Varied microbial community assembly and specialization patterns driven by early life microbiome perturbation and modulation in young ruminants.

Perturbations and modulations during early life are vital to affect gut microbiome assembly and establishment. In this study, we assessed how microbial communities shifted during calf diarrhea and with probiotic yeast supplementation (Saccharomyces cerevisiae var. boulardii, SCB) and determined the key bacterial taxa contributing to the microbial assembly shifts using a total of 393 fecal samples collected from 84 preweaned calves during an 8-week trial. Our results revealed that the microbial assembly patterns differed between healthy and diarrheic calves at 6- and 8-week of the trial, with healthy calves being stochastic-driven and diarrheic calves being deterministic-driven. The two-state Markov model revealed that SCB supplementation had a higher possibility to shift microbial assembly from deterministic- to stochastic-driven in diarrheic calves. Furthermore, a total of 23 and 21 genera were specific ecotypes to assembly patterns in SCB-responsive (SCB-fed calves did not exhibit diarrhea) and nonresponsive (SCB-fed calves occurred diarrhea) calves, respectively. Among these ecotypes, the area under a receiver operating characteristic curve revealed that Blautia and Ruminococcaceae UCG 014, two unidentified genera from the Ruminococcaceae family, had the highest predictiveness for microbial assembly patterns in SCB-responsive calves, while Prevotellaceae, Blautia, and Escherichia-Shigella were the most predictive bacterial taxa for microbial assembly patterns in SCB-nonresponsive calves. Our study suggests that microbiome perturbations and probiotic yeast supplementation serving as deterministic factors influenced assembly patterns during early life with critical genera being predictive for assembly patterns, which sheds light on mechanisms of microbial community establishment in the gut of neonatal calves during early life.

Mucosal Immunization with Spore-Based Vaccines against Mannheimia haemolytica Enhances Antigen-Specific Immunity.

BACKGROUND: Mannheimia haemolytica is a bovine respiratory pathogen commonly associated with bacterial bronchopneumonia. Current vaccine strategies have shown variable efficacy in feedlot cattle, and therefore novel vaccines are needed. Bacillus subtilis spores have been investigated as a mucosal vaccine platform, due to their ability to bind and present antigens to the mucosa and act as an adjuvant. The aim of this study was to develop two spore-based mucosal vaccines targeting M. haemolytica and evaluate their immunogenicity in mice. METHODS: Two antigen constructs composed of cholera toxin B subunit, M. haemolytica leukotoxin, and either the M. haemolytica outer membrane protein PlpE (MhCP1) or GS60 (MhCP2) were synthesized, purified and then bound to spores as vaccines. In two separate mice trials, the spore-bound vaccines (Spore-MhCP1 and Spore-MhCP2) were administered to mice through intranasal and intragastric routes, while free antigens were administered intranasally and intramuscularly. Unbound spores were also evaluated intranasally. Antigen-specific serum IgG and mucosal IgA from bronchoalveolar lavage, feces, and saliva were measured after vaccination. Mice sera from all treatment groups were assessed for their bactericidal activity against M. haemolytica. RESULTS: In both mice experiments, intramuscular immunization induced the strongest serum IgG antibody response. However, the intranasal administration of Spore-MhCP1 and Spore-MhCP2 elicited the greatest secretory IgA-specific response against leukotoxin, PlpE, and GS60 in bronchoalveolar lavage, saliva, and feces (p < 0.05). Compared to the intranasal administration of free antigen, spore-bound antigen groups showed greater bactericidal activity against M. haemolytica (p < 0.05). CONCLUSIONS: Since intranasally delivered Spore-MhCP1 and Spore-MhCP2 elicited both systemic and mucosal immune responses in mice, these vaccines may have potential to mitigate lung infection in cattle by restricting M. haemolytica colonization and proliferation in the respiratory tract. The efficacy of these mucosal spore-based vaccines merits further assessment against M. haemolytica in cattle.

In Silico Analysis of Shiga Toxin-Producing Escherichia coli O157:H7 Strains from Presumptive Super- and Low-Shedder Cattle.

Cattle are the primary reservoir for STEC O157, with some shedding >104 CFU/g in feces, a phenomenon known as super-shedding (SS). The mechanism(s) responsible for SS are not understood but have been attributed to the environment, host, and pathogen. This study aimed to compare genetic characteristics of STEC O157 strains from cattle in the same commercial feedlot pens with SS or low-shedding (LS) status. Strains from SS (n = 35) and LS (n = 28) collected from 11 pens in three feedlots were analyzed for virulence genes, Shiga toxin-carrying bacteriophage insertion sites, and phylogenetic relationships. In silico analysis showed limited variation regarding virulence gene profiles. Stx-encoding prophage insertion sites mrlA and wrbA for stx1a and stx2a, respectively, were all occupied, but two isolates had fragments of the stx-carrying phage in mrlA and wrbA loci without stx1a and stx2a. All strains screened for lineage-specific polymorphism assay (LSPA-6) were 111111, lineage I. Of the isolates, 61 and 2 were clades 1 and 8, respectively. Phylogenetic analysis revealed that pens with more than one SS had multiple distantly related clusters of SS and LS isolates. Although virulence genes and lineage were largely similar within and across feedlots, multiple genetic origins of strains within a single feedlot pen illustrate challenges for on-farm control of STEC.

Genotype-associated heritable rumen bacteria can be a stable microbiota passed to the offspring.

Recent studies have reported that some rumen microbes are "heritable" (those have significant narrow sense heritability) and can significantly contribute to host phenotype variations. However, it is unknown if these heritable rumen bacteria can be passed to the next generation. In this study, the rumen bacteria from mother cows (sampled in 2016) and their offspring (sampled in 2019) were assessed to determine if vertical transmission occurred between the two generations. The analysis of relationship between host genotypes and heritable bacterial abundances showed that potential of five host genotypes can affect the relative abundances of two unclassified species level heritable bacteria (Pseudoscardovia and p-251-o5). The G allele of BTB-01532239 and A allele of ARS-BFGL-NGS-8960 were associated with a higher relative abundance of p-251-o5. The A allele of BTB-00740910 and BovineHD1300021786 and G allele of BovineHD1900005868 were associated with a higher relative abundance of Pseudoscardovia. The mother-offspring comparison revealed that the heritable rumen bacteria had higher compositional similarity than nonheritable bacteria between two generations, and the predicted heritable microbial functions had higher stability than those from nonheritable bacteria. These findings suggest that a high stability exists in heritable rumen bacteria, which could be passed to the next generation in dairy cows.

- Invited Review - Translational gut microbiome research for strategies to improve beef cattle production sustainability and meat quality.

Advanced and innovative breeding and management of meat-producing animals are needed to address the global food security and sustainability challenges. Beef production is an important industry for securing animal protein resources in the world and meat quality significantly contributes to the economic values and human needs. Improvement of cattle feed efficiency has become an urgent task as it can lower the environmental burden of methane gas emissions and the reduce the consumption of human edible cereal grains. Cattle depend on their symbiotic microbiome and its activity in the rumen and gut to maintain growth and health. Recent developments in high-throughput omics analysis (metagenome, metatranscriptome, metabolome, metaproteome and so on) have made it possible to comprehensively analyze microbiome, hosts and their interactions and to define their roles in affecting cattle biology. In this review, we focus on the relationships among gut microbiome and beef meat quality, feed efficiency, methane emission as well as host genetics in beef cattle, aiming to determine the current knowledge gaps for the development of the strategies to improve the sustainability of beef production.

Comparative analysis of functional diversity of rumen microbiome in bison and beef heifers.

IMPORTANCE: Ruminants play a key role in the conversion of cellulolytic plant material into high-quality meat and milk protein for humans. The rumen microbiome is the driver of this conversion, yet there is little information on how gene expression within the microbiome impacts the efficiency of this conversion process. The current study investigates gene expression in the rumen microbiome of beef heifers and bison and how transplantation of ruminal contents from bison to heifers alters gene expression. Understanding interactions between the host and the rumen microbiome is the key to developing informed approaches to rumen programming that will enhance production efficiency in ruminants.

Cultivation of enteroids from fresh and cryopreserved bovine duodenal tissues.

This study aimed to develop a method for intestinal tissue cryopreservation and resuscitation for enteroid cultivation. Two different types of tissues, fresh duodenal tissues (n = 3, from Angus steers) and duodenal tissues cryopreserved in 90% fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO; n = 3, from Holstein calves), were collected to develop enteroids. Crypts were isolated using 2 mM EDTA/phosphate-buffered saline from both fresh and cryopreserved tissues and embedded in basement membrane extract. Embedded crypts were seeded in a 24-well plate and cultured in IntestiCult Organoid Growth Medium (Mouse) with inhibitors cocktail and Primocin. The upper opening of crypts became sealed, and crypts formed sphere structures (i.e., enteroids) within 24 h. Primary (passage 0) enteroids showed budding crypt domains from d 3 of cultivation at the earliest. After 7 d of cultivation, enteroids were passaged in a new 24-well plate. Fragments from passaged d 7 enteroids also formed sphere structures within 24 h after seeding and showed budding crypt domains from d 3 of cultivation at the earliest. The area of enteroids was measured in each animal during d 1 to 7 in passage 0 and 1, and the area of enteroids derived from both tissues increased during d 1 to 7 in passage 0 and 1. The area increased from d 1 to 7 of cultivation, and the area of passage 1 was greater than that of passage 0. F-actin staining using phalloidin revealed that brush border microvilli were distributed on the luminal side of the enteroids. In conclusion, a cryopreserved solution consisting of FBS and DMSO is useful for cryopreservation and resuscitation of bovine intestine for enteroid cultivation. This method allows researchers to investigate intestinal function and health in the laboratory using enteroids derived from fresh and cryopreserved tissues collected from cattle.

Saccharomyces cerevisiae boulardii accelerates intestinal microbiota maturation and is correlated with increased secretory IgA production in neonatal dairy calves.

Neonatal calves have a limited capacity to initiate immune responses due to a relatively immature adaptive immune system, which renders them susceptible to many on-farm diseases. At birth, the mucosal surfaces of the intestine are rapidly colonized by microbes in a process that promotes mucosal immunity and primes the development of the adaptive immune system. In a companion study, our group demonstrated that supplementation of a live yeast probiotic, Saccharomyces cerevisiae boulardii (SCB) CNCM I-1079, to calves from birth to 1 week of age stimulates secretory IgA (sIgA) production in the intestine. The objective of the study was to evaluate how SCB supplementation impacts the intestinal microbiota of one-week-old male calves, and how changes in the bacterial community in the intestine relate to the increase in secretory IgA. A total of 20 calves were randomly allocated to one of two treatments at birth: Control (CON, n = 10) fed at 5 g/d of carrier with no live yeast; and SCB (n = 10) fed at 5 g of live SCB per day (10 × 109 CFU/d). Our study revealed that supplementing calves with SCB from birth to 1 week of age had its most marked effects in the ileum, increasing species richness and phylogenetic diversity in addition to expediting the transition to a more interconnected bacterial community. Furthermore, LEfSe analysis revealed that there were several differentially abundant taxa between treatments and that SCB increased the relative abundance the family Eubacteriaceae, Corynebacteriaceae, Eggerthellaceae, Bacillaceae, and Ruminococcaceae. Furthermore, network analysis suggests that SCB promoted a more stable bacterial community and appears to reduce colonization with Shigella. Lastly, we observed that the probiotic-driven increase in microbial diversity was highly correlated with the enhanced secretory IgA capacity of the ileum, suggesting that the calf's gut mucosal immune system relies on the development of a stable and highly diverse microbial community to provide the necessary cues to train and promote its proper function. In summary, this data shows that supplementation of SCB promoted establishment of a diverse and interconnected microbiota, prevented colonization of Escherichia Shigella and indicates a possible role in stimulating humoral mucosal immunity.

Multi-omics revealed the long-term effect of ruminal keystone bacteria and the microbial metabolome on lactation performance in adult dairy goats.

BACKGROUND: The increased growth rate of young animals can lead to higher lactation performance in adult goats; however, the effects of the ruminal microbiome on the growth of young goats, and the contribution of the early-life rumen microbiome to lifelong growth and lactation performance in goats has not yet been well defined. Hence, this study assessed the rumen microbiome in young goats with different average daily gains (ADG) and evaluated its contribution to growth and lactation performance during the first lactation period. RESULTS: Based on monitoring of a cohort of 99 goats from youth to first lactation, the 15 highest ADG (HADG) goats and 15 lowest ADG (LADG) goats were subjected to rumen fluid microbiome and metabolome profiling. The comparison of the rumen metagenome of HADG and LADG goats revealed that ruminal carbohydrate metabolism and amino acid metabolism function were enhanced in HADG goats, suggesting that the rumen fluid microbiome of HADG goats has higher feed fermentation ability. Co-occurrence network and correlation analysis revealed that Streptococcus, Candidatus Saccharimonans, and Succinivibrionaceae UCG-001 were significantly positively correlated with young goats' growth rates and some HADG-enriched carbohydrate and protein metabolites, such as propionate, butyrate, maltoriose, and amino acids, while several genera and species of Prevotella and Methanogens exhibited a negative relationship with young goats' growth rates and correlated with LADG-enriched metabolites, such as rumen acetate as well as methane. Additionally, some functional keystone bacterial taxa, such as Prevotella, in the rumen of young goats were significantly correlated with the same taxa in the rumen of adult lactation goats. Prevotella also enriched the rumen of LADG lactating goats and had a negative effect on rumen fermentation efficiency in lactating goats. Additional analysis using random forest machine learning showed that rumen fluid microbiota and their metabolites of young goats, such as Prevotellaceae UCG-003, acetate to propionate ratio could be potential microbial markers that can potentially classify high or low ADG goats with an accuracy of prediction of > 81.3%. Similarly, the abundance of Streptococcus in the rumen of young goats could be predictive of milk yield in adult goats with high accuracy (area under the curve 91.7%). CONCLUSIONS: This study identified the keystone bacterial taxa that influence carbohydrate and amino acid metabolic functions and shape the rumen fluid microbiota in the rumen of adult animals. Keystone bacteria and their effects on rumen fluid microbiota and metabolome composition during early life can lead to higher lactation performance in adult ruminants. These findings suggest that the rumen microbiome together with their metabolites in young ruminants have long-term effect on feed efficiency and animal performance. The fundamental knowledge may allow us to develop advanced methods to manipulate the rumen microbiome and improve production efficiency of ruminants. Video Abstract.

Prophylactic feeding of neomycin to Holstein calves alters gut microbiota, bile acid metabolism, and expression of genes involved in immunometabolic regulation.

The objective of this study was to evaluate the effects of prophylactic neomycin administration on Holstein bull calves' intestinal microbiota, bile acid (BA) metabolism, and transcript abundance of genes related to BA metabolism. A total of 36 calves were blocked by body weight and assigned to either non-medicated milk replacer (CTL), or neomycin for 14 days (ST) or 28 days (LT) in their milk replacer. At the end of the study, calves were euthanized to collect tissue and digesta samples from the gastrointestinal tract, liver, and adipose tissue for analysis of intestinal microbial diversity, bile acid concentration and profile in various body tissues, and gene expression related to bile acid, lipid, carbohydrate metabolism, and inflammation. Calves that received prophylactic administration of neomycin for 28 d (LT) had reduced species richness (chao1 index), and tended to have reduced phylogenetic diversity in the ileum tissue. The relative abundance of Lactobacillus, and Bifidobacterium in ileum and colon digesta were decreased in LT compared with CTL. Concentrations of primary, secondary, and total BA were increased by ST in ileal tissue. In plasma, ST and LT treatments had lower concentrations of secondary BA. Gene expression of the BA receptor FXR was increased in ileum and liver by LT compared to CTL. The expression of FXR and TGR5 in the liver was increased in the ST group compared with CTL, and in adipose tissue, 5 genes related to triglyceride, gluconeogenesis, and immune activation were differentially expressed between CTL and ST. In conclusion, we provide evidence that prophylactic administration of neomycin leads to aberrant changes in BA concentration and profile in different compartments of the enterohepatic system through a process that possibly entails antimicrobial disruption of key bacterial groups, which persists even after cessation of neomycin administration. Additionally, we uncovered an apparent link between dysregulated BA metabolism and changes in lipid metabolism and immune activation in adipose tissue and liver.

Development of a spore-based mucosal vaccine against the bovine respiratory pathogen Mannheimia haemolytica.

Bovine respiratory disease (BRD) is a significant health issue in the North American feedlot industry, causing substantial financial losses due to morbidity and mortality. A lack of effective vaccines against BRD pathogens has resulted in antibiotics primarily being used for BRD prevention. The aim of this study was to develop a mucosal vaccine against the BRD pathogen, Mannheimia haemolytica, using Bacillus subtilis spores as an adjuvant. A chimeric protein (MhCP) containing a tandem repeat of neutralizing epitopes from M. haemolytica leukotoxin A (NLKT) and outer membrane protein PlpE was expressed to produce antigen for adsorption to B. subtilis spores. Adsorption was optimized by comparing varying amounts of antigen and spores, as well as different buffer pH and reaction temperatures. Using the optimal adsorption parameters, spore-bound antigen (Spore-MhCP) was prepared and administered to mice via two mucosal routes (intranasal and intragastric), while intramuscular administration of free MhCP and unvaccinated mice were used as positive and negative control treatments, respectively. Intramuscular administration of MhCP elicited the strongest serum IgG response. However, intranasal immunization of Spore-MhCP generated the best secretory IgA-specific response against both PlpE and NLKT in all samples evaluated (bronchoalveolar lavage, saliva, and feces). Since proliferation of M. haemolytica in the respiratory tract is a prerequisite to lung infection, this spore-based vaccine may offer protection in cattle by limiting colonization and subsequent infection, and Spore-MhCP warrants further evaluation in cattle as a mucosal vaccine against M. haemolytica.

Reduction in mucosa thickness is associated with changes in immune function in the colon mucosa during the weaning transition in Holstein bull dairy calves.

This study aims to characterize changes in the structure and the molecules related to immune function in the colon mucosa in dairy calves during the weaning transition (weaned at week 6 of age). Colon mucosa thickness, measured at week 5 to 8 and 12 of age, decreased for 2 weeks after weaning, but then recovered. Colon mucosa's transcriptome profiling at week 5, 7, and 12 of age was obtained using RNA-sequencing. Functional analysis showed that pathways related to immune function were up-regulated postweaning. A weighted gene co-expression network analysis identified 17 immune function related genes, expressed higher postweaning, which were negatively correlated with colon mucosa thickness, suggesting that these genes may be involved in colon mucosa inflammation and recovery from mucosa thickness decrement during the weaning transition. As such, it is important to determine the function of immune cells in the colon mucosa during the weaning transition in dairy calves.

Transcriptome profiling revealed that key rumen epithelium functions change in relation to short-chain fatty acids and rumen epithelium-attached microbiota during the weaning transition.

This study aims to characterize the functional changes of the rumen epithelium associated with ruminal short-chain fatty acid (SCFA) concentration and epithelium-attached microbes during the weaning transition in dairy calves. Ruminal SCFA concentrations were determined, and transcriptome and microbiota profiling in biopsied rumen papillae were obtained from Holstein calves before and after weaning using RNA- and amplicon sequencing. Metabolic pathway analysis showed that pathways related to SCFA metabolism and cell apoptosis were up- and down-regulated postweaning, respectively. Functional analysis showed that genes related to SCFA absorption, metabolism, and protective roles against oxidative stress were positively correlated with ruminal SCFA concentrations. The relative abundance of epithelium-attached Rikenellaceae RC9 gut group and Campylobacter was positively correlated with genes involved in SCFA absorption and metabolism, suggesting that these microbes can cooperatively affect host functions. Future research should examine the contribution of attenuated apoptosis on rumen epithelial functional shifts during the weaning transition.

Oral administration of Pinus koraiensis cone essential oil reduces rumen methane emission by altering the rumen microbial composition and functions in Korean native goat (Capra hircus coreanae).

This study aimed to investigate Pinus koraiensis cone essential oil (PEO) as a methane (CH4) inhibitor and determine its impact on the taxonomic and functional characteristics of the rumen microbiota in goats. A total of 10 growing Korean native goats (Capra hircus coreanae, 29.9 ± 1.58 kg, male) were assigned to different dietary treatments: control (CON; basal diet without additive) and PEO (basal diet +1 g/d of PEO) by a 2 × 2 crossover design. Methane measurements were conducted every 4 consecutive days for 17-20 days using a laser CH4 detector. Samples of rumen fluid and feces were collected during each experimental period to evaluate the biological effects and dry matter (DM) digestibility after PEO oral administration. The rumen microbiota was analyzed via 16S rRNA gene amplicon sequencing. The PEO oral administration resulted in reduced CH4 emission (eructation CH4/body weight0.75, p = 0.079) without affecting DM intake; however, it lowered the total volatile fatty acids (p = 0.041), molar proportion of propionate (p = 0.075), and ammonia nitrogen (p = 0.087) in the rumen. Blood metabolites (i.e., albumin, alanine transaminase/serum glutamic pyruvate transaminase, creatinine, and triglyceride) were significantly affected (p < 0.05) by PEO oral administration. The absolute fungal abundance (p = 0.009) was reduced by PEO oral administration, whereas ciliate protozoa, total bacteria, and methanogen abundance were not affected. The composition of rumen prokaryotic microbiota was altered by PEO oral administration with lower evenness (p = 0.054) observed for the PEO group than the CON group. Moreover, PICRUSt2 analysis revealed that the metabolic pathways of prokaryotic bacteria, such as pyruvate metabolism, were enriched in the PEO group. We also identified the Rikenellaceae RC9 gut group as the taxa potentially contributing to the enriched KEGG modules for histidine biosynthesis and pyruvate oxidation in the rumen of the PEO group using the FishTaco analysis. The entire co-occurrence networks showed that more nodes and edges were detected in the PEO group. Overall, these findings provide an understanding of how PEO oral administration affects CH4 emission and rumen prokaryotic microbiota composition and function. This study may help develop potential manipulation strategies to find new essential oils to mitigate enteric CH4 emissions from ruminants.

Microbial Interventions to Improve Neonatal Gut Health.

The diverse pioneer microbial community colonizing the mammalian gastrointestinal tract is critical for the developing immune system. Gut microbial communities of neonates can be affected by various internal and external factors, resulting in microbial dysbiosis. Microbial dysbiosis during early life affects gut homeostasis by changing metabolic, physiological, and immunological status, which increases susceptibility to neonatal infections and long-term pathologies. Early life is crucial for the establishment of microbiota and the development of the host immune system. Therefore, it provides a window of opportunity to reverse microbial dysbiosis with a positive impact on host health. Recent attempts to use microbial interventions during early life have successfully reversed dysbiotic gut microbial communities in neonates. However, interventions with persistent effects on microbiota and host health are still limited. This review will critically discuss microbial interventions, modulatory mechanisms, their limitations, and gaps in knowledge to understand their roles in improving neonatal gut health.

Competitive Analysis of Rumen and Hindgut Microbiota Composition and Fermentation Function in Diarrheic and Non-Diarrheic Postpartum Dairy Cows.

Postpartum dairy cows can develop nutritional diarrhea when their diet is abruptly changed for milk production. However, it is unclear whether nutritional diarrhea develops as a result of gut acidosis and/or dysbiosis. This study aimed to uncover changes in the gastrointestinal microbiota and its fermentation parameters in response to nutritional diarrhea in postpartum dairy cows. Rumen and fecal samples were collected from twenty-four postpartum cows fed with the same diet but with different fecal scores: the low-fecal-score (LFS: diarrheic) group and high-fecal-score (HFS: non-diarrheic) group. A microbiota difference was only observed for fecal microbiota, with the relative abundance of Defluviitaleaceae_UCG-011 and Lachnospiraceae_UCG-001 tending (p < 0.10) to be higher in HFS cows compared to LFS cows, and Frisingicoccus were only detected in HFS cows. The fecal bacterial community in LFS cows had higher robustness (p < 0.05) compared to that in HFS cows, and also had lower negative cohesion (less competitive behaviors) and higher positive cohesion (more cooperative behaviors) (p < 0.05) compared that in to HFS cows. Lower total volatile fatty acids and higher ammonia nitrogen (p < 0.05) were observed in LFS cows' feces compared to HFS cows. The observed shift in fecal bacterial composition, community networks, and metabolites suggests that hindgut dysbiosis could be related to nutritional diarrhea in postpartum cows.

Taxonomic and predicted functional signatures reveal linkages between the rumen microbiota and feed efficiency in dairy cattle raised in tropical areas.

Ruminants digest plant biomass more efficiently than monogastric animals due to their symbiotic relationship with a complex microbiota residing in the rumen environment. What remains unclear is the relationship between the rumen microbial taxonomic and functional composition and feed efficiency (FE), especially in crossbred dairy cattle (Holstein x Gyr) raised under tropical conditions. In this study, we selected twenty-two F1 Holstein x Gyr heifers and grouped them according to their residual feed intake (RFI) ranking, high efficiency (HE) (n = 11) and low efficiency (LE) (n = 11), to investigate the effect of FE on the rumen microbial taxa and their functions. Rumen fluids were collected using a stomach tube apparatus and analyzed using amplicon sequencing targeting the 16S (bacteria and archaea) and 18S (protozoa) rRNA genes. Alpha-diversity and beta-diversity analysis revealed no significant difference in the rumen microbiota between the HE and LE animals. Multivariate analysis (sPLS-DA) showed a clear separation of two clusters in bacterial taxonomic profiles related to each FE group, but in archaeal and protozoal profiles, the clusters overlapped. The sPLS-DA also revealed a clear separation in functional profiles for bacteria, archaea, and protozoa between the HE and LE animals. Microbial taxa were differently related to HE (e.g., Howardella and Shuttleworthia) and LE animals (e.g., Eremoplastron and Methanobrevibacter), and predicted functions were significatively different for each FE group (e.g., K03395-signaling and cellular process was strongly related to HE animals, and K13643-genetic information processing was related to LE animals). This study demonstrates that differences in the rumen microbiome relative to FE ranking are not directly observed from diversity indices (Faith's Phylogenetic Diversity, Pielou's Evenness, Shannon's diversity, weighted UniFrac distance, Jaccard index, and Bray-Curtis dissimilarity), but from targeted identification of specific taxa and microbial functions characterizing each FE group. These results shed light on the role of rumen microbial taxonomic and functional profiles in crossbred Holstein × Gyr dairy cattle raised in tropical conditions, creating the possibility of using the microbial signature of the HE group as a biological tool for the development of biomarkers that improve FE in ruminants.

Red seaweed extracts reduce methane production by altering rumen fermentation and microbial composition in vitro.

A series of in vitro batch culture incubations were carried out to investigate changes in rumen fermentation characteristics, methane (CH4) production, and microbial composition in response to supplementation with five different red seaweed species (Amphiroa anceps, AANC; Asparagopsis taxiformis, ATAX; Chondracanthus tenellus, CTEN; Grateloupia elliptica, GELL; and Gracilaria parvispora, GPAR). Prior to the incubations, the total flavonoid and polyphenol content of the red seaweed extracts was quantified. The incubated substrate consisted of timothy hay and corn grain [60:40 dry matter (DM) basis]. Treatments were substrate mixtures without seaweed extract (CON) or substrate mixtures supplemented with 0.25 mg/mL of red seaweed extract. Samples were incubated for 6, 12, 24, 36, and 48 h. Each sample was incubated in triplicates in three separate runs. In vitro DM degradability, fermentation parameters (i.e., pH, volatile fatty acids, and ammonia nitrogen), total gas production, and CH4 production were analyzed for all time points. Microbial composition was analyzed using 16S rRNA amplicon sequencing after 24 h of incubation. The highest CH4 reduction (mL/g DM, mL/g digested DM, and % of total gas production) was observed in ATAX (51.3, 50.1, and 51.5%, respectively, compared to CON; P < 0.001) after 12 h of incubation. The other red seaweed extracts reduced the CH4 production (mL/g DM; P < 0.001) in the range of 4.6-35.0% compared to CON after 24 h of incubation. After 24 h of incubation, supplementation with red seaweed extracts tended to increase the molar proportion of propionate (P = 0.057) and decreased the acetate to propionate ratio (P = 0.033) compared to the CON. Abundances of the genus Methanobrevibacter and total methanogens were reduced (P = 0.050 and P = 0.016) by red seaweed extract supplementation. The linear discriminant analysis effect size (P < 0.05, LDA ≥ 2.0) showed that UG Succinivibrionaceae, Anaeroplasma, and UG Ruminococcaceae, which are associated with higher propionate production, starch degradation, and amylase activity were relatively more abundant in red seaweed extracts than in the CON. Our results suggest that supplementation with red seaweed extracts altered the microbiota, leading to the acceleration of propionate production and reduction in CH4 production.

Metagenomic analysis provides bases on individualized shift of colon microbiome affected by delaying colostrum feeding in neonatal calves.

This study investigated the effect of colostrum feeding time on the colon digesta microbiome of 2-day-old dairy calves using whole-genome-based metagenome sequencing, aiming to understand the dynamic changes of the colon microbiome when the colostrum feeding is delayed. In total, 24 male Holstein calves were grouped to different pasteurized colostrum feeding time treatments randomly: TRT0h (45 min after birth, n = 7); TRT6h (6 h after birth, n = 8); and TRT12h (12 h after birth, n = 9). Bacteria, archaea, eukaryotes, and viruses were identified in the colon microbiome, with bacteria (99.20%) being the most predominant domain. Streptococcus, Clostridium, Lactobacillus, Ruminococcus, and Enterococcus were the top five abundant bacteria genera. For colon microbiome functions, 114 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were identified, with nutrients metabolism-related functions "carbohydrate metabolism," "amino acid metabolism," "metabolism of cofactors and vitamins," "metabolism of terpenoids and polyketides," and "metabolism of other amino acids" being the top five secondary level of KEGG hierarchy functions. When colon microbiomes were compared, they were not affected by delaying first colostrum feeding at both taxonomic and functional levels. However, distinct clusters of colon microbiome profiles were shown based on PERMANOVA analysis despite of different colostrum feeding treatment, suggesting the individualized responses. Moreover, the relative abundance of microbial taxa, microbial functions, and differentially expressed genes was compared between the two distinct clusters, and different relationships were observed among host differentially expressed genes, differential levels of microbial taxa, and microbial functions between the two clusters. Our results suggest that the host may play an important role in shaping the colon microbiome of neonatal dairy calves in response to the early life feeding management. Whether the observed colon microbiome shifts affect gut health and function in the long term requires further research.