In vitro rumen simulated (RUSITEC) metabolism of freshly cut or wilted grasses with contrasting polyphenol oxidase activities

In vitro rumen simulated (RUSITEC) metabolism offreshly cut or wilted grasses with contrastingpolyphenol oxidase activities

M. R. F. Lee*, A. Cabiddu†, F. Hou‡, V. Niderkorn§, E. J. Kim*, R. Fychan* and N. D. Scollan*

*Institute of Biological and Environmental Research, Aberystwyth University, Gogerddan Campus, Aberystwyth,

UK, †Dipartimento per la Ricerca nelle Produzioni Animali, AGRIS Sardegna, Olmedo, Italy, ‡College of Pastoral

Agriculture Science and Technology, Lanzhou University, Gansu Province, China, and §INRA, UR1213

Herbivores, F-63122, Saint-Genes-Champanelle, France

Abstract

The study investigated in vitro simulated rumen metab-

olism of freshly cut and wilted cocksfoot [Dactylis glom-

erata; high polyphenol oxidase (PPO)] and tall fescue

(Festuca arundinacea; low PPO). A 16-vessel RUSITEC was

used with the four treatment combinations: cocksfoot

wilted (Cw); cocksfoot fresh (Cf); tall fescue wilted (TFw)

and tall fescue fresh (TFf). Rumen liquor was collected

from four fistulated dairy cows maintained on perma-

nent pasture. The experiment ran for 12 d with sampling

of effluent for rumen parameters [volatile fatty acids

(VFA) and NH3-N] on days 10 and 11 at 0, 1, 2, 3, 4, 6, 8

and 24 h intervals. On days 9–12, gas production and

composition were measured using a plastic gas collection

bag attached to the effluent bottle. At the end of day 12,

the grass residue and effluent from the vessels were

collected and analysed for digestibility and lipid fraction-

ation. Lipolysis was calculated as the proportional loss of

glycerol-based membrane lipid between the forage and

residue. Polyphenol oxidase was higher (P < 0Æ001) in Cf

than the other treatments; Cw was higher than both TF

treatments, with no difference between TFw and TFf. The

level of protein-bound phenol (product of oxidation

reaction) tended to be higher for Cw and TFw than Cf and

TFf, (P < 0Æ1) and was higher for cocksfoot than tall

fescue (P < 0Æ001). As an average across the day, NH3-N

was lower (P < 0Æ001) in cocksfoot than tall fescue,

despite the lower nitrogen concentration of tall fescue,

and in fresh as opposed to wilted grass (P < 0Æ001). There

was a trend (P < 0Æ1) for lipolysis to be lower in cocksfoot

than tall fescue, and for both wilted treatments to

be lower than the fresh (P < 0Æ05). Total VFA concentra-

tion was not different across treatments, although there

were differences in molar proportions of individual

VFA. Cocksfoot as opposed to tall fescue showed a small

effect in lowering lipolytic and proteolytic activity

(release of NH3-N), although other differences between

the grasses other than just PPO activity such as lipase

activity, diphenol substrate content and digestibility may

have greater effects between the grasses confounding the

effect of PPO.

Keywords: cocksfoot, Dactylis glomerata, tall fescue,

Festuca arundinacea, polyphenol oxidase, rumen param-

eters, lipolysis, RUSITEC

Introduction

The group of enzymes that are collectively known as

polyphenol oxidase (PPO) have been associated pre-

dominately with the detrimental effect of browning

fruit and vegetables (Mayer, 2006). However, as Jones

et al. (1995) showed the potential of a browning extract

of red clover (Trifolium pratense) to inhibit proteolysis,

interest has been growing in its potential to improve

animal forage quality through greater N utilization. The

exact role of PPO in the plant is currently not

completely understood, although it has been associated

with plant defence against pathogens (Thipyapong and

Steffens, 1997), the biosynthesis of floral pigments

(Nakayama et al., 2001) or detoxifying oxygen species

in the chloroplast (Sherman et al., 1995). Polyphenol

oxidase catalyses the conversion of phenols to quinon-

es, which are extremely reactive and bind with cellular

nucleophiles such as proteins, to form protein-bound

phenol (PBP).

Following on from the findings of Jones et al. (1995),

PPO, through its formation of PBP in red clover, has

Correspondence to: M. R. F. Lee, Institute of Biological and

Environmental Research, Aberystwyth University,

Gogerddan Campus, Aberystwyth, UK.

E-mail: [email protected]

Received 24 June 2010; revised 23 November 2010

� 2011 Blackwell Publishing Ltd. Grass and Forage Science, 66, 196–205 doi: 10.1111/j.1365-2494.2010.00775.x196

Grass and Forage Science The Journal of the British Grassland Society The Official Journal of the European Grassland Federation

been shown to reduce both proteolysis and lipolysis in

silo (Albrecht and Muck, 1991; Lee et al., 2008) and the

rumen (Albrecht and Brodercik, 1992; Lee et al., 2007).

Lee et al. (2006) screened six grass species for PPO

activity and noted comparable activity in cocksfoot

(Dactylis glomerata) relative to red clover. They also

showed in vitro that grass PPO resulted in a reduction in

plant-mediated proteolysis and lipolysis similar to that

previously observed in red clover (Lee et al., 2004).

However, it is yet to be determined whether grass PPO

has any effect on proteolysis and lipolysis in the

presence of rumen micro-organisms.

Polyphenol oxidase activity has been associated with

extent of cell damage in red clover (Lee et al., 2009; Van

Ranst et al., 2010a), which is attributed to two processes:

i) loss of subcellular compartmentation resulting in the

mixing of enzyme (PPO, which resides in the chloroplast)

with substrate (diphenol, which predominately reside in

the vacuoles); ii) activation of the latent enzyme into the

active enzyme. Unlike red clover, where in fresh living

tissue PPO predominately resides in a latent form (ca.

80%), which during biotic or abiotic stress is activated to

the active form (Van Ranst et al., Submitted), grass PPO is

thought to only exist as an active form (Winters et al.,

2003). This in theory should increase the potential

activity of grass PPO to produce reactive quinones with

the subsequent formation of PBP, as it bypasses the need

for latent PPO conversion.

This study investigated rumen-simulated metabolism

of two grasses: cocksfoot (high PPO) and tall fescue

(Festuca arundinacea; low PPO) presented as freshly cut

or wilted for 24 h in order to determine the potential of

grass PPO and whether wilting, through extending the

time for the formation of PBP, will further increase

PPO’s potential to reduce proteolysis and lipolysis.

Materials and methods

Experimental design

The experiment consisted of a single period where two

eight-vessel RUSITEC apparatus (Czerkawski and

Breckeridge, 1977) were used to simulate a rumen

environment in vitro. There were four treatment com-

binations: cocksfoot wilted (Cw); cocksfoot fresh (Cf);

tall fescue wilted (TFw) and tall fescue fresh (TFf). The

combinations were split to assess two treatments: A)

wilted vs. fresh; B) tall fescue vs. cocksfoot.

Forages

Cocksfoot (cv. Abertop) and tall fescue (cv. Cochine)

were each sown in an area of 75 m2 on the 13th August

2007 at Bow Street, Aberystwyth, UK. A first cut was

taken on 9th June 2008 and Nitram (ICI Nitram,

Billingham, Teeside, UK) applied at a rate of 72 kg ha)1

(25 kg N ha)1) on the 16th June 2008. The plots were

split into three sub-plots, which were staggered to

provide 3-week re-growth periods for days: 1–4; 5–8

and 9–12, by taking a second cut on 14th, 18th and

22nd July 2008, respectively for each sub-plot. Fertil-

izer (25 kg N ha)1) was applied as before to each

subplot when it was cut.

The two wilted treatments (Cw and TFw) were cut [ca.

500 g fresh weight, (FW)] at 08:00 h and left at room

temperature in the laboratory for 24 h. A subsample

(ca. 50 g) was taken of each grass to determine dry

matter (DM) at 100�C for 24 h. The two fresh treat-

ments (Cf and TFf) were cut (ca. 500 g FW) the

following day at 08:00 h and collected on ice. All

grasses were cut 5 cm above soil level. Once transferred

to the laboratory, both fresh and wilted treatments were

passed through a garden shredder (Bioline 1000; Atika,

Ahlen, Germany) before being returned to ice. Each of

the four treatments were then weighed into four

Dacron bags to give 16 bags in total per d with ca.

10 g DM per bag, which was determined using the

previous day’s DM. A sample of each treatment ca.

100 g FW was collected for chemical analysis freeze-

dried, ground and maintained at )20�C. A further

sample collected in liquid nitrogen was stored at )80�Cprior to PPO and PBP analysis.

Rumen inoculum and artificial saliva

Four ruminally fistulated dairy cows (Bos p. taurus)

maintained on permanent pasture (predominately

perennial ryegrass; Lolium perenne) each provided 2 L

of hand-squeezed rumen liquor along with 400 g of

digesta solids. The samples from each cow were com-

bined and thoroughly mixed to provide a single 8 L

sample of rumen liquor and 1Æ6 kg of digesta solids.

These were transferred back to the laboratory in a

temperature-regulated container and then transferred

to a water bath at 39�C with continual CO2 purging

until the RUSITEC was ready to receive them.

Artificial saliva was made up by dissolving 3Æ6 g

anhydrous Na2HPO4 and 9Æ3 g Na2HCO3 per liter of

double distilled (dd) H2O. To this, 10 mL L)1 of salt mix

was added, which contained 47 g NaCl, 57 g KCl, 4 g

CaCl2 and 6 g MgCl2 dissolved in 1 L of dd H2O

(Mcdougall, 1948).

RUSITEC system

The RUSITEC consisted of eight air-tight 900-mL vessels

immersed in a water bath maintained at 39�C. Two

RUSITECs were used in the study to give 16 vessels in

total. Each vessel was charged with 500 mL of strained

rumen liquor, 200 mL of artificial saliva, one Dacron

RUSITEC metabolism of PPO grasses 197

� 2011 Blackwell Publishing Ltd. Grass and Forage Science, 66, 196–205

bag (pore size ca. 40 lm) containing 10 g DM of rumen

digesta solids and another containing the forage of

interest (10 g DM per bag), topped up with artificial

saliva and sealed. The motor-driven arm moved the

bags up and down through the liquor continuously.

Artificial saliva was infused by peristaltic pump (202U;

Watson-Marlow Ltd., Falmouth, Cornwall, UK) at a

rate of 0Æ5 mL min)1. The displaced effluent and

fermentation gases were collected in the effluent bottle

and gas collection bag respectively. After 24 h, each

vessel was opened and the rumen digesta solids

removed, squeezed and washed in artificial saliva. The

liquid fraction and washings were returned to the vessel

and a new feed bag inserted containing ca. 10 g DM of

the forage of interest. On subsequent days, the feed bag

that had been in the vessel for 48 h was replaced in a

similar way to the rumen digesta solids.

Sampling

The experiment ran for 12 d with sampling of liquor for

rumen parameters on days 10 and 11 at 0, 1, 2, 3, 4, 6, 8

and 24 h intervals from the addition of the new feed

bag. The pH was measured using a HI 8014 portable

pH metre (Hanna Instruments, Leighton Buzzard,

Bedfordshire, UK), 1 mL was taken for NH3-N analysis

and acidified with 100 lL of 2 MM HCl, and 1 mL for

volatile fatty acids (VFA) analysis and acidified with

100 lL of 15 MM orthophosphoric acid; these were then

stored ()20�C) prior to analysis. At the end of the

experiment (day 12), the grass residues and effluent

from the vessels were collected. The residues were

weighed to determine DM disappearance, freeze dried

and ground prior to analysis. The effluent volume was

measured and 10% subsampled, frozen and freeze dried

for fatty acid analysis. On days 9–12, gas was collected

using a plastic gas collection bag attached to the effluent

bottle. Following collection, gas volume was measured

directly using a dry test gas metre (Dry Test Gas Meter;

Shinagawa Corporation, Inagi, Japan), and methane ⁄carbon dioxide concentration determined by Infrared

gas analysis (5000 series gas analyser; Analytical

Development Co. Ltd., Hoddesdon, Hertfordshire, UK).

Chemical analysis and PPO assay

Water-soluble carbohydrate (WSC) was determined

spectrophotometrically using anthrone in sulphuric

acid on a Technicon Autoanalyser (Technicon

Corporation, New York, USA, Thomas, 1977). Ash

and by mass difference organic matter (OM) were

analysed by combusting the ground samples at 550�Cfor 6 h in a muffle furnace. Volatile fatty acids in the

effluent liquor were determined by gas chromatography

using Chrompack CP 9002 (CP-Sil 5CB column

10 m · 0Æ25 mm ID; Chrompack, London, UK) follow-

ing the method of Zhu et al. (1996). NH3-N was assessed

enzymatically using glutamate dehydrogenase on a

discrete analyser (FP-901M Chemistry Analyzer;

Labsystems Oy, Helsinki, Finland; Test kit No. 66-50;

Sigma-Aldrich Co. Ltd., Poole, Dorset, UK). Total N was

determined by micro-Kjeldahl technique using ‘Kjeltec’

equipment (Perstorp Analytical Ltd., Maidenhead,

Berkshire, UK). Neutral detergent fibre (NDF) was

determined as described by Van Soest et al. (1991) and

acid detergent fibre (ADF) was analysed according to

the method of Van Soest and Wine (1967) using the

Tecator Fibretec System equipment (Tecator Ltd.,

Thornbury, Somerset, UK). Lipid fractionation and fatty

acid analyses were carried out as described by Lee et al.

(2010) using thin layer and gas liquid chromatography.

For the PPO activity assay, plant tissue was extracted

according to the method of Winters and Minchin

(2001) and assayed according to the method of Robert

et al. (1995). In brief, material (ca. 0Æ5 g FW) was

extracted at 4�C in 2 mL of McIlvaine buffer (pH 7)

containing 0Æ1 MM ascorbic acid to inhibit PPO activity.

Extracts were centrifuged at 15 000 g for 10 min at 4�Cand the supernatant retained. Supernatants were

desalted by applying to columns (1Æ5 · 6 cm) containing

bio-Gel P6DG (Bio-Rad, Hertfordshire, UK) prepared in

McIlvaine buffer (pH 7) and centrifuging at 2500 g for

6 min at 4�C. Polyphenol oxidase activity was deter-

mined spectrophotometrically at 420 nm using 10 lL of

eluted fraction with 15 lL 0Æ001 mMM copper sulphate,

10 mMM methylcatechol and 1Æ5 mL of McIlvaine buffer.

Enzyme reaction rate was defined as the amount of

enzyme per g DM that produced 1 lmol of quinone per

second (lkatal) based on the absorption at k 420 nm of

a known concentration of quinones formed through the

reaction of methylcatechol and sodium periodate (Alder

and Magnusson, 1959) giving a conversion factor of:

lkatal = (0Æ0453 · D optical density). Protein-bound

phenol analyses were carried out using a modified

Lowry procedure described by Winters and Minchin

(2005), which takes into account the variable response

of diphenols with the Lowry assay and quantifies the

protein bound to phenol.

Statistical analysis

Chemical compositions of the grasses, residues and gas

emission were analysed using a general analysis of

variance with genotype (C vs. TF) · condition (fresh vs.

wilted) as the treatment effect and blocked according

d + RUSITEC for residues and gas emission. Lipolysis

was calculated as the proportional difference in

glycerol-based membrane lipid between the 0 h cut

and wilted grass samples and the 24 h ⁄ 48 h incubated

residues and statistically analysed as for chemical

198 M. R. F. Lee et al.


composition. Rumen parameters were analysed using a

repeated measures analysis of variance with genotype

(C vs. TF) · condition (fresh vs. wilted) · time (0, 1, 2,

3, 4, 6, 8, 24 h) and blocked according to RUSITEC,

with mean 24 h values reported in the table. All

statistical operations were performed with Genstat

Release 11.1 (PC ⁄ Windows, VSN International, Hemel

Hempstead, UK, 2008).

Results

Chemical composition of the forage treatmentsand residues

Cocksfoot had a higher proportion of ADF, NDF, N

(trend at P < 0Æ1), PBP and PPO activity and a lower

WSC and diacylglycerol content than tall fescue

(Table 1). Wilting reduced WSC and glycerol-based

membrane lipid and increased ADF, NDF, triacylglycerol

and free fatty acids, with a trend to also increase PBP

and diacylglycerol compared with fresh forage for both

cocksfoot and tall fescue. There was an interaction

between genotype and condition for DM, OM and PPO

activity. There was no difference in DM or OM between

genotypes when fresh but were higher in tall fescue

when wilted. Wilting significantly reduced PPO activity

in cocksfoot but had no effect on tall fescue (Table 1).

Following incubation for 24 h in the RUSITEC, there

was a trend (P < 0Æ1) for glycerol-based membrane lipid

and triacylglycerol to be higher and for free fatty acids

to be lower in cocksfoot compared with tall fescue.

Digestibility of WSC was higher (P < 0Æ05) in tall fescue

than cocksfoot. Wilting resulted in a lower (P < 0Æ001)

WSC digestibility and also tended (P < 0Æ1) to reduce

DM and OM digestibility than when offered as fresh.

There was no difference in NDF, ADF or N digestibility,

and diacylglycerol proportions across any of the

treatments at 24 h (Table 2).

Following incubation for 48 h in the RUSITEC,

cocksfoot exhibited a higher (P < 0Æ05) digestibility of

NDF than tall fescue. Wilting as opposed to fresh

resulted in a lower (P < 0Æ05) digestibility of N and WSC

with a trend (P < 0Æ1) for lower DM and OM digest-

ibility. There was no difference in ADF digestibility,

glycerol-based membrane lipid, diacylglycerol, triacyl-

glycerol and free fatty acids proportions across any of

the treatments at 48 h (Table 3).

Rumen parameters, gas emissions and lipolysis

Cocksfoot resulted in a higher concentration of

n-butyrate, n-valerate and a lower i-valerate, NH3-N

concentration with a trend (P < 0Æ1) for a lower 24 h

lipolysis than tall fescue. Condition had a greater effect

than genotype, with fresh grass resulting in lower

rumen pH, NH3-N, acetate, i-butyrate, i-valerate,

n-valerate and propionate, and a higher n-butyrate,

methane, carbon dioxide and 24 h lipolysis than wilted.

There was an interaction between genotype and con-

dition for the glucogenic:lipogenic VFA ratio [propio-

nate ⁄ (acetate + butyrate)] with fresh cocksfoot having

a lower ratio than wilted and no effect of condition in

tall fescue. There was no effect of treatment on total

VFA concentration or 48 h lipolysis (Table 4).

Diurnal rumen pH, NH3-N and total VFA in the

RUSITEC are shown in Figure 1. The Cf treatment

resulted in the lowest pH 8 h after feeding and also the

lowest NH3-N 2 h after feeding, with NH3-N for TFw

Table 1 Chemical composition (g kg)1 DM, unless stated), polyphenol oxidase activity and lipid profile of the four grass treatments

prior to incubation in the RUSITEC.

Cocksfoot Tall fescue

s.e.d.

P

Fresh Wilted Fresh Wilted G F ⁄ W Int.

Dry matter (g kg)1) 12Æ9 26Æ6 18Æ8 37Æ6 4Æ30 *** *** ***

Organic matter 898 892 898 896 1Æ0 * *** *

Acid detergent fibre 305 327 283 291 9Æ0 *** * NS

Neutral detergent fibre 586 611 534 569 5Æ6 *** *** NS

Nitrogen 28Æ1 28Æ6 26Æ2 26Æ0 1Æ44 † NS NS

Water soluble carbohydrate 55Æ4 36Æ1 97Æ6 84Æ8 4Æ74 *** *** NS

Polyphenol oxidase (lkatal) 15Æ7 8Æ27 1Æ26 1Æ57 1Æ738 *** ** **

Protein-bound phenol 1Æ81 2Æ11 0Æ21 0Æ52 0Æ287 *** † NS

Glycerol-based membrane lipid (g g)1 lipid) 0Æ89 0Æ78 0Æ88 0Æ79 0Æ027 NS *** NS

Diacylglycerol (g g)1 lipid) 0Æ05 0Æ07 0Æ07 0Æ12 0Æ025 † † NS

Triacylglycerol (g g)1 lipid) 0Æ02 0Æ04 0Æ02 0Æ04 0Æ008 NS ** NS

Free fatty acids (g g)1 lipid) 0Æ04 0Æ11 0Æ04 0Æ06 0Æ021 † * NS

G, Grass effect (cocksfoot vs. tall fescue); F ⁄ W, Condition effect (fresh vs. wilted); Int., Interaction (grass * condition).†P < 0Æ1; *P < 0Æ05; **P < 0Æ01; ***P < 0Æ001; NS, P > 0Æ1.



highest across the day compared with all treatments.

There were no differences in diurnal total VFA concen-

tration for any of the treatments.

Discussion

Chemical composition and influence of wilting

Fibre, N and OM compositions of the fresh grasses are

comparable to previous studies with cocksfoot (Davies

and Morgan, 1982a; Sanada et al., 2007) and tall fescue

(Davies and Morgan, 1982a; Smith et al., 1987; Hun-

tington et al., 2009). However, in the present study the

levels of WSC were lower in both forages than values

reported previously (Davies and Morgan, 1982a; Smith

et al., 1987). However, Sanada et al. (2007) for cocks-

foot and Davies and Morgan (1982a) for both cocksfoot

and tall fescue report a large annual variation in WSC

ranging between 39–133 and 85–152 g kg)1 DM for

cocksfoot and tall fescue, respectively, which are in the

range reported in the current study. The effect of

wilting on chemical composition in the present study

Table 2 Apparent digestibility (%) and lipid composition (g g)1 lipid) of the four grass treatments 24 h after incubation in the

RUSITEC


s.e.d.

P


Digestibility

Dry matter 55Æ5 42Æ4 44Æ6 35Æ1 9Æ12 NS † NS

Organic matter 59Æ5 44Æ6 47Æ9 36Æ9 3Æ41 NS † NS

Acid detergent fibre 24Æ6 36Æ3 31Æ7 24Æ1 7Æ99 NS NS NS

Neutral detergent fibre 38Æ9 40Æ9 37Æ2 29Æ6 6Æ69 NS NS NS

Nitrogen 50Æ9 41Æ8 41Æ9 35Æ2 9Æ47 NS NS NS

Water soluble carbohydrate 91Æ8 83Æ8 92Æ9 88Æ2 1Æ73 * *** NS

Lipid composition

Glycerol-based membrane lipid 0Æ15 0Æ15 0Æ12 0Æ13 0Æ019 † NS NS

Diacylglycerol 0Æ05 0Æ07 0Æ04 0Æ06 0Æ021 NS NS NS

Triacylglycerol 0Æ03 0Æ04 0Æ02 0Æ02 0Æ007 † NS NS

Free fatty acids 0Æ78 0Æ75 0Æ82 0Æ79 0Æ041 † NS NS

G, Grass effect (cocksfoot vs. tall fescue); F ⁄ W, Condition effect (fresh vs. wilted); Int., Interaction (grass * condition).†P < 0Æ1; *P < 0Æ05; ***P < 0Æ001; NS, P > 0Æ1.

Table 3 Apparent digestibility (%) and lipid composition (g g)1 lipid) of the four grass treatments 48 h after incubation in the

RUSITEC


s.e.d.

P


Digestibility

Dry matter 66Æ1 55Æ3 58Æ7 43Æ4 10Æ01 NS † NS

Organic matter 71Æ3 59Æ0 63Æ5 47Æ0 11Æ34 NS † NS

Acid detergent fibre 53Æ2 46Æ9 46Æ6 36Æ7 8Æ80 NS NS NS

Neutral detergent fibre 67Æ0 58Æ1 56Æ5 47Æ7 12Æ59 * NS NS

Nitrogen 59Æ1 47Æ1 59Æ7 40Æ8 9Æ01 NS * NS

Water soluble carbohydrate 90Æ9 87Æ4 94Æ1 92Æ5 1Æ51 ** * NS

Lipid composition

Glycerol-based membrane lipid 0Æ10 0Æ09 0Æ11 0Æ11 0Æ030 NS NS NS

Diacylglycerol 0Æ03 0Æ05 0Æ05 0Æ08 0Æ024 NS NS NS

Triacylglycerol 0Æ03 0Æ03 0Æ02 0Æ03 0Æ011 NS NS NS

Free fatty acids 0Æ84 0Æ83 0Æ82 0Æ79 0Æ052 NS NS NS

G, Grass effect (cocksfoot vs. tall fescue); F ⁄ W, Condition effect (fresh vs. wilted); Int., Interaction (grass * condition).†P < 0Æ1; *P < 0Æ05; **P < 0Æ01; NS, P > 0Æ1.



are as reported by Michalet-Doreau and Ould-Bah

(1992) in cocksfoot and Archibeque et al. (2002) for tall

fescue, characterized by a rise in fibre and drop in

digestibility. Water-soluble carbohydrate is an extre-

mely labile chemical constituent, which is lost during

drying (Kerepesi et al., 1996) and was subsequently

lower in the wilted material than fresh. The greater loss

of WSC in cocksfoot (35%) as opposed to tall fescue

(13%) is probably related to the higher proportion of

glucose and fructose typically found in cocksfoot (ca.

30%; Volaire and Gandoin, 1996) compared with that

typically found in tall fescue (ca. 19%; Mayland et al.,

2000). These mono- and disaccharides are readily used

during cellular respiration and are most volatile during

desiccation (Kerepesi et al., 1996).

The present study is consistent with previous work

(Lee et al., 2006; Marita et al., 2010) investigating PPO

in grasses, in which PPO is significantly higher in

cocksfoot than tall fescue and subsequently resulted in

higher levels of PBP. However, the size of the difference

in PPO activity between studies is variable. Lee et al.

(2006) reported PPO activity in cocksfoot to be ca. 113-

fold higher than tall fescue when using methylcatechol

as substrate; however, in the current study there was

only a 12Æ5-fold difference between the treatments

when using the same substrate. However, Marita et al.

(2010) reported a ca. 21-fold difference in greenhouse

Table 4 Rumen parameters (mean over 24 h), methane and carbon dioxide emission and lipolysis from the four grass treatments

during incubation in RUSITEC


s.e.d.

P


pH 6Æ96 7Æ01 7Æ00 7Æ01 0Æ018 NS * NS

NH3-N (lg ml)1) 77Æ6 82Æ3 82Æ9 94Æ1 2Æ14 *** *** *

Fermentation acids (mMM)

Acetate 19Æ1 21Æ0 19Æ7 20Æ8 0Æ96 NS * NS

i-Butyrate 0Æ45 0Æ46 0Æ43 0Æ48 0Æ020 NS * NS

n-Butryate 5Æ30 4Æ63 4Æ67 4Æ23 0Æ194 *** *** NS

i-Valerate 0Æ81 0Æ88 0Æ97 1Æ01 0Æ035 *** * NS

n-Valerate 1Æ54 1Æ63 1Æ45 1Æ57 0Æ057 * ** NS

Propionate 8Æ88 10Æ1 9Æ53 9Æ76 0Æ475 NS * NS

P ⁄ (A+B) 0Æ36 0Æ39 0Æ39 0Æ39 0Æ004 *** *** ***

Total volatile fatty acids 36Æ1 38Æ6 36Æ8 37Æ9 1Æ69 NS NS NS

Gas emission (ml g)1 DMD)

Methane 13Æ2 9Æ20 13Æ7 10Æ9 2Æ287 NS * NS

Carbon dioxide 76Æ6 53Æ8 81Æ5 63Æ8 13Æ63 NS * NS

Lipolysis

24 h (%) 83Æ3 79Æ9 87Æ1 81Æ7 2Æ29 † * NS

48 h (%) 88Æ6 89Æ1 86Æ2 86Æ6 3Æ58 NS NS NS

G, Grass effect (cocksfoot vs. tall fescue); F ⁄ W, Condition effect (fresh vs. wilted); Int., Interaction (grass * condition); DMD, Dry

matter digested; GM, dry matter.†P < 0Æ1; *P < 0Æ05; **P < 0Æ01; ***P < 0Æ001; NS, P > 0Æ1.

6·85

6·9

6·95

7

7·05

7·1

7·15

pH

CfTFw

CwTFf

50

60

70

80

90

100

110

NH

3N (µ

g m

L–1)

CfTFw

CwTFf

20

25

30

35

40

45

50

Tota

l VFA

(mm

ol L

–1)

Time (h)Time (h)Time (h)

CfTFw

CwTFf

0 2 4 6 8 10 12 14 16 18 20 22 24 0 2 4 6 8 10 12 14 16 18 20 22 24 0 2 4 6 8 10 12 14 16 18 20 22 24

(a) (b) (c)

Figure 1 Hourly rumen parameters of the four grass treatments during RUSITEC incubation: (a) pH, (b) NH3-N, (c) Total volatile

fatty acids.



material, whereas only a 6Æ25-fold difference in field-

grown material when using catechol as substrate. This

suggests an environmental difference in PPO activity as

Lee et al. (2006) used material grown under controlled

conditions, whereas in the present study grass from

external plots was used. This effect of environment on

PPO activity in red clover has been discussed in the

review by Van Ranst et al. (2010b) and confirms the

high variability in PPO activity within species governed

by maturity, abiotic and biotic stress as a consequence

of disease and environmental conditions. The choice of

substrate is also an important variable when determin-

ing PPO activity. Marita et al. (2010) reported signifi-

cant differences in PPO activity in 10 grass genotypes

when using three different substrates: caffeic acid,

chlorogenic acid and catechol. Parveen et al. (2010)

investigated the oxidative phenol substrates of a range

of forage crops containing PPO. They reported the only

substrate for tall fescue PPO was 5-caffeoylquinic acid,

whereas cocksfoot had a whole range of hydroxycin-

namate substrates based on caffeic acid. This may

suggest that caffeic acid as a substrate would be more

suitable than catechol for both tall fescue and cocksfoot.

This is borne out for the greenhouse-grown tall fescue,

reported by Marita et al. (2010) but not for the field

material where catechol showed the highest activity,

whereas chlorogenic acid showed the highest activity

for cocksfoot in both greenhouse-and field-grown

material.

The effect of wilting in both grasses resulted in an

increase in PBP as a result of PPO activity (Winters

et al., 2008; Lee et al., 2009), although it did not reach

significance (P < 0Æ1). Polyphenol oxidase activity

during wilting was significantly reduced in cocksfoot,

whereas there was no significant change in tall fescue.

This response is related to the higher original activity

of PPO in cocksfoot as during wilting it has been

shown that as a result of PPOs formation of quinones

the PPO protein itself becomes bound, forming

PBP resulting in a negative feedback temporal deac-

tivation.

Changes in lipid fractions

The lipid composition of the fresh forages is as reported

for cocksfoot (Lee et al., 2006) with no difference

between species in the current study. Wilting in both

species resulted in a decline in glycerol-based

membrane lipid, a rise in triacylglycerol and free fatty

acids and a tendency to increase diacylglycerol. These

changes are indicative of the process of plant-mediated

lipolysis as reported by Lee et al. (2004), although in the

current study the higher activity of PPO in cocksfoot did

not result in a lower plant-mediated lipolysis during

wilting as previously reported.

After 24 h in the RUSITEC, there was no difference

between fresh and wilted material with a tendency for

glycerol-based membrane lipid and triacylglycerol to be

higher and free fatty acids to be lower between

cocksfoot and tall fescue, although no differences were

observed after 48 h incubation. These differences are

reflected in the 24 h lipolysis values, which tended

(P < 0Æ1) to be lower in cocksfoot after 24 h incubation.

Previously, Lee et al. (2007) showed that red clover PPO

resulted in a reduction in lipolysis in the presence of

rumen microbial organisms. This was shown to be

related to an association of the glycerol-based mem-

brane lipid with PBP complexes (Lee et al., 2010). This

mechanism could explain the protection of lipid in

the current study in the presence of rumen micro-

organisms, despite the lack of difference in plant-

mediated lipolysis, suggesting a complexing of the lipid

within a PBP matrix. The loss of difference after 48 h

may be associated with the breakdown of these PBP

complexes.

The lower level of lipolysis in the wilted material as

opposed to the fresh may have been related to the

greater proportion of PBP in the wilted material, but

equally may reflect the lower levels of glycerol-based

membrane lipid in the wilted material as a consequence

of plant-mediated lipolysis.

It is important to highlight that these differences in

lipolysis at 24 h are small and as reported by Cabiddu

et al. (2010) when correlating polyphenolic content

with degree of lipolysis in Mediterranean forages PPO

only plays a contributing role to regulating lipolysis in

forages and the size of the role varies between species

depending on PPO activity, lipase activity, substrate

availability and other contributing plant secondary

metabolites.

In vitro apparent digestibility and rumenparameters

Apparent DM and OM digestibilities were not signifi-

cantly different between species at both 24 and 48 h

incubation. Although Miles et al. (1964) reported a

greater DM digestibility of cocksfoot over tall fescue,

more extensive studies have shown that the digestibilities

of the two species over the course of the growing season

are comparable (Minson et al., 1964; Davies and Morgan,

1982a). There was a trend for greater apparent DM and

OM digestibility in both species in the fresh as opposed to

the wilted treatment, potentially governed by the loss in

labile compounds such as WSC during wilting. This was

further evident in the lower apparent WSC digestibility in

the wilted material of both species as opposed to the fresh

material at both 24 and 48 h. The greater apparent WSC

digestibility in tall fescue than cocksfoot may also be

associated with the greater loss of low molecular weight



fructan during wilting of cocksfoot, as discussed previ-

ously, as in fresh grass the difference between species was

lower than for the wilted material. Apparent fibre

digestibilities were comparable across species and

treatments after 24 h incubation, whereas after 48 h

apparent NDF digestibility was higher in cocksfoot than

tall fescue, as previously reported by Mason et al. (1989).

Greater N use efficiency in cattle on cocksfoot has

previously been reported (Tyrrell et al., 1992; Huntington

et al., 2009) compared with gamagrass (Tripacum dactylo-

ides) and alfalfa (Medicago sativa) respectively. Both

studies reported a slower N digestion in the rumen as a

consequence of higher insoluble N on the cocksfoot

treatment. Mason et al. (1989) reported a greater N

retention in sheep offered cocksfoot hay as opposed to tall

fescue hay, despite a greater N faecal output on the

former as a consequence of higher faecal-insoluble N.

These responses could be associated with PBP formation.

In the present study, no difference in apparent N

digestibility between cocksfoot and tall fescue was

observed, although apparent N digestibility was lower

in the wilted material than the fresh, possibly as a

consequence of reduced protein solubility. The low

apparent N digestibilities in batch-culture experiments

are due to solid-associated bacteria strongly adhering to

the fibrous residues, which was evident in the present

study and may have masked differences in true N

digestibility. However, despite the similar N digestibili-

ties, NH3-N (as measure of dietary N metabolism) was

lower on cocksfoot in fresh and wilted material than tall

fescue. This response was observed despite the higher N

input into the system with cocksfoot and the higher

readily available energy from tall fescue in the form of

WSC, which has been shown to increase incorporation of

dietary N into microbial N and reduce NH3-N (Lee et al.,

2003; Merry et al., 2006). This would indicate a slower

degradation of dietary protein in cocksfoot as previously

observed, potentially as a consequence of PBP formation.

The higher levels of NH3-N in both forages in the wilted as

opposed to the fresh material are probably related to the

degree of protein breakdown during wilting as a conse-

quence of plant-mediated proteolysis.

Unlike the findings of Jayanegara et al. (2009) who

reported a significant retarding effect of condensed

tannins on methane formation in batch culture, PPO

appeared to have little effect. The pattern of fermen-

tation acids for both species are similar to those

reported by Grimes et al. (1967) in lambs grazing tall

fescue or cocksfoot, with a lower P ⁄ (A+B) ratio on

fresh cocksfoot in both studies. However, neither grass

had an influence on methane or carbon dioxide

emissions, but wilting resulted in a significant reduc-

tion in both. Wolin et al. (1997) stoichiometrically

determined that methanogenesis would be reduced

when the proportion of propionate increased, due to

its nature as a hydrogen sink. This was observed in the

present study when both forages were wilted, but

no difference in propionate was observed between

species.

Conclusions

Wilting produced a comparable effect during in vitro

incubation in both high-and low-PPO-containing grass

species and so would appear to have little effect in

increasing the potential of grass PPO. Cocksfoot tended

to have a lower lipolysis at 24 h incubation and resulted

in a lower proportion of NH3-N being produced in

the RUSITEC system, which is indicative of greater N

use efficiency than tall fescue. These responses could be

partly attributed to the higher PPO content of cocksfoot,

although when comparing forage species numerous

other confounding factors could have contributed to

the observed responses such as lipase and protease

activities, PPO substrate availability and other plant

secondary metabolites not measured. In addition,

differences in the potential of cocksfoot for animal

production in different climatic conditions (Davies and

Morgan, 1982b; Young et al., 1994) further complicate

the potential role of PPO-containing grasses.

Acknowledgments

The authors thank and acknowledge the financial

support from the Department for the Environment

Food and Rural Affairs; English Beef and Lamb Exec-

utive; Quality Meat Scotland and Hybu Cig Cymru. The

authors also thank Delma Jones and her team in

analytical chemistry for the chemical composition data;

John Tweed for his skilled lipid analysis; and Naomi

Gordon and Martin Leyland for the care of the

fistulated animals.

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In vitro rumen simulated (RUSITEC) metabolism of freshly cut or wilted grasses with contrasting polyphenol oxidase activities