The effect of lipid sources on intake, rumen fermentation parameters and microbial protein synthesis in Nellore steers supplemented with glycerol

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Comparison of fermentation characteristics and bacterial diversity in the rumen of sheep and batch cultures of rumen microorganisms

The objective of the current study was to assess how closely batch cultures (BC) of rumen microorganisms can mimic the dietary differences in fermentation characteristics found in the rumen, and to analyse changes in bacterial diversity over the in vitro incubation period. Four ruminally and duodenally cannulated sheep were fed four diets having forage : concentrate ratios (FCR) of 70 : 30 or 30 : 70, with either alfalfa hay or grass hay as forage. Rumen fluid from each sheep was used to inoculate BC containing the same diet fed to the donor sheep, and the main rumen fermentation parameters were determined after 24 h of incubation. There were differences between BC and sheep in the magnitude of most measured parameters, but BC detected differences among diets due to forage type similar to those found in sheep. In contrast, BC did not reproduce the dietary differences due to FCR found in sheep for pH, degradability of neutral detergent fibre and total volatile fatty acid (VFA) concentrations. There were differences between systems in the magnitude of most determined parameters and BC showed higher pH values and NH3–N concentrations, but lower fibre degradability and VFA and lactate concentrations compared with sheep. There were significant relationships between in vivo and in vitro values for molar proportions of acetate, propionate and butyrate, and the acetate : propionate ratio. The automated ribosomal intergenic spacer analysis (ARISA) of 16S ribosomal deoxyribonucleic acid showed that FCR had no effect on bacterial diversity either in the sheep rumen fluid used as inoculum (IN) or in BC samples. In contrast, bacterial diversity was greater with alfalfa hay diets than those with grass hay in the IN, but was unaffected by forage type in the BC. Similarity index between the bacterial communities in the inocula and those in the BC ranged from 67·2 to 74·7%, and was unaffected by diet characteristics. Bacterial diversity was lower in BC than in the inocula with 14 peaks out of a total of 181 detected in the ARISA electropherograms never appearing in BC samples, which suggests that incubation conditions in the BC may have caused a selection of some bacterial strains. However, each BC sample showed the highest similarity index with its corresponding rumen IN, which highlights the importance of using rumen fluid from donors fed a diet similar to that being incubated in BC when conducting in vitro experiments.

Efficiency of rumen microbial protein synthesis in cattle grazing tropical pastures as estimated by a novel technique

The efficiency of microbial protein synthesis (EMPS) in cattle grazing a range of tropical pasture types was examined using a new method of intra-jugular infusion of CrEDTA to estimate urinary excretion of purine derivatives (PD). Seven pasture types were studied in south-east Queensland, Australia, over a 13-month period. These included native tropical grass (C4) pasture (major species Heteropogon contortus and Bothriochloa bladhii) studied in the early wet, the wet/dry transition and the dry season; introduced tropical grass (C4) pasture (Bothriochloa insculpta) in the mid wet season; two introduced tropical legume species (C3), (Lablab purpureus and Clitoria ternatea); and the temperate grass (C3) pasture, ryegrass (Lolium multiflorum). There was a large range in EMPS across pasture types: 26-209 g microbial crude protein (MCP)/kg digestible organic matter intake (DOMI). Estimated rumen degradable protein (RDP) supply (42-525 g/kg DOMI) was the major factor associated with EMPS across the range of pasture types studied. EMPS in steers grazing all tropical grass pastures was low (<130 g/kg DOMI) and limited by RDP supply. Negative linear relationships (P<0.05) between EMPS and both neutral detergent fibre (NDF) and acid detergent fibre (ADF) concentrations in extrusa were evident. However, non-fibre carbohydrate in extrusa, total non-structural carbohydrate concentration in plucked pasture leaf, rumen fluid and particle dilution rate, protozoal concentration in rumen fluid and rumen fluid pH were not correlated with EMPS. It was concluded that EMPS was well below 130 g MCP/kg DOMI when cattle grazed unfertilised, tropical grass pastures in south-east Queensland and that RDP was the primary limiting nutrient. High EMPS was associated with very high RDP, vastly in excess of RDP requirements by microbes

Ruminal protein degradability of a range of tropical pastures

The rumen degradability parameters of the diet selected by two to four oesophageal-fistulated Brahman steers grazing a range of tropical pastures were determined by incubation of extrusa in nylon bags suspended in the rumen of rumen-fistulated (RF) Brahman steers. The effective protein degradability (Edg) was determined by measuring the rate of disappearance of neutral detergent insoluble nitrogen (NDIN) less acid detergent insoluble nitrogen (ADIN) in the incubated extrusa. Six to eight RF steers also grazed each of the pastures along with the oesophageal-fistulated steers, to allow determination of key rumen parameters and rumen particulate matter fractional outflow rates (FOR). The seven pastures studied included: native tropical grass (C4) pasture (major species Heteropogon contortus and Bothriochloa bladhii), studied in the early wet (NPEW), the wet/dry transition (NPT) and the dry (NPD) seasons; introduced tropical grass (C4) pasture (Bothriochloa insculpta), studied in the mid wet season (BB); the introduced tropical legumes (C3), Lablab purpureus (LL) and Clitoria ternatea (BP); and the temperate grass (C3) pasture, ryegrass (Lolium multiflorum, RG). Using the measured particle FOR values in calculations, the Edg estimates were very high for both C4 and C3 species: 0.82–0.91 and 0.95–0.98 g/g crude protein (CP), respectively. Substitution of an assumed FOR (kp = 0.02/h) for the measured values for each pasture type did not markedly affect estimates of Edg. However, C4 tropical grasses had much lower effective rumen degradable protein (ERDP) fractions (23–66 g/kg DM) than the C3 pasture species RG and LL (356 and 243 g/kg DM, respectively). This was associated with a lower potential degradability and degradation rate of organic matter (OM) in sacco, lower in vitro organic matter digestibility (IVOMD) and CP concentrations in the extrusa, and lower ammonia-N and branched-chain fatty acid concentrations in rumen fluid for the tropical grasses. As tropical grass pastures senesced, there was a decline in Edg, the ERDP and rumen undegradable protein (UDP) fractions, the potential degradability and degradation rate of OM and the IVOMD. These results provide useful data for estimating protein supply to cattle grazing tropical pastures.

Diversity of methanogens in ruminants in Queensland

Methane emissions from ruminant livestock represent a loss of carbon during feed conversion, which has implications for both animal productivity and the environment because this gas is considered to be one of the more potent forms of greenhouses gases contributing to global warming. Many strategies to reduce emissions are targeting the methanogens that inhabit the rumen, but such an approach can only be successful if it targets all the major groups of ruminant methanogens. Therefore, a thorough knowledge of the diversity of these microbes in different breeds of cattle and sheep, as well as in response to different diets, is required. A study was undertaken using the molecular techniques denaturing gradient gel electrophoresis, DNA cloning and DNA sequence analysis to define the extent of diversity among methanogens in ruminants, particularly Bos indicus cross cattle, on differing forages in Queensland. It was found that the diversity of methanogens in forage-fed cattle in Queensland was greater than in grain-fed cattle but there was little variability in methanogen community composition between cattle fed different forages. The species that dominate the rumen microbial communities of B. indicus cross cattle are from the genus Methanobrevibacter, although rumen-fluid inoculated digestors fed Leucaena leucocephala leaf were populated with Methanosphaera-like strains, with the Methanobrevibacter-like strains displaced. If ruminant methane emissions are to be reduced, then antimethanogen bioactives that target both broad groups of ruminant methanogens are most likely to be needed, and as a part of an integrated suite of approaches that redirect rumen fermentation towards other more useful end products.

Differences between the in vitro digestibility of extrusa collected from oesophageal fistulated steers and the forage consumed.

In a study that included C-4 tropical grasses, C-3 temperate grasses and C-3 pasture legumes, in vitro dry matter digestibility of extrusa, measured as in vitro dry matter loss (IVDML) during incubation, compared with that of the forage consumed, was greater for grass extrusa but not for legume extrusa. The increase in digestibility was not caused by mastication or by the freezing of extrusa samples during storage but by the action of saliva. Comparable increases in IVDML were achieved merely by mixing bovine saliva with ground forage samples. Differences were greater than could be explained by increases due to completely digestible salivary DM. There was no significant difference between animals in relation to the saliva effect on IVDML and, except for some minor differences, similar saliva effects on IVDML were measured using either the pepsin-cellulase or rumen fluid-pepsin in vitro techniques. For both C-4 and C-3 grasses the magnitude of the differences were inversely related to IVDML of the feed and there was little or no difference between extrusa and feed at high digestibilities (>70%) whereas differences of more than 10 percentage units were measured on low quality grass forages. The data did not suggest that the extrusa or saliva effect on digestibility was different for C-3 grasses than for C-4 grasses but data on C-3 grasses were limited to few species and to high digestibility samples. For legume forages there was no saliva effect when the pepsin-cellulase method was used but there was a small but significant positive effect using the rumen fluid-pepsin method. It was concluded that when samples of extrusa are analysed using in vitro techniques, predicted in vivo digestibility of the feed consumed will often be overestimated, especially for low quality grass diets. The implications of overestimating in vivo digestibility and suggestions for overcoming such errors are discussed.

Growth and carcass characteristics of cast-for-age Merino ewes fed sorghum-based feedlot diets

Grain feeding low bodyweight, cast-for-age (CFA) sheep from pastoral areas of eastern Australia at the end of the growing season can enable critical carcass weight grades to be achieved and thus yield better economic returns. The aim of this work was to compare growth and carcass characteristics for CFA Merino ewes consuming either simple diets based on whole sorghum grain or commercial feed pellets. The experiment also compared various sources of additional nitrogen (N) for inclusion in sorghum diets and evaluated several introductory regimes. Seventeen ewes were killed initially to provide baseline carcass data and the remaining 301 ewes were gradually introduced to the concentrate diets over 14 days before being fed concentrates and wheaten hay ad libitum for 33 or 68 days. Concentrate treatments were: (i) commercial feed pellets, (ii) sorghum mix (SM; whole sorghum grain, limestone, salt and molasses) + urea and ammonium sulfate (SMU), (iii) SMU + whole cottonseed at 286 g/kg of concentrate dry matter (DM), (iv) SM + cottonseed meal at 139 g/kg of concentrate DM, (v) SMU + virginiamycin (20 mg/kg of concentrate) for the first 21 days of feeding, and (vi) whole cottonseed gradually replaced by SMU over the first 14 days of feeding. The target carcass weight of 18 kg was achieved after only 33 days on feed for the pellets and the SM + cottonseed meal diet. All other whole grain sorghum diets required between 33 and 68 days on feed to achieve the target carcass weight. Concentrates based on whole sorghum grain generally produced significantly (P < 0.05) lower carcass weight and fat score than pellets and this may have been linked to the significantly (P < 0.05) higher faecal starch concentrations for ewes consuming sorghum-based diets (270 v. 72 g/kg DM on day 51 of feeding for sorghum-based diets and pellets, respectively). Source of N in whole grain sorghum rations and special introductory regimes had no significant (P > 0.05) effects on carcass weight or fat score of ewes with the exception of carcass weight for SMU + whole cottonseed being significantly lower than SM + cottonseed meal at day 33. Ewes finished on all diets produced acceptable carcasses although muscle pH was high in all ewe carcasses (average 5.8 and 5.7 at 33 and 68 days, respectively). There were no significant (P > 0.05) differences between diets in concentrate DM intake, rumen fluid pH, meat colour score, fat colour score, eye muscle area, meat pH or meat temperature.

Effect of varying the proportion of molasses in the diet on intake, digestion and microbial protein production by steers

The present experiment was conducted to determine the efficiency of microbial protein production in the rumen and intake by cattle fed high-molasses diets. Intake and microbial crude protein (MCP) production were measured along with the concentration of rumen ammonia-nitrogen (N) and volatile fatty acids (VFA), pH and the rate of digestion of roughage in the rumen. Eight Brahman crossbred steers weighing 211 ± 19.3 (± s.d.) kg were used in a double 4 × 4 Latin square design. Steers were allocated to one of four total mixed rations: control (pangola hay only), 25M (25% molasses/urea mix + 75% hay), 50M (50% molasses/urea + 50% hay), and 75M (75% molasses/urea + 25% hay). The production and efficiency of production of MCP (EMCP) of the diet increased quadratically as the level of molasses in the diet increased. The EMCP from the molasses/urea mix was estimated as 166 g MCP/kg digestible organic matter (DOM), a relatively high value. Intake of dry matter (DM) and DOM increased quadratically, reaching a peak when molasses was ~50% (as fed) of the ration. Digestibility of DM increased quadratically and that of neutral detergent fibre decreased linearly with increasing level of molasses in the diet. Molasses inclusion in the diet had no effect on rumen pH, ammonia and VFA concentration in the rumen fluid, plasma urea-N, urine pH or ruminal fractional outflow rate of ytterbium-labelled particles and Cr-EDTA. It was concluded that a diet with a high level of molasses (>50%) and supplemented with adequate N had high EMCP, and that low MCP production was not a factor limiting intake or performance of cattle consuming high-molasses diets.

Effect of varying the proportion of molasses in the diet on intake, digestion and microbial protein production by steers

The present experiment was conducted to determine the efficiency of microbial protein production in the rumen and intake by cattle fed high-molasses diets. Intake and microbial crude protein (MCP) production were measured along with the concentration of rumen ammonia-nitrogen (N) and volatile fatty acids (VFA), pH and the rate of digestion of roughage in the rumen. Eight Brahman crossbred steers weighing 211 ± 19.3 (± s.d.) kg were used in a double 4 x 4 Latin square design. Steers were allocated to one of four total mixed rations: control (pangola hay only), 25M (25% molasses/urea mix + 75% hay), 50M (50% molasses/urea + 50% hay), and 75M (75% molasses/urea + 25% hay). The production and efficiency of production of MCP (EMCP) of the diet increased quadratically as the level of molasses in the diet increased. The EMCP from the molasses/urea mix was estimated as 166 g MCP/kg digestible organic matter (DOM), a relatively high value. Intake of dry matter (DM) and DOM increased quadratically, reaching a peak when molasses was ∼50% (as fed) of the ration. Digestibility of DM increased quadratically and that of neutral detergent fibre decreased linearly with increasing level of molasses in the diet. Molasses inclusion in the diet had no effect on rumen pH, ammonia and VFA concentration in the rumen fluid, plasma urea-N, urine pH or ruminal fractional outflow rate of ytterbium-labelled particles and Cr-EDTA. It was concluded that a diet with a high level of molasses (>50%) and supplemented with adequate N had high EMCP, and that low MCP production was not a factor limiting intake or performance of cattle consuming high-molasses diets.

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

Kingston-Smith, A. H., Merry, R. J., Leemans, D. K., Thomas, Howard, Theodorou, M. K. (2005). Evidence in support of a role for plant-mediated proteolysis in the rumens of grazing animals. British Journal of Nutrition, 93(1), 73-79. Sponsorship: DEFRA / BBSRC RAE2008

Biomethane production from grass silage: laboratory assessment to maximise yields

On-farm biogas production is typically associated with forage maize as the biomass source. Digesters are designed and operated with the focus of optimising the conditions for this feedstock. Thus, such systems may not be ideally suited to the digestion of grass. Ireland has ca. 3.85 million ha of grassland. Annual excess grass, surplus to livestock requirements, could potentially fuel an anaerobic digestion industry. Biomethane associated with biomass from 1.1 % of grassland in Ireland, could potentially generate over 10 % renewable energy supply in transport. This study aims to identify and optimise technologies for the production of biomethane from grass silage. Mono-digestion of grass silage and co-digestion with slurry, as would occur on Irish farms, is investigated in laboratory trials. Grass silage was shown to have 7 times greater methane potential than dairy slurry on a fresh weight basis (107 m3 t-1 v 16 m3 t-1). However, comprehensive trace element profiles indicated that cobalt, iron and nickel are deficient in mono-digestion of grass silage at a high organic loading rate (OLR) of 4.0 kg VS m-3 d-1. The addition of a slurry co-substrate was beneficial due to its wealth of essential trace elements. To stimulate hydrolysis of high lignocellulose grass silage, particle size reduction (physical) and rumen fluid addition (biological) were investigated. In a continuous trial, digestion of grass silage of <1 cm particle size achieved a specific methane yield of 371 L CH4 kg-1 VS when coupled with rumen fluid addition. The concept of demand driven biogas was also examined in a two-phase digestion system (leaching with UASB). When demand for electricity is low it is recommended to disconnect the UASB from the system and recirculate rumen fluid to increase volatile fatty acid (VFA) and soluble chemical oxygen demand (SCOD) production whilst minimising volatile solids (VS) destruction. At times of high demand for electricity, connection of the UASB increases the destruction of volatiles and associated biogas production. The above experiments are intended to assess a range of biogas production options from grass silage with a specific focus on maximising methane yields and provide a guideline for feasible design and operation of on-farm digesters in Ireland.

In vitro biological activity of tannins from Acacia and other tree fruits: Correlations with colorimetric and gravimetric phenolic assays

This Study was designed to investigate impact of tannins on in vitro ruminal fermentation parameters as well as relationships between concentration and in vitro biological activity of tannins present in tree fruits. Dry and mature fruits of known phenolic content harvested from Acacia nilotica, A. erubescens, A. erioloba, A. sieberiana, Piliostigima thonningii and Dichrostachys cinerea tree species were fermented with rumen fluid in vitro with or without polyethylene glycol (PEG). Correlation between in vitro biological activity and phenolic concentration was determined. Polyethylene glycol inclusion increased Cumulative gas production from all fruit substrates. The largest Increase (225%) after 48 h incubation was observed in D. cinerea fruits while the least (12.7%) increase was observed in A. erubescens fruits. Organic matter degradability (48 h) was increased by PEG inclusion for all tree species except A. erubescens and P. thonningii. For D. cinerea fruits, colorimetric assays were poorly correlated to Increases In gas production due to PEG treatment. Ytterbium precipitable phenolics (YbPh) were also poorly correlated with response to PEG for A. erioloba and P. thonningii fruits. However, YbPh were strongly and positively correlated to the increase In Cumulative gas production due to PEG for A. erubescens and A. nilotica. Folin-Ciocalteau assayed phenolics (SPh) were not correlated to response to PEG in P. thonningii and A. sieberiana. It was Concluded that the PEG effect oil in vitro fermentation was closely related to some measures of phenolic concentration but the relationships varied with tree species.

Alternative methodologies - stretching the in vitro box

Current gas-based in vitro evaluation systems are extremely powerful research techniques. However they have the potential to generate a great deal more than simple fermentation dynamics. Details from four experiments are presented in which adaptation, and novel application, of an in vitro system allowed widely differing objectives to be examined. In the first two studies, complement methodologies were utilised. In such assays, an activity or outcome is inferred through the occurrence of a secondary event rather than by direct observation. Using an N-deficient incubation medium, the increase in starch fermentation, when supplemented with individual amino acids (i.e., known level of N) relative to that of urea (i.e., known quantity and N availability), provided an estimate of their microbial utilisation. Due to the low level of response observed with some arnino acids (notably methionine and lysine), it was concluded, that they may not need to be offered in a rumen-inert form to escape rumen microbial degradation. In another experiment, the extent to which degradation of plant cell wall components was inhibited by lipid supplementation was evaluated using fermentation gas release profiles of washed hay. The different responses due to lipid source and level of inclusion suggested that the degree of rumen protection required to ameliorate this depression was supplement dependent. That in vitro inocula differ in their microbial composition is of little interest per se, as long as the outcome is the same (i.e., that similar substrates are degraded at comparable rates and end-product release is equivalent). However where a microbial population is deficient in a particular activity, increasing the level of inoculation will have no benefit. Estimates of hydrolytic activity were obtained by examining fermentation kinetics of specific substrates. A number of studies identified a fundamental difference between rumen fluid and faecal inocula, with the latter having a lower fibrolytic activity, which could not be completely attributed to microbial numbers. The majority of forage maize is offered as an ensiled feed, however most of the information on which decisions such as choice of variety, crop management and harvesting date are made is based on fresh crop measurements. As such, an attempt was made to estimate ensiled maize quality from an in vitro analysis of the fresh crop. Fermentation profiles and chemical analysis confirmed changes in crop composition over the growing season, and loss of labile carbohydrates during ensiling. In addition, examination of degradation residues allowed metabolizable energy (ME) contents to be estimated. Due to difficulties associated with starch analysis, the observation that this parameter could be predicted by difference (together with an assumed degradability), allowed an estimate of ensiled maize ME to be developed from fresh material. In addition, the contribution of the main carbohydrates towards ME showed the importance of delaying harvest until maximum starch content has been achieved. (c) 2005 Elsevier B.V. All rights reserved.

In vitro microbial inoculum: A review of its function and properties

This review considers microbial inocula used in in vitro systems from the perspective of their ability to degrade or ferment a particular substrate, rather than the microbial species that it contains. By necessity, this required an examination of bacterial, protozoal and fungal populations of the rumen and hindgut with respect to factors influencing their activity. The potential to manipulate these populations through diet or sampling time are examined, as is inoculum preparation and level. The main alternatives to fresh rumen fluid (i.e., caecal digesta or faeces) are discussed with respect to end-point degradabilities and fermentation dynamics. Although the potential to use rumen contents obtained from donor animals at slaughter offers possibilities, the requirement to store it and its subsequent loss of activity are limitations. Statistical modelling of data, although still requiring a deal of developmental work, may offer an alternative approach. Finally, with respect to the range of in vitro methodologies and equipment employed, it is suggested that a degree of uniformity could be obtained through generation of a set of guidelines relating to the host animal, sampling technique and inoculum preparation. It was considered unlikely that any particular system would be accepted as the 'standard' procedure. However, before any protocol can be adopted, additional data are required (e.g., a method to assess inoculum 'quality' with respect to its fermentative and/or degradative activity), preparation/inoculation techniques need to be refined and a methodology to store inocula without loss of efficacy developed. (c) 2005 Elsevier B.V. All rights reserved.

A review and simplification of the in vitro incubation medium

The requirement to rapidly and efficiently evaluate ruminant feedstuffs places increased emphasis on in vitro systems. However, despite the developmental work undertaken and widespread application of such techniques, little attention has been paid to the incubation medium. Considerable research using in vitro systems is conducted in resource-poor developing countries that often have difficulties associated with technical expertise, sourcing chemicals and/or funding to cover analytical and equipment costs. Such limitations have, to date, restricted vital feed evaluation programmes in these regions. This paper examines the function and relevance of the buffer, nutrient, and reducing solution components within current in vitro media, with the aim of identifying where simplification can be achieved. The review, supported by experimental work, identified no requirement to change the carbonate or phosphate salts, which comprise the main buffer components. The inclusion of microminerals provided few additional nutrients over that already supplied by the rumen fluid and substrate, and so may be omitted. Nitrogen associated with the inoculum was insufficient to support degradation and a level of 25 mg N/g substrate is recommended. A sulphur inclusion level of 4-5 mg S/g substrate is proposed, with S levels lowered through omission of sodium sulphide and replacement of magnesium sulphate with magnesium chloride. It was confirmed that a highly reduced medium was not required, provided that anaerobic conditions were rapidly established. This allows sodium sulphide, part of the reducing solution, to be omitted. Further, as gassing with CO2 directly influences the quantity of gas released, it is recommended that minimum CO, levels be used and that gas flow and duration, together with the volume of medium treated, are detailed in experimental procedures. It is considered that these simplifications will improve safety and reduce costs and problems associated with sourcing components, while maintaining analytical precision. (c) 2005 Elsevier B.V. All rights reserved.