Review: Problems in determining metabolisable protein value of dairy cow diets and the impact on protein feeding.

Accurate determination of dairy cow's metabolisable protein (MP) requirements is essential for maximising milk yield and minimising N input in dairy production systems. The main objectives of feed evaluation systems are to predict accurately the nutritive value of feed resources and production responses to ingested nutrients. In recent years, protein evaluation systems have made progress in complexity but our analysis indicated that in comparison to simple MP models, or even models based on CP and metabolisable energy intake, new models have failed to improve milk protein yield predictions. Previous meta-analyses have indicated low marginal efficiencies for incremental CP intake even with high-quality feeds as protein supplements. Most current protein systems tend to overestimate MP supply from rumen undegradable protein and overlook the effects on rumen microbial protein synthesis. These findings suggest that there is scope for improving predictions of MP supply. This review highlights and discusses some evident problems in the current MP systems that may partly explain the modest progress achieved over the last few decades.

In Vitro Incubations Do Not Reflect In Vivo Differences Based on Ranking of Low and High Methane Emitters in Dairy Cows.

This study evaluated if ranking dairy cows as low and high CH4 emitters using the GreenFeed system (GF) can be replicated in in vitro conditions using an automated gas system and its possible implications in terms of fermentation balance. Seven pairs of low and high emitters fed the same diet were selected on the basis of residual CH4 production, and rumen fluid taken from each pair incubated separately in the in vitro gas production system. In total, seven in vitro incubations were performed with inoculums taken from low and high CH4 emitting cows incubated in two substrates differing in forage-to-concentrate proportion, each without or with the addition of cashew nutshell liquid (CNSL) as an inhibitor of CH4 production. Except for the aimed differences in CH4 production, no statistical differences were detected among groups of low and high emitters either in in vivo animal performance or rumen fermentation profile prior to the in vitro incubations. The effect of in vivo ranking was poorly replicated in in vitro conditions after 48 h of anaerobic fermentation. Instead, the effects of diet and CNSL were more consistent. The inclusion of 50% barley in the diet (SB) increased both asymptotic gas production by 17.3% and predicted in vivo CH4 by 26.2%, when compared to 100% grass silage (S) substrate, respectively. The SB diet produced on average more propionate (+28 mmol/mol) and consequently less acetate compared to the S diet. Irrespective of CH4 emitter group, CNSL decreased predicted in vivo CH4 (26.7 vs. 11.1 mL/ g of dry matter; DM) and stoichiometric CH4 (CH4VFA; 304 vs. 235 moles/mol VFA), with these being also reflected in decreased total gas production per unit of volatile fatty acids (VFA). Microbial structure was assessed on rumen fluid sampled prior to in vitro incubation, by sequencing of the V4 region of 16S rRNA gene. Principal coordinate analysis (PCoA) on operational taxonomic unit (OTU) did not show any differences between groups. Some differences appeared of relative abundance between groups in some specific OTUs mainly related to Prevotella. Genus Methanobrevibacter represented 93.7 ± 3.33% of the archaeal sequences. There were no clear differences between groups in relative abundance of Methanobrevibacter.

Residual carbon dioxide as an index of feed efficiency in lactating dairy cows.

High feed costs make feed conversion efficiency a desirable target for genetic improvement. Residual feed intake (RFI), calculated as the difference between observed and predicted intake, is a commonly used estimate of feed efficiency. However, determination of feed efficiency in dairy herds is challenging due to difficulties in measuring feed intake of individual animals reliably. Using residual CO2 (RCO2) production as an estimate of feed efficiency would allow ranking the cows according to feed efficiency, provided that CO2 production is closely related to heat production and feed intake. The objective of this study was to evaluate the potential of RCO2 as an index of feed efficiency using data from respiration calorimetry studies (289 cow per period observations). Heat production was precisely predicted from CO2 production [root mean square error (RMSE)] adjusted for random effects was 1.5% of observed mean]. Dry matter intake (DMI) was better predicted from energy-corrected milk (ECM) yield and CO2 production than from ECM yield and body weight in the model (adjusted RSME = 0.92 vs. 1.39 kg/d). Residual CO2 production estimated as the difference between actual CO2 production and that predicted from ECM yield, metabolic body weight was closely related to RFI (adjusted RMSE = 0.42) that was calculated as the difference between actual DMI and that predicted from ECM, metabolic body weight, and energy balance (EB). When the cows were categorized in 3 groups of equal sizes on the basis of RCO2 (low, medium, and high), low RCO2 cows had lower DMI, RFI, methane production and intensity (g/kg ECM), and heat production, but higher efficiency of metabolizable energy utilization for lactation than high RCO2 cows. When RFI was predicted from RCO2, the residuals (observed - predicted) were negatively related to EB and digestibility. Predicting RFI with a 2-variable model based on RCO2 and digestibility, adjusted RMSE decreased to 0.23 kg/d, and residuals were not significantly related to EB. The cows in low RCO2 group had a higher energy digestibility than the cows in the high RCO2 group, and differences in EB were observed between the groups. Error of the model predicting residual ECM production from RCO2 was 1.41 kg/d. The residuals were positively related to ECM yield and energy digestibility. Predicting residual ECM from RCO2 and ECM yield decreased adjusted RMSE to 1.07 kg/d, and further to 0.78 kg/d when digestibility was included in the 2-variable model. It is concluded that RCO2 has a potential for ranking individual cows based on feed efficiency.

Comparison of Methods to Measure Methane for Use in Genetic Evaluation of Dairy Cattle.

Partners in Expert Working Group WG2 of the COST Action METHAGENE have used several methods for measuring methane output by individual dairy cattle under various environmental conditions. Methods included respiration chambers, the sulphur hexafluoride (SF6) tracer technique, breath sampling during milking or feeding, the GreenFeed system, and the laser methane detector. The aim of the current study was to review and compare the suitability of methods for large-scale measurements of methane output by individual animals, which may be combined with other databases for genetic evaluations. Accuracy, precision and correlation between methods were assessed. Accuracy and precision are important, but data from different sources can be weighted or adjusted when combined if they are suitably correlated with the 'true' value. All methods showed high correlations with respiration chambers. Comparisons among alternative methods generally had lower correlations than comparisons with respiration chambers, despite higher numbers of animals and in most cases simultaneous repeated measures per cow per method. Lower correlations could be due to increased variability and imprecision of alternative methods, or maybe different aspects of methane emission are captured using different methods. Results confirm that there is sufficient correlation between methods for measurements from all methods to be combined for international genetic studies and provide a much-needed framework for comparing genetic correlations between methods should these become available.

A heritable subset of the core rumen microbiome dictates dairy cow productivity and emissions.

A 1000-cow study across four European countries was undertaken to understand to what extent ruminant microbiomes can be controlled by the host animal and to identify characteristics of the host rumen microbiome axis that determine productivity and methane emissions. A core rumen microbiome, phylogenetically linked and with a preserved hierarchical structure, was identified. A 39-member subset of the core formed hubs in co-occurrence networks linking microbiome structure to host genetics and phenotype (methane emissions, rumen and blood metabolites, and milk production efficiency). These phenotypes can be predicted from the core microbiome using machine learning algorithms. The heritable core microbes, therefore, present primary targets for rumen manipulation toward sustainable and environmentally friendly agriculture.

An in vitro evaluation of browser and grazer fermentation efficiency and microbiota using European moose spring and summer foods.

Evolutionary morphological and physiological differences between browsers and grazers contribute to species-specific digestion efficiency of food resources. Rumen microbial community structure of browsers is supposedly adapted to characteristic nutrient composition of the diet source. If this assumption is correct, domesticated ruminants, or grazers, are poor model animals for assessing the nutritional value of food consumed by browsing game species. In this study, typical spring and summer foods of the European moose (Alces alces) were combined with rumen fluid collected from both dairy cows (Bos taurus) and from moose, with the aim of comparing fermentation efficiency and microbial community composition. The nutritional value of the food resources was characterized by chemical analysis and advanced in vitro measurements. The study also addressed whether or not feed evaluation based on in vitro techniques with cattle rumen fluid as inoculum could be a practical alternative when evaluating the nutritional value of plants consumed by wild browsers. Our results suggest that the fermentation characteristics of moose spring and summer food are partly host-specific and related to the contribution of the bacterial phyla Firmicutes and Bacteriodetes to the rumen microbial community. Host-specific adaptations of the ruminal microbial community structure could be explained from the evolutionary adaptations related to feeding habitats and morphophysiological differences between browsers and grazers. However, the observed overall differences in microbial community structure could not be related to ruminal digestion parameters measured in vitro. The in vitro evaluation of digestion efficiency reveals that equal amounts of methane were produced across all feed samples regardless of whether the ruminal fluid was from moose or dairy cow. The results of this study suggested that the nutritional value of browsers' spring and summer food can be predicted using rumen fluid from domesticated grazers as inoculum in in vitro assessments of extent of digestion when excluding samples of the white water lily root, but not of fermentation characteristics as indicated by the proportions of individual fermentation fatty acids to the total of volatile fatty acids.

Effects of contrasting concentrate feeding strategies on meat quality of growing and finishing dairy bulls offered grass silage and barley based diets.

The objective was to examine the effects of concentrate level (barley grain 39 and 74 g dry matter/kg0.60 live weight) and allocation regime (steady, increased, decreased) on meat quality of growing dairy bulls fed grass silage ad libitum. Chemical, instrumental and sensory analyses were used for measuring quality of longissimus lumborum (LL). Greater concentrate level increased fat content (P = 0.035) and tenderness of sensory analysis (P = 0.009) of LL but did not affect pH, colour, drip loss, sarcomere length, shear force, juiciness or flavour. Periodic concentrate allocation reduced drip loss (P = 0.046) and tenderness (P = 0.001) compared to steady feeding. Observed effects on meat quality were minor and one explanation for this might be low carcass and meat fat content in all treatments. The experiment demonstrated the ability of growing bulls to adapt to different feeding regimes without major effects on meat quality, but simultaneously highlighted the challenge to affect beef quality by practically feasible diets.

Prediction of enteric methane production, yield, and intensity in dairy cattle using an intercontinental database.

Enteric methane (CH4 ) production from cattle contributes to global greenhouse gas emissions. Measurement of enteric CH4 is complex, expensive, and impractical at large scales; therefore, models are commonly used to predict CH4 production. However, building robust prediction models requires extensive data from animals under different management systems worldwide. The objectives of this study were to (1) collate a global database of enteric CH4 production from individual lactating dairy cattle; (2) determine the availability of key variables for predicting enteric CH4 production (g/day per cow), yield [g/kg dry matter intake (DMI)], and intensity (g/kg energy corrected milk) and their respective relationships; (3) develop intercontinental and regional models and cross-validate their performance; and (4) assess the trade-off between availability of on-farm inputs and CH4 prediction accuracy. The intercontinental database covered Europe (EU), the United States (US), and Australia (AU). A sequential approach was taken by incrementally adding key variables to develop models with increasing complexity. Methane emissions were predicted by fitting linear mixed models. Within model categories, an intercontinental model with the most available independent variables performed best with root mean square prediction error (RMSPE) as a percentage of mean observed value of 16.6%, 14.7%, and 19.8% for intercontinental, EU, and United States regions, respectively. Less complex models requiring only DMI had predictive ability comparable to complex models. Enteric CH4 production, yield, and intensity prediction models developed on an intercontinental basis had similar performance across regions, however, intercepts and slopes were different with implications for prediction. Revised CH4 emission conversion factors for specific regions are required to improve CH4 production estimates in national inventories. In conclusion, information on DMI is required for good prediction, and other factors such as dietary neutral detergent fiber (NDF) concentration, improve the prediction. For enteric CH4 yield and intensity prediction, information on milk yield and composition is required for better estimation.

Evaluation of a gas in vitro system for predicting methane production in vivo.

Methane production from ruminant livestock varies with the diet as a result of factors such as dry matter intake, diet composition, and digestibility. To estimate the effect of dietary composition and feed additives, CH4 production can be measured in vitro as a first step because large numbers of samples can be incubated and analyzed at the same time. This study evaluated a recently developed in vitro method for prediction of in vivo CH4 production by examining the relationship between predicted and observed CH4 production values. A total of 49 different diets (observations), used in previous 13 in vivo studies, were selected to include diets varying in nutrient composition. Methane production was measured in all in vivo studies by respiration chambers or the GreenFeed system (C-Lock Inc., Rapid City, SD). Overall, the in vitro system predicted CH4 production well (R2 = 0.96), but the values obtained were slightly underestimated compared with observed in vivo values (mean 399 L/d compared with 418 L/d: root mean square prediction error = 51.6 L/d or 12.3% of observed mean). Further analysis of the effect on residuals showed no significant relationship between CH4 production and most factors known to affect CH4 production such as dry matter intake, digestibility, and dietary concentrations of fat and starch. However, some factors included in the model were not well predicted by the system, with residuals negatively related to neutral detergent fiber concentration and positively related to concentrate proportion. The in vitro system can thus be useful for screening diets and evaluation of feed additives as a first step that can be best interpreted when feeding cows at maintenance level.

Short-term effects of soybean oil supplementation on performance, digestion, and metabolism in dairy cows fed sugarcane-based diets.

We aimed to quantify the productive and metabolic responses, and digestive changes in dairy cows fed various concentrations of soybean oil (SBO) in high-concentrate, sugarcane-based diets. Eight rumen-cannulated multiparous Holstein cows in mid lactation (574 ± 19.1 kg of body weight and 122 ± 6.9 d in milk), averaging 22.5 ± 1.22 kg/d of milk were assigned to replicated 4 × 4 Latin squares. The experimental period lasted 21 d as follows: 14 d for adaptation, followed by a sampling period from d 15 to 21. The diets were formulated with increasing concentrations of SBO [% of dry matter (DM)]: control (0%), low (LSBO; 1.57%), medium (MSBO; 4.43%), and high (HSBO; 7.34%). Dry matter intake decreased quadratically in response to SBO addition. The greatest decrease in DM intake was observed in MSBO and HSBO diets. Both milk and energy-corrected milk yield were quadratically affected by the SBO inclusion, with a slight decrease up to MSBO and substantial decrease in the HSBO diet. The milk fat concentration linearly decreased from 3.78% in the control to 3.50% in the HSBO diet. The potentially digestible neutral detergent fiber digestibility in the rumen decreased from 55.7% in the control to 35.2% in the HSBO diet. The fractional rate of digestion of potentially digestible neutral detergent fiber in the rumen decreased linearly from 3.13 to 1.39%/h from the control to HSBO diet. The fractional rate of indigestible neutral detergent fiber passage in the rumen was quadratically affected, with the lowest value (2.25%/h) for the HSBO diet. Rumen pH increased from 6.42 to 6.67, and ammonia nitrogen decreased from 28.1 to 21.4 mg/dL, in the control and HSBO diets, respectively. Rumen volatile fatty acids decreased quadratically, with the greatest decrease observed in MSBO and HSBO diets. Serum concentrations of glucose, fatty acids, and ß-hydroxybutyrate were unaffected by SBO inclusion. However, serum concentrations of total cholesterol and high- and low-density lipoproteins linearly increased with increasing concentrations of SBO in the diet. Inclusion of SBO at concentrations from 4.43 to 7.34% of the diet DM decreased DM intake, energy-corrected milk production, fiber digestibility, and rumen fermentation and was thus not recommended. Soybean oil supplementation at 1.57% of the diet DM proved to be a safe concentration for dairy cows fed high-concentrate diets with sugarcane as the sole forage.

Compartmental flux and in situ methods underestimate total feed nitrogen as judged by the omasal sampling method due to ignoring soluble feed nitrogen flow.

The objective of the present study was to estimate ruminal feed N outflow in lactating cows using the omasal sampling, compartmental flux or in situ method. A total of five ruminally fistulated Finnish Ayrshire dairy cows were used in a 5 × 5 Latin square study with 21 d periods. Experimental silages of grass or red clover harvested at two stages of maturity in addition to a supplement of 9·0 kg concentrate/d were fed to the cows. In vivo omasal N flow was determined using the omasal sampling technique. Ruminal in situ N flow was calculated from N intake and degradability (38 µm nylon bags). The samples of ruminal contents and faeces were divided into seven particle-size fractions by wet sieving; the concentrations of indigestible neutral-detergent fibre and N were used to calculate N flow in the compartmental flux method. In vivo omasal N flow was greater for the red clover silage diets than for the grass silage diets. The N flow calculated using the compartmental flux technique and that calculated using the in situ technique were highly correlated, but both were less than and poorly correlated with the in vivo N flow. In both in situ and compartmental flux techniques, forage maturity increased the particle-associated N flow, with the increase being significantly greater for the red clover diets than for the grass silage diets. In conclusion, the compartmental flux and in situ methods described the N flow associated with the particle fractions rather than the total ruminal outflow of feed N.

Dietary fish oil supplements modify ruminal biohydrogenation, alter the flow of fatty acids at the omasum, and induce changes in the ruminal Butyrivibrio population in lactating cows.

Four lactating cows fitted with ruminal cannulae and fed a grass silage-based diet were used in a 4 × 4 Latin square with 28-d periods to investigate the effects of incremental dietary fish oil (FO) supplementation (0, 75, 150, or 300 g/d) on the flow of fatty acids at the omasum and populations of rumen bacteria capable of biohydrogenation. FO decreased silage intake and ruminal volatile fatty acid concentrations and promoted an increase in molar butyrate and propionate proportions at the expense of acetate. Extensive ruminal biohydrogenation of 20:5(n-3) and 22:6(n-3) resulted in corresponding increases in numerous 20- and 22-carbon unsaturated fatty acids at the omasum. Omasal flow of several 20-, 21-, and 22-carbon all-cis (n-3) PUFA exceeded the intake from FO. Supplements of FO also induced a dose-dependent decrease in 18:0 and increased trans 18:1 and trans 18:2 flow at the omasum. Trans-11 was the major 18:1 intermediate in digesta, while FO induced quadratic increases in trans-10 18:1 flow, reaching a maximum of 300 g/d. FO had no substantial influence on omasal flow of CLA. Results suggest that one or more fatty acids in FO inhibit the reduction of trans-18:1 and trans-18:2 intermediates by ruminal microorganisms. qPCR based on 16S rRNA genes in omasal digesta indicated that key Butyrivibrio spp. declined linearly in response to FO. Dose-dependent increases in ruminal outflow of biohydrogenation intermediates containing one or more trans double bonds in response to FO has major implications for host metabolism and the nutritional quality of ruminant foods.

Ruminal infusions of cobalt-EDTA reduce mammary delta9-desaturase index and alter milk fatty acid composition in lactating cows.

Ruminal administration of a triple indigestible marker system comprised of cobalt EDTA (CoEDTA), ytterbium acetate (YbAc), and chromium-mordanted straw (CrS) decreases product:substrate ratios for Delta9-desaturase in bovine milk fat. This experiment was designed to identify the marker(s) responsible and develop an alternative system for simultaneous determination of nutrient flow in the gastro-intestinal tract and milk fatty acid composition. Five lactating dairy cows were used in a 5 x 5 Latin square with 21-d periods to evaluate the effects of YbAc, CoEDTA, and CrS independently or as part of a triple marker system (TMS), and CrEDTA as an alternative to CoEDTA on milk fat composition. Markers were administered in the rumen over a 7-d interval and samples of milk were collected on d -1, 3, 7, and 11. Both TMS and CoEDTA alone reduced the concentrations of milk fatty acids containing a cis-9 double bond, whereas YbAc, CrS, and CrEDTA had no effect. Reductions in product:substrate ratios for Delta9-desaturase were time dependent and evident within 3 d of administration. Ruminal infusion of CoEDTA for 7 d induced mean decreases in milk cis-9 14:1/14:0, cis-9 16:1/16:0, cis-9 18:1/18:0, and cis-9, trans-11 conjugated linoleic acid/trans-11 18:1 concentration ratios of 47.7, 26.7, 40.3, and 42.6%, respectively. In conclusion, ruminal infusion of CoEDTA alters milk fatty acid composition and appears to inhibit Delta9-desaturase activity in the bovine mammary gland. Results indicate that a TMS based on CrEDTA, YbAc, and indigestible neutral detergent fiber can be used for estimating nutrient flow without altering milk fat composition in lactating cows.

Transfer of absorbed cis-9, trans-11 conjugated linoleic acid into milk is biologically more efficient than endogenous synthesis from absorbed vaccenic acid in lactating cows.

Cis-9, trans-11, the major isomer of conjugated linoleic acid (CLA) in bovine milk fat, is derived from ruminal biohydrogenation of 18:2 (n-6) and endogenous conversion of trans-11 18:1 (vaccenic acid; VA) in the mammary gland. Most evidence to date suggests that endogenous synthesis is the major source of cis-9, trans-11 CLA, but the extent of VA desaturation is less well defined. Four lactating cows were used in consecutive 4 x 4 Latin squares to examine changes in milk fatty acid composition and secretion in response to abomasal infusions of lipid supplements enriched with cis-9, trans-11 CLA (88.8%) or VA (29.4%). Treatments were infused over 4-d, followed by a 3-d washout, during 7 d experimental periods and administered to deliver 0, 3, 6, and 12 g cis-9, trans-11 CLA/d (Expt. 1) or 0, 7.5, 15 and 30 g VA/d (Expt. 2). Infusions of cis-9, trans-11 CLA increased linearly milk cis-9, trans-11 CLA concentrations from 0.68 to 1.46 g/100 g fatty acids. Abomasal infusions of VA increased linearly milk VA and cis-9, trans-11 CLA content from 1.22 to 2.72 and 0.61 to 1.24 g/100 g fatty acids, respectively. Changes in milk fatty acid secretion indicated that 28.9% of VA was converted to cis-9, trans-11 CLA. Results provide evidence that conversion by Delta9-desaturase to cis-9, trans-11 CLA in the lactating cow is independent of postruminal VA supply. In conclusion, endogenous synthesis via VA was equivalent to approximately 21% of the response to increases in cis-9, trans-11 CLA available for absorption.

Effect of forage conservation method, concentrate level and propylene glycol on the fatty acid composition and vitamin content of cows' milk.

Based on potential health benefits, there is a need to develop effective strategies for enhancing milk fat concentrations of cis-9 18:1, 18:3 n-3 and conjugated linoleic (CLA) content in milk without compromising the sensory or storage characteristics of processed milk or dairy products. Sixteen Finnish Ayrshire dairy cows were used in a cyclic change-over experiment with four 21-d experimental periods and a 4 x 2 x 2 factorial arrangement of treatments to evaluate the effects of forage conservation method, concentrate level and supplements of propylene glycol (PG), and their interactions on milk fatty acid composition and vitamin content. Experimental treatments consisted of four conserved forages offered ad libitum, supplemented with two levels of a standard concentrate (7 or 10 kg/d) and PG (0 and 210 g/d) fed as three equal meals. Primary growths of timothy and meadow fescue sward were conserved by ensiling with none (NA), an inoculant enzyme preparation (IE) or a formic acid based (FORM) additive or as hay 1 week later. Conservation of grass by drying rather than ensiling resulted in lower forage 18:2n-6, 18:3n-3, total fatty acid and fat-soluble vitamin concentrations. In spite of lower intakes, milk fat 18:2n-6 and 18:3n-3 content was higher (P < 0.05) for hay than for silage diets (12.1, 9.6, 9.6 and 9.3 and 5.00, 3.51, 4.27 and 2.93 g/kg total fatty acids, for hay, NA, IE and FORM silages, respectively). Forage conservation method had no clear effects on milk trans 18:1 or CLA content. Compared with silage, hay diets resulted in milk containing lower (P < 0.001) riboflavin, alpha-tocopherol and beta-carotene concentrations, but had no effect on ascorbic acid, thiamine, pyridoxine or retinol content. Feeding more concentrates had no effect on milk fatty acid composition or milk vitamin content, other than lowering (P < 0.001) 16:0 concentrations from 348 to 338 g/kg fatty acids. Supplements of PG led to small (P < 0.05) increases in milk 13:0 anteiso and 15:0 content from 1.06 and 11.3 to 1.22 and 12.6 g/kg fatty acids and reduced (P < 0.05) the concentrations of ascorbic acid (16.1 v. 15.1 g/kg milk).

Determination of digesta flow entering the omasal canal of dairy cows using different marker systems.

Four studies were conducted to compare the effect of four indigestible markers (LiCoEDTA, Yb-acetate, Cr-mordanted straw and indigestible neutral-detergent fibre (INDF)) and three marker systems on the flow of digesta entering the omasal canal of lactating dairy cows. Samples of digesta aspirated from the omasal canal were pooled and separated using filtration and high-speed centrifugation into three fractions defined as the liquid phase, small particulate and large particulate matter. Co was primarily associated with the liquid phase, Yb was concentrated in small particulate matter, whilst Cr and INDF were associated with large particles. Digesta flow was calculated based on single markers or using the reconstitution system based on combinations of two (Co + Yb, Co + Cr and Co + INDF) or three markers (Co + Yb + Cr and Co + Yb + INDF). Use of single markers resulted in large differences between estimates of organic matter (OM) flow entering the omasal canal suggesting that samples were not representative of true digesta. Digesta appeared to consist of at least three phases that tended to separate during sampling. OM was concentrated in particulate matter, whilst the liquid phase consisted mainly of volatile fatty acids and inorganic matter. Yb was intimately associated with nitrogenous compounds, whereas Cr and INDF were concentrated in fibrous material. Current data indicated that marker systems based on Yb in combination with Cr or INDF are required for the accurate determination of OM, N and neutral-detergent fibre flow. In cases where the flow of water-soluble nutrients entering the omasal canal is also required, the marker system should also include Co.