Short communication
Comparison of the digestive ability of crop ¯uid
from the folivorous Hoatzin (
Opisthocomus hoazin
)
and cow rumen ¯uid with seven tropical forages
R.J. Jones
a,*, M.A. Garcia Amado
b, M.G. Dominguez-Bello
baCSIRO Davies Laboratory, PMB, Post Of®ce, Aitkenvale, Townsville, Qld. 4814, Australia bCentro de BiofõÂsica y BiochõÂmica, Instituto de Investigaciones Cientõ®cas (IVIC), Caracas, Venezuela
Received 5 October 1999; received in revised form 14 June 2000; accepted 10 August 2000
Abstract
We explored the hypothesis that the Hoatzin, a tropical bird which feeds on tree leaves and has a crop resembling a rumen, may have a better ability to digest tanniniferous feeds than do cattle. Comparisons were made between Hoatzin crop ¯uid (HCF) and cow rumen ¯uid (CRF) in in-vitro digestion studies using freeze dried leaves of six tropical tanniniferous shrub legumes and the
tropical grassPanicum maximum. In-vitro dry matter digestibility (IVDMD) and in-vitro nitrogen
digestibility (IVND) were measured in the presence and absence of polyethylene glycol (PEG) to minimise the effect of condensed tannin.
For all plant species, and in the presence or absence of PEG, the CRF gave higher IVDMD and
IVND than did HCF (P<0:01). The interaction of PEGanimal (P<0:01) for IVDMD was due
to an absence of response with CRF (62.1 v 63.0%) and a large response with HCF (43.5 v 37.4%). Similarly for IVND, the response to PEG with CRF was much smaller (83.4 v 75.6%) than for HCF
(51.9 v 26.5%) (P<0:01).
PEG had no signi®cant effect on IVDMD or IVND of the CT-freePanicum maximumby HCF or
CRF (P>0:05). However, values were much higher with CRF: 74.2 v 30.6% and 81.3 v 29.0%,
respectively.
The only legume to have reasonable levels (>55%) of IVDMD and IVND with HCF in the
absence of PEG was Gliricidia sepium, which has little or no free condensed tannins. Acacia
boliviana, Calliandra calothyrsus and Leucaena trichandra gave low values and those for L. leucocephalaandL. pallidawere intermediate.
The low IVDMD and IVND with HCF, together with the large response to PEG, clearly show that high tannin content in the diet cannot be handled by the Hoatzin. Low tannin content in the crop contents indicates that it avoids highly tanniniferous leaves. This supports other ®ndings that the Hoatzin is extremely selective of a high quality leafy diet.
87 (2000) 287±296
*Corresponding author. Tel.:61-747538500; fax:61-747538600.
E-mail address: raymond.jones@tag.csiro.au (R.J. Jones).
It is concluded that the Hoatzin is an unlikely source of bacteria to improve the feeding value of
tropical tanniniferous shrub legumes for cattle.#2000 Elsevier Science B.V. All rights reserved.
Keywords:Tanniniferous shrubs; Condensed tannins; In-vitro digestibility; Foregut fermentation; Hoatzin
1. Introduction
The Hoatzin (Opisthocomus hoazin) is a folivorous bird native to areas of South America. It is unique in having a digestion pattern similar to that in ruminants (Grajal et al., 1989). It feeds on the leaves of many riverine tree species. These trees belong to plant families known to have a variety of toxic principles. Among these toxic compounds are phenols and tannins (Dominguez-Bello et al., 1994). It has been hypothesised that these birds may have the ability to detoxify these antinutritive compounds by its foregut fermentation.
In this paper, we examine this hypothesis by comparing the digestion in-vitro of several tropical tanniniferous browse species which are being evaluated for cattle production in Australia. The aim of the work was to assess the potential of the Hoatzin for tannin or tannin±protein degrading bacteria that could be used in cattle to improve their utilisation of browse.
2. Materials and methods
The plant materials listed in Table 1 were grown at the CSIRO, Lansdown Pasture Research Station, 50 km south of Townsville in tropical north Queensland. The shrub samples consisted of the ®rst ®ve fully expanded leaves on actively growing shoots about 1 m long. The grass leaves were plucked from young actively growing tillers.
Table 1
The tropical forage species used and their nitrogen (N) and condensed tannin (CT) content
Species Cultivar or CPI no.a N (%) Free CT vc Free CT bc Total CT bc
Leucaena leucocephala cv. Cunningham 4.04 3.46 5.47 6.03
Leucaena pallida 84581 3.87 2.54 4.65 6.72
Leucaena trichandra 46568 3.89 2.54 7.58 9.25
Calliandra calothyrsus 115690 3.50 5.07 5.03 5.78
Gliricidia sepium 110395 4.07 0.26 0.01 4.07
Acacia boliviana 40175 4.24 4.32 1.75 1.75
Panicum maximum(grass) Local 2.97 NDb ND ND
aCommonwealth plant introduction number (Australia).
bND: no CT detected.
The leaves were collected and placed on dry ice, then freeze dried in the laboratory, ground to pass a 1 mm sieve and stored in sealed bottles in the laboratory. Samples were analysed for N (Kjeldahl) and condensed tannin (CT) by both the vanillin/HCl and the butanol/HCl assays (Jackson et al., 1996).
The in-vitro digestions were conducted at the Laboratorio de Fisiologia Gastro-intestinal, Centro de Bio®sica y Bioquimica, Instituto Venezolano de Investigaciones Cienti®cas (IVIC), Caracas, Venezuela. A modi®ed Tilley and Terry (1963) procedure was used to estimate in-vitro digestibility. In brief, the ®rst fermentation stage was extended from 48 to 72 h, which reduces variability with tanniniferous feeds, and the pepsin digestion stage was reduced from 48 to 24 h. In addition to dry matter digestion ((IVDMD), nitrogen digestion (IVND) was also measured by analysing the plant materials and the residues for N. The method is described in more detail by Jones et al. (1998).
Crop ¯uid was obtained from four mature birds collected at the Hato Pinero cattle ranch, Cojedes State, central Venezuela. The birds were captured at night, after their evening feeding period and brought live to IVIC the following morning. Crops were removed and the crop contents (which were rather dry compared with rumen contents) placed in 500 ml of McDougall buffer at 398C. Mixing was done in a blender under CO2,
and the contents were strained through four layers of cheesecloth, added to 2 l of buffer under CO2and maintained at 398C.
The rumen ¯uid was obtained from a Holstein cow ®stulated at the rumen. The cow had grazed tropical pastures at the Central University of Venezuela farm at Maracay. It was strained through four layers of cheesecloth and 500 ml added to 2 l of buffer as for the crop ¯uid.
Samples of the forages were weighed (0.25 g) into 50 ml capacity centrifuge tubes to which was added 1 ml of distilled water or 1 ml of polyethylene glycol 4000 (4 g/100 ml water) (PEG). Tubes then received 25 ml of either Hoatzin crop ¯uid/buffer (HCF) or cow rumen ¯uid/buffer (CRF). Tubes were ¯ushed with CO2 during these operations. The
capped tubes were then incubated in an anaerobic chamber for 72 h. Appropriate blank tubes containing no forage sample were also incubated. There were seven plant species2 animal species2 PEG treatments each in quadruplicate, plus 35 blanks, i.e. 147 tubes in the experiment.
Tubes were swirled three times a day. Caps were loosened to release any pressure each day. After 3 days the tubes were centrifuged, the supernatant poured off and 25 ml of acid pepsin solution (4 g/l of 0.1 M HCl) added. Tubes were then incubated at 398C for 24 h, and swirled three times during this period. They were then centrifuged, the supernatant discarded and 37.5 ml water at 608C added. Tubes were shaken vigorously and again centrifuged to wash the samples. The supernatant was discarded and the tubes dried at 1008C for 72 h before weighing. The residues in the tubes were ground in a pestle and mortar to reduce particle size for estimation of N by a micro-Kjeldahl technique.
Dry matter and N loss was calculated from initial and ®nal weights after correction for the blank values. These values expressed as a percentage were referred to as IVDMD and IVND, respectively. The analytical mean data were analysed in the general linear model package (SPSS) as a factorial layout. The highest order interaction was used as the error term.
3. Results
The feeds varied in N and CT content. All species except the grass had N contents higher than 3%. G. sepium had the lowest free CT and L. trichandra the highest as estimated by the butanol/HCl assay with the other species intermediate. With the vanillin/ HCl assay,G. sepiumhad again the lowest value andC. calothyrsusthe highest (Table 1). In general, the vanillin/HCl values were lower than the butanol/HCl values but the notable exception was A. boliviana. For this species, the vanillin/HCl values were far higher than the butanol/HCl values (Table 1).
Samples inoculated with both CRF and HCF appeared to digest normally with active fermentation apparent on days 1 and 2. The samples incubated with HCF stopped digesting some 12 h earlier than did the samples incubated with CRF. The grass sample was the ®rst to stop with HCF and the last to stop digesting with the CRF.
For IVDMD, plant species and animal species gave signi®cant effects (P<0:01) as did PEG (P<0:05). In addition, the two-way interactions of animalplant species (Table 2) and animalPEG (Table 3) were also signi®cant (P<0:01). CRF gave higher IVDMD than HCF for all feeds, but the response of 44% units withP. maximumwas far
Table 2
Effect of animal and plant species on the IVDMD (%) of seven tropical foragesa
Plant species Animal species
The effect of animal (Hoatzin and cow) and PEG on the mean IVDMD (%) and mean IVND (%) of seven tropical foragesa
PEG treatment IVDMD (%) IVND (%)
Hoatzin Cow Hoatzin Cow
No PEG 37.4 63.0 26.5 75.6
Plus 160 mg PEG/g DM 43.5 62.1 51.9 83.4
greater than with the other feeds (P<0:01) (Table 2). There was no signi®cant effect of PEG on IVDMD with CRF but a highly signi®cant (P<0:01) improvement with HCF (Table 3).
Overall, the IVDMD with CRF was 22% units higher than with HCF (P<0:01). There was a positive response to PEG with HCF of about 6 units overall. The grass was very well digested by CRF (74%) but very poorly digested by HCF (30%) (P<0:01). In the absence of PEG, the relation between IVDMD with CRF and HCF was poor (r20:407)
mainly because of two species (Panicum maximumandAcacia boliviana). Omitting these values resulted in a good relation (r2 0:867). In the presence of PEG, only one species,
P. maximum, departed from a linear relation between CRF and HCF. Omitting this species resulted in an excellent relation between IVDMD values obtained with CRF and HCF (r20:954) (Fig. 1).
The differences between shrub species were large (P<0:01) (Table 2). Of the shrub legumes,G. sepiumhad the highest IVDMD with both inoculum sources andA. boliviana
had the lowest digestibility. Overall the ranking of the shrubs at P0:05 was
G:sepium>L:leucocephala>P:maximum>L:pallidaL:trichandra>C:calothyrsus
>A:boliviana.
For IVND, the effects of PEG (51.7 v 67.6%;P<0:001) and animal species (79.5 v 39.2%; P<0:001) were far greater than for IVDMD. In addition, the animalPEG (Table 3) and the PEGplant species (Table 4) interactions were also signi®cant (P<0:01±0.02), but the animalplant species interaction was not signi®cant (P>0:05). In the absence of PEG, the HCF gave very low IVND values for all feeds (Table 3). In the presence of PEG, IVND was almost doubled, whereas for the CRF, IVND increased only by about 10% with PEG (Table 3). With both inoculum sources, PEG had little effect on the IVND of Panicum maximum which contains no tannins (Table 4). However, with HRF the mean IVND was only about 30% compared with 80% for CRF.A. bolivianaandC. calothyrsusgave IVND values <40% (Table 4). Differences due to PEG were relatively small for P. maximum and G. sepium, but large for A. bolivianaandC. calothyrsusgiving rise to the signi®cant plant speciesPEG interaction (P<0:02) (Table 4).
Table 4
The effect of plant species and PEG on the IVND (%) of seven tropical foragesa
Plant species PEG treatment
The relation between IVND for HCF (x) and CRF (y) without PEG was generally linear (r20:643). Omitting P. maximumandA. bolivianagave a better ®t for the remaining species (y65:30:427x;r2 0:779) (Fig. 2A).
PEG reduced the IVND differences between legume species from 25.6 to 7.6% units with the CRF and from 46.9 to 15.4% units with HCF. However, there was still a large difference between the CRF and the HRF for the legumes in the presence of PEG (mean of 28.3% units). The relation between IVND with HCF and CRF with PEG was generally
linear overall (r20:476). However, omitting P. maximum improved the relationship (y58:70:452x;r20:792) (Fig. 2B).
4. Discussion
The range in CT content of the legumes varied with the assay technique as has been reported elsewhere (Jackson et al., 1996). The low free and high total CT forG. sepium Fig. 2. The relation between in-vitro nitrogen digestibility (IVND) measured with cow rumen ¯uid (CRF) and measured with Hoatzin crop ¯uid (HCF) in the absence A, and presence B, of PEG 4000 at 160 mg/g DM. The data forP maximumwere not included in the regressions. The relationships are described by the equations: A:
y65:30:472x(r20:779); B:y58:670:452x(r20:792).
with the butanol/HCl technique is explained by the high protein-bound CT found in this species (Jackson et al., 1996). Estimates of CT by the 14C PEG binding method
(Silanikove et al., 1996) show that the range of values is wider than that reported here for the same species (Jones and Palmer, 2000).
The differences between leguminous browse species in IVDMD were similar to those reported in earlier work using nylon bags suspended in the rumen (Bamualim, 1981; Balogun et al., 1998,). These differences will not be discussed here. The centre of interest for this study was the difference between the two inoculum sources.
Clearly the IVDMD of all feeds was lower with the Hoatzin crop ¯uid and particularly so with the grass andA. boliviana. With the latter species, this poor performance could well be due to the nature of the tannin present, since the addition of PEG brought this species into line with the other shrubs (Fig. 2), or to the presence of some other toxin. The poor performance with the grass, however, remained even in the presence of PEG. The grass has no tannins, hence some other factor must be involved. Hoatzins do not eat grass, so it is perhaps not surprising that with HCF the tropical grass was not well digested. It is likely that the ®bre in this C4species was not well digested. In other studies, cellulolytic
bacteria were only detected in small numbers in HCF (Dominguez-Bello et al., 1993). The results for IVDMD in the presence of PEG need to be viewed with caution. In the light of work using 14C-labelled PEG, these results are underestimates. This bias is
caused by the presence of PEG/tannin complexes in the residue after digestion (Palmer and Jones, 2000). The values, if corrections for these insoluble complexes are made, could result in the estimates being 2±10 digestibility units higher for the plant species used (Jones and Palmer, 2000). No such problem exists when IVND is calculated.
Tannin-tolerant bacteria from ruminants may protect the host from tannins (Brooker et al., 1994; Nelson et al., 1995). Although tannin-tolerant bacteria have been isolated from the Hoatzin (Woolston et al., 1999), our results for IVND do not support the hypothesis that the Hoatzin is capable of handling plant tannins by virtue of its foregut fermentation. It is possible, though perhaps unlikely in a bird, that salivary secretions may bind to tannins. The large improvement in both IVDMD and IVND in the presence of PEG, which is known to preferentially bind to tannins (Jones, 1965; Jones and Mangan, 1977), indicate that tannins have a marked depressing effect on digestibility in the Hoatzin. The higher IVDMD and IVND ofG. sepium, which contains little or no free tannins, also supports this conclusion. Although the genera on which the Hoatzin feeds are known to contain tannin (Dominguez-Bello et al., 1994), there are no records of the tannin contents of the tree species selected by these birds. Our analysis of the crop contents indicates low levels of free condensed tannin. Also the feeding habits indicate extreme selectivity of a high quality leafy diet (Grajal et al., 1989) which would be relatively low in ®bre and possibly low in CT.
binding plant proteins. In particular,C. calothyrsusin the absence of PEG had an IVND of only 8% compared with 48% in the presence of PEG. It is noteworthy that the tannin in this species has been shown to have adverse effects on rumen bacteria (Mwendia, 1994; McSweeney et al., 1998) and it is possible that the effect on bacteria in the crop of the Hoatzin may be even greater.
It could be argued that the digestibility of the grass was favoured by CRF since the inoculum was obtained from a cow eating mainly grass. By a similar argument, the digestibility of the shrub leaves should have been favoured by HCF since they were feeding on shrub and tree leaves. Despite this, lower digestibility of the shrub leaves was measured with HCF. In the light of these results, it is concluded that the Hoatzin will not be a suitable source of bacteria capable of metabolizing condensed tannins, or tannin± protein complexes, to improve the feeding value of tropical tanniniferous shrub legumes for cattle.
Acknowledgements
We thank Dr Luis Quintero, Universidad Central de Venezuela, Nucleo Maracay for provision of the rumen ¯uid, the Ministerio del Ambiente for permit No 11-001237 to capture the Hoatzins from the wild and Mr. Don Antonio Branger for allowing us to use the facilities at the Hato Pinero ranch, and acknowledge the ®nancial support from CONICIT through grant S1 2720. We also thank the Australian Meat and Livestock Authority (MLA) who provided ®nancial support for the senior author to travel to and conduct the study in Venezuela.
References
Balogun, R.O., Jones, R.J., Holmes, J.H.G., 1998. Digestibility of some tropical browse species varying in tannin content. Anim. Feed Sci. Technol. 76, 77±88.
Bamualim, A., 1981. Nutritive value of some tropical browse species in the wet and dry season. M.Sc. Thesis, James Cook University of North Queensland, Townsville, Australia, pp. 1±103.
Dominguez-Bello, M.G., Lovera, A., Suarez, P., Michelangeli, F., 1993. Microbial digestive symbionts of the crop of the Hoatzin (Opisthocomus hoazin): an avian foregut fermenter. Physiol. Zool. 66 (3), 374±383.
Dominguez-Bello, M.G., Michelangeli, F., Ruis, M.C., Garcia, A., Rodriguez, E., 1994. Ecology of the folivorous Hoatzin (Opisthocomus hoazin) on the Venezuelan plains. The Auk 111 (3), 643±651. Grajal, A., Strahl, S.D., Parra, R., Dominguez, M.G., Neher, N., 1989. Foregut fermentation in the Hoatzin, a
neotropical leaf eating bird. Science 245, 1236±1238.
Jackson, F.S., Barry, T.N., Lascano, C., Palmer, B., 1996. The extractable and bound condensed tannin content of leaves from tropical tree shrub and forage legumes. J. Sci. Food Agric. 71, 103±110.
Jones, R.J., Stoltz, M.A., Meyer, J.H.F., Bechaz, F.M., 1998. The effect of rumen ¯uid storage time on digestive capacity with ®ve forage/browse species. Trop. Grassl. 32, 270±272.
Jones, R.J., Palmer, B., 2000. In-vitro digestion studies using14C-labelled polyethylene glycol 4000 (PEG):
comparison of six tanniniferous shrub legumes and the grassPanicum maximum. Anim. Feed Sci. Technol.
85, 215±221.
McSweeney, C.S., Palmer, B., Kennedy, P.M., Krause, D., 1998. Effects of Calliandra tannins on rumen microbial function. Proc. Aust. Soc. Anim. Prod. 22, 289.
Mwendia, C.W., 1994. Tannins in tropical legumes: characterization and effects on ruminal biological activity. Ph.D. Thesis, University of Guelph, Ont., Canada.
Silanikove, N., Shinder, D., Gilboa, N., Eyal, M., Nitsan, Z., 1996. Binding of poly(ethylene glycol) to samples of forage plants as an assay of tannins and their negative effects on ruminal degradation. J. Agric. Food Chem. 44, 3230±3234.
