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SID 5 Research Project Final Report

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NoteIn line with the Freedom of Information Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. The SID 5 (Research Project Final Report) is designed to capture the information on the results and outputs of Defra-funded research in a format that is easily publishable through the Defra website. A SID 5 must be completed for all projects.

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Project identification

1. Defra Project code LS3654

2. Project title

Exploiting the beneficial effects of PPO on the utilisation of protein and lipids in grazed forages

3. Contractororganisation(s)

Institute of Grassland and Environmental ResearchPlas GogerddanAberystwythCeredigionSY23 3EB

54. Total Defra project costs £ 250,726

5. Project: start date................ 01 September 2004

end date................. 31 August 2007

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6. It is Defra’s intention to publish this form. Please confirm your agreement to do so...................................................................................YES NO (a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They

should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow.Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer.In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.

(b) If you have answered NO, please explain why the Final report should not be released into public domain

Executive Summary7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the

intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.The plant enzyme polyphenol oxidase (PPO) has been demonstrated to reduce both proteolysis and lipolysis during the ensiling of red clover. The purpose of this work was to explore the potential for exploiting this beneficial trait in other dietary regimes and determine its effectiveness across the rumen to improve N use efficiency and the incorporation of beneficial polyunsaturated fatty acids in ruminant products. The rate of proteolysis of forages in the rumen is a key determinant of the efficiency of conversion of feed nitrogen into products and pollutants. The efficiency of conversion of feed N into milk (20-30%) or meat (10-20%) by ruminants is often well below potential (>40%), and is particularly low with diets based on poor grass silages or grazed herbage. At the same time, the biohydrogenation of fatty acids in the rumen, following lipolysis of plant phospho-lipids, reduces the polyunsaturated: saturated fatty acid (P: S) ratio of both meat and milk. This work identified current opportunities and future goals in developing new strategies for forage management and livestock feeding by increasing the efficiency of feed protein utilisation and increasing polyunsaturated fatty acid production by ruminants, thereby reducing pollutants and enhancing the nutritional status of milk and meat.

By comparing a red clover mutant, unique to IGER with a low PPO activity mutant, with a normal red clover, high PPO activity, we have confirmed, under laboratory conditions, the role of PPO in protein protection. These differences have also been shown to carry through during the ensiling of normal and low PPO lines. We have increasing evidence of a further beneficial effect of PPO in relation to lipid metabolism. Biohydrogenation of lipids in the rumen of cattle fed a red clover silage diet is lower than for grass silage, which results in increased levels of unsaturated fatty acids (particularly linolenic acid) being incorporated into milk (and meat). Recent laboratory studies with leaf extracts of normal and low PPO red clovers demonstrated increased levels of lipolysis with the low PPO mutant. This strongly suggests that PPO can also have a role in reducing lipid degradation. Earlier work has concentrated on effects of PPO during the ensiling of red clover and in the resultant silages. This project focused on its effects in cut and fresh forage regimes and tested 3 main hypotheses:

1. That PPO activity, protein polymerisation and lipid protection occurs in grazed fresh forage.

PPO activity requires the presence of oxygen, whilst the rumen is a largely anaerobic environment. Freshly-ingested feed boluses retained oxygen for a period of time (5-10min), whilst incubation in rumen fluid resulted in rapid scavenging of oxygen within 2 min. Oxygen content of the rumen was also investigated during ingestion of red clover boluses. The entrance of a bolus into the rumen caused a peak in oxygen concentration (0.28 +/- 0.199 mg O2/l, 5.9 (+/- 0.277) seconds after swallowing, returning to the initial undetectable level within 6.4 (+/- 0.194) seconds. Nevertheless, we have shown that the quinone-protein binding process in red clover is extremely rapid (2 minutes) and PPO was shown to be activated in red clover boluses caught in ruminally fistulated animals at the oesophageal-reticulo-rumen orifice. The activated PPO was shown to have resulted in a significant elevation in bound-phenol-protein complexes which were shown to reduce protein solubility and consequently reduce proteolysis and lipolysis when the boluses were incubated in rumen fluid. However, the extent of PPO activation and subsequent protein and glycerol based lipid protection was governed by the maturity of the forage and a balance was required

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between PPO concentration and fibre content required to increase mastication and consequent activation of the PPO through aeration of the damaged red clover. These difficulties in juggling the window of opportunity to activate PPO by the extent of mastication and lushness of the crop and hence PPO activity were overcome by a prior wilting which resulted in PPO activation prior to mastication and a greater level of protein and glycerol-based lipid protection

2. That PPO activity, protein polymerisation and lipid protection occurs during the conditioning of forage.

Cell damage was highly correlated with PPO activity and activation and subsequently activation was highly correlated with bound-phenol production. Cell damage is thought to have three mechanisms for increasing PPO activity: i) decompartmentation of intracellular membranes with a subsequent mixing of enzyme and substrate resulting in activation of latent PPO; ii) up-regulating of substrate formation induced through signal compounds, resulting in a substrate induced activation of the latent PPO; iii) up-regulation of the PPO gene increasing enzyme production. These three mechanisms suggest slow rather than rapid cell damage such as a field wilt would result in the greatest level of PPO activation and subsequent protection of protein and glycerol-based lipid.

3. That polymerised proteins are degraded slowly in the rumen and the feeding of red clover conditioned to elevate PPO activation will result in an improved N utilisation and fatty acid profile of ruminant products.

The rate of protein break down and whether rumen microbial populations adapt to allow utilisation of polymerised protein were examined. Ultimately two animal performance studies were carried out to show the potential of PPO in increasing N use efficiency and improving the fatty acid profile of ruminant products. A series of in vitro studies with the high and low PPO lines of red clover showed retardation in protein and glycerol based lipid degradation as a result of PPO protection. It was also confirmed through the use of red clover adapted rumen micro-organisms that these rumen micro-organisms are unable to adapt to increase their utilisation of the phenol-bound protein produced through the action of PPO and this protection of protein also conferred a protection to the glycerol-based lipid. The feeding of red clover silage as opposed to grass silage to cull cows was shown as a successful feeding regime to reach the EBLEX finishing grade and also to increase the fatty acid composition quality of the meat, as the animals fed on red clover showed a significant elevation in the n-3 PUFA in their muscle and increased the P:S ratio as opposed to animals finished on grass silage. However, levels of vitamin-E in their tissues were significantly reduced leading to a reduction in shelf life and a vitamin E supplementation would be required to increase the protection of the enhanced lelve of n-3 PUFA in animal products when feeding red clover. A study with dairy cows investigated the effect of the extent of PPO activity and activation on N utilisation and the fatty acid quality of milk. Cows were fed either cut grass, red clover or conditioned red clover (cut and crushed followed by chilling in a blast freezer for 2 h). Nitrogen output in milk was significantly higher in the two red clover treatments, although the efficiency of feed N conversion into milk was not significantly different across treatments and more work is required to understand the utilisation of PPO protected protein. The quality of the fatty acid profile of the milk on both red clover dietary regimes was significantly enhanced over the grass fed animals. C18:2n-6 and C18:3n-3 concentrations were significantly higher and whilst there was a trend for a higher P:S ratio on the conditioned red clover treatment over the cut red clover treatment it was not significant. This suggests that feeding the cut red clover resulted in significant PPO activation to result in protection of the plant protein and the glycerol-based lipid as shown in the bolus studies.

This work has important linkages to studies on PPO within the forage breeding programmes at IGER. Whilst the area of red clover that is grazed is currently low, it will increase both as the sustainability benefits of red clover are appreciated and as varieties that are more tolerant to grazing are produced. In addition, we have recently shown PPO activity in grass, which also have the ability to protect the soluble protein fractions and glycerol-based lipid from proteolytic and lipolytic degradation, respectively. We have also shown cultivar/species differences in PPO activity within grasses, indicating the potential for up-regulating this activity through selective breeding with possible benefits of reduced lipid degradation. Thus, the techniques and findings of this red clover project could be extended into studies on forage grasses. We have also formed links with the United States Dairy Forage Research Institute to collaborate on mechanistic approaches for PPO using plants with point-mutations at the PPO1 gene loci. These studies could lead to a greater understanding of the mechanism of protein and glycerol-based lipid conservation and also potential use of the mechanism for the protection of other forage crops.

Project Report to Defra

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8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include: the scientific objectives as set out in the contract; the extent to which the objectives set out in the contract have been met; details of methods used and the results obtained, including statistical analysis (if appropriate); a discussion of the results and their reliability; the main implications of the findings; possible future work; and any action resulting from the research (e.g. IP, Knowledge Transfer).

IntroductionThe plant enzyme polyphenol oxidase (PPO) has been demonstrated to reduce both proteolysis and lipolysis during the ensiling of red clover. The purpose of this work was to explore the potential for exploiting this beneficial trait in other dietary regimes and determine its effectiveness across the rumen to improve N use efficiency and the incorporation of beneficial polyunsaturated fatty acids (PUFA) in ruminant products. These studies focused on the potential of PPO activation and consequent protein and lipid protection in fresh/cut red clover and also how this could be improved through conditioning techniques. Ultimately these new strategies for forage management and livestock feeding by increasing the efficiency of feed protein-N utilisation and increasing polyunsaturated fatty acid production by ruminants would reduce pollutants and enhance the nutritional status of milk and meat. This work contributed directly to the DEFRA objective of reducing the output of waste (potential pollutants) per unit of product. Although the project has generic relevance to all ruminant livestock, it is particularly focused on N-excretion problems associated with the dairy sector. The potential to deliver improved product quality (and associated economic advantages) represents added value.

ObjectivesThe project had six main objectives:

1. Evaluate the effect of PPO on proteolysis and lipolysis in freshly ingested red clover using in sacco and in vitro techniques.

2. Investigate the effect of cropping techniques on PPO activity in red clover.3. Investigate the effects of growth stage and time of day on red clover PPO activity and its phenolic

substrates.4. Investigate the rate at which polymerised red clover proteins are degraded by an adapted population of

rumen microbes. 5. Conduct a feeding study with dairy cows based on the optimal crop and/or feeding management strategy

identified in objectives (1) to (4).6. Conduct a feeding study with cull cows designed to enhance meat quality, based on the optimal crop

and/or feeding management strategy identified in (1) to (4)

Extent to which the project objectives have been met Following discussions with the DEFRA project manager in September 2006, the following objectives were amendedObjective 5: Briefly, this study sought to compare four treatments: (1) zero-grazed grass, (2) low v. (3) high PPO red clover and (4) mechanically-chopped red clover (to alter PPO activation). However, due to problems with seed multiplication of the low PPO mutant, it was not possible to sow plots large enough to conduct a grazing study. Hence the low v. high PPO red clover option was not available. The experiment was changed to a 3-period changeover design using 6 lactating dairy cows (spring calvers) comparing (1) grass (2) red clover and (3) mechanically chopped red clover.Objective 6: This study sought to assess the benefits of grazed red clover versus grass for finishing cull dairy cows. The study was changed to use silage rather than grazing and was conducted in the winter 06/07. Numbers of animals remained unchanged. This was to ensure completion of all experimentation within the contract period. With these amendments taken into account the objectives of the project were fully met and surpassed.

Evaluate the effect of PPO on proteolysis and lipolysis in freshly ingested red clover using in sacco and in vitro techniques.INTRODUCTION

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The effect of PPO on proteolysis and lipolysis in animal fodder has the potential to improve nitrogen use efficiency (Broderick et al. 2001, Merry et al. 2006) and improve the PUFA composition of ruminant products (Lee et al. 2003, Dewhurst et al. 2003). Studies on the potential benefits of high PPO forage crops such as red clover so far have concentrated on conserved feed such as silage, due to the requirements of both oxygen and cell damage for enzyme activation, and, as a consequence, little work has been done on the potential benefit of grazing high PPO crops. This study investigated the activity, activation and efficacy under grazing of non-conserved, cut red clover PPO.

Three experiments were carried out on cut red clover to investigate i) red clover bolus and rumen oxygen contents, ii) PPO activity during bolus formation and its effect on protein and lipid profiles and iii) effect of fresh red clover PPO on proteolysis and protein solubilisation.

MATERIALS AND METHODSExperiment 1 - PPO efficacy under grazing – Bolus and rumen oxygen content. The rumen of a

Holstein × Friesian dairy cow fitted with a rumen cannula was emptied with a sample (2 l) of muslin strained rumen fluid collected and stored at 39oC. The cow was then offered freshly cut red clover and ingested boluses caught at the oesophageal orifice in the reticulo-rumen. Each bolus was immediately transferred to an air-tight jar containing de-oxygenated water maintained at 39oC. A dissolved oxygen probe (Orion Research Inc, MA, USA, calibrated according to the manufacturer’s instructions) was inserted into the bolus and the oxygen concentration measured at 30 second intervals over a time course of 10 min. This procedure was repeated twice more (n=3). A second set of red clover boluses (n=3) were then incubated in the retained strained rumen fluid and the oxygen concentration measured as before.

In vivo measurements were taken using two rumen fistulated dairy cows. The oxygen probe was positioned inside the reticulo-rumen by the oesophageal orifice and a reading taken as the initial oxygen concentration. The animals were then allowed to eat for one mouthful before the red clover was removed. The time was recorded between swallowing and observing the oxygen peak as the bolus entered the rumen and then the time taken to return to a stable reading. After returning to stable oxygen reading this was recorded as the initial value for the second reading and the animal offered the red clover once again for one more mouthful. This was repeated several times for each cow (cow 1, n=6; cow 2, n=4).

Experiment 2 - PPO efficacy under grazing – Bolus formation and lipid changes during incubation. The experiment involved two rumen fistulated Holstein × Friesian dairy cows over two periods. Period 1 used mature red clover at 8 weeks of regrowth and period 2 used lush new growth material 4 weeks into regrowth. At the start of each period the rumens were emptied and the animals offered 3 types of red clover in turn, each cut from field plots: i) fresh red clover from plants of a mutant low PPO line (LRC), ii) fresh red clover from wild-type plants with a high PPO activity (HRC) and iii) plants which were frozen, thawed and wilted for 24 h to fully activate the PPO red clover (WRC). Samples (100 g) of each herbage type (H) were flash-frozen in liquid N 2 and stored at -20oC prior to analysis.

Once the cows were feeding, the initial bolus caught at the oesophageal orifice in the reticulo-rumen was discarded to avoid rapid swallowing with the first herbage and cross-contamination with the 2 later herbages. Replicate boluses (B, n=6) were collected from each herbage type, flash-frozen in liquid N2 and stored at -20oC prior to analysis. A second set of boluses (n=6) was then collected as before and a sub-sample (ca. 20 g portion) incubated in rumen fluid for 60 min at 39oC (IB). The rumen fluid was collected prior to feeding, strained through muslin and 100 ml poured into 36 labelled Duran bottles (6 replicates (boluses) × 3 treatments × 2 cows) which were incubated at 39oC. At the end of each incubation the Duran bottles were emptied into pre-prepared extraction tubes containing 100 ml iso-propanol : chloroform (1:1; v/v) and 1ml of internal standard (C23:0 15mg/ml in chloroform) and lipid extracted as described by Lee et al. (2004). The collected extract was then fractionated by thin layer chromatography (TLC) as described by Nichols et al. (1963). The separated lipid classes (membrane lipid, ML; diacylglycerol, DAG; triacylglycerol, TAG and free fatty acids, FFA) were scraped from the TLC plate, extracted from the silica and methylated using toluene containing C21:0 (0.4mg/ml) as an internal standard and 5% methanolic HCl at 60oC for 2 h. Methylated samples were analysed by gas chromatography on a CP-Select chemically bonded for FAME column (100 m × 0.25 mm i.d., Varian Inc., California, USA) with split injection (30:1) and He as the carrier gas. Peaks were identified from standards and quantified using the internal standard (C21:0).

Herbage (H) and bolus (B) samples were analysed for PPO activity, protein and bound phenols as described in Experiment 3. Freeze-dried and ground samples of H and B were analysed for organic matter (OM), total nitrogen (TN), WSC, acid detergent fibre (ADF) and neutral detergent fibre (NDF) as described by Lee et al. (2007). Lipid was extracted from 1 g of freeze-dried and ground sample in 3 × 5 mL chloroform : methanol (2:1; v/v) and the extract fractionated as described for the incubated bolus (IB) extract above. Lipolysis was calculated by expressing the decrease in fatty acid content of the ML fraction between B and IB samples, as a proportion of total fatty acid content (ML + TAG + FFA + DAG) in B. Biohydrogenation was calculated as the proportional loss of C18 PUFA between the B and IB samples. Changes in lipid fractions, PPO activity, protein and phenol content, lipolysis and biohydrogenation for H, B and IB (where appropriate) were analysed statistically using a general ANOVA with herbage type (LRC v HRC v WRC)* regrowth period (8wk v 4wk) as the treatment effect and blocking by animal, performed with Genstat Release 8.11 (PC/Windows XP; Lawes Agricultural Trust, 2005, Rothamsted, UK).

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Experiment 3 - Effect of fresh red clover PPO on proteolysis and protein solubilisation. Leaves from mutant plants with low PPO (LP) and wild type (WT) plants grown in controlled environmental conditions were ground in liquid nitrogen and sub-samples (ca. 0.5 g) were extracted at 0oC in 2 ml of either McIlvaine buffer (pH 7.0) alone, or McIlvaine buffer (pH 7.0) including 75 mM ascorbic acid and 0.3% polyvinylpyrrolidone to block PPO activity. Extracts were cleared by centrifugation at 15,000 × g for 10 min at 4oC. For each replicate leaf extract, 250 l aliquots were put into 4 micro tubes. A 250 l volume of 20% trichloroacetic acid (TCA), 0.4% phosphotungstic acid (PTA) was added immediately to the first tube (to precipitate protein and prevent any further PPO activity) which was then stored at 4oC. The three remaining tubes were incubated in the dark at 30oC for 6, 12 or 24 h after which time a 250 l volume of the 20% TCA, 0.4% PTA solution was added to the tubes, which were subsequently stored at 4oC. Following incubation for a minimum of 30 min at 4oC (to ensure complete protein precipitation), all tubes were spun at 15,000 × g for 10 min at 4oC. The supernatant was retained for analysis of free amino acids and phenols whilst the pellet was resuspended in 1.5 ml 0.1 M NaOH and analysed for protein content.

Amino acid and protein analyses. Supernatant fractions were adjusted to neutral pH, by combining 480 l volumes with 265 l 1M NaOH. These samples were then analysed for free amino acid content by the method of Winters et al. (2002) and for phenols by the method described by Veltman et al. (1999) using tyrosine as a standard. Proteins and phenol-bound proteins were assayed using a modified Lowry procedure described by Winters and Minchin (2005) which takes into account the variable response of ortho-diphenols with the Lowry assay and estimates the quantity of protein-bound phenol.

PPO assays. Red clover leaf protein was extracted in McIlvaine buffer (pH 7.0) including 75 mM ascorbic acid and desalted with Bio-Gel P6DG (Bio-rad, Hertfordshire, UK) as described by Winters et al. (2003). The eluted fractions served as crude enzyme preparations in which PPO activity was analysed spectrophotometrically (Biotech ultraspec, Pharmacia Biotech, St. Albans, Hertfordshire, UK) at 420 nm with methylcatechol as a substrate. Standard reactions were carried out in a volume of 1.5 ml containing either 10 l of WT red clover enzyme or 20 l LP red clover enzyme, 0.001 mM copper sulphate and 10 mM methylcatechol in McIlvaine buffer (pH 7.0) alone or including 0.25% sodium dodecylsulphate (SDS) for measurement of active and total (active + latent) enzyme activity, respectively. The PPO reaction was initiated by the addition of the methylcatechol substrate and quinone product was monitored by measuring increase in absorption at 420 nm. Reactions were monitored over a 40-60 s period (10 readings s-1) and rates were calculated from the linear phase of the curve with SWIFT II software (Pharmacia Biotech; Biochrom Ltd., Cambridge, Cambridgeshire, UK). For the purpose of investigating the effect of pre-incubation with o-diphenol (caffeic acid, chlorogenic acid, catechol and methylcatechol) on PPO activity, desalted WT red clover extract was incubated with an equal volume of o-diphenol (10mM) in McIlvaine buffer (pH 7.0) and pH was adjusted to 7 where necessary. After various time intervals 20 l volumes were assayed for PPO activity with methylcatechol as described above

Substrate extraction Fresh leaves were rapidly removed from the plant material and 2.5 g were immersed in 25 ml boiling 70% methanol for 10 min. The liquid volume was subsequently topped up to 25 ml with 70% methanol and left on a gel shaker for 2hr at room temperature. Supernatant was decanted and the residual plant material was rinsed with two further 10 ml volumes of 70% methanol which were pooled with the first fraction. Extracts were concentrated by rotary evaporation to a volume of 5ml. A 1ml volume was applied to a C18 Sep-Pak cartridge (Waters). Phenols were eluted with 4 ml methanol and analysed by reverse phase HPLC (Waters- Nova-Pak C18, 8 x 100mm radial-Pak cartridge) with a 35 min gradient 0-70% methanol, 2ml/min flow and detected by PDA.

Protein gel-electrophoresis To demonstrate changes in PPO isoforms, proteins were extracted at 0oC in 2 ml of McIlvaine buffer (pH 7.0) including 75 mM ascorbic acid with and without 1mM phenylmethylsulfonyl fluoride (PMSF). Proteins in fresh extracts and extracts incubated for 24 hours at 25oC, were separated by electrophoresis on ready-cast IEF gradient gels pH5-8, (Biorad, Hertfordshire, UK) according to the manufacturer’s instructions. PPO activity was detected by incubation of gels in 10 mM L-3,4-dihydroxyphenylalanine (L-DOPA) in McIlvaine buffer (pH 7.0) with and without 0.25% SDS. For the purpose of detecting formation of high molecular-weight protein-phenol complexes, leaf material (ca. 0.5 g) was extracted at 0oC in 2 ml of McIlvaine buffer (pH 7.0). Extracts were centrifuged at 15,000 × g for 10 min at 4oC and the supernatant was retained and incubated at 30oC for up to 24 h. Samples were taken at various time points and further enzymatic activity was stopped by boiling for 3 min. Soluble protein extracts were applied to 4-20% gradient polyacrylamide gels (Biorad, Hertfordshire, UK) and proteins were separated by electrophoresis according to the method of Laemmli (1970).

RESULTSThe oxygen depletion from the red clover boluses incubated in de-oxygenated water, reached completion

after 5 min (Figure 1a). When the red clover bolus was immersed in strained rumen fluid the oxygen disappeared within 2 min (Figure 1b).

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0

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00.10.20.30.40.50.60.70.80.9

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Figure 1. Dissolved oxygen content of red clover boluses incubated in a) water or b) rumen liquor at 39oC.

In the in vivo study (Table 1) there were no significant differences between cows, the initial concentration of oxygen in the reticulo-rumen being negligible. The entrance of a bolus into the rumen caused a peak in oxygen concentration of 0.28 (±0.199) mg O2/l, 5.9 (±0.28) seconds after swallowing, returning to the initial oxygen concentration within 6.4 (±1.94) seconds.

Table 1. Oxygen concentration in the reticulo-rumen of two dairy cows prior to and during ingestion of red clover.

Initial [O2] (mg O2/l)

Peak [O2] (mg O2/l)

Time to reach peak (s)

Time to return to initial (s)

n

Cow 1 0.00(±0.001) 0.18(±0.030) 5.9(±0.27) 6.0(±0.18) 6Cow 2 0.00(±0.001) 0.44(±0.200) 5.9(±0.29) 6.8(±0.12) 4

The chemical composition, protein content, PPO status and lipid composition for herbage (H) and bolus (B) samples of the three red clover treatments (HRC, LRC and WRC) at two levels of regrowth (8 and 4 weeks) are given in tables 2 and 3, respectively. In the H samples there were significant regrowth and treatment effects for dry matters with 8wk regrowth and WRC significantly higher than 4wk and HRC and LRC, respectively. Organic matter was not significantly different across regrowth and treatments whereas total nitrogen was significantly lower in the 8wk regrowth material compared with the 4wk regrowth material, in contrast with WSC and fibre concentration. Treatment effects were smaller with total nitrogen significantly higher in LRC as opposed

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to the other two treatments in the 8wk regrowth, and WSC and fibre significantly higher and lower, respectively, in the WRC 4wk regrowth, compared to the other treatments. Bound phenol concentrations were significantly higher for the WRC treatment than either HRC or LRC with no effect on regrowth period. PPO activity showed no significant difference between 8 and 4wk of regrowth but was significantly higher in HRC compared to WRC which in turn was significantly higher than LRC, whereas activation of the PPO was higher in WRC than both HRC and LRC. No significance was found between the two regrowth periods for the WRC treatment in terms of activation whereas in both HRC and LRC treatments activation was significantly reduced for the 4wk regrowth. Total lipid concentration were similar across treatment types but were significantly lower in the 8wk regrowth material. Significant interaction effects were observed between regrowth and treatment types for composition of ML, FFA and TAG, whereas DAG showed a significant regrowth effect being significantly higher in the 4wk regrowth than the 8wk regrowth material.

Table 2. Chemical composition (g/kg DM), bound-phenol (mg/g protein), PPO activity (∆OD/gDW/min), activation (%), total lipid (g/kg DM) and lipid content (%) of the three red clover treatments as herbage (H) at 8 and 4 week regrowth.

HRC LRC WRC P8wk 4wk 8wk 4wk 8wk 4wk s.e.d. wk treat I

Dry Matter 220.0c 145.6a 216.2c 149.4a 304.1d 186.2b 8.93 *** *** NSOrganic Matter 917.3b 886.8a 912.1b 894.5a 912.3b 886.8a 2.17 *** NS NSTotal Nitrogen 24.7a 42.0c 30.3b 42.7c 26.0a 42.5c 1.23 *** ** *Water Soluble Carbohydrate

96.4c 66.2b 92.1c 61.0ab 94.5c 55.7a 3.49 *** * NS

Acid Detergent Fibre

300.2bc 162.1a 280.1b 181.0a 313.5c 175.3a 13.13 *** NS NS

Neutral Detergent Fibre

431.1c 268.4a 395.3c 258.4a 408.6c 321.1b 17.64 *** * NS

Phenol 18.6a 18.3a 10.2a 10.0a 61.2b 77.2b 4.70 NS *** NSPPO activity 347.6c 463.6d 26.2a 54.3a 123.6b 217.7b 59.97 NS *** NSPPO activation 41.2b 31.6b 33.3b 35.6b 100.0d 100.0d 8.54 NS *** NSTotal lipid 15.6a 40.9b 14.3a 36.9b 19.7a 33.2b 5.02 *** NS NSMembrane lipid 91.1c 91.0c 86.2b 91.4c 85.1b 79.6a 2.26 * *** **Diacylglycerol 2.4a 6.5bc 4.1ab 5.3bc 4.1ab 7.4c 1.16 * NS NSFree Fatty Acid 5.5b 2.1a 9.3c 2.2a 9.9c 11.4c 1.63 ** *** ***Triacylglycerol 1.1b 0.4a 0.5a 1.1b 0.9ab 1.6c 0.27 NS * **

In the B samples the difference in dry matter concentration between regrowths was reduced with only the LRC treatment showing a significant difference but, as with the H samples, the WRC treatment remained significantly higher than the other two treatments. Organic matter showed an interaction effect between regrowth and treatment. Total nitrogen, WSC and fibre mirrored the effects seen in the H samples (Table 2) but the differences between regrowth periods were reduced. Bound phenol concentration was similar at both regrowths and across treatments for LRC and HRC whereas the WRC treatment was significantly higher and also higher in the 8wk regrowth than the 4wk regrowth. PPO activity was significantly lower in the LRC treatment than either HRC or WRC, with the latter being significantly higher than the former for PPO activity, and vice versa for PPO activation. Regrowth period significantly effected PPO activation for the HRC and LRC treatments with 8wk significantly higher than 4wk. Total lipid compositions were similar across treatments but, as with the H samples, were significantly lower in the 8wk regrowth than the 4wk regrowth. ML and FFA showed an interaction effect between treatment and regrowth whereas DAG concentration was significantly higher in the 4wk regrowth than the 8wk regrowth for the HRC treatment. There were no significant differences in TAG concentration in the B samples across either regrowth period or treatment.

Table 4 shows the changes in lipid composition which occurred when B material were incubated in rumen fluid for 1 h at 39oC. Total lipid composition in the IB samples was similar to H and B samples with the 8wk regrowths significantly lower than the 4wk regrowths across the treatments, however the 4wk WRC treatment was significantly lower than the corresponding HRC and LRC treatments. There was a significant interaction effect for ML composition across regrowth period and treatment. DAG and TAG concentrations were significantly higher in the 4wk regrowths than the 8wk regrowths across the three treatments types with WRC significantly higher than HRC and LRC. FFA concentration, lipolysis and biohydrogenation showed the opposite response to DAG and TAG with lower levels in the 4wk regrowth as opposed to the 8wk regrowth, with WRC being significantly lower than the other two treatments.

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Table 3. . Chemical composition (g/kg DM), bound-phenol (mg/g protein), PPO activity (∆OD/gDW/min), activation (%), total lipid (g/kg DM) and lipid content (%) of the three red clover treatments as boluses (B) at 8 and 4 week regrowth.


Dry Matter 82.2a 92.1ab 88.5a 106.5b 134.2c 145.2c 8.72 * *** NSOrganic Matter 845.3a 857.4ab 843.9a 867.2bc 873.1c 861.3c 6.54 * ** **Total Nitrogen 29.4ab 38.4c 31.4b 42.5d 28.0a 38.7c 1.32 *** ** NSWater Soluble Carbohydrate

74.6c 68.3b 79.6c 58.5b 77.6c 49.4a 3.99 ** * NS

Acid Detergent Fibre

235.4b 155.4a 239.2b 176.5a 260.9b 188.2a 31.75 * NS NS

Neutral Detergent Fibre

320.9b 294.8ab 331.5bc 256.6a 353.2c 320.2b 31.27 * NS NS

Phenol 36.6a 37.4a 25.2a 28.7a 58.6b 111.2c 7.71 *** *** ***PPO activity 583.7c 705.0c 66.9a 126.1a 134.5a 334.1b 81.31 * *** NSPPO activation 97.1b 49.0a 98.8b 41.7a 100.0b 100.0b 13.78 ** ** NSTotal lipid 18.2a 34.4c 24.6ab 31.9c 22.5ab 28.7bc 4.29 *** NS NSMembrane lipid 85.3bc 87.2bc 85.4bc 89.2c 83.1ab 79.7a 20.34 NS *** **Diacylglycerol 3.9a 6.4b 3.4a 4.2ab 5.1ab 5.4ab 1.17 * NS NSFree Fatty Acid 9.9b 4.4a 10.2b 6.0a 11.0b 14.8c 1.49 * *** ***Triacylglycerol 0.8 2.0 1.1 0.6 0.9 0.5 0.65 NS NS NS

Table 4. Lipid content (g/kg DM), fractions (%), lipolysis (%) and C18 polyunsaturated fatty acid biohydrogenation (%) of the three red clover treatments after incubation of boluses (IB) in rumen fluid at 39oC for 1h.


Total Lipid 17.4a 35.7c 19.1a 35.1c 16.0a 26.9b 2.38 *** * NSMembrane lipid 16.7ab 18.6b 12.0a 14.9a 19.0b 16.9ab 1.45 NS *** *Diacylglycerol 2.7a 4.8ab 2.9a 4.5ab 5.2b 8.2c 1.15 *** ** NSFree Fatty Acid 79.7b 74.2a 84.0c 79.2b 74.2a 72.1a 1.94 *** *** NSTriacylglycerol 0.9a 2.2c 1.1ab 1.4ab 1.7b 2.8c 0.31 *** *** NSLipolysis (%) 80.2bc 78.9ab 86.0d 82.9cd 77.1ab 75.6a 1.84 * *** NSC18:2 Biohydrogenation

84.0c 56.1a 80.1bc 58.0a 78.0b 51.2a 3.60 *** * NS

C18:3 Biohydrogenation

90.2d 78.8b 87.3cd 79.5b 83.4bc 66.8a 2.46 *** *** NS

Changes in protein and free amino acid content were monitored during incubation of WT and LP red clover leaf extracts at 30oC for 24 h (Figures 2 and 3) to study the effect of PPO on protein breakdown. Both LP extracts and WT extracts, in which PPO activity had been blocked with ascorbic acid, showed extensive protein breakdown with concomitant increases in free amino acid content. Indeed, LP extracts with ascorbic acid showed significantly greater protein breakdown after 24 h compared with all other treatments whilst free amino acid content was significantly higher at all sampling times. Conversely, the extent of protein breakdown and free amino acid accumulation were greatly reduced in WT extracts from which ascorbic acid was omitted. The degree of phenol binding to protein (protein-bound phenol, Figure 4) showed a negative relationship with protein degradation parameters; highest levels were observed in WT extracts and lowest levels in LP extracts with ascorbic acid, with other treatments showing intermediate levels. Analysis by SDS-PAGE (Figure 5a) clearly shows the rapid formation (within 3 minutes) of high molecular weight protein-phenol complexes in WT but not LP extracts. There is a marked change in the protein profile of WT extracts over the 24 h period with an obvious decrease in low molecular weight proteins and a concomitant increase in high molecular weight complexes (Figure 5b). By 24 h the de novo appearance of high molecular complexes can also be observed in LP extracts.

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0 6 12 18 24

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otein

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)

Figure 2. Protein breakdown during incubation of wild type () and low PPO mutant () red clover leaf extracts. Extracts incubated in the presence of ascorbic acid are represented by open symbols and extracts in the absence of ascorbic acid by closed symbols. Vertical bars represent the standard error of the mean.

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Figure 3. Increase in free amino acids (FAAs) during incubation of wild type () and low PPO mutant () red clover leaf extracts. Extracts incubated in the presence of ascorbic acid are represented by open symbols. Vertical bars represent the standard error of the mean.

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enol

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Figure 4. Changes in protein-bound phenol during incubation of wild type () and low PPO mutant () red clover leaf extracts. Extracts incubated in the presence of ascorbic acid are represented by open symbols. Units are based on tyrosine equivalents. Vertical bars represent the standard error of the mean.

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LP WT

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Figure 5. Separation of soluble protein by SDS-PAGE following incubation of WT and LP red clover crude extracts for (a) 0-15 minutes and (b) 0-24 h. Arrows indicate newly formed protein-phenol complexes.

DISCUSSIONEffective PPO activity requires two factors: latent PPO activation and oxygen. The rumen is widely

considered to be anaerobic; nevertheless, rumen gas contains between 0.5 and 1.0 % oxygen (McArthur and Miltimore, 1962). Czerkawski (1969) calculated that oxygen transfer from saliva, food and diffusion from the blood of the host animal might account for 38 litres of oxygen entering the rumen of a sheep per day. Within grass boluses oxygen levels were recorded at 5.8 mg O2/l declining to 0.05 mg O2/l after 10 min, however, in the presence of rumen fluid oxygen levels were significantly lower and depletion of oxygen occurred at a much quicker rate (2.5 min) (Lee et al. 2006). The current study with red clover showed a much lower bolus oxygen level of 1.75 mg O2/l and a more rapid decline within 5 min to undetectable levels but showed a similar pattern in the presence of rumen fluid. Lee et al. (2006) postulated that differences in oxygen content of boluses may be related to the coarseness of the forage resulting in greater mastication which may explain the differences between the two studies. The level to which oxygen levels must fall to inhibit PPO is one of debate. Zibilske and

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Bradford (2007) showed PPO activity in soil at levels as low as 0.5 %, similar to what would be found in rumen gas, whereas Radler and Torokfalvy (1973) reported an oxygen requirement for grape PPO of 3.5 mg O 2/l. In addition Gomez et al. (2006) showed inhibition of mushroom PPO at a level of 0.12 mg O 2/l. This would be in line with the oxygen content of boluses but below that found in the rumen liquor shortly after swallowing. Given these results it is apparent that any occurrence of PPO activity during grazing of red clover would be largely confined to the period of mastication and that the amount of oxygen brought in from the boluses would be rapidly scavenged or insufficient to maintain PPO activity.

In experiment two the chemical composition of the red clovers were as expected with significantly higher dry matter, organic matter, WSC and fibre in the more mature forages with significantly higher total nitrogen in the less mature 4wk regrowth (Radojevic et al. 1994). Differences between red clover treatments were smaller and were mainly a consequence of wilting in the WRC treatment with a subsequent increase in dry matter and decrease in WSC. The effect of mastication in the chemical composition of the samples was apparent in the boluses. Dry matter was significantly reduced due to saliva contamination. This was particularly apparent with the 8wk regrowths as these samples were chewed to a greater extent as a result of the higher fibre content. Organic matter and WSC contents were also significantly reduced on the 8wk regrowth as a consequence of increased cell damage and loss of cell content.

The level of bound phenol within the herbages was significantly higher in the WRC treatment for both regrowths and is indicative of activated PPO as confirmed with 100% activation of PPO with WRC and the lower levels of activation with HRC and LRC with a mean across treatments and regrowth of 35.4%. Activity of PPO was significantly higher in the HRC treatment as expected and lowest in the LRC treatment (Winter et al. 2007) in both herbage and boluses. The intermediate nature of WRC may be related to denaturation of the enzyme during its 24 h wilt due to the activity of plant proteases. Although not significant there was a trend (P<0.1) for activities to be higher in the 4wk regrowth than the 8wk regrowth in both herbage and boluses. Within the boluses levels of bound phenol increased in all treatments compared to the herbage samples. This indicates a level of PPO activation across all treatments and although not significant there was a trend (P<0.1) for bound phenols in HRC to be higher than LRC with no difference between regrowths, although PPO activation was significantly higher in the 8wk regrowth as a potential consequence of the greater mastication aerating the bolus (Lee et al. 2006).

Changes in lipid profiles in the current study were examined from herbage (H) to bolus (B) to a bolus incubated in rumen fluid (IB) for 6 h to determine whether there was any PPO enhanced protection of lipid in grazed red clover. Lipid content and fractions were similar in the H and B samples and showed a significantly higher total lipid content in the less mature red clover, due to the higher content of leaf material. ML was significantly lower in the WRC treatment than the other treatments with a corresponding increase in FFA relating to plant mediated lipolysis which occurs during wilting (Lee et al. 2004). When the boluses were incubated in rumen liquor for 6 h significant changes in lipid fractions occurred although the content of total lipid remained relatively constant across H, B and IB. Incubation resulted in a significant loss of ML and rise in FFA similar to that which occurred during wilting but at a much greater extent due to a higher rate of lipolysis induced by microbial lipases (Lee et al. 2007). Although there are interaction effects between treatment and regrowth period LRC had the lowest ML composition after incubation and WRC and HRC were similar. FFA proportions were higher on the LRC treatments than the other two and higher on the 8wk regrowths than the 4 wk regrowths. Lipolysis as a measure of the breakdown of ML in the samples and the subsequent biohydrogenation of C18 PUFA were significantly lower on WRC than LRC and the 8 wk HRC treatment. This indicates that red clover required conditioning (a prior wilt) to activate PPO to protect glycerol based lipid. However, there also appears to be a maturity issue in that less mature herbage resulted in a lower level of lipolysis than the more mature herbage despite a lower PPO activation. This appears to be related to the degree of cell damage induced through mastication of the more fibrous mature forage.

This third experiments confirms that red clover PPO inhibits proteolysis in crude leaf extracts irrespective of the low levels of constitutive PPO occurring in the active form The relationship between protein-bound phenol and protein degradation parameters is convincing evidence for a link between PPO activity and protein degradation, as originally proposed by Jones et al. (1994). The appearance of high molecular weight protein species in the presence of PPO may be attributed to the formation of protein-phenol complexes similar to those observed by Kroll et al. (2000) in studies on the interactions of quinones with myoglobin. Kroll et al (2000) also suggested that these protein-phenol complexes are resistant to proteolytic attack.

Investigate the effect of cropping techniques on PPO activity in red clover.INTRODUCTION

The level of red clover PPO activity can be greatly increased following cell damage. This is probably due to activation of latent PPO by quinone products of phaselic acid produced by the constitutive active PPO, which is normally at a much lower level than the latent PPO. This “self activation” of PPO following tissue damage could act as an indicator of the level of tissue damage in a conditioned red clover crop. However, this needs to be calibrated by comparing a range of tissue damage levels with the PPO activation state and activation with bound-phenol formation.

MATERIALS AND METHODSRed clover herbage (cv. Milvus) grown in controlled conditions as previously described was harvested at

approximately 8 weeks regrowth. Leaf tissue (200 g FW) was removed and 50 g sub samples subjected to one of

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four levels of damage: i) undamaged (UD), ii) lightly damaged (crushed with a rolling pin, LD), iii) heavily damaged with a blender (HD), iv) frozen in liquid nitrogen and thawed (FT). Each set of samples was left at room temperature for 30 min and then split for analysis of degree of cell damage, PPO activation and production of bound phenols.

Cell damage was measured using two models: i) loss of electrolytic cellular components measured through conductivity or ii) loss of water-soluble carbohydrate (g/kg DM). Sub-samples (1 g) in triplicate of each damage level were shaken in 25 ml of ddH2O on an Orbital shaker (IKA Labortechnik, Werke, Germany) for 60 min, after which 3 replicate 8 ml samples of the shaken fluid (S) were taken. The tissue was then transferred to a clean tube and 25 ml of fresh ddH2O added, which was boiled for 10 min and, again, 3 replicate 8 ml samples of the boiled fluid (B) taken. Conductivity and WSC content of the S and B samples were measured using a conductivity meter and Technicon Autoanalyser (Technicon Corporation, New York, USA), respectively. In each case percentage damage of the tissue was calculated by the formula:Tissue Damage (%) = S/(S + B)*100 Where: S = conductivity (micro Siemens) or WSC lost due to the level of tissue damage imposed and B = conductivity or WSC remaining in the tissue. Both models of tissue damage were compared and correlated with PPO activation and bound phenol formation.

For the PPO activity the damaged tissue was extracted according to the method of Winters et al. (2003) and assayed according to the method of Robert et al (1995). Samples taken for analysis of bound phenol fractions were centrifuged at 15,000 × g for 10 min at 4oC and the supernatant retained. Soluble protein was precipitated by addition of 1 ml 20% trichloroacetic acid, 0.4% phosphotungstic acid. Following incubation for 30 min at 0 oC, precipitated protein was centrifuged at 15,000 × g for 10 min at 4oC. The protein pellet, which included bound phenol, was dissolved in 6 ml 0.1 M NaOH and analysed for protein and phenol content according to the method Winters and Minchin (2005). Protein concentration was based on the difference in response using Folin’s reagent with and without copper. The assay response without copper is mainly due to bound phenol content, whilst the response with copper is due to a combination of protein and bound phenol content. By estimating the protein component of this response it was possible to calculate the concentration of bound phenol.

Comparisons across levels of cell damage were subject to ANOVA performed with Genstat Release 8.11 (PC/Windows XP; Lawes Agricultural Trust, 2005, Rothamsted, UK).

RESULTSFigures 6 show the extent of tissue damage imposed by the four treatments measured through change in

conductivity and show a significant elevation (P<0.05) in conductivity with increasing cell damage moving from UD to FT. LD and HD were both significantly (P<0.05) greater than UD and significantly (P<0.05) lower than FT but were not significantly different (P=0.876) from each other in both models used for cell damage assessment. PPO activity and activation for the four levels of tissue damage are shown in Figure 7. Activity and activation is increased with increasing cell damage from UD to FT although there is no significant difference between LD and HD. The relationship between PPO activation and cell damage correlated strongly using both cell damage models: Activation (%) = 0.7425 × Cell damage (% conductivity loss) + 20.454 (R2 = 0.876) and Activation (%) = 0.7243 × Cell damage (% WSC loss) + 21.933 (R2 = 0.863). There was also a high correlation between PPO activation and bound phenol production with an R2 of 0.975.

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uctiv

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icro

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Figure 6. Degree of cell damage as measured by change in conductivity and % conductivity imposed by the four levels of tissue processing.

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Figure 7. PPO activity and activation of the induced by the four tissue damage processes.

DISCUSSIONCell damage activation of PPO is the most studied; be it through pathogen invasion, attack of herbivorous

insects, cutting or freeze/chilling cell damage. In the present study cell damage was highly correlated with PPO activity and activation and, subsequently, activation was highly correlated with bound-phenol production. Cell damage is thought to have three mechanisms for increasing PPO activity: i) decompartmentation of intracellular membranes with a subsequent mixing of enzyme and substrate resulting in activation of latent PPO as discussed above; ii) up-regulating of substrate formation induced through signal compounds, such as jasmonic acid, polyamines and hydrogen peroxide, resulting in a substrate-induced activation of the latent PPO; iii) up-regulation of the PPO gene increasing enzyme production. These three mechanisms may explain the greatest activation of PPO through freezing and thawing as this technique would increase PPO activity by all three mechanisms, whereas a rapid maceration may prevent an increase in PPO gene expression and thus limit overall activity.

Investigate the effects of growth stage and time of day on red clover PPO activity and its phenolic substrates.INTRODUCTION

In this experiment we examined levels of both active and latent PPO enzyme activity and substrate concentrations in wild-type red clover (WT) and a mutant with greatly reduced levels of PPO activity (low-PPO mutant, LP) over a growing season to determine if seasonality may be important in the feeding of red clover to optimise the effects of PPO.

MATERIAL AND METHODSRed clover (1.4 ha, WT) was sown on the 31st August 2004 at Trawscoed Research Farm (52o25'N,

4o05'W). The WT plot surrounded a sub-plot of 0.3 ha of LP red clover sown at the same time. Plots were maintained as silage leys and for the purpose of the present study, were sampled during the second year of growth. Approximately 200g of WT and LP plant material (6 replicates sampled at random points) was cut ca. 5 cm above soil level mid-morning at 2 weekly intervals, between May 16th and October 5th, 2005. The sward was cut back 3 times during this period. Fully expanded leaves were removed from plant material and either extracted immediately for determination of PPO substrate content or flash-frozen in liquid nitrogen and stored at -80 oC and subsequently sampled for PPO activity and substrate concentration as described previously.

RESULTS Assays for total PPO activity in leaves of WT and LP red clover plants gave mean values (three

replicates) of 2443 ± 193 U gFW-1 min-1 versus 51 ± 16 U gFW-1 min-1, respectively. Crosses of WT and LP mutant plants produced the WT phenotype in all F1 progeny and all backcrosses between F1 progeny and WT. Backcrosses between F1 progeny and LP mutants segregated 41% LP phenotype and 59% WT whilst the F2 progeny segregated 21% LP phenotype and 79% WT.

PPO activity and PPO substrate (phaselic acid) levels were measured in a WT and LP red clover pasture (close to Aberystwyth, 52o25'N, 4o05'W) over a growing season in 2005. Figure 8 shows mean levels of active and latent enzyme activity over the sampling period. Latent activity was estimated by subtracting constitutive activity (measured in the absence of SDS) from total potential activity which was measured in the presence of SDS. Levels of latent enzyme in WT red clover were greater than the active form at all but one sampling time and exceeded active enzyme by an average of 3-fold varying from 1 - 6.5 fold. LP plants showed greatly reduced levels of activity, however the ratios between latent and active forms were similar to those in WT red clover (mean of 2.35: 1). Whilst levels of active enzyme in WT red clover were low throughout the season, total potential activity remained relatively high despite showing some fluctuation in the same period (Figure 9). Figure 10 shows phaselic acid (endogenous PPO substrate in red clover) content over the growing season in WT and LP red

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clover. Substrate was present at all sampling points but showed variation during this period, whilst substrate profiles did not vary significantly between WT and LP red clover. There was an apparent relationship between phaselic acid content and the level of solar radiation to which plants were exposed in the preceding 72 h period, with the exception of forage harvested in late September and October when growth rates were reduced (see Figure 10).

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ity (

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Figure 8. Mean levels of active and latent red clover PPO enzyme in leaves over a growing season. Latent and active enzyme levels are represented by solid and hatched bars respectively.

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Figure 9. Total red clover PPO enzyme activity in leaves over a growing season in wild-type (solid bars) and low PPO mutant (hatched bars) plants. Arrows indicate cutting dates.

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Figure10. Phaselic acid content of red clover leaves over the growing season in wild-type (solid bars) and low PPO (hatched bars) mutant plants. Mean levels of solar radiation over the 3 preceding days are indicated by the solid triangles. Arrows indicate cutting times.

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DISCUSSION In this study, constitutive red clover PPO was always present in both the active and latent form, with the latter form predominating. Total levels of PPO activity showed seasonal fluctuations, however, as this study was only carried out over one season, it cannot be ruled out that variation was due to environmental effects. This is supported by the finding that PPO levels in September 2004 were approximately 5-fold lower than levels in the equivalent month in 2005 (Fothergill and Rees, 2005) and levels in May 2006 were almost 2-fold higher than in May 2005 (unpublished observations). It is notable that red clover growth rates were high in the mild, wet conditions of September 2004 compared with the much lower rates observed in the drier conditions of 2005.

The major red clover PPO substrate is the caffeic-malic ester, phaselic acid and levels observed over a growing season (equivalent to 5-20 mol caffeic acid g-1 FW) are in good agreement with the range of 10-15 mol g-1 FW predicted by Sullivan and Hatfield (2006). The caffeic amide, trans caffeoyl L-DOPA (clovamide) was also observed but at much lower concentrations with values equivalent to 1-4 mol caffeic acid g-1 FW (unpublished observations). An apparent relationship with solar radiation may indictate that phaselic acid accumulates when the rate of carbon fixation exceeds carbon demand for growth and primary metabolism. There was no clear relationship between enzyme and substrate levels and this, coupled with the observation of phenotypically normal levels of phaselic acid in LP red clover, would indicate that PPO expression is independent of substrate accumulation.

Investigate the rate at which polymerised red clover proteins are degraded by an adapted population of rumen microbes. INTRODUCTION

PPO had been shown to reduce both proteolysis and lipolysis in incubated red clover (Lee et al. 2004). However it has not been determined whether rumen microbes can adapt to utilise PPO protected protein and lipid. This study investigated whether rumen inoculum from cows offered red clover silage resulted in higher levels of proteolysis and lipolysis in red clover (+PPO) and also in red clover with the PPO1 gene silenced (–PPO) (Sullivan and Hatfield, 2006), than rumen inoculum from cows offered grass silage, due to microbial adaptation.

MATERIALS AND METHODSPPO1 gene silenced (PPO-) and wild type (PPO+) plants grown in controlled conditions were harvested

at 5cm above soil level, crushed and cut into 5mm strips and wilted for 1h. Two rumen fistulated cows were offered ad libitum either red clover silage (RC) or grass silage (G) for two 2 week periods in a 2 x 2 Latin Square. One litre of hand squeezed rumen inoculum was collected from each cow at the end of each period and transferred back to the laboratory in a temperature regulated flask (39oC). At the end of the first period the cows were given 2 weeks rest before starting the alternative silage in the second period. Red clover (2.5 g) was weighed into 32 tubes, 16 PPO+ and 16 PPO- (for each period) which provided duplicate tubes at each time point (0, 2, 6, and 24 h). Into each tube 7.5ml of anaerobic buffer, 0.35 ml reducing agent and 2.5 ml of either RC or G strained rumen inoculum were added before being purged with CO2 and incubated at 39oC. At each time point the supernatant was sub-sampled for free amino acid analyses. The tubes were then frozen with liquid N2, freeze-dried and the lipid extracted with 3 × 5ml of chloroform : methanol (2:1; v/v) and dried down under N 2 at 50oC. The sample was then fractionated by thin layer chromatography and the lipid fractions bimethylated before being run on GC as previously described. Lipolysis was calculated as the proportional loss of membrane lipid, and rise in free amino acids used as a predictor of proteolysis.

RESULTSFigure 11 and 12 show lipolysis and rise in free amino acids (proteolysis), respectively of the two red

clovers + and – PPO in either RC or G silage fed rumen inoculum. In both rumen inoculums the level of lipolysis and proteolysis were lower (P<0.05) in the PPO+ treatment than the PPO- treatment over time. RC inoculum did not result in a higher level of proteolysis or lipolysis than the G inoculum.

DISCUSSION Lipolysis and proteolysis in PPO+ was significantly lower than PPO- in both inoculum regimes with no

difference between RC or G inoculum. The results suggest that rumen-micro-organisms grown in an environment containing PPO protected phenol-bound protein do not adapt to increase the utilisation of the protected protein to any great extent. These results suggest that phenol-bound protein acts as a sustainable strategy to protect protein in the rumen to increase N use efficiency and decrease N pollution. The protection of protein by the PPO induced chemical cascade also results in a protection of glycerol-based lipid and more work is required to determine this mechanism. However, glycerol-based lipid is also protected by PPO and is not degraded to any greater extent in the presence of red clover adapted and hence PPO adapted rumen fluid.

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Figure 11. Lipolysis of glycerol based membrane lipid in wild type (+) and PPO1 gene silenced (-) red clover incubated in grass silage or red clover silage fed cows donated rumen liquor.

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Figure 12. Proteolysis as measured by the release of free amino acids in wild type (+) and PPO1 gene silenced (-) red clover incubated in grass silage or red clover silage fed cows donated rumen liquor.

Conduct a feeding study with dairy cows based on the optimal crop and/or feeding management strategy identified in objectives (1) to (4).INTRODUCTION

The rate of proteolysis of forages in the rumen is a key determinant of the efficiency of conversion of feed nitrogen into products and pollutants. The efficiency of conversion of feed N into milk (20-30%) or meat (10-20%) by ruminants is often well below potential (>40%), and is particularly low with diets based on poor grass silages (Dewhurst et al., 1996) or grazed herbage (Beever et al., 1986). At the same time, the biohydrogenation of fatty acids in the rumen, following lipolysis of plant phospho-lipids, reduces the P:S ratio of both meat and milk. PPO has been demonstrated to reduce both proteolysis and lipolysis during the ensiling of red clover and this has been shown to reduce nitrogen and PUFA losses when red clover silage has been fed to dairy cows (Dewhurst et al. 2003). However, whether PPO is active when animals graze fresh red clover is uncertain and the need to pre-activate through chopping prior to feeding was investigated. This work identified current opportunities and future goals in developing new strategies for forage management and livestock feeding by increasing the efficiency of feed protein utilisation and increasing polyunsaturated fatty acid production by ruminants. Thereby reducing pollutants and enhancing the nutritional status of milk and meat, through feeding forages with high levels of PPO.

MATERIALS AND METHODSThe experiment involved 3 dietary treatments in a changeover-design experiment (3 × 3 Latin Square):

(a) Grass, low PPO, (b) Red clover, high PPO, (c) Conditioned (chopped/bruised) red clover, high PPO and activated pre-feeding. Measurements will include (i) nitrogen partitioning into milk, with forage intake, urine and faeces and (ii) milk fatty acid profiles (which will be used to evaluate effects on biohydrogenation of alpha-linolenic acid (C18:3n-3)).

Perennial ryegrass (Lolium perenne) and red clover (Trifolium pratense) were sown in plots each with approximate areas of 0.7 and 1.5 ha, respectively. Six multiparous Holstein × Friesian dairy cows in mid-lactation (with similar milk yields, and stage of pregnancy) were maintained on a grass silage diet prior to allocation to treatment and during the covariate period (23rd April – 30th April). Mean milk yields were recorded during the covariate, and the three cows with the three highest mean yields were allocated at random within Square 1 (cows

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1-3), and the cows with the three lowest yields allocated at random within Square 2 (cows 4-6). Within each Latin square, the cows were allocated at random to one of three treatments: (a) zero grazed grass (G), (b) zero grazed red clover (R), (c) zero grazed conditioned red clover (CR). Each period consisted of 3 weeks adaptation followed by a 1 week measurement period.

In March 2007, each plot was split into 4 subplots. Each of these four groups of subplots were subsequently managed to provide sufficient forage to be zero grazed for each week of the experiment to give circa 4 weeks regrowth. Fresh grass (G) and red clover (R) were harvested daily in the early afternoon using a Haldrop 1500 plot harvester (J. Haldrop, Løgstør, Denmark). The CR treatments were cut using a circular mower and chopper and left as a swath in the field for circa 1 h before collection. All forage was cut to a height of 5 cm above soil level. The cut forage (c. 200 kg fresh matter for each treatment) were then transported from the field and circa 50 kg placed into 12 large weld mesh containers (120 × 80 × 100 cm, length × depth × height), to give 4 baskets of each treatment. The grass treatments were transferred to a blast freezer kept at -15 oC for 2 h. A short piece of perforated drainage tube was placed into the centre of each basket to aid with the chilling of the forage. After 2 h in the blast freezer half the feed was offered to the cows (c. 16:00) and the remainder kept chilled in the airlock at 4oC for the morning feed (c. 09:00). Both morning and afternoon red clover feeds were kept in the air lock. All animals received enough fresh forage for the measurement of ad libitum intakes with refusals of at least 0.1 of the forage offered. Samples of forage taken as cut and as fed were collected for each feeding and bulked per day during the measurement week. Samples for PPO analysis were collected daily during the measurement period from material placed in front of the cows, frozen immediately (liquid N) and kept frozen.

Animal measurements were concentrated in the third week of each period. Cow weights and body condition score were recorded at the start of the experiment as well as at the beginning and end of each period (before changing treatments).Cows were milked in a D-unit and received 2.0 kg/day of standard 18% protein dairy concentrate, sub sampled daily and bulked across the measurement week for each period (1.0 kg per milking). Milk yield was recorded across the experiment. Milk from the last two days of each period (separate am and pm samples) were collected and submitted for NMR analysis of fat, protein and lactose content. Additional milk samples from these milkings (frozen without preservative) were retained for fatty acid analysis. Nitrogen balance was carried out on all animals for total faecal and urine output for five days recorded and sub-samples stored frozen for subsequent analysis. 5 ml milk per kg milk produced were subsampled from each cow at each milking through the 6-d N balance period, bulked and stored at 4°C without preservative.

Chemical composition of the feed were analysed as described by Lee et al. (2007). PPO analysis and substrate analysis were carried out as previously described. Milk fatty acids were extracted using a single step extraction and base methylation as described by Lee ant Tweed (2007). Methyl esters were run on GC as previously described.

RESULTSChemical composition of the three forages as fed are given in Tables 5 and 6. Dry matter, water-soluble

carbohydrate and fibre were significantly higher in the grass than the two red clover diets, whilst the opposite was true for total nitrogen. PPO activity and soluble phenol concentration were significantly higher on the RC treatment than the other two treatments. Both G and CRC had complete PPO activation and CRC had the highest level of bound phenol. Substrate o-diphenols phaselic acid and clovamide were highest in RC. Chlorogenic acid was found to be the o-diphenol substrate in grass.

Total fatty acid content of the forages was similar although the red clovers had higher concentrations of C18:2n-6 whilst G had a higher concentration of C18:3n-3. These differences are shown in the intake of the individual fatty acids show in Table 7. Total fatty acid and dry matter intakes were similar across dietary treatments, whereas animals consuming red clover consumed significantly more C18:0, C18:2n-6 and C20:0 than animals on the G diet.

Milk yield, somatic cell count and fat composition were not significantly different from cows fed on the three different forages. Protein and lactose composition were higher in milk from the animals offered G compared to RC or CRC. Milk from cows offered either red clover diet resulted in significantly higher levels of C14:0, C18:2n-6, C18:3n-3 and total long chain fatty acids than animals offered G.

Table 5. Chemical composition, polyphenol oxidase activity and o-diphenol concentrations in the forages (g/kg DM unless stated)

G RC CRC s.e.d. PDM (g/kg) 16.0 13.9 14.5 0.60 **OM 905 904 904 6.89 NSWSC 223 109 105 28.0 **TN 22.1 32.6 33.1 4.69 †NDF 441 309 320 37.0 *PPO activity (U/g DM) 17.2 158 22.1 31.1 ***PPO activation (%) 100 29.1 100 4.84 ***Bound phenol 1.72 5.48 10.5 1.612 ***Soluble phenol 37.0 90.4 68.9 5.78 ***Phaselic (ug/g DM) - 1922 119 102.1 ***Clovamide (ug/g DM) - 117 - 20.8 ***

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Chlorogenic (ug/g DM) 125 - - 32.8 ***

Table 6. Fatty acid compositions (g/kg DM) of the dietary forages and concentrateG RC CRC Conc

C12:0 0.04 0.01 0.03 2.35C14:0 0.07 0.06 0.08 0.98C16:0 3.52 3.53 4.43 13.9C16:1c9 0.34 0.12 0.04 0.16C18:0 0.25 0.43 0.55 1.66C18:1c9 0.48 0.44 0.61 17.6C18:2c9c12 3.22 5.07 6.11 21.1C18:3c9c12c15 16.5 13.3 14.7 2.10C20:0 0.08 0.15 0.21 0.19Total 25.1 24.1 28.4 62.8

Table 7. Dry matter (kg/d) and fatty acid intake (g/d) in cows fed the three forage dietsG RC CRC s.e.d. P

DM 18.7 19.6 18.2 0.747 NSC12:0 4.76 4.37 4.84 0.167 *C14:0 2.90 2.74 3.31 0.183 *C16:0 84.3 87.3 95.5 4.03 †C16:1c9 0.84 0.49 1.13 0.259 NSC18:0 7.09 10.6 12.0 0.399 ***C18:1c9 39.0 38.5 38.7 1.95 NSC18:2c9c12 92.0 127 135 5.48 ***C18:3c9c12c15 288 240 245 27.9 NSC20:0 1.64 3.00 3.99 0.198 ***Total FA 541 540 569 37.7 NS

Table 8. Milk yield, composition (%) and fatty acid concentration (mg/ml) from cows fed the three forage dietsG RC CRC s.e.d. P

Yield 32.1 32.7 32.9 1.39 NSSCC 65.6 83.8 64.2 12.77 NSProtein 3.18 3.13 3.11 0.016 ***Lactose 4.70 4.63 4.66 0.022 *Fat 3.97 4.00 3.88 0.157 NSC12:0 1.12 1.02 0.96 0.057 †C14:0 3.21 4.12 4.13 0.143 ***C16:0 10.5 9.28 9.31 0.415 *C16:1c9 0.54 0.44 0.51 0.042 NSC18:0 4.18 3.74 3.77 0.423 NSC18:1c9 8.06 8.14 8.74 0.504 NSC18:1 total cis 8.39 8.53 9.15 0.517 NSC18:1 total trans 1.55 1.74 1.58 0.119 NSC18:2c9c12 0.47 0.81 0.85 0.028 ***C18:2 CLA 0.37 0.37 0.33 0.047 NSC18:2 NC 0.18 0.23 0.20 0.026 NSC18:3c9c12c15 0.29 0.50 0.53 0.027 ***C20:0 0.05 0.04 0.05 0.007 NSTotal SC 1.93 1.78 1.83 0.077 NSTotal LC 0.18 0.24 0.24 0.006 ***Total BOC 1.38 1.33 1.36 0.060 NSTotal FA 35.7 34.1 34.6 1.24 NS

Table 9. Nitrogen intake and portioning in dairy cows fed the three forage dietsG RC CRC s.e.d. P

Nitrogen Intake (g/d)Grass 385 581 561 52.5 *Concentrate 56 56 56 - -Total 441 637 618 52.5 *Nitrogen OutputUrine (g/d) 168 293 277 23.1 **Proportion of N Intake 0.39 0.48 0.45 0.066 NSFaeces (g/d) 183 198 224 15.4 †Proportion of N Intake 0.45 0.32 0.37 0.047 †

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Milk (g/d) 122 154 163 14.4 †Proportion of N Intake 0.28 0.25 0.26 0.024 NSRetention/loss (g/d) -31.1 -24.0 -46.1 34.6 NSProportion of N Intake -0.12 -0.05 -0.08 0.011 NS

DISCUSSIONPPO activity of the CRC treatment was surprisingly low and comparable to the G treatment. This was due

to proteolytic deactivation of the enzyme as a consequence of the level of cell damage. However all of the PPO was in the active form and had resulted in a significant depletion of the soluble phenol and an increase in the level of bound phenol, indicating that the PPO had catalysed the conversion of the substrate to quinones and these had bound with protein. Consequently the level of PPO activity and substrate in CRC as fed was comparable to that of G. Intakes of dry matter and total fatty acids were similar across diets. Milk PUFA concentrations were significantly higher in milk from animals fed the red clover treatments but were not different between RC and CRC. As previous studies with silage have indicated (Lee et al. 2003, Lee et al. 2006) red clover PPO resulted in a significant protection of C18 PUFA across the rumen with a subsequently higher concentration in animal product (milk) compared to G. This study confirms that the feeding of cut red clover as well as conserved forage can result in a PPO protection of glycerol-based lipid. The lack of difference between CRC and RC may be due to significant activation of the RC during mastication so that the protein and lipid were as protected as the already activated CRC. On the other hand how maceration alters PPO action is not known. For example a slow cell damage procedure such as wilting may result in a gradual o-diphenol oxidation which could favour nucleophilic coupling of o-quinones to proteins followed by oxidative cross linking of the o-diphenol-protein complexes. Such cross-linking would protect proteins from plant and microbial protease action. By contrast a more severe cell damage such as cutting and crushing followed by chilling as with CRC may have resulted in a more rapid oxidation of o-diphenols favouring coupling of o-quinones with proteins at the expense of continued coupling reactions to cross link o-diphenol-protein complexes. Under this scenario the protein would be protected from plant proteases but only partially from microbial proteases.

Nitrogen intake was significantly higher on the red clover diets than the grass and this resulted in higher levels of Nitrogen in milk, urine and faeces, although no difference was found between the two red clover diets. Proportion of N in milk and urine were not significantly different across diets with a trend towards a higher proportionally higher N output in the faeces of animals offered grass. These results are surprising and contradict the findings of Dewhurst et al. (2003) and Broderick et al. (2001) who both found a greater portioning of dietary N into product (milk and retained) when red clover was fed as opposed to grass. The results may have been due to the significantly higher level of water-soluble carbohydrate in the grass than the red clover diets resulting in a elevation in the N use efficiency of the grass diet as a consequence of the greater balance of N and energy in the rumen (Miller et al. 2001). This may suggest that in the current experiment animals offered the red clover diet were energy limiting and thus unable to show elevations in N use efficiency due to the poor incorporation of dietary N into microbial N. This may explain the high levels of urinary N on the red clover diet as ammonia released in the rumen failed to be incorporated into microbial protein and was lost as urea in the urine. Further investigation is required to fully understand the protection of protein by PPO in the rumen as to whether increasing energy to the diet in line with the grass fed animals will result in a significant elevation in N use efficiency an whether certain amino acids are made unavailable to the animal as a consequence of the phenol covalent attachment to sulpho-amino acids.

Conduct a feeding study with cull cows designed to enhance meat quality, based on the optimal crop and/or feeding management strategy identified in (1) to (4)INTRODUCTION

Effective cull cow marketing again becomes important as the ending of the Over Thirty Month Scheme (OTMS) allows all stock born after July 1996 and testing negative for BSE back into the food chain. While the value of cull cow finishing depends upon specific market conditions, marketing dairy culls with the best possible combination for the least possible cost is always advisable. Natural forage systems are an attractive opportunity to achieve this. This experiment aims to compare the finishing of dairy cull cows using grass or red clover silage in terms of days to finish and final meat quality.

MATERIALS AND METHODSSixteen Holstein × Friesian cull cows were blocked on live weight and randomly allocated to one of two

diets grass silage or red clover silage sourced from Trawsgoed farm. The animals were weighed and condition scored at the start of the experiment and then every 3 weeks, on two consecutive days at the same time of day. Dry matter intake was recorded per animal per day through the Calan gate system. Samples of silage were taken every day and bulked per week. This was kept frozen (-20oC) and stored before chemical analysis. Back muscle / Fat scanning were measured using Ultrasound - 2 measurements made at the beginning of treatments and then before being sent to slaughter. The animals were maintained on diet until reaching an adequate level of finish (Grade 1-2 EBLEX Grading of cull cows). Animals on reaching the required finishing were transported to Bristol for slaughter and Meat fatty acid and quality analysis as described by Warren et al (2007).

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Grade 1: Produces a very good commercial carcase. Well fleshed throughout; fat cover even, not patchy or excessive.

Grade 2: Average to good quality carcase, not as good as grade 1. May benefit from further finishing.

RESULTSChemical composition of the two silages were significantly different with dry matter (DM), total N (TN),

and pH significantly higher on the RC silage than the G silage and vice versa for Organic matter (OM), water-soluble carbohydrate (WSC), neutral detergent fibre (NDF), dry matter digestibility (DMD), ammonia-N and acetic acid (Table 10). Fatty acid compositions were also different with G silage having significantly higher levels of C12:0, C14:0, C16:1c9, C18:3n-3 and total fatty acids whereas RC had higher levels of C18:0, C18:2n-6 and C20:0. These differences were mirrored in the intake of fatty acids and were attenuated due to the greater DM intake on the RC silage diet (Table 11). Fatty acid profiles of the longissimus dorsi steaks are shown in Table 12. RC fed animals had higher concentrations of C18:3n-3 and total n-3 fatty acids compared to animals offered the G silage that had higher levels of C12:0 and C16:1c9. All other fatty acids were similar but there were tendencies for C14:0, C18:1c9, CLA and total MUFA to be higher in the muscle of G fed animals and total PUFA to be higher in the tissues of RC fed animals.

Carcass weights were not significantly different across treatments with left side and right side weights on average 187.8 and 177.8, respectively. Days on treatment were also similar (75 +/- 5 days) as were terminal pH (5.4) and grade (P+-O-; 4L-4H). Longissimus dorsi lengths and widths were longer in the GS fed animals 135.1 and 69.9 versus 120.4 and 63.3 for G and RC, respectively.

Table 10. Chemical composition (g/kg DM) and fatty acid profile of the experimental silagesG RC s.e.d P

DM (g/kg) 352 571 14.6 ***OM 923 899 3.1 ***TN 26.8 29.8 1.29 *NDF 451 405 16.5 *ADF 283 302 13.4 NSDMD (%) 88.0 80.2 1.12 ***pH 3.72 4.77 0.063 ***NH3-N 1.52 1.33 0.040 ***Acetic 9.13 4.33 1.049 ***Propionic 0.04 tr 0.026 NSButyric 0.05 0.02 0.040 NSValeric 0.01 0.01 0.017 NSC12:0 0.07 0.04 0.005 ***C14:0 0.11 0.07 0.007 ***C16:0 3.64 3.75 0.207 NSC16:1c9 0.06 0.04 0.003 ***C18:0 0.31 0.48 0.021 ***C18:1c9 0.37 0.47 0.021 ***C18:2c9c12 3.20 3.96 0.168 ***C18:3c9c12c15 13.5 8.11 0.936 ***C20:0 0.10 0.18 0.007 ***Total 22.8 18.6 1.42 *

Table 11. Dry matter (kg/d) and fatty acid intake (g/d) in cows fed the experimental silages

Table 12. Fatty acid composition of the Longissimus dorsi muscle (mg/100g tissue) from the cows fed the experimental silages

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G RC s.e.d. PDM 10.9 13.3 1.01 *C12:0 0.99 0.52 0.070 ***C14:0 1.50 0.87 0.108 ***C16:0 48.5 49.9 4.35 NSC16:1c9 0.746 0.486 0.056 ***C18:0 4.08 6.43 0.476 ***C18:1c9 4.96 6.22 0.498 *C18:2c9c12 42.7 52.7 4.24 *C18:3c9c12c15 179 108 13.1 ***C20:0 1.34 2.42 0.173 ***Total FA 304 247 24.5 *

Colour saturation of steaks in modified atmosphere packs under retail conditions were similar up to day 7 after which the steaks from animals fed the RC silage lost colour at a faster rate than those animals fed G silage (Figure 12).

101112131415161718

0 2 4 6 8 10

days displayed

colo

ur s

atur

atio

n (c

hrom

a) Grass silage Red clover

Figure 12. Colour shelf life of Longissimus dorsi steaks in modified atmosphere packs under retail conditions from cull cows offered either grass or red clover silage

TBARS in the steaks from animals offered RC were significantly higher and vitamin E levels significantly lower (Figure 13) whereas tenderness of the steaks was higher in the RC steaks as measured by shear force 3.81 versus 4.04 kg for RC and G produced steaks, respectively. Sensory attributes of the two steak types were similar with the only difference being a higher score on a 1-100 sliding scale for fishy in the RC steaks (1.01 versus 1.57; P<0.01 for G and RC, respectively).

0123456789

10

TBARS Vit E

mg/

g

Grass Red clover

Figure 13. Fatty acid oxidation and vitamin E content of Longissimus dorsi steaks from cull cows offered either grass or red clover silage

DISCUSSION

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G RC s.e.d. PC12:0 1.82 1.28 0.239 *C14:0 89.0 66.5 10.80 †C16:0 954 759 120.8 NSC16:1c9 156 101 21.8 *C18:0 468 428 78.0 NSC18:1c9 1364 957 204.9 †C18:2c9c12 65.7 69.0 6.86 NSC18:2 CLA 8.05 4.61 1.652 †C18:3c9c12c15 24.6 39.8 2.88 ***C20:0 3.18 2.79 0.46 NSTotal MUFA 1606 1116 238.3 †Total PUFA 162 180 10.2 †Total SAT 1569 1302 205.8 NSTotal n-3 60.5 77.2 4.35 **Total n-6 107 103 8.85 NSTotal FA 3564 2769 466.2 NS

The experiment set out to determine if red clover as opposed to grass silage could result in EBLEX grade 1 or 2 class carcasses of cull cows within 100 days of trail and whether the feeding of red clover would improve the quality of the meat produced. Red clover and grass silage feeding resulted in a good standard of finish after approximately 75 days. Although grass silage feeding resulted in slightly larger carcasses and rib steaks the fatty acid composition of the red clover fed animals was significantly enhanced. C18:3n-3 concentrations were significantly higher in red clover silage fed animals than the grass silage fed animals despite the greater intake of C18:3n-3 on the latter. This is a response to PPO protecting the PUFA across the rumen with a subsequent increased deposition in muscle and neutral lipid (Lee et al. 2003, Dewhurst et al. 2003). This elevated C18:3n-3 also resulted in an increase in total PUFA and n-3 fatty acids and may be responsible for the slight fishy score given to the steaks by the trained taste panel. This higher level of PUFA also resulted in a greater oxidation of the steaks and a reduced shelf life, although both are within the range required by retailers. A disadvantage of feeding red clover to ruminants is the relative poor uptake of vitamin E, (Scollan et al. 2006) which as an anti oxidant would protect the higher levels of PUFA in red clover fed animal products. Feeding strategies which incorporate red clover may also require a supplement of vitamin E to ensure that the beneficial increase in PUFA in the product is protected against oxidation.

Overall Conclusion

Red clover PPO exists in two forms; active and latent, which can be activated in vivo during cell damage through plant proteases and/or an induced activation by the natural substrates phaselic acid and clovamide. Activation of the PPO occurs during mastication of red clover but activity is prevented in the rumen through the rapid scavenging of oxygen by the rumen micro-organisms. Factors such as maturity of the forage also play a role in PPO concentration and activation. Protection of protein and glycerol-based lipid by PPO can be increased with a conditioning (wilting) step to increase the window of opportunity for activation, ortho-quinone production and formation of protein-bound-phenol complexes. These complexes once formed protect the protein from microbial degradation with the microbiota being unable to adapt to increase its degradation. This PPO induced protection of protein and glycerol-based lipid results in increased N utilisation, decreases in N excretion and environmental pollution and enhances final product quality in terms of the fatty acid profile.

Future Work

This work has important linkages to studies on PPO within the forage breeding programmes at IGER. Whilst the area of red clover that is grazed is currently low, it will increase both as the sustainability benefits of red clover are appreciated and as varieties that are more tolerant to grazing are produced. In addition, we have recently shown PPO activity in grass, which also have the ability to protect the soluble protein fractions and glycerol-based lipid from proteolytic and lipolytic degradation, respectively. We have also shown cultivar/species differences in PPO activity within grasses, indicating the potential for up-regulating this activity through selective breeding with possible benefits of reduced lipid degradation. Thus, the techniques and findings of this red clover project could be extended into studies on forage grasses. We have also formed links with the United States Dairy Forage Research Institute to collaborate on mechanistic approaches for PPO using plants with point-mutations at the PPO1 gene loci. These studies could lead to a greater understanding of the mechanism of protein and glycerol-based lipid conservation and also potential use of the mechanism for the protection of other forage crops.

In addition further work is required to fully investigate the effect of PPO on N utilisation in the ruminant, both across the rumen and across the whole gastro-intestinal tract (GIT). This will validate PPOs protection of dietary N, the effect on microbial protein synthesis and thus total N flow to the abomasum. It will also determine the bioavailability of PPO protected protein across the GIT as some concern has been raised with availability of sulphur-amino acids which act as the covalent adhesion site of ortho-quinones in the formation of protein-phenol complexes.

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Sci. 2003, 76, 491-501.Lee, M. R. F.; Winters, A. L.; Scollan, N. D.; Dewhurst, R. J.; Theodorou, M. K.; Minchin, F. R. Plant-mediated lipolysis and proteolysis in red clover with different polyphenol oxidase activities. J. Sci. Food Agric. 2004, 84, 1639-1645.Lee, M. R. F.; Parfitt, L. J.; Scollan, N. D.; Minchin, F. R. Lipolysis in red clover with different polyphenol oxidase activities in the presence and absence of rumen fluid. J. Sci Food Agric. 2007, 87, 1308-1314.Lee, M. R. F.; Dewhurst, R. J.; Minchin, F. R. The concentration of oxygen and its depletion in bovine grass-boluses. Proc. Brit. Soc. Anim. Sci. 2006, 181.Lee, M. R. F.; Tweed. J. K. S. Evolution of artefact trans-9trans-11 conjugated linoleic acid (CLA) from cis-9 trans-11 CLA and its reduction by a rapid base catalysed methylation of milk fat. J. Dairy Res. In pressLloyd, D.; Ellis, J. E.; Hillman, K.; Williams, A. G. Membrane inlet mass spectrometry: probing the rumen ecosystem. J. App. Bacteriology. 1992, 73, 155S-163S.McArthur, J. M.; Miltimore, J. E. Rumen gas analysis by gas-solid chromatography. Can. J. Anim. Sci. 1962, 41, 187-196.Merry, R. J.; Lee, M. R. F.; Davies, D. R.; Dewhurst, R. J.; Moorby, J. M.; Scollan, N. D.; Theodorou, M. K.; Effects of high-sugar ryegrass silage and mixtures with red clover silage on ruminant digestion . 1. In vitro and in vivo studies of nitrogen utilization. J. Anim. Sci. 2006, 84, 3049-3060.Miller, L.A.; Moorby, J.M; Davies, D.R.; Humphreys, M.O.; Scollan, N.D.; MacRae, J.C.; Theodorou, M.K. Increased concentration of water-soluble carbohydrate in perennial ryegrass (Lolium perenne L.): milk production from late-lactation cows. Grass Forage Sci. 2001, 56, 383-394.Nichols, B. W. Separation of the lipids of photosynthetic tissues: improvement in analysis by thin-layer chromatography. Biochim. Biophys. Acta. 1963, 70, 417-422.Radler, F.; Torokfalvy, E. The affinity for oxygen of polyphenoloxidase in grapes. Zeitschrift fur Lebensmitteluntersuchung und- Forschung A 1973, 152, 38-41.Radojevic, I.; Simpson, R. J.; St John, J. A.,; Humphreys, M. O. Chemical composition and in vitro digestibility of lines of Lolium perenne selected for high concentrations of WSC. Aust. J. Agric. Sci. 1994, 86, 443-452.Robert, C. M.; Cadet, F.R.; Rouch, C. C.; Pabion, M.; Richard-Forget, F. Kinetic study of the irreversible thermal deactivation of palmito (Acanthophoenix rubra) polyphenol oxidase and effect of pH. J. Sci. Food Chem. 1995, 43, 2375-2380.Scollan, N. D. , Wood, J. D. Enhancing the nutritional value of beef and its relationship with meat quality Linking up the meat chain: Ensuring quality and safety for consumers International Conference, Krakow, Poland, 19-20 October 2006 83-84 Sullivan, M. L.; Hatfield, R. D. Polypehnol oxidase and o-diphenols inhibit postharvest proteolysis in red clover and alfalfa. Crop Sci. 2006, 46, 662-670Warren, H. E. , Scollan, N. D. , Nute, G. R. , Hughes, S. I. , Richardson, R. I. , Wood, J. D. Effects of breed and a concentrate or grass silage diet on beef quality in cattle of 3 ages: II. Meat stability and flavour Meat Science 2007, In the press Winters, A. L.; Minchin, F. R.; Michaelson-Yates, T. P. T.; Lee, M. R. F.; Morris, P. Characterisation of polyphenol oxidase (PPO) activity in red clover (Trifolium pratense) and use of a low-PPO mutant to study the role of PPO in proteolysis reduction. J. Agric. Food Chem. 2007, In the press. Winters, A. L.; Minchin, F. R.; Merry, R. L.; Morris, P.; Comparison of polyphenol oxidase activity in red clover and perennial ryegrass. Asp. Appl. Biol. 2003, 70, 121-128. Winters, A. L.; Lloyd, J.D.; Jones, R.; Merry, R. J. Evaluation of a rapid method for estimating free amino acids in silages. Anim. Feed Sci. Technol., 2002, 99, 177-187.Winters A. L.; Minchin F. R. Modification of the Lowry assay to measure proteins and phenols in covalently bound complexes. Analytical Biochemistry, 2005, 346, 43-48.Veltman, R.H.; Larrigaudiere, C.; Wichers, H.J.; van Schaik, A.C.R.; van der Plas, L.H.W.; Oosterhaven, J. (1999) PPO activity and polyphenol content are not limiting factors during brown core development in pears (Pyrus communis L. cv. Conference). J. Plant Physiol. 1999, 154, 697-702.Zibilske, L. M.; Bradford, J. M. Oxygen effects on carbon, polyphenols, and nitrogen mineralization potential in soil. Soil Sci. Soc. Am. J. 2007, 71, 133-139.

References to published material

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9. This section should be used to record links (hypertext links where possible) or references to other published material generated by, or relating to this project.

Lee, M. R. F., Tweed, J. K. S., Scollan, N. D., Sullivan, M. L. Ruminal micro-organisms do not adapt to increase utilisation of polyphenol oxidase protected red clover protein and glycerol-based lipid Journal of Agriculture and Food Chemistry In the press

Lee, M. R. F., Tweed, J. K. S., Minchin, F. R., Winters, A. L. Red clover polyphenol oxidase: activation, activity and efficacy under grazing Journal of Agriculture and Food Chemistry In the press

Winters, A. L., Minchin, F. R., Michaelson-Yates, P.T., Lee, M. R. F., Lee, Morris, P. Latent and active PPO in red clover ( Trifolium pratense ) and use of a low-PPO mutant to study the role of PPO in proteolysis reduction Journal of Agriculture and Food Chemistry In the press

Lee, M. R. F. , Scott, M. B. , Tweed, J. K. S. , Minchin, F. R. , Davies, D. R. The effect of polyphenol oxidase on lipolysis and proteolysis of red clover silage with and without a silage inoculant (Lactobacillus plantarum L54) Animal Feed Science and Technology In the press

Lee, M. R. F. , Parfitt, L. J. , Scollan, N. D. , Minchin, F. R. (2007) Lipolysis in red clover with different polyphenol oxidase activities in the presence and absence of rumen fluid Online at: http://www3.interscience.wiley.com/cgi-bin/fulltext/114189578/PDFSTART Journal of the Science of Food and Agriculture 87 (7) 1308-1314

Winters, A. L. , Robbins, M. P. , Gallagher, J. A. , Lee, M. R. F. , Minchin, F. R. , Parveen, I. et al. (2007) Biochemistry and molecular biology of polyphenol oxidases in grasses Abstracts,'Highlights in the evolution of phytochemistry', Phytochemical Society of Europe, 50th Anniversary Meeting, Cambridge, 11-14 April 2007

Lee, M. R. F. , Scott, M. B. , Davies, D. R. , Minchin, F. R. The effect of polyphenol oxidase on lipolysis and proteolysis of red clover silage with and without live culture inoculum 2nd International Symposium on Energy and Protein Metabolism and Nutrition, Vichy, France, 9-13 September 2007

Lee, M. R. F. , Minchin, F. R. , Hatfield, R. D. , Sullivan, M. L. (2007) Red clover polyphenol oxidase reduces ruminal lipolysis in in vitro batch culture Proceedings of BSAS Annual General Conference, Southport Theatre & Floral Hall Complex, Southport, 2-4 April 2007 025

Lee, M. R. F. , Minchin, F. R. (2007) The concentration of oxygen and its depletion in bovine red clover-boluses Proceedings of BSAS Annual General Conference, Southport Theatre & Floral Hall Complex, Southport, 2-4 April 2007 234

Theodorou, M. K. , Kingston-Smith, A. H. , Winters, A. L. , Lee, M. R. F. , Minchin, F. R. , Morris, P. et al. (2006) Polyphenols and their influence on gut function and health in ruminants: A review Online at: http://dx.doi.org/doi:10.1007/s10311-006-0061-2 Environmental Chemistry Letters 4 (3) 121-126

Winters, A. L. , Bryant, D. N. , Gallagher, J. A. , Lee, M. R. F. , Minchin, F. R. , Parveen, I. et al. (2006) Biochemistry and molecular biology of polyphenol oxidases in grasses International Conference on Polyphenols XXIII, University of Manitoba, Winnepeg, Canada, 22-25 August 2006 p303-304

Lee, M. R. F. , Minchin, F. R. (2006) Reducing nitrogen pollution and increasing the healthiness of livestock products: Does red clover hold the key? Presentations by Britain's Top Younger Scientists, Engineers and Technologists for UK Nationals Science Week, House of Commons, Westminster, 13 March 2006 L2-43

Lee, M. R. F. , Winters, A. L. , Minchin, F. R. (2007) Plant mediated lipolysis of grass genotypes with varying concentrations of polyphenol oxidase 'High Value Grassland: providing biodiversity, a clean environment and premium products': Proceedings BGS/BES/BSAS Conference, Keele University, Staffs, 17-19 April 2007. British Grassland Society Occasional Symposium, 38 Hopkins, J. J. , Duncan, A. J. , McCracken, D. I. , Peel, S. , Tallowin, J. R. B., eds. 253-256

Lee, M. R. F. , Parfitt, L. J. , Minchin, F. R. (2006) Lipolysis of red clover with differing Polyphenol oxidase activities in batch culture Journal of Animal Science American Society of Animal Science, Minneapolis, 9-13 July 2006 84 (Supplement) p101

Dewhurst, R. J. , Shingfield, K. J. , Lee, M. R. F. , Scollan, N. D. (2006) Increasing the concentrations of beneficial polyunsaturated fatty acids in milk produced by dairy cows in high-forage systems Online at: http://dx.doi.org/10.1016/j.anifeedsci.2006.04.016 Animal Feed Science and Technology 131 (3-4) 168-206

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http://dx.doi.org/doi:10.1007/s10311-006-0061-2

http://dx.doi.org/doi:10.1007/s10311-006-0061-2












Lee, M. R. F. , Winters, A. L. , Dewhurst, R. J. , Minchin, F. R. (2006) Polyphenol oxidase activity, protein complexing and lipid profiles in bovine red clover-boluses Proceedings of the British Society of Animal Science Annual Conference, York, 27-29 March 2006 p45

Lee, M. R. F. , Dewhurst, R. J. , Minchin, F. R. (2006) The concentration of oxygen and its depletion in bovine grass-boluses Proceedings of the British Society of Animal Science Annual Conference, York, 27-29 March 2006 p181

Lee, M. R. F. , Olmos Colmenero, J. de J. , Winters, A. L. , Scollan, N. D. , Minchin, F. R. (2006) Polyphenol oxidase activity in grass and its effect on plant-mediated lipolysis and proteolysis of Dactylis glomerata (Cocksfoot) in a simulated rumen environment Online at: http://www3.interscience.wiley.com/ Journal of the Science of Food and Agriculture 86 (10) 1503-1511

Theodorou, M. K. , Kingston-Smith, A. H. , Winters, A. L. , Lee, M. R. F. , Minchin, F. R. , Morris, P. et al. (2005) Polyphenols and their influence on gut function and health in ruminants International Conference on Polyphenols, XXII, Helsinki, Finland, 25-28 August 2004 5-6

Lee, M. R. F. , Winters, A. L. , Scollan, N. D. , Dewhurst, R. J. , Theodorou, M. K. , Minchin, F. R. (2004) Plant mediated lipolysis and proteolysis in red clover with different polyphenol oxidase activities Online at: http://dx.doi.org/doi:10.1002/jsfa.1854> Journal of the Science of Food and Agriculture 84 (13) 1639-1645

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http://www3.interscience.wiley.com/cgi-bin/fulltext/112649761/PDFSTART








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