[show abstract][hide abstract] ABSTRACT: We employed most probable numbers (MPNs) enumeration of enrichment cultures, combined with the use of a range of carbon sources (glucose, cellobiose, cellulose, xylan and wheat straw), to recover and identify morphologically different groups of anaerobic fungi (monocentric rhizoidal [Neocallimastix, Piromyces spp.], polycentric rhizoidal [Anaeromyces, Orpinomyces spp.], bulbous non-rhizoidal [Caecomyces, Cyllamyces spp.]) from rumen digesta, and fresh or frozen–thawed faeces of silage-fed cattle. Highest MPN counts (>106 thallus forming units [TFU] g−1 dry matter (DM)) were obtained using wheat straw but use of other carbon sources revealed large variation in the relative abundance of the morphotypes recovered in culture. Polycentric morphotypes were overall the most abundant fungi, comprising ca. 60 % of observations and recovered most frequently with xylan and wheat straw. Bulbous morphotypes showed a reciprocal pattern of occurrence, being most frequently observed on glucose, cellobiose and cellulose. Monocentric morphotypes were surprisingly the least abundant (<10 % overall), occurring mostly on glucose and wheat straw. Freezing of faeces (−20 °C/5 weeks) and thawing prior to enrichment culture reduced MPN counts by ca. 40 % from a mean of 1.8 × 105 TFU g−1 DM, but greater relative abundance of polycentric morphotypes in frozen–thawed faeces suggested differential survival in response to environmental stresses. PCR–RFLP demonstrated the simultaneous presence of seven ribotypes in one animal, but not all ribotypes could be associated with a particular genus.
[show abstract][hide abstract] ABSTRACT: To develop an automated ribosomal intergenic spacer region analysis (ARISA) method for the detection of anaerobic rumen fungi and also to demonstrate utility of the technique to monitor colonization and persistence of fungi, and diet-induced changes in community structure.
The method could discriminate between three genera of anaerobic rumen fungal isolates, representing Orpinomyces, Piromyces and Neocallimastix species. Changes in anaerobic fungal composition were observed between animals fed a high-fibre diet compared with a grain-based diet. ARISA analysis of rumen samples from animals on grain showed a decrease in fungal diversity with a dominance of Orpinomyces and Piromyces spp. Clustering analysis of ARISA profile patterns grouped animals based on diet. A single strain of Orpinomyces was dosed into a cow and was detectable within the rumen fungal population for several weeks afterwards.
The ARISA technique was capable of discriminating between pure cultures at the genus level. Diet composition has a significant influence on the diversity of anaerobic fungi in the rumen and the method can be used to monitor introduced strains.
Through the use of ARISA analysis, a better understanding of the effect of diets on rumen anaerobic fungi populations is provided.
Letters in Applied Microbiology 01/2009; 47(6):492-9. · 1.63 Impact Factor
[show abstract][hide abstract] ABSTRACT: Home grown forages offer the livestock farmer high quality and traceable feeds for his or her animals. As animal genetics change to give faster growth rates and higher milk yields, the genetics of forage crops also need to improve to match animal requirements. Breeding is essentially a process of exploitation of genetic variation. This has to be either selected or created and then incorporated into genetic stock that already contains a number of desirable characteristics, combining new genes while maintaining other required traits. For forage improvement, this genetic variation must then be fixed into new plant varieties. This is a more complex task in out-breeding species that have incompatibility mechanisms, such as most forage grasses and legumes, compared with inbreeding species such as most cereal crops.
Recent Advances in Animal Nutrition. 12/2008; 2008(1):39-65.
[show abstract][hide abstract] ABSTRACT: Summary 37I. INTRODUCTION 37II. DIGESTION OF PLANTS IN THE RUMEN: OLD AND NEW CONCEPTS 39III. RUMEN-INDUCED PLANT METABOLISM: CELL DEGRADATION AND DEATH 411. Summary of plant cell death processes 412. Anaerobic stress and flooding tolerance of plants 423. Plant cell responses to elevated temperatures 454. Wounding responses/pathogen attachment 455. Senescence in the rumen? 47IV. FUTURE PROSPECTS 50Acknowledgements 51References 51It is generally assumed that breakdown of plant material in the rumen is a process mediated by gut microorganisms. This view arose because of the identification of a pre-gastric fermentation in the rumen, brought about by a large and diverse microbial population. The extensive use of dried and ground feed particles in forage evaluation might have helped to promote this assumption. However, although the assumption might be correct in animals feeding on conserved forage (hay and silage) where the cells of ingested forage are dead, it is possible that with grazed (living) forage, the role played by plant enzymes in the rumen has been overlooked. In a grazing situation, plant cells that remain intact on entering the rumen are not inert, but will respond to the perceived stresses of the rumen environment for as long as they are metabolically viable. Metabolic adjustments could include anaerobic and heat-shock responses that could promote premature senescence, leading to remobilization of cell components, especially proteins. Moreover, contact of plant cells with colonizing microorganisms in the rumen might promote a type of hypersensitive response, in much the same way as it does outside the rumen. After fresh plant material enters the rumen and prior to extensive plant cell-wall degradation, there is often a phase of rapid proteolysis providing N in excess of that required to maintain the rumen microbial population. The inefficient use of this ingested N results in generation of ammonia and urea in exhaled breath and urine, which promotes welfare and environmental pollution concerns. Therefore an important research goal in livestock agriculture is to find ways of decreasing this initial rate of proteolysis in the rumen. This will benefit the farmer financially (through decreased use of feed supplements), but will also benefit the environment, as N pollution can adversely affect pasture diversity and ecology. This review considers the possible responses of plant metabolism to the rumen environment, and how such considerations could alter current thinking in ruminant agriculture.
New Phytologist 07/2008; 148(1):37 - 55. · 6.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Plant breeding has developed perennial ryegrass varieties with increased concentrations of water-soluble carbohydrates (WSCs) compared with conventional varieties. Water-soluble carbohydrates are major metabolic and storage components in ryegrass. Therefore, if perennial ryegrass herbage is allowed to grow to greater heights it should contain higher water-soluble carbohydrates concentrations, for example as under rotational grazing rather than continuous grazing by livestock. This study investigated this hypothesis and measured the performance of lambs grazed rotationally and continuously. Replicated plots of the variety AberDart (bred to express high WSC concentrations) or the variety Fennema were grazed by a core group of ten male Cheviot lambs for 10 weeks. Lambs were weighed and replicate forage samples were taken every 7 d. Concentrations of WSC in AberDart herbage were significantly (P < 0·05), but not substantially, higher than those in Fennema herbage. Rotational grazing did not increase the differential in WSC concentration between the AberDart and Fennema varieties. However, there was a tendency (P = 0·07) for lambs rotationally grazing the AberDart swards to have a higher final live weight than lambs grazing the Fennema swards. Overall, lamb performance was increased when either perennial ryegrass variety was rotationally rather than continuously grazed (P < 0·001).
[show abstract][hide abstract] ABSTRACT: 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.
Journal of Animal Science 12/2006; 84(11):3049-60. · 2.09 Impact Factor
[show abstract][hide abstract] ABSTRACT: A range of temperate grasses species including orchard grass (Dactylis glomerata), timothy (Phleum pratense), tall fescue (Festuca arundinacea), meadow fescue (Festuca pratense), Italian ryegrass (Lolium multiflorum), perennial ryegrass (Lolium perenne) and hybrid ryegrass were tested for polyphenol oxidase (PPO) activity. Levels of PPO activity were relatively high in orchard grass and Italian, perennial and hybrid ryegrasses but were low in fescues and timothy. Chlorogenic acid was the major endogenous PPO substrate in timothy, meadow fescue and Italian, perennial and hybrid ryegrasses. The major PPO substrate in orchard grass was identified as caffeoylisocitric acid. Genetic analysis indicates that perennial ryegrass contains three genes which encode for PPOs. These genes were isolated, sequenced and shown to have high sequence homology with other monocot PPOs.
XXIII International Conference on Polyphenols, Winnipeg, Manitoba, Canada; 08/2006
[show abstract][hide abstract] ABSTRACT: 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.
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.
Vacuum-packed silos do provide a realistic model system for lab-scale silage fermentations.
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.
Journal of Applied Microbiology 02/2005; 98(1):106-13. · 2.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
British Journal Of Nutrition 02/2005; 93(1):73-9. · 3.30 Impact Factor
[show abstract][hide abstract] ABSTRACT: A study was designed to examine the relationships between protein, condensed tannin and cell wall carbohydrate content and composition and the nutritional quality of seven tropical legumes (Desmodium ovalifolium, Flemingia macrophylla, Leucaena leucocephala, L pallida, L macrophylla, Calliandra calothyrsus and Clitoria fairchildiana). Among the legume species studied, D ovalifolium showed the lowest concentration of nitrogen, while L leucocephala showed the highest. Fibre (NDF) content was lowest in C calothyrsus, L Leucocephala and Lpallida and highest in L macrophylla, which had no measurable condensed tannins. The highest tannin concentration was found in C calothyrsus. Total non-structural polysaccharides (NSP) varied among legumes species (lowest in C calothyrsus and highest in D ovalifolium), and glucose and uronic acids were the most abundant carbohydrate constituents in all legumes. Total NSP losses were lowest in F macrophylla and highest in L leucocephala and L pallida. Gas accumulation and acetate and propionate levels were 50% less with F macrophylla and D ovalifolium as compared with L leucocephala. The highest levels of branched-chain fatty acids were observed with non-tanniniferous legumes, and negative concentrations were observed with some of the legumes with high tannin content (D ovalifolium and F macrophylla). Linear regression analysis showed that the presence of condensed tannins was more related to a reduction of the initial rate of gas production (0–48 h) than to the final amount of gas produced or the extent (144 h) of dry matter degradation, which could be due to differences in tannin chemistry. Consequently, more attention should be given in the future to elucidating the impact of tannin structure on the nutritional quality of tropical forage legumes.
Journal of the Science of Food and Agriculture 09/2003; 83(12):1256-1266. · 1.76 Impact Factor
[show abstract][hide abstract] ABSTRACT: Programmed plant cell death is a widespread phenomenon resulting in the formation of xylem vessels, dissected leaf forms, and aerenchyma. We demonstrate here that some characteristics of programmed cell death can also be observed during the cellular response to biotic and abiotic stress when plant tissue is ingested by grazing ruminants. Furthermore, the onset and progression of plant cell death processes may influence the proteolytic rate in the rumen. This is important because rapid proteolysis of plant proteins in ruminants is a major cause of the inefficient conversion of plant to animal protein resulting in the release of environmental N pollutants. Although rumen proteolysis is widely believed to be mediated by proteases from rumen microorganisms, proteolysis and cell death occurred concurrently in clover leaves incubated in vitro under rumenlike conditions (maintained anaerobically at 39 degrees C) but in the absence of a rumen microbial population. Under rumenlike conditions, both red and white clover cells showed progressive loss of DNA, but this was only associated with fragmentation in white clover. Cell death was indicated by increased ionic leakage and the appearance of terminal deoxynucleotidyl transferase-mediated dUTP-nick-end-labelled nuclei. Foliar protein decreased to 50% of the initial values after 3 h incubation in white clover and after 4 h in red clover, while no decrease was observed in ambient (25 degrees C, aerobic) incubations. In white clover, decreased foliar protein coincided with an increased number of protease isoforms.
[show abstract][hide abstract] ABSTRACT: The effect of adding water-soluble carbohydrate (WSC) on the microbial fermentation of fresh perennial ryegrass was examined in an in vitro RUSITEC system over two 10 day periods. Four treatment levels of WSC were used: a basal grass and a basal grass plus a sugar infusion to raise the WSC basal grass level by approximately 1.25×; 1.5× and 1.75×. The infusion was a mixture of inulin and sucrose (80:20) infused over the first 14 h of each 24 h cycle, based on a preliminary study which indicated a linear disappearance of forage WSC over this period. With increasing WSC inclusion there were linear reductions in pH and ammonia-N (NH3N) (P=0.001) but only a small increase in total volatile fatty acid concentration. There was also a decrease in the proportion of acetate and an increase in propionate with increasing WSC inclusion (P=0.02 and 0.009, respectively). Microbial nitrogen production was similar for the first three treatments of Basal to Basal × 1.5 but was lower at the highest level of WSC inclusion (P<0.001). The efficiency of microbial protein synthesis increased from Basal to Basal × 1.5 (9.9, 10.8, 12.7 g N/kg organic matter apparently digested (OMAD), respectively) but at the highest level of WSC inclusion (Basal × 1.75) there was a reduction to 7.1 g N/kg OMAD. This may have been related to the low pH values (<6.0) at certain times during incubation and/or futile bacterial energy cycles as a result of the low nitrogen concentration in the vessels. With increasing WSC inclusion there were also significant reductions in OMAD from 14.4 g per day at Basal to 12.0 g per day at Basal × 1.75.This study demonstrated the effect of increasing the concentration of WSC of Lolium perenne in batch culture on the rumen microbial population. Elevation of WSC caused a drop in the pH of the effluent, ammonia-N concentration and an increase in the efficiency of microbial protein synthesis, up to Basal × 1.5, however, this was due to a drop in the organic matter apparently digested. This suggests a switch in substrate specificity of the micro-organisms which may be associated with changes in the microbial population.
[show abstract][hide abstract] ABSTRACT: During the preclinical phase of bovine spongiform encephalopathy (BSE), significantly increased concentrations of lactic acid were measured in the blood of infected dairy cows. Other plasma metabolites, including alanine, leucine, serine, and glutamic acid, also showed significantly altered concentrations in the preclinical BSE animals compared to a control group. This appears consistent with the exaggerated stress response observed in clinical BSE, and precedes the development of clinical signs and overt behavioural changes. A number of other plasma metabolites including other amino acids and components of the plasma fatty acid content showed no association with BSE status.
Research in Veterinary Science 11/2002; 73(2):191-3. · 1.77 Impact Factor
[show abstract][hide abstract] ABSTRACT: This research considers the effects of feedstuff processing (drying, fractionation and particle size) on the resultant fermentation characteristics of the feed. The effect of drying was investigated using fresh perennial ryegrass (Lolium perenne) or samples that had been microwave, freeze or oven dried. Methods of drying had no effect on dry matter (DM) loss, gas and volatile fatty acid (VFA) production profiles, although fresh grass produced significantly less gas (P<0.05) but with similar values for DM loss and VFA production. The effect of particle size was investigated using naked oats (Avena nuda) and perennial ryegrass hay. Naked oats were divided into whole, half, quarter, coarsely- and finely-ground particles, whilst the hay sample was dry-ground through a 4 mm mesh and then fractionated by wet-sieving through the following mesh sizes; 2.4, 1.2, 0.6, 0.3 and 0.15 mm. With the exception of whole naked oats, where fermentation was minimal, there was little difference between gas production profiles for the different particle sizes of naked oats. In contrast, gas production from the hay particles showed distinct differences in that the smallest particles produced most gas with lesser amounts produced as particle size increased. Similarly, DM loss and VFA production were reduced after incubation of the larger hay particles compared to the smaller particles. From these results, we have concluded that sample processing (grinding and fractionation) can have a significant affect on DM loss, VFA production and the gas production profiles obtained using the pressure transducer technique (PTT) and this must be taken into account when extrapolating to the situation in vivo.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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.
Applied and Environmental Microbiology 05/2000; 66(4):1435-43. · 3.68 Impact Factor
[show abstract][hide abstract] ABSTRACT: This paper describes an automated system that has been developed to measure the production of fermentation gas from ruminant livestock feeds inoculated with rumen fluid. The design of the apparatus and its method of use enables gas production to be determined from fresh, unprocessed plant material, as well as the more commonly used ground, particulate substrates, thus representing a closer simulation of forages consumed in vivo. The system consists of 48×140 ml bottles containing 100 ml buffered rumen fluid and 1 g of test substrate. Gas is produced as a consequence of the fermentation of the substrate. Gas, accumulating in the head-space of bottles, is released automatically, by use of pressure sensitive switches and solenoid valves, when a pre-determined pressure is reached. This prevents any build up of pressure in the fermentation bottle, which can affect the behaviour of the gas and the fermentation process. Gas accumulation profiles are produced as the fermentation proceeds and give information on forage digestibility and fermentation kinetics. In this paper, we describe the principles of the gas production technique and provide examples of how the automated system has been used in the evaluation of forages for ruminants. The results obtained show that the automated system is a useful tool for the determination of fermentation kinetics of ruminant feeds. It is simple to use and is considerably less labour intensive than manual gas measurement techniques.
[show abstract][hide abstract] ABSTRACT: The gut fungi are an unusual group of zoosporic fungi occupying a unique ecological niche, the anaerobic environment of the rumen. They exhibit two basic forms, with nuclear migration throughout the hyphal mass for polycentric species and with concentration of nuclear material in a zoosporangium for monocentric species. Differentiation between isolates of these fungi is difficult using conventional techniques. In this study, DNA-based methodologies were used to examine the relationships within and between two genera of monocentric gut fungi gathered from various geographical locations and host animals. The ribosomal ITS1 sequence from 16 mono- and 4 polycentric isolates was PCR-amplified and sequenced; the sequences obtained were aligned with published sequences and phylogenetic analyses were performed. These analyses clearly differentiate between the two genera and reflect the previously published physiological conclusions that Neocallimastix spp. constitute a more closely related genus than the relatively divergent genus Piromyces. The analyses place two type species N. frontalis and N. hurleyensis together but, contrary to a recent suggestion in the literature, place them apart from the other agreed species N. patriciarum. In situ hybridization and slot-blotting were investigated as potential methods for detection of and differentiation between monocentric gut fungi. DNA slot-blot analysis using ribosomal sequences is able to differentiate between gut fungal genera and thus has considerable potential for use in ecological studies of these organisms.