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.

Intake in sugar-rich diets can be limited either via rumen fill or excessive rumen fermentation and source of non-fibre carbohydrate (NFC) in the diet can affect both factors. The aim of the current study was to quantify the effect of partially replacing ground maize (GM) with steam-rolled maize (SRM) or pelleted citrus pulp (PCP) at two concentrate levels in sugarcane-based diets on digestibility, rumen ecosystem and metabolism of Nellore steers. Six rumen-cannulated steers were assigned to a 6 × 6 Latin square, replicated in time, in a 2 × 3 factorial arrangement of treatments with two levels of concentrate (600 or 800 g concentrate/kg dry matter [DM]) and three NFC sources. Each steer within a period was considered an experimental unit. Feeding more concentrate increased total tract digestibility of organic matter and decreased fibre intake and passage rate. It also reduced rumen populations of Fibrobacter succinogenes and Streptococcus bovis and increased Ruminococcus flavefaciens. Substituting PCP for GM increased rumen pH, acetic acid and organic matter digestibility. Feeding PCP also reduced R. flavefaciens and R. amylophilus rumen populations. Substituting SRM for GM increased starch digestibility and rumen propionic acid, but decreased rumen ammonia concentration. Feeding SRM increased rumen populations of Megasphaera elsdenii with the high-concentrate diet but reduced Ruminococcus albus populations at both concentrate levels. In conclusion, partial replacement of GM by PCP decreased intake in sugar-rich diets, while increasing total tract neutral detergent fibre digestibility. Replacement of GM with SRM increases rumen fermentation and total tract digestibility of starch.

The objective of this study was to determine whether a combination of crude glycerin (CG) and soyabean oil (SO) could be used to partially replace maize in the diet of Nellore steers while maintaining optimum feed utilisation. Eight castrated Nellore steers fitted with ruminal and duodenal cannulas were used in a double 4×4 Latin square design balanced for residual effects, in a factorial arrangement (A×B), when factor A corresponded to the provision of SO, and factor B to the provision of CG. Steers feed SO and CG showed similar DM intake, DM, organic matter and neutral-detergent fibre digestibility to that of steers fed diets without oil and without glycerine (P>0·05). Both diets with CG additions reduced the acetate:propionate ratio and increased the proportion of iso-butyrate, butyrate, iso-valerate and valerate (P<0·05). Steers fed diets containing SO had less total N excretion (P<0·001) and showed greater retained N expressed as % N intake (P=0·022). SO and CG diet generated a greater ruminal abundance of Prevotella, Succinivibrio, Ruminococcus, Syntrophococcus and Succiniclasticum. Archaea abundance (P=0·002) and total ciliate protozoa were less in steers fed diets containing SO (P=0·011). CG associated with lipids could be an energy source, which is a useful strategy for the partial replacement of maize in cattle diets, could result in reduced total N excretion and ruminal methanogens without affecting intake and digestibility.

Description of α-linolenic acid (cis-9,cis-12,cis-15-18 : 3, ALA) metabolism in the rumen is incomplete. Ruminal digesta samples were incubated with ALA and buffer containing water or deuterium oxide to investigate the products and mechanisms of ALA biohydrogenation. Geometric Δ9,11,15-18 : 3 isomers were the main intermediates formed from ALA. An increase in the n+1 isotopomers of Δ9,11,15-18 : 3 was due to 2H labelling at C-13. Isomers of Δ9,11,13-18 : 3, cis-7,cis-12,cis-15-18 : 3 and cis-8,cis-12,cis-15-18 : 3 were also formed. No increase in n+1 isotopomers of Δ7,12,15-18 : 3 or Δ8,12,15-18 : 3 was detected. Enrichment in n+2 isotopomers of 18 : 2 products indicated that ALA metabolism continued via the reduction of 18 : 3 intermediates. Isomers of Δ9,11,15-18 : 3 were reduced to Δ11,15-18 : 2 labelled at C-9 and C-13. ALA resulted in the formation of Δ11,13-18 : 2 and Δ12,14-18 : 2 containing multiple 2H labels. Enrichment of the n+3 isotopomer of Δ12,15-18 : 2 was also detected. Metabolism of ALA during incubations with rumen contents occurs by one of three distinct pathways. Formation of Δ9,11,15-18 : 3 appears to be initiated by H abstraction on C-13. Octadecatrienoic intermediates containing cis-12 and cis-15 double bonds are formed without an apparent H exchange with water. Labelling of Δ9,11,13-18 : 3 was inconclusive, suggesting formation by an alternative mechanism. These findings explain the appearance of several bioactive fatty acids in muscle and milk that influence the nutritional value of ruminant-derived foods.

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.

An experiment was carried out to examine the effect of increasing the proportion of Wimmera ryegrass hay in a lucerne hay-based diet on net transfer of nutrients to the intestine, and on the disappearance of n-alkanes in the reticulo-rumen and the hindgut of sheep. Following a latin square design, four adult ewes were fed 1:0, 0·33:0·67, 0·67:0·33 and 0:1 proportions of legume and grass. Increasing the proportion of ryegrass in the diet linearly decreased the intake of DM (P= 0·017), organic matter (P= 0·021) and N (P= 0·001). However, neutral-detergent fibre intake was not affected (P= 0·148), nor was its digestibility coefficient (P>0·10). Diet had no effect on duodenal flows of nutrients (P>0·10), although the proportion of N intake (NI) recovered at the duodenum as non-NH3 N (NAN) increased linearly with Lolium rigidum in the diet (P= 0·002). Full recovery of NI as NAN was achieved at NH3 concentrations in the rumen below 110 g/l. Microbial N contribution to NAN varied in a quadratic manner (P< 0·05) with the proportion of grass in the diet, although efficiency of microbial synthesis was not affected (P>0·10). Duodenal recovery of consumed n-alkanes was not affected by diet and was complete for those present in higher concentrations in the forages. Isolated rumen bacteria contained significant amounts of n-alkanes, contributing to the duodenal flow of these hydrocarbons in variable proportions depending on the diet consumed.

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.

Ruminal bacterial counts and in vivo digestibility were determined on four Mediterranean buffalo bulls and four Friesian bulls, all fistulated at the rumen, and given at maintenance level (50 g/kg M0·75 per day of dry matter) four different diets with the same crude protein content (N ✕ 6·25 = 140 g/kg dry matter) and with forage: concentrate ratios as follows: diet D12·5 = 0·875: 0·125; diet D25·0 = 0·75: 0·25; diet D37·5 = 0·625: 0·375; diet D50·0 = 0·5: 0·5. All the animals received the diets during four consecutive periods in a Latin-square design. Buffaloes had higher total microbial counts (10·78 v. 10·08 log10 cells per g dry rumen content, P < 0·01) as compared with cattle; differences in total ruminal bacterial counts among the diets were only observed within the buffalo species (diet D12·5 v. diets D25·0, D37·5, D50·0: 10·04 v. 10·92, 10·98, 11·17 log10 cells per g dry rumen content, P 0·01) and when comparing the two species for each diet, significantly higher values for bacterial counts in buffaloes were found for diets D25·0: 10·92 v. 10·28 (P 0·05), D37·5: 10·98 v. 10·08 (P 0·01) and D50·0: 11·17 v. 9·76 (P 0·01) log10 cells per g dry rumen content. Cattle showed significantly higher digestibility values for: organic matter (0·696 v. 0·676, P 0·05), neutral-detergent fibre (NDF; 0·548 v. 0·511, P 0·05) and cellulose (0·621 v. 0 · 509, P 0·01), while the crude protein digestibility (CPD) values were similar (0·667 and 0·671). Comparing the two species for each diet, cattle showed significantly higher digestibility values for organic matter in diet D50·0 only (0·714 v. 0·688, P 0·01), for NDF in diet D12·5 only (0·578 v. 0·531, P 0·05) and for cellulose in all diets (0·660 v. 0·546, 0·630 v. 0·525, 0·605 v. 0·505, 0·588 v. 0·460, P 0·01); in contrast buffaloes showed higher values of the CPD for diet D12·5 (0·662 v. 0·632, P 0·05).

A study was conducted to determine the effect of the feed intake on the chemical composition of bacteria associated with the solid (solid-associated bacteria; SAB) and liquid (liquid-associated bacteria; LAB) fractions of rumen digesta, the digestive passage kinetics and their relationships. Whole rumen contents were sampled after a period of continuous infusion of 15NH3 from four ruminally-cannulated wethers provided successively with a hay–concentrate diet (2: 1 w/w on a DM basis) at two rates of feed intake: 40 and 80 g DM/kg body weight0·75. SAB had a higher content of organic matter and total lipids (P < 0·001) and a similar N content as compared with LAB. The concentration of purines and 15N was lower (P = 0·011 and P < 0·001 respectively) in SAB than LAB, whereas the opposite was observed for the concentration of amino acids (mg/g DM; P = 0·031). An increase in feed intake produced an increase in the N (P = 0·034) and purine (P = 0·066) concentrations in bacteria and a decrease (P = 0·033) in their amino acid concentrations. Significant increases of rumen outflow rates of liquid and particles were also observed with increased feed intake. Rates of rumen outflow showed positive and negative linear relationships (P < 0·001) with the purine: N ratio and the proportion of amino acid on total N of bacteria respectively. SAB contained significantly higher proportions of leucine, isoleucine, lysine and phenylalanine and lower proportions of alanine, methionine and valine than LAB. The increase in feed intake also induced significant changes in the amino acid profile of bacteria, increasing arginine and methionine and decreasing alanine and glycine proportions. Results show that the outflow rate of rumen contents is a major factor in determining the proportion of nucleic acids and protein in rumen bacteria and explains some of the differences observed between LAB and SAB.

Four ewes fitted with ruminal and duodenal T-piece cannulae were each given six diets in a 6 × 4 factorial design. Diets or experimental treatments consisted of two ratios of forage: concentrate (700:150 (LC) and 400: 600 (HO). Forage was ammonia-treated straw and the concentrate was formulated with barley supplemented with one of three protein sources: sunflower meal, soya-bean meal or fish meal. Duodenal flows ofdigesta were estimated by the dual-phase technique using Co-EDTA and Yb acetate as liquid and solid markers. Microbial nitrogen (N) was estimated from the digesta flow of purine bases and 15N enrichment using as reference samples, bacterial isolates from the liquid (LAB) or solid (SAB) phase of rumen digesta.

Duodenal flow of purine bases (mmol/day) was lower on LC (12·9) than HC (17·7) diets but in both treatments it was depressed by fish meal (12·3) compared with either soya-bean (17·3) or sunflower meal (16·3) as supplements (s.e. 1·13). Urinary excretion of purine derivatives showed a similar trend, 8·6 v. III mmol/day in LC and HC respectively and 8·8 v. 10·4 and 10·5 mmol/day in fish meal, soya-bean and sunflower meal diets (s.e. 0·56), respectively. Variation in excretion of urinary purine derivatives was mainly associated with digestible organic matter intake with an average ratio of 1·7 (s.e. 0·11) mmol per 100 g digestible organic matter intake. Irrespective of the microbial marker used, microbial yield was higher in animals offered HC than in those offered LC and with soya-bean or sunflower meal compared with fish meal supplemented diets. The microbial purine bases/N (mmol/g) ratio varied between LAB (1·99, s.e. 0·092) and SAB (1·69, s.e. 0·071) isolates leading to different estimates of microbial-N yield (g) from duodenal purine bases (7·76 (s.e. 2·84) v. 9·13 (s.e. 3·24)), urinary excretion of allantoin (5·57 (s.e. 2·0) v. 6·57 (s.e. 2·03)) or total purine derivatives (6·43 (s.e. 2·39) v. 7·56 (s.e. 2·77)). Urinary excretion of allantoin or total purine derivatives provided consistently lower estimates of duodenal microbial-N than duodenal purine bases or 15N, although it closely reflected the pattern observed in direct measurements.

The effect of energy and nitrogen (N) supply pattern on rumen bacterial growth was investigated in vitro. In experiment 1, glucose was was fed to batch cultures of mixed rumen bacteria according to three patterns namely a pulse dose at time zero (P); even increments at 0·5-h intervals (G) or an intermediate pattern (I), whilst N was supplied in excess. In experiment 2, glucose and N (not in excess) were fed to batch cultures according to four patterns namely glucose and N as pulse doses at time zero, (EPNP); glucose as a pulse dose at time zero and N in 24 even increments at 0·5-h intervals (EPNG); glucose in 24 even increments at 0·5-h intervals and N as a pulse dose at time zero (EGNP) or both glucose and N in 24 even increments at 0·5-h intervals (EGNG). Fermentaton was studied over a 12-h period for both experiments.

In experiment 1, bacterial growth efficiency and specific growth rate (39·8,35·5 and 29·9 (g bacterial dry matter (DM) per mol glucose utilized) and 0·33, 0·27 and 0·20 (fraction per h) for treatments P, I, and G respectively) differed significantly between glucose supply patterns. In experiment 2, bacterial growth efficiency and specific growth rate (33·8, 34·7, 25·9 and 22·5 (g baterial DM per mol glucose) and 0·21, 0·18, 0·14 and 0·13 (fraction per h) for treatments EPNP, EPNG, EGNP and EGNG respectively) differed significantly only between glucose supply patterns.

It is concluded that the pattern according to which a given amount of energy becomes available affects bacterial growth efficiency, with the fastest supply rate giving the highest efficiency and that, within accepted levels of N supply, synchronization between energy and N availability may be of less importance to bacterial growth efficiency than the energy supply pattern.

The lipid distribution and fatty acid (FA) composition of total lipids, polar lipids and free fatty acids (FFA) were determined in liquid-associated bacteria (LAB) and solid-adherent bacteria (SAB) isolated from the rumen contents of seven dairy cows fitted with rumen fistulas. Two experiments, arranged according to a 4 x 4 and 3 x 3 Latin Square design, were performed using two basal diets consisting of one part hay and one part barley-based concentrate, and five lipid-supplemented diets consisting of the basal diet plus (g/kg dry matter): 53 or 94 rapeseed oil, 98 tallow, 87 soya-bean oil or 94 palmitostearin. For all diets used, total lipids were 1.7–2.2 times higher in SAB than in LAB (P < 005); this probably resulted from a preferential incorporation of dietary FA adsorbed onto food particles. Addition of oil or fat to the diets did not modify the polar lipid content but increased the FFA content of SAB and LAB by 150%. Lipid droplets were observed in the cytoplasm in SAB and LAB using transmission electron microscopy, which suggested that part of the additional FFA was really incorporated into the intracellular FFA rather than associated with the cell envelope by physical adsorption. Linoleic acid was specifically incorporated into the FFA of SAB, which emphasized the specific role of this bacterial compartment in the protection of this acid against rumen biohydrogenation.