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The potential for tree forage supplements to manipulate rumen protozoa to enhance protein to energy ratios in ruminants fed on poor quality forages.
by R.A. Leng, S.H. Bird, A. Klieve, B.S. Choo, F.M. Ball, G. Asefa, P. Brumby, V.D.Mudgal, U.B. Chaudhry, S.U.Haryono and N. Hendratno


It is probable that there are large numbers of forages with secondary plant compounds that may manipulate digestive function in the rumen to the benefit of the animal. There is a need to survey such forages as they could represent cost-effective supplements for ruminants on poor quality pastures/crop residues. The first priority is to balance the rumen to allow an effective use of the basal diet which requires fermentative digestion. The main point to be emphasised is the need to extract, by microbial digestion, the maximum nutrients from the basal diet. At the same time, the growth efficiency of the rumen microbes must be optimised to provide the animal with a high protein (amino acids) to energy ratio (P/E ratio) in the nutrients absorbed. This can be achieved by supplementation with a combination of non-protein nitrogen and minerals. Once this has been achieved then the animal appears to need a small amount of extra supplementary protein that escapes to the lower tract to optimise the P/E ratio in the nutrients absorbed and maximize the animal's efficient use of the diet. The major problem for most developing countries is the availability of the resources needed to provide these supplements. Tree forages, and in addition seeds and pods, are potential sources of supplements that could provide an array of minerals and soluble nitrogen for the rumen microorganisms. The same materials can potentially also provide the bypass protein either naturally, when they contain tannins, or when induced by simple processing.

The P/E ratio in the nutrients absorbed by ruminants on forage-based diets depends largely on the microbial growth efficiency in the rumen and on the amount of bypass protein supplement included in the diet.

It has been demonstrated recently that the presence of rumen protozoa reduces the protein to energy ratio in the nutrients absorbed (Bird, 1991) through:

“With respect to the protein economy of the animal the advantages of removing the protozoa from the rumen and maintaining the defaunated state are:

It appears that it can now be stated definitely that there will be more microbial and dietary protein available to the ruminant when protozoa are absent from the rumen. It follows therefore that in the absence of protozoa in the rumen there is an increase in the total protein available relative to the VFA produced and absorbed from the rumen. The improved P/E ratio will benefit the animal where it is fed diets low in true protein. In such a feeding situation, the increased P/E ratio will increase the efficiency of nutrient utilisation by the animal. Thus removal of protozoa or a decrease in protozoal density in the rumen can be expected to increase ruminant production under most feeding conditions pertaining to roughage fed ruminants (Leng, 1990).

Following considerable research that tested the above statements, research began about 1980 to seek natural defaunating agents.


Research commenced with the establishment of a bioassay system to assess the effects of various additives on the viability of rumen protozoa in culture medium.

Culturing and maintaining protozoa in vitro

A stock culture protozoa isolated from the rumen was maintained in vitro in caudatum-type salt media using techniques of Coleman (1978) as follows:

Four Hungate roll tubes containing 3 ml of fresh media were prepared. To each tube of fresh media a mixed protozoal population was added. The cultures were fed daily with 0.1mg dry ground lucerne and 0.05 ml of corn starch suspension, 1.5% (w/v), the tubes were gassed with CO2, sealed tightly with a rubber bung and incubated at 39°C. After three days, 3ml of fresh media was added to each tube, and culturing was continued for a further 4 days. After this time, cultures were examined for protozoal growth and were transferred to 50 ml conical flasks with 25ml fresh media. The daily amount of feed was increased to 1.5mg lucerne and 0.2ml of the starch suspension.

Four stock cultures were maintained in this way. These cultures were found to consist of mixed Entodinia spp., as other genera present in the original rumen fluid rapidly disappeared from the mixed culture.

From this point onward each stock culture was maintained by removing 15ml of the culture media every three days and replacing this with fresh media.

Anti-protozoal assay

60ml of protozoal culture was available twice a week for assaying material for potential anti-protozoal toxins. Protozoa were fed with the starch suspension and a mixture of the test forage and lucerne.

Assay of anti-protozoal effects of forages

A number of forages were obtained; these had been dried at 60°C and ground through a 1mm sieve. All samples were assayed, mixed with the lucerne and the nutrients added to the cultures daily. In most cases, the test forages were at levels to replace 1, 10 or 100% of the lucerne.

Fresh caudatum-type salt media was prepared and 8ml of media was added to each of a number of Hungate roll tubes which were incubated at 39°C for at least 1 hour prior to inoculation. To each tube, 2ml of well mixed stock culture was injected and fed with 0.1mg of the mixture of lucerne and test forage and 0.1ml starch (1.5 (w/w) suspension. The tubes were incubated at 39°C for three days. On the fourth day, a 1ml sample of well mixed culture was taken and added to 1ml of formal saline (10% (v/v) formaldehyde; 0.9% (w/v) NaCl), with a few drops of iodine solution. This solution (0.1ml) was placed on a microscopic slide and all the protozoa present were counted using a dissecting microscope at 40x magnification. The protozoa count multiplied by 20 gave the number of protozoa in 1ml of the original culture.

Results of the anti-protozoal assay

The number of protozoa cultured, when starch plus a fibre source of either 100% lucerne or 100% Leucaena leucocephala was included, were similar (3.5×103 respectively).

In preliminary studies, a sample of powdered leaves of Lotus pendunculatus and also Acacia dealbata showed anti-protozoal activity. The numbers of protozoa in culture were depressed by increasing the Lotus proportion of the forage source in the growth medium. Lotus pendunculatus foraget at 100% of the forage apparently killed all protozoa in the medium.

Anti-protozoal properties of forages

The results of assays of forages which showed anti-protozoal activity are shown in the following tables. Forages that stimulated protozoal growth, had no effect on the number of protozoa in culture, or inhibited protozoal growth are shown in Tables 1, 2 and 3 respectively.

TABLE 1. Forages that apparently promote protozoal growth
Name of plant Forage substitution rate for lucerne leaf powder in the substrate added to incubation medium
1% 10%
  protozoa numbers as % of control
Acacia leveocloala 121 109
Brachychiton australis 111 113
Casuarina cunninghamia 114 130
Albizia lebbeck 112 102
Commersonia bastramia 124 106
Desmanthus unantiem 112 114
Gliricidia sepium 113 138
Samanea saman 110 114
Casuarina cunninghamiana 123 130
Cajanus cajan 126 104
Desmanthus virgatus 112 108
Soya bean (Glycine max) 118 122
Desmodium intortum (green leaf) 139 114
Centrosema viaginium 110 142
Macroptilium atropurpureum 112 121
Macroptilium lathyroides 111 114
Cassia brewsteria 112 115
Gejera parviflora 122 111
TABLE 2. Forages with no effects on protozoal growth
Name of plant Forage substitution rate for lucerne leaf powder in the substrate added to incubation medium
1% 10%
  protozoa numbers as % of control
Casuarina cristata 107 128
Alphitonia excelsa 108 123
Acacia excelsa 91 85
Acacia harpophylla 109 113
Brachychiton populeus 109 98
Acacia salicina 94 88
Acacia glavocarpa 109 143
Acacia bidwilli 107 108
Alphiltonia petriei 106 124
Albizia chimensis 108 90
Acacia harpophylla 102 110
Desmanthus virgatus 96 99
Sesbania sesban 107 130
Indigofera schemperi 98 97
Macroptilium lathyroides 95 73
Arachis hypogea 106 117

Identification of a potent anti-protozoal component in Enterolobium cyclocarpum leaf

Enterolobium cyclocarpum was identified as the most likely forage to be used as a rumen manipulator. It was quickly established that all ruminants would consume the leaf materials after an initial introductory period. Over the next 6 years, a series of trials were carried out to test the value of Enterolobium spp. as a potential rumen anti protozoal agent and the response of animals to consumption of small quantities.


The following experiments illustrate the potential usefulness of this material in small quantities to stimulate ruminant productivity.

TABLE 3. Forages with apparent anti-protozoal properties at 1% and 10% substitution rate for lucerne pasture of the in a culture medium
Name of plant Forage substitution rate for lucerne leaf powder in the substrate added to incubation medium
1% 10%
  protozoa numbers as % of control
Acacia deanei 38 79
Acacia crassa 38 74
Acacia semilunata 72 77
Acacia spectabilis 56 73
Acacia chinchillensis 75 103
Desmanthus intortum 63 73
Centrosema pubescens 78 84
Casuarina ronophlora 79 111
Black wattle 51 8
Fern leaf 81 97
Indigofera schemperi 75 75
Macroptilium lathyroides 88 100
Leucaena leucocephala 69 99
Vigna parteri 55 86
Lotononis bainesii 75 78
Desmodium uncinatum 78 108
Aeschynomena falcata 82 99
Cassia rotundifolia 88 106
Enterolobium cyclocarpum 0 0
Enterolobium timbouva 0 0

Enterolobium cyclocarpum: Anti-protozoal activity in vivo

Four rumen fistulated buffaloes in Indonesia (Batan) (350kg LWt) were fed cut/carry grass with minerals. Samples of rumen fluid were removed daily through the rumen cannula for 3 days prior to the commencement of feeding the leaf forage. The buffaloes were then given 500 g of fresh leaves (30% DM) daily for seven days. The effects of feeding the leaf material is shown in Figure 1. Although rumen protozoa were markedly depressed over the 7 days when these leaves were provided, protozoa were not removed totally and rapidly returned to normal densities in rumen fluid following cessation of feeding of the leaf material.

FIGURE 1. The effects of feeding 500g of fresh leaves from the tropical tree Enterolobium cyclocarpum to buffaloes given cut and carry grass in Indonesia


In India (CIRB, Hisar), a trial was conducted feeding young buffaloes on chopped wheat straw based diets. Dried leaves (375g) of Enterolobium timbova fed for 3 days apparently totally removed protozoa from the rumen but defaunation was not complete (or security failed) as periodically protozoa appeared in the rumen within one week after the cessation of feeding the leaf materials. In Australia, fistulated sheep were fed small amounts of Enterolobium cyclocarpum leaves at various rates in a diet of oaten chaff. The results on protozoal densities in the rumen indicated that Enterolobium leaf materials at 75 g/d could reduce protozoal numbers in rumen fluid to negligible numbers but that it was most difficult to remove them entirely and they returned to normal population densities within 4 days of cessation of feeding the leaf-meal. It was concluded that it was unlikely that defaunation in ruminants would be effected by feeding the leaf meal from this tree.

Effects of leaf forage defaunation in buffaloes on productivity

No research has been reported of the effects of the presence of protozoa in the rumen of large ruminants fed poor quality forages. The potential for manipulating rumen protozoa and stimulating productivity is illustrated by research undertaken in the Central Institute for Research in Buffaloes, Hisar, India, which was carried prior to using the leaf meal as a potential rumen modifier.

Forty-eight Murrah buffalo heifers (average 105 kg live weight) were allocated to one of eight treatments (4 diets x 2 fauna states). Feed intake and live weight changes were monitored over a 106-day period. A basal diet of urea ensiled wheat straw, fresh green cut/carry grass (3 kg/day) and minerals was supplemented with groundnut cake (GNC) at four levels (g/day); 0, 250,500 and 750. The buffalo were defaunated by dosing with a surface active agent.

The fauna-free condition which was created by drenching the animals with a surface active agent (Bird and Leng, 1978) increased live weight gain and improved feed conversion efficiency of heifers on all diets with the response being greatest in animals receiving the most GNC supplement (Table 4). Supplementing the basal diet with 750 g/day GNC increased the growth rate of the faunated animals from 220 g/day to 400 g/day (82%) and the growth rate of the defaunated animals from 263 g/day to 477 g/day (82%). A combination of defaunation and supplementation with 750 g/day GNC increased the growth rate of heifers from 220 g/day to 477 g/day (117 %).

TABLE 4. Growth rate (g/day) and feed conversion (FCE) of faunated (+F) and defaunated (-F) buffalo given straw-based diets.
Diet Total DM intake (kg/day) Growth rate (g/day) FCE g DMI/g gain
+F -F +F -F +F -F
Basala 3.13 2.57 220 263 18.2 11.1
Basal + GNCb (250g/d) 3.16 3.08 277 370 12.9 9.8
Basal + GNC (500g/day) 3.50 3.24 360 434 10.1 8.2
Basal + GNC (750g/day) 3.48 3.14 400 477 9.8 7.2

a Basal ration: urea-ensiled wheat straw, green fed 3kg, minerals.

b GNC = Groundnut cake.

Continual re-infection with protozoa during the study period meant it was necessary to drench each heifer (on average) four times during the 106-day period. During each drenching treatment (2–3 days), feed was not offered and animals took several days to regain full appetite following treatment. Regular drenching therefore greatly reduced feed intake of the heifers in the fauna-free group indicating that the differences reported may be minimal.

Results from these studies clearly show that defaunation can improve ruminant production from straw-based diets. Unfortunately there is no satisfactory method currently available for defaunating animals under field conditions. In these studies a detergent (sodium lauryl diethoxy sulphate) drenched directly into the rumen was used to defaunate animals. This method is not suitable for commercial use. The application of this technology is therefore dependent upon the development of a specific anti-protozoal agent. An alternative approach using leaf material is still being tested. However, the testing has been restricted to sheep because of the quantities of leaf material necessary to feed cattle.

Production of sheep given low quality forages supplemented with Enterolobium leaf material.

Cross-bred lambs, 10–11 months of age were dye-branded (technique for determining wool growth) while still in the paddock and then were individually penned in an animal house for 3 weeks prior to the experimental period. A pre-experimental rate of wool production was determined for all animals. Animals were divided into four dietary groups according to weight. The basal diet was oaten chaff (ad lib.), 1% urea and a mineral and vitamin mix. The experimental diets were:

  1. Basal diet (oaten chaff + 1% urea)
  2. Basal + 125 g/d lupins (cracked)
  3. Basal + 110 g/d lupins + (25 g/d) Enterolobium cyclocarpum (E.C.) leaves
  4. Basal + (90 g/d) lupins + (75 g/d) Enterolobium cyclocarpum (E.C.) leaves (diets 2,3,4 were iso-nitrogenous)

Bodyweight change was monitored over an 8 week period and wool growth over the last 6 weeks of the experimental period. There were 9 animals in dietary groups 1 and 2 but only 4 animals in groups 3 and 4 (there was insufficient E.C. leaves to feed 9 animals/group). The results are shown in Table 5.

TABLE 5. Effects of feeding Enterolobium cyclocarpum leaves (EC) on the rate of live weight gain and wool growth of lambs.
  Dietary Group
  Urea Lupins Lupins + 25g/d EC Lupins + 75g/d EC
initial weight 35 35 34 34
Final weight 39 40 41 41
Oaten chaff intake (g/d) 876 853 909 901
Total intake (g/d) 896 894 1029 1021
Growth rate (g/d) 63 93 103 115
FCR (g feed/g gain) 14.3 10.6 10.0 8.9
Wool growth* (clean wool) (g/d) 6 7 7.5 8.8
Rumen protozoa popn. 105/ml 3.1 202 2.6 3.1

* Wool growth rates have been adjusted using pre-experimental wool growth as a covariate.

Discussion of results with sheep

Supplements of EC leaves were readily accepted by sheep. There was no apparent detrimental effects of EC leaves on rumen function even to the extent that protozoal numbers in the rumen did not change. Intake of oaten chaff increased with the addition of leaf.

Supplements of EC leaves significantly increased the rate of body weight gain (24%) and wool growth (27%). These responses appear to be due largely to an improved efficiency of feed utilisation and therefore probably represent a change in the protein to energy ratio in the nutrients absorbed. The supplements (25 and 75 g/d) of EC leaves did not apparently alter the numbers of protozoa in the rumen, so it appears that the leaf had either altered the behaviour of the protozoa (making them more efficient) or affected some other (unknown) aspect of rumen function. This is an area that requires considerable further study to determine whether there are other effects of the leaf meal on the rumen protozoa.


Studies over a period of 15 years at the University of New England have shown that, on low true protein forage based diets in particular, removal of protozoa from the rumen of sheep and cattle and preservation of the fauna free state has improved animal productivity (see Bird et al., 1990; Bird, 1991). The increased productivity appears to be associated with a greater supply of essential amino acids from microbial protein but, where the basal forage is high in protein, extra dietary protein escapes the rumen for digestion in the intestines in fauna free ruminants (Ushida and Jouany, 1986). The increased amino acid supply appears to stimulate productivity of traits that are limited by amino acid supply such as body weight gain, wool growth and milk production. The improved P/E ratio in the nutrients absorbed increases the efficiency of feed utilisation for production.

The identification of a naturally occurring anti-protozoal forage that is relatively easily obtained in tropical countries or can be grown as a crop would open up new potential strategies for increasing animal productivity from poor quality forages.

In the research presented here, only a small range of the forages that are potentially available in tropical countries have been examined and there is a real need for a survey of different groups of plants. At the present time, the active ingredient in the forage has been identified and is being prepared as a single compound for use in many intensive animal production systems.

One of the important aspects of study concerns protozoal ecology in the rumen. It is apparent that many factors in the feed affect the capacity of protozoa to lower the P/E ratio in the nutrients absorbed. Perhaps protozoal activity can be modified by inclusion of other compounds in feed that have been identified through research. Bentonite, a clay mineral, has an overall effect similar to defaunation when added to the diet of sheep in small quantities (15–20g/d) (Fenn and Leng, 1990). In the studies now presented, sub-optimal levels of this anti-protozoal compound in the diet were almost as effective as the defaunated state in stimulating protein availability to sheep and increasing productivity.


Secondary plant compounds are chemicals produced by plants that create an advantage for that plant in the ecosystem within which it has evolved. They are produced as protective agents in a number of plants to modify, limit or prevent damage to the plant against consumption or attack by insects, fungi bacteria, protozoa or grazing animals, and also may be produced to reduce competition by other plants.

In recent times it has been suggested that some plants respond to invasive actions in stimulating an increased synthesis of particular secondary plant compounds which then decrease consumption of the plant by graziers.

It would be surprising that the wide variety of secondary plant compounds produced by a diversity of tree forages were all detrimental to ruminants. It could also be expected that, where forages have been components of the diet of ruminants, microbial diversity and genetic selection would provide the rumen microorganisms with the ability to modify or degrade these secondary compounds.

In the same way, with plants seldom consumed by ruminants because of location of growth, growth habit or taste, it would be surprising if they did not affect selectively or totally the growth of microbes in the rumen and that, where intake of such plants/compounds is controlled, there is great potential to manipulate rumen function.


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