Livestock Research for Rural Development 7 (3) 1995

Citation of this paper

A chick assay method for the evaluation of non-conventional protein sources derived from nacedero (Trichanthera gigantea) and azolla (Azolla filiculoides)

Patricia Sarria & T R Preston (1)

(1) Finca Ecológica, University of Agriculture and Forestry, Ho Chi Min city, Vietnam

Fundación Centro para la Investigación en Sistemas Sostenibles de Producción Agropecuaria - CIPAV, A.A. 20591 Cali, Colombia (


Three experiments using 120 day-old broiler chicks, were carried out using a method designed to evaluate quality of protein from non-conventional sources, such as aquatic plants and leaves of trees. This work was done at the senior author's home located in Cali (Colombia) from June to August of 1995. The area is 1000 m above sea level with 24 ?C daily mean temperature, 75% humidity and 1000 mm annual rainfall. A chick growth asay is an attractive biological test because results are obtained within two to three weeks after giving the test protein. Energy sources free of protein were used to ensure that the growth response reflected the quality of the test protein. The protein sources to be evaluated were included at levels that permitted replacement of between 0 and 15% of the protein in soya bean meal.

The first experiment compared a basal diet based on cereals with one in which the energy source was provided by raw sugar and palm oil; in both diets the protein source was soya bean meal. Intake and growth on the sugar/oil diet were low, compared with the cereal diet (21 vs. 38 g/d of intake; 4 vs. 24 g/d growth). In the subsequent experiments cassava starch was included in the test diet (21%). In the second experiment the treatments were 0, 10 and 15% replacement of the protein from soya bean meal with ground, oven-dried leaves of Trichanthera gigantea. There were no differences in intake (1.49, 1.43, 1.5 and 1.51 g/hour, respectively) nor in growth rate (0.39, 0.36, 0.45 and 0.36 g/hour). In the third experiment the test protein was derived from Azolla filiculoides included at the same levels as in Experiment 2. There were no differences in intake ( g/d) nor in growth rate (24.6, 29.4, 34.0 g/d).

It is concluded that: (i) leaf meals from Trichanthera gigantea and Azolla filiculoides can replace up to 15% of the protein normally derived from soya bean meal in diets of monogastric animals; (ii) the chick growth assay with protein-free energy sources is an appropriate biological test to assess the nutritional value of potential protein-rich supplements.

Key words: Chick growth assay, trees, Trichanthera gigantea, aquatic plants, Azolla filiculoides, non-conventional protein sources.


In Colombia, agricultural production from small farms located in rain-fed, mountaineous areas supplies half the national food supply and yet occupies only 15% of the land area (CIPAV 1992, unpublished data). There have been many attempts, at national and international level, to increase animal production in these regions, but the results in general have been meagre. Perhaps the most pertinent explanation of this phenomenon is the lack of understanding of the ecological, socio-economical and cultural limitations inherent in these countries and which constrain severely the application of conventional development models based on external inputs (Preston and Murgueitio 1994). It is very important to promote alternative production systems which make better use of local resources, and which will support sustainable development in these ecologically fragile areas.

Multipurpose trees are considered to be an essential feature of agricultural production systems for sustainable rural development in all less developed countries, but especially those in the tropics (Preston and Murgueitio 1994). The tree known in Colombia as "Nacedero" (Trichanthera gigantea), which is native to the coffee growing areas, is well adapted for inclusion in integrated production systems since the leaves are readily consumed by pigs and poultry (Sarria et al 1992).

Water plants such as Azolla filiculoides, are highly productive sources of protein-rich biomass and are an ideal complement for the low-fibre tropical feed resources being developed as alternatives to cereals in poultry and pig diets (Becerra 1991). Aquatic plants are also good for the environment since they decontaminate (extract nutrients) from livestock and human wastes (Preston and Murgueitio 1994).

Conventional methods to evaluate nutritional quality of feeds are based on Weende and Van Soest analyses, which aim to determine the principal nutrients present in feeds. The utilization of these fractions by the animal is assessed by measuring their digestibity which is done indirectly by subtracting nutrients excreted from those ingested. While broadly useful as a means of measuring energy supply, digestibility is less useful for assessing the protein fraction since animal needs are for a range of amino acids, both essential and non-essential, and protein in the Weende system is measured as total nitrogen multiplied by a constant representing the average N content of most proteins (N x 6.25). A further disadvantage of the conventional analytical methods is that they take no account of secondary metabolites (principally tannins and alkaloids) which affect animal response (Kass 1994).

Biological methods seem more appropriate because in most developing countries it is easier and cheaper to aquire animals and feeds than laboratory equipment and chemicals both of which also require foreign exchange. Furthermore, biological response data are more useful, and more easily understood, as a basis of making recommendations to farmers.

In this paper, preliminary data are reported using a chick growth assay to evaluate non-conventional protein sources derived from forage trees and water plants. The method is based on experiences in Cuba (Juana Rodriguez and Vilda Figueroa 1994, unpublished data) and Colombia (Julio Vargas 1995, unpublished data)).



This study was conducted from June to August, 1995, at the researcher's home located in Cali (Colombia), which is at an altitude of 1000 m above sea level with annual mean values of air temperature 24°C, relative humidity 75% and rainfall 1000 mm.

Experiment 1: evaluation of the basal energy source

The objective was to compare a basal test diet composed of protein-free energy sources (raw sugar and palm oil) with a conventional one based on cereals. The composition of the diets is in Table 1.

Table 1: Composition of diets and calculated nutrient content (Experiment 1)
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Maize (Control) Sugar-oil
% %
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Yellow maize 60.0
Raw sugar 39.0
Soya been meal * 27.2 49.0
Fish meal ** 6.0
Vegetal oil 3.4 4.0
Biofos 1.2 2.0
CaCO3 1.0 1.0
Mineral-vitamins 0.4 1.0
Soya bean hulls 4.0
Additives # 0.8
N x 6.25, % 22.9 23.1
Energy, MJ/kg 13.6 13.6
Fiber, % 2.68 3.30
Ca, % 0.80 0.80
P, % 0.50 0.37
BLGIF.GIF (44 bytes)


* 45%protein; ** 63% protein; # anti-microbial drug and antibiotics


Treatments, animals and design

Fourty day-old broiler chicks were used. The two treatments varied in source of energy: the control was based on maize and the test diet on protein-free energy sources (raw sugar and palm oil). Each treatment had six replications with three chicks in each experimental unit.


Twelve wire cages were located in a 3 x 4 m room with natural ventilation. The cage dimensions were 0.23 x 0.25 x 0.30 m, and they were elevated 1.1 m from the floor. Each cage had two external rectangle metallic boxes for water and the experimental diet. All chickens were allocated in the cages from the first day. During the first week, six 100 watt lamps were turned-on 24 hours per day, after this, only during the night.

Diets and feeding

During the first seven days the chicks received a comercial grain- based diet. They were then fasted for 16 hours prior to introducing the test diets. The objective of the fasting period was to stimulate the intake of the experimental diets during the test period.


The chickens were weighed at 0, 96 and 120 hours after starting the trial. Feed consumption was recorded at 0.5, 2, 4, 6, 24, 48, 72, 96, 120 and 144 hours.

Results and discussion

The results are shown in Table 2. Feed intake and growth rate during the test period were much worse for the test diet. It was hypothezised that the absence of starch in the test diet was the reason for the poor performance. In subsequent experiments it was decided to include pure starch derived from cassava roots.

Table 2: Mean values for growth and intake on control and test diets (Experiment 1)
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Control Test SE/Prob
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Live weight, g
Initial 115 115
Final 260 139
Daily gain 24.1 4.05
Increase/hour 1.01 0.20 ±0.07/0.00
Intake, g
Daily 38.1 21.1
Hourly 2.49 1.67 ±0.15/0.00
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Experiments 2 and 3: Trichanthera gigantea and Azolla filiculoides as a partial replacement of soya bean meal

Treatments, design and animals

The objective was to observe the effect of including leaf meals of Trichanthera gigantea and Azolla filiculoides as a partial replacement for soya bean meal. There were four treatments; a positive control with 21% protein from soya bean meal and two levels of the leaf meals to replace 10 and 15% of the soya bean protein. The negative control was intended to contain the same level of soya bean as the 10% leaf meal diets but without the leaf meal. However, due to an error in formulation the soya bean level in the negative control for the trichanthera was the same as in the positive control and the only difference was in the ratio of sugar to starch. For the Azolla treatments the negative control was correctly formulated.

The birds and housing were the same as in Experiment 1, except that in one replicate there were 4 instead of 3 birds per cage.

Feeds and management

Table 3: Composition of the diets containing Trichanthera gigantea (Experiment 2)
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Soya:Trich (protein contribution, %)

100:0* 100:0# 90:10 85:15
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Ingredients, %
Soya bean meal 42.0 41.5 38.0 36.0
Raw sugar 21.0 17.0 16.0 15.0
Cassava starch 21.5 26.5 17.0 10.0
Palm oil 3.0 3.0 8.0 12.0
Trich meal 0 0 14.0 23.0
Biofos 2.0 2.0 2.0 2.0
CaCO3 1.0 1.0 1.0 1.0
Min-vit mix 1.0 1.0 1.0 1.0
Soya bean hulls 8.5 8.0 3.0 0.0
Nutrient content:
N x 6.25, % 21.1 20.9 20.9 21.0
Energy, MJ/kg 13.0 12.9 12.6 12.5
Fibre, % 5.34 5.29 5.38 5.50
Ca, % 0.80 0.80 0.80 0.80
P, % 0.40 0.47 0.40 0.4
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* Positive control; # Negative control


On the basis of results from Experiment 1, cassava root starch was incorporated into the basal test diet (Tables 3 and 4). Soya bean hulls were included in all the diets except the 15% leaf meal so as to have equal levels of fibre. The fasting period was only twelve hours because, in the first experiment, the birds were unduly stressed and one chick died. During this fasting period, a small amount of crude sugar (about 4%) was dissolved in the drinking water.

Table 4: Composition of diets containing Azolla (Experiment 2)
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Soya:Azolla (protein contributing, %)

100:0* 90:0# 90:10 85:15
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Ingredients, % :
Soya been meal 42.0 38.0 38.0 36.0
Raw sugar 19.5 23.0 17.0 14.0
Cassava starch 24.0 24.0 18.5 18.0
Palm oil 3.0 3.0 9.0 11.0
Azolla meal 0 0 11.0 17.0
Biofos 2.0 2.0 2.0 2.0
CaCO3 1.0 1.0 1.0 1.0
Min-vit mix 1.0 1.0 1.0 1.0
Soya bean hulls 7.5 8.0 2.5 0.0
Nutrients, %
N x 6.25, % 20.6 18.8 20.7 20.8
Energy, MJ/kg 13.6 13.8 13.8 13.6
Fibre, % 4.47 4.49 4.45 4.54
Ca, % 0.80 0.80 0.80 0.80
P, % 0.36 0.35 0.35 0.34
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The chickens were weighed at 0, 48, 72 and 120 hours. The consumption was registered at 2, 4, 6, 24, 48, 72, 96 and 120 hours.

Results and discussion

The results for the test diets containing Trichanthera are shown in Table 5 and those for Azolla in Table 6. There were no significant differences for growth and intake between the control and the diets with 10 and 15% of protein from Trichanthera (Table 5). In contrast, there were significant increases in growth (P=0.02) and a tendency for intake to be higher (P=0.09) when soya bean protein was replaced by Azolla up to the 15% level (Table 6).

Table 5: Intake and growth of chicks given Trichanthera gigantea as partial replacement of soya been meal
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Soya:Trichanthera (protein contributing, %)

100:0 100:0 90:10 85:15 SE/Prob
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Live weight, g
Initial 178 179 180 180
Final 225 223 236 224
Daily gain 9.4 8.7 10.8 8.74
Hourly gain 0.39 0.36 0.45 0.364 ± 0.030/0.24
Intake, g
Daily 35.8 34.2 36.7 36.2
Hourly 1.49 1.43 1.53 1.51 ±0.56/0.004
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There were marked differences in response between the two experiments. In Experiment 3 (Azolla) intake was almost twice and growth three times what was recorded in Experiment 2. This was true for both the control and experimental diets. There are no obvious reasons for these differences as the housing and basal diet were similar for both experiments. The chicks in experiment were three days older and were 30% heavier than in experiment 2, but this is only a small difference compared with the differences in performance.

Table 6: Intake and growth of chicks given Azolla filiculoides as partial replacement of soya been meal
BLGIF.GIF (44 bytes)

Soya:Azolla (protein contribution, %)

100:0 100:0 90:10 85:15 SE/Prob
BLGIF.GIF (44 bytes)
Live weight, g
Initial 259 265 250 235
Final 407 399 426 439
Daily gain 21.8 23.0 29.5 35.5
Hourly gain 0.91 0.96 1.23 1.48 ± 0.12/0.05
Intake, g
Daily 56.6 59.0 58.3 67.0
Hourly 2.36 2.46 2.43 2.79 ±0.10/0.09
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The aim of having a negative control diet was to separate the effects due to protein level and to those that might be caused by secondary compounds in the test foliages. In experiment 2, a formulation error prevented such an analysis. However, in experiment 3 there was a significant difference between the negative control diet having a soya bean level of 90% of the positive control and the test diet with the same soya bean level but with the balance of protein (10%) coming from the Azolla.

The results of experiments 2 and 3 indicate that for monogastric animals: (i) Azolla is superior to Trichanthera as a source of supplementary protein; (ii) a low level of azolla (15% of the protein supply) may improve performance compared with soya bean alone. Part of the explanation for these effects may be the higher fibre and lower digestibility of the Trichanthera leaves compared with Azolla (Maricel Becerra and Julio Vargas 1995, unpublished data). The other factor is the superior balance of amino acids in Azolla compared with soya beans and Trichanthera (Table 7).

Table 7: Amino acid balance in soya, Azolla and Trichanthera compared with the ideal protein (expressed as % of lysine=100)
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Opt Soya Azolla Trich
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Lys 100 100 100 100
Met+Cys 63 48 63 61
Threo 72 61 72 93
Tryp 18 22 31 0
Val 75 83 103 118
Isoleu 60 90 83 97
Leuc 110 133 139 162
Phen+Tyr 120 139 149 193
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Use of a chick growth assay and a protein-free basal diet provides useful information concerning the value of leaves from trees and water plants as protein supplements for monogastric animals. The water plant Azolla filiculoides was superior to leaves from the tree Trichanthera gigantea when given at levels equivalent to the replacement of 15% of the dietary protein supplied by soya bean meal. The advantage of Azolla may be related to its lower fibre content and superior amino acid pattern compared with Trichanthera.

The main advantages of the chick growth assay are that it uses locally available resources and is relatively inexpensive. Only about 2 kg of dried foliage is needed and the results are obtainable in a period of two weeks.


This research was partially supported by the International Foundation for Science through a grant (B/1627-2) to the senior author.


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(Received 20 October 1995)