Animal Feed Science and Technology 210 (2015) 37–45

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Animal Feed Science and Technology

journal homepage: www.elsevier.com/locate/anifeedsci

Digestibility, ruminal fermentation and duodenal flux of

amino acids in steers fed grass forage plus concentrate
containing increasing levels of Acacia mearnsii tannin extract
T. Orlandi, G.V. Kozloski ∗ , T.P. Alves, F.R. Mesquita, S.C. Ávila
Departamento de Zootecnia (Animal Science Department), Universidade Federal de Santa Maria, Santa Maria, RS 97105-900, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Four Holstein steers (156 ± 33 kg of body weight (BW)) fitted with duodenal cannula and
Received 3 April 2015 rumen catheter, were housed in metabolism pens and used in a 4 × 4 Latin square trial
Received in revised form to evaluate the effect of dietary inclusion of levels of Acacia mearnsii tannin extract on
16 September 2015
rumen fermentation, digestion and duodenal flux of amino acids. The diet was offered at
Accepted 18 September 2015
a restricted amount of 20 g dry matter (DM)/kg BW and consisted of oat (Avena strigosa)
plus concentrate, in a proportion of 0.55:0.45 (DM basis) respectively. Treatments were
Keywords:
no tannin (Control) or inclusion of tannin extract in the concentrate at a rate of 20, 40 or
Avena strigosa
60 g/kg DM (i.e. 9, 18 or 27 g/kg of total dietary DM). The apparent total-tract organic matter
Amino acids
Cattle (OM) digestibility was not affected whereas the neutral detergent fiber (aNDF) digestibility
Digestibility and the OM true total-tract digestibility tended to linearly decrease (P < 0.10) at increased
Ruminal levels of tannin extract inclusion. Tannin extract linearly reduced (P < 0.05) the total-tract
Fermentation digestibility of N compounds, as well as the urinary N excretion, and linearly increased
Tannins (P < 0.05) the fecal N excretion, N retention and the efficiency of N utilization by steers. The
ruminal pH was similar for all treatments whereas the concentration of ammonia N and
reducing sugars in ruminal fluid linearly decreased (P < 0.05) with tannin extract inclusion.
Linear positive responses (P < 0.05) to tannin treatments were observed for duodenal flux
of total N, ␣-amino N and non-ammonia non-microbial N. The microbial N supply tended
to be negatively affected (P < 0.10) whereas both the ruminal OM digestibility and ruminal
degradability of feed N compounds linearly decreased (P < 0.05) with increasing tannin
extract levels. The efficiency of ruminal microbial protein synthesis tended to increase
quadratically (P < 0.10) at increased levels of tannin extract. The dietary inclusion of tannin
extract positively impacted (P < 0.05) or tended to impact (P < 0.10) the duodenal flux of
all amino acid groups, as well as of most individual amino acids. The amino acid profile in
duodenal digesta was more closely related to the profile in feed at increased tannin extract
inclusion. In conclusion, dietary inclusion of Acacia mearnsii tannin extract up to a level
of 18 g/kg DM decreased the urinary N excretion and improved the amino acid supply in
steers fed fresh-frozen oat forage plus concentrate without significantly affecting the total
OM digestibility.
© 2015 Elsevier B.V. All rights reserved.

Abbreviations: ADF, acid detergent fiber; BW, body weight; DM, dry matter; EMPS, efficiency of microbial protein synthesis; EE, ether extract; EAA,
essential amino acids; GAA, glucogenic amino acids; INDF, indigestible NDF; NAN, non-ammonia N; aNDF, neutral detergent fiber; NANMN, non-ammonia
non-microbial N; NEAA, non-essential amino acids; NFC, non-fiber carbohydrate; NDIN, neutral detergent insoluble N; OM, organic matter; PD, purine
derivatives; RDP, ruminal degradability of feed N compounds.
∗ Corresponding author.
E-mail address: gilberto.kozloski@ufsm.br (G.V. Kozloski).

http://dx.doi.org/10.1016/j.anifeedsci.2015.09.012
0377-8401/© 2015 Elsevier B.V. All rights reserved.
38 T. Orlandi et al. / Animal Feed Science and Technology 210 (2015) 37–45

1. Introduction

Animal performance depends, among other factors, on the metabolizable energy and protein supply. Metabolizable
protein is defined as the amino acids absorbed from the small intestine, which originate from rumen microbial, undegraded
feed and endogenous true protein (NRC, 2001). When there is no lack of N for rumen bacteria, the supply of rumen microbial
protein is directly related to the amount of organic matter (OM) fermented in the rumen (Van Soest, 1994). In turn, when
N availability is above rumen bacteria requirements, the excess is absorbed as ammonia, metabolized to urea in liver and
partially excreted in urine. Ingestion of excessive amounts of rumen degradable protein increases urinary N losses, which
in turn negatively impacts the environment, decreases the efficiency of feed N use and, may limit the metabolizable protein
supply and performance of high productive animals (Rossi et al., 2007). This undesired nutritional condition is common in
animals fed N-rich high digestible forages and/or concentrate containing oil-seed meals as protein sources, where ruminal
degradability is usually higher than 0.65 (NRC, 2001). To reduce the ruminal degradability of these protein sources without
affecting its intestinal digestibility is among the current research challenges in ruminant nutrition.
Tannins are plant polyphenols with the capacity to form complexes mainly with proteins reducing their degradation in
the rumen (Waghorn et al., 1987). However, the chemical structure, concentration and the biological effects of tannins in
forage legumes, and consequently their nutritional impact, show large variability (McSweeney et al., 2001). Alternatively,
industrial tannin extracts, as from Acacia mearnsii, show the potential to be used as a feed additive for ruminants. Carulla
et al. (2005) observed that supplementing this extract at a rate of 41 g/kg of diet dry matter (DM) to lambs fed ryegrass
increased forage intake but depressed the OM digestibility. However, because methane emission and urinary N excretion
also decreased, energy and N retention were not affected. Grainger et al. (2009) reported that offering this extract at a
rate of 19 g/kg DM also reduced methane emissions and urinary N excretion, however, tannins also reduced feed intake,
OM digestibility and milk production by dairy cows grazing a ryegrass pasture. Griffiths et al. (2013), in turn, reported the
positive potential of the tannin extract in reducing urinary N losses without affecting milk production in dairy cows grazing
high-N ryegrass pastures when supplemented, as an oral drench, with no more than 185 g/day of a black wattle powder
containing 601 g/kg of condensed tannins.
All of the above studies, however, were conducted with animals fed forage based diets without or with small amounts
of supplementary concentrate feedstuffs. Tannins reduce not only protein degradation but also carbohydrate degradation
by rumen bacteria (Frutos et al., 2004). Most OM in forages is comprised of fibrous carbohydrates, with limited digestibility
in the lower gastrointestinal tract, thus the total OM digestibility is usually decreased by tannins in ruminants fed forage
based diets (Kozloski et al., 2012). In turn, assuming that most starch and true protein that escapes ruminal degradation
and flows to the duodenum is digested in the lower gastrointestinal tract, it was hypothesized that there is a level of tannin
extract inclusion in diets containing medium to high levels of starch and true protein sources, offered to cattle, where the
metabolizable protein supply is improved without or with a minor impact on the total OM digestibility. Moreover, none of
previous studies tested the effect of the tannin extract on duodenal flux of individual amino acids.
The present study was conducted to evaluate the impact of increased dietary inclusion of tannin extract from Acacia
mearnsii on digestibility, ruminal fermentation, rumen microbial protein synthesis, N utilization and duodenal flux of indi-
vidual amino acids in steers fed a temperate grass forage plus concentrate containing soybean meal as the main protein
source.

2. Materials and methods

2.1. Feedstuffs, animals, housing and experimental design

Research protocols followed the guidelines recommended by the Animal Care and Ethical Committee of the Universidade
Federal de Santa Maria. Four Holstein steers (156 ± 33 kg of body weight (BW)) housed in metabolism pens were used in a
4 × 4 Latin square experiment. The steers were fitted with a chronic rumen catheter (siliconized PVC, 35 cm length, 10 mm
o.d. × 7 mm i.d.) and a duodenal silicone rubber T-type cannula, and the experiment started after approximately 30 days after
surgeries. To minimize the effects of feed intake level on digestion processes, the experimental diet was offered at restricted
amount of 20 g dry matter (DM)/kg BW and consisted of vegetative oat (Avena strigosa) plus concentrate, in a proportion of
0.55:0.45 (DM basis) respectively. To ensure that the same forage type was offered throughout the experimental periods,
all forage was harvested from a pasture fertilized with urea at a rate of 100 kg/ha, when the grass sward reached a height
of approximately 30 cm. It was then cut 10 cm above the ground level and immediately stored in a cooling chamber at
−20 ◦ C until offered in each experimental period. The concentrate was a mixture of soybean meal (0.30), cracked corn grain
(0.35) and rice bran (0.35). The chemical composition of forage and concentrate is shown in Table 1. With the exception of
the cracked corn grain, all other dietary components were reported to have protein with rumen degradability above 0.60
(Valadares Filho et al., 2010). Diets were formulated to contain high levels of crude protein, above steer requirement, as to
simulate the diet usually offered for dairy cows grazing temperate grass pastures and given concentrate supplementation in
Southern of Brazil. Treatments were zero tannin (Control) or inclusion of Acacia mearnsii tannin extract (Weibull Black, Tanac
S. A., Montenegro, RS, Brazil) in the concentrate at a rate of 20, 40 or 60 g/kg DM (i.e. 9, 18 or 27 g/kg of total dietary DM).
The extract was the same previously used by Kozloski et al. (2012) and contained 694 g/kg DM of total tannins, which was
analyzed using the Folin-Ciocalteu method after aqueous acetone (70%, v/v) extraction following the procedures of Makkar
 T. Orlandi et al. / Animal Feed Science and Technology 210 (2015) 37–45 39

Table 1
Chemical composition of dietary components.

Components Avena strigosa Concentratea

Dry matter (g/kg) 112 863

Composition (g/kg dry matter)
Organic matter 875 913
Crude protein 175 200
Neutral detergent fiber 477 220
Acid detergent fiber 285 107
Lignin (sa) 18 50
Non-fiber carbohydratesb 216 464
Ether extract 49 26
a
Composed of soybean meal (0.30), rice bran (0.35) and cracked corn grain (0.35).
b
Organic matter − [(neutral detergent fiber − (neutral detergent insoluble N × 6.25)) + (total N × 6.25) + ether extract],
all expressed as g/kg dry matter.

(2000). Tanic acid was used as standard for this analysis. Using a Sephadex LH-20 purified tannin extract as standard for the
HCl-butanol analysis, the concentration of condensed tannins in this extract was reported to be approximately 610 g/kg DM
(Carulla et al., 2005; Grainger et al., 2009). Forage was offered separately from concentrate twice daily at 08.00 and 17.00 h.
The concentrate was offered three times daily at 08.00, 13.00 and 17.00 h. Steers had free access to water and to a mineral
salt containing (g/kg): Ca: 165, P: 73, Na: 117, Mg: 15, Mn: 1.5, Zn: 3.0, Fe: 2.0, Cu: 0.65, Co: 0.025, I: 0.04, Se: 0.01, F: 0.74.
Experimental periods lasted for 16 d, with 11 d adaptation and 5 d collection period. Feed offered was weighed and
sampled daily from day 12 to 16 of each experimental period. Total feed refusals and feces were taken daily throughout the
collection period and stored at −20 ◦ C. At the end of each experimental period they were weighed and a sample (100 g/kg of
wet weight from each steer) was collected. All samples were oven-dried at 55 ◦ C for at least 72 h and ground through a 1 mm
screen (Willey mill, Arthur H. Thomas, Philadelphia, PA) for subsequent chemical analysis. Urine was collected daily during
the collection period, in buckets containing 500 ml of 1.8 M H2 SO4 . The volume of urine was measured and a sample of 10 ml
was taken, diluted to 50 ml with distilled water and stored frozen (−20 ◦ C) until analysis. From day 12 to 15 of experimental
periods, duodenal digesta samples (200 ml) were collected at 2 h intervals over a 24 h period, composited within animal and
period and stored frozen (−20 ◦ C). For analysis, these samples were thawed, an aliquot of the supernatant (10 ml) was taken
for ammonia-N analysis and stored at −20 ◦ C, and the remaining was dried in a forced-air oven (55 ◦ C) and ground to pass
through a 1 mm screen. On day 16 of each period, rumen fluid samples (approximately 100 ml) were collected at 0, 2, 4, 6,
8, 10, 12, 18 and 24 h after the morning meal (i.e. 08.00 h). These samples were filtered through a 50 ␮m nylon filter, the
pH was immediately measured with a digital pH meter (Marte Ltda, MG, Brazil) and two aliquots (18 ml) of filtered fluid
were removed. In one aliquot, 2 ml of 1.8 M H2 SO4 and, in the other, 2 ml of 500 g/L trichloroacetic acid were added. Samples
were centrifuged (4000 × g for 20 min) at room temperature and the supernatants collected and stored frozen at −20 ◦ C. The
supernatant of trichloroacetic acid acidified samples were assumed to contain free amino acids and short chain peptides
(Greenberg and Shipe, 1979).

2.2. Chemical analysis

Samples of feed, refusals, feces, duodenal digesta and urine were pooled on a 5-day basis within each animal and exper-
imental period. Dry matter content was determined by oven drying at 105 ◦ C for at least 16 h. Ash was determined by
combusting at 600 ◦ C for 3 h and OM by mass difference. Total N was assayed by a Kjeldahl method (Method 984.13) of AOAC
(1997). The neutral detergent fiber (aNDF) analysis was based on the procedures described by Mertens (2002) without using
sodium sulphite and with use of heat-stable ␣-amylase, and the concentration of acid detergent fiber (ADF) was analyzed
according to Method 973.18 of AOAC (1997) except that the samples were weighed in polyester filter bags (porosity of
16 ␮m) and treated with neutral or acid detergent in an autoclave at 110 ◦ C for 40 min (Senger et al., 2008). For lignin (sa)
analysis the bags containing residual ADF were treated with 12 M H2 SO4 for 3 h (Method 973.18 of AOAC (1997)). Analysis of
neutral detergent insoluble N (NDIN) was performed according to Licitra et al. (1996). Ether extract (EE) concentration was
determined in a reflux system (Soxtherm 2000 S 306 M, Gerhardt; Königswinter, Germany) with ethyl ether at 180 ◦ C for
2 h. The content of non-fiber carbohydrate (NFC, g/kg) was calculated as: OM – [(aNDF − (NDIN × 6.25)) + (N × 6.25) + EE],
according to Van Soest et al. (1991). Total N in urine samples was assayed by the Kjeldahl method and allantoin and
uric acid concentrations were determined by colorimetric according to Chen and Gomes (1992). Uric acid was determined
using a commercial kit (Labtest, Lagoa Santa MG, Brazil). Total purine derivatives (PD) were the sum of allantoin and uric
acid.
Duodenal supernatant samples were analyzed for ammonia N using the phenol-hypochlorite method described by
Weatherburn (1967). Rumen fluid samples acidified with H2 SO4 were analyzed for ammonia N (Weatherburn, 1967) and
reducing sugars (Dubois et al., 1956). The trichloroacetic-acidified rumen fluid samples were treated with 6 M HCl (2 ml of
sample and 2 ml of 6 M HCl) at 110 ◦ C for 24 h, neutralized by addition the 16 ml of 0.75 M NaOH, filtered and analyzed for
␣-amino N. The analysis of ␣-amino N was also performed in dried duodenal samples. Approximately 0.1 g of dried duodenal
samples were weighed into screw-cap tubes and treated with 2 ml of HCl 6 M at 110 ◦ C for 24 h. After hydrolysis, 8 ml of 1.5 M
40 T. Orlandi et al. / Animal Feed Science and Technology 210 (2015) 37–45

NaOH was added into the tubes, the content diluted to 50 ml with distilled water and then filtered through a filter paper. The
concentration of ␣-amino N in rumen fluid and duodenal filtrates was analyzed using a non-automated colorimetric method
adapted from Palmer and Peters (1969) as described by Hentz et al. (2012). For individual amino acids analysis, feed and
duodenal dried samples were hydrolyzed with 6 M HCl solution containing 3 g/L of phenol during 24 h at 110 ◦ C. Free amino
acids were determined by reverse-phase HPLC (Agilent Model 1200, Santa Clara, CA, USA, with column Pico Tag (Waters,
Milford, MA, USA) of 3.9 × 300 mm, detector UV) at 254 nm following pre-column derivatization with phenylisothiocyanate
(Hagen et al., 1989). The ␣-aminobutyric acid was used as internal standard. Tryptophan was not included among detected
amino acids.

2.3. Calculations

The apparent digestibility of feed fractions were calculated as follows: [intake (g/day) − fecal excretion (g/day)]/intake
(g/day). The true digestibility of OM was estimated considering that only the aNDFom fraction of feces is originated from
feed (Van Soest, 1994) as follows: [OM intake (g/day) − fecal aNDF (g/day)]/OM intake (g/day). The true digestibility of N
compounds was estimated considering that only the NDIN fraction of feces is originated from feed (Van Soest, 1994) as
follows: [N intake (g/day) − fecal NDIN (g/day)]/N intake (g/day). The amount (g/day) of retained N was calculated as: N intake
(g/day) − fecal N (g/day) − urinary N (g/day).
The duodenal flow of DM (g/day), and of its respective components, was initially calculated based on either marker
ADF or indigestible NDF (INDF) concentration in duodenal digesta and feces as follows: [fecal DM (g/day) × fecal ADF or
INDF (g/kg of DM)]/duodenal ADF or INDF (g/kg of DM). The use of the first marker was based on a previous study reporting
no significant disappearance of ADF at the lower gastrointestinal tract (Kozloski et al., 2014). The INDF concentration in
duodenal and fecal samples was determined by 288 h ruminal incubation in nylon bags with a pore size of 16 ␮m (Huhtanen
et al., 1994). Each marker yielded similar effects among treatments, therefore, duodenal digesta flux was estimated from
the average of individual paired values based on each marker which reduced the standard deviation within treatments. The
apparent ruminal OM digestibility was calculated as follows: [OM intake (g/day) − duodenal OM (g/day)]/OM intake (g/day).
The amount of microbial N flowing to the small intestine was estimated based on the urinary excretion of PD according to
Chen and Gomes (1992). The chemical composition of rumen microbial samples was not measured for individual animals
as the purine to N ratio was assumed to be constant. The efficiency of microbial protein synthesis (EMPS, g microbial N/kg
digested OM) was calculated as: microbial N (g/day)/apparent rumen digestible OM (kg/day). Duodenal flux (g/day) of non-
ammonia non-microbial N (NANMN) was calculated as: duodenal N (g/day) − [microbial N (g/day) + ammonia N (g/day)]. The
ruminal degradability of dietary N compounds (RDN) was calculated without correction for N from endogenous origin as:
1 − [NANMN (g/day)/N intake (g/day)].

2.4. Statistical analysis

Data was analyzed using the MIXED procedures of SAS (2002) following the model: Yijk =  + Ai + Pj + Tk + εijk , where Y
is the dependent variable,  is the overall mean, A is the random effect of the animal, P is the random effect of the
period, T is the fixed effect of the treatment and ε is the residual error. Data derived from rumen samples collected
at each sampling interval were analyzed as repeated measurements using the PROC MIXED of SAS according to model:
Yijkl =  + Ai + Pj + Tk + A(P × T)ijk + Tpl + (T × Tp)kl + εijkl where Tp is time, T × Tp is treatment by time interaction, A(P × T) is the
between unit random effect and εijkl is the within units residual error. The compound symmetry (CS) covariance structure
was used for this analysis. Orthogonal contrasts were used to test the control against tannin treatments, as well as to test
the linear and quadratic effects of tannin levels. Results were presented as least square means. The amino acids profile of
duodenal digesta was compared to profile in feed through linear regression. A paired t test was used to evaluate whether the
intercept was different from zero or the slope was different from 1. Values of P ≤ 0.05 were considered significant. Tendency
of treatment effect was considered when 0.05 < P ≤ 0.10.

3. Results

The intake of OM and aNDF, and the total-tract apparent OM digestibility were similar for all treatments (Table 2).
The aNDF digestibility (P < 0.06), as well as the total-tract OM true digestibility (P < 0.07) tended to linearly decrease at
increased levels of tannin extract inclusion. Tannin extract linearly decreased (P < 0.05) the apparent and true digestibility
of N compounds, as well as the urinary N excretion, whereas it linearly increased (P < 0.05) fecal N excretion, N retention
and the efficiency of N utilization by steers (Table 3).
The interaction time by treatment was not significant for any ruminal variable whereas all ruminal variables were affected
(P < 0.05) by time after meal (results not shown). Ruminal pH was similar for all treatments whereas the concentration of
ammonia N and reducing sugars in ruminal fluid linearly decreased (P < 0.05) with tannin extract inclusion (Table 4). A
quadratic response (P < 0.05) to treatments was observed for ␣-amino N concentration in rumen fluid, as maximal mean
value was obtained for the 9 g/kg DM treatment.
Linear positive responses (P < 0.05) to tannin extract treatments were observed for duodenal flux of total N, ␣-amino
N and NANMN (Table 5). The microbial N supply tended (P < 0.10) to be negatively affected whereas both the ruminal
 T. Orlandi et al. / Animal Feed Science and Technology 210 (2015) 37–45 41

Table 2
Effect of tannin extracta level on intake and total-tract digestibility of organic matter (OM) and neutral detergent fiber (aNDF) in steers fed fresh-frozen
Avena strigosa (0.55) plus concentrate (0.45).

Item Level of tannin extract (g/kg of diet DM) SEMb P-value

0 9 18 27 C vs. Tc Linear Quadratic

Total intake (g/d)

OM 2792 2858 2853 2769 300.0 0.566 0.744 0.178
aNDF 1185 1214 1201 1155 129.6 0.855 0.354 0.152
Apparent total-tract digestibility
OM 0.82 0.81 0.80 0.79 0.013 0.163 0.128 0.731
aNDF 0.74 0.71 0.68 0.67 0.024 0.079 0.057 0.626
d
OM true digestibility 0.89 0.88 0.87 0.86 0.010 0.092 0.069 0.632
a
Acacia mearnsii tannin extract (Weibull Black, Tanac S. A., Montenegro, Brazil).
b
Standard error of means, where n = 4 per treatment.
c
Contrast analysis, where: C vs. T = control × average of all tannin treatments.
d
[OM intake (g/d) − fecal NDF (g/d)]/OM intake (g/d).

Table 3
Effect of tannin extracta level on intake, total-tract digestibility, excretion, retention and efficiency of N use in steers fed fresh-frozen Avena strigosa (0.55)
plus concentrate (0.45).

Item Level of tannin extract (g/kg of diet DM) SEMb P-value

0 9 18 27 C vs. Tc Linear Quadratic

Intake (g/d) 93.2 94.5 93.3 90.0 9.94 0.716 0.155 0.188
Total-tract digestibility
Apparent 0.83 0.79 0.78 0.77 0.015 0.036 0.035 0.387
Trued 0.94 0.90 0.88 0.87 0.011 0.003 0.003 0.147
Excretion (g/d)
Fecal 16.2 19.5 20.7 20.7 2.22 0.037 0.048 0.265
Urinary 62.1 51.8 39.4 41.0 10.52 0.009 0.005 0.132
Retention (g/d) 17.3 23.2 33.2 28.3 4.84 0.026 0.019 0.102
ENUe 0.20 0.27 0.35 0.31 0.061 0.046 0.044 0.151
a
Acacia mearnsii tannin extract (Weibull Black, Tanac S. A., Montenegro, Brazil).
b
Standard error of means, where n = 4 per treatment.
c
Contrast analysis, where: C vs. T = control × average of all tannin treatments.
d
[N intake (g/d) − fecal neutral detergent insoluble N (g/d)]/N intake (g/d).
e
Efficiency of N utilization by animal (g of retained N/g of ingested N).

OM digestibility and RDN linearly decreased (P < 0.05) with increasing tannin extract levels. The EMPS tended to increase
quadratically (P < 0.10) at increased levels of tannin extract showing maximal mean values at tannin extract inclusion of
18 g/kg DM.
The dietary inclusion of tannin extract positively impacted (P < 0.05) or tended to impact (P < 0.10) the duodenal flux of
all amino acids groups, as well as of most individual amino acids (Table 6). No treatment effect was observed on duodenal
flux of Arg, His, Lys, Ser and Thr. The amino acids profile in duodenal digesta was not affected by tannin extract treatments
(g of individual amino acid/100 g of total amino acids; P > 0.10, data not presented), although it was linearly related (P < 0.05)
to the amino acids profile in feed in all treatments (Fig. 1). However, the linear regression for the 18 g/kg DM tannin extract
treatment showed the highest determination coefficient (i.e. R2 = 0.83). Moreover, in this treatment neither was the intercept
different from zero nor was the slope different from unity.

Table 4
Effect of tannin extracta level on ruminal fluid parameters in steers fed fresh-frozen Avena strigosa (0.55) plus concentrate (0.45).

Item Level of tannin extract (g/kg of diet DM) SEMb P-value

0 9 18 27 C vs. Tc Linear Quadratic

pH 6.73 6.76 6.85 6.75 0.066 0.337 0.470 0.166

Ruminal fluid compounds (mg/dl)
Ammonia N 30.4 25.1 25.6 23.2 2.60 <0.001 <0.001 0.253
␣-Amino N 31.6 39.4 33.1 26.8 3.70 0.540 0.025 <0.001
Reducing sugars 42.0 39.3 35.5 34.2 3.35 0.036 0.006 0.734
a
Acacia mearnsii tannin extract (Weibull Black, Tanac S. A., Montenegro, Brazil).
b
Standard error of means, where n = 4 per treatment.
c
Contrast analysis, where: C vs. T = control × average of all tannin treatments.
42 T. Orlandi et al. / Animal Feed Science and Technology 210 (2015) 37–45

Table 5
Effect of tannin extracta level on duodenal flow of N compounds, ruminal digestibility of organic matter (OM) and N compounds, and efficiency of rumen
microbial protein synthesis (EMPS) in steers fed fresh-frozen Avena strigosa (0.55) plus concentrate (0.45).

Item Level of tannin extract (g/kg of diet DM) SEMb P-value

0 9 18 27 C vs. Tc Linear Quadratic

Duodenal flow of N compounds (g/d)

Total 33.8 49.6 57.6 56.5 7.21 0.012 0.020 0.140
␣-Amino 22.8 41.0 45.4 44.3 5.91 0.017 0.037 0.141
Ammonia 2.1 2.8 2.3 2.1 0.58 0.467 0.854 0.283
Non-ammonia, non-microbial 13.6 26.9 38.1 41.1 6.22 0.033 0.024 0.429
Microbial 16.2 19.9 17.1 13.1 3.92 0.383 0.080 0.204
Ruminal OM apparent digestibility 0.65 0.55 0.45 0.45 0.022 <0.001 <0.001 0.056
RDNd 0.85 0.71 0.59 0.54 0.052 0.017 0.009 0.484
EMPSe 9.5 12.4 13.4 10.7 1.50 0.287 0.778 0.095
a
Acacia mearnsii tannin extract (Weibull Black, Tanac S. A., Montenegro, Brazil).
b
Standard error of means, where n = 4 per treatment.
c
Contrast analysis, where: C vs. T = control × average of all tannin treatments.
d
Ruminal degradability of feed N compounds.
e
g of microbial N/kg of apparent rumen degradable OM.

Table 6
Effect of tannin extracta level on duodenal flow of amino acids (g/d) in steers fed fresh-frozen Avena strigosa (0.55) plus concentrate (0.45).

Amino acid Level of tannin extract (g/kg of diet DM) SEMb P-value

0 9 18 27 C vs. Tc Linear Quadratic

Ala 13.5 15.7 17.4 18.0 1.68 0.098 0.095 0.704

Arg 18.1 17.7 22.3 24.7 3.49 0.430 0.130 0.689
Asxd 27.9 25.2 40.9 37.8 1.89 0.022 0.111 0.983
Cys 5.7 7.1 8.5 9.4 1.57 0.091 0.095 0.862
Glxe 33.4 35.9 51.0 43.4 4.62 0.097 0.139 0.452
Gly 17.7 20.2 21.6 25.1 2.87 0.211 0.074 0.861
His 7.6 7.3 8.1 8.2 1.44 0.889 0.651 0.875
Ile 7.9 10.3 11.7 11.5 0.77 0.010 0.095 0.419
Leu 13.4 17.3 20.4 20.9 1.16 0.004 0.041 0.526
Lys 22.6 24.5 23.5 29.9 3.25 0.401 0.250 0.580
Met 8.0 10.1 10.9 11.8 1.15 0.071 0.077 0.709
Phe 12.7 25.1 19.0 18.9 5.46 0.138 0.585 0.096
Pro 20.6 22.9 28.1 30.4 3.45 0.153 0.043 0.997
Ser 17.3 18.3 22.4 20.1 2.80 0.392 0.409 0.643
Thr 14.7 15.9 18.2 16.5 2.45 0.477 0.518 0.592
Tyr 24.2 28.7 32.6 32.7 4.10 0.092 0.361 0.763
Val 10.0 12.8 13.7 15.6 1.17 0.025 0.038 0.828
EAAf 115.0 140.9 147.7 158.0 14.84 0.056 0.115 0.682
NEAAg 160.2 173.9 222.6 217.0 11.74 0.017 0.049 0.686
GAAh 179.8 192.9 244.8 244.6 14.73 0.031 0.059 0.813
Total 275.2 314.7 370.1 375.0 24.93 0.035 0.060 0.671
a
Acacia mearnsii tannin extract (Weibull Black, Tanac S. A., Montenegro, Brazil).
b
Standard error of means, where n = 4 per treatment.
c
Contrast analysis, where: C vs. T = control × average of all tannin treatments.
d
Asp and Asn.
e
Glu and Gln.
f
Essential amino acids (Arg, His, Ile, Leu, Lys, Met, Phe, Thr and Val).
g
Nonessential amino acids (Ala, Asp, Asn, Cys, Glu, Gln, Gly, Pro, Ser and Tyr).
h
Glucogenic amino acids (Ala, Arg, Asp, Asn, Cys, Gly, Glu, Gln, His, Met, Pro, Ser and Val).

4. Discussion

The expected impact of the tannin extract on protein and carbohydrate degradation by rumen bacteria (Makkar, 2003;
Min et al., 2003; Jayanegara et al., 2012) was clearly observed in the present study. The ruminal concentrations of ammonia
N, ␣-amino N and reducing sugars, as well as the RDN were reduced whereas the duodenal flux of NANMN was enhanced at
increased dietary levels of tannin extract. Similar results were observed in steers fed high-concentrate diet containing 0 or
4 g/kg DM of quebracho tannin (Mezzomo et al., 2011). Moreover, as also expected, increasing the dietary levels of tannin
extract significantly decreased the apparent ruminal OM digestibility. However, there was a trend toward reduced true
total-tract OM digestibility, indicating a shift of digestion site from rumen to intestines. However the tendency for reduced
total tract OM digestibility (i.e. feed OM digestibility) suggests that there was a slight reduction in the digestible energy to
the steers with increasing tannin extract.
 T. Orlandi et al. / Animal Feed Science and Technology 210 (2015) 37–45 43

Fig. 1. Relationship between the amino acids profile in feed with the amino acids profile in duodenal digesta of steers fed grass forage plus concentrate
containing levels of Acacia mearnsii tannin extract: 0 (Panel A); 9 (Panel B); 18 (Panel C) and 27 (Panel D) g/kg feed DM. The intercept was different from 0
(P < 0.05) and the slope was different from 1 (P < 0.05) for the linear regressions in Panel A, B and D.

As consequence of decreased rumen ammonia N concentration, tannin extract also shifted N excretion from urine to
feces which is an effect consistently reported in literature (Aufrère et al., 2008; Theodoridou et al., 2010). This is positive
from the environment point of view if it reduces nitrous oxide emissions from cattle manure (Schils et al., 2013). Moreover,
as consequence of the stronger negative impact on urinary N excretion compared to the minor positive impact on fecal
N excretion, the inclusion of tannin extract at a level of up to 18 g/kg DM improved N retention by steers. Previous older
(Driedger and Hatfield, 1972) and more recent studies (Carulla et al., 2005) have reported similar positive impact of the tannin
extract on N retention by sheep. In the present study, in which the digestible OM intake was similar for all treatments, the
positive impact of tannin extract on N retention by steers was clearly consequence of its positive impact on duodenal flux
of ␣-amino N compounds.
Tannins have been reported to exert a positive effect on the EMPS (Makkar, 2003). In the present study the EMPS tended
to improve, reaching a maximal mean value at inclusion of 18 g/kg DM of tannin extract. However, at the highest level of
tannin extract inclusion (i.e. 27 g/kg DM) rumen OM digestibility did not decrease despite a drastic reduction of the amount
of microbial N flowing to duodenum. As consequence, the EMPS and the duodenal flux of ␣-amino N compounds decreased
when the dietary inclusion of tannin extract increased from 18 to 27 g/kg DM. These results indicate that, at dietary inclusion
above 18 g/kg DM, tannin extract exerts a stronger negative impact on nutrients uptake by rumen bacteria compared to a
minor negative effect on the hydrolytic activity of bacterial membrane-associated enzymes, which are involved in rumen OM
degradation. Alternatively, a possible and plausible impact of tannins on purines flux and absorption from small intestines
and, consequently, on urinary excretion of PD might not be discarded. This hypothesis, however, needs to be tested.
The duodenal flux of all amino acids groups was clearly improved by tannin extract, increasing in average 30% above the
mean value observed in control treatment. Particularly relevant, mainly for lactating dairy cows, was the positive impact
on the intestinal supply of essential and glucogenic amino acids groups, as well as on the intestinal flux of Met and Phe
(Lapierre et al., 2012). The impact of tannin extract treatments on duodenal flux of amino acids was clearly consequence of
the increased duodenal flux of residual feed true protein (i.e. NANMN). Even though no treatment effect on the amino acids
44 T. Orlandi et al. / Animal Feed Science and Technology 210 (2015) 37–45

profile in duodenal digesta was detected, the amino acids profile in duodenal digesta was more closely related to profile in
feed at the higher levels of tannin extract inclusion. This result indicates the opportunity to improve the supply of limiting
amino acids to animals by using the tannin extract in diets containing true proteins of higher biologic value as, for example,
canola meal (Piepenbrink and Schingoethe, 1998; Brito and Broderick, 2007).
In conclusion, the dietary inclusion of Acacia mearnsii tannin extract up to a level of 18 g/kg DM reduced the urinary N
excretion and improved the amino acids supply in steers fed fresh-frozen oat forage plus concentrate without significantly
affecting the total OM digestibility.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgment

The authors thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brasília, DF, Brazil) for
support funding.

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