Christo CHRISTEV1, Matina NICKOLOVA1, Dimo PENKOV1, Rumiana IVANOVA1,
Desislava ABADJIEVA2, Svetlana GRIGOROVA2
1Agricultural University, Mendeleev Str. 12, 40000 Plovdiv, Bulgaria
2Institute of Animal Science – Kostinbrod
The aim of the present research was to investigate the effect of Bulgarian dry extract from the annual plant Tribulus terrestris – L (Vemoherb PT), produced by Vemo 99 Ltd Company, Sofia on main biochemical characteristics and hematological parameters of the blood in guinea fowl (Numida meleagris). An experiment was carried out with 30 Pearl-gray Guinea fowl (32 weeks old), distributed in two groups – a control and an experimental, 12 female and 3 male each. All birds were fed the same mixture for breeder guinea fowl. Bulgarian Tribulus terrestris extract was supplemented to the compound feed of the experimental group in a daily dose of 10 mg/kg body weight for a period of 12 weeks.
The tested product decreased significantly the levels of total triglycerides (P < 0.05), total cholesterol (P <0.01) and glucose (P < 0.01; P < 0.001 in male and female birds respectively) in the blood serum. It was established significantly higher total protein- (P < 0.001) and calcium (P<0.01) values in the blood serum of the treated birds.
The addition of Bulgarian Tribulus terrestris extract increased significantly hemoglobin level, the number of erythrocytes and leukocytes and decreased the number of eosinophils in guinea fowl from the both sexes.
KEYWORDS: guinea fowl, Tribulus terrestris, biochemical characteristics, hematological parameters of blood
Extensive use of antibiotics results in residual medication of poultry products as well as bacterial antibiotic resistance and tolerance. This situation represents a considerable risk to human health . The banning of the use of nutritive antibiotics has accelerated and led to investigations of alternative feed additives in poultry production . Plant extracts can play a role in supporting both performance and health status of the poultry. Beneficial effect of herbal extract or active substances in poultry nutrition may include the stimulation of appetite and feed intake, the improvement of endogenous digestive enzyme secretion, activation of immune response and antibacterial antiviral antioxidant and antihelminthic actions. They are also seen to have a great potential in practical application.
One of the most popular phytoproducts is the extract of the flowering annual herb Tribulus terrestris Linn. (Zygophilaceae). This extract is harmless for humans and animals and contains biologically active substances as saponins, flavonoids, [17,20], glycosides, phytosterols , alkaloids and other constituents . Its main active components are saponins of the furostanol type, termed protodioscin . Figure 1 presents the mechanism of protodioscin action. Flavonoids have anti-inflammatory effect and improve the overall physiological status of the animals. Tannins influence gastrointestinal microflora and exert astringent effect due to their antibacterial properties . Our earlier investigations have shown that dry extract of Tribulus terrestris increase significantly calcium level in the blood serum in broilers parent ; decrease significantly glucose blood level in laying hens ; decline total serum cholesterol in laying hens and broiler parents [7,8]. These changes are related with increased growth rate and egg performance. On the other hand, it is well known that growth performance is closely related with poultry health status. There are no data concerning the effect of Tribulus terrestris extract on blood biochemical parameters in other poultry species.
The objective of this research was to investigate how the addition of Bulgarian dry extract from Tribulus terrestris commercially known as Vemoherb PT produced by Vemo 99 Ltd Company, Sofia changes main biochemical characteristics and hematological parameters of the blood in guinea fowl (Numida meleagris).
Figure 1. Mechanism of protodioscin action
MATERIAL AND METHODS
The used in the current study herbal product is standardized and contains (in percent of dry matter): not less than 60% saponins of furostanol type defined as protodioscin; not less than 10% tannins; not less than 10% flavonoids determined as rutin. The heavy metals content of this extract is ≤ 0.001%.
The present investigation was conducted in the period April – June, 2008 in the Experimental base of Agricultural University, Plovdiv, Bulgaria with 30 Pearl gray guinea fowl (32 weeks old), raised under extensive conventional method of production on a deep litter pen . The birds were randomly distributed in two groups – a control and an experimental, 12 female and 3 male each. The trial duration was 13 weeks – one week adaptation and 12 weeks experimental period. All birds were fed the same mixture for breeder guinea fowl (Table 1). The nutritive value
Table 1. Ingredients and chemical composition of compound feed for breeder Guinea fowl
Ingredients % %
Soybean meal, solvent 44% CP 22.00
Sunflower meal 5.00
D-C-P (Dicalcium Phosphate) 1.60
Vitamin Premix 0.50
Mineral premix 0.20
Chemical composition %
Metabolizable energy, MJ/kg 11.80
Crude protein 17.77
Crude fat 4.21
Crude fiber 5.41
Methionine + Cystine 0.82
Phosphorus, available 0.43
The nutritive value of the diet was determined by traditional Weende analysis. The metabolizable energy was calculated according to WPSA, 1989 .Vemoherb PT (dry extract from Bulgarian Tribulus terrestris) was supplemented to the compound feed of the experimental group in a daily dose of 10mg/kg body weight for a period of 12 weeks.
The blood for this study was taken from vena cutanea ulnaris at the end of the trial, morning before feeding. The biochemical parameters in the blood serum (triglycerides, total cholesterol, glucose, total protein and calcium) were measured with Pentra 400 biochemical analyzer. The red and white blood cells, the erythrocyte indices (mean corpuscular haemoglobin or MCH and mean corpuscular hemoglobin concentration or MCHC) were measured by Angelov et al., 1998 . The hemoglobin concentration was determined by the cyanmethemoglobin method, the eosinophils – by the method of Dunker, described by Christev, 2007 .
Statistical analysis of obtained results was performed by Excel 2000, single factor, Anova program. Values are expressed as means ± SEM.
RESULTS AND DISCUSSION
The triglycerides, total cholesterol, glucose, total protein and calcium values measured in the blood serum of guinea fowl are shown in Table 2. The levels of total triglycerides and total cholesterol in the blood serum of the treated birds were significant lower (P < 0.05; P < 0.01 in male and female birds respectively) in comparison with the control group. Similar date reported other authors in mice , in bulls  and in poultry [7,8]. The Tribulus terrestris mode of action might be explained with the enhancement of testosterone level, which has a proven cholesterol decline effect . The cholesterol, witch synthesis is taken principally in the liver is a precursor of all steroid hormones including the sexual hormones.
The serum glucose value in treated group was significant lower (P < 0.01; P < 0.001 in male and female birds respectively) compared to the control birds. Analogical results have been obtained by Grigorova et al.  in hens given 10 mg/kg body weight/day Vemoherb PT (dry extract from Bulgarian Tribulus terrestris) for a period of 12 weeks. The extract of Tribulus terrestris reduces the serum glucose level perhaps by inhibiting gluconeogenesis [1,9].
It was found a significant enhancement of the serum total protein content in male and female guinea fowl from experimental group (P < 0.001) in relation to the control group. The established increase of serum total protein level in treated guinea fowl was in agreement with the data of Petkova et al. , who observed higher serum protein value in bulls fed Vemoherb PT (dry extract from Bulgarian Tribulus terrestris) supplemented diet in dose 3 mg/kg body weight/day for 40 days. Probably the increased level of this parameter is due to the main effect of protodioscin contained in Tribulus terrestris extract, which is expressed in improvement of vitality and metabolism .
The significant increase of calcium content in the blood serum of female guinea fowl from the treated group might be explained with significant higher egg productivity of the treated group  as well as with the stimulating of calcium metabolism under the influence of the tested herbal extract. It is widely accepted that egg productivity correlates positively with blood estrogens level . It is well known that estrogens modulate calcium metabolism . About the level of calcium in the blood serum there is not difference between male guinea fowl from the both groups. Similar results are reported by Grigorova et al.  in broilers’ parent given 10 mg/kg body weight/day Vemoherb PT (dry extract from Bulgarian Tribulus terrestris) to the diet.
Table 2. Levels of triglycerides, total cholesterol, glucose, total protein and calcium in blood serum of Guinea fowl
The hematological parameters in the blood of guinea fowl from the control and experimental groups are given in Table 3. The hemoglobin level in the blood of experimental guinea fowl from both sexes was significantly higher (P < 0.05; P < 0.01 in male and female birds respectively) than those of the control group. Similar results were observed about number of erythrocytes (P < 0.01; P < 0.001 in male and female birds respectively), about MCH (P < 0.001) and MCHC (P < 0.05; P < 0.01 in male and female birds respectively).The positive effect of the tested product on erythropoiesis could be explained by the fact, that protodioscin in the Tribulus increases blood testosterone level and its transformation into dihydrotestosterone . In turn dihydrotestosterone stimulates erythropoiesis, which leads to increase the hemoglobin level (see Figure 1).
In the current research was established significant increase of the leukocytes number in the treated birds (P < 0.06; P < 0.01 in male and female birds respectively). The experimental group had significantly lower number of eosinophils (P < 0.01; P < 0.001) compared to the control group. The leukocytosis and eosinopenia are accepted as reliable indices of stress . Therefore, it could be suggested that the observed changes in leukocytes and eosinophils in female experimental birds due to their higher egg performance (P < 0.05) reported in our earlier investigation.
Bearing in mind that Tribulus terrestris extract has stimulating effect on the reproducibility, we assume that it improves metabolic interaction. Ultimately leads to the observed chances in our case.
Table 3. Main hematological indices in Guinea fowl from the both groups
The supplementation of 10 mg/kg body weight/day Vemoherb PT (dry extract from Bulgarian Tribulus terrestris) to the mixture of breeder guinea fowls for a period of 12 weeks decreased significantly the levels of total triglycerides (P < 0.05), total cholesterol (P < 0.01) and glucose (P < 0.01; P < 0.001 for male and female birds respectively) in the blood serum. It was observed significant enhancement of the total protein- (P < 0.001) and calcium (P < 0.01) values in the blood serum of the treated birds. The addition of the tested phytoproduct increased significantly hemoglobin level (P < 0.05; P < 0.01 for male and female birds respectively), the number of erythrocytes (P < 0.01; P < 0.001for male and female birds respectively) and leukocytes (P < 0.05; 0.01 for male and female birds respectively). It was found, that the number of eosinophils decreased significantly in birds from the experimental group (P < 0.05; P < 0.01 for male and female guinea fowls respectively) in comparison to the control group.
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