Final Report Summary - REDNEX (Innovative and practical management approaches to reduce nitrogen excretion by ruminants)
Executive Summary:
Dairying is an important sector of EU agriculture, but intensification has been accompanied by an increase in N surplus. This has a negative environmental impact on groundwater (pollution with nitrates), surface water (eutrophication) and on the atmosphere (de-nitrification and ammonia volatilisation). The EU seeks to stimulate measures that improve management of nutrients, waste and water as a start to move to management practices beyond “usual good-farming practice”. The objective of RedNEx has been to develop innovative and practical management approaches for dairy cows that reduce N excretion into the environment through the optimization of rumen function, an improved understanding and prediction of dietary N utilization for milk production and excretion in urine and faeces. Novel tools for monitoring these processes and predicting the consequences in terms of N losses on–farm have been developed. At the centre of the project are two mathematical models. One model of N utilization by the cow acts to integrate results from previous work and from new research carried out in the project. This interlinked research aims to improve the supply of amino acids to be absorbed relative to the quantity and quality of amino acids and carbohydrates in feed allowing a reduction in N intake. Research to understand amino acid absorption, intermediary utilization and the processes involved in the transfer of urea N from blood to the gastro-intestinal tract has further underpinned model development and indicate strategies to reduce N losses. To predict N losses on-farm and the impact on profitability, a harmonised applied model has been developed . Impact of the research has been enabled by dissemination and knowledge interaction using a participatory approach to include the views of stakeholders and recognition of the need to provide support to EU neighbours.
Problem (background):
In the EU, 25 million dairy cows produce more than 130.1012 kg of milk per year, providing a total annual income of the 2.8 million dairy farmers in the EU of more ca. € 40.109. However, dairying within the EU is becoming more intensive and more specialised. As intensification increases, the level of application of (N) fertilisers and manures usually rises to levels that are greater than crop requirements or than the ability of the soil to retain them. Increasing milk yield from 8,700 kg to 20,500 kg per hectare in Dutch farming situations, raised the N surplus from 376 to 650 kg of N per hectare. Thus, an increased intensification of dairy production and hence N use will have potentially impact on groundwater (pollution with nitrates) and surface water (eutrophication) as well as on the atmosphere (de-nitrification and ammonia volatilisation). Therefore measures that improve management of nutrients, waste and water should be implemented. One step to improve nutrient management is EU Council Directive 91/676/EEC aiming at reducing water pollution caused or induced by nitrates from agricultural sources and at preventing further such pollution. EU Council Directive 91/676/EEC obliges Member States to establish action programmes to realize the aims of this directive. RedNEx contributes to the scientific and technical data that are required to impose measures to reduce N input and consequently N losses during the production of milk and meat by ruminants and provides a methodology to translate this knowledge into practice.
Aim (objectives):
The objective of the RedNEx project was to develop innovative and practical management approaches for dairy cows that reduce N excretion into the environment through the optimization of rumen function, an improved understanding and prediction of dietary N utilization for milk production and excretion in urine and faeces, and the development of novel tools for monitoring these processes. The central focus of the project was to provide the dairy production sector with approaches that will reduce N input on dairy farms without a substantial reduction in N output via consumable proteins in the form of milk. The specific objectives within this project were:
• develop standardised rapid tools to measure fermentation characteristics of feeds
• improve feeding strategies to provide low-N rations to high-producing dairy cattle
• better understanding of amino acid absorption, metabolism and secretion in milk
• stimulate N recycling within the animal while reducing total N inputs
• developing biomarkers in urine, plasma or milk to monitor the nutrient status in dairy cows
• develop and expand predictive models of N output at cow and herd levels
• disseminate knowledge of N management on dairy farms
Results
Rapid analytical techniques (FTIR and NIR spectroscopy) have been successfully developed to estimate degradation characteristics of protein and fibre. For starch degradation characteristics rapid techniques have given reasonable results. To obtain reasonable predictions of degradation rates by using NIRS it was necessary to divide feeds into forages and non-forages.
Lowering dietary N far below present recommendations decreased OM and fibre digestibility and consequently energy intake and animal performance. The rumen microbial population does not adapt to protein underfeeding by increasing the efficiency of microbial protein synthesis.
Attempts to reduce the protein degradation by influencing the ruminal microbial population were unsuccessful.
Compared to feeding fibre-rich diets, feeding starch-rich diets resulted in a more than 8% increase in N use efficiency (milk N output / N intake) in dairy cattle. Correcting the amino acid profile of metabolisable protein to obtain an “ideal” amino acid profile increased the efficiency of metabolisable protein, but results were inconsistent.
Balancing the amino acid profile of metabolisable protein allows for lowering dietary N content to some extent, resulting in reduced N excretion, but other correlating factors influence the response on animal performance.
A large proportion of ammonia hydrolysed from recycled urea never reaches the rumen pool, and this may be an obstacle to the use of recycled urea as a method to improve N efficiency of dairy cattle. Dairy cows appear unable to increase transport of urea from blood to gastrointestinal tract in periods with low rumen ammonia concentrations and thus urea transport is apparently not effective in buffering infrequent N supply.
Advanced mechanistic models of gut wall and liver metabolism were successfully developed and evaluated.
Analysis of samples using a variety of analytical methods such as chromatography, spectroscopy and nuclear magnetic resonance spectroscopy, has been done in the search for new biomarkers of the efficiency of N utilisation and N sufficiency. This has highlighted the use of previous biomarkers, such as purine derivative excretion in urine and the secretion of odd and branched-chain fatty acids in milk, as well has indicating the potential of several new markers.
N excretion per cow may vary from 107 to 138 kg per year, in relation to annual N content in the diet and level of production. Nitrogen excretion per 1000 kg of milk varied from 10 to 18 kg and this variation is significantly linked to urinary N excretion. These results clearly show that it is possible to make significant progress for reducing N output by a dairy herd.
Potential applications:
Two mathematical models resulted from this project.
One model will give insight in protein flows within the dairy cow, which will be a helpful tool for nutritionists in formulating low protein diets without disturbing essential pathways and processes in the digestion, absorption and metabolism of amino acids.
COWNEX estimates the effect of herd and feeding management on the excretion of nitrogen at herd level. This can be used as a tool for strategic and operational management and as an estimator of nitrogen utilisation.
Two other tools to be used by farmers and nutritionist have been developed.
Rapid analytical methods have been developed to measure the nutritive value of forages and other feed material. Parameters for a more precise estimation of the nutritive value include ruminal degradation characteristics for protein, starch and fibre. This can be incorporated in “precision feeding” approaches and smaller “insecurity” margins in diet formulation.
Indicators have been developed to measure the response of dietary changes concerning protein utilisation. Although milk urea has been used as an indicator of surplus dietary nitrogen already before the start of the RedNEx project, new indicators also provide more control at protein utilisation.
Project Context and Objectives:
Dairying is an important sector of EU agriculture, but intensification is accompanied by an increase in nitrogen surplus in the nutrient cycle on dairy farms. This nitrogen surplus has a negative environmental impact on groundwater (pollution with nitrates), surface water (eutrophication) and on the atmosphere (ammonia volatilisation, nitrous oxide). The EU seeks to stimulate measures that improve management of nutrients, waste and water as a start to move to management practices beyond “usual good farming practice”.
The objective of the EU-FP7 project REDNEX was to develop innovative and practical management approaches for dairy cows that reduce N excretion into the environment through the optimization of rumen function, an improved understanding and prediction of dietary N utilization for milk production, and excretion in urine and faeces. The project developed more-accurate feed characterisation tools and a detailed mathematical model of N utilisation by the cow to integrate results and aimed to improve the supply and efficiency of absorbed amino acids, allowing a reduction in N intake. Practical tools have been developed to predict N losses at a whole-herd level and to monitor the cow’s N status using biomarkers. Dissemination and knowledge interaction have been used in a participatory approach to include the views of stakeholders and recognition of the need to provide support to EU neighbours.
Project Results:
Description of work performed and main results
Work has been performed on feed characterisation, ruminal protein digestion, conversion of metabolisable protein into milk, urea recycling, mechanistic modelling of amino acid metabolism, biomarkers to monitor rumen protein supply and predicting and reducing urinary nitrogen excretion.
Feed characterisation
Precision feeding of ruminants requires an accurate prediction of ruminal behaviour of feed components (protein, cell walls, and starch). Usually, ruminal behaviour is predicted from the chemical composition and established relationships between chemical composition and degradation characteristics are obtained by using in situ incubations. Without chemical analyses standard chemical composition from feed tables is used.
Within REDNEX (1) the database on ruminal degradation characteristics of feed components has been expanded not only for protein, but also for fibre and starch, (2) prediction of ruminal behaviour of these feed component from the chemical composition of feeds have been further improved and (3) cheap and rapid techniques (near-infrared reflection spectroscopy – NIRS; Fourier transform infrared spectroscopy – FTIR; nuclear magnetic resonance - NMR) to improve the accuracy of feed evaluation have been developed and evaluated.
Ruminal protein digestion
Lowering dietary protein content below present recommendations decreases organic matter and fibre digestibility, energy intake and animal performance. One approach to minimize these negative responses is to improve protein digestion in the rumen by reducing microbial degradation of feed protein or by improving ruminal microbial protein synthesis.
Various attempts were made to reduce ruminal protein hydrolysis and deamination. A preliminary analysis of the literature showed that the most efficient additives for decreasing ruminal protein degradation are various essential oils, but their effect in vivo remained unclear. A first innovative approach was the inclusion of essential oils in silages. Essential oils in ryegrass silage are efficient in reducing silage protein degradation but the dose required and thus the costs may be too high for practical use. A second innovative approach was the inclusion of polyclonal antibodies against ruminal hyper ammonia producing bacteria. Several attempts failed to decrease protein degradation. As these innovative ways were not efficient, the most promising ways arising from literature analysis were used. Capsicum oil and propyl-propyl thiosulphate, derived from garlic oil, were used. Both essential oils tended to increase nitrogen use efficiency, but these results need to be confirmed with more animals.
A first attempt to optimise microbial protein synthesis in the rumen was by modifying the nature of forages. In vitro, microbial protein yield did not differ between ryegrass and red clover, but microbes were more efficient with ryegrass for capturing N and in efficiency of N utilization in the rumen. Comparison of varieties (ryegrass varying in sugar content, red clover varying in polyphenol oxidase) needs further research. The effect of source of dietary carbohydrate (starch or fibre) on the response of reducing dietary crude protein was tested in another in vivo experiment.. The source of dietary carbohydrates, starch or fibre, had a minor effect on ruminal protein metabolism; a trend to a higher microbial protein flow and efficiency of synthesis was observed with starch. In another experiment the effect of niacin supplementation to low-protein diets was studied.
Reducing dietary crude protein tended to decrease microbial protein synthesis. Niacin supplementation did not change microbial protein flow and efficiency of synthesis but increased rumen protozoa population.
Ruminal dietary protein use efficiency is reduced by the presence of protozoa. On one hand ruminal protozoa generate an extra nitrogen loss by devouring ruminal bacteria but on the other hand protozoa play a role in fibre degradation. Therefore, the microbial ecosystem, especially protozoa, was further characterised for a better knowledge of the role of different populations. The role of different protozoa in bacterial breakdown has been specified. Entodinium and Epidinium are especially active, whereas Holotrichs have a minor predatory activity. Therefore, lowering numbers of Entodiniomorphids in the rumen may be a strategy for improving microbial synthesis. In normal- or low-protein diets, defaunation (or faunation with an Holotrich species) did not change rumen ammonia in sheep suggesting a better use of N by rumen microbes, which results in a lower urinary N. Cows are able to adapt themselves to fibrous diets by increasing the complexity of the rumen microbial community and the concentrations of protozoa, anaerobic fungi, and methanogens. The identification of key bacteria involved in protein metabolism by using DNA-Stable Isotope Probing was faced with large challenges and results were inconsistent.
Conversion of metabolisable protein into milk
Within REDNEX, experimental investigations addressing hypotheses concerning the effects of dietary carbohydrate and metabolisable protein supply on the metabolism of nutrients by the splanchnic tissues of lactating dairy cows have been conducted at the University of Reading and at INRA Theix. The work at Reading focused on providing metabolisable protein at three levels of predicted supply, below, at, and above requirement, with basal diets formulated to contain differing amounts of maize or grass silage as forage sources. The work at INRA Theix used a similar approach, with diets formulated at two levels of metabolisable protein and basal diets containing differing types of highly digestible carbohydrates, testing the potential effect of energy yielding substrates arising from the digestion of starch- or fibre-based concentrates on nutrient metabolism and the efficiency of dietary protein utilization. Related work at INRA Rennes used a duodenal infusion protocol to further define the ideal essential amino acid profile in metabolisable protein in terms of requirements for arginine, isoleucine, and valine for milk protein synthesis. Subsequent studies determined the effects of providing an ideal profile of essential amino acids when metabolisable protein was provided at levels below and above requirement to determine if the response to amino acid profile was altered by metabolisable protein supply relative to requirement and the extent to which nutrient metabolism by the mammary gland was affected.
The focus of the experiments was to improve understanding of amino acid and nutrient absorption and utilization in lactating dairy cows and in so doing describe the impact of amino acid supply relative to requirement and dietary carbohydrate source on the utilization of nutrients for milk protein production and N excretion in urine and faeces. A second objective was to improve our understanding of the optimum balance of essential amino acids for milk protein production by dairy cows and how amino acid balance impacts mammary gland metabolism. The descriptions of the integrated responses observed will be of value in the development of models of dietary nutrient utilization and the development and refinement of dietary strategies that improve the efficiency of dietary nitrogen utilization.
Concerning the effects of carbohydrate sources in the concentrates fed to lactating dairy cows, results from 3 experiments using similar diets (two levels of metabolisable protein with high starch or high fibre concentrates) at INRA Theix were aggregated for analysis of production response and the efficiency of dietary nitrogen utilization. Overall, diets rich in starch significantly increased dietary protein use efficiency compared to diets rich in fibre, without a significant interaction between concentrate carbohydrate and level of metabolisable protein supply. This suggests a higher microbial protein supply to the small intestine than the theoretical values with diets rich in starch vs. fibre. Measurements of nutrient absorption and metabolism confirm that the improved efficiency of dietary nitrogen utilization for higher starch diets was associated with differences in nutrient absorption, including greater glucose and lower acetate absorption, as well as greater amino acid absorption at lower dietary protein concentration. These findings confirm that a part of the starch effect was attributable to greater metabolisable protein supply, which likely reflects greater microbial protein synthesis. However, a reduction in the relative rate of removal of amino acids by the liver was also observed, confirming that the positive effects of starch on dietary nitrogen utilization were also attributable to differences in post-absorptive metabolism. Results suggest that the higher milk N yield found with diets rich in starch vs fibre could be the result of both digestive (likely via a higher microbial protein synthesis) and metabolic (via a decrease in liver AA uptake) adaptations.
As regards the effects of forage carbohydrate type and metabolisable protein supply, the experiment at the University of Reading found increasing dietary protein concentration linearly increased feed intake, milk yield, and milk protein production, but decreased dietary nitrogen use efficiency. In contrast forage source had no effect on feed intake, milk production or dietary nitrogen use efficiency, but diets were formulated to minimize differences in total starch and fibre concentrations across treatments. Importantly and as for the effects of concentrate type, there were no interactions between forage carbohydrate type and dietary protein supply in terms of production responses and dietary nitrogen use efficiency. With increasing dietary protein there were linear increases in rumen ammonia concentration, net absorption of ammonia, liver production of urea, and concentrations of urea in arterial blood, milk, and urine, as well as arterial concentrations of most essential and many nonessential amino acids. In addition, the increase in feed intake and milk yield with increased dietary protein supply was associated with a linear increase in liver production of glucose and β-hydroxybutyrate. There was a positive relationship between N intake and rumen ammonia, arterial urea, and milk urea concentration, all of which were negatively related to N utilization efficiency.
Work on the effects balancing essential amino acid profile, found there were no important interactions between the effect of increasing metabolisable protein supply and the effect of balancing the EAA profile. As in other experiments for WP3, increasing metabolisable protein supply increased milk protein yield, but increased nitrogen absorption and arterial urea concentration and decreased dietary nitrogen use efficiency. In contrast, balancing amino acid profile increased milk protein yield, but at equal nitrogen absorption, thus dietary protein use efficiency was increased. This was associated with increased arterial concentrations of most essential amino acids, but decreased arterial concentration of urea. In both cases, increased milk protein yield was associated with increased uptake of essential amino acids by the mammary gland, but not nonessential amino acids. Balancing the essential amino acid profile increased milk protein yield and dietary protein use efficiency at both levels of metabolisable protein supply by increasing mammary uptake of essential amino acids and adjusting mammary uptake of nonessential amino acids, leading to less availability of ‘excess’ amino acids for catabolism and urea synthesis.
Other research examined the milk protein responses to changes in the supply of arginine, isoleucine, and valine relative to other essential amino acids. The study found that the concentration of arginine currently recommended for metabolisable protein was not limiting. In contrast, changing the supply of isoleucine altered the milk protein to fat ratio, whilst there was evidence that valine may be limiting milk protein synthesis when requirements for methionine, lysine, and histidine are met. However, the effects of valine and isoleucine in this experiment may be related to the concentration of leucine. Therefore, in future it is proposed that possible antagonisms between the branched chain amino acids should be considered when establishing their requirements.
Urea recycling
To expand our understanding of the mechanisms regulating urea recycling across the portal-drained visceral (PDV) tissue and ruminal epithelia, five experiments with multi-catheterized and rumen-cannulated lactating Holstein cows were conducted at the intensive barn facility at Department of Animal Science, Aarhus University, Foulum, Denmark. More knowledge on the physiological adaptation and the functional properties of urea transport across the PDV and ruminal epithelium is a valuable tool in order to be able to model the internal N cycling and to investigate if the expectations we have for urea recycling in increasing N utilization can be kept alive.
Evaluating the regulatory effect of varying N intake on the functional properties of urea recycling from these intensive studies, the conclusion is clear: the total amount of urea transferred from blood to GI tract, including the rumen is not increased with decreasing N supply. In fact the opposite seems to be the trend, that is, increased urea transfer with increasing N supply. Still, an actual up-regulation of the extraction of arterial urea across the ruminal and PDV epithelia with decreasing dietary N supply was evident, and is an indication that decreasing N supply will increase the permeability of the epithelium for urea and that the permeability of the epithelia adapts to given dietary conditions. However, this increase in the permeability of the epithelium for urea is insufficient to compensate for the concomitant decreases in blood urea concentrations. Hence, one of the main limiting factors for utilizing the ability of the dairy cow to recycle urea in order to increase the N efficiency is that the permeability of the epithelium for urea appears to start declining, or is not up-regulated enough when N status of the cow is still insufficient to sustain optimal rumen microbial synthesis and production performance. Thus, urea recycling had no potential for compensating low N supply to maintain production performance neither when cows were fed varying N concentration nor with infrequent N supplementation over the day. Based on this, we conclude that the regulatory mechanism of urea transport appears only partly effective or controlled by something else than the ammonia requirements of ruminal fermentation.
Efforts to manipulate urea recycling by increasing the pull of urea on the hindgut via abomasal oligofructose infusion for hindgut fermentation resulted in decreased blood urea concentration which could not be explained by increased urea recycling but appeared instead to be a result of increased sequestration of ammonia in the hindgut and thus a relative increased N excretion in faeces. Increased hindgut fermentation did not impair rumen fermentation and shifted some N excretion from urine to faeces.
Using dietary NaCl to increase water intake and to possible increase the pull of urea on the kidneys to compete with urea recycling to the GI tract did not lead to changes in total urea recycling, nor in blood urea concentration, and urinary urea excretion. Dietary adaptation of the kidneys to high NaCl supply reduced the effect on blood urea concentration, indicating that the priority of the kidneys is to keep circulating amounts of urea within a given homeostatic range. Ruminal fermentation was not impaired by high dietary supply NaCl. Milk urea concentration was unchanged and ECM increased slightly by high dietary NaCl supply.
It has not been possible to detect any up-regulation of the urea transporter protein, UT-B which is speculated to be involved in the regulation of the permeability of the epithelia for urea with decreasing N supply. Nor could any of the investigated aquaporins explain the observed changes in rumen epithelial permeability for urea.
Mechanistic modelling of amino acid metabolism
Based on literature data and on the observations within REDNEX studies on amino acid utilisation and urea recycling, mechanistic models of amino acid metabolism in gut wall, liver and mammary gland of dairy cattle were developed and subsequently integrated into a single modelling framework with the nutrient flows between the models connected accordingly. A user interface has been developed and programmed to allow the integrated post-absorptive model to be used by a broad audience.
Biomarkers to monitor rumen protein supply
Blood, milk and urine samples were collected within the in vivo experiments of the REDNEX project and samples were analysed using a number of methodologies with the aim of confirming the use of established biomarkers and discovering new and novel biomarkers of dietary protein use efficiency and rumen N sufficiency. Targeted analyses (i.e. of known compounds) has helped validate the use of odd and branched chain fatty acid (OBCFA) secretion in milk and of purine derivative excretion in urine from lactating dairy cows. Both of these markers have been demonstrated to be related to the duodenal flow of microbial protein from the rumen, and therefore offer non-invasive estimation of rumen N use. Metabolomic, or untargeted, analysis of samples by nuclear magnetic resonance spectroscopy, Fourier-transform infrared spectroscopy, and various forms of chromatography coupled with mass spectrometry, has demonstrated that various compounds correlate well with duodenal microbial protein flow. These include compounds previously known to be of such use, e.g. purine derivatives, and also compounds not previously known to correlate this way, e.g. para-cresol sulphate. Some currently unidentified compounds have also been found to be of potential use, but these require further work to confirm their identities and validate their utility.
Predicting and reducing urinary nitrogen excretion
Within REDNEX, a large database was produced comprising data of most N balance trials conducted in Europe on dairy cows giving new insights on the fate of N intake. N excreted in faeces is primarily a function of DM intake, the effect of DM intake being higher for grass silage-based diets and high-concentrate diets than for fresh grass and maize silage-based diets whereas N excreted in urine primarily depends of total N intake for a given level of milk yield. Some nitrogen is not recovered in milk, faeces or urine. This difference is partly explained by animal characteristics (pregnancy and N retention as affected by the energy balance); external temperature as losses of N as urea excreted in sweat may occur and by some methodological problems.
Reduce dietary protein content without affecting performances, depends on various factors which resulted in variable responses in the experiments performed within REDNEX. In some experiments it was possible to compensate the reduction in protein supply by adjusting the essential amino acid profile, but the room to manoeuvre to economize protein utilisation remains limited in most of the current situations. In another experiment the reduction in dietary protein content reduced dry matter (energy intake) and consequently milk (protein) yield. Supplementing the low-protein diets with methionine and lysine, partly compensated the reduction in dry matter intake, but had no significant effect on milk (protein) secretion.
A simulator (CowNex) was developed to calculate protein synthesis in milk and meat and nitrogen excretion in dairy herd, including heifers, depending on herd management. The simulator performs these calculations throughout the year and measures the impact of management changes, particularly nutrition, on the dietary protein use efficiency by the herd for milk and meat production and the release of faecal or urine nitrogen. The amount of nitrogen to manage as manure is considered separately from the quantities directly returned to pasture.
Knowledge dissemination
The main mission of knowledge dissemination in REDNEX is to ensure that the project and its results meet the needs of the end-users, i.e. those that are involved in dairy production. This is reached by creating a participatory framework that will allow meaningful dialogue between the Work Package coordinators and the stakeholders and to transfer the information from the project to the intended stakeholders / end-users in an effective manner. Another objective is to extend the consultation and dissemination to newly-entered and candidate states and to train professionals from these countries.
Stakeholder platform
Representatives of the animal feed industry and dairy farmers, and representatives involved in mathematical modelling of biological processes, environment impact of livestock production, animal feed analyses, soil N cycling, technology transfer, and of dairy systems participate in the REDNEX Stakeholder platform. This platform is used to gain advice and feedback from the stakeholders to assist in the development of the research and dissemination process. Individual platform members have been linked with work packages to stimulate the interaction between researchers and end-users. Brian Cook, one of the members of the platform, passed away 8 May 2012.
Websites
A public website www.rednex-fp7.eu has been created and maintained. At this website, presentations of all REDNEX scientific meetings can be accessed and the link to CowNex can be found. The website also contains publishable summaries of public REDNEX deliverables.
Meetings
REDNEX organised and supported eight scientific meetings: at Vilnius, Lithuania (EAAP satellite workshop; 23 August 2008), Barcelona, Spain (EAAP Session, 24 August 2009), Heraklion, Greece (EAAP Session, 24 August 2010), Parma, Italy (ISEP Session, 6 September 2010), Stavanger, Norway (EAAP Session, 23 August 2011), Aberystwyth (oral and poster presentations at 8th ISNH, September 2011) , Bratislava (EAAP Session, 27 August 2012), and Zaragoza (Course on Environmental Assessment of Livestock production Systems, April 2013) The project was completed with a final conference in Paris on 20 August 2013. It was attended by 109 delegates, including 19 early career scientists supported by the project.
Publications
Besides abstracts and other contributions in conference proceedings, articles in regional and national magazines, more than 35 research papers have been submitted or published in peer-reviewed international scientific journals.
Dissemination to newly-entered and candidate EU states
The project has financially (up to € 1,000 per person) supported 63 early career scientists from Central and Eastern Europe and the Mediterranean to attend the REDNEX meetings and to act as contacts for future dissemination of REDNEX research within their region.
Regional meetings were organised in Croatia, Romania and Lithuania involving a total of 366 delegates and supporting 12 early career scientists from disadvantaged countries to attend. The discussions agreed that the project had made an important contributing to reducing the environmental impact of milk production. The main barriers to translation of this research were the low herd size. Standardisation of feeding systems supported by low cost rapid feed analysis were seen as a priority together with better links between research organisations and the feed industry.
Potential Impact:
IMPACT
Feed characterisation
Results from REDNEX on degradation rates for starch and part of the model for starch digestion have been implemented in the NorFor ration evaluation system. A number of feed samples have been examined for in situ degradation characteristics of protein, which will be incorporated in feed tables. The NIRS calibrations for degradation parameters for protein will be published and made freely available for the feed industry.
Ruminal protein digestion
Although some additives (essential oils) may reduce the microbial degradation of feed protein in the rumen, REDNEX obtained insufficient data to confirm this. Reducing crude protein in dairy cow rations to 11-12% of DM resulted in a moderate decrease in milk production under some circumstances. Reducing dietary crude protein had no effect on microbial protein synthesis per unit of fermentable organic matter and it was therefore concluded that the rumen microbial population does not adapt to protein underfeeding by increasing the efficiency of microbial protein synthesis. Rumen protozoa, fungi, methanogens and certain bacterial species are sensitive to N shortage which can result in a decrease in OM digestibility.
Conversion of metabolisable protein into milk
Results demonstrate that improvements in dietary protein use efficiency attributable to supplemental starch are not due solely to digestive and fermentation responses, but also due to metabolic effects on the utilization of absorbed nutrients. Importantly, there were few interactions observed between dietary carbohydrate and protein supply, which has implication for diet formulation strategies.
Current recommendations for the balance of essential amino acid concentrations in metabolisable protein are sufficient for arginine, but that potential antagonisms exist for the branched chain amino acids that should be described more clearly and may have implications for their recommended concentrations.
Even at low metabolisable protein supply, balancing the amino acid profile increased nitrogen use efficiency. The is explained by an increased uptake of essential amino acids by the mammary gland. This suggests that amino acid supply can be expressed in percentage of metabolisable protein regardless of the level of protein supply.
Urea recycling
The amount of urea recycled showed to be relatively unaffected by even major changes in dietary protein concentrations. Therefore, the possibilities for increasing dietary protein use efficiency of the dairy cow through urea recycling by dietary N intervention strategies seem limited. The outcome of this series of elegant intensive studies performed prevents to formulate a new feeding strategy that utilize urea recycling, although this was the original idea at the start of the project.
Mechanistic modelling of amino acid metabolism
The integrated post-absorptive model that was developed may potentially replace current empirical protein evaluation systems. The integrated model enhances understanding of N utilization, assist researchers in developing diets to improve N utilization by cattle and promote reductions in N excretion to the environment.
Biomarkers to monitor rumen protein supply
Across all the studies the clear relationship between dietary protein use efficiency and urea concentrations in blood, milk, and urine confirm the potential value of urea measurement as management tool for improving dietary protein use efficiency on farm. Multivariate analysis (especially partial least squares analysis) of other metabolomics data has demonstrated the ability to predict with reasonable accuracy parameters such as dietary protein use efficiency for milk production, and urinary N excretion, but more work is required to confirm these results.
The ability of a dairy farmer to estimate dietary protein use efficiency from a simple blood, milk or urine sample, particularly following a change in feeding regime, has significant potential in helping to refine dairy cow diet formulation, optimising diet nutrient use, and reducing N excretion from dairy cows and biomarkers to monitor rumen protein supply.
Predicting and reducing urinary nitrogen excretion
In the validating experiments, reducing dietary protein resulted in a lower milk protein yield, but nevertheless improved dietary protein use efficiency and reduced urinary urea excretion. The developed CowNex software to estimate N balances on a herd level can be accessed without restriction via an Internet network with a multilingual Web interface (French, English) at the address: http://www.cownex-record.inra.fr/.
List of Websites:
http://www.rednex-fp7.eu
Dairying is an important sector of EU agriculture, but intensification has been accompanied by an increase in N surplus. This has a negative environmental impact on groundwater (pollution with nitrates), surface water (eutrophication) and on the atmosphere (de-nitrification and ammonia volatilisation). The EU seeks to stimulate measures that improve management of nutrients, waste and water as a start to move to management practices beyond “usual good-farming practice”. The objective of RedNEx has been to develop innovative and practical management approaches for dairy cows that reduce N excretion into the environment through the optimization of rumen function, an improved understanding and prediction of dietary N utilization for milk production and excretion in urine and faeces. Novel tools for monitoring these processes and predicting the consequences in terms of N losses on–farm have been developed. At the centre of the project are two mathematical models. One model of N utilization by the cow acts to integrate results from previous work and from new research carried out in the project. This interlinked research aims to improve the supply of amino acids to be absorbed relative to the quantity and quality of amino acids and carbohydrates in feed allowing a reduction in N intake. Research to understand amino acid absorption, intermediary utilization and the processes involved in the transfer of urea N from blood to the gastro-intestinal tract has further underpinned model development and indicate strategies to reduce N losses. To predict N losses on-farm and the impact on profitability, a harmonised applied model has been developed . Impact of the research has been enabled by dissemination and knowledge interaction using a participatory approach to include the views of stakeholders and recognition of the need to provide support to EU neighbours.
Problem (background):
In the EU, 25 million dairy cows produce more than 130.1012 kg of milk per year, providing a total annual income of the 2.8 million dairy farmers in the EU of more ca. € 40.109. However, dairying within the EU is becoming more intensive and more specialised. As intensification increases, the level of application of (N) fertilisers and manures usually rises to levels that are greater than crop requirements or than the ability of the soil to retain them. Increasing milk yield from 8,700 kg to 20,500 kg per hectare in Dutch farming situations, raised the N surplus from 376 to 650 kg of N per hectare. Thus, an increased intensification of dairy production and hence N use will have potentially impact on groundwater (pollution with nitrates) and surface water (eutrophication) as well as on the atmosphere (de-nitrification and ammonia volatilisation). Therefore measures that improve management of nutrients, waste and water should be implemented. One step to improve nutrient management is EU Council Directive 91/676/EEC aiming at reducing water pollution caused or induced by nitrates from agricultural sources and at preventing further such pollution. EU Council Directive 91/676/EEC obliges Member States to establish action programmes to realize the aims of this directive. RedNEx contributes to the scientific and technical data that are required to impose measures to reduce N input and consequently N losses during the production of milk and meat by ruminants and provides a methodology to translate this knowledge into practice.
Aim (objectives):
The objective of the RedNEx project was to develop innovative and practical management approaches for dairy cows that reduce N excretion into the environment through the optimization of rumen function, an improved understanding and prediction of dietary N utilization for milk production and excretion in urine and faeces, and the development of novel tools for monitoring these processes. The central focus of the project was to provide the dairy production sector with approaches that will reduce N input on dairy farms without a substantial reduction in N output via consumable proteins in the form of milk. The specific objectives within this project were:
• develop standardised rapid tools to measure fermentation characteristics of feeds
• improve feeding strategies to provide low-N rations to high-producing dairy cattle
• better understanding of amino acid absorption, metabolism and secretion in milk
• stimulate N recycling within the animal while reducing total N inputs
• developing biomarkers in urine, plasma or milk to monitor the nutrient status in dairy cows
• develop and expand predictive models of N output at cow and herd levels
• disseminate knowledge of N management on dairy farms
Results
Rapid analytical techniques (FTIR and NIR spectroscopy) have been successfully developed to estimate degradation characteristics of protein and fibre. For starch degradation characteristics rapid techniques have given reasonable results. To obtain reasonable predictions of degradation rates by using NIRS it was necessary to divide feeds into forages and non-forages.
Lowering dietary N far below present recommendations decreased OM and fibre digestibility and consequently energy intake and animal performance. The rumen microbial population does not adapt to protein underfeeding by increasing the efficiency of microbial protein synthesis.
Attempts to reduce the protein degradation by influencing the ruminal microbial population were unsuccessful.
Compared to feeding fibre-rich diets, feeding starch-rich diets resulted in a more than 8% increase in N use efficiency (milk N output / N intake) in dairy cattle. Correcting the amino acid profile of metabolisable protein to obtain an “ideal” amino acid profile increased the efficiency of metabolisable protein, but results were inconsistent.
Balancing the amino acid profile of metabolisable protein allows for lowering dietary N content to some extent, resulting in reduced N excretion, but other correlating factors influence the response on animal performance.
A large proportion of ammonia hydrolysed from recycled urea never reaches the rumen pool, and this may be an obstacle to the use of recycled urea as a method to improve N efficiency of dairy cattle. Dairy cows appear unable to increase transport of urea from blood to gastrointestinal tract in periods with low rumen ammonia concentrations and thus urea transport is apparently not effective in buffering infrequent N supply.
Advanced mechanistic models of gut wall and liver metabolism were successfully developed and evaluated.
Analysis of samples using a variety of analytical methods such as chromatography, spectroscopy and nuclear magnetic resonance spectroscopy, has been done in the search for new biomarkers of the efficiency of N utilisation and N sufficiency. This has highlighted the use of previous biomarkers, such as purine derivative excretion in urine and the secretion of odd and branched-chain fatty acids in milk, as well has indicating the potential of several new markers.
N excretion per cow may vary from 107 to 138 kg per year, in relation to annual N content in the diet and level of production. Nitrogen excretion per 1000 kg of milk varied from 10 to 18 kg and this variation is significantly linked to urinary N excretion. These results clearly show that it is possible to make significant progress for reducing N output by a dairy herd.
Potential applications:
Two mathematical models resulted from this project.
One model will give insight in protein flows within the dairy cow, which will be a helpful tool for nutritionists in formulating low protein diets without disturbing essential pathways and processes in the digestion, absorption and metabolism of amino acids.
COWNEX estimates the effect of herd and feeding management on the excretion of nitrogen at herd level. This can be used as a tool for strategic and operational management and as an estimator of nitrogen utilisation.
Two other tools to be used by farmers and nutritionist have been developed.
Rapid analytical methods have been developed to measure the nutritive value of forages and other feed material. Parameters for a more precise estimation of the nutritive value include ruminal degradation characteristics for protein, starch and fibre. This can be incorporated in “precision feeding” approaches and smaller “insecurity” margins in diet formulation.
Indicators have been developed to measure the response of dietary changes concerning protein utilisation. Although milk urea has been used as an indicator of surplus dietary nitrogen already before the start of the RedNEx project, new indicators also provide more control at protein utilisation.
Project Context and Objectives:
Dairying is an important sector of EU agriculture, but intensification is accompanied by an increase in nitrogen surplus in the nutrient cycle on dairy farms. This nitrogen surplus has a negative environmental impact on groundwater (pollution with nitrates), surface water (eutrophication) and on the atmosphere (ammonia volatilisation, nitrous oxide). The EU seeks to stimulate measures that improve management of nutrients, waste and water as a start to move to management practices beyond “usual good farming practice”.
The objective of the EU-FP7 project REDNEX was to develop innovative and practical management approaches for dairy cows that reduce N excretion into the environment through the optimization of rumen function, an improved understanding and prediction of dietary N utilization for milk production, and excretion in urine and faeces. The project developed more-accurate feed characterisation tools and a detailed mathematical model of N utilisation by the cow to integrate results and aimed to improve the supply and efficiency of absorbed amino acids, allowing a reduction in N intake. Practical tools have been developed to predict N losses at a whole-herd level and to monitor the cow’s N status using biomarkers. Dissemination and knowledge interaction have been used in a participatory approach to include the views of stakeholders and recognition of the need to provide support to EU neighbours.
Project Results:
Description of work performed and main results
Work has been performed on feed characterisation, ruminal protein digestion, conversion of metabolisable protein into milk, urea recycling, mechanistic modelling of amino acid metabolism, biomarkers to monitor rumen protein supply and predicting and reducing urinary nitrogen excretion.
Feed characterisation
Precision feeding of ruminants requires an accurate prediction of ruminal behaviour of feed components (protein, cell walls, and starch). Usually, ruminal behaviour is predicted from the chemical composition and established relationships between chemical composition and degradation characteristics are obtained by using in situ incubations. Without chemical analyses standard chemical composition from feed tables is used.
Within REDNEX (1) the database on ruminal degradation characteristics of feed components has been expanded not only for protein, but also for fibre and starch, (2) prediction of ruminal behaviour of these feed component from the chemical composition of feeds have been further improved and (3) cheap and rapid techniques (near-infrared reflection spectroscopy – NIRS; Fourier transform infrared spectroscopy – FTIR; nuclear magnetic resonance - NMR) to improve the accuracy of feed evaluation have been developed and evaluated.
Ruminal protein digestion
Lowering dietary protein content below present recommendations decreases organic matter and fibre digestibility, energy intake and animal performance. One approach to minimize these negative responses is to improve protein digestion in the rumen by reducing microbial degradation of feed protein or by improving ruminal microbial protein synthesis.
Various attempts were made to reduce ruminal protein hydrolysis and deamination. A preliminary analysis of the literature showed that the most efficient additives for decreasing ruminal protein degradation are various essential oils, but their effect in vivo remained unclear. A first innovative approach was the inclusion of essential oils in silages. Essential oils in ryegrass silage are efficient in reducing silage protein degradation but the dose required and thus the costs may be too high for practical use. A second innovative approach was the inclusion of polyclonal antibodies against ruminal hyper ammonia producing bacteria. Several attempts failed to decrease protein degradation. As these innovative ways were not efficient, the most promising ways arising from literature analysis were used. Capsicum oil and propyl-propyl thiosulphate, derived from garlic oil, were used. Both essential oils tended to increase nitrogen use efficiency, but these results need to be confirmed with more animals.
A first attempt to optimise microbial protein synthesis in the rumen was by modifying the nature of forages. In vitro, microbial protein yield did not differ between ryegrass and red clover, but microbes were more efficient with ryegrass for capturing N and in efficiency of N utilization in the rumen. Comparison of varieties (ryegrass varying in sugar content, red clover varying in polyphenol oxidase) needs further research. The effect of source of dietary carbohydrate (starch or fibre) on the response of reducing dietary crude protein was tested in another in vivo experiment.. The source of dietary carbohydrates, starch or fibre, had a minor effect on ruminal protein metabolism; a trend to a higher microbial protein flow and efficiency of synthesis was observed with starch. In another experiment the effect of niacin supplementation to low-protein diets was studied.
Reducing dietary crude protein tended to decrease microbial protein synthesis. Niacin supplementation did not change microbial protein flow and efficiency of synthesis but increased rumen protozoa population.
Ruminal dietary protein use efficiency is reduced by the presence of protozoa. On one hand ruminal protozoa generate an extra nitrogen loss by devouring ruminal bacteria but on the other hand protozoa play a role in fibre degradation. Therefore, the microbial ecosystem, especially protozoa, was further characterised for a better knowledge of the role of different populations. The role of different protozoa in bacterial breakdown has been specified. Entodinium and Epidinium are especially active, whereas Holotrichs have a minor predatory activity. Therefore, lowering numbers of Entodiniomorphids in the rumen may be a strategy for improving microbial synthesis. In normal- or low-protein diets, defaunation (or faunation with an Holotrich species) did not change rumen ammonia in sheep suggesting a better use of N by rumen microbes, which results in a lower urinary N. Cows are able to adapt themselves to fibrous diets by increasing the complexity of the rumen microbial community and the concentrations of protozoa, anaerobic fungi, and methanogens. The identification of key bacteria involved in protein metabolism by using DNA-Stable Isotope Probing was faced with large challenges and results were inconsistent.
Conversion of metabolisable protein into milk
Within REDNEX, experimental investigations addressing hypotheses concerning the effects of dietary carbohydrate and metabolisable protein supply on the metabolism of nutrients by the splanchnic tissues of lactating dairy cows have been conducted at the University of Reading and at INRA Theix. The work at Reading focused on providing metabolisable protein at three levels of predicted supply, below, at, and above requirement, with basal diets formulated to contain differing amounts of maize or grass silage as forage sources. The work at INRA Theix used a similar approach, with diets formulated at two levels of metabolisable protein and basal diets containing differing types of highly digestible carbohydrates, testing the potential effect of energy yielding substrates arising from the digestion of starch- or fibre-based concentrates on nutrient metabolism and the efficiency of dietary protein utilization. Related work at INRA Rennes used a duodenal infusion protocol to further define the ideal essential amino acid profile in metabolisable protein in terms of requirements for arginine, isoleucine, and valine for milk protein synthesis. Subsequent studies determined the effects of providing an ideal profile of essential amino acids when metabolisable protein was provided at levels below and above requirement to determine if the response to amino acid profile was altered by metabolisable protein supply relative to requirement and the extent to which nutrient metabolism by the mammary gland was affected.
The focus of the experiments was to improve understanding of amino acid and nutrient absorption and utilization in lactating dairy cows and in so doing describe the impact of amino acid supply relative to requirement and dietary carbohydrate source on the utilization of nutrients for milk protein production and N excretion in urine and faeces. A second objective was to improve our understanding of the optimum balance of essential amino acids for milk protein production by dairy cows and how amino acid balance impacts mammary gland metabolism. The descriptions of the integrated responses observed will be of value in the development of models of dietary nutrient utilization and the development and refinement of dietary strategies that improve the efficiency of dietary nitrogen utilization.
Concerning the effects of carbohydrate sources in the concentrates fed to lactating dairy cows, results from 3 experiments using similar diets (two levels of metabolisable protein with high starch or high fibre concentrates) at INRA Theix were aggregated for analysis of production response and the efficiency of dietary nitrogen utilization. Overall, diets rich in starch significantly increased dietary protein use efficiency compared to diets rich in fibre, without a significant interaction between concentrate carbohydrate and level of metabolisable protein supply. This suggests a higher microbial protein supply to the small intestine than the theoretical values with diets rich in starch vs. fibre. Measurements of nutrient absorption and metabolism confirm that the improved efficiency of dietary nitrogen utilization for higher starch diets was associated with differences in nutrient absorption, including greater glucose and lower acetate absorption, as well as greater amino acid absorption at lower dietary protein concentration. These findings confirm that a part of the starch effect was attributable to greater metabolisable protein supply, which likely reflects greater microbial protein synthesis. However, a reduction in the relative rate of removal of amino acids by the liver was also observed, confirming that the positive effects of starch on dietary nitrogen utilization were also attributable to differences in post-absorptive metabolism. Results suggest that the higher milk N yield found with diets rich in starch vs fibre could be the result of both digestive (likely via a higher microbial protein synthesis) and metabolic (via a decrease in liver AA uptake) adaptations.
As regards the effects of forage carbohydrate type and metabolisable protein supply, the experiment at the University of Reading found increasing dietary protein concentration linearly increased feed intake, milk yield, and milk protein production, but decreased dietary nitrogen use efficiency. In contrast forage source had no effect on feed intake, milk production or dietary nitrogen use efficiency, but diets were formulated to minimize differences in total starch and fibre concentrations across treatments. Importantly and as for the effects of concentrate type, there were no interactions between forage carbohydrate type and dietary protein supply in terms of production responses and dietary nitrogen use efficiency. With increasing dietary protein there were linear increases in rumen ammonia concentration, net absorption of ammonia, liver production of urea, and concentrations of urea in arterial blood, milk, and urine, as well as arterial concentrations of most essential and many nonessential amino acids. In addition, the increase in feed intake and milk yield with increased dietary protein supply was associated with a linear increase in liver production of glucose and β-hydroxybutyrate. There was a positive relationship between N intake and rumen ammonia, arterial urea, and milk urea concentration, all of which were negatively related to N utilization efficiency.
Work on the effects balancing essential amino acid profile, found there were no important interactions between the effect of increasing metabolisable protein supply and the effect of balancing the EAA profile. As in other experiments for WP3, increasing metabolisable protein supply increased milk protein yield, but increased nitrogen absorption and arterial urea concentration and decreased dietary nitrogen use efficiency. In contrast, balancing amino acid profile increased milk protein yield, but at equal nitrogen absorption, thus dietary protein use efficiency was increased. This was associated with increased arterial concentrations of most essential amino acids, but decreased arterial concentration of urea. In both cases, increased milk protein yield was associated with increased uptake of essential amino acids by the mammary gland, but not nonessential amino acids. Balancing the essential amino acid profile increased milk protein yield and dietary protein use efficiency at both levels of metabolisable protein supply by increasing mammary uptake of essential amino acids and adjusting mammary uptake of nonessential amino acids, leading to less availability of ‘excess’ amino acids for catabolism and urea synthesis.
Other research examined the milk protein responses to changes in the supply of arginine, isoleucine, and valine relative to other essential amino acids. The study found that the concentration of arginine currently recommended for metabolisable protein was not limiting. In contrast, changing the supply of isoleucine altered the milk protein to fat ratio, whilst there was evidence that valine may be limiting milk protein synthesis when requirements for methionine, lysine, and histidine are met. However, the effects of valine and isoleucine in this experiment may be related to the concentration of leucine. Therefore, in future it is proposed that possible antagonisms between the branched chain amino acids should be considered when establishing their requirements.
Urea recycling
To expand our understanding of the mechanisms regulating urea recycling across the portal-drained visceral (PDV) tissue and ruminal epithelia, five experiments with multi-catheterized and rumen-cannulated lactating Holstein cows were conducted at the intensive barn facility at Department of Animal Science, Aarhus University, Foulum, Denmark. More knowledge on the physiological adaptation and the functional properties of urea transport across the PDV and ruminal epithelium is a valuable tool in order to be able to model the internal N cycling and to investigate if the expectations we have for urea recycling in increasing N utilization can be kept alive.
Evaluating the regulatory effect of varying N intake on the functional properties of urea recycling from these intensive studies, the conclusion is clear: the total amount of urea transferred from blood to GI tract, including the rumen is not increased with decreasing N supply. In fact the opposite seems to be the trend, that is, increased urea transfer with increasing N supply. Still, an actual up-regulation of the extraction of arterial urea across the ruminal and PDV epithelia with decreasing dietary N supply was evident, and is an indication that decreasing N supply will increase the permeability of the epithelium for urea and that the permeability of the epithelia adapts to given dietary conditions. However, this increase in the permeability of the epithelium for urea is insufficient to compensate for the concomitant decreases in blood urea concentrations. Hence, one of the main limiting factors for utilizing the ability of the dairy cow to recycle urea in order to increase the N efficiency is that the permeability of the epithelium for urea appears to start declining, or is not up-regulated enough when N status of the cow is still insufficient to sustain optimal rumen microbial synthesis and production performance. Thus, urea recycling had no potential for compensating low N supply to maintain production performance neither when cows were fed varying N concentration nor with infrequent N supplementation over the day. Based on this, we conclude that the regulatory mechanism of urea transport appears only partly effective or controlled by something else than the ammonia requirements of ruminal fermentation.
Efforts to manipulate urea recycling by increasing the pull of urea on the hindgut via abomasal oligofructose infusion for hindgut fermentation resulted in decreased blood urea concentration which could not be explained by increased urea recycling but appeared instead to be a result of increased sequestration of ammonia in the hindgut and thus a relative increased N excretion in faeces. Increased hindgut fermentation did not impair rumen fermentation and shifted some N excretion from urine to faeces.
Using dietary NaCl to increase water intake and to possible increase the pull of urea on the kidneys to compete with urea recycling to the GI tract did not lead to changes in total urea recycling, nor in blood urea concentration, and urinary urea excretion. Dietary adaptation of the kidneys to high NaCl supply reduced the effect on blood urea concentration, indicating that the priority of the kidneys is to keep circulating amounts of urea within a given homeostatic range. Ruminal fermentation was not impaired by high dietary supply NaCl. Milk urea concentration was unchanged and ECM increased slightly by high dietary NaCl supply.
It has not been possible to detect any up-regulation of the urea transporter protein, UT-B which is speculated to be involved in the regulation of the permeability of the epithelia for urea with decreasing N supply. Nor could any of the investigated aquaporins explain the observed changes in rumen epithelial permeability for urea.
Mechanistic modelling of amino acid metabolism
Based on literature data and on the observations within REDNEX studies on amino acid utilisation and urea recycling, mechanistic models of amino acid metabolism in gut wall, liver and mammary gland of dairy cattle were developed and subsequently integrated into a single modelling framework with the nutrient flows between the models connected accordingly. A user interface has been developed and programmed to allow the integrated post-absorptive model to be used by a broad audience.
Biomarkers to monitor rumen protein supply
Blood, milk and urine samples were collected within the in vivo experiments of the REDNEX project and samples were analysed using a number of methodologies with the aim of confirming the use of established biomarkers and discovering new and novel biomarkers of dietary protein use efficiency and rumen N sufficiency. Targeted analyses (i.e. of known compounds) has helped validate the use of odd and branched chain fatty acid (OBCFA) secretion in milk and of purine derivative excretion in urine from lactating dairy cows. Both of these markers have been demonstrated to be related to the duodenal flow of microbial protein from the rumen, and therefore offer non-invasive estimation of rumen N use. Metabolomic, or untargeted, analysis of samples by nuclear magnetic resonance spectroscopy, Fourier-transform infrared spectroscopy, and various forms of chromatography coupled with mass spectrometry, has demonstrated that various compounds correlate well with duodenal microbial protein flow. These include compounds previously known to be of such use, e.g. purine derivatives, and also compounds not previously known to correlate this way, e.g. para-cresol sulphate. Some currently unidentified compounds have also been found to be of potential use, but these require further work to confirm their identities and validate their utility.
Predicting and reducing urinary nitrogen excretion
Within REDNEX, a large database was produced comprising data of most N balance trials conducted in Europe on dairy cows giving new insights on the fate of N intake. N excreted in faeces is primarily a function of DM intake, the effect of DM intake being higher for grass silage-based diets and high-concentrate diets than for fresh grass and maize silage-based diets whereas N excreted in urine primarily depends of total N intake for a given level of milk yield. Some nitrogen is not recovered in milk, faeces or urine. This difference is partly explained by animal characteristics (pregnancy and N retention as affected by the energy balance); external temperature as losses of N as urea excreted in sweat may occur and by some methodological problems.
Reduce dietary protein content without affecting performances, depends on various factors which resulted in variable responses in the experiments performed within REDNEX. In some experiments it was possible to compensate the reduction in protein supply by adjusting the essential amino acid profile, but the room to manoeuvre to economize protein utilisation remains limited in most of the current situations. In another experiment the reduction in dietary protein content reduced dry matter (energy intake) and consequently milk (protein) yield. Supplementing the low-protein diets with methionine and lysine, partly compensated the reduction in dry matter intake, but had no significant effect on milk (protein) secretion.
A simulator (CowNex) was developed to calculate protein synthesis in milk and meat and nitrogen excretion in dairy herd, including heifers, depending on herd management. The simulator performs these calculations throughout the year and measures the impact of management changes, particularly nutrition, on the dietary protein use efficiency by the herd for milk and meat production and the release of faecal or urine nitrogen. The amount of nitrogen to manage as manure is considered separately from the quantities directly returned to pasture.
Knowledge dissemination
The main mission of knowledge dissemination in REDNEX is to ensure that the project and its results meet the needs of the end-users, i.e. those that are involved in dairy production. This is reached by creating a participatory framework that will allow meaningful dialogue between the Work Package coordinators and the stakeholders and to transfer the information from the project to the intended stakeholders / end-users in an effective manner. Another objective is to extend the consultation and dissemination to newly-entered and candidate states and to train professionals from these countries.
Stakeholder platform
Representatives of the animal feed industry and dairy farmers, and representatives involved in mathematical modelling of biological processes, environment impact of livestock production, animal feed analyses, soil N cycling, technology transfer, and of dairy systems participate in the REDNEX Stakeholder platform. This platform is used to gain advice and feedback from the stakeholders to assist in the development of the research and dissemination process. Individual platform members have been linked with work packages to stimulate the interaction between researchers and end-users. Brian Cook, one of the members of the platform, passed away 8 May 2012.
Websites
A public website www.rednex-fp7.eu has been created and maintained. At this website, presentations of all REDNEX scientific meetings can be accessed and the link to CowNex can be found. The website also contains publishable summaries of public REDNEX deliverables.
Meetings
REDNEX organised and supported eight scientific meetings: at Vilnius, Lithuania (EAAP satellite workshop; 23 August 2008), Barcelona, Spain (EAAP Session, 24 August 2009), Heraklion, Greece (EAAP Session, 24 August 2010), Parma, Italy (ISEP Session, 6 September 2010), Stavanger, Norway (EAAP Session, 23 August 2011), Aberystwyth (oral and poster presentations at 8th ISNH, September 2011) , Bratislava (EAAP Session, 27 August 2012), and Zaragoza (Course on Environmental Assessment of Livestock production Systems, April 2013) The project was completed with a final conference in Paris on 20 August 2013. It was attended by 109 delegates, including 19 early career scientists supported by the project.
Publications
Besides abstracts and other contributions in conference proceedings, articles in regional and national magazines, more than 35 research papers have been submitted or published in peer-reviewed international scientific journals.
Dissemination to newly-entered and candidate EU states
The project has financially (up to € 1,000 per person) supported 63 early career scientists from Central and Eastern Europe and the Mediterranean to attend the REDNEX meetings and to act as contacts for future dissemination of REDNEX research within their region.
Regional meetings were organised in Croatia, Romania and Lithuania involving a total of 366 delegates and supporting 12 early career scientists from disadvantaged countries to attend. The discussions agreed that the project had made an important contributing to reducing the environmental impact of milk production. The main barriers to translation of this research were the low herd size. Standardisation of feeding systems supported by low cost rapid feed analysis were seen as a priority together with better links between research organisations and the feed industry.
Potential Impact:
IMPACT
Feed characterisation
Results from REDNEX on degradation rates for starch and part of the model for starch digestion have been implemented in the NorFor ration evaluation system. A number of feed samples have been examined for in situ degradation characteristics of protein, which will be incorporated in feed tables. The NIRS calibrations for degradation parameters for protein will be published and made freely available for the feed industry.
Ruminal protein digestion
Although some additives (essential oils) may reduce the microbial degradation of feed protein in the rumen, REDNEX obtained insufficient data to confirm this. Reducing crude protein in dairy cow rations to 11-12% of DM resulted in a moderate decrease in milk production under some circumstances. Reducing dietary crude protein had no effect on microbial protein synthesis per unit of fermentable organic matter and it was therefore concluded that the rumen microbial population does not adapt to protein underfeeding by increasing the efficiency of microbial protein synthesis. Rumen protozoa, fungi, methanogens and certain bacterial species are sensitive to N shortage which can result in a decrease in OM digestibility.
Conversion of metabolisable protein into milk
Results demonstrate that improvements in dietary protein use efficiency attributable to supplemental starch are not due solely to digestive and fermentation responses, but also due to metabolic effects on the utilization of absorbed nutrients. Importantly, there were few interactions observed between dietary carbohydrate and protein supply, which has implication for diet formulation strategies.
Current recommendations for the balance of essential amino acid concentrations in metabolisable protein are sufficient for arginine, but that potential antagonisms exist for the branched chain amino acids that should be described more clearly and may have implications for their recommended concentrations.
Even at low metabolisable protein supply, balancing the amino acid profile increased nitrogen use efficiency. The is explained by an increased uptake of essential amino acids by the mammary gland. This suggests that amino acid supply can be expressed in percentage of metabolisable protein regardless of the level of protein supply.
Urea recycling
The amount of urea recycled showed to be relatively unaffected by even major changes in dietary protein concentrations. Therefore, the possibilities for increasing dietary protein use efficiency of the dairy cow through urea recycling by dietary N intervention strategies seem limited. The outcome of this series of elegant intensive studies performed prevents to formulate a new feeding strategy that utilize urea recycling, although this was the original idea at the start of the project.
Mechanistic modelling of amino acid metabolism
The integrated post-absorptive model that was developed may potentially replace current empirical protein evaluation systems. The integrated model enhances understanding of N utilization, assist researchers in developing diets to improve N utilization by cattle and promote reductions in N excretion to the environment.
Biomarkers to monitor rumen protein supply
Across all the studies the clear relationship between dietary protein use efficiency and urea concentrations in blood, milk, and urine confirm the potential value of urea measurement as management tool for improving dietary protein use efficiency on farm. Multivariate analysis (especially partial least squares analysis) of other metabolomics data has demonstrated the ability to predict with reasonable accuracy parameters such as dietary protein use efficiency for milk production, and urinary N excretion, but more work is required to confirm these results.
The ability of a dairy farmer to estimate dietary protein use efficiency from a simple blood, milk or urine sample, particularly following a change in feeding regime, has significant potential in helping to refine dairy cow diet formulation, optimising diet nutrient use, and reducing N excretion from dairy cows and biomarkers to monitor rumen protein supply.
Predicting and reducing urinary nitrogen excretion
In the validating experiments, reducing dietary protein resulted in a lower milk protein yield, but nevertheless improved dietary protein use efficiency and reduced urinary urea excretion. The developed CowNex software to estimate N balances on a herd level can be accessed without restriction via an Internet network with a multilingual Web interface (French, English) at the address: http://www.cownex-record.inra.fr/.
List of Websites:
http://www.rednex-fp7.eu