Search

Summary Proliferation of filamentous fungi following ingress of oxygen to silage is an important cause of dry matter losses, resulting in significant waste. In addition, the production of mycotoxins by some filamentous fungi poses a risk to animal health through mycotoxicosis. Quantitative assessment of fungal growth in silage, through measurement of ergosterol content, colony‐forming units or temperature increase is limiting in representing fungal growth dynamics during aerobic spoilage due to being deficient in either representing fungal biomass or being able to identify specific genera. Here, we conducted a controlled environment aerobic exposure experiment to test the efficacy of a monoclonal antibody‐based enzyme‐linked immunosorbent assay (ELISA) to detect the proliferation of fungal biomass in six silage samples. We compared this to temperature which has been traditionally deployed in such experiments and on‐farm to detect aerobic deterioration. In addition, we quantified ergosterol, a second marker of fungal biomass. After 8 days post‐aerobic exposure, the ergosterol and ELISA methods indicated an increase in fungal biomass in one of the samples with a temperature increase observed after 16 days. A comparison of the methods with Pearson's correlation coefficient showed a positive association between temperature and ergosterol and both markers of fungal biomass. This work indicates that the technology has potential to be used as an indicator of microbial degradation in preserved forage. Consequently, if it developed as an on‐farm technique, this could inform forage management decisions made by farmers, with the goal of decreasing dry matter losses, improving resource and nutrient efficiency and reducing risks to animal health., Current techniques for detection of aerobic spoilage in silage include measurement of changes in temperature and ergosterol concentration. Here, we investigate a novel approach to detection of fungal proliferation in silage through application of a monoclonal antibody based method previously described for detection of fungi in soil and medical settings.

Goat milk is globally consumed but nutritional profiling at retail level is scarce. This study compared the nutrient composition of retail cow and goat milk (basic solids, fatty acids, minerals, and phytoestrogens) throughout the year and quantified the potential implications on the consumers’ nutrient intakes. When compared to cow milk, goat milk demonstrated nutritionally desirable traits, such as lower concentrations of C12:0, C14:0, C16:0 and Na: K ratio, and the higher concentrations of cis polyunsaturated fatty acids (PUFA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), isoflavones, B, Cu, Mg, Mn, P and I, although the latter may be less desirable in cases of high milk intakes. However, in contrast with nutritional targets, it had lower concentrations of omega-3 PUFA, vaccenic acid, lignans, Ca, S and Zn. The extent of these differences was strongly influenced by season and may demonstrate a combination of differences on intrinsic species metabolism, and farm breeding/husbandry practices.

Context Selenium (Se) is a trace element essential for cellular function in animals as a component of the enzymes glutathione peroxidase and iodothyronine-5-deiodinase. In many parts of Europe, Se is often deficient in livestock diets due to the low Se status of soil. Supplementation of diets with selenised yeast (predominately as seleno-methionine) or inorganic sodium selenite is common practice in most livestock systems, including ruminants. Lactic acid bacteria have been shown to convert inorganic Se into predominantly elemental nano-Se, which has been used recently in human pro-biotics as a less toxic form of Se. Therefore, silage lactic acid bacteria may provide a supplementation route of bioavailable nano-Se for ruminants. Aim Here, we report on the effect of feeding inoculated silage enriched with a supra-nutritional level of nano-Se (Selage) versus control inoculated silage (Silage) on the Se status of finishing lambs and their products, followed by a second study where blood parameters were investigated in ewes. Methods In the first study, 40 Charollais × Suffolk lambs (42 ± 1.7 kg) were paired according to weight and sex, then allocated to the two treatments for 8 or 10 weeks. Uptake of Se into wool was temporally assessed, as well as excretion of Se into faeces. Selenium concentrations in blood and muscle, carcass characteristics and meat quality are reported postmortem. In the second study, individually penned Suffolk × Mule ewes (n = 12; 76 ± 4.5 kg) were offered the same diets as in the first study. Blood parameters were assessed at the start and after 6 weeks, with intake and excretion into faeces and urine assessed temporally throughout the study. Key results In the first study, dry-matter (DM) intake was similar in both treatment groups, at 0.8 ± 0.03 kg/day, but Se concentrations of the diets were significantly different, resulting in intakes of ~0.14 and 1.60 mg/day on the Silage and Selage diets, respectively. This was reflected in higher Se concentrations in faeces (0.4 vs 2.0 mg/kg DM; P < 0.001), wool (0.11 vs 0.25 mg/kg DM; P < 0.001), blood (0.19 vs 0.46 mg/L; P < 0.001) and muscle (0.31 vs 0.41 mg/kg: P < 0.01) on the Selage than on the Silage diet. Colour (chroma) shelf life of the meat was significantly higher on the Selage treatment (8.05 vs 9.2 days; P < 0.05). In the second trial, for ewes fed Selage, blood seleno-methionine increased from 0.21 to 0.25 mg/L and seleno-cysteine from 0.25 to 0.35 mg/L after 6 weeks on the treatment, whereas there was no change in ewes fed Silage. Glutathione peroxidase increased, whereas haematocrit, haemoglobin and platelet count were decreased across time during the study, but there was no difference between the treatments. Conclusions Nano-Se provided by the Selage treatment was shown to be available to sheep and improve shelf life, with no adverse haematological effects. Implications There is potential to use silage inoculants to provide bioavailable Se to ruminants. Further research is required to determine the most appropriate dose for animal performance and product quality.

The role of beef in human diets has been questioned over the last few decades, due largely to its typically high mass-based carbon footprint. However, recent advancements in sustainability literature challenge this paradigm based on the new theory that climate impacts of food commodities should be measured relative to their overall nutritional value rather than their nominal mass. This shift has opened a new opportunity for the global beef industry, and especially for pasture-based systems that can avoid food-feed competition for land and other resources, as beef is a nutritionally dense food. Nonetheless, the sector’s true capability to supply a wide range of nutrients for humans, consistently across multiple systems under multiple weather patterns, has not been well-documented. Using whole-system datasets from the North Wyke Farm Platform in the South West of England, we investigated the nutritional value of beef produced from the three most common pasture systems in temperate regions: permanent pasture (PP), grass and white clover (GWC) and a short-term monoculture grass ley (MG). Beef produced from these three pasture systems was analysed for key nutrients (fatty acids, minerals and vitamin E) over three production cycles (2015–2017) to determine potential differences between systems. Fatty acid, mineral and vitamin E profiles of the pasture and silage fed to each group were also assessed, with subtle differences between pastures reported. For beef, subtle differences were also observed between systems, with GWC having higher omega-6 polyunsaturated fatty acid (PUFA) concentrations than PP and MG. However, the overall nutritional quality of beef was found to be largely comparable across all systems, suggesting that temperate pasture-based beef can be classified as a single commodity in future sustainability assessments, regardless of specific sward types. A 100 g serving of temperate pasture-based beef was found to be a high source (>20% recommended daily intake: RDI) of protein, monounsaturated fatty acids, saturated fatty acids, vitamins – B2, B3, B12 and minerals – Fe, P, Zn; a good source (10–19% RDI) of vitamin – B6 and mineral – K; and a complementary source (5–9% RDI) of omega-3 PUFA, vitamin – B9 and minerals – Cu, Mg, Se. The nutritional value of a food item should be used in defining its environmental cost (e.g. carbon footprint) to make fair comparisons across different food groups (e.g. protein sources). Here, we showed that pasture-based beef had a nutrient indexed carbon footprint of between 0.19 and 0.23 Kg CO2-eq/1% RDI of key nutrients.

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.

Bovine mycotoxicosis is a disorder caused by the ingestion of fungal toxins. It is associated with chronic signs, such as reduced growth rate and milk yield, and causes significant economic cost to the dairy industry. The mycotoxins deoxynivalenol (DON), zearalenone (ZEN), and fumonisin B1 (FB1) are commonly found in grain fed to cattle. Patulin (PA) is a common grass silage contaminant but is also found in grain. The effects of these mycotoxins on cellular function at low concentrations are not well understood. Using Madin&ndash, Darby bovine kidney cells we evaluated the cellular response to these mycotoxins, measuring cytotoxicity, de novo protein synthesis, cell proliferation, cell cycle analysis, and also metabolic profiling by 1H NMR spectroscopy. DON, ZEN, and PA induced cytotoxicity, and PA and FB1 induced a decrease in metabolic activity in surviving cells. DON was the only mycotoxin found to have a significant effect on the metabolic profile, with exposed cells showing increased cellular amino acids, lactate, 2-oxoglutarate, 3-hydroxybutyrate, and UDP-N-acetylglucosamine and decreased &beta, alanine, choline, creatine, taurine, and myo-inositol. Cells exposed to DON also showed reductions in protein synthesis. DON has previously been documented as being a ribotoxin, the results here suggest that exposure of bovine cells to DON causes a decrease in protein synthesis with corresponding cellular accumulation of precursors. Cell proliferation was also arrested without causing apoptosis. It is likely that exposure triggers hypoxic, hypertonic, and ribotoxic responses in bovine cells, and that these responses contribute to reduced productivity in exposed cattle.

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.

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.

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.

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.

Although often investigated within the context of plant growth and development and/or seed composition, plant lipids have roles in responses to environment. To dissect changes in lipid and fatty acid composition linked to drought tolerance responses in oats, we performed a detailed profiling of ( > 90) different lipids classes during a time course of water stress. We used two oat cultivars, Flega and Patones previously characterized as susceptible and tolerant to drought, respectively. Significant differences in lipid classes (mono, di and triacylglycerols; [respectively MAG, DAG, and TAG] and free fatty acids [FFA]) and in their fatty acid (FA) composition was observed between cultivars upon drought stress. In Flega there was an increase of saturated FAs, in particular 16:0 in the DAG and TAG fractions. This led to significant lower values of the double bond index and polyunsaturated/saturated ratio in Flega compared with Patones. By contrast, Patones was characterized by the early induction of signaling- related lipids and fatty acids, such as DAGs and linolenic acid. Since the latter is a precursor of jasmonates, we investigated further changes of this signaling molecule. Targeted measurements of jasmonic acid (JA) and Ile-JA indicated early increases in the concentrations of these molecules in Patones upon drought stress whereas no changes were observed in Flega. Altogether, these data suggest a role for jasmonates and specific fatty acids in different lipid classes in coping with drought stress in oat.

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 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.

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.

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.

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

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

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.

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

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.

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.

This study investigated the evolution of trans-9 trans-11 conjugated linoleic acid (CLA) from cis-9 trans-11 CLA during methylation and its avoidance through a rapid base methylation of milk fat. The study examined three conditions shown to result in loss of cis-9 trans-11 CLA during methylation namely: temperature, methylation time, water contamination in old reagents and acidic conditions. Three techniques currently used for the conversion of milk fat into fatty acid methyl esters for analysis of CLA content by gas liquid chromatography and a fourth procedure designed to eliminate acidic conditions and to limit methylation temperature and time were used. The four methods were: (i) acidic methylation (AM); (ii) acidic and basic bimethylation with fresh reagents (FBM); (iii) acidic and basic bimethylation with pre-prepared reagents (PBM) and (iv) basic methylation (BM). Each regime was carried out on six milk samples over two periods and methylated 1 ml freeze-dried milk (n=12 per regime). Total CLA was not different across methylation regimes (0·30 mg/ml). Isomer cis-9 trans-11 was higher (PPP

Polyphenol oxidase (PPO) is predominately associated with the detrimental effect of browning fruit and vegetables, however, interest within PPO containing forage crops (crops to be fed to animals) has grown since the browning reaction was associated with reduced nitrogen (N) losses in silo and the rumen. The reduction in protein breakdown in silo of red clover (high PPO forage) increased the quality of protein, improving N-use efficiency [feed N into product N (e.g., Milk): NUE] when fed to ruminants. A further benefit of red clover silage feeding is a significant reduction in lipolysis (cleaving of glycerol-based lipid) in silo and an increase in the deposition of beneficial C18 polyunsaturated fatty acid (PUFA) in animal products, which has also been linked to PPO activity. PPOs protection of plant protein and glycerol based-PUFA in silo is related to the deactivation of plant proteases and lipases. This deactivation occurs through PPO catalyzing the conversion of diphenols to quinones which bind with cellular nucleophiles such as protein reforming a protein-bound phenol (PBP). If the protein is an enzyme (e.g., protease or lipase) the complexing denatures the enzyme. However, PPO is inactive in the anaerobic rumen and therefore any subsequent protection of plant protein and glycerol based-PUFA in the rumen must be as a result of events that occurred to the forage pre-ingestion. Reduced activity of plant proteases and lipases would have little effect on NUE and glycerol based-PUFA in the rumen due to the greater concentration of rumen microbial proteases and lipases. The mechanism for PPOs protection of plant protein in the rumen is a consequence of complexing plant protein, rather than protease deactivation per se. These complexed proteins reduce protein digestibility in the rumen and subsequently increase undegraded dietary protein flow to the small intestine. The mechanism for protecting glycerol-based PUFA has yet to be fully elucidated but may be associated with entrapment within PBP reducing access to microbial lipases or differences in rumen digestion kinetics of the forage and therefore not related to PPO activity.

Plant phenols can influence utilization of forages by ruminants through binding with proteins either by complexing with oxidized phenols (products of polyphenol oxidase activity or formed in oxidizing conditions) or by hydrogen binding with tannins. These protein-bound phenols (PBPs) affect rumen metabolism by inhibition of both proteolysis and lipolysis. The aim of this work was to assess the level of polyphenol oxidase (PPO) activity (determined with a range of diphenolic substrates), PBP content, and to identify the main source of variation in different forage ecotypes harvested at different phenological stages. PPO activity was strongly influenced by substrate type with a significant effect of forage ecotype. Forage ecotype and phenological stage also influenced PPO and PBP content. PPO showed a strong relationship with dry matter and phenol content of plants.

Red clover and fish oil (FO) are known to alter ruminal lipid biohydrogenation leading to an increase in the polyunsaturated fatty acid (PUFA) and conjugated linoleic acid (CLA) content of ruminant-derived foods, respectively. The potential to exploit these beneficial effects were examined using eight Hereford X Friesian steers fitted with rumen and duodenal cannulae. Treatments consisted of grass silage or red clover silage fed at 90% of ad libitum intake and FO supplementation at 0, 10, 20 or 30 g/kg diet dry matter (DM). The experiment was conducted with two animals per FO level and treatments formed extra-period Latin squares. Flows of fatty acids at the duodenum were assessed using ytterbium acetate and chromium ethylene diamine tetra-acetic acid as indigestible markers. Intakes of DM were higher (P

Ruminant products are criticised for their SFA content relative to PUFA, although n-6:n-3 PUFA is desirable for human health ( n-3 PUFA flow due to intracellular chloroplast content. A total of six Holstein × Friesian steers were fed, in a two-period changeover design, either straw:concentrate (S:C, 60:40; DM basis; S:C, low chloroplast) or fresh perennial ryegrass (PRG; high chloroplast). Following 12 d adaptation to diet, ruminal protozoal and whole duodenal samples were obtained. N and fatty acid content of whole duodenum and rumen protozoal samples were assessed and protozoal 18S rDNA quantitative PCR performed, enabling calculation of protozoal N flow. The ratio of individual fatty acids:N in rumen protozoal samples was calculated to obtain protozoal fatty acid flows. Based on total fatty acid flow, contribution (%) of protozoa to individual fatty acid flows was calculated. Protozoal fatty acid data and microscopical observations revealed that protozoa were enriched with 18 : 3n-3 following PRG feeding, compared with the S:C diet, due to increased intracellular chloroplast content. However, duodenal protozoal 18S rDNA concentration post PRG feeding was low, indicating rumen retention of the protozoa. Nutrition influences the 18 : 3n-3 content of protozoa; the challenge is to increase protozoal flow to the small intestine, while maintaining sustainable rumen densities.

When fractionation of meat lipids is not required, procedures such as saponification can be used to extract total fatty acids, reducing reliance on toxic organic compounds. However, saponification of muscle fatty acids is laborious, and requires extended heating times, and a second methylation step to convert the extracted fatty acids to fatty acid methyl esters prior to gas chromatography. Therefore the development of a more rapid direct methylation procedure would be of merit. The use of freeze-dried material for analysis is common and allows for greater homogenisation of the sample. The present study investigated the potential of using freeze-dried muscle samples and a direct bimethylation to analyse total fatty acids of meat (beef, chicken and lamb) in comparison with a saponification procedure followed by bimethylation. Both methods compared favourably for all major fatty acids measured. There was a minor difference in relation to the C18:1 trans 10 isomer with a greater (P < 0.05) recovery with saponification. However, numerically the difference was small and likely as a result of approaching the limits of isomer identification by single column gas chromatography. Differences (P < 0.001) between species were found for all fatty acids measured with no interaction effects. The described technique offers a simplified, quick and reliable alternative to saponification to analyse total fatty acids from muscle samples.

A facile and efficient Cu(I)-catalyzed cross-coupling method is reported for the preparation of N-alkynyl or N-bromoalkenyl heteroarenes from bromoalkynes. Generally superior yields and functional group tolerance were obtained with microwave (MW) irradiation using imidazole, benzimidazole, pyrazole, and indazole substrates and poly(ethylene glycol) 400 (PEG400) as an additive. We speculate that PEG400 acts as both a Cu(I)-stabilizing ligand as well as a phase transfer solvent.

Despite evidence supporting improved incorporation of beneficial polyunsaturated fatty acids (PUFA) into ruminant products, such as meat and milk, following red clover and fish oil (FO) inclusion in the ruminant diet, little is known regarding the concomitant bacterial diversity. We evaluated the effects of feeding grass vs. red clover silage with incremental FO inclusion on known lipolytic, biohydrogenating, cellulolytic and proteolytic rumen bacterial communities of steers. Following 14 days of dietary adaptation, liquid-associated (LAB) and solid-associated (SAB) bacterial communities were harvested, DNA extracted and bacterial denaturing gradient gel electrophoresis (DGGE) and specific-bacterial quantitative PCR (QPCR) were undertaken. DGGE-derived dendrograms showed that diet caused the greatest change in LAB and SAB bacterial diversity, with FO inclusion at the 2% and 3% dry matter intake also causing some changes. QPCR revealed that diet resulted in changes in the DNA concentration of Anaerovibrio lipolytica, the Butyrivibrio proteoclasticus group, Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens. FO inclusion caused changes in A. lipolytica, F. succinogenes and R. flavefaciens DNA concentration only. In the B. proteoclasticus group, which are the only known bacteria with the capacity to biohydrogenate PUFA to 18:0, DNA concentration did not correlate to 18:0 flow to the duodenum, however, suggesting that other bacteria may play a role in biohydrogenation. A greater understanding of microbial changes that accompany beneficial dietary changes will lead to novel strategies to improve ruminant product quality.

More accurate and sensitive visualisation of faecal contamination in the abattoir would significantly reduce the risk posed by harbouring pathogenic micro-organisms. We carried out a preliminary investigation of the range of fluorophores found naturally in faeces from typical ruminant diets. Sixteen ewes were offered either: i) fresh forage (FF), ii) grass silage (GS), iii) grass hay (GH) or iv) concentrate and barley straw (CB). Animals offered FF diets had a greater concentration (Pb0.001) of chlorophyll based compounds in their faeces and subsequent fluorescent emission spectra. In a second experiment we investigated a range of fluorescent markers against a basal concentrate and barley straw diet. Ten Cheviot sheep were split into five treatment groups during a duplicate 5 × 5 Latin square design. Four of the groups received a chlorophyll based marker at a rate of 2 g/d: i) Mg–Chlorophyllin (MgC), ii) Fe–Chlorophyllin (FeC), iii) Zn–Chlorophyllin (ZnC) or iv) Spirulina (Chlorophyll a extract from blue green algae, Sp). The last group received no supplement as the control (Con). The appearance of chlorophyllin markers and their derivatives in faeces was similar with mean concentrations of 3.1 and 7.2 μg/g DM, respectively. The most intense fluorescent signal was shown with MgC followed by ZnC, FeC, Sp and Con at 685 nm. The use of markers in pre-slaughter diets would improve the accuracy of faecal detection as a result of greater fluorescence and specific emission wavelengths which do not overlap with natural meat components to help with visualisation

A facile and efficient Cu(I)-catalyzed cross-coupling method is reported for the preparation of N-alkynyl or N-bromoalkenyl heteroarenes from bromoalkynes. Generally superior yields and functional group tolerance were obtained with microwave (MW) irradiation using imidazole, benzimidazole, pyrazole, and indazole substrates and poly(ethylene glycol) 400 (PEG400) as an additive. We speculate that PEG400 acts as both a Cu(I)-stabilizing ligand as well as a phase transfer solvent.

Polyphenol oxidase (PPO) in conditioned red clover (ensiled or cut and crushed) reduces both proteolysis and lipolysis in the herbage, which has led to increases in N use efficiency and polyunsaturated fatty acid (PUFA) content of milk when offered to dairy cows. In damaged plant cells, PPO is activated and binds protein through the formation of protein-bound phenols. This study investigated a) whether freshly cut red clover could increase N use efficiency and milk PUFA concentrations in dairy cows or whether PPO enzymes require prior activation before feeding to elicit a response, and b) apparent whole-tract amino acid digestibility to help determine the effect of PPO on amino acid utilization. Six multiparous Holstein x Friesian dairy cows in mid-lactation were allocated at random to 1 of 3 dietary treatments in a 3 x 3 Latin square: a control treatment of grass (low PPO, G); red clover (high PPO, RC), and conditioned red clover (high fully activated PPO, CRC). The CRC herbage was cut and chopped in the field and then transported with the G and RC herbages to the animal house. Each period consisted of a 2-wk adaptation to diet and a week of measuring dietary effects (N balance and milk collection). The PPO activity was greatest in the RC treatment as fed, whereas activation of latent PPO enzyme and protein-bound phenol levels were greatest in the CRC diet. Dry matter and total fatty acid intakes were comparable across treatments (18.8 kg/d and 550 g/d, respectively). Milk yields and total fatty acid content were similar across treatments (32.6 kg/d and 34.8 mg/mL, respectively). Cows offered either RC or CRC had greater levels of protein, C18 PUFA and total long-chain PUFA in their milk than animals offered grass with no difference between RC and CRC. Nitrogen intakes, and output in milk, urine, and feces were greater in cows offered the 2 red clover treatments than G, with no difference between RC and CRC. However, there were no differences in N use efficiency among diets as measured by the proportion of feed N converted into milk N, possibly as the result of the excessive supply of N with the red clover diets. Amino acid apparent whole-tract digestibilities were greater when on RC than G diets and intermediate when on CRC for all amino acids, with the exception of Met, which was reduced in cows on both red clover diets compared with G. It is proposed that the PPO trait could show more benefit to ruminants if red clover was fed in combination with lesser N-containing forages or if red clover was bred to contain less N.

Sixteen Holstein–Friesian dairy cull cows were offered either ad libitum grass (G) or red clover (RC) silage for 12 weeks. Dry matter (DM), total nitrogen, and pH was higher for the RC than the G silage and organic matter, water-soluble carbohydrate, fibre, DM digestibility, ammonia-N, vitamin E and acetic acid higher for the G silage (P

Polyphenol oxidase (PPO) activity in leaf extracts of wild type (WT) red clover and a mutant line expressing greatly reduced levels of PPO (LP red clover) has been characterized. Both latent and active forms of PPO were present, with the latent being the predominant form. PPO enzyme and substrate (phaselic acid) levels fluctuated over a growing season and were not correlated. Protease activation of latent PPO was demonstrated; however, the rate was too low to have an immediate effect following extraction. A novel, more rapid PPO activation mechanism by the enzyme’s own substrate was identified. Rates of protein breakdown and amino acid release were significantly higher in LP red clover extracts compared with WT extracts, with 20 versus 6% breakdown of total protein and 1.9 versus 0.4 mg/g FW of free amino acids released over 24 h, respectively. Inclusion of ascorbic acid increased the extent of protein breakdown. Free phenol content decreased during a 24 h incubation of WT red clover extracts, whereas protein-bound phenol increased and high molecular weight protein species were formed. Inhibition of proteolysis occurred during wilting and ensilage of WT compared with LP forage (1.9 vs 5 and 17 vs 21 g/kg of DM free amino acids for 24 h wilted forage and 90 day silage, respectively). This study shows that whereas constitutive red clover PPO occurs predominantly in the latent form, this fraction can contribute to reducing protein breakdown in crude extracts and during ensilage.

The declining consumption of ruminant products has been partly associated with their high proportion (but not necessarily content) of saturated fatty acids. Recent studies have focused on the less prominent fact that they are also important sources of beneficial fatty acids, includingn-3 fatty acids and conjugated linoleic acids. α-Linolenic acid (18: 3n-3) is of particular interest because it also contributes to improved flavour of beef and lamb. Many recent studies showed large effects of special concentrates on levels of fatty acids in milk and meat. However, the ‘rumen protection’ treatments, needed to ensure a worthwhile level of fatty acid in products, are expensive. Herbage lipids are the cheapest and safest source of these fatty acids and so breeding to increase delivery of fatty acids from plants into ruminant products is an important long-term strategy. Plant lipids usually contain high levels of polyunsaturated fatty acids, particularly 18: 2n-6 and 18:3n-3 which are the precursors of beneficial fatty acids. Whilst some plants are particularly rich in individual fatty acids (e.g. 18:3n-3 in linseed), there are also useful levels in grass and clover (TrifoliumSpp.). Levels of fatty acids in forages in relation to species and varieties are considered, as well as management and conservation methods. Relationships between levels of fatty acids and existing traits and genetic markers are identified. The effects of forage treatments on the fatty acid content of ruminant products are reviewed. The higher levels of polyunsaturated fatty acids in milk from cows fed clover silages show that the level of fatty acids in herbage is not the only factor affecting levels of fatty acids in ruminant products. Further effort is needed to characterise susceptibility of unsaturated fatty acids to oxidative loss during field wilting and biohydrogenation losses in the rumen, and the relative importance of plant and microbial processes in these losses. The pathways of lipolysis and lipid oxidation are reviewed and other plant factors which offer potential to breed for reduced losses are considered.

The effect of adding water-soluble carbohydrate (WSC) on the microbial fermentation of fresh perennial ryegrass was examined in an in vitro RUSITEC system over two 10 day periods. Four treatment levels of WSC were used: a basal grass and a basal grass plus a sugar infusion to raise the WSC basal grass level by approximately 1.25×; 1.5× and 1.75×. The infusion was a mixture of inulin and sucrose (80:20) infused over the first 14 h of each 24 h cycle, based on a preliminary study which indicated a linear disappearance of forage WSC over this period. With increasing WSC inclusion there were linear reductions in pH and ammonia-N (NH3N) (P = 0.001) but only a small increase in total volatile fatty acid concentration. There was also a decrease in the proportion of acetate and an increase in propionate with increasing WSC inclusion (P = 0.02 and 0.009, respectively). Microbial nitrogen production was similar for the first three treatments of Basal to Basal × 1.5 but was lower at the highest level of WSC inclusion (P < 0.001). The efficiency of microbial protein synthesis increased from Basal to Basal × 1.5 (9.9, 10.8, 12.7 g N/kg organic matter apparently digested (OMAD), respectively) but at the highest level of WSC inclusion (Basal × 1.75) there was a reduction to 7.1 g N/kg OMAD. This may have been related to the low pH values (