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Simple Summary Globally, the livestock sector is responsible for 37% of total anthropogenic methane emissions, most of which are produced from enteric fermentation of ruminants. Livestock is also responsible for 65% anthropogenic nitrous oxide and 64% of anthropogenic ammonia emissions. The literature reports several dietary management options to reduce greenhouse gas emissions from ruminants, and potentially improve productivity. However, strategies that aim to reduce the emissions of one specific greenhouse gas can have side effects (increase) on other pollutant gases. In this study, we evaluated the effect of two types of perennial ryegrass (PRG) pastures differing in their concentration of water-soluble carbohydrates (WSC, high (HS) and low (LS)) on the in vitro nitrogen use efficiency in the rumen and on methane emissions. The greater WSC and lower crude protein (CP) concentrations of high sugar pastures modified in vitro rumen fermentation, tending to increase total volatile fatty acids (VFA) production, reduce acetate:propionate ratio and methane (CH4) concentration, and improve nitrogen (N) use efficiency through lower rumen ammonia-N (NH3-N) concentrations. In vivo studies with cattle are required to confirm the potential of these measures to increase the sustainability and reduce the environmental impact of grazing livestock production systems. Abstract The objective of this study was to determine the effect of perennial ryegrass (PRG) forages differing in their concentration of water-soluble carbohydrates (WSC) and crude protein (CP), and collected in spring and autumn, on in vitro rumen fermentation variables, nitrogen (N) metabolism indicators and methane (CH4) output, using a batch culture system. Two contrasting PRG pastures, sampled both in autumn and spring, were used: high (HS) and low (LS) sugar pastures with WSC concentrations of 322 and 343 g/kg for HS (autumn and spring), and 224 and 293 g/kg for LS in autumn and spring, respectively. Duplicates were incubated for 24 h with rumen inocula in three different days (blocks). Headspace gas pressure was measured at 2, 3, 4, 5, 6, 8, 10, 12, 18, and 24 h, and CH4 concentration was determined. The supernatants were analysed for individual volatile fatty acids (VFA) concentrations, and NH3-N. The solid residue was analysed for total N and neutral detergent insoluble N. Another set of duplicates was incubated for 4 h for VFA and NH3-N determination. The HS produced more gas (218 vs. 204 mL/g OM), tended to increase total VFA production (52.0 mM vs. 49.5 mM at 24 h), reduced the acetate:propionate ratio (2.52 vs. 3.20 at 4 h and 2.85 vs. 3.19 at 24 h) and CH4 production relative to total gas production (15.6 vs. 16.8 mL/100 mL) and, improved N use efficiency (22.1 vs. 20.9). The contrasting chemical composition modified in vitro rumen fermentation tending to increase total VFA production, reduce the acetate:propionate ratio and CH4 concentration, and improve N use efficiency through lower rumen NH3-N.

Sustainable intensification is a process by which agricultural productivity is enhanced whilst also creating environmental and social benefits. We aimed to identify practices likely to deliver sustainable intensification, currently available for UK farms but not yet widely adopted. We compiled a list of 18 farm management practices with the greatest potential to deliver sustainable intensification in the UK, following a well-developed stepwise methodology for identifying priority solutions, using a group decision-making technique with key agricultural experts. The list of priority management practices can provide the focal point of efforts to achieve sustainable intensification of agriculture, as the UK develops post-Brexit agricultural policy, and pursues the second Sustainable Development Goal, which aims to end hunger and promote sustainable agriculture. The practices largely reflect a technological, production-focused view of sustainable intensification, including for example, precision farming and animal health diagnostics, with less emphasis on the social and environmental aspects of sustainability. However, they do reflect an integrated approach to farming, covering many different aspects, from business organization and planning, to soil and crop management, to landscape and nature conservation. For a subset of ten of the priority practices, we gathered data on the level of existing uptake in English and Welsh farms through a stratified survey in seven focal regions. We find substantial existing uptake of most of the priority practices, indicating that UK farming is an innovative sector. The data identify two specific practices for which uptake is relatively low, but which some UK farmers find appealing and would consider adopting. These practices are: prediction of pest and disease outbreaks, especially for livestock farms; staff training on environmental issues, especially on arable farms.

The objective was to evaluate water-soluble carbohydrate (WSC) and crude protein (CP) concentration of perennial ryegrass (PRG) cultivars with different genetic potential for producing WSC under two contrasting agronomic managements in temperate climate (southern Chile). A 4 × 2 factorial design was randomly allocated to 24 plots (31 m2 each, three blocks): four PRG cultivars (diploid standard cultivar, “2nSt”; tetraploid standard cultivar, “4nSt”; diploid high sugar cultivar developed in New Zealand, “2nHSNZ”; and tetraploid high sugar cultivar developed in Europe, “4nHSEU”) and two agronomic managements (“favourable,” defoliations at three leaves per tiller and nitrogen (N) fertilization rate of 83.3 kg N ha−1year−1; “unfavourable,” defoliations at two leaves per tiller and N fertilization rate of 250 kg N ha−1year−1). Herbage samples were collected in early spring, spring, summer and autumn. Concentration of WSC did not differ among cultivars in spring and summer, averaging 194 and 251 g/kg DM, respectively. The cultivar 4nHSEU had the greatest WSC concentration in early spring and autumn (187 and 266 g/kg DM, respectively) and the greatest CP concentration across samplings (average 230 g/kg DM). Favourable management improved WSC concentrations in early spring and summer and decreased CP in spring, summer and autumn. Annual DM yield did not vary with cultivar or management, averaging 8.43 t/ha. Within a 12-month study at one site in a temperate environment in southern Chile, PRG cultivars have not shown a consistent expression of the “high sugar” trait, where a genetic × environment interaction might be operating.

Polyphenol oxidase (PPO) in red clover (RC) has been shown to reduce both lipolysis and proteolysis in silo and implicated (in vitro) in the rumen. However, all in vivo comparisons have compared RC with other forages, typically with lower levels of PPO, which brings in other confounding factors as to the cause for the greater protection of dietary nitrogen (N) and C18 polyunsaturated fatty acids (PUFA) on RC silage. This study compared two RC silages which when ensiled had contrasting PPO activities (RC+ and RC−) against a control ofperennial ryegrass silage (PRG) to ascertain the effect of PPO activity on dietary N digestibility and PUFA biohydrogenation. Two studies were performed the first to investigate rumen and duodenal flow with six Hereford×Friesian steers, prepared with rumen and duodenal cannulae, and the second investigating whole tract N balance using six Holstein-Friesian non-lactating dairy cows. All diets were offered at a restricted level based on animal live weight with each experiment consisting of two 3×3 Latin squares using big bale silagesensiled in 2010 and 2011, respectively. For the first experiment digesta flow at the duodenum was estimated using a dual-phase marker system with ytterbium acetate and chromium ethylenediaminetetraacetic acid as particulate and liquid phase markers, respectively. Total N intake was higher on the RC silages in both experiments and higher on RC− than RC+. Rumen ammonia-N reflected intake with ammonia-N per unit of N intake lower on RC+ than RC−. Microbial N duodenal flow was comparable across all silage diets with nonmicrobial N higher on RC than the PRG with no difference between RC+ and RC−, even when reported on a N intake basis. C18 PUFA biohydrogenation was lower on RC silage diets than PRG but with no difference between RC+ and RC−. The N balance trial showed a greater retention of N on RC+ over RC−; however, this response is likely related to the difference in N intake over any PPO driven protection. The lack of difference between RC silages, despite contrasting levels of PPO, may reflect a similar level of protein-boundphenol complexing determined in each RC silage. Previously this complexing has been associated with PPOs protection mechanism; however, this study has shown that protection is not related to total PPO activity.

Global agriculture is at a critical juncture where competing requirements for maximal production and minimal pollution impose various patterns of economic-environmental trade-offs. Livestock production is central to this debate, as it is estimated to generate 7.1 Gt CO2-e of greenhouse gases (GHG) each year, and to occupy 30% of the Earth’s surface, of which one-fifth is subject to land degradation due to inappropriate management and overproduction. These statistics, together with the notion that cereals and pulses currently being fed to livestock could potentially be consumed by humans to alleviate hunger, have frequently led to a criticism that livestock farming is an unethical industry.On the other hand, ruminants can utilise land unsuitable for arable crop production and convert fibre and low quality nitrogen sources to nutritionally-valuable human-edible protein. Furthermore, grazing animals have been shown to be net providers of a range of ecosystem services and, depending on the natural conditions, capable of not only maintaining but also regenerating soil fertility by facilitating nutrient cycling.Using high-resolution primary data from an intensively instrumented farm-scale ruminant research facility located in southwest UK a novel, information-driven approach to carry out comprehensive assessments of economic-environmental trade-offs inherent within pasture-based production systems is proposed. The results suggest that a potentially systematic interaction exists between ‘soil health’, ecological surroundings and livestock grazing, whereby a higher level of soil organic carbon (SOC) stock is associated with a better animal performance and less nutrient losses into watercourses, and a higher stocking density with greater botanical diversity and elevated SOC. We contend that a combination of farming system-wide trials and environmental instrumentation provides an ideal setting for enrolling scientifically sound and biologically informative metrics for agricultural sustainability, through which agricultural producers could obtain guidance to manage soils, water, pasture and livestock in an economically and environmentally acceptable manner.

Life cycle assessment (LCA) is widely regarded as a useful tool for comparing the environmental impacts of multiple livestock production systems. While LCA results are typically communicated in the form of environmental burdens per mass unit of the end product, it is increasingly becoming recognized that the product quality also needs to be accounted for to truly understand the value of a farming system to society. To date, a number of studies have examined environmental consequences of different food consumption patterns at the diet level; however, few have addressed nutritional variations of a single commodity attributable to production systems, leaving limited insight into how on‐farm practices can be improved to better balance environment and human nutrition. Using data from seven livestock production systems encompassing cattle, sheep, pigs, and poultry, this paper proposes a novel framework to incorporate nutritional value of meat products into livestock LCA. The results of quantitative case studies demonstrate that relative emissions intensities associated with different systems can be dramatically altered when the nutrient content of meat replaces the mass of meat as the functional unit, with cattle systems outperforming pig and poultry systems in some cases. This finding suggests that the performance of livestock systems should be evaluated under a whole supply chain approach, whereby end products originating from different farm management strategies are treated as competing but separate commodities.

There is a hypothesis in the anthropological literature (e.g. Milton, 2003) that our complex human brains would not have developed without hominoids having routine access to foods of animal origin. In many countries, therefore, the consumption of animal source foods, and meat in particular, became part of a cultural identity, while in others (e.g. India) vegetarianism has long been part of the cultural/religious identity (approx. 30% of Indians are vegetarian, although the majority of those consume milk products). In the twenty first century, humans can, theoretically, have access to non animal- derived sources of nutrients required for brain development, so animal products are no longer as important for nutrition as they were for our ancestors. There are also other issues, however, associated with livestock production which impact on the consumption of animal products. Culture in many countries has evolved to give a higher priority to the welfare of farm animals and there are additional ethical issues associated with the negative environmental impacts of livestock production. We read a lot, therefore, about the rise of veganism, with calls for diets free of animal products, yet there is also evidence that the consumption of meat at a country level increases as the income of that country increases (FAO, 2009). The average global per capita meat consumption at over 40 kg, has, therefore, doubled from the consumption in 1960. However, the annual global rate of increase in both meat and milk consumption is projected (OECD/FAO, 2017) to decrease slightly in the decade from 2017-2026, to 1.5% per annum for meat and 1.9% per annum for milk.

Bulk collagen δ15N analysis is widely used to investigate past diet and trophic positions, but these values average the δ15N values of the constituent amino acids. Compound–specific isotope analysis of amino acids (AAs) can help elucidate the complex metabolic effects underpinning bulk δ15N values. Although trophic level effects on individual AA δ15N values have been investigated in aquatic and terrestrial invertebrate food webs, most archaeological applications involve terrestrial herbivores, hence a greater understanding of these effects between diet and consumer in this food chain is required. The North Wyke Farm Platform provided baseline nitrogen isotope information for cattle grazing on a Lolium perenne- dominated pasture. Bulk dentine δ15N values show a shift expected for a one trophic level increase, but obscure insight into the underlying metabolic processes that cause this change in value. However, determination of AA δ15N values of hydrolysable plant protein and cattle tooth dentine clarifies the trophic effect on consumer AA δ15N values. The observed trophic shift in the studied system is different from previously studied food webs, with a trophic enrichment factor, based on the δ15N values of glutamate and phenylalanine, of 4.0‰ compared to 7.6‰ commonly used in ecological and archaeological studies. This emphasises the need to understand the trophic shifts in the particular food web being investigated in order to apply isotopic investigations in archaeological contexts.

For livestock production systems to play a positive role in global food security, the balance between their benefits and disbenefits to society must be appropriately managed. Based on the evidence provided by field-scale randomised controlled trials around the world, this debate has traditionally centred on the concept of economic-environmental trade-offs, of which existence is theoretically assured when resource allocation is perfect on the farm. Recent research conducted on commercial farms indicates, however, that the economic-environmental nexus is not nearly as straightforward in the real world, with environmental performances of enterprises often positively correlated with their economic profitability. Using high-resolution primary data from the North Wyke Farm Platform, an intensively instrumented farm-scale ruminant research facility located in southwest United Kingdom, this paper proposes a novel, information-driven approach to carry out comprehensive assessments of economic-environmental trade-offs inherent within pasture-based cattle and sheep production systems. The results of a data-mining exercise suggest that a potentially systematic interaction exists between 'soil health', ecological surroundings and livestock grazing, whereby a higher level of soil organic carbon (SOC) stock is associated with a better animal performance and less nutrient losses into watercourses, and a higher stocking density with greater botanical diversity and elevated SOC. We contend that a combination of farming system-wide trials and environmental instrumentation provides an ideal setting for enrolling scientifically sound and biologically informative metrics for agricultural sustainability, through which agricultural producers could obtain guidance to manage soils, water, pasture and livestock in an economically and environmentally acceptable manner. Priority areas for future farm-scale research to ensure long-term sustainability are also discussed.

The kynurenine pathway is the major route of L-tryptophan (L-Trp) catabolism in biology, leading ultimately to the formation of NAD(+). The initial and rate-limiting step of the kynurenine pathway involves oxidation of L-Trp to N-formylkynurenine. This is an O2-dependent process and catalyzed by indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase. More than 60 years after these dioxygenase enzymes were first isolated (Kotake, Y., and Masayama, I. (1936) Z. Physiol. Chem. 243, 237-244), the mechanism of the reaction is not established. We examined the mechanism of substrate oxidation for a series of substituted tryptophan analogues by indoleamine 2,3-dioxygenase. We observed formation of a transient intermediate, assigned as a Compound II (ferryl) species, during oxidation of L-Trp, 1-methyl-L-Trp, and a number of other substrate analogues. The data are consistent with a common reaction mechanism for indoleamine 2,3-dioxygenase-catalyzed oxidation of tryptophan and other tryptophan analogues.

Citrus is one of the major fruit crops of the world, with production in subtropical and tropical areas of six continents. Citrus originated in south eastern Asia. Many of the forests in this region where natural populations of wild citrus likely occur have been developed for agriculture. Climate change threatens to further reduce the remaining habitat of wild citrus. The spread of modern citrus cultivars to these areas is displacing local selections and landraces. Therefore conservation of citrus germplasm is a priority, as recognized by development of a global citrus germplasm network in 1997. The International Treaty on Plant Genetic Resources for Food and Agriculture (ITPGRFA) has recognized Citrus as an Annex 1 crop, and an initial meeting in 2012 began work to establish a global conservation strategy for citrus. To characterize the current state of citrus germplasm collections, a survey of citrus genebanks was developed and distributed worldwide. Eleven germplasm collections responded to the survey. These collections vary in size, age, location in developed vs developing countries, and emphasis of the collection. Most collections are expanding, but they vary in rates of turnover of accessions, with some having high rates of loss. Most collections maintain germplasm primarily as field collections, but greenhouses, nurseries, and cryopreservation are used by some. Pest and disease problems are an economic problem for most collections and affect a wide range of accessions. Loss of accessions due to these problems affects only two collections. Relatively few collections distribute germplasm beyond the country in which they are located. Most collections report having inadequate funding, and need new trained staff to better manage citrus germplasm. We conclude that a global citrus germplasm network is needed with resources to provide stable funding to important but imperilled collections.

Forage type and management influences the nutritional quality and fatty acid composition of ruminant milk. Replacing grass silage with red clover (RC; L.) silage increases milk fat 18:3-3 concentration. Red clover has a higher polyphenol oxidase (PPO) activity compared with grasses, which has been suggested to decrease lipolysis and . The present study characterized the abundance and fatty acid composition of esterified lipid and NEFA before and after ensiling of grass and RC to investigate the influence of forage species, growth stage, and extent of fermentation on lipolysis. A randomized block design with a 2 × 3 × 4 factorial arrangement of treatments was used. Treatments comprised RC or a mixture of timothy ( L.) and meadow fescue ( Huds.) harvested at 3 growth stages and treated with 4 levels of formic acid (0, 2, 4, and 6 L/t). Lipid in silages treated with 0 or 6 L/t formic acid were extracted and separated into 4 fractions by TLC. Total PPO activity in fresh herbage and the content of soluble bound phenols in all silages were determined. Concentrations of 18:3-3 and total fatty acids (TFA) were higher ( < 0.001) for RC than for grass. For both forage species, 18:3-3 and TFA content decreased linearly ( < 0.001) with advancing growth stage, with the highest abundance at the vegetative stage. Most of lipid in fresh RC and grass herbage (97%) was esterified, whereas NEFA accounted for 71% of TFA in both silages. Ensiling resulted in marginal increases in TFA content and the amounts of individual fatty acids compared with fresh herbages. Herbage total PPO activity was higher ( < 0.001) for RC than grass (11 vs. 0.11 μkatal/g leaf fresh weight). Net lipolysis during ensiling was extensive for both forage species (660 to 759 g/kg fatty acid for grass and 563 to 737 g/kg fatty acid for RC). Formic acid application (0 vs. 6 L/t) resulted in a marked decrease ( = 0.026) in net lipolysis during the ensiling of RC, whereas the opposite was true ( = 0.026) for grass. In conclusion, results suggest that formic acid addition during the ensilage of RC decreases lipolysis . For both plant species, total PPO activity was not associated with the extent of lipolysis . However, bound phenols formed via PPO activity appear to have a role in protecting lipid and protein against degradation in grass and lowering proteolysis of RC during ensiling.

Tocopherols are lipid-soluble antioxidants that contribute to plant resistance to abiotic stresses. However, it is still unknown to what extent alterations in tocopherol composition can affect the plant response to biotic stresses. The response to bacterial and fungal attack of the vte1 mutant of Arabidopsis thaliana, which lacks both α- and γ-tocopherol, was compared to that of the vte4 mutant (which lacks α- but accumulates γ-tocopherol) and the wild type (with accumulates α-tocopherol in leaves). Both mutants exhibited similar kinetics of cell death and resistance in response to Pseudomonas syringae. In contrast, both mutants exhibited delayed resistance when infected with Botrytis cinerea. Lipid and hormonal profiling was employed with the aim of assessing the underlying cause of this differential phenotype. Although an altered tocopherol composition in both mutants strongly influenced fatty acid composition, and strongly altered jasmonic acid and cytokinin contents upon infection with B. cinerea, differences between genotypes in these phytohormones were observed during late stages of infection only. By contrast, genotype-related effects on lipid peroxidation, as indicated by malondialdehyde accumulation, were observed early upon infection with B. cinerea. We conclude that an altered tocopherol composition in chloroplasts may negatively influence the plant response to biotic stress in Arabidopsis thaliana through changes in the membrane fatty acid composition, enhanced lipid peroxidation and delayed defence activation when challenged with B. cinerea.

After significant increases in livestock productivity, China now needs to improve efficiency and environmental performance., China’s livestock industry has experienced a vast transition during the last three decades, with profound effects on domestic and global food provision, resource use, nitrogen and phosphorus losses, and greenhouse gas (GHG) emissions. We provide a comprehensive analysis of the driving forces around this transition and its national and global consequences. The number of livestock units (LUs) tripled in China in less than 30 years, mainly through the growth of landless industrial livestock production systems and the increase in monogastric livestock (from 62 to 74% of total LUs). Changes were fueled through increases in demand as well as, supply of new breeds, new technology, and government support. Production of animal source protein increased 4.9 times, nitrogen use efficiency at herd level tripled, and average feed use and GHG emissions per gram protein produced decreased by a factor of 2 between 1980 and 2010. In the same period, animal feed imports have increased 49 times, total ammonia and GHG emissions to the atmosphere doubled, and nitrogen losses to watercourses tripled. As a consequence, China’s livestock transition has significant global impact. Forecasts for 2050, using the Shared Socio-economic Pathways scenarios, indicate major further changes in livestock production and impacts. On the basis of these possible trajectories, we suggest an alternative transition, which should be implemented by government, processing industries, consumers, and retailers. This new transition is targeted to increase production efficiency and environmental performance at system level, with coupling of crop-livestock production, whole chain manure management, and spatial planning as major components.

Production of pork, the most consumed meat globally, is estimated to emit 668 m tonnes CO2-eq of greenhouse gases each year. Amongst various production systems that comprise the pig industry, grain-based intensive production is widely regarded as the largest polluter of the environment, and thus it is imperative to develop alternative systems that can provide the right balance between sustainability and food security. Using an original dataset from the Republic of Ireland, this paper examines the life-cycle environmental impacts of representative pig farms operating under varying production efficiencies. For the baseline farm with an average production efficiency, global warming potential (GWP), acidification potential (AP) and eutrophication potential (EP) per kg carcass weight departing the slaughterhouse were estimated to be 3.5 kg CO2-eq, 43.8 g SO2-eq and 32.1 g PO4-eq, respectively. For herds with a higher production efficiency, a 9% improvement in feed conversion ratio was met by 6%, 15% and 12% decreases in GWP, EP, AP, respectively. Scenario and sensitivity analyses also revealed that (a) a switch to high-protein diets results in lower GWP and higher AP and EP, and (b) reducing transportation distances by sourcing domestically produced wheat and barley does not lower environmental impacts in any notable manner. To improve cross-study comparability of these findings, results based on an auxiliary functional unit, kg liveweight departing the farm gate, are also reported., Highlights • Environmental footprints of Irish commercial pig production systems were examined. • Dual-functional unit approach was adopted to enhance cross-study comparability. • Trade-offs were identified between global warming and acidification/eutrophication. • ‘Feed mileage’ was found to have little impact on environmental performance. • Economic and environmental performances were shown to be positively correlated.

Degraded animal fats, characterized by the presence of palmitic (C16:0) and stearic (C18:0) fatty acids and related glycerolipids are the most common class of preserved lipids in organic residues trapped in the porous clay matrix of archaeological ceramic vessels. The ubiquitous presence of fatty acids in animal fats and plant oils precludes identification of fat types by the solely molecular composition of residues. Hence, animal fats are identified by determining their fatty acyl lipid distributions and stable carbon (δ13C) values allowing distinctions to be drawn between non-ruminant and ruminant, and dairy and adipose fats. The Δ13C proxy (= δ13C18:0 - δ13C16:0) originally proposed in the 1990s by Evershed and co-workers was based on modern reference fats sampled from animals raised in Britain on C3 plant diets. Further analyses on adipose and dairy fats from ruminants grazing in a wide range of isoscapes have shown that the Δ13C proxy can be applied in mixed C3/C4 environments, such as in Africa. Here we show, however, through the investigation of milk fats, how the Δ13C proxy can be perturbed when animals are reared on modern diets, specifically maize silage. It is thus shown that extreme care has to be taken when choosing modern reference fats for archaeological studies, and especially that insecurely-sourced animal fats should be precluded from such databases. Keywords: Modern reference materials, animal fats, lipid residue analyses, dairy fats, silage

Polyphenol oxidase (PPO) activity in red clover (Trifolium pratense) has been reported to reduce both proteolysis and lipolysis, resulting in greater N use efficiency and protection of PUFA across the rumen. Although high levels of PPO have been reported in grasses such as cocksfoot (orchard grass; Dactylis glomerata), no in vivo research has determined whether grass PPO elicits the same response as red clover PPO. To test the hypothesis that silage ensiled from grass with high levels of PPO protects N and PUFA across the rumen, 6 steers with ruminal and duodenal cannulas were offered cocksfoot silage (CO; high-PPO grass), perennial ryegrass silage (PR; Lolium perenne; low-PPO grass), or red clover silage (RC; high-PPO control) at 16 g DM/kg BW daily with the experiment consisting of two 3 × 3 Latin squares with 21-d periods, consisting of 12 d of diet adaptation, 6 d of duodenal marker infusion, 2 d of duodenal sampling, and 1 d of ruminal sampling. All silages were well preserved, with DM of 34.4, 55.3, and 45.4% for CO, PR, and RC. Activity of PPO in silages was low due to deactivation but was greater in CO than either PR or RC (0.15 vs. 0.05 and 0.08 μkatal/g DM). Protein-bound phenol (mg/g DM) as a measure of the degree of oxidation and an indication of PPO protection was greatest for RC (15.9) but comparable for PR (10.1) and CO (12.2). Biohydrogenation of C18 PUFA was significantly lower on RC compared to the 2 grass silages with CO greater than PR. Despite lower levels of total fatty acid intake and subsequent duodenal flow, CO resulted in greater levels of phytanic acid and total branched and odd chain fatty acids in duodenal digesta than RC or PR. Ruminal ammonia concentration was greatest for RC, with no difference between the grasses. Duodenal flow of microbial N and efficiency of microbial protein synthesis were lowest for CO and comparable for RC and PR. The CO (high-grass PPO) did not result in elevated levels of C18 PUFA escaping the rumen or improve efficiency of total N transfer through the rumen compared to PR. The RC resulted in a greater flow of N and nonmicrobial N to the duodenum than the 2 grasses with PR greater than CO.

Diets based on red clover silage (RCS) typically increase the concentration of polyunsaturated fatty acids (PUFA) in ruminant meat and milk and lower the efficiency of N utilization compared with grass silages (GS). Four multiparous Finnish Ayrshire cows (108 d postpartum) fitted with rumen cannulas were used in a 4 × 4 Latin square design with 21-d periods to evaluate the effect of incremental replacement of GS with RCS on milk production, nutrient digestion, whole-body N metabolism, and milk fatty acid composition. Treatments comprised total mixed rations offered ad libitum, containing 600 g of forage/kg of diet dry matter (DM), with RCS replacing GS in ratios of 0:100, 33:67, 67:33, and 100:0 on a DM basis. Intake of DM and milk yield tended to be higher when RCS and GS were offered as a mixture than when fed alone. Forage species had no influence on the concentration or secretion of total milk fat, whereas replacing GS with RCS tended to decrease milk protein concentration and yield. Substitution of GS with RCS decreased linearly whole-tract apparent organic matter, fiber, and N digestion. Forage species had no effect on total nonammonia N at the omasum, whereas the flow of most AA at the omasum was higher for diets based on a mixture of forages. Replacing GS with RCS progressively lowered protein degradation in the rumen, increased linearly ruminal escape of dietary protein, and decreased linearly microbial protein synthesis. Incremental inclusion of RCS in the diet tended to lower whole-body N balance, increased linearly the proportion of dietary N excreted in feces and urine, and decreased linearly the utilization of dietary N for milk protein synthesis. Furthermore, replacing GS with RCS decreased linearly milk fat 4:0 to 8:0, 14:0, and 16:0 concentrations and increased linearly 18:2n-6 and 18:3n-3 concentrations, in the absence of changes in cis-9 18:1, cis-9, trans-11 18:2, or total trans fatty acid concentration. Inclusion of RCS in the diet progressively increased the apparent transfer of 18-carbon PUFA from the diet into milk, but had no effect on the amount of 18:2n-6 or 18:3n-3 at the omasum recovered in milk. In conclusion, forage species modified ruminal N metabolism, the flow of AA at the omasum, and whole-body N partitioning. A lower efficiency of N utilization for milk protein synthesis with RCS relative to GS was associated with decreased availability of AA for absorption, with some evidence of an imbalance in the supply of AA relative to requirements. Higher enrichment of PUFA in milk for diets based on RCS was related to an increased supply for absorption, with no indication that forage species substantially altered PUFA bioavailability.

Published research relevant to the UK dairy goat industry is scarce. Current practices and concerns within the UK dairy goat industry must be better understood if research is to have optimal value. A postal survey was conducted of the farmer membership of the Milking Goat Association as a first step in addressing gaps in knowledge. Questions were asked about husbandry practices, farmer observations of their goats and their priorities for further research. Seventy-three per cent of Milking Goat Association members responded, representing 38 per cent of commercial dairy goat farms and 53 per cent of the commercial dairy goat population in England and Wales. Findings were comprehensive and showed extensive variation in farm practices. Farmers reported pneumonia and scours (diarrhoea) as the most prevalent illnesses of their kids. Pneumonia, diarrhoea, failure to conceive and poor growth were the most prevalent observations of youngstock. Overly fat body condition, assisted kidding, failure to conceive and difficulty drying off were the most prevalent observations of adult milking goats. Farmers' top priorities for further research were kid health (79.5 per cent of farmers), Johne's disease (69.5 per cent of farmers), tuberculosis (59 per cent of farmers) and nutrition (47.7 per cent of farmers).

Diets based on red clover silage (RCS) typically increase the concentration of polyunsaturated fatty acids (PUFA) in ruminant milk and meat compared with grass silages (GS), an effect that has been attributed to higher activity of polyphenol oxidase in red clover, promoting ruminal escape of dietary lipid. Four multiparous Finnish Ayrshire cows in mid lactation fitted with rumen cannulas were used in a 4×4 Latin Square design with 21-d experimental periods to evaluate the effects of incremental replacement of GS with RCS on ruminal lipid metabolism, using the omasal sampling technique in combination with Cr-EDTA, Yb acetate, and indigestible neutral detergent fiber as markers. Treatments comprised total mixed rations offered ad libitum containing 600 g of forage/kg of diet dry matter, with RCS replacing GS in a ratio of 0:100, 33:67, 67:33, and 100:0 on a dry matter basis. Silages contained a high proportion of lipid as nonesterified fatty acids (NEFA), with no difference between forage species (75 and 73% for GS and RCS, respectively). Substitution of GS with RCS had no influence on the intakes of NEFA, polar lipid, triacylglycerol, diacylglycerol, monoacylglycerol, or total fatty acids (FA), but altered the ingestion of specific FA. Replacing GS with RCS decreased linearly 18:3n-3 and increased linearly 18:2n-6 intakes. Changes in the proportion of RCS in the diet had no effect on the amounts or on the relative proportions of different lipid fractions at the omasum. On average, NEFA, polar lipid, triacylglycerol, diacylglycerol, and monoacylglycerol accounted for 80, 12, 4.4, 2.4, and 0.8% of total FA in omasal digesta, respectively. Replacement of GS with RCS increased linearly the amount of esterified and nonesterified 18:3n-3 at the omasum. Flows of cis -9 18:1 and 18:2n-6 were also increased linearly in response to RCS in the diet, whereas 3,7,11,15-tetramethyl-16:0 at the omasum was decreased. Replacing GS with RCS in the diet decreased linearly the lipolysis of dietary esterified lipids in the rumen from 85 to 70%. Effects on lipolysis due to forage species were also associated with linear decreases in apparent ruminal 18:3n-3 biohydrogenation from 93 to 85% and a trend toward lowered biohydrogenation of cis -9 18:1 and 18:2n-6 in the rumen. However, forage species had no effect on the flow of bound phenols formed as a consequence of polyphenol oxidase activity at the omasum. In conclusion, despite minimal differences in the extent of lipolysis in silo, lipid and constituent FA in RCS were less susceptible to ruminal lipolysis and biohydrogenation compared with GS.

The use of fecal fluorescence to improve detection of contamination of carcasses in the abattoir was previously reported. However, incidents of false negatives can result when animals are offered diets that contain little chlorophyll (e.g., concentrate). Here, we investigated the potential of incorporating a high-chlorophyll-containing feed ingredient (concentrated alfalfa extract; CAE) into the diets of sheep and cattle to improve fecal fluorescence intensity. The sheep experiment evaluated the fecal fluorescence of animals from pasture, when fed a concentrate-barley straw diet and when the concentrate diet incorporated CAE (100 g of dry matter a day). Fecal chlorophyll and metabolite content was highest on the pasture-fed animals and increased significantly over the concentrate diet when CAE was included. Subsequently fluorescent intensity was increased from 15,000 to 36,000 arbitrary units for concentrate and CAE-concentrate diets, respectively, compared with 59,000 for the pasture-fed animals. The cattle experiment investigated the potential of CAE to improve fluorescence of feces from a concentrate diet as well as a silage diet at two levels of incorporation (75 and 150 g CAE/kg of dry matter intake). This study also determined the fluorescence of digesta and carcass contamination in the abattoir on a subset of carcasses. In agreement with the sheep study, CAE significantly improved fluorescence of feces and digesta when added to a concentrate diet, but had little effect on improving fecal fluorescence from the silage-fed animals. This was thought to be related to greater chlorophyll degradation in the rumen or/ and the dark nature of the silage feces acting as a quencher of emitted fluoresced light. Incorporating high-chlorophyll-containing plant ingredients into ruminant concentrate diets will improve detection of fecal contamination by reducing false-negative readings. However, they will have little effect on false-positive readings due to the range of wavelengths emitted by natural chlorophyll and its metabolites. Implications and potential solutions for this are discussed.

The North Wyke Farm Platform (NWFP) generates large volumes of temporally-indexed data that provides a valuable test-bed for agricultural mathematical models in temperate grasslands. In our study, we used the primary datasets generated from the NWFP (https://nwfp.rothamsted.ac.uk/) to validate the SPACSYS model in terms of the dynamics of water loss and forage dry matter yield estimated through cutting. The SPACSYS model is capable of simulating soil water, carbon (C) and nitrogen (N) balance in the soil-plant-atmosphere system. The validated model was then used to simulate the responses of soil water, C and N to reseeding grass cultivars with either high sugar (Lolium perenne L. cv. AberMagic) or deep rooting (Festulolium cv. Prior) traits. Simulation results demonstrated that the SPACSYS model could predict reliably soil water, C and N cycling in reseeded grassland. Compared to AberMagic, the Prior grass could fix more C in the second year following reseeding, whereas less C was lost through soil respiration in the first transition year. In comparison to the grass cultivar of the permanent pasture that existed before reseeding, both grasses reduced N losses through runoff and contributed to reducing water loss, especially Prior in relation to the latter. The SPACSYS model could predict these differences as supported by the rich dataset from the NWFP, providing a tool for future predictions on less characterized pasture., Graphical abstract Image 1, Highlights • SPACSYS could simulate forage cutting dry matter, soil movement and nutrient cycling. • Simulation showed that deep-rooting grass may reduce water loss from soil. • Deep-rooting grass increased soil carbon storage from simulation results. • Simulation demonstrated deep-rooting grass lowered soil denitrification loss.

Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) are heme-containing enzymes that catalyze the O2-dependent oxidation of l-tryptophan (l-Trp) in biological systems. Although many decades have passed since their discovery, the mechanism of tryptophan oxidation has not been established. It has been widely assumed that IDO and TDO react using the same mechanism, although there is no evidence that they do. For IDO, a Compound II (ferryl) species accumulates in the steady state and is implicated in the mechanism; in TDO, no such species has ever been observed. In this paper, we examine the kinetics of tryptophan oxidation in TDO. We find no evidence for the accumulation of Compound II during TDO catalysis. Instead, a ternary [Fe(II)-O2, l-Trp] complex is detected under steady state conditions. The absence of a Compound II species in the steady state in TDO is not due to an intrinsic inability of the TDO enzyme to form ferryl heme, because Compound II can be formed directly through a different route in which ferrous heme is reacted with peroxide. We interpret the data to mean that the rate-limiting step in the IDO and TDO mechanisms is not the same.

Layers diets typically contain 15–20% soya due to its high crude protein content (ca. 36%). Reliance on soya for protein can result in large increases in cost of feed due to the law of supply and demand as a global commodity. Lupin grains have high protein content (35–40%) but previous experience with white lupins has shown toxic effects in poultry due to high levels alkaloids and poor performance due to anti-nutritional Non-starch polysaccharides (NSP). Here blue lupins either processed or whole were trialled for their potential as a protein source. Point of lay chickens (64) at 16 weeks of age were weighed and allocated to 16 coops of four hens. Coops, as the experimental unit, were randomly allocated to four treatments: layers mash with soya (Control); or layers mash with 150 g of lupin/kg diet with the lupin either: whole (Whole); dehulled (Dehulled) or dehulled + a solid state fermentation enzyme extract (SSF; 150 g/tonne DM). All diets were ground and formulated to be balanced for energy, crude protein and essential amino acids using NIRS. No difference in growth rate, final hen weight, DM and water intake, eggs per day, mean egg weight, yellowness of yolk or chroma was found between treatments. There was a trend (P

Lee, M. R. F., Tweed, J. K. S., Sullivan, M. L. (2012). Oxidation of ortho-diphenols in red clover with and without polyphenol oxidase (PPO) activity and their role in PPO activation and inactivation. Grass and Forage Science, 68 (1), 83-92.

Because of the potential benefits to hu- man health, there is interest in increasing 18:3n-3, 20:5n- 3, 22:6n-6, and cis-9,trans-11 CLA in ruminant foods. Four Aberdeen Angus steers (406 ± 8.2 kg of BW) fit- ted with ruminal and duodenal cannulas were used in a 4 × 4 Latin square experiment with 21-d periods to ex- amine the potential of fish oil (FO) and linseed oil (LO) in the diet to increase ruminal outflow of trans-11 18:1 and total n-3 PUFA in growing cattle. Treatments con- sisted of a control diet (60:40; forage:concentrate ratio, on a DM basis, respectively) based on maize silage, or the same basal ration containing 30 g/kg of DM of FO, LO, or a mixture (1:1, wt/wt) of FO and LO (LFO). Diets were offered as total mixed rations and fed at a rate of 85 g of DM/(kg of BW 0.75 /d). Oils had no ef- fect (P = 0.52) on DMI. Linseed oil had no effect (P > 0.05) on ruminal pH or VFA concentrations, whereas FO shifted rumen fermentation toward propionate at the expense of acetate. Compared with the control, LO increased (P < 0.05) 18:0, cis 18:1 (Δ9, 12-15), trans 18:1 (Δ4-9, 11-16), trans 18:2, geometric isomers of Δ9,11, Δ11,13, and Δ13,15 CLA, trans-8,cis-10 CLA, trans-10,trans-12 CLA, trans-12,trans-14 CLA, and 18:3n-3 flow at the duodenum. Inclusion of FO in the diet resulted in greater (P < 0.05) flows of cis-9 16:1, trans 16:1 (Δ6-13), cis 18:1 (Δ9, 11, and 13), trans 18:1 (Δ6-15), trans 18:2, 20:5n-3, 22:5n-3, and 22:6n-3, and decreased (P < 0.001) 18:0 at the duodenum relative to the control. For most fatty acids at the duodenum, re- sponses to LFO were intermediate of FO and LO. How- ever, LFO resulted in greater (P = 0.04) flows of total trans 18:1 than LO and increased (P < 0.01) trans-6 16:1 and trans-12 18:1 at the duodenum compared with FO or LO. Biohydrogenation of cis-9 18:1 and 18:2n-6 in the rumen was independent of treatment, but both FO and LO increased (P < 0.001) the extent of 18:3n-3 biohydrogenation compared with the control. Ruminal 18:3n-3 biohydrogenation was greater (P < 0.001) for LO and LFO than FO, whereas biohydrogenation of 20:5n-3 and 22:6n-3 in the rumen was marginally less (P = 0.05) for LFO than FO. In conclusion, LO and FO at 30 g/kg of DM altered the biohydrogenation of un- saturated fatty acids in the rumen, causing an increase in the flow of specific intermediates at the duodenum, but the potential of these oils fed alone or as a mixture to increase n-3 PUFA at the duodenum in cattle ap- pears limited.

Lee, M. R. F., Cabiddu, A., Niderkorn, V., Kim. E. J., Fychan, A. R., Scollan, N. D. (2011). In vitro rumen simulated (RUSITEC) metabolism of freshly cut or wilted grasses with contrasting polyphenol oxidase activities. Grass and Forage Science, 66 (2), 196-205. IMPF: 01.09 RONO: 05262

Increasing the polyunsaturated fatty acid (PUFA) composition of milk is acknowledged to be of benefit to consumer health. Despite the high PUFA content of forages, milk fat contains only about 3% of PUFA and only about 0.5% of n-3 fatty acids. This is mainly due to intensive lipid metabolism in the rumen (lipolysis and biohydrogenation) and during conservation (lipolysis and oxidation) such as drying (hay) and ensiling (silage). In red clover, polyphenol oxidase (PPO) has been suggested to protect lipids against degradation, both in the silage as well as in the rumen, leading to a higher output of PUFA in ruminant products (meat and milk). PPO mediates the oxidation of phenols and diphenols to quinones, which will readily react with nucleophilic binding sites. Such binding sites can be found on proteins, resulting in the formation of protein-bound phenols. This review summarizes the different methods that have been used to assess PPO activity in red clover, and an overview on the current understanding of PPO activity and activation in red clover. Knowledge on these aspects is of major importance to fully harness PPO's lipid-protecting role. Furthermore, we review the studies that evidence PPO-mediated lipid protection and discuss its possible importance in lab-scale silages and further in an in vitro rumen system. It is demonstrated that high (induction of) PPO activity can lead to lower lipolysis in the silage and lower biohydrogenation in the rumen. There are three hypotheses on its working mechanism: (i) protein-bound phenols could directly bind to enzymes (e. g. lipases) as such inhibiting them; (ii) binding of quinones in and between proteins embedded in a lipid membrane (e. g. in the chloroplast) could lead to encapsulation of the lipids; (iii) direct binding of quinones to nucleophilic sites in polar lipids also could lead to protection. There is no exclusive evidence on which mechanism is most important, although there are strong indications that only lipid encapsulation in protein-phenol complexes would lead to an effective protection of lipids against ruminal biohydrogenation. From several studies it has also become apparent that the degree of PPO activation could influence the mode and degree of protection. In conclusion, this review demonstrates that protein-bound phenols and encapsulation in protein-phenol complexes, induced by PPO-mediated diphenol oxidation, could be of interest when aiming to protect lipids against pre-ruminal and ruminal degradation.

Diet composition can be estimated in free-ranging animals by the use of n-alkane and long-chain fatty alcohol concentrations in feces. However, this technique involves relatively laborious and costly analytical techniques. Two spectroscopy techniques were investigated as a way of determining whether dietary differences are likely, thus indicating whether the more expensive and labor-intensive techniques for more detailed analysis are justified. Fourier-transform infrared spectroscopy (FTIR) and front-face fluorescence emission spectroscopy (lambda(excitation) = 380 nm, lambda(emission) = 600 to 760 nm) were used to analyze fecal samples collected from 2 different breeds of cattle and sheep (4 groups in total, n = 6 per group) grazing moorland plants in 2 grazing sessions. These fecal samples were also analyzed for alkane and alcohol concentrations. Fourier-transform infrared spectra, particularly in the alkane regions, demonstrated clear separation between animal species. Fluorescence emission spectra showed similar separation; fluorophores were most likely chlorophylls and their derivatives. Multivariate analysis of all 3 data sets showed similar variation within and between groups of cattle and sheep, indicating differences in diet selection particularly between species, but also between breed and grazing session. Both spectroscopy methods showed utility in suggesting differences in diet composition that would be worth investigating using more detailed chemical analyses. Of the 2 techniques, the FTIR spectroscopy gave the better comparative results, being able to detect differences in sampling months that were detected with alkanes and alcohols that the fluorescence emission spectroscopy did not detect.

Based on the potential benefits to human health, there is interest in developing sustainable nutritional strategies to enhance the concentration of long-chainn-3 fatty acids in ruminant-derived foods. Four Aberdeen Angus steers fitted with rumen and duodenal cannulae were used in a 4 × 4 Latin square experiment with 21 d experimental periods to examine the potential of fish oil (FO) in the diet to enhance the supply of 20 : 5n-3 and 22 : 6n-3 available for absorption in growing cattle. Treatments consisted of total mixed rations based on maize silage fed at a rate of 85 g DM/kg live weight0·75/d containing 0, 8, 16 and 24 g FO/kg diet DM. Supplements of FO reduced linearly (P P P trans-16 : 1,trans-18 : 1,trans-18 : 2, 20 : 5n-3 and 22 : 6n-3, and decreased linearly (P n-3 at the duodenum. Increases in the flow oftrans-18 : 1 were isomer dependent and were determined primarily by higher amounts oftrans-11 reaching the duodenum. In conclusion, FO alters ruminal lipid metabolism of growing cattle in a dose-dependent manner consistent with an inhibition of ruminal biohydrogenation, and enhances the amount of long-chainn-3 fatty acids at the duodenum, but the increases are marginal due to extensive biohydrogenation in the rumen.

Cabiddu, A., Sallis, L., Tweed, J. K. S., Molle, G., Decandia, M., Lee, M. R. F. (2010). The influence of plant polyphenols on lipolysis and biohydrogenation in dried forages at different phenological stages in vitro. Journal of the Science of Food and Agriculture, 90, (5), 829-835 Sponsorship: University Research Fund; Makira Fund; Livery Guild IMPF: 01.36

BACKGROUND: The enzyme polyphenol oxidase (PPO) reduces the extent of proteolysis and lipolysis within red clover fed to ruminants. PPO catalyses the conversion of phenols to quinones, which can react with nucleophilic cellular constituents (e.g. proteins) forming protein–phenol complexes that may reduce protein solubility, bioavailability to rumen microbes and deactivate plant enzymes. In this study, we localized PPO in red clover leaf tissue by immunogold labelling and investigated whether red clover lipid was protected in the absence of PPO-induced protein–phenol complexes and plant enzymes (lipases). RESULTS: PPO protein was detected to a greater extent (P < 0.001) within the chloroplasts of mesophyll cells in stressed (cut/crushed and wilted for 1 h) than freshly cut leaves for both palisade (61.6 and 25.6 Au label per chloroplast, respectively) and spongy mesophyll cells (94.5 and 40.6 Au label per chloroplast, respectively). Hydrolysis of lipid and C18 polyunsaturated fatty acid biohydrogenation during in vitro batch culture was lower (P < 0.05) for wild-type red clover than for red clover with PPO expression reduced to undetectable levels but only when cellular matrices containing protein–phenol complexes were present. CONCLUSION: Damaging of the leaves resulted in over a doubling of PPO detected within mesophyll cells, potentially as a consequence of conversion of the enzyme from latent to active form. PPO reduction of microbial lipolysis was apparent in macerated red clover tissue but not in the absence of the proteinaceous cellular matrix, suggesting that the PPO mechanism for reducing lipolysis may be primarily through the entrapment of lipid within protein–phenol complexes. Copyright © 2009 Society of Chemical Industry

Within this study, we investigated whether the polyunsaturated fatty acids (PUFA)-rich nature of rumen protozoa is a consequence of ingestion of PUFA-rich chloroplasts. Four Hereford x Friesian steers were offered hay [low 18:3 (n-3) and low chlorophyll concentration] followed by freshly cut perennial ryegrass [high 18:3 (n-3) and high chlorophyll concentration] for 16 days. On the 14th and 16th days, rumen protozoa as well as attached and planktonic bacteria were fractionated 1 h before (-1 h), 2 and 6 h postfeeding, and their fatty acid concentrations determined. Protozoa fractionated from fresh grass-fed steers were richer (P

Synopsis An account is given of the general anatomy, histology and physiology of the anterior mid-gut of Periplaneta americana and of the formation of the peritrophic membrane.

This study investigated red clover polyphenol oxidase (PPO), its: activity, activation from latent into active form, and whether grazing at different levels of herbage maturity could result in sufficient PPO activation to protect proteins and glycerol-based lipid in the rumen. Wild-type red clover with high PPO activity and a mutant red clover with low PPO activity were used in five experiments. Firstly to investigate activation of the latent red clover PPO enzyme either through: protease, substrate (ortho-diphenols), cell damage or chemically (sodium dodecylsulphate detergent) induced mediation. Secondly PPO's activity at a range of pHs were determined to show how activation method altered the enzymes activity. Finally whether grazing could activate latent PPO and ultimately result in protected protein and glycerol-based lipid in the rumen was investigated. Incubation of extracted red clover PPO enzyme with a range of ortho-diphenols in the presence of oxygen resulted in rapid conversion of the latent to the active form of PPO. This conversion was blocked by the addition of ascorbic acid suggesting that this mechanism requires formation of ortho-quinones. Cell damage was highly correlated with activation of latent PPO and the formation of protein-bound phenols due to mixing of the chloroplastic enzyme with the vacuolar substrate (ortho-diphenols). The potential for PPO activity to occur during grazing was investigated by measuring the oxygen concentration in red clover boluses and within the rumen and PPO's effect on protein and glycerol-based lipid within red clover boluses. There was a rapid depletion of oxygen from the rumen, limiting the potential for PPO activation and activity to occur to the period of mastication. PPO was shown to be activated in both wild-type and mutant red clover boluses, however, factors Such as fibre content and maturity of the red clover herbage (PPO concentration) altered the efficacy of PPO to produce phenol-bound protein. As a result greater protection of glycerol-based lipid in this study was achieved with a prior conditioning (wilting) of the red clover.

Lee, M. R. F., Tweed, J. K. S., Scollan, N. D., Sullivan, M. L. (2008). Ruminal micro-organisms do not adapt to increase utilization of polyphenol oxidase protected red clover protein and glycerol-based lipid. Journal of the Science of Food and Agriculture, 88 (14), 2479-2485. Sponsorship: EU(PROSAFEBEEF)FOOD-CT-2006-36241:DEFRA IMPF: 01.33 RONO: 1920 6624 Sponsorship: BBSRC RONO: BBS/E/W/00003135A; BBS/E/W/00003135B

Microbial transformations in the rumen ecosystem have a major impact on our ability to meet the challenge of reducing the environmental footprint of ruminant livestock agriculture, as well as enhancing product quality. Current understanding of the rumen microbial ecosystem is limited, and affects our ability to manipulate rumen output. The view of ruminal fermentation as the sum of activities of the dominant rumen microbiota is no longer adequate, with a more holistic approach required. This paper reviews rumen functionality in the context of the microbiota of the rumen ecosystem, addressing ruminal fermentation as the product of an ecosystem while highlighting the consequences of this for ruminant agriculture. Microbial diversity in the rumen ecosystem enhances the resistance of the network of metabolic pathways present, as well as increasing the potential number of new pathways available. The resulting stability of rumen function is further promoted by the existence of rumen microbiota within biofilms. These protected, structured communities offer potential advantages, but very little is currently known about how ruminal microorganisms interact on feed-surfaces and how these communities develop. The temporal and spatial development of biofilms is strongly linked to the availability of dietary nutrients, the dynamics of which must also be given consideration, particularly in fresh-forage-based production systems. Nutrient dynamics, however, impact not only on pathway inputs but also the turnover and output of the whole ecosystem. Knowledge of the optimal balance of metabolic processes and the corresponding microbial taxa required to provide a stable, balanced ecosystem will enable a more holistic understanding of the rumen. Future studies should aim to identify key ecosystem processes and components within the rumen, including microbial taxa, metabolites and plant-based traits amenable to breeding-based modification. As well as gaining valuable insights into the biology of the rumen ecosystem, this will deliver realistic and appropriate novel targets for beneficial manipulation of rumen function.

This experiment aims to determine whether polyphenol oxidase (PPO) can reduce the extent of lipolysis and the consequent polyunsaturated fatty acid loss through microbial biohydrogenation in red clover when incubated in the presence of rumen fluid. PPO is involved in the browning reaction of red clover leaves when cut or crushed and exposed to air. It starts the browning process by oxidizing endogenous phenols to quinones, which contain electrophilic sites. These sites react with nucleophilic sites of other compounds such as proteins and have been shown to reduce proteolysis and lipolysis in silo. Two lines of red clover (cv. Milvus), a genotypic mutant with reduced PPO activity (L) and the wild type (H) with a high level of PPO activity, were cut 3 cm above soil level, crushed and cut into 1 cm strips before being loaded into incubation bottles. These were then incubated in anaerobic buffer at 39 °C in either the absence (−) or the presence (+) of rumen microorganisms. The incubations were then compared over a 24 h time course in terms of lipolytic activity. Characterization of the tissues showed PPO activities of 25.3 and 5.13 U g−1 fresh weight for H and L, respectively. Lipolysis, measured as the proportional decline in the membrane lipid, was reduced (P < 0.001) with increasing PPO activity in both the presence (+) and absence (−) of rumen microorganisms. However, values were significantly higher (P < 0.001) in the presence of rumen microorganisms, with values after the 24 h incubation of 0.28, 0.42, 0.72 and 0.82 for H−, L−, H+ and L+, respectively. Biohydrogenation of C18:2 and C18:3 polyunsaturated fatty acids were significantly lower in the H+ treatment than the L+ treatment, with mean values after 24 h incubation of 53% and 57% (P < 0.05) for C18:2 and 65% and 74% (P < 0.01) for C18:3, respectively. Changes that occurred in the lipid fractions (membrane lipid, diacylglycerol, triacylglycerol and free fatty acids) during the incubations are also reported and discussed. These results support the selection of forages high in PPO activity to reduce polyunsaturated fatty acid loses in the rumen.

BackgroundTwo dairy goat farms with high level of lameness in lactating animals were presented for further investigation. Farm 1 and Farm 2 presented with 37 and 67 % morbidity, respectively. Both farms had an all year round indoor system, feeding ad libitum concentrate with forage available at all times.Case presentationThe lameness was found to be based in the foot. Previous treatments consisting of biweekly footbathing with zinc sulphate, spraying lesions with oxytetracycline spray and packing lesions with copper crystals on a single occasion and single injections with long acting oxytetracycline had not been successful. Mild cases had signs of haemorrhaging in the white line or on the sole of the foot. Moderate cases showed under running of the wall horn or small areas of exposed sole corium. Severe cases would consist of horn or wall separation with the corium exposed and infected. In extreme cases only the wall horn of the claw remained, with a large area of necrotic tissue in the centre and no healthy corium visible. Only one animal was seen to have interdigital lesions. Polymerase chain reaction (PCR) and culture of swabs taken from exposed corium and the interdigital space were negative for Dichelobacter nodosus but PCR for treponemes were positive in both the adults and the youngstock tested. Due to the high level of concentrate in the diet of these goats, nutrition was thought to contribute to the problem. Transcutaneous rumen fluid samples were taken and pH was measured on both farms, with 35 % of the samples below pH value 5.5.ConclusionNo definite diagnosis could be made. However, the results suggest both treponemes and nutrition play a role in the aetiology of the lameness. The initial sole or wall horn lesions were thought to be secondarily infected by treponemes. Further investigation is needed to definitively diagnose the cause and contributing factors for this lameness.

Forage type and management influences the nutritional quality and fatty acid composition of ruminant milk. Replacing grass silage with red clover (RC; L.) silage increases milk fat 18:3-3 concentration. Red clover has a higher polyphenol oxidase (PPO) activity compared with grasses, which has been suggested to decrease lipolysis and . The present study characterized the abundance and fatty acid composition of esterified lipid and NEFA before and after ensiling of grass and RC to investigate the influence of forage species, growth stage, and extent of fermentation on lipolysis. A randomized block design with a 2 × 3 × 4 factorial arrangement of treatments was used. Treatments comprised RC or a mixture of timothy ( L.) and meadow fescue ( Huds.) harvested at 3 growth stages and treated with 4 levels of formic acid (0, 2, 4, and 6 L/t). Lipid in silages treated with 0 or 6 L/t formic acid were extracted and separated into 4 fractions by TLC. Total PPO activity in fresh herbage and the content of soluble bound phenols in all silages were determined. Concentrations of 18:3-3 and total fatty acids (TFA) were higher (0.001) for RC than for grass. For both forage species, 18:3-3 and TFA content decreased linearly (0.001) with advancing growth stage, with the highest abundance at the vegetative stage. Most of lipid in fresh RC and grass herbage (97%) was esterified, whereas NEFA accounted for 71% of TFA in both silages. Ensiling resulted in marginal increases in TFA content and the amounts of individual fatty acids compared with fresh herbages. Herbage total PPO activity was higher (0.001) for RC than grass (11 vs. 0.11 μkatal/g leaf fresh weight). Net lipolysis during ensiling was extensive for both forage species (660 to 759 g/kg fatty acid for grass and 563 to 737 g/kg fatty acid for RC). Formic acid application (0 vs. 6 L/t) resulted in a marked decrease ( = 0.026) in net lipolysis during the ensiling of RC, whereas the opposite was true ( = 0.026) for grass. In conclusion, results suggest that formic acid addition during the ensilage of RC decreases lipolysis . For both plant species, total PPO activity was not associated with the extent of lipolysis . However, bound phenols formed via PPO activity appear to have a role in protecting lipid and protein against degradation in grass and lowering proteolysis of RC during ensiling.

Lee, M. R. F., Connelly, P. L., Tweed, J. K. S., Dewhurst, R. J., Merry, R. J., Scollan, N, D. (2006). Effects of high-sugar ryegrass silage and mixtures with red clover silage on ruminant digestion. 2. Lipids . Journal of Animal Science, 84, (11), 3061-3070. Key Words: beef steer • biohydrogenation • fatty acid • high-sugar grass silage • phytanic acid • red clover silage Sponsorship: European Commission Key Action 1 project, HealthyBeef QLRT-CT-2000-31423. Partly funded DEFRA

Little is known about the level or activity of polyphenol oxidase (PPO) in grasses and its potential impact on proteolysis and lipolysis. Six grass species were initially screened for PPO activity (740.6, 291.9, 213.6, 119.0, 16.3 and 6.5 U g(-1) fresh weight (FW) for cocksfoot, hybrid ryegrass, Italian ryegrass, perennial ryegrass, timothy and tall fescue respectively). Cocksfoot, which expressed the highest activity, was then used to determine the effect of PPO on plant-mediated proteolysis and lipolysis in a simulated rumen environment. Sourced cocksfoot was macerated and incubated in an antibiotic-containing anaerobic medium with or without ascorbate to deactivate PPO in the dark at 39 degrees C over five time points. At each time point (0, 1, 2, 6 and 24h), six replicate samples were destructively harvested; three of the replicates were used for lipid analysis and the other three for protein, free amino acid and bound phenol determination. Characterisation of the herbage showed PPO activities of 649.6 and 0 U g(-1) FW, which were reflected in the extent of phenol (derived from quinones) binding to protein after 24h of incubation, namely 65.1 and 29.6mg bound phenol g(-1) protein (P

Duodenally and ruminally fistulated steers were offered grass silage and one of three concentrates at a ratio of 60: 40 (forage: concentrate on a dry-matter basis) : F0, F1 or F4 at 14 g/kg live weight. The concentrates were designed to be iso-lipid and to provide the same amount of sunflower oil but increasing amounts of fish oil : 0, 1 and 4 g per 100 g, respectively. Ruminal characteristics were measured along with fatty acid intakes and duodenal flows to determine the effect of fish oil on : ruminal pH, ammonia-N concentration, volatile fatty acid (VFA) concentration and polyunsaturated fatty acid (PUFA) metabolism. Fish oil had no significant effect on ruminal pH, ammonia-N concentration or the molar proportions of the major VFA, although total VFA concentration was significantly reduced at the highest level of fish oil inclusion. Fish oil significantly increased the flow of long chain PUFA, total conjugated linoleic acid and vaccenic acid to the duodenum and decreased the flow of stearic acid. Biohydrogenation, as determined by the net loss of fatty acid between the mouth and duodenum, of oleic and linolenic acid was not affected by fish oil inclusion and averaged 0·64 and 0·92, respectively. There was a small increase in the biohydrogenation of linoleic acid with increasing fish oil from 0·89 to 0·92 (P< 0·01) on F0 and F4, respectively. Biohydrogenation of the long chain PUFA C20 : 5(n-3) and C22 : 6(n-3) increased from 0·49 and 0·74 to 0·79 and 0·86 (P< 0·01), respectively when fish oil in the concentrate increased from 1 to 4 g per 100 g. The net effect of fish oil on lipid metabolism appears to inhibit the transition of vaccenic acid to stearic acid in the rumen resulting in a build up of this intermediate in the biohydrogenation pathway of C18 PUFA.

Polyphenol oxidase (PPO) is an enzyme involved in the browning reaction of red clover leaves, when cut or crushed and exposed to air. PPO starts the browning process by oxidizing endogenous phenols to quinones, which contain electrophilic sites. These sites react with nucleophilic sites of other compounds such as proteins. The leaf tissue of two lines of red clover (cv. Milvus, a genotypic mutant with reduced PPO activity (LowPPO) and the wild-type, NormalPPO) were extracted in phosphate-citrate buffer, and a third treatment was prepared by extracting the LowPPO leaves in phosphate-citrate buffer plus 50 mm ascorbate to inhibit PPO activity (AscPPO). These extracts were compared over a 12 h time course in terms of proteolytic and lipolytic activity. Characterization of the tissues showed PPO activities of 9.11, 1.85 and 0 Delta optical density g(-1) fresh weight min(-1), which were reflected in the extent of phenol (derived from quinones) binding to protein after 12 h incubation 102.3, 83.2 and 45.8 mg bound phenol g(-1) protein (p

Duodenal fistulated steers were offered grass silage at 14 g/kg liveweight and one of three concentrates at a ratio of 60:40 (forage:concentrate on a dry matter basis): FISH0, FISH1 or FISH2 designed to be iso-lipid and to provide the same amount of sunflower oil but increasing amounts of fish oil: 0, 1 and 4%, respectively. Fatty acid intakes and duodenal flows were examined to determine the effects of fish oil on polyunsaturated fatty acid (PUFA) metabolism. Fish oil significantly increased the flow of long chain PUFA, conjugated linoleic acid and trans vaccenic acid to the duodenum and decreased the flow of stearic acid. Biohydrogenation of linoleic and linolenic acids was not affected by fish oil inclusion.

Grass is a rich source of alpha-linolenic acid (18: 3n-3) and in this study the effects on ruminal fatty acid metabolism of feeding beef steers zero-grazed Lolium perenne containing elevated levels of water-soluble carbohydrate (WSC) were investigated. Eight Hereford x Friesian steers were offered ad libitum access to one of two varieties of Lolium perenne, Ba11353, high WSC (HS) or AberElan, intermediate WSC ( experimental control) harvested at different times of the day ( 14: 00 and 10: 00 h, respectively) to accentuate WSC differentials. The grass was zero-grazed and fed for 21 days, after which the animals were offered ad libitum grass silage for 14 days to provide a covariate intake. The dry matter ( 202 vs. 167 g per kg fresh weight), WSC( 243 vs. 161 g per kg DM), total fatty acids (21.4 vs. 17.9 g per kg DM) and proportion of 18: 3n-3 (0.54 vs. 0.43) were greater and fibre content was lower ( 251 vs. 296 g ADF per kg DM) for HS compared with the control. DM intake and intake of total fatty acids and 18: 3n-3 was higher for HS (9.3 vs. 6.7 kg per d; 201 vs. 117 and 108.5 vs. 51.3 g per d, respectively). There was a trend ( P

Ten Hereford ✕ Friesian steers prepared with rumen, duodenal and ileal cannulae were offered ad libitum access to either grass (G), white clover (W) or red clover (R) silage or mixtures of the grass silage with the clover silages (GW and GR; 60: 40 dry matter (DM) basis). The experiment was conducted as a two-period change-over design within each clover treatment, with grass silage as an experimental control. The clover silages had higher concentrations of DM, nitrogen (N), and total fatty acids and lower concentrations of fibre (P < 0·05) than the grass silage. Rumen fermentation characteristics were similar between treatments apart from a slight elevation of n-butyric acid levels with white clover silage diets (P < 0·05). Duodenal flows of DM, organic matter (OM), total and microbial nitrogen (MN) were significantly higher (P < 0·05) with the white clover silage treatments compared with the grass silage diet. However there were no significant differences in the efficiency of microbial protein synthesis with a mean value of 27·1 g MN per kg OM apparently digested in the rumen. Duodenal flows of total fatty acids per unit intake were not significantly different, although there was a trend (P < 0·1) for increased flows with the white clover silage diets. Duodenal flows of cis-vaccenic acid (P < 0·01), linoleic acid (P < 0·05), α-linolenic acid (P < 0·05) and cis-9 trans-11 conjugated linoleic acid (CLA) (P < 0·05) were higher for the clover silages, particularly white clover silage. The increased flows of α-linolenic and linoleic acids remained significant after correcting for differences in DM intake. Biohydrogenation of linoleic (mean = 0·83) and α-linolenic (mean = 0·86) acids was extensive for all diets but the latter was significantly lower (P < 0·01) for the diets based on red clover silage. These results suggest potential for modifying the fatty acid composition of ruminant products by feeding clover silages.

Polyphenol oxidase (PPO) activity in red clover (Trifolium pratense) has been reported to reduce both proteolysis and lipolysis, resulting in greater N use efficiency and protection of PUFA across the rumen. Although high levels of PPO have been reported in grasses such as cocksfoot (orchard grass; Dactylis glomerata), no in vivo research has determined whether grass PPO elicits the same response as red clover PPO. To test the hypothesis that silage ensiled from grass with high levels of PPO protects N and PUFA across the rumen, 6 steers with ruminal and duodenal cannulas were offered cocksfoot silage (CO; high-PPO grass), perennial ryegrass silage (PR; Lolium perenne; low-PPO grass), or red clover silage (RC; high-PPO control) at 16 g DM/kg BW daily with the experiment consisting of two 3 × 3 Latin squares with 21-d periods, consisting of 12 d of diet adaptation, 6 d of duodenal marker infusion, 2 d of duodenal sampling, and 1 d of ruminal sampling. All silages were well preserved, with DM of 34.4, 55.3, and 45.4% for CO, PR, and RC. Activity of PPO in silages was low due to deactivation but was greater in CO than either PR or RC (0.15 vs. 0.05 and 0.08 μkatal/g DM). Protein-bound phenol (mg/g DM) as a measure of the degree of oxidation and an indication of PPO protection was greatest for RC (15.9) but comparable for PR (10.1) and CO (12.2). Biohydrogenation of C18 PUFA was significantly lower on RC compared to the 2 grass silages with CO greater than PR. Despite lower levels of total fatty acid intake and subsequent duodenal flow, CO resulted in greater levels of phytanic acid and total branched and odd chain fatty acids in duodenal digesta than RC or PR. Ruminal ammonia concentration was greatest for RC, with no difference between the grasses. Duodenal flow of microbial N and efficiency of microbial protein synthesis were lowest for CO and comparable for RC and PR. The CO (high-grass PPO) did not result in elevated levels of C18 PUFA escaping the rumen or improve efficiency of total N transfer through the rumen compared to PR. The RC resulted in a greater flow of N and nonmicrobial N to the duodenum than the 2 grasses with PR greater than CO.

With improved breeding and cultivation, ruminant animals can yield food that is better for people and the planet, say Mark C. Eisler, Michael R. F. Lee and colleagues.