Integrated multi-omics reveals the relationship between growth performance, rumen microbes and metabolic status of Hu sheep with different residual feed intakes.

Residual feed intake (RFI) is a metric that provides a more accurate measure of feed efficiency. The lower the RFI, the higher the feed efficiency. The changes in the host microbiome and metabolome contribute to the greater feed efficiency of low RFI (LRFI) animals. The aim of this study was to explore the differences in rumen microorganisms, rumen metabolites and plasma metabolites of Hu sheep with differing RFI through the microbiome and metabolome. A total of 80 Hu sheep were used. The experiment consisted of a 15-d pretrial period and a 128-d experimental period. The RFI in the experimental period was calculated for all sheep, and the sheep were screened into high RFI (HRFI, n = 8) and LRFI (n = 8) groups. The HRFI and LRFI sheep did not differ in their initial and final body weights, average daily gain and body measurements, but the dry matter intake of LRFI sheep was significantly decreased (28.4%, P < 0.001). The sheep with LRFI had higher digestibility of crude protein (P = 0.010) and ether extract (P = 0.010) compared to HRFI group. The concentrations of acetate (P = 0.036), propionate (P = 0.010), valerate (P = 0.027) and total volatile fatty acids (P = 0.048) in rumen of LRFI group were higher compared to HRFI group. The results of 16S rDNA sequencing indicated that the sheep with LRFI had higher proportions of Prevotella genus in rumen liquid (P = 0.031). The rumen metabolome and plasma metabolome results showed that the citrate cycle, pyruvate metabolism and alanine, aspartate and glutamate metabolism processes were more active for sheep in LRFI group, which provided more energy substrate such as malic acid, oxoglutaric acid and citric acid. In conclusion, sheep with LRFI can utilize feed more efficiently, and the more active energy metabolism pathway and the production of energy substances may account for the higher feed efficiency.

Tea Polyphenols Inhibit Methanogenesis and Improve Rumen Epithelial Transport in Dairy Cows.

This study systematically investigated the effects of tea polyphenols on methane (CH4) production and the rumen epithelial cell transport capability in cattle using both in vitro and animal experiments, employing multi-omics techniques. The in vitro results demonstrated that, compared to the control group, tea polyphenols significantly reduced CH4 production and the acetate/propionate ratio (p < 0.05). Tea polyphenols reduced CH4 production by inhibiting the relative abundance of unclassified_d_Archaea methanogens and the protozoa Pseudoentodinium and g__Balantioides. The animal experiments showed that tea polyphenols significantly increased the concentrations of T-AOC and GSH-PX in bovine blood (p < 0.05). In addition, microbial groups such as Rikenellaceae_RC9_gut_group, Ruminococcaceae_NK4A214_group, and Butyrivibrio_2 were significantly enriched in the ruminal fluid of the tea polyphenol group (p < 0.05). The proteomic results indicated significant upregulation of proteins such as COIII, S100A8, FABP1, SLC2A8, and SLC29A1 (p < 0.05) and downregulation of proteins including HBB, RAB4A, RBP4, LOC107131172, HBA, and ZFYVE19 (p < 0.05), with FABP1 showing a positive correlation with propionate concentration, and RAB4A had a negative correlation (p < 0.05). Overall, tea polyphenols modulate the microbial composition within the rumen, inhibiting CH4 production and enhancing the host's rumen epithelial cell transport capacity for volatile fatty acids.

Sward type alters enteric methane emissions, nitrogen output and the relative abundance of the rumen microbial ecosystem in sheep.

Observed improvements in animal and sward performance, coupled with a desire for more sustainable pasture-based feeding systems, has triggered a surge in the implementation of more botanically diverse pastures. However, thus far, there has been limited research investigating the effects of botanically diverse sward types on enteric methane (CH4) or nitrogen (N) excretion, alongside the ruminal microbiota and fermentation profile, in sheep. Hence, this study investigates the effect of sward type on CH4 production and N excretion, in addition to assessing the rumen microbiome, volatile fatty acid proportions, and ammonia nitrogen (NH3-N) concentration in sheep. A 5 × 5 Latin square design experiment was implemented to investigate 5 dietary treatments; perennial ryegrass (Lolium perenne L.; PRG) only or PRG plus white clover (Trifolium repens L.; PRG + WC), red clover (Trifolium pratense L.; PRG + RC), chicory (Chicorium intybus L.; PRG + Chic) or plantain (Plantago lanceolata L.; PRG + Plan). Diets were mixed at a ratio of 75% PRG and 25% of the respective companion forage and 100% PRG for the PRG treatment, on a dry matter basis. Twenty castrated male sheep were housed in metabolism crates across 5 feeding periods. Methane measurements were acquired utilizing portable accumulation chambers. Rumen fluid was harvested using a transoesophageal sampling device. Microbial rumen DNA was extracted and subjected to 16S rRNA amplicon sequencing and fermentation analysis. Data were analyzed using PROC MIXED in SAS. Results show that animals consuming PRG + WC ranked lower for CH4 production (g/d) than sheep offered PRG, PRG + Chic or PRG + Plan (P < 0.01) while the addition of any companion forage ranked CH4 yield (g/kg dry matter intake (DMI)) lower (P < 0.001) than PRG. There was a moderate positive correlation between DMI and CH4 (g/d; r = 0.51). Ruminal NH3-N was lowest in animals consuming the PRG diet (P < 0.01). There was a greater abundance of Methanobrevibacter and reduced abundance of Methanosphaera (P < 0.001) in sheep offered PRG, compared with any binary sward. On average, herb diets (PRG + Chic or PRG + Plan) reduced the urinary nitrogen concentration of sheep by 34% in comparison to legume diets (PRG + WC or PRG + RC) and 13% relative to the PRG diet (P < 0.001). Sheep offered PRG + Chic had a greater dietary nitrogen use efficiency than PRG + RC (P < 0.05). This study demonstrates the potential for sward type to influence rumen function and the microbial community, along with CH4 and N output from sheep.

Mitigating greenhouse gas emissions from ruminants fed forage diets will reduce the carbon footprint of livestock production and the agricultural sector globally, thereby improving the overall environmental sustainability of ruminant production. In the current study, sheep housed in metabolism crates were offered 5 differing zero-grazed sward types. Methane production and methane yield from animals offered diets containing white clover ranked 14% and 27% lower, respectively, in comparison to the perennial ryegrass monoculture. The inclusion of herbs (chicory or plantain) led to an average reduction of 13% and 34% in urinary nitrogen concentration when compared to perennial ryegrass or perennial ryegrass and legume (white clover or red clover) treatments, respectively. Results from the current study support the implementation of binary sward mixtures (perennial ryegrass plus white clover, red clover, chicory, or plantain) as a viable strategy for mitigating methane emissions and nitrogen excretion from pasture-based sheep production systems.

Inhibiting methanogenesis by targeting thermodynamics and enzymatic reactions in mixed cultures of rumen microbes in vitro.

Mitigation of enteric methane (CH4) emissions from ruminant livestock represents an opportunity to improve the sustainability, productivity, and profitability of beef and dairy production. Ruminal methanogenesis can be mitigated via two primary strategies: (1) alternative electron acceptors and (2) enzymatic inhibition of methanogenic pathways. The former utilizes the thermodynamic favorability of certain reactions such as nitrate/nitrite reduction to ammonia (NH3) while the latter targets specific enzymes using structural analogs of CH4 and methanogenic cofactors such as bromochloromethane (BCM). In this study, we investigated the effects of four additives and their combinations on CH4 production by rumen microbes in batch culture. Sodium nitrate (NaNO3), sodium sulfate (Na2SO4), and 3-nitro-1-propionate (3NPA) were included as thermodynamic inhibitors, whereas BCM was included as a enzymatic inhibitor. Individual additives were evaluated at three levels of inclusion in experiments 1 and 2. Highest level of each additive was used to determine the combined effect of NaNO3 + Na2SO4 (NS), NS + 3NPA (NSP), and NSP + BCM (NSPB) in experiments 3 and 4. Experimental diets were high, medium, and low forage diets (HF, MF, and LF, respectively) and consisted of alfalfa hay and a concentrate mix formulated to obtain the following forage to concentrate ratios: 70:30, 50:50, and 30:70, respectively. Diets with additives were placed in fermentation culture bottles and incubated in a water bath (39°C) for 6, 12, or 24h. Microbial DNA was extracted for 16S rRNA and ITS gene amplicon sequencing. In experiments 1 and 2, CH4 concentrations in control cultures decreased in the order of LF, MF, and HF diets, whereas in experiments 3 and 4, CH4 was highest in MF diet followed by HF and LF diets. Culture pH and NH3 in the control decreased in the order of HF, MF, to LF as expected. NaNO3 decreased (p < 0.001) CH4 and butyrate and increased acetate and propionate (p < 0.03 and 0.003, respectively). Cultures receiving NaNO3 had an enrichment of microorganisms capable of nitrate and nitrite reduction. 3NPA also decreased CH4 at 6h with no further decrease at 24 h (p < 0.001). BCM significantly inhibited methanogenesis regardless of inclusion levels as well as in the presence of the thermodynamic inhibitors (p < 0.001) while enriching succinate producers and assimilators as well as propionate producers (p adj < 0.05). However, individual inclusion of BCM decreased total short chain fatty acid (SCFA) concentrations (p < 0.002). Inhibition of methanogenesis with BCM individually and in combination with the other additives increased gaseous H2 concentrations (p < 0.001 individually and 0.028 in combination) while decreasing acetate to propionate ratio (p < 0.001). Only the cultures treated with BCM in combination with other additives significantly (padj < 0.05) decreased the abundance of Methanobrevibacter expressed as log fold change. Overall, the combination of thermodynamic and enzymatic inhibitors presented a promising effect on ruminal fermentation in-vitro, inhibiting methanogenesis while optimizing the other fermentation parameters such as pH, NH3, and SCFAs. Here, we provide a proof of concept that the combination of an electron acceptor and a methane analog may be exploited to improve microbial efficiency via methanogenesis inhibition.

High concentrate diets altered the structure and function of rumen microbiome in goats.

This study used metatranscriptomics to investigate the effects of concentrate diet level on rumen microbiome composition and function in goats. A total of 12 healthy 120-day-old Da'er goats were randomly allotted into two treatments: L group (low dietary concentrate level group, concentrate: forage ratio was 25: 75) and H group (high dietary concentrate level group, concentrate: forage ratio was 80: 20). The study included a 10-day pre-feeding period and a 60-day growth experiment. The results showed that compared with the L group, the average daily gain and the slaughter rate in the H group were increased, while the F/G was decreased; the concentration of lactate and ammonia nitrogen, and the proportion of butyrate and valerate in the rumen of the H group were increased, while the proportion of acetate, and the ratio of acetate to propionate were decreased (p < 0.05). Among rumen bacteria, compared with the L group, the H group significantly decreased the relative abundance of Firmicutes and Fibrobacteria at the phylum level, decreased the relative abundance of Bacteroidetes, Fibrobacter, and Sarcina and increased the relative abundance of Clostridium at the genus level, and decreased the relative abundance of Fibrobacter succinogenes, Sarcina sp. DSM 11001, Oscillibacter sp. KLE 1728, and Ruminococcus flavefaciens and increased the relative abundance of Clostridium sp. ND2 and Firmicutes bacteria CAG: 103 at the species level (p < 0.05). Among rumen fungi, the relative abundance of Basidiomycota, Neocallimastigomycota, Mortierella, Mortierella elongata, and Gonapodyna prolifera was lower in the H group than that in the L group (p < 0.05). Functional annotation results showed that the abundance of Glycoside hydrolases genes in rumen microbiome was significantly decreased in the H group compared to the L group (p < 0.05). The result of KEGG DEGs enrichment analysis showed that the gene expression of cellulose 1,4-ß-cellobiosidase, acetyl-CoA hydrolase, lactate dehydrogenase, succinate-semialdehyde dehydrogenase, D-malate dehydrogenase and related genes in methane production pathways of rumen microbiome was decreased in the H group. In summary, feeding high concentrate diets improved the production performance of goats, altered the structure and composition of rumen microbiome and changed the function of rumen microbiome.

Deciphering functional groups of rumen microbiome and their underlying potentially causal relationships in shaping host traits.

Over the years, microbiome research has achieved tremendous advancements driven by culture-independent meta-omics approaches. Despite extensive research, our understanding of the functional roles and causal effects of the microbiome on phenotypes remains limited. In this study, we focused on the rumen metaproteome, combining it with metatranscriptome and metabolome data to accurately identify the active functional distributions of rumen microorganisms and specific functional groups that influence feed efficiency. By integrating host genetics data, we established the potentially causal relationships between microbes-proteins/metabolites-phenotype, and identified specific patterns in which functional groups of rumen microorganisms influence host feed efficiency. We found a causal link between Selenomonas bovis and rumen carbohydrate metabolism, potentially mediated by bacterial chemotaxis and a two-component regulatory system, impacting feed utilization efficiency of dairy cows. Our study on the nutrient utilization functional groups in the rumen of high-feed-efficiency dairy cows, along with the identification of key microbiota functional proteins and their potentially causal relationships, will help move from correlation to causation in rumen microbiome research. This will ultimately enable precise regulation of the rumen microbiota for optimized ruminant production.

Integrated Multi-Omics Reveals New Ruminal Microbial Features Associated with Peanut Vine Efficiency in Dairy Cattle.

The aim of this study was to improve the utilization of peanut vines as forage material for ruminants by investigating the degradation pattern of peanut vines in the dairy cow rumen. Samples of peanut vine incubated in cow rumens were collected at various time points. Bacterial diversity was investigated by scanning electron microscopy (SEM) and 16S rRNA gene sequencing. Carbohydrate-active enzymes (CAZymes) were analyzed by metagenomics. The peanut vines degraded rapidly from 2 to 24 h, before slowing from 24 to 72 h. SEM images confirmed dynamic peanut vine colonization. Firmicutes and Bacteroidetes were the two most dominant bacterial phyla throughout. Principal coordinates analysis indicated significant microbial composition changes at 6 and 24 h. This may be because, in the early stage, soluble carbohydrates that are easily degradable were degraded, while in the later stage, fibrous substances that are difficult to degrade were mainly degraded. Glycoside hydrolases (GHs) were the most abundant CAZymes, with peak relative abundance at 6 h (56.7 trans per million, TPM), and reducing at 24 (55.9 TPM) and 72 h (55.3 TPM). Spearman correlation analysis showed that Alistipes_sp._CAG:435, Alistipes_sp._CAG:514, Bacteroides_sp._CAG:1060, Bacteroides_sp._CAG:545, Bacteroides_sp._CAG:709, Bacteroides_sp._CAG:770, bacterium_F082, bacterium_F083, GH29, GH78, and GH92 were important for plant fiber degradation. These findings provide fundamental knowledge about forage degradation in the cow rumen, and will be important for the targeted improvement of ruminant plant biomass utilization efficiency.

Effects of diets for three growing stages by rumen inocula donors on in vitro rumen fermentation and microbiome.

Hanwoo and Jeju Black cattle (Jeju Black) are native breeds of Korean cattle. Jeju Black cattle are recognized as natural monuments and are known to exhibit slower growth rates compared to Hanwoo. While several studies have analyzed the genetic characteristics of these cattle, there has been limited research on the differences in their microbiome. In this study, rumen fluid was obtained from three Hanwoo steers and three Jeju Black steers, and three different diets (total mixed rations [TMRs] for growing, early fattening, and late fattening periods) were used as substrates for in vitro fermentation. The in vitro incubation was conducted for 3 h and 24 h following a 2 × 3 factorial arrangement. After both incubation periods, fermentation characteristics were analyzed, and ruminal microbiome analysis was performed using 16S rRNA gene sequencing, employing both QIIME2 and PICRUSt2. The results revealed significant differences in the ruminal microbiota due to the inoculum effect. At the phylum level, Patescibacteria and Synergistota were found to be enriched in the Jeju Black inoculum-treated group. Additionally, using different inocula also affected the relative abundance of major taxa, including Ruminococcus, Pseudoramibacter, Ruminococcaceae CAG-352, and the [Eubacterium] ruminantium group. These microbial differences induced by the inoculum may have originated from varying levels of domestication between the two subspecies of donor animals, which mainly influenced the fermentation and microbiome features in the early incubation stages, although this was only partially offset afterward. Furthermore, predicted commission numbers of microbial enzymes, some of which are involved in the biosynthesis of secondary metabolites, fatty acids, and alpha amylase, differed based on the inoculum effect. However, these differences may account for only a small proportion of the overall metabolic pathway. Conversely, diets were found to affect protein biosynthesis and its related metabolism, which showed differential abundance in the growing diet and were potentially linked to the growth-promoting effects in beef cattle during the growing period. In conclusion, this study demonstrated that using different inocula significantly affected in vitro fermentation characteristics and microbiome features, mainly in the early stages of incubation, with some effects persisting up to 24 h of incubation.

Rumen microbiome-driven insight into bile acid metabolism and host metabolic regulation.

Gut microbes play a crucial role in transforming primary bile acids (BAs) into secondary forms, which influence systemic metabolic processes. The rumen, a distinctive and critical microbial habitat in ruminants, boasts a diverse array of microbial species with multifaceted metabolic capabilities. There remains a gap in our understanding of BA metabolism within this ecosystem. Herein, through the analysis of 9371 metagenome-assembled genomes and 329 cultured organisms from the rumen, we identified two enzymes integral to BA metabolism: 3-dehydro-bile acid delta4,6-reductase (baiN) and the bile acid:Na + symporter family (BASS). Both in vitro and in vivo experiments were employed by introducing exogenous BAs. We revealed a transformation of BAs in rumen and found an enzyme cluster, including L-ribulose-5-phosphate 3-epimerase and dihydroorotate dehydrogenase. This cluster, distinct from the previously known BA-inducible operon responsible for 7α-dehydroxylation, suggests a previously unrecognized pathway potentially converting primary BAs into secondary BAs. Moreover, our in vivo experiments indicated that microbial BA administration in the rumen can modulate amino acid and lipid metabolism, with systemic impacts underscored by core secondary BAs and their metabolites. Our study provides insights into the rumen microbiome's role in BA metabolism, revealing a complex microbial pathway for BA biotransformation and its subsequent effect on host metabolic pathways, including those for glucose, amino acids, and lipids. This research not only advances our understanding of microbial BA metabolism but also underscores its wider implications for metabolic regulation, offering opportunities for improving animal and potentially human health.

Potential to reduce methane production of using cultivated seaweeds supplementation to reshape the community structure of rumen microorganisms.

Methane is a short-lived greenhouse gas but has a far greater warming effect than carbon dioxide. At the same time, the livestock sector serves as a large contributor to global emissions of anthropogenic methane. Herein, this work aimed to use cultivated seaweed supplementation to reduce methane emissions and investigate the potential influencing mechanism. To evaluate the feasibility, two cultivated seaweeds, Laminaria japonica Aresch, and Porphyra tenera, along with the enzymatic hydrolysates derived from L. japonica, underwent in vitro trials, and they were both added into corn silage feed (CSF) with different concentrations (1%, 5%, and 10% of CSF) for methane reduction evaluation. The results indicated that >75% and 50% reductions in methane production were observed for the seaweeds and seaweed enzymatic hydrolysates in 9- and 30-day, respectively. Combined high-throughput sequencing and multivariate analysis revealed that supplementation with seaweed and seaweed enzymatic hydrolysates had a notable impact on the prokaryotic community structure. Mantel tests further revealed that significant correlations between the prokaryotic community and methane accumulation (P < 0.05), implying the prokaryotic community plays a role in reducing methane emissions within the rumen. Correspondingly, the networks within the prokaryotic community unveiled the crucial role of propionate/butyrate-producing bacteria in regulating methane emissions through microbial interactions. The predicted function of the prokaryotic community exhibited a significant reduction in the presence of the narB gene in seaweed-supplemented treatments. This reduction may facilitate an increased rate of electron flow toward the nitrate reduction pathway while decreasing the conversion of H2 to methane. These results indicated the supplementation of cultivated seaweeds and the enzymatic hydrolysates has the potential to reshape the community structure of rumen microbial communities, and this alteration appears to be a key factor contributing to their methane production-reduction capability.

Analysis of differences in the rumen microbiome and metabolic function in prepartum dairy cows with different body condition scores.

BACKGROUND: The rumen is a crucial digestive organ for dairy cows. The rumen microbiota assists in the digestion of plant feed through microbe-mediated fermentation, during which the plant feed is transformed into nutrients for the cow's use. Variations in the composition and function of the rumen microbiome affect the energy utilization efficiency of dairy cows, which is one of the reasons for the varying body condition scores (BCSs). This study focused on prepartum Holstein dairy cows to analyze differences in rumen microbiota and metabolites among cows with different BCSs. Twelve prepartum dairy cows were divided into two groups, low BCS (LBCS, BCS = 2.75, n = 6) and high BCS (HBCS, BCS = 3.5, n = 6), to explore differences in microbial composition and metabolites. RESULTS: In the HBCS group, the genera within the phylum Firmicutes exhibited stronger correlations and greater abundances. Phyla such as Firmicutes, Patescibacteria, Acidobacteriota, Euryarchaeota, and Desulfobacterota, in addition to most of their constituent microbial groups, were significantly more abundant in the HBCS group than in the LBCS group. At the genus level, the abundances of Anaerovibrio, Veillonellaceae_UCG_001, Ruminococcus_gauvreauii_group, Blautia, Eubacterium, Prevotellaceae_YAB2003_group, Schwartzia, and Halomonas significantly increased in the HBCS group. The citrate cycle, involved in carbohydrate metabolism, exhibited a significant enrichment trend, with a notable increase in the abundance of its key substrate, citrate, in the HBCS group. This increase was significantly positively correlated with the differential bacterial genera. CONCLUSION: In this study, prepartum dairy cows with higher BCS exhibited greater abundance of Firmicutes. This study provides theoretical support for microbiological research on dairy cows with different BCSs and suggests that regulating the rumen microbiome could help maintain prepartum dairy cows within an optimal BCS range.

In vitro fermentation end-products and rumen microbiome as influenced by microencapsulated phytonutrient pellets (LEDRAGON) supplementation.

The objective of this study was to investigate the effect of microencapsulated bioactive compounds from lemongrass mixed dragon fruit peel pellet (MiEn-LEDRAGON) supplementation on fermentation characteristics, nutrient degradability, methane production, and the microbial diversity using in vitro gas production technique. The study was carried out using a completely randomized design (CRD) with five levels of MiEn-LEDRAGON supplementation at 0, 1, 2, 3, and 4% of the total dry matter (DM) substrate. Supplementation of MiEn-LEDRAGON in the diet at levels of 3 or 4% DM resulted in increased (p < 0.05) cumulative gas production at 96 hours (h) of incubation time, reaching up to 84.842 ml/ 0.5 g DM. Furthermore, supplementation with 3% MiEn-LEDRAGON resulted in higher in vitro nutrient degradability and ammonia-nitrogen concentration at 24 h of the incubation time when compared to the control group (without supplementation) by 5.401% and 11.268%, respectively (p < 0.05). Additionally, supplementation with MiEn-LEDRAGON in the diet led to an increase in the population of Fibrobacter succinogenes at 24 h and Butyrivibrio fibrisolvens at 12 h, while decreasing the population of Ruminococcus albus, Ruminococcus flavefaciens, and Methanobacteriales (p < 0.05). Moreover, supplementation of MiEn-LEDRAGON in the diet at levels of 2 to 4% DM resulted in a higher total volatile fatty acids (VFA) at 24 h, reaching up to 73.021 mmol/L (p < 0.05). Additionally, there was an increased proportion of propionic acid (C3) and butyric acid (C4) at 12 h (p < 0.05). Simultaneously, there was a decrease in the proportion of acetic acid (C2) and the ratio of acetic acid to propionic acid (C2:C3), along with a reduction of methane (CH4) production by 11.694% when comparing to the 0% and 3% MiEn-LEDRAGON supplementation (p < 0.05). In conclusion, this study suggests that supplementing MiEn-LEDRAGON at 3% of total DM substrate could be used as a feed additive rich in phytonutrients for ruminants.

Expression and characterization of a novel microbial GH9 glucanase, IDSGLUC9-4, isolated from sheep rumen.

OBJECTIVE: This study aimed to identify and characterize a novel endo-ß-glucanase, IDSGLUC9-4, from the rumen metatranscriptome of Hu sheep. METHODS: A novel endo-ß-glucanase, IDSGLUC9-4, was heterologously expressed in Escherichia coli and biochemically characterized. The optimal temperature and pH of recombinant IDSGLUC9-4 were determined. Subsequently, substrate specificity of the enzyme was assessed using mixed-linked glucans including barley ß-glucan and Icelandic moss lichenan. Thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), matrix assisted laser desorption ionization time of flight mass spectrometry analyses were conducted to determine the products released from polysaccharides and cello-oligosaccharides substrates. RESULTS: The recombinant IDSGLUC9-4 exhibited temperature and pH optima of 40°C and pH 6.0, respectively. It exclusively hydrolyzed mixed-linked glucans, with significant activity observed for barley ß-glucan (109.59±3.61 µmol/mg min) and Icelandic moss lichenan (35.35±1.55 µmol/mg min). TLC and HPLC analyses revealed that IDSGLUC9-4 primarily released cellobiose, cellotriose, and cellotetraose from polysaccharide substrates. Furthermore, after 48 h of reaction, IDSGLUC9-4 removed most of the glucose, indicating transglycosylation activity alongside its endo-glucanase activity. CONCLUSION: The recombinant IDSGLUC9-4 was a relatively acid-resistant, mesophilic endo-glucanase (EC 3.2.1.4) that hydrolyzed glucan-like substrates, generating predominantly G3 and G4 oligosaccharides, and which appeared to have glycosylation activity. These findings provided insights into the substrate specificity and product profiles of rumen-derived GH9 glucanases and contributed to the expanding knowledge of cellulolytic enzymes and novel herbivore rumen enzymes in general.

Short- and long-term effects of different forage types supplemented in preweaning dairy calves on performance and milk production into first lactation.

We investigated the short- and long-term effects of different forage types supplemented in preweaning dairy calves on growth performance, blood metabolites, rumen fermentation, bacterial community, and milk production during first lactation. A total of 60 healthy 30-d-old female Holstein calves were blocked by birth date and body weight and randomly assigned to 1 of 3 groups (n = 20): normal milk and pelleted starter feeding (CON), supplemented with chopped oat hay (OAH; 75.0 g/d per calf [DM basis]), or alfalfa hay (ALF; 75.0 g/d per calf [DM basis]). The forage supplementation started when calves were 30 d old (d 1 of the experimental period) and ended when they were 73 d old (d 44 of the experimental period, when calves were weaned). Milk and feed intakes and fecal consistency scores were recorded daily. Growth performance, rumen fluid, and blood samples were collected biweekly. After weaning, all the calves were integrated with the same barn and diets. After calving, the milk production was recorded daily. During the experimental period, the OAH group had greater solid feed and total DM intakes and greater rumen pH than the CON group (P ≤ 0.04), but had lower forage intake and CP digestibility than the ALF group (P ≤ 0.04). The ALF group had higher rumen pH and blood BHB concentration (P ≤ 0.04), lower fecal score (P = 0.02), and greater ether extract digestibility (P = 0.02) than the CON group. The ALF and OAH groups had lower concentrations of ruminal total VFA (P = 0.01). Still, the ALF group had a greater proportion of acetate and a relative abundance of cellulose degradation-related bacteria (Lachnoclostridium_1 and Oribacterium) and a lower relative abundance of inflammation-related bacteria (Erysipelotrichaceae_UCG-009) in the rumen compared with CON. Interestingly, the average milk production from 6 to 200 DIM was greater in the ALF group (P < 0.01), even though no significant effects were found on the rumen fermentation parameters and blood metabolites at 200 DIM. Generally, alfalfa hay supplementation in preweaning dairy calves had positive effects in the short- and long-term for rumen development, health status, and future milk production.

Effects of different combinations of antibacterial compound supplements in calf pellets on growth performance, health, blood parameters, and rumen microbiome of dairy calves.

This study investigated the effects of different combinations of antibacterial compounds (attapulgite, plant essential oils, and chitosan oligosaccharides) on growth performance, blood biochemical parameters, and rumen microbiome of calves. A total of 48 preweaning calves were randomly divided into four groups (n = 12 per group), and fed the following full mixed-ration granule diets for the 67-d-feeding trial: (1) basal diet (control group); (2) basal diet +1,000 g/t attapulgite, plant essential oils, and chitosan oligosaccharide (AEOCO group); (3) basal diet +1,000 g/t attapulgite and chitosan oligosaccharide (ACO group); and (4) basal diet +1,000 g/t attapulgite and plant essential oil (AEO group). The results showed that the daily weight gain of the AEOCO and AEO groups significantly increased (p < 0.05), whereas the feed conversion ratio decreased compared with that of the control group. Among the three treatment groups, AEO group showed the most positive effect, with the diarrhea rate reduced by 68.2% compared with that of the control group. Total protein and globulin levels were lower in the AEO group than in the control group. Albumin levels were higher in the AEOCO and AEO groups than in the control group. Immunoglobulin A, immunoglobulin G, and immunoglobulin M concentrations were higher in the AEOCO group (p < 0.05) than in the control group. The interleukin-6 concentration was lower in the AEOCO and AEO groups than in the control group (p < 0.05). The Chao 1 richness and ACE indices were higher in the AEOCO group than in the control group (p < 0.05). The ACO group had a significantly lower (p < 0.05) relative abundance of Firmicutes than the control group. The relative abundance of Bacteroidetes was the lowest in the control group, whereas that of Spirochaetota and Fibrobacteriota was the highest (p < 0.05). The relative abundance of Succiniclasticum was higher in the ACO and AEO groups (p < 0.05). These findings indicate that the combination of attapulgite, plant essential oils, and chitosan oligosaccharides has ameliorative effects on the growth performance, blood parameters, and rumen microbiome of calves.

Structural and functional analysis of the active cow rumen's microbial community provides a catalogue of genes and microbes participating in the deconstruction of cardoon biomass.

BACKGROUND: Ruminal microbial communities enriched on lignocellulosic biomass have shown considerable promise for the discovery of microorganisms and enzymes involved in digesting cell wall compounds, a key bottleneck in the development of second-generation biofuels and bioproducts, enabling a circular bioeconomy. Cardoon (Cynara cardunculus) is a promising inedible energy crop for current and future cellulosic biorefineries and the emerging bioenergy and bioproducts industries. The rumen microbiome can be considered an anaerobic "bioreactor", where the resident microbiota carry out the depolymerization and hydrolysis of plant cell wall polysaccharides (PCWPs) through the catalytic action of fibrolytic enzymes. In this context, the rumen microbiota represents a potential source of microbes and fibrolytic enzymes suitable for biofuel production from feedstocks. In this study, metatranscriptomic and 16S rRNA sequencing were used to profile the microbiome and to investigate the genetic features within the microbial community adherent to the fiber fractions of the rumen content and to the residue of cardoon biomass incubated in the rumen of cannulated cows. RESULTS: The metatranscriptome of the cardoon and rumen fibre-adherent microbial communities were dissected in their functional and taxonomic components. From a functional point of view, transcripts involved in the methanogenesis from CO2 and H2, and from methanol were over-represented in the cardoon-adherent microbial community and were affiliated with the Methanobrevibacter and Methanosphaera of the Euryarchaeota phylum. Transcripts encoding glycoside hydrolases (GHs), carbohydrate-binding modules (CBMs), carbohydrate esterases (CEs), polysaccharide lyases (PLs), and glycoside transferases (GTs) accounted for 1.5% (6,957) of the total RNA coding transcripts and were taxonomically affiliated to major rumen fibrolytic microbes, such as Oscillospiraceae, Fibrobacteraceae, Neocallimastigaceae, Prevotellaceae, Lachnospiraceae, and Treponemataceae. The comparison of the expression profile between cardoon and rumen fiber-adherent microbial communities highlighted that specific fibrolytic enzymes were potentially responsible for the breakdown of cardoon PCWPs, which was driven by specific taxa, mainly Ruminococcus, Treponema, and Neocallimastigaceae. CONCLUSIONS: Analysis of 16S rRNA and metatranscriptomic sequencing data revealed that the cow rumen microbiome harbors a repertoire of new enzymes capable of degrading PCWPs. Our results demonstrate the feasibility of using metatranscriptomics of enriched microbial RNA as a potential approach for accelerating the discovery of novel cellulolytic enzymes that could be harnessed for biotechnology. This research contributes a relevant perspective towards degrading cellulosic biomass and providing an economical route to the production of advanced biofuels and high-value bioproducts.

Associations between the rumen microbiota and carcass merit and meat quality in beef cattle.

The rumen microbiota is important for energy and nutrient acquisition in cattle, and therefore its composition may also affect carcass merit and meat quality attributes. In this study, we examined the associations between archaeal and bacterial taxa in the rumen microbiota of beef cattle and 12 different attributes, including hot carcass weight (HCW), dressing percentage, ribeye area (REA), intramuscular fat content, marbling score, fat thickness, yield grade, moisture content, purge loss, and shear force. There were significant correlations between the relative abundance of certain archaeal and bacterial genera and these attributes. Notably, Selenomonas spp. were positively correlated with live weight and HCW, while also being negatively correlated with purge loss. Members of the Christensenellaceae R-7, Moryella, and Prevotella genera exhibited positive and significant correlations with various attributes, such as dressing percentage and intramuscular fat content. Ruminococcaceae UCG-001 was negatively correlated with live weight, HCW, and dressing percentage, while Acidaminococcus and Succinivibrionaceae UCG-001 were negatively correlated with intramuscular fat content, moisture content, and marbling score. Overall, our findings suggest that specific changes in the rumen microbiota could be a valuable tool to improve beef carcass merit and meat quality attributes. Additional research is required to better understand the relationship between the rumen microbiota and these attributes, with the potential to develop microbiome-targeted strategies for enhancing beef production. KEY POINTS: • Certain rumen bacteria were associated with carcass merit and meat quality • Moryella was positively correlated with intramuscular fat in beef carcasses • Acidaminococcus spp. was negatively correlated with marbling and intramuscular fat.

Linkages between rumen microbiome, host, and environment in yaks, and their implications for understanding animal production and management.

Livestock on the Qinghai-Tibetan Plateau is of great importance for the livelihood of the local inhabitants and the ecosystem of the plateau. The natural, harsh environment has shaped the adaptations of local livestock while providing them with requisite eco-services. Over time, unique genes and metabolic mechanisms (nitrogen and energy) have evolved which enabled the yaks to adapt morphologically and physiologically to the Qinghai-Tibetan Plateau. The rumen microbiota has also co-evolved with the host and contributed to the host's adaptation to the environment. Understanding the complex linkages between the rumen microbiota, the host, and the environment is essential to optimizing the rumen function to meet the growing demands for animal products while minimizing the environmental impact of ruminant production. However, little is known about the mechanisms of host-rumen microbiome-environment linkages and how they ultimately benefit the animal in adapting to the environment. In this review, we pieced together the yak's adaptation to the Qinghai-Tibetan Plateau ecosystem by summarizing the natural selection and nutritional features of yaks and integrating the key aspects of its rumen microbiome with the host metabolic efficiency and homeostasis. We found that this homeostasis results in higher feed digestibility, higher rumen microbial protein production, higher short-chain fatty acid (SCFA) concentrations, and lower methane emissions in yaks when compared with other low-altitude ruminants. The rumen microbiome forms a multi-synergistic relationship among the rumen microbiota services, their communities, genes, and enzymes. The rumen microbial proteins and SCFAs act as precursors that directly impact the milk composition or adipose accumulation, improving the milk or meat quality, resulting in a higher protein and fat content in yak milk and a higher percentage of protein and abundant fatty acids in yak meat when compared to dairy cow or cattle. The hierarchical interactions between the climate, forage, rumen microorganisms, and host genes have reshaped the animal's survival and performance. In this review, an integrating and interactive understanding of the host-rumen microbiome environment was established. The understanding of these concepts is valuable for agriculture and our environment. It also contributes to a better understanding of microbial ecology and evolution in anaerobic ecosystems and the host-environment linkages to improve animal production.

Host and rumen microbiome contributions to feed efficiency traits in Holstein cows.

It is now widely accepted that dairy cow performance is influenced by both the host genome and rumen microbiome composition. The contributions of the genome and the microbiome to the phenotypes of interest are quantified by heritability (h2) and microbiability (m2), respectively. However, if the genome and microbiome are included in the model, then the h2 reflects only the contribution of the direct genetic effects quantified as direct heritability (hd2), and the holobiont effect reflects the joint action of the genome and the microbiome, quantified as the holobiability (ho2). The objectives of this study were to estimate h2, hd2,m2, and ho2 for dry matter intake, milk energy, and residual feed intake; and to evaluate the predictive ability of different models, including genome, microbiome, and their interaction. Data consisted of feed efficiency records, SNP genotype data, and 16S rRNA rumen microbial abundances from 448 mid-lactation Holstein cows from 2 research farms. Three kernel models were fit to each trait: one with only the genomic effect (model G), one with the genomic and microbiome effects (model GM), and one with the genomic, microbiome, and interaction effects (model GMO). The model GMO, or holobiont model, showed the best goodness-of-fit. The hd2 estimates were always 10% to 15% lower than h2 estimates for all traits, suggesting a mediated genetic effect through the rumen microbiome, and m2 estimates were moderate for all traits, and up to 26% for milk energy. The ho2 was greater than the sum of hd2 and m2, suggesting that the genome-by-microbiome interaction had a sizable effect on feed efficiency. Kernel models fitting the rumen microbiome (i.e., models GM and GMO) showed larger predictive correlations and smaller prediction bias than the model G. These findings reveal a moderate contribution of the rumen microbiome to feed efficiency traits in lactating Holstein cows and strongly suggest that the rumen microbiome mediates part of the host genetic effect.

Enhancing the Conventional Culture: the Evaluation of Several Culture Media and Growth Conditions Improves the Isolation of Ruminal Bacteria.

The rumen microbiota is critical in cattle digestion. Still, its low cultivability makes it difficult to study its ecological function and biotechnological potential. To improve the recovery of ruminal microorganisms, this study combined the evaluation of several cultivation parameters with metabarcoding analysis. The parameters tested comprised eight media cultures, three sample dilutions (10-2, 10-6, 10-12), and two incubation times (3 and 7 days). Bacterial populations were determined through Illumina sequencing of 16S rRNA from three biological replicates. The results indicate that none of the culture media recovered all rumen populations and that there was an altered relative abundance of the dominant phyla. In the rumen, Bacteroidetes and Firmicutes comprised 75% and 15% of the relative abundance, respectively, while in the culture media, these were 15% and 60%, respectively. Principal coordinate analysis (PCoA) of the bacterial community revealed significant shifts in population composition due to dilution, with 10-2 and 10-6 dilutions clustered closely while the 10-12 dilution differed markedly. In contrast, incubation duration did not influence population diversity. According to the results, two media, CAN and KNT, were selected based on their ability to recover more similar populations compared to the rumen sample. The metataxonomic study showed that CAN media had consistent reproducibility over time, while KNT showed enrichment of different taxa due to the use of rumen fluid as a substrate. From these, 64 pure cultures were obtained and 54 were identified through 16S rRNA gene sequencing. Being Streptococcus the most frequently isolated genus, this prevalence contrasts with the liquid media composition, underscoring the importance of refining single colony isolation strategies. Although no culture medium could replicate the native rumen bacterial population perfectly, our findings highlight the potential of CAN and KNT media in recovering populations that are more closely aligned to natural rumen conditions. In conclusion, our study emphasizes the importance of integrating molecular approaches in selecting suitable cultivation media and parameters to depict rumen bacteria accurately.