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ABSTRACT: The nutritional components and quality of milk are influenced by the rumen microbiota and its metabolites at different lactation stages. Hence, rumen fluid and milk samples from 6 dairy cows fed the same diet were collected during peak lactation, early mid-lactation, and later mid-lactation. Untargeted metabolomics and 16S rRNA sequencing were applied for analyzing milk and rumen metabolites, as well as rumen microbial composition, respectively. The levels of lipid-related metabolites, l-glutamate, glucose-1-phosphate, and acetylphosphate in milk exhibited lactation-dependent attenuation. Maltol, N-acetyl-d-glucosamine, and choline, which are associated with milk flavor or coagulation properties, as well as l-valine, lansioside A, clitocine, and ginsenoside La, increased significantly in early mid-lactation and later mid-lactation, especially in later mid-lactation. The obvious increase in rumen microbial diversities (ACE and Shannon indices) were observed in early mid-lactation compared with peak lactation. Twenty-one differential bacterial genera of the rumen were identified, with Succinivibrionaceae_UCG-001, Candidatus Saccharimonas, Fibrobacter, and SP3-e08 being significantly enriched in peak lactation. Rikenellaceae_RC9_gut_group, Eubacterium_ruminantium_group, Lachnospira, Butyrivibrio, Eubacterium_hallii_group, and Schwartzia were most significantly enriched in early mid-lactation. In comparison, only 2 bacteria (unclassified_f__Prevotellaceae and Prevotellaceae_UCG-001) were enriched in later mid-lactation. For rumen metabolites, LysoPE(16:0), l-glutamate, and l-tyrosine had higher levels in peak lactation, whereas PE(17:0/0:0), PE(16:0/0:0), PS(18:1(9Z)/0:0), l-phenylalanine, dulcitol, 2-(methoxymethyl)furan, and 3-phenylpropyl acetate showed higher levels in early mid-lactation and later mid-lactation. Multiomics-integrated analysis revealed that a greater abundance of Fibrobacter contributed to phospholipid content in milk by increasing ruminal acetate, l-glutamate, and LysoPE(16:0). Prevotellaceae_UCG-001 and unclassified_f_Prevotellaceae provide substrates for milk metabolites of the same category by increasing ruminal l-phenylalanine and dulcitol contents. These results demonstrated that milk metabolomic fingerprints and critical functional metabolites during lactation, and the key bacteria in rumen related to them. These findings provide new insights into the development of functional dairy products.

ABSTRACT: Some cellulolytic bacteria cannot transport branched-chain AA (BCAA) and do not express complete synthesis pathways, thus depending on cross-feeding for branched-chain volatile fatty acid (BCVFA) precursors for membrane lipids or for reductive carboxylation to BCAA. Our objective was to assess BCVFA uptake for BCAA synthesis in continuous cultures administered high forage (HF) and low forage (LF) diets without or with corn oil (CO). We hypothesized that BCVFA would be used for BCAA synthesis more in the HF than in LF diets. To help overcome bacterial inhibition by polyunsaturated fatty acids in CO, BCVFA usage for bacterial BCAA synthesis was hypothesized to decrease when CO was added to HF diets. The study was an incomplete block design with 8 dual-flow fermenters used in 4 periods with 8 treatments (n = 4) arranged as a 2 × 2 × 2 factorial. The factors were: HF or LF (67 or 33% forage, 33:67 alfalfa:orchardgrass pellets), without or with supplemental CO (3% of dry matter), and without or with 2.15 mmol/d (5 mg/d 13C) each of isovalerate, isobutyrate, and 2-methylbutyrate for one combined BCVFA treatment. The flow of bacterial BCAA increased by 10.7% by supplementing BCVFA and 9.14% with LF versus HF; similarly, dosing BCVFA versus without BCVFA increased BCAA by 1.98% in total bacterial AA, whereas LF increased BCAA by 1.92% versus HF. Additionally, BCVFA supplementation increased bacterial AA flow by 16.6% when supplemented in HF − CO and 12.4% in LF + CO diets, but not in the HF + CO (−1.5%) or LF − CO (+6.7%) diets (Diet × CO × BCVFA interaction). The recovery of 13C in bacterial AA flow was 31% lower with LF than with HF. Of the total 13C recovered in bacteria, 13.8, 17.3, and 30.2% were recovered in Val, Ile, and Leu, respectively; negligible 13C was recovered in other AA. When fermenters were dosed with BCVFA, nonbacterial and total effluent flows of AA, particularly of alanine and proline, suggest decreased peptidolysis. Increased ruminal outflow of bacterial AA, especially BCAA, but also nonbacterial AA could potentially support postabsorptive responses from BCVFA supplementation to dairy cattle.

ABSTRACT: This research aimed to investigate the effects of replacing soybean meal with high-oil pumpkin seed cake (HOPSC) on ruminal fermentation, lactation performance, milk fatty acid, and ruminal bacterial community in Chinese dairy cows. Six multiparous Chinese Holstein cows at 105.50 ± 5.24 d in milk (mean ± standard deviation) and 36.63 ± 0.74 kg/d of milk yield were randomly allocated, in a 3 × 3 Latin square design, to 3 dietary treatments in which HOPSC replaced soybean meal. Group 1 was the basal diet with no HOPSC (0HOPSC); group 2 was a 50% replacement of soybean meal with HOPSC and dried distillers grains with solubles (DDGS; 50HOPSC), and group 3 was a 100% replacement of soybean meal with HOPSC and DDGS (100HOPSC). We found no difference in the quantity of milk produced or milk composition among the 3 treatment groups. Feed efficiency tended to increase linearly as more HOPSC was consumed. In addition, rumen fermentation was not influenced when soybean meal was replaced with HOPSC and DDGS; the relative abundance of ruminal bacteria at the phylum and genus levels was altered. We also observed that as the level of HOPSC supplementation increased, the relative abundance of Firmicutes and Tenericutes linearly increased, whereas that of Bacteroidetes decreased. However, with increasing HOPSC supplementation, the relative abundance of Ruminococcus decreased linearly at the genus level in the rumen, and the relative abundance of Prevotella showed a linear downward tendency. Changes in dietary composition and rumen bacteria had no significant effect on the fatty acid composition of milk. In conclusion, our results indicated that replacing soybean meal with a combination of HOPSC and DDGS can meet the nutritional needs of high-yielding dairy cows without adversely affecting milk yield and quality; however, the composition of rumen bacteria could be modified. Further study is required to investigate the effects of long-term feeding of HOPSC on rumen fermentation and performance of dairy cows.

Sweet sorghum silage (SS; Sorghum dochna ‘Dochna’) has been extensively studied in recent years as a supplementary forage-to-corn silage (CS; Zea mays L.), but there are still relatively few studies on its effects on the rumen environment of sheep. Determining the short-term impact of converting roughage from corn straws to SS compared to CS on rumen fermentation and bacterial population dynamics was the main goal of the current study. Twelve female thin-tailed Han sheep (29.8 ± 1.34 kg) were randomly divided into one of two treatments: concentrate supplemented with SS or CS, respectively. During the 15-day pretest period, concentrate was fed in two separate feedings at 0800 h and 1800 h, and ensure that the animals were all consumed within an hour of being fed. Thereafter, the animals had free access to corn straw. The feeding procedures during the pretest period were the same as during the measurement period. Rumen fluid was collected via sheep esophageal tube on the last day of adaptation phase (1–7 days) and stabilisation phase (8–30 days), respectively. The results showed that there was a similarity in the total concentration of VFA (volatile fatty acid) and the proportions of acetate, propionate, butyrate, and branched-chain VFA (P > 0.05) and microbial diversity indices (P > 0.05) between the two silage groups throughout the experimental period. The concentration of Ammonia nitrogen (P = 0.001) and proportion of valerate (P = 0.028) decreased in the CS and SS groups, respectively. The abundance and predicted function of rumen bacteria in the SS group did not differ significantly (P > 0.05) between the two measurement phases. However, the abundance of Prevotella_1 (P = 0.038) was higher in the CS group than in the SS group at 7 d. The abundances of Firmicutes (P = 0.005) and Ruminococcaceae_NK4A214_group (P = 0.002) increased, while the abundances of Bacteroidetes (P = 0.044), Proteobacteria (P = 0.046), and Prevotella_1 (P = 0.009) decreased in the CS group at 30 d. Genes related to pyruvate metabolism (P = 0.020) were significantly higher at 30 d than at 7 d, whereas purine metabolism (P = 0.007), pyrimidine metabolism (P = 0.007), and metabolic pathways (P = 0.010) were lower at 30 d in the CS group. In conclusion, this study indicated that SS maintained a steady rumen environment, while CS caused high fluctuations in bacterial abundance and predicted function for sheep.

ABSTRACT: Forty-eight newborn Holstein dairy calves [40 ± 3.4 (SD) of kg of body weight (BW); 24 females and 24 males] were used in a completely randomized design to investigate the effects of a mixture of phytobiotic-rich herbal extract (Immunofin, IMPE) incorporated into milk on performance, ruminal fermentation, bacterial population, and serum biochemical metabolites during the preweaning period. Calves had free access to calf starter and clean water from d 6 until weaning. The treatments were the control (CON; without additive) and IMPE at 4, 8, and 12 mL/d. The treatments had no significant effect on total dry matter intake, weight gain, and BW at weaning. The incidence of diarrhea was lower in calves fed 8 mL of IMPE/d compared with CON. At weaning, body measurements (except for front leg circumference) were not affected by IMPE treatment. Relative to the CON group, front leg circumference was significantly decreased by IMPE supplementation. Serum IgG concentration was not significantly increased by IMPE supplementation compared with the CON group. Triglyceride concentration decreased in calves receiving 4, 8, and 12 mL/d of IMPE compared with the CON groups. In contrast to the CON group, serum albumin and total serum protein concentrations increased with IMPE supplementation. Calves receiving 4 mL/d of IMPE had a greater abundance of total bacteria, Ruminococcus albus, Ruminococcus flavefaciens, and Fibrobacter succinogenes compared with the other treatments. Molar proportions of acetate increased in calves fed IMPE (at 12 mL/d) compared with calves fed CON. Ruminal N-NH3 concentrations decreased linearly with the increase in IMPE supplementation. The results of the present study suggest that the addition of IMPE to milk may improve some health and immunity conditions, blood metabolite concentrations, and increase the abundance of some cellulolytic bacteria in the rumen of Holstein dairy calves. The use of IMPE may be an alternative to feeding antibiotics at subtherapeutic concentrations to improve calf health and immunity status.

A greater understanding of the rumen microbiota and its function may help find new strategies to improve feed efficiency in cattle. This study aimed to investigate whether the cattle breed affects specific ruminal taxonomic microbial groups and functions associated with feed conversion ratio (FCR), using two genetically related Angus breeds as a model. Total RNA was extracted from 24 rumen content samples collected from purebred Black and Red Angus bulls fed the same forage diet and then subjected to metatranscriptomic analysis. Multivariate discriminant analysis (sparse partial least square discriminant analysis (sPLS-DA)) and analysis of composition of microbiomes were conducted to identify microbial signatures characterizing Black and Red Angus cattle. Our analyses revealed relationships among bacterial signatures, host breeds and FCR. Although Black and Red Angus are genetically similar, sPLS-DA detected 25 bacterial species and 10 functions that differentiated the rumen microbial signatures between those two breeds. In Black Angus, we identified bacterial taxa Chitinophaga pinensis, Clostridium stercorarium and microbial functions with large and small subunits ribosomal proteins L16 and S7 exhibiting a higher abundance in the rumen microbiome. In Red Angus, nonetheless, we identified the poorly characterized bacterial taxon Oscillibacter valericigenes with a higher abundance and pathways related to carbohydrate metabolism. Analysis of composition of microbiomes revealed that C. pinensis and C. stercorarium exhibited a higher abundance in Black Angus compared to Red Angus associated with FCR, suggesting that these bacterial species may play a key role in the feed conversion efficiency of forage-fed bulls. This study highlights how the discovery of signatures of bacterial taxa and their functions can be used to harness the full potential of the rumen microbiome in Angus cattle.

Quorum sensing (QS) is a type of microbe-microbe communication system that is widespread among the microbial world, particularly among microorganisms that are symbiotic with plants and animals. Thereby, the cell-cell signalling is likely to occur in an anaerobic rumen environment, which is a complex microbial ecosystem. In this study, using six ruminally fistulated Liuyang black goats as experimental animals, we aimed to detect the activity of quorum sensing autoinducers (AI) both in vivo and in vitro and to clone the luxS gene that encoded autoinducer-2 (AI-2) synthase of microbial samples that were collected from the rumen of goats. Neutral detergent fiber (NDF) and soluble starch were the two types of substrates that were used for in vitro fermentation. The fermented fluid samples were collected at 0, 2, 4, 6, 8, 12, 24, 36, and 48 h of incubation. The acyl-homoserine lactones (AHLs) activity was determined using gas chromatography-mass spectrometer (GC-MS) analysis. However, none of the rumen fluid extracts that were collected from the goat rumen showed the same or similar fragmentation pattern to AHLs standards. Meanwhile, the AI-2 activity, assayed using a Vibrio harveyi BB170 bioassay, was negative in all samples that were collected from the goat rumen and from in vitro fermentation fluids. Our results indicated that the activities of AHLs and AI-2 were not detected in the ruminal contents from six goats and in ruminal fluids obtained from in vitro fermentation at different sampling time-points. However, the homologues of luxS in Prevotella ruminicola were cloned from in vivo and in vitro ruminal fluids. We concluded that AHLs and AI-2 could not be detected in in vivo and in vitro ruminal fluids of goats using the current detection techniques under current dietary conditions. However, the microbes that inhabited the goat rumen had the potential ability to secrete AI-2 signaling molecules and to communicate with each other via AI-2-mediated QS because of the presence of luxS.

Ruminant-derived methane (CH4), a potent greenhouse gas, is a consequence of microbial fermentation in the digestive tract of livestock. Development of mitigation strategies to reduce CH4 emissions from farmed animals is currently the subject of both scientific and environmental interest. Methanogens are the sole producers of ruminant CH4, and therefore CH4 abatement strategies can either target the methanogens themselves or target the other members of the rumen microbial community that produce substrates necessary for methanogenesis. Understanding the relationship that methanogens have with other rumen microbes is crucial when considering CH4 mitigation strategies for ruminant livestock. Genome sequencing of rumen microbes is an important tool to improve our knowledge of the processes that underpin those relationships. Currently, several rumen bacterial and archaeal genome projects are either complete or underway. Genome sequencing is providing information directly applicable to CH4 mitigation strategies based on vaccine and small molecule inhibitor approaches. In addition, genome sequencing is contributing information relevant to other CH4 mitigation strategies. These include the selection and breeding of low CH4-emitting animals through the interpretation of large-scale DNA and RNA sequencing studies and the modification of other microbial groups within the rumen, thereby changing the dynamics of microbial fermentation.

This in vitro study aimed at estimating the disappearance rates of 14 terpenes and terpenoids after 24-h incubation with mixed bacteria from caprine rumens. These compounds comprised nine monoterpene hydrocarbons (δ-3-carene, p-cymene, β-myrcene, (E)- and (Z)-β-ocimene, α-phellandrene, α-terpinene, γ-terpinene and α-terpinolene), four oxygenated monoterpenes ((E)- and (Z)-linalool oxide, 4-terpinenol, α + γ terpineol) and one sesquiterpene hydrocarbon (β-cedrene). They were individually exposed to goat rumen microflora for 24 h in 70 ml culture tubes at an input level of 0.5 ml/l. Terpenoids were the least degraded, 100% of (E)-linalool oxide, 95% of (Z)-linalool oxide, 91% of 4-terpinenol and 75% of terpineol remained intact after 24-h incubation. In contrast, α-terpinolene concentration in fermentation broth extracts was below quantification limit, thus indicating an extensive, if not complete, degradation by rumen bacteria. Only 2% of the initial amounts of α-phellandrene were recovered. The other monoterpenes and β-cedrene were partly degraded, with losses ranging from 67% for δ-3-carene to 90% for (E)-β-ocimene. The corresponding rates of disappearance were between 2.67 and 4.08 μmol/ml inoculum per day.

Previous studies have indicated that 15N enrichment of solid-associated bacteria (SAB) may be predicted from the same value in liquid-associated bacteria (LAB). The aims of this study were to confirm this and to measure the error in the nutrient supply from SAB, when LAB are used as the reference sample. For this purpose, the chemical and amino acid (AA) compositions of both the bacterial populations were studied in four experiments carried out on different groups of three rumen cannulated wethers. Diets (one in Experiments 1 and 4 and three in Experiments 2 and 3) had forage-to-concentrate ratios (dry matter (DM) basis) between 2 : 1 and 40 : 60, and were consumed at intake levels between 40 and 75 g DM/kg (BW)0.75. The bacteria samples were isolated after continuous infusion of (15NH4)2SO4 (40, 18, 30 and 25 mg 15N/day, in Experiments 1 to 4, respectively) for at least 14 days. In all experiments, SAB had consistently higher concentrations of organic matter (826 v. 716 g/kg DM, as average) and total lipids (192 v. 95 g/kg DM, as average) than LAB. Similar CP concentrations of both populations were observed, except a higher concentration in SAB than in LAB in Experiment 3. A consistent (in Experiment 4 only as tendency) higher AA-N/total N ratio (on average 17.5%) was observed in SAB than in LAB. The 15N enrichment in SAB was systematically lower than in LAB. On the basis of the results of all studies a close relationship was found between the 15N enrichment in SAB and LAB, which was shown irrespective of experiments. This relationship was established from Experiments 1 and 2 and the above cited previous results (n = 20; P < 0.001; R2 = 0.996), and then confirmed from the results of Experiments 3 and 4. These relationships between SAB and LAB demonstrate that CP supply from SAB is underevaluated by, on average, 21.2% when LAB are used as the reference. This underevaluation was higher for true protein and even higher for the lipid supply (32.5% and 59.6%, respectively, as an average of the four experiments). Large differences in AA profile were observed between SAB and LAB. The prediction equation obtained using 15N as the marker may be used to correct the errors associated with the traditional use of LAB as the reference sample, and therefore to obtain more accurate estimates of the microbial nutrient supply to the ruminants.

Whether the rumen microbes are able to synthesize and/or degrade long-chain alkanes in anaerobic conditions remains a question to be answered before these hydrocarbons can be confidently used as duodenal flow or rumen transit markers. In this context, an experiment in vitro was carried out to establish whether within a rumen liquor fermentation system, n-alkanes can be derived from de-waxed structures of the plant or from non-alkane wax components (long-chain fatty alcohols, long-chain fatty acids and esters), or may be metabolized by bacteria to other components or to shorter-chain hydrocarbons. Ryegrass was labelled with 14C in growth chambers under controlled conditions in order to use it as a substrate. The labelled material obtained was separated in three fractions: labelled alkanes, labelled de-waxed plant and labelled wax components without the alkanes. These fractions were used for three different incubations in vitro, which objectives were as follows: 1. To check whether rumen bacteria can synthesize alkanes from carbon structures other than waxes (e.g. sugars). 2. To verify whether rumen bacteria can metabolize the n-alkanes to other compounds. 3. To check whether rumen bacteria can synthesize n-alkanes from other carbon compounds from waxes. The results showed that there was neither bacterial synthesis nor metabolism of the n-alkanes in in vitro conditions.

Yeast α-mannan (YM) is a densely branched N-linked glycan that decorates the surface of yeast cell walls. Owing to the high degree of branching, cleavage of the backbone of YM appears to rely on the coupled action of side-chain-cleaving enzymes. Upon examining the genome sequences of bovine-adapted Bacteroides thetaiotaomicron strains, isolated for their ability to degrade YM, we have identified a tandem pair of genes inserted into an orphan pathway predicted to be involved in YM metabolism. Here, we investigated the activity of one of these enzymes, a predicted endo-mannanase from glycoside hydrolase (GH) family 76 (BtGH76-MD40). Purified recombinant BtGH76-MD40 displayed activity on structurally distinct YMs from Saccharomyces cerevisiae and Schizosaccharomyces pombe. Linkage analysis of released oligosaccharide products from S. cerevisiae and S. pombe mannan determined BtGH76-MD40 targets a specific linkage that is conserved in structurally diverse YM substrates. In addition, using two differential derivatization methods, we have shown that there is an absolute requirement for undecorated d-mannopyranose in the -1 subsite. Determination of the BtGH76-MD40 X-ray crystal structure and structural superimposition and molecular docking of a branched alpha-mannopentatose substrate supported these findings. In contrast, BtGH76-MD40 can accommodate extended side chains in the +1 and -2 subsites, highlighting that a single alpha-1,6-mannosyl residue is a prerequisite for activity, and cleavage occurs at the reducing end of the undecorated monosaccharide. Collectively these results demonstrate how acquisition of new enzymes within extant pathways contributes to the functional abilities of saccharolytic bacteria persisting in complex digestive ecosystems., (Copyright © 2020. Published by Elsevier Ltd.)