on RC silage. This study compared two RC silages which when ensiled had contrasting PPO activities (RC+ and RC−) against a control of

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chromium EDTA (2 797 ± 120.6 ppm) infusions commenced
(WSC) was determined spectrophotometrically (Thomas, in Tables 1 and 2. For experiment 1 DM, organic matter
Threonine and serine were corrected for loss (5% and 10%, silages were higher in the two RC silages than PRG, with
acids in silage were extracted and methylated as described reported in Supplementary Tables S1 to S3.
Table 1 Chemical composition, polyphenol oxidase (PPO) activity and
of true digesta to calculate ﬂow (correction factors (R) ranged Hydroxycinnamates 0.04 0.12 ND 0.382
Table 2 Chemical composition, polyphenol oxidase (PPO) activity and
dase red clover silage; PRG = perennial ryegrass silage; OM = organic matter; ammonia-N across the day is reported in Table 3 with a
higher on RC+ than RC− for both experiments. Total phe- (38.6% and 54.2%, respectively). Total N, non-microbial
(Table 3). Temporal rumen ammonia-N is shown in Figure 1 higher on PRG as opposed to the two RC silages, with no
Table 4 Intake and duodenal ﬂow of N and amino acids (AA) in steers OMTD = organic matter truly digested; NS = not signiﬁcant; EMPS = efﬁciency
OMTD (%) 55.4 55.8 51.5 2.43 NS Table 6. For cis isomers, cis-9 was the most dominant aver-
Total N 280b 291b 197a 15.3 *** aging 77.7% of all cis isomers, with PRG showing the highest
b diets contributing <1% of total cis isomers. trans-11 was the
(Table 7). Total N, total AA, aspartic acid and valine intake
MN/NI6 (%) 45.2a 46.7a 72.3b 6.49 ** For glutamic acid, glycine and alanine intake on RC− was
NMN/NI7 (%) 63.8 57.9 70.3 5.29 NS higher than PRG but there was no difference between RC+
Table 5 Intake and duodenal ﬂow (g/day, unless otherwise stated) of Table 6 Proportions (%) of C18:1 isomers in the duodenal digesta of
were comparable across the three silage diets (Table 7).
Means within row not bearing a common letter differ (P < 0.05). comparable for PRG and RC+ which showed a small N gain
Table 7 Intake and faecal output of N and amino acids (AA) in steers
OMAD (%) 65.3a 63.6a 76.4b 0.68 *** PPO = polyphenol oxidase; RC + = high PPO red clover silage; RC− = low
Means within row not bearing a common letter differ (P < 0.05). on a FW basis, if we assume a DM of 20% to compare with
NS = NS at P < 0.05; tr = trend P < 0.1. the current study, Winters et al. (2008) reported a range of 5
*P < 0.05, **P < 0.01 and ***P < 0.001. to 18.8 g/kg DM compared to the present range across the
Flythe et al., 2013) and although not a substrate for PPO may reﬂect the low N content of the grass (ca. 10% CP). The
In the current study, all silages were limited to 14 to 15 and (2010) which predicted values of 42% and 74%, respectively,
which should equate to a range of intakes between 8.20 to Higher total N duodenal ﬂows with RC silage than PRG
the range and were not different to each other, and whilst RC 2009; Lee et al., 2014), and is related to the greater N con-
Rumen pH mean values relate to values reported on previously reported (Dewhurst et al., 2003; Merry et al.,
and RC silage (64.1). Apparent total AA digestibility was also 66%, these values compare with 180, 214 and 168 g/kg DM for
comparable to previously reported values of 55% and 67%, RC+, RC− and PRG in the current study and highlights the
mean of 32% and 38% for Moorby et al. (2009) and Lee et al. within protein complexes as a consequence of PPO activity,
was also comparable in the previous studies averaging 40% ecology. The current study suggests that PPO has little effect
and 43% (both studies were on lactating animals, hence the on biohydrogenation as RC+ failed to show any protection of

Methods (brief)

2011). However, reduced activity of plant lipases would have opening in April 2011. Samples of RC were taken as cut for
potential mechanisms for protecting PUFA were assessed as Sample collection methods – experiment 1
either-or combination of entrapment within PBP reducing A representative silage sample of each forage per period was
access to microbial lipases or differences in rumen digestion collected by sub-sampling (ca. 200 g FW) daily and bulking
increase NUE (N retention) and reduce PUFA biohydrogena- ground samples (250 ml) were collected and a whole and a
tion compared to a RC with lower PPO activity. centrifuged digesta sample prepared, freeze-dried and ana-
Experimental design – experiment 1 – rumen nitrogen each collection) was collected on days 19 and 20, manually
A similar design was previously reported by Lee et al. (2014) and a whole and centrifuged sample prepared as described
in brief: six Hereford × Friesian steers (623 ± 33.6 kg), pre- above. Hourly rumen ﬂuid (10 ml) was collected on day 21
sub-sampled so that actual DM intake could be determined. Sample collection methods – experiment 2
second cut on 10 August 2010 wilted for 48-h and ensiled Subsamples of urine (1% of daily production) were stored at
conditioner and then spread and baled after a 24-h wilt with Faeces were weighed and a representative sub-sample taken
wrap 750 mm wide 25 µm thick and stored on farm until ﬁrst determination of DM. A further sub-sample (3% of daily
atomic absorption spectrophotometry (Williams et al., 1962). with PRG higher than the two RC silages. This was reﬂected
Purine and pyrimidine bases were determined by HPLC (Cozzi in the fermentation acids which were lower in PRG than the
et al., 1993). Amino acids (AAs) were determined by HPLC. two RC silages, which were comparable. Ammonia-N in the
in RC samples before baling was assessed using a modiﬁed
sample as the repeated measure. Biohydrogenation of C18 Propionate 0.53 0.31 0.09 0.206

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The five most recent substantive edits to this page, classified major (evidence or structure moved), correction (a published value or statement was wrong and has been fixed), or minor (narrative rewritten without changing the underlying evidence). Each description is derived from what the edit did to this page; the linked commit is the authoritative record, routine regeneration passes are excluded, and the full version history lives in git. When DOI minting comes online (see schema docs), each entry below will also link to a version-pinned DataCite DOI.

Commit	Date	Change	Description
b01ec52c	2026-08-04	major	2 sections added
d49e450f	2026-08-03	major	5 sections added; narrative text revised