Rumen Fluid Samples Research Articles

Disorders of acid-base balance promote rumen lipopolysaccharide biosynthesis in dairy cows by modulating the microbiome

IntroductionDisorders of acid-base balance in the rumen of dairy cows have a significant impact on their health and performance. However, the effect of transient differences in pH on susceptibility to subacute ruminal acidosis (SARA) and lipopolysaccharide (LPS) biosynthesis in dairy cows remains unclear.MethodsIn this study, milk, serum, and rumen fluid samples from 40 Holstein dairy cows (on d 56 postpartum) with different rumen pH (2–4 h after morning feeding) were explored to investigate the difference of susceptibility to SARA and the correlation between microbiome, LPS and inflammation. These cows were categorized into low pH (LPH, pH ≤ 6.0, n = 20) and high pH (HPH, pH ≥ 6.5, n = 20) groups.ResultsThe results showed that LPH group increased the concentrations of total volatile fatty acids, acetate, propionate, butyrate and valerate. However, milk yield and milk compositions were unaffected. Compared to the HPH group, the LPH group increased the concentrations of serum BHBA, NEFA, LPS, HIS, IL-2, IL-6, TNF-α, and MDA, and decreased the concentrations of serum IgA, IgM, IgG, SOD, T-AOC, and mTOR. In addition, the LPH group decreased the copies of Ruminococcus flavefaciens and increased the copies of Fibrobacter succinogenes. Microbial community analysis isupplendicated a significant difference in bacterial composition between the two groups. At the phylum level, Bacteroidota and Firmicutes were enriched in the LPH and HPH groups, respectively. At the genus level, the dominant bacteria in the LPH group were Prevotella. Additionally, the LPH group increased the proportions of Gram-negative phenotypes, potentially pathogenic phenotypes and LPS biosynthesis. The close correlation between two key enzymes for LPS synthesis LpxL and LpxM with rumen pH, inflammatory markers, and microorganisms indicates that low pH may increase the risk of inflammation by facilitating the lysis of Gram-negative bacteria and the release of penta-acylated LPS. Penta-acylated and hexa-acylated LPS may be mainly derived from Prevotella and Succinivibrionaceae_UCG-001, respectively.DiscussionOverall, these results support the notion that transient low pH could reflect the risk of cows suffering from SARA and associated inflammation and is strongly associated with penta-acylated LPS. Our findings provide new insights into ruminant health improvement and disease prevention strategies.

Exploring the rumen microbial function in Angus bulls with divergent residual feed intake

This study leverages Shotgun metagenomics to assess the rumen microbial community and functionality in Angus bulls with differing residual feed intake-expected progeny difference (RFI-EPD) values, aiming to elucidate the microbial contributions to feed efficiency. Negative RFI-EPD bulls (NegRFI: n=10; RFI-EPD= -0.3883 kg/d) and positive RFI-EPD bulls (PosRFI: n=10; RFI-EPD=0.2935 kg/d) were selected from a group of 59 Angus bulls (average body weight (BW) = 428 ± 18.8 kg; 350 ± 13.4 d of age) fed a high-forage total mixed ration after a 60-d testing period. At the end of the 60-d period, rumen fluid samples were collected for bacterial DNA extraction and subsequent shotgun metagenomic sequencing. Results of the metagenome analysis revealed greater gene richness in NegRFI bulls, compared to PosRFI. Analysis of similarity revealed a small but noticeable difference (P =0.052; R-value = 0.097) in the rumen microbial community of NegRFI and PosRFI bulls. Linear Discriminant Analysis effect size (Lefse) was utilized to identify the differentially abundant taxa. The Lefse results showed that class Fibrobacteria (LDA = 5.1) and genus Fibrobacter (LDA = 4.8) were greater in NegRFI bulls, compared to PosRFI bulls. Relative abundance of the carbohydrate-active enzymes was also compared using Lefse. The results showed greater relative abundance of glycoside hydrolases and carbohydrate-binding modules such as GH5, CBM86, CBM35, GH43, and CBM6 (LDA > 3.0) in NegRFI bulls whereas GH13 and GT2 were greater in PosRFI bulls. The distinct metabolic and microbial profiles observed in NegRFI, compared to PosRFI bulls, characterized by greater gene richness and specific taxa such as Fibrobacter, and variations in carbohydrate-active enzymes, underscore the potential genetic and functional differences in their rumen microbiome. These findings contribute to a deeper understanding of the interplay between rumen microbiota and feed efficiency in Angus bulls, opening avenues for targeted interventions and advancements in livestock management practices.

Comparative Analysis of Microbiota in Jiani Yaks with Different Rib Structures

The Jiani yak is a nationally renowned species that is known for its meat which is rich in various minerals, amino acids, and proteins. The rumen microbiota plays a critical role in gastrointestinal health and feed degradation, contributing proteins, lipids, and volatile fatty acids (VFAs) essential for milk and meat production. However, there is limited knowledge about the microbiota of free-ranging Jiani yaks, especially those with 15 ribs. Rumen fluid samples were collected from yaks with 14 (PL) ribs and 15 (DL) ribs from a slaughterhouse in Jiani County, China. The total DNA of rumen fluid microorganisms was extracted for microbiota sequencing. Our results revealed 643,713 and 656,346 raw sequences in DL and PL animals, respectively, with 611,934 and 622,814 filtered sequences in these two yak groups. We identified 13,498 Amplicon Sequence Variants (ASVs), with 2623 shared between DL and PL animals. The ratio of Bacteroidota to Firmicutes differed between PL (3.04) and DL (2.35) animals. Additionally, 6 phyla and 21 genera showed significant differences between yaks with 14 and 15 ribs, leading to altered microbiota functions, with 51 and 35 notably different MetaCyc and KEGG pathways, respectively. Hence, the microbiota of yaks with 15 ribs differs from those with 14 ribs. Therefore, these microbiota-related comparative investigations will provide insights into yak husbandry practices and genetic selection strategies for their improved productivity in harsh environments.

Dietary supplementation with citrus peel extract in transition period improves rumen microbial composition and ameliorates energy metabolism and lactation performance of dairy cows

BackgroundDuring the transition period, excessive negative energy balance (NEB) lead to metabolic disorders and reduced milk yield. Rumen microbes are responsible for resolving plant material and producing volatile fatty acids (VFA), which are the primary energy source for cows. In this study, we aimed to investigate the effect of citrus peel extract (CPE) supplementation on rumen microbiota composition, energy metabolism and milk performance of peripartum dairy cows.MethodsDairy cows were fed either a basal diet (CON group) or the same basal diet supplemented with CPE via intragastric administration (4 g/d, CPE group) for 6 weeks (3 weeks before and 3 weeks after calving; n = 15 per group). Samples of serum, milk, rumen fluid, adipose tissue, and liver were collected to assess the effects of CPE on rumen microbiota composition, rumen fermentation parameters, milk performance, and energy metabolic status of dairy cows.ResultsCPE supplementation led to an increase in milk yield, milk protein and lactose contents, and serum glucose levels, while reduced serum concentrations of non-esterified fatty acid, β-hydroxybutyric acid, insulin, aspartate aminotransferase, alanine aminotransferase, and haptoglobin during the first month of lactation. CPE supplementation also increased the content of ruminal VFA. Compared to the CON group, the abundance of Prevotellaceae, Methanobacteriaceae, Bacteroidales_RF16_group, and Selenomonadaceae was found increased, while the abundance of Oscillospiraceae, F082, Ruminococcaceae, Christensenellaceae, Muribaculaceae UCG-011, Saccharimonadaceae, Hungateiclostridiaceae, and Spirochaetaceae in the CPE group was found decreased. In adipose tissue, CPE supplementation decreased lipolysis, and inflammatory response, while increased insulin sensitivity. In the liver, CPE supplementation decreased lipid accumulation, increased insulin sensitivity, and upregulated expression of genes involved in gluconeogenesis.ConclusionsOur findings suggest that CPE supplementation during the peripartum period altered rumen microbiota composition and increased ruminal VFA contents, which further improved NEB and lactation performance, alleviated lipolysis and inflammatory response in adipose tissue, reduced lipid accumulation and promoted gluconeogenesis in liver. Thus, CPE might contribute to improve energy metabolism and consequently lactation performance of dairy cows during the transition period.

Hydroxy-selenomethionine helps cows to overcome heat stress by enhancing antioxidant capacity and alleviating blood-milk barrier damage

Heat stress can lead to decreased feed intake, apoptosis of mammary epithelial cells, and decreased milk yield and quality. Selenium is an important element in the composition of at least 25 selenoproteins. Hydroxy-selenomethionine (HMSeBA; Andy Su Life Science Products Co., Ltd., Shanghai, China) is a novel organic selenium that has been shown to have a better deposition effect. However, whether HMSeBA alleviates damage to the mammary gland blood-milk barrier caused by heat stress and how this affects the performance of dairy cows remain largely unexplored. Therefore, 32 healthy Holstein cows with similar gestation days (150.41 ± 20.07 d), milk yield (36.15 ± 3.02 kg) and parity (3.25 ± 0.51) were selected and randomly divided into two total mixed rations with different selenium (Se) sources: sodium selenite (SSe) and HMSeBA. This study evaluated the outcomes of HMSeBA on antioxidant capacity, immunity, and blood-milk barrier damage in dairy cows during heat stress by collecting the samples of blood, rumen fluid and mammary gland biopsy. The experiment was conducted over 35 d, including a 5-day pre-feeding period and a 30-day experimental period. The temperature and humidity index (THI) were all above 80 throughout the experiment period. The results showed that HMSeBA decreased the respiratory rate (P < 0.001) and the content of inflammatory cytokines in the serum and increased the content of immune factors and antioxidant capacity (P < 0.05). In addition, HMSeBA reduced the expression of inflammatory cytokines and heat shock proteins in mammary gland (P < 0.05). Hematoxylin-eosin-stained pathological sections showed massive thickening of acinar walls and severe destruction of glandular structures in the SSe group, but the structure of the acinar mammary gland in the HMSeBA group was intact. Furthermore, HMSeBA promoted the expression of the phosphatidylinositol 3-kinase (PI3K, P < 0.001)/protein kinase B (AKT, P = 0.011)/mammalian target of rapamycin (mTOR, P = 0.008) pathway and improved the expression of zonula occludens-1 (ZO-1, P = 0.014) and occluding (OCLN, P = 0.012) in the mammary gland, suggesting less damage caused by heat stress to the blood-milk barrier. Our results demonstrated that HMSeBA can improve the antioxidant capacity and immunity of dairy cows and the expression of tight junction proteins in mammary gland to help alleviate the blood-milk barrier damage by heat stress, which could reduce the damage of heat stress on milk yield.

Early-life Clostridium butyricum supplementation improved rumen development and immune by promoting the maturation of intestinal microbiota

This study evaluated the effects of Clostridium butyricum supplementation on rumen fermentation, serum immunoglobulins, and fecal microbiota in dairy calves. Seventy-five 3-days-age calves were divided into five groups (n = 15 per group) receiving 0, 0.2, 0.4, 0.8, and 1.6 g/day of C. butyricum freeze-dried powder (10ˆ10 CFU/g) in milk replacer. Rumen fluid and blood samples were collected at various intervals, and fecal samples from eight calves per group were collected at 3, 28, and 42 days. C. butyricum supplementation showed a negative correlation with rumen pH (P = 0.032) and positive correlations with acetate/propionate (P = 0.007) and acetate + butyrate/propionate (P = 0.022). It had quadratic effects on rumen acetate (P = 0.029) and isobutyrate (P = 0.021). IgA concentration increased with dose (P = 0.002), and dose-time interactions affected serum IgM (P = 0.013) and IgG (P < 0.001). C. butyricum did not influence fecal microbiota diversity but increased the abundance of Helicobacter japonicus, Weissella viridescens, Leuconostoc mesenteroides, Leuconostoc citreum, while negatively correlating with Streptococcus hyointestinalis (P < 0.05). Microbiota functions related to galactose metabolism, and fatty acid degradation decreased with age (P < 0.05), while fatty acid biosynthesis and glycolysis/gluconeogenesis increased (P < 0.05). L. mesenteroides, and L. citreum, and negatively correlated with galactose metabolism and fatty acid degradation but positively with fatty acid biosynthesis (P < 0.01). C. butyricum supplementation variably impacted rumen pH, acetate/propionate, and acetate + butyrate/propionate, particularly on day 42 versus day 60, affecting acetate, butyrate, and isovalerate differently over time. Our findings suggest that early-life C. butyricum supplementation may promote the maturation of immunity and intestinal microbiota of pre-weaning dairy calves, the optimal dose was 0.8 to 1.6 x 10ˆ10 CFU/d.

Programming rumen microbiome development in calves with the anti-methanogenic compound 3-NOP

The aim of this study was to establish a distinctive rumen microbial and fermentation profile using the anti-methanogenic compound 3-NOP to assess dam effect, and nutritional intervention of the juvenile offspring on microbial structure and function of rumen up to 12 months of age, once the treatment was withdrawn. Forty-eight pregnant heifers (H) and their future offspring (C) were allocated to either Control (-) or 3-NOP (+) treatment resulting in four experimental groups: H+/C+, H+/C-, H-/C + and H-/C-. Animals were treated from 6 weeks prior to calving until weaning, with the offspring monitored until 12 months of age. Rumen fluid samples and methane measurements using the Greenfeed system were collected during the trial. Results supported the mode of action of the compound, with a shift in fermentation from acetate to propionate, increases in branched chain fatty acids and formic acid in the 3-NOP treated animals. Similar shifts in microbial populations occurred in 3-NOP treated animals with lower abundances of rumen methanogen populations, increases of bacterial groups Succiniclasticum spp, Candidatus Saccharimonas. Fibrobacter and the families Prevotellaceae and Succinivibrioacea. and the protozoa Entodinium. Early life intervention had an enduring impact on the rumen microbial structure of young animals up to 28 weeks post weaning, however the effect was diminished once 3-NOP was withdrawn. Interestingly, a group of young animals emitted significantly less methane (15%) than the animals that did not receive the treatment during their juvenile stage. Our results suggest a higher resemblance of the young calf microbiome to a low methane adult and that early life colonisation of the rumen persists through to later life with the pre-weaning microbiome comprising ~ 65% of the yearling animal. Further research needs to be performed to determine the timing and dose of 3-NOP for new-born calves that can sustain a reduction in methane emissions after the treatment is withdrawn, under extensive grazing or controlled conditions.

Gender and age-related variations in rumen fermentation and microbiota of Qinchuan cattle.

Our study aimed to investigate the gender and age-related variations in rumen fermentation, serum metabolites, and microbiota in Qinchuan cattle. A total of 38 Qinchuan beef cattle were selected and maintained on a uniform diet for three months. Rumen fluid and blood samples were collected to determine rumen fermentation, serum metabolites, and microbial 16S rRNA sequencing. The results revealed that the concentration of rumen butyrate in female Qinchuan cattle was significantly higher than in males (P<0.05). Isobutyrate, butyrate, and isovalerate exhibited significant age-related differences. Females exhibited lower serum GLU and higher TG, NEFA levels compared to males (P<0.05). Serum ALB and UREA levels increased with age (P<0.05). Furthermore, the alpha diversity of rumen bacteria improved with age (P<0.05), with no gender differences observed. Males had higher relative abundances of Bacteroidota, Verrucomicrobiota, and Cyanobacteria, while females had higher Firmicutes and Desulfobacterota (P<0.05). The cellulose-degrading genus Ruminococcus and propionate-producing genus Succiniclasticum were more abundant in females, whereas the anti-inflammatory genus Lachnospiraceae_NK4A136_group and the hemicellulose-degrading genus Prevotella were more abundant in males (P<0.05). Age-related differences in bacteria were found in Pseudobutyrivibrio and several members of the Lachnospiraceae. Functional prediction indicated that "Amino acid metabolism" and "Lipid metabolism" were mainly enriched in females, whereas "Carbohydrate metabolism" and "Glycan biosynthesis and metabolism" were enriched in males (P<0.05). RDA analysis highlighted butyrate as a key factor influencing the rumen bacterial community. NK4A214_group and Ruminococcus were positively correlated with butyrate, while Prevotella and Pseudobutyrivibrio were negatively correlated with butyrate (P<0.05). We observed a significant improvement in the diversity and stability of rumen microbiota as age increased. Ruminococcus, NK4A214_group, and Prevotella were likely contributors to variations in energy utilization and fat deposition between male and female Qinchuan cattle.

Effect of a Bacillus subtilis-based direct-fed microbial, on milk yield, milk components, feed intake and plasma hormones and metabolites in lactating Holstein cows

To investigate the effects of a Bacillus-based direct-fed microbial on milk production and related factors, a study was conducted on Holstein cows (n = 28) starting at 90 ± 11 DIM. The cows were divided into two dietary groups: a control group (CON) that received a total mixed ration (TMR), and a Bacillus-fed group (DFM) that received the same TMR along with a Bacillus subtilis product containing two strains (747 and 1781) in equal amounts. The study lasted for 25 wk and included both primiparous and multiparous cows. The cows were housed in a free-stall barn and were provided with ad libitum TMR, which was fed twice a day. Their daily meals and dry matter intake (DMI) were recorded during wk 1–4 and wk 19–25 using electronic feeders. Milk samples were collected weekly during morning and evening milkings and analyzed for milk fat, protein, lactose, and milk urea nitrogen (MUN). Blood samples were also collected weekly and analyzed for plasma glucose, insulin, cholesterol, insulin-like growth factor 1 (IGF1), and progesterone concentrations. Additionally, rumen fluid samples were collected and evaluated for various species of bacteria. One of the key findings was that daily 4 % fat-corrected milk production (FCM) was influenced by the interaction between treatment (DFM vs. CON) and parity (primiparous vs. multiparous), with multiparous DFM cows producing 11 % more FCM compared to CON. The DFM cows also had a higher milk fat percentage (4.41 % vs. 4.02 %) and MUN concentrations were 1.0 mg/dL higher in the DFM group. In terms of blood metabolites and hormones, multiparous DFM cows had lower plasma cholesterol, glucose, and insulin concentrations compared to CON multiparous cows. However, there were no significant differences between the groups in terms of plasma IGF1 concentrations, insulin-to-glucose ratios, and luteal phase plasma progesterone concentrations. The analysis of rumen fluid samples revealed that the abundance of Ruminococcus albus and Fibrobacter succinogenes group I was greater in the DFM cows compared to CON cows. Furthermore, during the periods when feed intake was measured, the DFM cows had a 9 % reduction in feed intake and a 14 % improvement in feed efficiency (FCM per DMI) compared to CON cows. In conclusion, the two-strain Bacillus product used in this study (Certillus) showed potential as an effective direct-fed microbial. It was found to increase feed efficiency and milk production by altering the composition of ruminal microbiota and metabolism in the cows.

Effects of water extracts of Artemisia annua L. on rumen immune and antioxidative indexes, fermentation parameters and microbials diversity in lambs.

The present study investigated the effects of water extracts of Artemisia annua L. (WEAA) on rumen immune and antioxidative indexes, fermentation parameters and microbial diversity in lambs. A total of 32 3-month-old Dorper × Han female lambs having comparable body weights (24±0.09 kg) were selected and were randomly assigned to four treatments, with eight repetitions for each treatment. The basal diet, consisting of 45% concentrate and 55% forage, was solely provided to the control group. For the other treatment groups, the basal diet was supplemented with WEAA at dosages of 500, 1000, and 1500 mg/kg diet, respectively. Rumen tissue samples were collected for the analysis of immune and antioxidative parameters, as well as related gene expression. Rumen fluid samples were collected to assess rumen fermentation parameters on days 30 and 60 and to evaluate the microbiota on day 60. Results showed that WEAA supplementation linearly or quadratically increased the content of sIgA, IL-4, IL-2 and the gene expression level of MyD88, IκB-α, IL-4, COX-2, iNOS in rumen tissue (p < 0.05), as well as the bacteria negatively associated with IL-6 (g_ [Eubacterium] _cellulosolvens_group). Furthermore, the addition of WEAA linearly or quadratically increased rumen T-SOD, GSH-Px (p < 0.05) and the gene expression level of Nrf2, SOD2, GSH-Px, HO-1 (p < 0.05), and decreased the rumen concentration of malondialdehyde (MDA) and gene expression level of Keap1 (p < 0.05), as well as the bacteria positively associated with T-AOC, T-SOD and GSH-Px (g_Lachnospiraceae_NK3A20_group, g_Saccharofermentans, g__Marvinbryantia, g_unclassified_f_Eggerthellaceae). The supplementation of WEAA caused the concentration of microprotein (MCP), total volatile fatty acids (TVFA), propionate to increase either linearly or quadratically, while reducing the concentration of NH3-N and the acetate/propionate ratio (A:P) in rumen fluid (p < 0.05). The addition of WEAA linearly or quadratically increased the abundance of Actinobacteriota, Cyanobacteria and Lachnospiraceae_NK3A20_group (p < 0.10), and g__Lachnospiraceae_NK3A20_group, g_Saccharofermentans, g_Marvinbryantia, g_Bifidobacterium were significantly abundant as specific microflora in the 1000 mg/kg WEAA supplementation group. In conclusion, dietary inclusion of 1000 mg/kg WEAA improved the rumen immune function, antioxidant status, rumen fermentation, and composition of rumen microbes in lambs.

Impact of Forage Sources on Ruminal Bacteriome and Carcass Traits in Hanwoo Steers During the Late Fattening Stages.

This study examined the effects of different forage sources on the ruminal bacteriome, growth performance, and carcass characteristics of Hanwoo steers during the fattening stage. In Korea, where high-concentrate feeding is common, selecting suitable forage is crucial for sustainable beef production. Fifteen 23-month-old Hanwoo steers, weighing an average of 679.27 ± 43.60 kg, were fed the following five different forage sources: oat hay (OAT), rye silage (RYE), Italian ryegrass (IRS), barley forage (BAR), and rice straw silage (RSS), alongside 1.5 kg of dry matter concentrate daily for five months. Carcass traits were evaluated post-slaughter, and rumen fluid samples were analyzed using full-length 16S rRNA gene sequencing to determine the bacteriome composition. The forage source significantly affected the alpha-diversity indices and bacteriome biomarkers linked to the feed efficiency and ruminal fermentation. Differences in the backfat thickness and meat yield index were noted, with alpha-diversity indices correlating with carcass traits. The phylum Planctomycetota, especially the family Thermoguttaceae, was linked to nitrogen fixation in high-protein diets like IRS, while the genus Limimorpha emerged as a biomarker for the meat yield. These findings highlight the importance of forage selection during late fattening to optimize beef production, considering diet and bacteriome shifts.

Preweaning Megasphaera elsdenii Supplementation in Dairy-Beef Calves: Impact on Performance, Behavior, and Rumen Development

This study evaluates the effects of an oral probiotic capsule containing a live culture of M. elsdenii NCIMB 41125 on performance, feeding behavior, rumen pH VFA, and development of dairy-beef crossbred calves. Thirty-one male dairy-beef crossbred calves (Holstein x Angus; mean ± SD; 45.3 ± 7.1 kg; 8.2 ± 2.0 d old) were enrolled in a blinded, 76-d randomized trial. Calves were randomly assigned to one of 3 treatments: placebo, probiotic capsule administration on d 15, or probiotic capsule on d 15 plus a second capsule on d 39 of the study. Calves were housed individually with ad libitum access to water and calf starter and were fed 7L/d of milk replacer (1050g of powder/d) in 2 meals until d 41, then 3.5L/d in 2 meals until weaning on d 56. Behavioral observations were recorded in 1-min intervals using a wall-mounted camera. Rumen fluid samples were collected on d 14, 35, 49, 58, and 70, and analyzed for pH and VFA. Upon euthanasia on d 77, forestomach weights were recorded, and rumen papillae dimensions were measured. Mixed linear models were used for statistical analysis. Probiotic treatment resulted in greater daily solid feed DMI and ADG, particularly during weaning and postweaning periods. Additionally, probiotic treated calves spent more time drinking water and tended to have lower rumen pH compared with control. Empty rumen weight and papillae area were greater in calves supplemented with the probiotic capsule on d 15 compared with the other treatments. These findings suggest that preweaning M. elsdenii supplementation enhances performance and rumen development in dairy-beef crossbred calves. However, the impacts of timing and number of capsule applications on rumen development and calf performance should be further investigated. Further research should also investigate the probiotic's impact on the rumen microbiome and fermentation dynamics throughout the rearing period using detailed microbiome analysis.

New insights into the enteric methane production based on the archaeal genome atlas of ruminant gastrointestinal tract

IntroductionAs one of the important components of ruminant gastrointestinal tract (GIT) microbiome, archaea are involved in many biological processes, especially methanogenesis. In spite of being a well-recognised member of the mammalian gut microbiome, it remains poorly characterized, partly due to the lack of a unified reference genome catalog. ObjectivesThis study aimed to construct a unified genome atlas that captures the wider diversity in archaea and is thus more appropriate for functional and taxonomic exploration of ruminant GIT archaea. MethodsWe collected archaeal genomes from public sources and new data of this study. We performed phylogenetic and functional genomics analysis, prophage identification based on the genomes. Using collected genomes as a reference, we conducted metagenomic and metatranscriptomic analysis on rumen fluid samples from 18 dairy cows, and investigated the correlation between rumen archaeal communities and methane (CH4) production profiles. ResultsWe constructed the ruminant GIT archaeal genomes (RGAG) by compiling 405 strain-level (160 species) non-redundant archaeal genomes from more than 10 ruminant species. Investigating the functional heterogeneity and methanogenic structure within RGAG revealed that it possessed 1,124 (99.5%) unknown microbial biosynthetic gene clusters. A survey of RGAG-borne prophages identified 63 prophages with 122 host-beneficial genes and 18 auxiliary metabolic genes. The pipeline for both metagenomics and metatranscriptomics generated in the study revealed the roles of archaeal genomes under-assessed in general multi-omics analysis. The highly expressed genus Methanosphaera was negatively correlated with CH4 production at the RNA level. ConclusionA unified genome atlas of ruminant GIT archaea is constructed in the study. Our analyses revealed the advantages of metatranscriptomics over metagenomics in studying rumen archaeal communities and further demonstrated that the multifaceted functions of ruminant archaea remain undiscovered. Differences in rumen archaeal community structure among cattle with different CH4 production profiles may reflect the balance between rumen hydrogen production and methanogenesis. Our work provides a new resource for interrogating archaeal functions in the ruminant GIT and potential targets for future CH4 reduction.

PSIX-30 Interaction between rumen microbiome and host feed efficiency phenotype across contrasting breeds and dietary sources

Abstract Provision of feed accounts for a high proportion of the variable cost in beef production systems, and consequently is a major determinant of overall profitability. Thus, improving the conversion of feed to animal product by identifying and breeding cattle with improved feed efficiency potential provides a method through which feed input costs may be reduced, whilst also contributing to more environmentally sustainable beef production. The rumen microbiome dictates the feed degradation capacity and subsequent nutrient supply in ruminants and is thus potentially impacted by feed efficiency phenotype. However, the relationship between rumen microbiota and host feed efficiency phenotype is yet to be fully elucidated. This is further complicated when contrasting diets, cattle breeds as well as various stages of animal development are considered. The objective of this study was to undertake network analysis on rumen microbiome data generated from two contrasting steer breed types (Charolais and Holstein-Friesian), divergent within-breed for residual feed intake (RFI), and offered contrasting diets during different stages of development: i) high-concentrate during the growing phase; ii) grass silage during the growing phase; iii) zero-grazed grass during the growing phase, and iv) high-concentrate during the finishing phase. From approximately 10 mo of age, Charolais (n = 90) and Holstein-Friesian (n = 77) steers were individually fed each of the aforementioned diets for 70 d, following an adaptation period between each diet. At the end of each dietary phase, RFI was determined for every steer and rumen fluid was sampled using a transesophageal sampling device from the 10 most-efficient (Low-RFI) and the 10 least-efficient (High-RFI) steers within each breed. Rumen fluid samples were then subjected to 16S rRNA sequencing and a network-based systems biology analysis subsequently undertaken using PCIT incorporating the 16S sequencing data and individual RFI values across each breed and dietary phase. A total of 110 microbial taxa were connected (P &amp;lt; 0.05; r &amp;gt; 0.8) to the breed and diet contrasts examined; however, no single microbe was commonly connected to all contrasts. The Pyramidobacter genus was the most represented phylotype, displaying a positive connection with the Charolais steers during the grass silage diet, as well as a negative association with Charolais steers during the first high-concentrate diet and Holstein-Friesian steers during the zero-grazed grass diet. Similarly, of the 35 microbial taxa significantly connected to more than one breed/diet contrast, 12 displayed altered direction of association, indicating a differential relationship between the rumen microbiome and RFI phenotype depending on the prevailing dietary management. Acknowledgement: The animal model used in this study was funded by the Irish Department of Agriculture, Food and the Marine (RSF13/S/519). Kate Keogh received funding for this work from the Research Leaders 2025 program (co-funded by Teagasc and the European Union’s Horizon 2020, Marie Skłodowska-Curie grant agreement number 754380).

PSLBII-13 Bioinformatics of rumen fluid from steers fed alfalfa-bermudagrass mixtures in the Deep South

Abstract When harvesting high-quality, high-water content forages like legumes, there is often a greater percentage of nutritive loss due to the drying process. Alternative feeding strategies like baleage provide a way to mitigate these losses. However, there is not much adoption of baleage in the Deep South. Therefore, our objective was to assess the influence of conserved forage type on rumen metabolomes of beef cattle systems in the Deep South. Three treatments including bermudagrass (Cynodon dactylon) hay, alfalfa (Medicago sativa) baleage, and alfalfa-bermudagrass baleage were supplied to four fistulated steers with a calculated daily forage allowance based on intake, body weight, and feed refusals. Each diet was allocated to one of three periods in which all four animals received the same diet due to potential feed spoilage. Each period had a 21-d adaptation period followed by a 5-d collection period to analyze rumen fluid, total fecal weight, feed intake, feed refusal, and total urine. This study integrated 16S RNA sequencing and LC-MS-based metabolomics to evaluate rumen bacterial diversity and metabolome of beef steers. Sequencing data from the rumen fluid samples were then analyzed using QIIME2. Bermudagrass hay had a reduced alpha diversity compared with alfalfa baleage (Shannon: H = 5.19, P = 0.02). Additionally, we saw a change in beta diversity (P &amp;lt; 0.001), measured using the unweighted UniFrac distance, between conserved forage types and distinct clustering in the PCoA analysis. The most abundant taxa in all microbial communities were from the orders Lachnospirales, Bacteroidales, and Oscillospirales. Previous analysis in this study evidenced that baleage had an increased dry matter digestibility (DMD; P = 0.02). The increase in DMD was associated with the increase in diversity of the metabolome which could influence animal health and performance. Thus, baleage can serve as a beneficial conserved forage type that mitigates the losses of high-quality forages and diversifies the gut microbiome of ruminant animals.

PSXII-16 Effect of including sugar cane molasses on rumen fermentation characteristics in young beef finishing heifers

Abstract In ruminant animals, the use of molasses has benefits related to improved voluntary intake and increased production of short-chain fatty acids (SCFA) in the rumen, especially butyrate. Butyrate is closely related to gastrointestinal tract health, as it may contribute to the increase in the absorption area of the ruminal epithelium and improve the utilization of diet components, resulting in improved voluntary intake and immune response capacity in stressful situations, such as weaning. Hence, our objective was to assess the effect of sugar cane molasses on rumen fermentation characteristics in young beef finishing heifers. Newly weaned Nellore × Red Angus heifers (n = 12) averaging 238 ± 5.1 kg body weight (BW), were used. All heifers were housed in individual pens with concrete floors, covered feeders, and drinkers. The basal diet was 50 % concentrate, and consisted of corn silage, sorghum silage, ground corn, soybean meal, urea, and a mineral premix. The diet was provided twice daily at 0700 and 1600 h, allowing up to 10% in orts (as fed). The following treatments were evaluated: control (no addition of powdered molasses) or addition of 50 g/kg of diet [dry matter (DM) basis] of powdered molasses. The experiment lasted for a total of 80 d, including a 14-d adaptation period. On d 68 and 76, rumen fluid samples were collected both before and 4 h after the morning feeding using an oral probe coupled to a vacuum pump. The initial fluid samples were discarded to prevent saliva contamination. The rumen fluid was filtered through four layers of cheesecloth, and then pH was measured. Subsequently, samples were taken and stored in an ultra-freezer (-80°C) for subsequent SCFA analysis. The data were subjected to analysis of variance using the GLM procedure of SAS 9.4 (α = 0.05). Providing molasses increased ruminal pH compared with control heifers (P &amp;lt; 0.032). Adding molasses did not affect SCFA concentration (P &amp;gt; 0.90). Conversely, we detected an effect of molasses on SCFA molar proportion. The molar proportion of both acetate (P &amp;lt; 0.013) and butyrate (P &amp;lt; 0.012) were greater in heifers fed molasses than with controls. On the other hand, dietary molasses addition decreased (P &amp;lt; 0.001). the propionate molar proportion. This resulted in an increased (P &amp;lt; 0.002) acetate-to-propionate ratio in heifers fed diets containing molasses. In conclusion, providing sugar cane molasses enhances rumen fermentation characteristics in young beef finishing heifers.

PSVI-6 Genomic determinants of the rumen microbiome and metabolome in Angus steers

Abstract The rumen microbiome provides approximately 70% of energy required for the ruminant host through fermentation byproducts. Due to microbial impacts on host health and productivity, several studies have associated microbiome and metabolome variation with important sustainability phenotypes, such as feed efficiency and methane emissions. Recent studies have indicated the microbial communities present in rumen fluid are under low-to-moderate host genetic control. However, microorganisms present on the rumen wall have been underrepresented in research, negating their unique role in host-microbiome interactions. Current studies investigating heritability of the rumen planktonic microorganisms are plagued by small sample sizes, high management and breed heterogeneity, and low resolution for classifying microbial communities. To determine the heritability of the rumen fluid and epithelium-associated microbiome and metabolome, Angus steers (n = 350) were enrolled in a 70-d feed efficiency trial, where real-time dry matter intake was measured. Body weights were collected throughout the trial to calculate average daily gain and residual feed intake. Mid-trial, a novel surgery method was used to swab the rumen wall to capture epithelium-associated microorganisms. On d 70, 50 mL of rumen fluid was obtained from steers using orogastric tubing for collection of planktonic microbial communities. Metagenomic DNA extracted from both rumen fluid and epimural samples was used for reduced representation sequencing to predict microbial features such as diversity, composition, and putative function. Genomic DNA isolated from whole blood was used to perform low-pass whole genome sequencing for use in downstream heritability estimates and variant association analyses. Many core microbial abundances are under low-to-moderate host genetic control (h2 &amp;gt; 0.15; P &amp;lt; 0.05); however, many microorganisms had low heritability estimates (h2 &amp;lt; 0.10). Preliminary genome-wide associations studies are vastly underpowered to detect host genetic variants associated with microbial features, but data generated using the total number of animals will better refine these polymorphisms. Several key heritable metabolites were identified in amino acid metabolism pathways in rumen fluid (P &amp;lt; 0.05). The rumen microbiome appears to act as a complex polygenic trait and incorporating microbial features into prediction models may improve feed efficiency estimations. Understanding host genetic control of the rumen microbiome and metabolome may empower downstream applications to enhance genomic and phenotypic predictions for feed efficiency and other sustainability traits in beef cattle.

PSLBI-27 Rumen microbiome dynamics in feedlot steers receiving free-choice garlic-infused mineral supplements

Abstract Garlic (Allium sativum) in its dehydrated or distillate (garlic oil) form has been used as a feed additive owing to its insecticidal, antimicrobial, and organoleptic properties. Commercial mineral mixtures have been formulated to contain a low proportion of dehydrated garlic powder as a strategy to deter pest flies and increase mineral intake in cattle. As an antimicrobial, garlic powder has the potential to alter the rumen microbiome, with possible implications for feed digestibility and the growth performance of beef cattle. This study evaluated the effect of short-term feeding of a garlic-mineral mixture in feedlot steers receiving high-grain diets. Four groups of twenty steers [total = 80, ~14 mo old, body weight (BW) = 599 ± 32 kg] were randomly assigned to either a 5% garlic-mineral (5DGP) or plain mineral supplement (MS). The individual feed and mineral intakes of the steers were measured with an automatic feeding system. Feed and mineral supplements were tracked in different bunks for each group. Baseline rumen fluid samples were collected from all steers at the end of an acclimatization period before administering the trial supplements. Rumen samples were later collected from 10 steers in each group with the greatest mineral supplement intake over the trial period. Total DNA and RNA were extracted from all rumen samples and subjected to metagenomic and metatranscriptomic sequencing, respectively. None of the bacterial species with an overall relative abundance of greater than 0.1% were differentially abundant between the two supplement treatments, nor did the rumen microbial community structures differ between the two groups (PERMANOVA: R2 = 0.02, P = 0.43). Regardless of the dietary supplement, the rumen microbiome of the steers was dominated by uncultured Prevotella spp. (in addition to Prevotella lacticifex) as well as the uncharacterized UBA2810 sp900317945 and UBA2810 sp002351705. Overall, these results show that a 5DGP can be effectively administered to cattle without significantly disrupting the rumen microbiome.

402 The effect of supplemental protein degradability and frequency of supplementation on nitrogen metabolism of beef cattle consuming low-quality forage

Abstract Rumen degradable protein (RDP) is required for cattle consuming low-quality forage to ensure adequate fiber digestion. However, in situations where cattle are not supplemented daily, supplementing RDP could overwhelm the nitrogen recycling mechanisms, leading to increased nitrogen excretion. The objective of this experiment was to determine the effect of rumen degradability and frequency of supplemental protein in beef cattle consuming low-quality forage. Treatments were arranged in a 2 × 2 factorial with protein degradability (68.5% or 32.8% RDP) and supplementation frequency (daily or biweekly) as the two factors. Ruminally cannulated Angus steers [n = 8; body weight (BW) = 328 ± 12.2 kg] were used in a Latin rectangle. The low RDP treatment was composed of 50% soybean hulls and 50% corn gluten meal (40.3% CP; 32.8% RDP), and the high RDP treatment consisted of 45.4% soybean hulls, 41.4% soybean meal, 4.1% urea, and 9.1% molasses (40% CP; 68.5% RDP). The biweekly steers were supplemented on d 0, 4, 7, 11, 14, 18, and 21 of each period. Steers were given ad libitum access to low-quality (7.4% CP) native grass hay. Daily blood samples were collected from d 14 to 17 at 0630 h and d 21 at -2, 3, and 7 h relative to supplementation. Rumen fluid samples were collected every 4 h on d 18 to 21. Daily NEFA concentrations decreased (P &amp;lt; 0.001) post-supplementation in the biweekly supplemented steers and then returned to baseline values, while the daily supplemented steers had no change. Hourly NEFA concentrations in steers supplemented with high RDP biweekly increased from 2 h pre-supplementation to 3 h post-supplementation (P &amp;lt; 0.001), then decreased at 7 h post-supplementation back to pre-supplementation concentrations. Plasma urea nitrogen (PUN) increased in biweekly high RDP supplemented steers 1 d after supplementation (P &amp;lt; 0.001) and decreased from d 15 to d 17. PUN concentrations increased from 2 h pre-supplementation through 7 h post-supplementation in both high RDP treatments, while low RDP treatments did not change (P &amp;lt; 0.001). Rumen fluid lactate and ammonia concentrations rapidly increased in the biweekly high RDP-supplemented steers in response to each supplementation (P &amp;lt; 0.001). There were spikes in ammonia concentrations for the daily high RDP treatment post-supplementation; however, they were smaller in magnitude compared with the biweekly high RDP. The low RDP treatments remained steady across all hours of ammonia sampling, where daily and biweekly supplemented steers decreased slightly in the time points following supplementation and then gradually increased. Rumen pH displayed drastic changes in the biweekly high RDP-supplemented group with values ranging from 6.99 to 5.97 (P &amp;lt; 0.001), but the low RDP treatments remained consistent. Supplementing biweekly with high RDP resulted in fluctuations in metabolites not observed in the other treatments, which could reduce forage utilization and increase nitrogen excretion.

93 Ruminal archaea and bacteria metatranscriptomic responses to supplementation in steers fed low-quality forage

Abstract Forage comprises the majority of beef cattle diets globally, and its quality can vary. Supplementation presents a significant opportunity to improve the efficiency of production. Supplementation has been shown to increase ruminally available nitrogen, improve forage utilization, and reduce livestock methane (CH4) emissions. However, corresponding effects of supplementation on gene expression of rumen microbiota, in particular metabolic pathways involved in methanogenesis, have yet to be studied. This study aimed to evaluate the effect of supplement composition before and after feeding on rumen archaeal and bacterial gene expression in steers consuming a low-quality forage diet. Ruminal samples were collected from five ruminally cannulated Angus steers [body weight (BW) = 375 ± 45 kg] used in a 4 × 4 Latin square fed a basal diet of King Ranch Bluestem Hay (3.5% CP, 73% NDF), and one of four isonitrogenous (130 mg N/kg BW) supplements. Steers provided the two most divergent supplements (2% starch, 43% CP; 56% starch, 21% CP) were used for metatranscriptomic analysis. Rumen fluid samples were collected via rumen cannula 0 and 4 h post-feeding. Total RNA was extracted and sequenced. Differentially expressed genes (DEGs) were identified and functionally annotated. Pathway enrichment analysis was completed to determine enriched pathways (broad metabolic pathways) and modules (specific metabolic reactions). Only main effects are presented in this abstract. Archaeal hydrogenotrophic and methylotrophic methanogenesis reactions and tryptophan biosynthesis reactions were upregulated in the 2% starch supplement (P &amp;lt; 0.05). The 2% starch supplement resulted in fewer bacterial total upregulated carbohydrate-active enzymes compared with the 56% starch supplement (957 vs. 1,695 DEGs, respectively; P &amp;lt; 0.05), and an increased proportion of cellulose and hemicellulose-specific enzymes (35 vs. 29%, respectively; P &amp;lt; 0.05). The 56% starch supplement resulted in upregulation of the bacterial 3-Hydroxypropionate bi-cycle reaction (P &amp;lt; 0.05). Collection time exerted a larger effect than supplement composition on both archaeal and bacterial gene expression, with 83% and 63% of DEGs associated with time, respectively (P &amp;lt; 0.05). Archaeal hydrogenotrophic methanogenesis and carbon metabolism were upregulated 4 h post-feeding. In addition, 1,704 bacterial carbohydrate-active enzymes were upregulated 4 h post-feeding, as compared with 975 upregulated at 0 h (P &amp;lt; 0.05). However, these differences in DEGs did not correlate with significantly enriched pathways of bacterial carbohydrate metabolism. These findings demonstrate improvements to low-quality forage utilization, through CP and starch supplementation, can be correlated to relevant changes in gene expression of rumen microbiota. Furthermore, they demonstrate that supplementation influences the expression of methanogenesis reactions, suggesting an opportunity to affect CH4 production. Additional studies quantifying forage utilization, rumen microbiota gene expression and CH4 production are needed to further optimize supplementation strategies for improved efficiency of beef production whilst maintaining or reducing its carbon footprint.