Small Ruminant Research 61 (2006) 3544

Influence of condensed tannins from Brazilian semi-arid legumes

on ruminal degradability, microbial colonization and ruminal
enzymatic activity in Saanen goats
P.M. Guimaraes-Beelen a, , T.T. Berchielli b , R. Beelen c , A.N. Medeiros a
b
c

a Animal Science Department, Universidade Federal Da Paraba, 58397-000 Areia, PB, Brazil
Animal Science Department, Universidade Estadual Paulista, 14 870-000 Jaboticabal, SP, Brazil
Animal Science Department, Universidade Federal De Alagoas, 57 100-000 Rio Largo, AL, Brazil

Received 11 December 2003; received in revised form 12 November 2004; accepted 3 January 2005
Available online 19 March 2005

Abstract
The present study aimed at determining the influence of condensed tannins present in the Brazilian legume species Mimosa
hostilis, Mimosa caesalpinifolia and Bauhinia cheilantha on ruminal degradability, microbial colonization and enzymatic activity. Polyethylene glycol (PEG) was used to reduce the astringency and concentration of soluble condensed tannins. Four
ruminally-cannulated Saanen goats (60 8 kg BW) were fed, in two experimental periods, with a hay diet based on the studied
legumes treated or non-treated with PEG. Voluntary intake, microbial colonization, DM, CP, NDF, and ruminal degradability of
PEG treated and non-treated forage leaves, as well as pH, ammonia and 1,4 -endoglucanase activity of the rumen content were
evaluated. Astringency and soluble tannin concentration of the studied legumes were reduced by approximately 70% and 50%, respectively, with PEG treatment. Average DM intake was higher for the treated diet (16.76 g DM/kg BW/day against 13.06 g DM/kg
BW/day). Percentile values for degradation parameters and for potential and effective degradabilities of DM, CP and NDF were
also affected by the tannins, but at different intensities. Electron microscopic observations of ruminally-incubated legume leaves
showed a more effective microbial colonization of PEG-treated leaves for all legume species. A decrease in pH and an increase in
ammonia concentration and in endoglucanase activity in the ruminal content was also observed for PEG-treated diets at all sampling periods. Condensed tannins of the studied legume species have influenced the adhesion conditions, colonization and enzymatic activity of the microbial ecosystem, and consequently the ruminal degradation of the different dietary fractions. For this reason, the reduction in condensed tannin would be of great importance to improve the nutrition of ruminant feeding of these species.
2005 Elsevier B.V. All rights reserved.
Keywords: Rumen microbial colonization; Astringency; Endoglucanase activity; Native legumes; Brazilian semi-arid region; Saanen goats

1. Introduction

Corresponding author. Tel.: +55 83 362 2504;

fax: +55 83 362 2504.
E-mail address: pbeelen@cca.ufpb.br (P.M. Guimaraes-Beelen).
0921-4488/$ see front matter 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.smallrumres.2005.01.007

Animal production in the Brazilian northeastern

semi-arid region is almost exclusively based on pastures of the native plants from Caatinga vegetation, with

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P.M. Guimaraes-Beelen et al. / Small Ruminant Research 61 (2006) 3544

legume species being of great importance as protein

sources.
Caatinga is the low-thorn forest that covers the semiarid region of North-eastern Brazil, comprising an area
of about one million sq km. This biome sustains more
than 90% and 35% of the countrys goat and sheep
production, respectively (Araujo Filho et al., 1995).
Despite a CP content usually higher than 15%, the
legume species from Caatinga have low in vitro and
in situ degradabilities in goats and sheep, which could
be, in part, attributed to their high concentrations of
condensed tannins (Berchielli et al., 2003).
Studies have suggested that the high concentrations
of condensed tannins present in tropical forages are associated with a decrease in intake, digestibility in vivo,
in situ and in vitro degradability of nutrients (Carulla,
1994; Salawu et al., 1997; Ramrez et al., 2000; Baba
et al., 2002).
Considering that cell wall degradation and the subsequent release of the cellular content in the rumen depends on the activity of the microbial ecosystem, where
the microbial population adhered to feed particles is
predominant and directly responsible for their digestion, condensed tannins may engender important nutritional consequences to ruminants feeding on tanninrich feeds.
Very few studies have investigated the effects of tannins present in Brazilian semi-arid forages on ruminal
degradability, microbial adhesion and on the activity of
microbial enzymes. Therefore, the goal of the present
study was to assess, with the use of polyethylene glycol
(PEG), the influence of tannins present in the legume
species Sabia (Mimosa caesalpinifolia), Jurema preta
(Mimosa hostilis) and Mororo (Bauhinia cheilantha)
on DM, CP, and NDF degradability, on the activity of
the enzyme 1,4 -endoglucanase (cellulase) of the rumen content, and on the microbial colonization of these
legumes in the rumen of goats.
2. Materials and methods
2.1. Intake, DM, CP and NDF degradability
To assess the influence of tannins on voluntary intake, DM, CP, and NDF degradability of the three
legume species, an in situ degradability assay was conducted at the Animal Science Department facility, Sao
Paulo State University, FCAV-UNESP.

2.1.1. Animals, lodging and feeding regime

Four ruminally-cannulated Saanen male goats
(60 8 kg BW) were used. The animals were individually housed and fed ad libitum with a diet containing 40% concentrate (35% corn, 3% molasses, 1%
mineral premix and 1% oil) and 60% roughage, which
was formulated to supply the animals requirement for
maintenance according to the AFRC (1998). A hay
mixture composed of equal proportions of leaves of
the studied forages, harvested at the full-growth phase
(FebruaryMarch 2000) and shade-dried, was used as
roughage. The roughage was either treated or nontreated with PEG to reduce tannin contents. Voluntary
intake was determined daily and corresponded to the
difference between what was offered and the left-over.
2.1.2. Harvest and sample preparation for
incubation
Legume species were harvested in a Caatinga area
belonging to the National Center of Goat Research
CNPC, EMBRAPA, Sobral-CE. Twelve plants of each
species were randomly chosen. Trying to simulate animal grazing, an equal proportion of terminal leaves
were collected from the plants. Once collected, samples
were immediately frozen, lyophilized, ground at 2 mm
(Michalet-Doreau and Nozi`ere, 1999), and pooled to
form a single representative sample of each species.
2.1.3. PEG treatment
PEG treatment consisted in spraying roughage and
lyophilized leaves with a solution of 90 g of PEG 4000
in 300 mL of water per kg of DM (Ben Salem et al.,
1999), which was done when preparing the diets and
the nylon bags, respectively.
2.1.4. Nylon bag degradability
Two in sacco degradability periods were conducted.
In each period two animals were used to evaluate the
PEG-treated legumes and two to assess the non-treated
ones, which were fed with the corresponding (treated
or non-treated) roughage. Animals were adapted to the
diet for 10 days, and the incubation period in both periods was 20 days. Nylon bags (14 cm 7 cm with pore
size of 50 m) containing 4 g of lyophilized ground
(2 mm) leaves were incubated for 0, 6, 12, 24, 48 and
96 h. Bags were always inserted into the rumen around
8:00 am, i.e. before feeding the animals. The incubation time zero was determined by immersing two bags

P.M. Guimaraes-Beelen et al. / Small Ruminant Research 61 (2006) 3544

per legume species in water at 39 C for an hour. On removal nylon bags were immediately placed in cold water, washed in a washing machine, oven-dried at 55 C
for 72 h and weighed, and their content was analyzed.
2.1.5. Laboratory analyses
Original samples and incubation residues were analyzed for DM, CP according to the DUMAS method
with the aid of a Leco 528LC protein analyzer (Leco
Corp., St. Joseph, MI, USA), and for NDF using a
fiber analyzer (ANKOM Technology Co., Fairport,
NY, USA). Potential and effective degradability determination followed the model proposed by rskov
and McDonald (1979), with potential degradability
(PD) and effective degradability (ED) represented
by the following equations: PD = a + b(1 ekdt ) and
ED = a + ((bkd)/(kd + kp)). Ruminal pH and ammonia
levels were also monitored.
Levels of condensed tannins (soluble and bound
to solid residue) of roughage and lyophilized legume
leaves, treated or non-treated with PEG, were determined using the method described by Terrill et al.
(1992). Soluble condensed tannins were extracted from
samples of 10 mg, in duplicate, using a mixture containing 2.5 mL of aqueous acetone 70%, ascorbic acid
0.1% and 2.5 mL of diethyl ether. Upon evaporation of
the solvents the extracts were brought to 5 mL with distilled water, centrifuged and separated from the residue.
Subsequently, 1.8 mL of butanolHCl (5%, v/v) was
added to extract aliquots of 0.5 mL, which were placed
in a water bath at 95 C for 70 min. Condensed tannins bound to the solid residue were determined by
the addition of 0.7 mL of distilled water and 4.2 mL of
butanolHCl (5%) in a water bath as described above.
Absorbance was read in a spectrophotometer at a wavelength of 550 nm, and the results were converted to
percentage of condensed tannins based on the tannin
standard regression curve of each species. These regression curves were obtained using Sephadex LH-20
purified tannins as described by Rosales (1999). The
total concentration of tannins was obtained by the addition of the soluble fraction and the fraction bound to
the residue.
Astringency of condensed tannins was measured
by the radial diffusion method (Hangerman, 1987).
Aliquots of 8 L of plant extract were placed in Petri
dishes containing 9.5 mL of a mixture of agarose (1%,
w/v) and BSA (0.1%, w/v) in acetic acid buffer solu-

37

tion (0.3%, v/v) containing 0.001 g of ascorbic acid (pH

5.0). Tannins diffused through the agarose gel forming a ring of precipitate. The diameter of the ring was
considered proportional to their capacity to precipitate
proteins (astringency).
2.1.6. Experimental design
Species and tannin level effects on the legume degradation characteristics were analyzed by a randomized
block design (period) with sub-subdivided parcels. The
animal (4) effect was controlled in the parcel; the time
of incubation (7) in the sub-parcel; and tannin level
in the sub-sub parcel in a 3 2 factorial arrangement
(3 legume species 2 PEG levels). ANOVA analysis
and comparison of means by the Tukeys test were conducted using the PROC GLM of the Statistical Analysis
System (SAS, 1990).
2.2. Activity of the enzyme 1,4 -endoglucanase of
the ruminal content
The activity of the enzyme 1,4 -endoglucanase
(cellulase) of the ruminal content was evaluated in a
colorimetric assay. The assay was based on the enzymatic degradation of soluble polysaccharides covalently labeled with dye (AZO-CM-Cellulose, Remazolbrillant Blue R (Megazyme, Ireland)), and followed
the protocol described by Megazyme (2000). Sodium
acetate (pH 4.6) was used as buffer in order to get better
simulate ruminal pH.
2.2.1. Sampling
Samples of ruminal content were collected during
the first 3 days of the second in situ degradability experimental period (1st day at 8, 14 and 20 h; 2nd day at
10, 16 and 22h and 3rd day at 12, 18 and 24 h), totaling
nine samples per animal. Once collected the samples
were immediately frozen and placed in liquid nitrogen
until needed for analysis.
2.2.2. Enzymatic assay
Approximately 5 g of each sample was homogenized with 20 mL of phosphate buffer (pH 6; 0.2 M).
The enzymes were solubilized by sonication (Branson Sonifer 250) on ice for 30 s at 70% output and
recovered by centrifugation (15,000 g) at 4 C for
15 min. 0.5 mL of the supernatant (solution containing

38

P.M. Guimaraes-Beelen et al. / Small Ruminant Research 61 (2006) 3544

the enzymes) was added to 0.5 mL of substrate solution (AZO-CM-Celulose 4M, Remazolbrillant Blue R
(Megazyme, Ireland) in phosphate buffer pH 6), homogenized in a vortex mixer and incubated in a water bath at 39 C for 10 min. The reaction was stopped
and the high-molecular weight substrate was precipitated by the addition of 2.5 mL of precipitant solution
(200 mL of zinc acetate (4 g) and trihydrated sodium
acetate (40 g) solution (pH 5) in 800 mL of ethanol
95%, v/v) with vigorous stirring for 10 s in a vortex
mixer. The reaction tubes were allowed to equilibrate
to room temperature for 10 min and were then centrifuged at 1000 g for 10 min (Megazyme, 2000).
The supernatant was then recovered and the absorbance read against a blank in a spectrophotometer at
590 nm wavelength. The blank was prepared by adding
precipitant solution to the substrate before addition of
the enzyme. Results were expressed as the relative percentile activity of the enzyme.
2.3. Effect of tannins on microbial colonization
Microscopic observations of digestion residues
were made aiming at characterizing the effect of tannins on microbial colonization of incubated leaves.
Nylon bags (7 cm 3.5 cm with pore size of 50 m)
containing approximately 1 g of lyophilised-ground
forage leaves, treated and non-treated with PEG,
were ruminally incubated for the same time as described in the in sacco digestibility assay. Subsequently,
residue samples were analyzed by scanning electron
microscopy (Grenet, 1997).
At the right incubation time nylon bags were removed and their content was randomly sampled at different regions of the residue. The samples obtained
were fixed with 3% glutaraldehyde in 0.05 M cacody-

late buffer (pH 7.4) for 6 h at 4 C, post fixed with 1%

OsO4, dehydrated in graded alcohol series, and critical point dried (Tousimis Samdri PVT3) as described
by Santos (1996). Samples were mounted onto an aluminum stub, metalized with goldpalladium of approximately 30 nm, and studied in a JEOL JSM5410 scanning electron microscope.

3. Results
3.1. Intake, DM, CP and NDF degradability
Polyethylene glycol treatment of roughage and
lyophilized leaves allowed a reduction in condensed
tannin levels and astringency of approximately 50%
and 70%, respectively, without affecting DM, CP and
NDF composition (Table 1).
Tannins influenced animals DMI (Fig. 1). The average DMI of the PEG-treated hay diet was superior to
the non-treated one (16.76 and 13.06 g DM/kg BW/day,
respectively).
PEG treatment also affected degradation parameter
percentile values, as well as DM, CP and NDF potential
and effective degradabilities of lyophilized leaves of the
three legumes (Table 2).
The non-digested residue fraction (C) decreased
(P < 0.01) in all studied species while the potentially
degradable insoluble fraction (B) as well as the potential (PD) and effective (ED) degradabilities increased
in M. hostilis and M. caesalpinifolia, in response to
PEG application. However, an interaction (legume
species PEG) was observed for B, PD and ED, because PEG addition did not affect these parameters in
B. cheilantha.

Table 1
Condensed tannin levels, tannin biological activity, DM, CP and NDF contents of the roughage (Roug) and the lyophilized leaves of Mimosa
caesalpinifolia (MC), Mimosa hostilis (MH) and Bauhinia cheilantha (BC), treated and non-treated with polyethylene glycol (PEG)
Variables

Soluble tannin (%)

Tannin residue (%)
Astringency (% PR)
DM (%)
CP (%)
NDF (%)

PEG-treated

Non-treated

Roug

MC

MH

BH

Roug

MC

MH

BC

8.1
5.5
5.6
90.98
15.61
32.27

8.0
3.2
6.3
88.96
18.13
46.93

13.2
6.5
7.8
89.77
16.82
34.36

4.4
3.9
4.7
90.12
19.03
42.08

15.9
3.1
19.3
91.35
16.14
35.27

15.4
2.3
20.5
89.12
17.85
47.64

25.5
4.6
22.9
89.95
16.45
34.98

8.2
2.5
14.1
90.55
19.46
42.01

P.M. Guimaraes-Beelen et al. / Small Ruminant Research 61 (2006) 3544

39

Fig. 1. Voluntary intake of goats ingesting a hay diet based on semi-arid native legume species treated and non-treated with polyethylene glycol.

Ruminal pH and ammonia levels measured are

shown in Fig. 2 and Fig. 3, respectively. The type of
diet ingested influenced ruminal pH values. Higher
values were obtained for the animals consuming
non-treated hay. Despite being systematically lower,
the variation in the animals consuming the PEG-

treated hay was not high, the minimum point being

6.2.
Analysis of ruminal ammonia (NNH3 ) concentration showed that the reduction of condensed tannins by
PEG treatment increased ammonia levels in the rumen.
A mean concentration of 10.5 mg NNH3 /dl, against

Table 2
DM, CP and NDF degradation characteristics of Mimosa hostilis, Mimosa caesalpinifolia and Bauhinia cheilantha leaves, treated (PEG) and
non-treated (control) with PEG
Item

M. hostilis

M. caesalpinifolia

B. cheilantha

Significance

Control

PEG

Control

PEG

Control

PEG

S.E.

Leg

PEG

Leg PEG

DM
A (%)
B (%)
C (%)
kD (%/h)
PD (%)
ED (0.02 h1 )

21.7
33.0c
45.4
3.5b
53.5b
42.7b

26.8
37.5d
35.8
2.9a
62.1c
49.1c

24.1
21.8a
54.1
2.4a
43.7a
36.0a

28.3
26.7b
45.0
5.1c
54.8b
47.7c

20.2
59.6e
20.2
3.9b
78.4d
59.7d

25.7
57.4e
16.9
3.6b
81.3d
62.7d

3.4
1.2
2.0
0.7
2.1
2.7

0.14
<0.01
<0.01
<0.01
<0.01
<0.01

0.07
<0.01
<0.01
<0.01
<0.01
<0.01

0.63
<0.01
0.48
<0.01
<0.05
<0.05

CP
A (%)
B (%)
C (%)
kD (%/h)
PD (%)
ED (0.02 h1 )

17.8
30.9b
51.3
2.9
46.7b
36.0b

20.0
35.1c
44.9
3.1
53.4c
41.4c

15.1
25.9a
59.0
3.2
39.8a
31.0a

18.9
31.0b
50.1
4.2
49.4b
39.9c

18.6
65.6d
15.8
3.5
81.9d
60.4d

20.2
65.3d
14.5
3.7
83.6d
62.4d

1.3
4.2
3.2
0.9
2.4
1.7

<0.01
<0.01
<0.01
0.38
<0.01
<0.01

<0.01
<0.01
<0.01
<0.05
<0.01
<0.01

0.12
<0.01
0.47
0.51
<0.05
<0.05

NDF
B (%)
C (%)
kD (%/h)
PD (%)
ED (0.02 h1 )

41.7
58.3
3.3a
37.9a
26.0a

52.6
47.4
4.0c
49.6c
35.0c

38.1
61.9
3.7ab
35.4a
24.6a

47.1
52.9
4.0c
44.5b
31.5b

61.1
38.9
4.7d
62.4d
42.8d

65.7
34.3
3.9bc
64.2d
43.4d

3.9
2.7
0.6
3.0
1.8

<0.01
<0.01
<0.01
<0.01
<0.01

<0.01
<0.01
<0.01
<0.01
<0.01

0.72
0.32
<0.01
<0.05
<0.05

Leg: legume species; A: soluble fraction; B: potentially degradable insoluble fraction; C: residue fraction in the digesta; kd: degradation rate of
B; PD: potential degradability; ED: effective degradability; means within a row without a common letter in their superscript (a, b, c, d, e) are
different (P < 0.05).

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P.M. Guimaraes-Beelen et al. / Small Ruminant Research 61 (2006) 3544

Fig. 2. Mean evolution of ruminal pH in goats consuming a hay diet based on semi-arid native legume species treated and non-treated with
polyethylene glycol.

6 mg NNH3 /dl of ruminal fluid for the animals consuming the non-treated hay diet was observed. The
presence of tannins in the diet was able to reduce ammonia concentration to the level of 3.7 mg NNH3 /dl
of ruminal fluid.
3.2. Activity of the enzyme 1,4 -endoglucanase of
the ruminal content

of PEG treatment at all analyzed times, and showed

variability along the day. Points of maximum activity were observed approximately 4 h after feeding the
treated diet and 2 h after feeding the non-treated one.
Points of minimum activity occurred just after feeding,
and were more pronounced for the animals consuming
the treated diet (66% reduction).
3.3. Effect of tannins on microbial colonization

The activity of the enzyme 1,4 -endoglucanase

present in the ruminal content of the animals consuming the PEG-treated and non-treated hay diet is shown
in Fig. 4. Endoglucanase activity increased in function

Results of microscopic observations of incubation

residues are shown in Fig. 5. These followed the
same tendency demonstrated for the values of DM

Fig. 3. Mean evolution of ruminal ammonia in goats consuming a

hay diet based on semi-arid native legume species treated and nontreated with polyethylene glycol.

Fig. 4. Activity of the enzyme 1,4 -endoglucanase present in the

ruminal content of goats consuming a hay diet based on semi-arid native legume species treated and non-treated with polyethylene glycol.
Arrows indicate the feeding periods.

P.M. Guimaraes-Beelen et al. / Small Ruminant Research 61 (2006) 3544

41

Fig. 5. Scanning electron micrographs of the leaves of native semi-arid legumes incubated in the rumen of goats. (A) Mimosa hostilis, treated
with PEG, after 12 h of incubation; (B) Mimosa hostilis, not treated, after 24 h of incubation; (C) Mimosa caesalpinifolia, treated with PEG,
after 12 h of incubation; (D) Mimosa caesalpinifolia, not treated, after 12 h of incubation; (E) Bauhinia cheilantha, treated with PEG, after 12 h
of incubation; (F) Bauhinia cheilantha, not treated, after 12 h of incubation. Bars = 5 m.

degradability. M. hostilis leaves treated with PEG

presented higher as well as precocious colonization
and degradation levels, when compared to the nontreated leaves of the same species (Fig. 5A and
B). Similar conditions were observed for M. caesalpinifolia and B. cheilantha (Fig. 5C, D and E, F
respectively).

4. Discussion
Polyethylene glycol treatment reduced both astringency and soluble tannin concentration of hay and
lyophilized forage leaves. Nevertheless, PEG addition
did not modify DM, NDF and CP levels of the treated
material (Table 1). Therefore, the differences in intake,

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P.M. Guimaraes-Beelen et al. / Small Ruminant Research 61 (2006) 3544

degradability, endoglucanase activity and microbial

colonization of PEG-treated forages were attributed to
a lower astringency and soluble tannin concentration.
On the other hand, PEG treatment allowed an increase in the concentration of tannin bound to the
residue in all analyzed samples. Terrill et al. (1992)
suggested that the tannin fraction bound to the residue
would be the tannin bound to protein and fiber. However, in the present study the increase of the bound
tannin fraction after PEG treatment was considered as
being the tannin bound to PEG. At least in part, it seems
that this tannin has not been solubilized during the analysis.
The literature shows variable effects of tannins
on voluntary intake, even when tannins defense role
against herbivore consumption is considered. Some
studies report no effect of tannins on voluntary intake
(McNabb et al., 1993; Wang et al., 1994). As observed
in the present study, other authors (Carulla, 1994;
Silanikove et al., 1994; Barahona et al., 1997) reported
an increase in voluntary intake following PEG utilization in the diet. Kumar and Vaithiyanathan (1990) suggested that high tannin concentrations could reduce the
intake in the following ways: (1). physical distension
of the rumen, resulting in a decrease in dry matter digestion; (2). hormonal response due to the binding of
tannins to the gut wall; (3). reduction of the diet palatability caused by its astringency; and (4). binding of
tannins to salivary and mucosal proteins.
However, when assessing the acceptance of different Caatingas legume species by Santa Ines sheep,
Silva et al. (1998) observed that M. hostilis was
the most consumed species both at the beginning
(JanuaryFebruary) and the end (MayJune) of the
rainy season. This may indicate that not only the concentration of tannins but also the adaptation of the animals and microorganisms to their inhibitory effects
must be considered when predicting the effects of tannins on voluntary intake and digestibility. Barry (1985)
reported that deleterious effects of Lotus penduculatus tannin consumption were lower in sheep previously
conditioned to the diet.
The effects of tannins on nutrient degradability depends essentially on the formation of complexes between tannins and the components of the diet, i.e. primarily with proteins and to a lesser extent with amino
acids and polysaccharides, as well as on their effect on
the microbial population and on its enzymatic activity

(Kumar and Vaithiyanathan, 1990; Mcsweeney et al.,

2001).
In the present study, polyethylene glycol treatment
allowed an increase in voluntary intake, a better microbial colonization of the legume leaves, and an increase
in the activity of the enzyme 1,4 -endoglucanase.
Microscopic observations of M. hostilis, M. caesalpinifolia and B. cheilantha leaves at different ruminal incubation periods demonstrated that the microbial colonization was more effective when the abovementioned species were treated with PEG (Fig. 5).
After 12 h of incubation, M. hostilis leaves treated
with PEG showed a higher colonization than the nontreated leaves of the same species, even when those
were submitted to 24 h of incubation. The epidermis of
non-treated M. hostilis leaves seemed to be more resistant to the adhesion and penetration of rumen microbes than the leaves of M. caesalpinifolia and B.
cheilantha. Eventhough the PEG-treated leaves of the
two latter species presented a better colonization, a reasonable colonization of non-treated leaves could also
be observed after 12 h of incubation. Chiquette et al.
(1988) pointed out that adherent bacteria penetrate easily into tissues containing lower tannin levels. The
localization of tannin vesicles just above the epidermis and close to the stomata would inhibit microbial
colonization.
The rumen content of the animals consuming the
PEG-treated hay diet presented a higher enzymatic activity, which suggests that the tannins present in the
non-treated diet have inhibited the endoglucanase activity. Other studies have shown tannin inhibitory effects on ruminal enzymes (Makkar et al., 1988; Bae et
al., 1993; Jones et al., 1994), which were attributed to
the formation of tanninenzyme complexes with consequent inactivation of the enzyme.
In both treatments we observed a variation in the
enzymatic activity along the day (Fig. 4). Points of
maximum activity were observed approximately 4 h
after feeding the treated diet and 2 h after feeding the
non-treated one. Points of minimum activity occurred
in both cases just after feeding, and were more pronounced for the animals consuming the treated diet,
which was probably due to a more intense dilution effect, due to the higher intake by those animals.
Colonization characteristics and endoglucanase activity were reflected in the degradability results. The
addition of PEG increased the soluble and the poten-

P.M. Guimaraes-Beelen et al. / Small Ruminant Research 61 (2006) 3544

tially degradable insoluble fraction, as well as the potential and effective DM, CP and NDF degradabilities
of M. hostilis and M. caesalpinifolia leaves (Table 2).
The high tannin concentrations (Table 1) and low effective degradabilities (Table 2), observed when those
species were not treated with PEG, point to the fact
that tannin levels contribute considerably to their low
ruminal degradability.
B. cheilantha leaves, which contain lower tannin
concentration among the three species studied (8.2%),
presented a good effective degradability, even in their
native form (59.74, 60.35 and 42.84 for DM, CP and
NDF, respectively). The ruminal degradability of B.
cheilantha was slightly influenced by PEG treatment,
showing that the condensed tannin affected to a lesser
extent the ruminal degradability of this species. Carulla
(1994) obtained a similar result. The addition of PEG
(5% of DM) did not increase Desmodium ovalifolium
(6.8% of tannin) degradability.
Low tannin concentrations protect the dietary protein from ruminal degradation, increasing its availability in the lower intestine, without compromising microbial digestion efficiency. Nevertheless, at high concentrations, the protection of protein exerted by the tannins may critically reduce ammonia levels, compromising the proper digestion of the diet (Mcsweeney et al.,
2001). This probably happened to the animals consuming the non-treated diet where a higher pH was systematically observed, indicating lower fermentation, when
compared to the animals receiving the PEG-treated diet
(Fig. 2).
The low pH observed in the animals consuming the
PEG-treated diet of 6.2 was slightly above the limit
value (pH 6) considered as inhibitory to fiber digestion
by Russell and Wilson (1996).
The average ammonia concentration in the animals
consuming the non-treated diet attained a minimum
value of 3.7 mg/dl, staying, therefore, below the 5 mg/dl
suggested for the maximization of DM ruminal digestion. These values help to explain the difference observed in the degradability of treated and non-treated
leaves, which happened to be 32.2% higher for the DM
of treated M. caesalpinifolia.
The potential cell wall degradation and the subsequent release of cellular content in the rumen depend
on adhesion conditions, colonization and enzymatic activity of the microbial ecosystem. Condensed tannins
of the studied legume species have influenced those

43

factors, and consequently the ruminal degradation of

the different dietary fractions. For this reason, the reduction in condensed tannin, mainly of M. hostilis and
M. caesalpinifolia, would be of great importance to improve the nutrition of tropical ruminant feeding of these
species.
5. Conclusion
Condensed tannins present in the legume species M.
hostilis, M. caesalpinifolia and B. cheilantha have negatively influenced DM, CP and NDF ruminal degradability of these plants. Tannins have inhibited the intake
and microbial adhesion to forage leaves and have reduced the endoglucanase activity in the rumen content.
M. hostilis was the more affected forage, while B.
cheilantha practically preserved its ruminal degradation values in the presence of tannin. B. cheilantha
showed to be the most promising forage for animal
grazing among the three species studied.
Acknowledgements
The research was funded by Fundaca o de Amparo a` Pesquisa do Estado de Sao Paulo (FAPESP),
Project No. 00/03666-0. P. Guimaraes-Beelen was supported by a fellowship provided by Capes Foundation,
Braslia, DF, Brazil. The author personally wishes to
thank Dr. Joao Ambrosio Araujo Filho for providing
the legume samples, as well as for his interest and support in this research.
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