Nitrogen partitioning and isotopic fractionation in dairy cows consuming diets based on a range of contrasting forages.

Nine multiparous Holstein-Friesian cows (initially 97 d in milk), were used in a 3×3 lattice square design experiment with 4-wk periods. All cows received 4 kg/d concentrates and dietary treatments were based on silages offered ad libitum: perennial ryegrass (PRG); timothy (TIM); tall fescue (TF); red clover (RC); red clover/corn silage mixture [40/60 on a dry matter (DM) basis; RCC]; red clover/whole-crop oat silage mixture (40/60 on a DM basis; RCO); or red clover/whole-crop oat silage mixture (25/75 on a DM basis; ORC). The remaining treatments were based on RCO with feed intake restricted to the level of PRG (RCOr) or with a low protein concentrate (50/50 mixture of barley and molassed sugar beet pulp; RCOlp). Experiment objectives were to evaluate diet effects on N partitioning and N isotopic fractionation. Yields of milk and milk protein were consistently high for diets RC, RCC, and RCO and low for the diets based on poorly ensiled grass silages. Restriction of intake (RCOr) and inclusion of a higher proportion of whole-crop oat silage (ORC) and the low-protein concentrate (RCOlp) led to some loss of production. Diet had little effect on milk fat, protein, and lactose concentrations: low concentrations of milk protein and lactose reflect the restricted energy intakes for all treatments. The highest diet digestibilities were measured for RC and PRG, whereas increasing inclusion of the whole-crop oat silage (0, 60, and 75% of forage DM) led to a marked decrease in diet digestibility (0.717, 0.624, and 0.574 g/g, respectively). Urinary excretion of purine derivatives, an indicator for rumen microbial protein synthesis, was significantly higher for RCC than for TIM and TF. Nitrogen intake ranged between 359 and 626 g/d (treatment means). Partitioning of N intake to feces and urine was closely related to N intake, although urinary N losses were less than predicted from N intake for the 60/40 mixtures of cereal silage and red clover silage. The 15N content of milk, urine, and feces were all influenced by diet 15N content. Isotopic fractionation meant that feces and milk were enriched and urine was depleted in 15N relative to the diet. Significant relationships were observed between the extent of enrichment of urine, feces, and milk, suggesting some commonality in fractionation pathways. The trend for the lowest 15N enrichment in milk protein occurring in diets with low N-use efficiency (milk N/feed N) was contrary to expectations, possibly because of endogenous contributions to milk protein or fractionation when dietary ammonia was incorporated into microbial protein.

Effects of high-sugar ryegrass silage and mixtures with red clover silage on ruminant digestion. 1. In vitro and in vivo studies of nitrogen utilization.

Two experiments were carried out to determine the effects of feeding grass silages differing in their water-soluble carbohydrate content, with or without red clover silage, on the efficiency of nutrient use. High-sugar grass, control grass, and red clover were ensiled in laboratory silos for use in an in vitro experiment (Exp. 1). For an in vivo experiment (Exp. 2), the same forage types were baled and ensiled. All silages were well preserved; within experiments the grass silages had similar composition, except for greater (P < 0.05) water-soluble carbohydrate concentrations in the high-sugar than the control grass silage. In Exp. 1, high-sugar grass, control grass, and red clover silages were fed alone or as mixtures (30:70, 50:50, or 70:30 on a DM basis, respectively) of each grass with the red clover silage to a simulated rumen culture system. There were no significant differences in microbial N flow or efficiency of microbial protein synthesis between individual forages. However, the corresponding values for the 70:30 ratio of high-sugar grass:red clover silage were greater (P < 0.05) than for the red clover silage. The value for the efficiency of N use (g of microbial N/g of feed N) was greater (0.86; P < 0.05) for high-sugar grass silage than the control grass silage. In addition, the high-sugar grass:red clover silage mixtures all gave greater (P < 0.05) values for the efficiency of N use than red clover silage alone; this difference was not achieved with the control grass mixture. Experiment 2 was an incomplete Latin square design conducted with 6 Here-ford x Friesian steers (163 +/- 5.9 kg of BW) with rumen and duodenal cannulas fed the following 5 silage diets: high-sugar grass silage; control grass silage; high-sugar grass and red clover silage (50:50 DM basis); control grass and red clover silage (50:50 DM basis); and red clover silage. Rumen NH3-N concentration was lowest (P < 0.05) with the high-sugar grass silage. Microbial N flows to the duodenum and efficiency of microbial protein synthesis were greater (P < 0.05) for steers fed the high-sugar grass silage than for control grass and red clover silages, and mixing red clover with grass silages increased (P < 0.05) these values compared with red clover silage alone. In both experiments, the efficiency of incorporation of silage N into microbial N was more than 20% greater (P < 0.05) for high-sugar grass than for control grass silage. These data suggest that grass silage with high-sugar content provides a forage-based strategy for balancing N and energy supply and improving the efficiency of use of grass silage N in the rumen.

Effects of high-sugar ryegrass silage and mixtures with red clover silage on ruminant digestion. 2. Lipids.

The experiment investigated the digestion of lipids from different forage silages in beef steers. Six Hereford x Friesian steers prepared with rumen and duodenal cannulas were given ad libitum access to a high-sugar grass silage, control grass silage, red clover silage, or mixtures of the red clover and each of the grass silages (50:50, DM basis). The experiment was conducted as an incomplete 5 x 5 Latin square, with an additional randomly repeated sequence. Total fatty acid and C18:3n-3 concentrations were greater (P < 0.05) for the high-sugar grass silage than the control grass silage or the red clover silage. Dry matter and total fatty acid intake were less (P < 0.05) for steers fed the control grass silage than for steers fed the other diets. Duodenal flow of C18:3n-3 was greater (P < 0.05), and flows of C18:0 and total C18:1 trans were less (P < 0.05), for the red clover silage compared with the 2 grass silage diets, with the mixtures intermediate. These results were supported by a reduction (P < 0.05) in biohydrogenation of C18:3n-3 for the red clover silage, with the mixtures again being intermediate. Flows of total branched- and odd-chain fatty acids were greater (P < 0.05) for the high-sugar grass silage diet, possibly as a result of greater microbial flow, because these fatty acids are associated with bacterial lipid. Duodenal flows of the chlorophyll metabolite, phytanic acid, were greater (P < 0.05) for animals fed the high-sugar grass silage treatments compared with the other treatments. These results confirm the potential for modifying the fatty acid composition of ruminant products by feeding red clover silage.

Evidence in support of a role for plant-mediated proteolysis in the rumens of grazing animals.

The present work aimed to differentiate between proteolytic activities of plants and micro-organisms during the incubation of grass in cattle rumens. Freshly cut ryegrass was placed in bags of varying permeability and incubated for 16 h in the rumens of dairy cows that had previously grazed a ryegrass sward, supplemented with 4 kg dairy concentrate daily. Woven polyester bags (50 microm pore size) permitted direct access of the micro-organisms and rumen fluid enzymes to the plant material. The polythene was impermeable even to small molecules such as NH(3). Dialysis tubing excluded micro-organisms and rumen enzymes/metabolites larger than 10 kDa. DM loss was 46.3 % in polyester, 36.2 % in polythene and 38.1 % in dialysis treatments. It is possible that the DM loss within polythene bags occurred due to a solubilisation of plant constituents (e.g. water-soluble carbohydrates) rather than microbial attachment/degradation processes. The final protein content of the herbage residues was not significantly different between treatments. Regardless of bag permeability, over 97 % of the initial protein content was lost during incubations in situ. Electrophoretic separation showed that Rubisco was extensively degraded in herbage residues whereas the membrane-associated, light-harvesting protein remained relatively undegraded. Protease activity was detected in herbage residues and bathing liquids after all incubation in situ treatments. Although rumen fluid contains proteases (possibly of plant and microbial origin), our results suggest that, owing to cell compartmentation, their activity against the proteins of intact plant cells is limited, supporting the view that plant proteases are involved in the degradation of proteins in freshly ingested herbage.

Vacuum packing: a model system for laboratory-scale silage fermentations.

AIMS: To determine the utility of vacuum-packed polythene bags as a convenient, flexible and cost-effective alternative to fixed volume glass vessels for lab-scale silage studies. METHODS AND RESULTS: Using perennial ryegrass or red clover forage, similar fermentations (as assessed by pH measurement) occurred in glass tube and vacuum-packed silos over a 35-day period. As vacuum-packing devices allow modification of initial packing density, the effect of four different settings (initial packing densities of 0.397, 0.435, 0.492 and 0.534 g cm(-3)) on the silage fermentation over 16 days was examined. Significant differences in pH decline and lactate accumulation were observed at different vacuum settings. Gas accumulation was apparent within all bags and changes in bag volume with time was observed to vary according to initial packing density. CONCLUSIONS: Vacuum-packed silos do provide a realistic model system for lab-scale silage fermentations. SIGNIFICANCE AND IMPACT OF THE STUDY: Use of vacuum-packed silos holds potential for lab-scale evaluations of silage fermentations, allowing higher throughput of samples, more consistent packing as well as the possibility of investigating the effects of different initial packing densities and use of different wrapping materials.

Comparison of grass and legume silages for milk production. 2. In vivo and in sacco evaluations of rumen function.

Two experiments were conducted to investigate the basis for higher voluntary intakes and increased alpha-linolenic acid content in milk from cows offered clover silages. Six cows with rumen and duodenal cannulae were used in a four-period changeover-design experiment. Cows received 8 kg/d of dairy concentrate and had ad libitum access to one of six silage treatments: grass, red clover, white clover, alfalfa, and 50/50 (dry matter basis) mixtures of grass with red clover or white clover. The rumen fermentability of grass, red clover, white clover, and grass/red clover silages was also evaluated in a nylon bag study. Legume silages led to increased dry matter intake and milk production in comparison with grass silage. There was no significant effect of legume silages on rumen pH and volatile fatty acid concentrations, but a significant increase in rumen ammonia concentration with the legume silages, reflecting their higher protein content. The inclusion of white clover or alfalfa silage, but not red clover silage, in diets led to an increase in molar proportions of isobutyric, iso-valeric, and n-valeric acids in comparison with diets based on grass silage. Rumen fill was significantly lower, and rumen passage rates were significantly higher for cows offered alfalfa or white clover silages. However, the markedly different particle size distribution of rumen contents with these feeds suggests very different mechanisms for the high intake characteristics: high rates of particle breakdown and passage with alfalfa, and high rates of fermentation and passage with white clover. Microbial energetic efficiency (grams microbial N per kilogram organic matter apparently digested in the rumen) was highest for cows offered alfalfa silage, intermediate for clover silage, and lowest for cows offered grass silage. These differences reflect the higher rumen outflow rates for legume silages in comparison with grass silage. However, the effect of these differences on N-use efficiency (feed to milk) was probably quite small in comparison with effects of N intake. Although the biohydrogenation of alpha-linolenic acid was still high for red clover silage (86.1% compared with 94.3% for grass silage), there was a 240% increase in the proportion of alpha-linolenic acid passing through the rumen. This explains the increased recovery of alpha-linolenic acid from feed into milk with diets based on red clover silage.

Alternative forages--back to the future.

The BSE crisis, restrictions over export of UK beef and bans on animal-derived concentrate feeds are driving agriculture in the UK 'back' towards more sustainable, low input systems for feeding cattle and sheep using 'natural' home-grown forages. Research is in progress to answer some of the questions surrounding the use of protein- and energy-rich alternative forages as feeds for ruminants.

Effects of lactic acid bacteria in inoculants on changes in amino acid composition during ensilage of sterile and non-sterile ryegrass.

A study was carried out on the changes occurring in the amino acid fraction of a hybrid ryegrass during ensilage in laboratory-scale silos to help to establish the relative roles of plant and microbial proteases on protein degradation in the silo. Herbage treatments included (i) normal grass without treatment (ii) lambda-irradiated grass (sterile) without treatment (iii) sterile, inoculated with a strain of Lactobacillus plantarum and (iv) sterile, inoculated with a strain of Lactobacillus paracasei subsp. paracasei. These treatments had a significant effect on silage amino acid profiles. Concentrations of free amino acids and the extent of amino acid catabolism varied with treatment. However, levels were notably higher in control silages after 90 days (free amino acid nitrogen constituting 54% of total amino acid nitrogen compared with 37, 32 and 22% for treatments i, ii and iv, respectively). These results indicate that the extent of protein hydrolysis during ensilage is influenced by factors other than rate of pH decline and plant protease activity, and that microbial proteases play a role.

Efficient improvement of silage additives by using genetic algorithms.

The enormous variety of substances which may be added to forage in order to manipulate and improve the ensilage process presents an empirical, combinatorial optimization problem of great complexity. To investigate the utility of genetic algorithms for designing effective silage additive combinations, a series of small-scale proof of principle silage experiments were performed with fresh ryegrass. Having established that significant biochemical changes occur over an ensilage period as short as 2 days, we performed a series of experiments in which we used 50 silage additive combinations (prepared by using eight bacterial and other additives, each of which was added at six different levels, including zero [i.e. , no additive]). The decrease in pH, the increase in lactate concentration, and the free amino acid concentration were measured after 2 days and used to calculate a "fitness" value that indicated the quality of the silage (compared to a control silage made without additives). This analysis also included a "cost" element to account for different total additive levels. In the initial experiment additive levels were selected randomly, but subsequently a genetic algorithm program was used to suggest new additive combinations based on the fitness values determined in the preceding experiments. The result was very efficient selection for silages in which large decreases in pH and high levels of lactate occurred along with low levels of free amino acids. During the series of five experiments, each of which comprised 50 treatments, there was a steady increase in the amount of lactate that accumulated; the best treatment combination was that used in the last experiment, which produced 4.6 times more lactate than the untreated silage. The additive combinations that were found to yield the highest fitness values in the final (fifth) experiment were assessed to determine a range of biochemical and microbiological quality parameters during full-term silage fermentation. We found that these combinations compared favorably both with uninoculated silage and with a commercial silage additive. The evolutionary computing methods described here are a convenient and efficient approach for designing silage additives.

Prevention of yeast spoilage in feed and food by the yeast mycocin HMK.

The yeast Williopsis mrakii produces a mycocin or yeast killer toxin designated HMK; this toxin exhibits high thermal stability, high pH stability, and a broad spectrum of activity against other yeasts. We describe construction of a synthetic gene for mycocin HMK and heterologous expression of this toxin in Aspergillus niger. Mycocin HMK was fused to a glucoamylase protein carrier, which resulted in secretion of biologically active mycocin into the culture media. A partial purification protocol was developed, and a comparison with native W. mrakii mycocin showed that the heterologously expressed mycocin had similar physiological properties and an almost identical spectrum of biological activity against a number of yeasts isolated from silage and yoghurt. Two food and feed production systems prone to yeast spoilage were used as models to assess the ability of mycocin HMK to act as a biocontrol agent. The onset of aerobic spoilage in mature maize silage was delayed by application of A. niger mycocin HMK on opening because the toxin inhibited growth of the indigenous spoilage yeasts. This helped maintain both higher lactic acid levels and a lower pH. In yoghurt spiked with dairy spoilage yeasts, A. niger mycocin HMK was active at all of the storage temperatures tested at which yeast growth occurred, and there was no resurgence of resistant yeasts. The higher the yeast growth rate, the more effective the killing action of the mycocin. Thus, mycocin HMK has potential applications in controlling both silage spoilage and yoghurt spoilage caused by yeasts.

Evidence of a role for plant proteases in the degradation of herbage proteins in the rumen of grazing cattle.

Protein breakdown in the rumen is generally regarded as a two-stage process in which proteases produced by rumen microorganisms cleave plant protein into peptides and amino acids. However, many of the fiber-degrading cellulolytic species in the rumen are not in fact proteolytic, and the proteolytic activity of the entire rumen microbial population is only moderate when compared to the gastric and pancreatic secretions in the abomasum. Moreover, plant cell walls remain largely intact after initial chewing (particularly in cattle), presenting a physical barrier that must be breached prior to their effective colonization. The present study considers the hypothesis that the plant enzymes are at least partly responsible for herbage protein degradation in grazing ruminants. Ryegrass, red clover, white clover, and bird's-foot trefoil were incubated in the presence and absence of rumen microorganisms. The production of volatile fatty acids indicated the level of microbial activity, whereas the relative disappearance of the large subunit of ribulose 1,5 bisphosphate carboxylase/oxygenase (Rubisco LSU) indicated proteolytic activity. In all incubations, the relative abundance of the Rubisco LSU decreased as the incubation progressed. When rumen microorganisms were absent, low molecular weight peptides (below 20 kDa) accumulated as the incubation progressed. This accumulation was not observed in the presence of rumen microorganisms. Therefore we suggest that the intrinsic plant proteases contribute to the initial stages of proteolysis of grazed herbage.

Proteolysis during ensilage of forages varying in soluble sugar content.

The effect of contrasting concentrations of water-soluble carbohydrates of herbage on silage fermentation and composition was examined using grass with high [250 g/kg of dry matter (DM)] concentrations of water-soluble carbohydrates and grass and clover with low (66 g/kg of DM) concentrations of water-soluble carbohydrates. Herbages were ensiled untreated, after inoculation with lactic acid bacteria, or after treatment with formic acid. Good quality silages were produced from herbage with high concentrations of water-soluble carbohydrates, regardless of treatment, and all pH values were below 3.7 after 90 d of ensilage. However, the silage formed from inoculated herbage had a significantly lower concentration of ammonia N and a significantly higher proportion of residual ribulose-1,5-bisphosphate carboxylase compared with the other two silages. Fast protein liquid chromatography (Pharmacia, Uppsala, Sweden) was used to measure ribulose-1,5-bisphosphate carboxylase, and measurement of true plant protein fractions in herbage and silage showed benefits over traditional measurements such as the measurement of N and ammonia N. Herbages with low concentrations of water-soluble carbohydrates produced inferior quality silages that had lower ribulose-1,5-bisphosphate carboxylase contents and higher ammonia N contents, regardless of treatment; few significant differences were observed among treatments. Under good ensiling conditions, when available water-soluble carbohydrate is adequate, the use of inoculants can improve fermentation characteristics and increase the ribulose-1,5-bisphosphate carboxylase content of silages. However, when the herbage has low concentrations of water-soluble carbohydrates, even in inoculated herbages, lactic acid bacteria may follow a heterofermentative pathway instead of a homofermentative pathway, which can result in a decrease in silage quality and a reduction in intact ribulose-1,5-bisphosphate carboxylase.

Viability of Cryptosporidium parvum during ensilage of perennial ryegrass.

The survival of Cryptosporidium parvum during ensilage of perennial ryegrass was examined in laboratory silos with herbage prepared in one of three different ways; either untreated, inoculated with a strain of Lactobacillus plantarum or by direct acidification with formic acid. The pH values of all silages initially fell below 4.5, but only formic acid-treated silage remained stable at less than pH 4 after 106 d, with the pH of the untreated and inoculant-treated silages rising to above 6. The formic acid-treated silage had a high lactic acid concentration (109 g kg-1 dry matter (DM)) and low concentrations of propionic and butyric acids after 106 d. However, the untreated and inoculant-treated silages showed an inverse relationship, with low lactic acid concentrations and high concentrations of acetic, propionic and butyric acids. These silages also contained ammonia-N concentrations in excess of 9 g kg-1 DM. In terms of the viability of Cryptosporidium parvum oocysts very few differences were seen after 14 d of ensilage with ca 50% remaining viable, irrespective of treatment and total numbers had declined from the initial level of 5.9 x 10(4) to 1 x 10(4) g(-1) fresh matter. Total oocyst numbers remained approximately the same until the end of the ensiling period, with the percentage of viable oocysts declining to 46, 41 and 32% respectively for formic acid, inoculant and untreated silages. The results are discussed in terms of changes occurring during the silage fermentation, in particular the products which may influence the survival of Cryptosporidium and implications for agricultural practice and the health of silage fed livestock.

Studies of rumen function in an in vitro continuous culture system.

An in vitro continuous culture system to simulate processes in the rumen is described. This comprises a culture vessel (Fig. 2) fed continuously with pelleted solid feed (Fig. 1); artificial saliva also enters the vessel while effluent leaves it continuously in two streams, one is filtered and the other simply overflows (Fig. 1). In this way liquid and solid turnover times may be manipulated independently; in a trial experiment the former and latter were 6.3% and 3.4% per hour respectively. In four replicated experiments a steady-state was achieved in 5-6 days with a feed of barley, tapioca, fishmeal, urea and straw and maintained for a further 7 to 14 days. During a steady-state period, rumen characteristics in terms of protozoal count, pH, redox potential, total volatile fatty acid concentration and ammonia concentration were stable and similar to those found in the rumens of animals given similar diets. Flows of microbial N compounds were assessed by infused Na2H32PO4 with the artificial saliva and estimating the 32P label incorporated in bacterial fractions. The system demonstrably provided a satisfactory means for studying many aspects of rumen function.

Effect of different levels of phosphorus on rumen microbial fermentation and synthesis determined using a continuous culture technique.

A continuous culture technique was used to study the phosphorus requirements of rumen micro-organisms. Solutions of artificial saliva containing 120, 80, 40 and 0 mg inorganic phosphorus (Pi)/l were infused into the reaction vessels previously inoculated with rumen contents, resulting in Pi concentrations in the vessel contents of 48, 28, 4 and less than 1 mg/l respectively. Various fermentative and synthetic characteristics were examined. In the vessel contents, concentrations of protozoa (about 0.9 X 10(5)/ml) were not significantly affected by Pi concentration. Total volatile fatty acids (VFA) produced averaged about 6.83 mmol/h with Pi levels of 48 and 28 mg/l. Reduction in Pi concentrations to 4 and less than 1 mg/l resulted in significant reductions in total VFA to approximately 6.25 and 3.75 mmol/h respectively, accompanied by a rise in pH from 6.5 to 7.3. Ammonia-nitrogen values, which averaged about 131 mg/l at the higher Pi concentrations, also increased with the lowest level of Pi to about 240 mg/l. ATP concentrations averaged about 14 mumol/l at the highest Pi concentration and fell progressively with each reduction in Pi concentration to a final value of 2.5 mumol/l with the Pi level less than 1 mg/l. At Pi concentrations of 48 and 28 mg/l, the digestibilities of xylose, arabinose and cellulose-glucose were maintained at about 0.90, 0.62 and 0.70 g/g input respectively. At lower Pi concentrations these digestibilities fell significantly and corresponding values at Pi less than 1 mg/l were 0.73, 0.41 and 0.31 respectively. Starch digestion was unaffected by Pi concentration and remained at about 0.90 g/g input. The amount of microbial-N synthesized averaged 0.48 g/d and was maintained with Pi concentrations down to 4 mg/l. There was, however, a significant reduction to 0.26 g/d with Pi concentrations of less than 1 mg/l. The efficiency of microbial protein synthesis was variable but averaged approximately 25 g N/kg total carbohydrate fermented. It was estimated that the minimum Pi concentrations required in rumen fluid in vivo to maintain maximum degradative and synthetic microbial activities was in the range 75-100 mg/l and that over-all P requirement of the microbes was of the order of 5.1 g/kg apparently digested organic matter intake.

A comparison of the chemical composition of mixed bacteria harvested from the liquid and solid fractions of rumen digesta.

Steers, equipped with simple rumen cannulas, were given diets of approximately equal parts of rolled barley and straw supplemented with urea. The diets provided sufficient estimated rumen degradable nitrogen (RDN; RDN:metabolizable energy values of 1:3) to maintain maximum microbial synthesis. In some experiments Na235SO4 was introduced into the rumen to label microbial protein. Rumen digesta samples were taken before feeding and mixed rumen bacteria were separated from the solid (solid-associated bacteria; SAB) and liquid (liquid-associated bacteria; LAB) fractions of digesta. The most effective method of removing SAB from the fibre was a combination of homogenizing and pummelling. This process did not affect the physical form or chemical composition of the bacteria. Samples of SAB contained significantly (P less than or equal to at least 0.05) less ash, total N, RNA and diaminopimelic acid (DAP) and significantly (P less than or equal to 0.01) more lipid than samples of LAB. Concentrations (g/kg dry matter) of ash, total N, RNA, DAP and lipid in SAB were approximately 87, 70, 35, 2.2 and 245 respectively. Corresponding values for LAB were 157, 80, 50, 3.8 and 124 respectively. RNA-N:total N and DAP-N:total N values in SAB were significantly lower than those in LAB (P less than or equal to 0.05 and 0.02 respectively). 35S:total N values were similar in both groups of bacteria. The importance of differences in constituent:total N values in the two groups of bacteria in relation to their use as indices of microbial protein synthesis is discussed.

Glycosyl ureides in ruminant nutrition. 2. In vitro studies on the metabolism of glycosyl ureides and their free component molecules in rumen contents.

1. The fate of glucosyl urea (GU), lactosyl urea (LU) and corresponding mixtures of the free sugars and urea and their degradation products were examined during in vitro incubation of the compounds with rumen contents taken from donor sheep and steers at various stages of adaptation to these compounds. 2. The sugar-urea bond was virtually unattacked in rumen contents from unadapted sheep and steers but generally a slow release of the galactose moiety occurred. After feeding LU or GU to animals for a period of approximately 10 d, the rates of disappearance of both bound urea and sugar had increased, but were still markedly slower than those of the corresponding free sugars and urea. In vitro rates of degradation of both free lactose and urea also increased in response to the feeding of lactose and urea to rumen content donor animals. 3. Ammonia accumulation in rument contents when GU or LU were the substrates was notably lower than when equivalent amounts of glucose and urea or lactose and urea were the substrates. 4. Bacterial growth was estimated using an vitro method based on incorporation of 32P into bacterial nucleic acids. Markedly different patterns of bacterial growth were observed depending on whether LU or lactose and urea were the substrates.

Glycosyl ureides in ruminant nutrition. 1. Preparation and estimation of lactosyl urea and other glycosyl ureides.

1. Glucosyl urea, lactosyl urea and galactosyl urea were prepared from pure sugars and urea and their purity confirmed by determination of their melting points, specific rotations and by mass spectrometry. 2. Using whey as a lactose source, a range of conditions were examined for the preparation of lactosyl urea on a laboratory scale. Yields of 60% were achieved when lactose and urea (molar ratio, urea: lactose 0:6) were reacted for 15 h in sulphuric acid at pH 2.0 and a temperature of 70 degrees. 3. Methods of detection and estimation of all three ureides in whey preparations and ruminant digesta samples were developed. Two quantitative methods, one involving acid-hydrolysis, the other ion-exchange chromatography, were used. The latter method enabled determination of individual quantities of ureides in mixtures.

Glycosyl ureides in ruminant nutrition. 3. In vivo studies on the metabolism of glycosyl ureides and corresponding mixtures of their free component molecules.

1. Steers and sheep were given basal diets of barley and straw (1:1, w/w), usually containing urea, which for certain experimental periods were supplemented with pure glucosyl urea (GU), pure lactosyl urea (LU) or a product prepared from whey concentrate (EW) which contained 65-80% of the lactose in the form of LU. 2. On the morning of an experiment ureide (or EW) was omitted from the feed and a dose of either ureide (or EW) or equivalent amounts of free lactose and urea (L + U) was added to the rumen within 30 min of feeding, together in some experiments with polyethylene glycol (PEG) as a fluid-phase marker. Samples of rumen contents, and in some experiments abomasal contents, were taken at intervals for up to 8 h. 3. For both steers and sheep given GU and LU for the first time (unadapted animals) there was little or no accumulation of ammonia in the rumen or cleavage of the sugar-urea bond. Galactose was, however, slowly liberated from LU. 4. For steers and sheep which had been given GU, LU or EW for approximately 7-10 d or more (adapted animals) some accumulation of ammonia occurred after adding GU or LU to the rumen, but for LU it occurred less rapidly and to a lower peak concentration than when L + U was added. In adapted animals cleavage of the sugar-urea bond in LU was virtually complete in 2-4 h. Degradation of the components of L + U was virtually complete within 1 h. 5. Recovery at the abomasum of ureide (present either as GU or LU) estimated from ureide: PEG values, appeared to be complete in experiments with unadapted sheep given a dose of EW. In adapted sheep only very small amounts of ureide in an EW dose (on average 6%) entered the abomasum undegraded. Amounts lost in this way appeared to be positively correlated with the rate of fluid turnover in the rumen.