thereby facilitating the production of high-quality protein, albeit with suboptimal efficiency and producing methane. Despite extensive research delineating associations between the rumen microbiome and ruminant production traits, the functional roles of the pervasive and diverse rumen virome remain to be determined. RESULTS: Leveraging a recent comprehensive rumen virome database, this study analyzes virus-microbe linkages, at both species and strain levels, across 551 rumen metagenomes, elucidating patterns of microbial and viral diversity, co-occurrence, and virus-microbe interactions. Additionally, this study assesses the potential role of rumen viruses in microbial diversification by analyzing prophages found in rumen metagenome-assembled genomes. Employing CRISPR-Cas spacer-based matching and virus-microbe co-occurrence network analysis, this study suggests that the viruses in the rumen may regulate microbes at strain and community levels through both antagonistic and mutualistic interactions. Moreover, this study establishes that the rumen virome demonstrates responsiveness to dietary shifts and associations with key animal production traits, including feed efficiency, lactation performance, weight gain, and methane emissions. CONCLUSIONS: These findings provide a substantive framework for further investigations to unravel the functional roles of the virome in the rumen in shaping the microbiome and influencing overall animal production performance. Video Abstract.

Rumen protozoa and viruses: The predators within and their functions-A mini-review.

The rumen microbiome digests plant feedstuff that would be otherwise indigestible and provides most of the metabolizable energy and protein the host animals need. Until recently, research efforts have primarily been directed to bacteria and archaea, leaving the protozoa, fungi, and viruses much less understood. Protozoa contribute to feed digestion and fermentation, but as predators, they affect the microbiome and its function by regulating the abundance and activities of other rumen microbes both in a top-down (by directly killing the prey) and bottom-up (by affecting the metabolism of other microbes) manner. Rumen viruses (or phages, used interchangeably below) are diverse and abundant but the least understood. They are also predators (intracellular "predators") because of their lytic lifecycle, although they can co-exist peacefully with their hosts and reprogram host metabolism, buttressing host ecological fitness. In doing so, rumen viruses also affect the rumen microbiome in both a top-down and a bottom-up manner. Here we review the recent advancement in understanding both types of predators, focusing on their potential impact on the rumen microbiome and functions.

A green tea extract confection decreases circulating endotoxin and fasting glucose by improving gut barrier function but without affecting systemic inflammation: A double-blind, placebo-controlled randomized trial in healthy adults and adults with metabolic syndrome.

Anti-inflammatory activities of catechin-rich green tea extract (GTE) in obese rodents protect against metabolic endotoxemia by decreasing intestinal permeability and absorption of gut-derived endotoxin. However, translation to human health has not been established. We hypothesized that GTE would reduce endotoxemia by decreasing gut permeability and intestinal and systemic inflammation in persons with metabolic syndrome (MetS) compared with healthy persons. A randomized, double-blind, placebo-controlled, crossover trial in healthy adults (n = 19, 34 ± 2 years) and adults with MetS (n = 21, 40 ± 3 years) examined 4-week administration of a decaffeinated GTE confection (890 mg/d total catechins) on serum endotoxin, intestinal permeability, gut and systemic inflammation, and cardiometabolic parameters. Compared with the placebo, the GTE confection decreased serum endotoxin (P = .023) in both healthy persons and those with MetS, while increasing concentrations of circulating catechins (P < .0001) and γ-valerolactones (P = .0001). Fecal calprotectin (P = .029) and myeloperoxidase (P = .048) concentrations were decreased by GTE regardless of health status. Following the ingestion of gut permeability probes, urinary lactose/mannitol (P = .043) but not sucralose/erythritol (P > .05) was decreased by GTE regardless of health status. No between-treatment differences (P > .05) were observed for plasma aminotransferases, blood pressure, plasma lipids, or body mass nor were plasma tumor necrosis factor-α, interleukin-6, or the ratio of lipopolysaccharide-binding protein/soluble cluster of differentiation-14 affected. However, fasting glucose in both study groups was decreased (P = .029) by the GTE confection compared with within-treatment arm baseline concentrations. These findings demonstrate that catechin-rich GTE is effective to decrease circulating endotoxin and improve glycemic control in healthy adults and those with MetS, likely by reducing gut inflammation and small intestinal permeability but without affecting systemic inflammation.

Methane prediction equations including genera of rumen bacteria as predictor variables improve prediction accuracy.

Methane (CH4) emissions from ruminants are of a significant environmental concern, necessitating accurate prediction for emission inventories. Existing models rely solely on dietary and host animal-related data, ignoring the predicting power of rumen microbiota, the source of CH4. To address this limitation, we developed novel CH4 prediction models incorporating rumen microbes as predictors, alongside animal- and feed-related predictors using four statistical/machine learning (ML) methods. These include random forest combined with boosting (RF-B), least absolute shrinkage and selection operator (LASSO), generalized linear mixed model with LASSO (glmmLasso), and smoothly clipped absolute deviation (SCAD) implemented on linear mixed models. With a sheep dataset (218 observations) of both animal data and rumen microbiota data (relative sequence abundance of 330 genera of rumen bacteria, archaea, protozoa, and fungi), we developed linear mixed models to predict CH4 production (g CH4/animal·d, ANIM-B models) and CH4 yield (g CH4/kg of dry matter intake, DMI-B models). We also developed models solely based on animal-related data. Prediction performance was evaluated 200 times with random data splits, while fitting performance was assessed without data splitting. The inclusion of microbial predictors improved the models, as indicated by decreased root mean square prediction error (RMSPE) and mean absolute error (MAE), and increased Lin's concordance correlation coefficient (CCC). Both glmmLasso and SCAD reduced the Akaike information criterion (AIC) and Bayesian information criterion (BIC) for both the ANIM-B and the DMI-B models, while the other two ML methods had mixed outcomes. By balancing prediction performance and fitting performance, we obtained one ANIM-B model (containing 10 genera of bacteria and 3 animal data) fitted using glmmLasso and one DMI-B model (5 genera of bacteria and 1 animal datum) fitted using SCAD. This study highlights the importance of incorporating rumen microbiota data in CH4 prediction models to enhance accuracy and robustness. Additionally, ML methods facilitate the selection of microbial predictors from high-dimensional metataxonomic data of the rumen microbiota without overfitting. Moreover, the identified microbial predictors can serve as biomarkers of CH4 emissions from sheep, providing valuable insights for future research and mitigation strategies.

Simultaneous Covalent Modification of K-Ras(G12D) and K-Ras(G12C) with Tunable Oxirane Electrophiles.

Owing to their remarkable pharmaceutical properties compared to those of noncovalent inhibitors, the development of targeted covalent inhibitors (TCIs) has emerged as a powerful method for cancer treatment. The K-Ras mutant, which is prevalent in multiple cancers, has been confirmed to be a crucial drug target in the treatment of various malignancies. However, although the K-Ras(G12D) mutation is present in up to 33% of K-Ras mutations, no covalent inhibitors targeting K-Ras(G12D) have been developed to date. The relatively weak nucleophilicity of the acquired aspartic acid (12D) residue in K-Ras may be the reason for this. Herein, we present the first compound capable of covalently engaging both K-Ras(G12D) and K-Ras(G12C) mutants. Proteome profiling revealed that this compound effectively conjugates with G12C and G12D residues, modulating the protein functions in situ. These findings offer a unique pathway for the development of novel dual covalent inhibitors.

Interrogating the viral dark matter of the rumen ecosystem with a global virome database.

The diverse rumen virome can modulate the rumen microbiome, but it remains largely unexplored. Here, we mine 975 published rumen metagenomes for viral sequences, create a global rumen virome database (RVD), and analyze the rumen virome for diversity, virus-host linkages, and potential roles in affecting rumen functions. Containing 397,180 species-level viral operational taxonomic units (vOTUs), RVD substantially increases the detection rate of rumen viruses from metagenomes compared with IMG/VR V3. Most of the classified vOTUs belong to Caudovirales, differing from those found in the human gut. The rumen virome is predicted to infect the core rumen microbiome, including fiber degraders and methanogens, carries diverse auxiliary metabolic genes, and thus likely impacts the rumen ecosystem in both a top-down and a bottom-up manner. RVD and the findings provide useful resources and a baseline framework for future research to investigate how viruses may impact the rumen ecosystem and digestive physiology.

Integrated meta-omics analyses reveal a role of ruminal microorganisms in ketone body accumulation and ketosis in lactating dairy cows.

The extent to which a nutrition-related disorder such as ketosis alters the ruminal microbiota or whether microbiota composition is related to ketosis and potential associations with host metabolism is unknown. We aimed to evaluate variations occurring in the ruminal microbiota of ketotic and nonketotic cows in the early postpartum period, and how those changes may affect the risk of developing the disease. Data on milk yield, dry matter intake (DMI), body condition score, and blood ß-hydroxybutyrate (BHB) concentrations at 21 d postpartum were used to select 27 cows, which were assigned (n = 9 per group) to a clinical ketotic (CK, 4.10 ± 0.72 mmol BHB/L, DMI 11.61 ± 0.49 kg/d, ruminal pH 7.55 ± 0.07), subclinical ketotic (SK, 1.36 ± 0.12 mmol BHB/L, DMI 15.24 ± 0.34 kg/d, ruminal pH 7.58 ± 0.08), or control (NK, 0.88 ± 0.14 mmol BHB/L, DMI 16.74 ± 0.67/d, ruminal pH 7.61 ± 0.03) group. Cows averaged 3.6 ± 0.5 lactations and a body condition score of 3.11 ± 0.34 at the time of sampling. After blood serum collection for metabolomics analysis (1H nuclear magnetic resonance spectra), 150 mL of ruminal digesta was collected from each cow using an esophageal tube, paired-end (2 × 300 bp) sequencing of isolated DNA from ruminal digesta was performed via Illumina MiSeq, and sequencing data were analyzed using QIIME2 (v 2020.6) to measure the ruminal microbiota composition and relative abundance. Spearman correlation coefficients were used to evaluate relationships between relative abundance of bacterial genera and concentrations of serum metabolites. There were more than 200 genera, with approximately 30 being significant between NK and CK cows. Succinivibrionaceae UCG 1 taxa decreased in CK compared with NK cows. Christensenellaceae (Spearman correlation coefficient = 0.6), Ruminococcaceae (Spearman correlation coefficient = 0.6), Lachnospiraceae (Spearman correlation coefficient = 0.5), and Prevotellaceae (Spearman correlation coefficient = 0.6) genera were more abundant in the CK group and were highly positively correlated with plasma BHB. Metagenomic analysis indicated a high abundance of predicted functions related to metabolism (37.7%), genetic information processing (33.4%), and Brite hierarchies (16.3%) in the CK group. The 2 most important metabolic pathways for butyrate and propionate production were enriched in CK cows, suggesting increased production of acetyl coenzyme A and butyrate and decreased production of propionate. Overall, the combined data suggested that microbial populations may be related to ketosis by affecting short-chain fatty acid metabolism and BHB accumulation even in cows with adequate feed intake in the early postpartum period.

Metabolic influence of core ciliates within the rumen microbiome.

Protozoa comprise a major fraction of the microbial biomass in the rumen microbiome, of which the entodiniomorphs (order: Entodiniomorphida) and holotrichs (order: Vestibuliferida) are consistently observed to be dominant across a diverse genetic and geographical range of ruminant hosts. Despite the apparent core role that protozoal species exert, their major biological and metabolic contributions to rumen function remain largely undescribed in vivo. Here, we have leveraged (meta)genome-centric metaproteomes from rumen fluid samples originating from both cattle and goats fed diets with varying inclusion levels of lipids and starch, to detail the specific metabolic niches that protozoa occupy in the context of their microbial co-habitants. Initial proteome estimations via total protein counts and label-free quantification highlight that entodiniomorph species Entodinium and Epidinium as well as the holotrichs Dasytricha and Isotricha comprise an extensive fraction of the total rumen metaproteome. Proteomic detection of protozoal metabolism such as hydrogenases (Dasytricha, Isotricha, Epidinium, Enoploplastron), carbohydrate-active enzymes (Epidinium, Diplodinium, Enoploplastron, Polyplastron), microbial predation (Entodinium) and volatile fatty acid production (Entodinium and Epidinium) was observed at increased levels in high methane-emitting animals. Despite certain protozoal species having well-established reputations for digesting starch, they were unexpectedly less detectable in low methane emitting-animals fed high starch diets, which were instead dominated by propionate/succinate-producing bacterial populations suspected of being resistant to predation irrespective of host. Finally, we reaffirmed our abovementioned observations in geographically independent datasets, thus illuminating the substantial metabolic influence that under-explored eukaryotic populations have in the rumen, with greater implications for both digestion and methane metabolism.

The inhibition of high ammonia to in vitro rumen fermentation is pH dependent.

Ammonia is an important rumen internal environment indicator. In livestock production, feeding a large amount of non-protein nitrogen to ruminants will create high ammonia stress to the animals, which increases the risk of ammonia toxicity. However, the effects of ammonia toxicity on rumen microbiota and fermentation are still unknown. In this study, an in vitro rumen fermentation technique was used to investigate the effects of different concentrations of ammonia on rumen microbiota and fermentation. To achieve the four final total ammonia nitrogen (TAN) concentrations of 0, 8, 32, and 128 mmol/L, ammonium chloride (NH4Cl) was added at 0, 42.8, 171.2, and 686.8 mg/100 mL, and urea was added at 0, 24, 96, and 384 mg/100 mL. Urea hydrolysis increased, while NH4Cl dissociation slightly reduced the pH. At similar concentrations of TAN, the increased pH of the rumen culture by urea addition resulted in a much higher free ammonia nitrogen (FAN) concentration compared to NH4Cl addition. Pearson correlation analysis revealed a strong negative correlation between FAN and microbial populations (total bacteria, protozoa, fungi, and methanogens) and in vitro rumen fermentation profiles (gas production, dry matter digestibility, total volatile fatty acid, acetate, propionate, etc.), and a much weaker correlation between TAN and the above indicators. Additionally, bacterial community structure changed differently in response to TAN concentrations. High TAN increased Gram-positive Firmicutes and Actinobacteria but reduced Gram-negative Fibrobacteres and Spirochaetes. The current study demonstrated that the inhibition of in vitro rumen fermentation by high ammonia was pH-dependent and was associated with variations of rumen microbial populations and communities.

Functional gene-guided enrichment plus in situ microsphere cultivation enables isolation of new crucial ureolytic bacteria from the rumen of cattle.

BACKGROUND: Ruminants can utilize urea as a dietary nitrogen source owing to their ability to recycle urea-N back to the rumen where numerous ureolytic bacteria hydrolyze urea into ammonia, which is used by numerous bacteria as their nitrogen source. Rumen ureolytic bacteria are the key microbes making ruminants the only type of animals independent of pre-formed amino acids for survival, thus having attracted much research interest. Sequencing-based studies have helped gain new insights into ruminal ureolytic bacterial diversity, but only a limited number of ureolytic bacteria have been isolated into pure cultures or studied, hindering the understanding of ureolytic bacteria with respect to their metabolism, physiology, and ecology, all of which are required to effectively improve urea-N utilization efficiency. RESULTS: We established and used an integrated approach, which include urease gene (ureC) guided enrichment plus in situ agarose microsphere embedding and cultivation under rumen-simulating conditions, to isolate ureolytic bacteria from the rumen microbiome. We optimized the dilutions of the rumen microbiome during the enrichment, single-cell embedding, and then in situ cultivation of microsphere-embedded bacteria using dialysis bags placed in rumen fluid. Metabonomic analysis revealed that the dialysis bags had a fermentation profile very similar to the simulated rumen fermentation. In total, we isolated 404 unique strains of bacteria, of which 52 strains were selected for genomic sequencing. Genomic analyses revealed that 28 strains, which were classified into 12 species, contained urease genes. All these ureolytic bacteria represent new species ever identified in the rumen and represented the most abundant ureolytic species. Compared to all the previously isolated ruminal ureolytic species combined, the newly isolated ureolytic bacteria increased the number of genotypically and phenotypically characterized ureolytic species by 34.38% and 45.83%, respectively. These isolated strains have unique genes compared to the known ureolytic strains of the same species indicating their new metabolic functions, especially in energy and nitrogen metabolism. All the ureolytic species were ubiquitous in the rumen of six different species of ruminants and were correlated to dietary urea metabolism in the rumen and milk protein production. We discovered five different organizations of urease gene clusters among the new isolates, and they had varied approaches to hydrolyze urea. The key amino acid residues of the UreC protein that potentially plays critical regulatory roles in urease activation were also identified. CONCLUSIONS: We established an integrated methodology for the efficient isolation of ureolytic bacteria, which expanded the biological resource of crucial ureolytic bacteria from the rumen. These isolates play a vital role in the incorporation of dietary nitrogen into bacterial biomass and hence contribute to ruminant growth and productivity. Moreover, this methodology can enable efficient isolation and cultivation of other bacteria of interest in the environment and help bridge the knowledge gap between genotypes and phenotypes of uncultured bacteria. Video abstract.

Interactions between Entodinium caudatum and an amino acid-fermenting bacterial consortium: fermentation characteristics and protozoal population in vitro.

Ruminal protozoa, especially entodiniomorphs, engulf other members of the rumen microbiome in large numbers; and they release oligopeptides and amino acids, which can be fermented to ammonia and volatile fatty acids (VFAs) by amino acid-fermenting bacteria (AAFB). Studies using defaunated (protozoa-free) sheep have demonstrated that ruminal protozoa considerably increase intraruminal nitrogen recycling but decrease nitrogen utilization efficiency in ruminants. However, direct interactions between ruminal protozoa and AAFB have not been demonstrated because of their inability to establish axenic cultures of any ruminal protozoan. Thus, this study was performed to evaluate the interaction between Entodinium caudatum, which is the most predominant rumen ciliate species, and an AAFB consortium in terms of feed degradation and ammonia production along with the microbial population shift of select bacterial species (Prevotella ruminicola, Clostridium aminophilum, and Peptostreptococcus anaerobius). From an Ent. caudatum culture that had been maintained by daily feeding and transfers every 3 or 4 days, the bacteria and methanogens loosely associated with Ent. caudatum cells were removed by filtration and washing. An AAFB consortium was established by repeated transfers and enrichment with casamino acids as the sole substrate. The cultures of Ent. caudatum alone (Ec) and AAFB alone (AAFB) and the co-culture of Ent. caudatum and AAFB (Ec + AAFB) were set up in three replicates and incubated at 39°C for 72 h. The digestibility of dry matter (DM) and fiber (NDF), VFA profiles, ammonia concentrations, pH, and microscopic counts of Ent. caudatum were compared among the three cultures. The co-culture of AAFB and Ent. caudatum enhanced DM degradation, VFA production, and Ent. caudatum cell counts; conversely, it decreased acetate: propionate ratio although the total bacterial abundance was similar between Ec and the Ec + AAFB co-culture after 24 h incubation. The ammonia production and relative abundance of C. aminophilum and P. anaerobius did not differ between AAFB alone and the Ec + AAFB co-culture. Our results indicate that Ent. caudatum and AAFB could have a mutualistic interaction that benefited each other, but their interactions were complex and might not increase ammoniagenesis. Further research should examine how such interactions affect the population dynamics of AAFB.

Maternal Rumen Bacteriota Shapes the Offspring Rumen Bacteriota, Affecting the Development of Young Ruminants.

The maternal rumen microbiota can affect the infantile rumen microbiota and likely offspring growth, and some rumen microbes are heritable and are associated with host traits. However, little is known about the heritable microbes of the maternal rumen microbiota and their role in and effect on the growth of young ruminants. From analyzing the ruminal bacteriota from 128 Hu sheep dams and their 179 offspring lambs, we identified the potential heritable rumen bacteria and developed random forest prediction models to predict birth weight, weaning weight, and preweaning gain of the young ruminants using rumen bacteria as predictors. We showed that the dams tended to shape the bacteriota of the offspring. About 4.0% of the prevalent amplicon sequence variants (ASVs) of rumen bacteria were heritable (h2 > 0.2 and P < 0.05), and together they accounted for 4.8% and 31.5% of the rumen bacteria in relative abundance in the dams and the lambs, respectively. Heritable bacteria classified to Prevotellaceae appeared to play a key role in the rumen niche and contribute to rumen fermentation and the growth performance of lambs. Lamb growth traits could be successfully predicted using some maternal ASVs, and the accuracy of the predictive models was improved when some ASVs from both dams and their offspring were included. IMPORTANCE Using a study design that enabled direct comparison of the rumen microbiota between sheep dams and their lambs, between littermates, and between sheep dams and lambs from other mothers, we identified the heritable subsets of rumen bacteriota in Hu sheep, some of which may play important roles in affecting the growth traits of young lambs. Some maternal rumen bacteria could help predict the growth traits of the young offspring, and they may assist in breeding of and selection for high-performance sheep.

Catechin Bioavailability Following Consumption of a Green Tea Extract Confection Is Reduced in Obese Persons without Affecting Gut Microbial-Derived Valerolactones.

Obesity-related cardiometabolic disorders are driven by inflammation, oxidative stress, and gut dysbiosis. Green tea catechins protect against cardiometabolic disorders by anti-inflammatory, antioxidant, and prebiotic activities. However, whether obesity alters catechin bioavailability remains unknown. We hypothesized that obesity would decrease catechin bioavailability due to altered gut microbiota composition. Obese and healthy persons completed a pharmacokinetics trial in which a confection formulated with green tea extract (GTE; 58% epigallocatechin gallate, 17% epigallocatechin, 8% epicatechin, 6% epicatechin gallate) was ingested before collecting plasma and urine at timed intervals for up to 24 h. Stool samples were collected prior to confection ingestion. Catechins and γ-valerolactones were assessed by LC-MS. Obesity reduced plasma area under the curve (AUC0-12h) by 24-27% and maximum plasma concentrations by 18-36% for all catechins. Plasma AUC0-12h for 5'-(3',4'-dihydroxyphenyl)-γ-valerolactone and 5'-(3',4',5'-trihydroxyphenyl)-γ-valerolactone, as well as total urinary elimination of all catechins and valerolactones, were unaffected. ⍺-Diversity in obese persons was lower, while Slackia was the only catechin-metabolizing bacteria that was altered by obesity. Ascorbic acid and diversity metrics were correlated with catechin/valerolactone bioavailability. These findings indicate that obesity reduces catechin bioavailability without affecting valerolactone generation, urinary catechin elimination, or substantially altered gut microbiota populations, suggesting a gut-level mechanism that limits catechin absorption.

Yak rumen microbiome elevates fiber degradation ability and alters rumen fermentation pattern to increase feed efficiency.

Rumen microbes play an important role in ruminant energy supply and animal performance. Previous studies showed that yak (Bos grunniens) rumen microbiome and fermentation differ from other ruminants. However, little is understood about the features of the rumen microbiome that make yak adapted to their unique environmental and dietary conditions. This study was to investigate the rumen microbiome and metabolome to understand how yak adapt to the coarse forage and harsh environment in the Qinghai-Tibetan plateau. Nine female Qaidam yellow cattle (Bos taurus), 9 dzomo (hybrids of cattle and yak) and 9 female plateau yak (B. grunniens), about 5 to 6 years old, were used in this study. Rumen fermentation parameters, fibrolytic enzyme activities, and rumen metataxonomic were determined. Then 18 (6 samples per group) were selected for rumen metagenomic and metabolome analysis. Metataxonomic analysis revealed that the rumen microbiota was significantly different among plateau yak, Qaidam yellow cattle, and dzomo (P < 0.05). Metagenomic analysis displayed a larger gene pool encoding a richer repertoire of carbohydrate-active enzymes in the rumen microbiome of plateau yak and dzomo than Qaidam yellow cattle (P < 0.05). Some of the genes encoding glycoside hydrolases that mediate the digestion of cellulose and hemicellulose were significantly enriched in the rumen of plateau yak than Qaidam yellow cattle, but glycoside hydrolase 57 that primarily includes amylases was abundant in Qaidam yellow cattle (P < 0.05). The rumen fermentation profile differed also, Qaidam yellow cattle having a higher molar proportion of acetate but a lower molar proportion of propionate than dzomo and plateau yak (P < 0.05). Based on metabolomic analysis, rumen microbial metabolic pathways and metabolites were different. Differential metabolites are mainly amino acids, carboxylic acids, sugars, and bile acids. Changes in rumen microbial composition could explain the above results. The present study showed that the rumen microbiome of plateau yak helps its host to adapt to the Qinghai-Tibetan plateau. In particular, the plateau yak rumen microbiome has more enzymes genes involved in cellulase and hemicellulase than that of cattle, resulting higher fibrolytic enzyme activities in yak, further providing stronger fiber degradation function.

Fecal microbiota dynamics and its relationship to diarrhea and health in dairy calves.

BACKGROUND: Diarrhea is a major cause of morbidity and mortality in young calves, resulting in considerable economic loss for dairy farms. To determine if some gut microbes might have resistance to dysbiotic process with calf diarrhea by dictating the microbial co-occurrence patterns from birth to post-weaning, we examined the dynamic development of the gut microbiota and diarrhea status using two animal trials, with the first trial having 14 Holstein dairy calves whose fecal samples were collected 18 times over 78 d from birth to 15 d post-weaning and the second trial having 43 Holstein dairy calves whose fecal samples were collected daily from 8 to 18 days of age corresponding to the first diarrhea peak of trial 1. RESULTS: Metataxonomic analysis of the fecal microbiota showed that the development of gut microbiota had three age periods with birth and weaning as the separatrices. Two diarrhea peaks were observed during the transition of the three age periods. Fusobacteriaceae was identified as a diarrhea-associated taxon both in the early stage and during weaning, and Clostridium_sensu_stricto_1 was another increased genus among diarrheic calves in the early stage. In the neonatal calves, Prevotella_2 (ASV4 and ASV26), Prevotella_9 (ASV43), and Alloprevotella (ASV14) were negatively associated with Clostridium_sensu_stricto_1 (ASV48), the keystone taxa of the diarrhea-phase module. During weaning, unclassified Muribaculaceae (ASV28 and ASV44), UBA1819 (ASV151), Barnesiella (ASV497), and Ruminococcaceae_UCG-005 (ASV254) were identified being associated with non-diarrheic status, and they aggregated in the non-diarrhea module of co-occurrence patterns wherein unclassified Muribaculaceae (ASV28) and Barnesiella (ASV497) had a direct negative relationship with the members of the diarrhea module. CONCLUSIONS: Taken together, our results suggest that the dynamic successions of calf gut microbiota and the interactions among some bacteria could influence calf diarrhea, and some species of Prevotella might be the core microbiota in both neonatal and weaning calves, while species of Muribaculaceae might be the core microbiota in weaning calves for preventing calf diarrhea. Some ASVs affiliated with Prevotella_2 (ASV4 and ASV26), Prevotella_9 (ASV43), Alloprevotella (AVS14), unclassified Muribaculaceae (ASV28 and ASV44), UBA1819 (ASV151), Ruminococcaceae_UCG-005 (ASV254), and Barnesiella (ASV497) might be proper probiotics for preventing calf diarrhea whereas Clostridium_sensu_stricto_1 (ASV48) might be the biomarker for diarrhea risk in specific commercial farms.

Genomic insights into the phylogeny and biomass-degrading enzymes of rumen ciliates.

Understanding the biodiversity and genetics of gut microbiomes has important implications for host physiology and industrial enzymes, whereas most studies have been focused on bacteria and archaea, and to a lesser extent on fungi and viruses. One group, still underexplored and elusive, is ciliated protozoa, despite its importance in shaping microbiota populations. Integrating single-cell sequencing and an assembly-and-identification pipeline, we acquired 52 high-quality ciliate genomes of 22 rumen morphospecies from 11 abundant morphogenera. With these genomes, we resolved the taxonomic and phylogenetic framework that revised the 22 morphospecies into 19 species spanning 13 genera and reassigned the genus Dasytricha from Isotrichidae to a new family Dasytrichidae. Comparative genomic analyses revealed that extensive horizontal gene transfers and gene family expansion provided rumen ciliate species with a broad array of carbohydrate-active enzymes (CAZymes) to degrade all major kinds of plant and microbial carbohydrates. In particular, the genomes of Diplodiniinae and Ophryoscolecinae species encode as many CAZymes as gut fungi, and ~80% of their degradative CAZymes act on plant cell-wall. The activities of horizontally transferred cellulase and xylanase of ciliates were experimentally verified and were 2-9 folds higher than those of the inferred corresponding bacterial donors. Additionally, the new ciliate dataset greatly facilitated rumen metagenomic analyses by allowing ~12% of the metagenomic sequencing reads to be classified as ciliate sequences.

Effects of supplementation with Saccharomyces cerevisiae products on dairy calves: A meta-analysis.

Saccharomyces cerevisiae products (SCP) have the potential to promote the growth and development of the gastrointestinal tract and immunity in young livestock animals. However, the effects of SCP supplementation on calves are inconsistent among the reported studies in the literature. Hence, we performed a meta-analysis to comprehensively assess the effects of SCP on the growth performance, ruminal fermentation parameters, nutrients digestibility, ruminal histological morphology, serum immune response, and fecal pathogen colony counts in calves. We searched the Web of Science, ScienceDirect, PubMed, and China National Knowledge Infrastructure for relevant studies published up to October 1, 2021. After screening against a set of criteria, the data of 36 studies were included in our meta-analysis (2,126 calves in total). We evaluated the quality of the data using sensitivity analysis and assessed publication bias. Our meta-analysis revealed several important findings. First, SCP supplementation increased the ruminal short-chain fatty acid concentration, ruminal papilla height, and fiber digestibility, pointing toward stimulation of the development of the rumen in calves. Second, SCP supplementation increased the serum concentrations of total protein, IgA, and IgG but decreased fecal pathogen colony counts, suggesting that SCP could help calves to promote immunity (especially maintaining circulating concentrations of immunoglobulins in preweaning calves) and resistance to pathogens. Third, a subgroup analysis between preweaning and postweaning calves showed that SCP increased average daily gain and dry matter intake preweaning but not postweaning, suggesting that SCP is better supplemented to preweaning calves to achieve the best results. Forth, based on the dose-response curve, 24 to 25 g/d might be the optimal dose range of SCP supplementation (into starter feed) preweaning to achieve the best overall effect, meanwhile, we need more studies to improve the consistency and accuracy of the dose-response curve prediction. Overall, SCP supplementation improved growth performance, rumen development, and immunocompetence in calves, particularly in preweaning calves.

Heat stress impacts the multi-domain ruminal microbiota and some of the functional features independent of its effect on feed intake in lactating dairy cows.

BACKGROUND: Heat stress (HS) affects the ruminal microbiota and decreases the lactation performance of dairy cows. Because HS decreases feed intake, the results of previous studies were confounded by the effect of HS on feed intake. This study examined the direct effect of HS on the ruminal microbiota using lactating Holstein cows that were pair-fed and housed in environmental chambers in a 2 × 2 crossover design. The cows were pair-fed the same amount of identical total mixed ration to eliminate the effect of feed or feed intake. The composition and structure of the microbiota of prokaryotes, fungi, and protozoa were analyzed using metataxonomics and compared between two thermal conditions: pair-fed thermoneutrality (PFTN, thermal humidity index: 65.5) and HS (87.2 for daytime and 81.8 for nighttime). RESULTS: The HS conditions altered the structure of the prokaryotic microbiota and the protozoal microbiota, but not the fungal microbiota. Heat stress significantly increased the relative abundance of Bacteroidetes (primarily Gram-negative bacteria) while decreasing that of Firmicutes (primarily Gram-positive bacteria) and the Firmicutes-to-Bacteroidetes ratio. Some genera were exclusively found in the heat-stressed cows and thermal control cows. Some co-occurrence and mutual exclusion between some genera were also found exclusively for each thermal condition. Heat stress did not significantly affect the overall functional features predicted using the 16S rRNA gene sequences and ITS1 sequences, but some enzyme-coding genes altered their relative abundance in response to HS. CONCLUSIONS: Overall, HS affected the prokaryotes, fungi, and protozoa of the ruminal microbiota in lactating Holstein cows to a different extent, but the effect on the structure of ruminal microbiota and functional profiles was limited when not confounded by the effect on feed intake. However, some genera and co-occurrence were exclusively found in the rumen of heat-stressed cows. These effects should be attributed to the direct effect of heat stress on the host metabolism, physiology, and behavior. Some of the "heat-stress resistant" microbes may be useful as potential probiotics for cows under heat stress.

Infection with white spot syndrome virus affects the microbiota in the stomachs and intestines of kuruma shrimp.

Maintaining eubiosis of the gastrointestinal (GI) microbiota is essential for animal health. White spot syndrome virus (WSSV) is the most lethal viral pathogen because it causes extremely high mortality in shrimp farming. However, it remains poorly understood how WSSV infection affects the microbiota in different regions of the GI tract of shrimp. In the present study, we established an experimental model of kuruma shrimp (Marsupenaeus japonicus) infection with WSSV and then investigated the effects of WSSV infection on the microbiota in the cardiac stomach, pyloric stomach, and intestines using metataxonomics. We identified 34 phyla and 576 genera of bacteria collectively. At the phylum level, Proteobacteria and Firmicutes were the most abundant in all the three GI segments. The WSSV infection decreased microbial diversity to a different extent in the stomachs and in a time-dependent manner. The infection with WSSV affected the microbiota composition in the two stomachs, but not the intestines. Firmicutes increased significantly, while Actinobacteria, Bacteroidetes, and Cyanobacteria decreased in the two stomachs of the WSSV-infected shrimp. At the genus level, Trichococcus and Vibrio increased, but Bradyrhizobium and Roseburia decreased in the cardiac stomach of the WSSV-infected shrimp. Trichococcus and Photobacterium increased in the pyloric stomach. Although Vibrio showed a slight downward trend, Aliivibrio (formerly Vibrio) increased in the pyloric stomach. Thiothrix, Fusibacter, and Shewanella decreased in the pyloric stomach, but no significant differences in these genera were detected in the cardiac stomach. Analysis of the predicted functions of the GI microbiota indicated that the WSSV infection resulted in losses of some microbiota functions. The new information from this study may help better understand the bacteria-virus interaction in the GI tract of shrimp and other crustacean species, and inform pathogen prevention/control and sustainable aquaculture production.

Full adoption of the most effective strategies to mitigate methane emissions by ruminants can help meet the 1.5 °C target by 2030 but not 2050.

To meet the 1.5 °C target, methane (CH4) from ruminants must be reduced by 11 to 30% by 2030 and 24 to 47% by 2050 compared to 2010 levels. A meta-analysis identified strategies to decrease product-based (PB; CH4 per unit meat or milk) and absolute (ABS) enteric CH4 emissions while maintaining or increasing animal productivity (AP; weight gain or milk yield). Next, the potential of different adoption rates of one PB or one ABS strategy to contribute to the 1.5 °C target was estimated. The database included findings from 430 peer-reviewed studies, which reported 98 mitigation strategies that can be classified into three categories: animal and feed management, diet formulation, and rumen manipulation. A random-effects meta-analysis weighted by inverse variance was carried out. Three PB strategies­namely, increasing feeding level, decreasing grass maturity, and decreasing dietary forage-to-concentrate ratio­decreased CH4 per unit meat or milk by on average 12% and increased AP by a median of 17%. Five ABS strategies­namely CH4 inhibitors, tanniferous forages, electron sinks, oils and fats, and oilseeds­decreased daily methane by on average 21%. Globally, only 100% adoption of the most effective PB and ABS strategies can meet the 1.5 °C target by 2030 but not 2050, because mitigation effects are offset by projected increases in CH4 due to increasing milk and meat demand. Notably, by 2030 and 2050, low- and middle-income countries may not meet their contribution to the 1.5 °C target for this same reason, whereas high-income countries could meet their contributions due to only a minor projected increase in enteric CH4 emissions.