MATERIALS AND METHODS

Plant Material
Ruta graveolens and S. argel leaves were purchased from a local market, separately ground and then mixed in a basal diet (Table 1).

Experimental Design
One day-old Bovans chicks were obtained from Coral Company Ltd., Khartoum, and reared in pens within the premises of the College of Veterinary Medicine and Animal Production, Sudan University of Science and Technology, Khartoum North. The chicks were allowed free access to drinking water and feed. The pens were illuminated at night and early morning throughout the experimental period. After 2 weeks, the chicks were allotted at random to 4 groups, each of 10 chicks. Group1 was the control and fed normal basal diet. Groups 2 and 3 were fed diets containing 10% (w/w) of R. graveolens leaves and 10% (w/w) of S. argel leaves, respectively. Group 4 received a diet containing a mixture of 5% (w/w) of each plant. All chicks were fed the designated experimental diets for 2 weeks.

Average body weight and body weight gain were measured weekly for each group. The chicks in each group were slaughtered after 2 weeks of treatment. Blood samples were collected from each of the killed chicks for hematology and serum analysis.

Blood Analyses
Hemoglobin (Hb) concentration, Packed Cell Volume (PCV), Red Blood Cell (RBC) counts, Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH) and Mean Corpuscular Hemoglobin Concentration (MCHC) were estimated by standard methods (Schalm et al., 1975). Sera were analysed for the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and for concentrations of total protein, albumin, cholesterol and uric acid using commercial kits (Linear Chemicals, Barcelona, Spain).

Pathological Examinations
Necropsies were performed on all chicks immediately after slaughter to identify gross lesions and specimens of liver, kidneys, spleen, intestines and heart were fixed in 10% neutral buffered formalin, embedded in praffin wax, sectioned at 5μm and stained with hematoxylin and eosin (H and E).

Statistical Analysis
The significance of differences between means was compared at each time point using Duncan’s multiple range test after ANOVA for one-way classified data (Snedecor and Cochran, 1989).

Table 1:	Percent composition of basal diet fed

RESULTS

Effect on Growth
The effects of diet containing 10% R. graveolens (group 2), 10% S. argel (group 3) or 5% mixture of two plants (group 4) on body weight and body weight gain of the chicks are shown in Table 2. The chicks fed diets containing 5% mixture of two plants (group 4) had the lowest growth rate over the 2 week period but none of the chicks died during the course of the experiment.

Hematological Changes
Hematological data are summarized in Table 3. In group 3, there were decreases in MCV and MCH while in group 2, the values of Hb, PCV and RBC decreased but those of MCV increased with no adverse effects on MCH or MCHC values. In group 4, the values of Hb, RBC, PCV and MCHC did not change but those of MCH decreased.

Serobiochemical Changes
In groups 2, 3 and 4, there were decreases in the concentration of total protein, albumin and globulin. The concentration of serum cholesterol decreased in the chicks fed a diet containing 10% R. graveolens leaves (group 2). Uric acid concentration decreased in groups 2 and 4. Neither AST nor ALT activity was found to increase in any of the chicks in groups 2-4 (Table 4).

Pathological Changes
There was mild fatty change in the liver of the chicks in groups 3 and 4. Hepatic fatty change was marked in group 2 with congestion of the blood vessels or hemorrhage especially in the heart. On microscopy, fatty cytoplasmic vacuolation and individual-cell necrosis of the centrilobular hepatocytes were observed but degeneration of the epithelial cells of the renal proximal convoluted tubules was mild. Other organs of the test chicks did not show significant lesions. There were no changes in the control (group 1).

Table 2:	Growth changes in Bovans chicks fed R. graveolens, S. argel or their mixture for 2 weeks
Values are means±SE; means within columns with no common letters (a-c) differ significantly (p<0.05)

Table 3:	Hematological changes in Bovans chicks fed diets containing R. graveolens, S. argel or their mixture for 2 weeks
Values are means±SE; means within rows with no common letters (a-c) differ significantly (p<0.05)

Table 4:	Serobiochemical changes in Bovans chicks fed diet containing R. graveolens, S. argel or their mixture for 2 weeks
Values are means±SE; means within rows with no common letters (a-c) are significantly different (p<0.05)

DISCUSSION

Although the R. graveolens and S. argel leaves are commonly used in Sudan and other countries for the treatment of various ailments, toxicological information on chicks or rodents is unavailable. The results of the present study indicated that 10% R. graveolens and 10% S. argel or 5% mixture of two plants in the diet were toxic but not lethal to Bovans chicks when fed for 2 weeks. Inappetence as well as the damage to the liver could explain the depression in growth.

Phytochemical studies on the aerial parts of R. graveolens have shown that rutin, furocoumarin and lemonin are the major active constituents (Srivastava et al., 1998). Volatile oils, flavonoids, tannins, sterols, monoterpenes, triterpenes, glycosides and saponins are the main constituents in S. argel (Kamel et al., 2000). It has been suggested that the susceptibility of chicks, rodents and livestock to plant material is at least dependent on the type of active constituents and concentration in the amount added as well as the rate of their metabolic conversion to metabolites and consequent excretion (Barri et al., 1983; Bakhiet and Adam, 1996a; Adam et al., 2000).

The widespread congestion and hemorrhage could be due to altered permeability of the capillaries by the active constituents in R. graveolens leaves. The vascular changes in the chicks fed 10% S. argel leaves or 5% mixture of two plants were less intense. The hypoproteinemia detected in chicks fed 10% R. graveolens leaves, 10% S. argel leaves, or 5% mixture of two plants might have been due to hepatotoxicity. This as well as the occurrence of hypocholesterolemia particularly in chicks fed R. graveolens is further evidence of liver damage. The absence of change in the activity of serum AST or ALT in the chicks fed R. graveolens and S. argel leaves singly or combined might have been due to enzyme excretion.

In R. graveolens-fed Nubian goats, there were decreases in the concentration of serum cholesterol and total protein and increases in AST activity and urea concentration indicating hepatorenal malfunction (El Agraa et al., 2002). In the present study, serum uric acid concentration did not show significant changes; this finding might indicate the absence of significant nephrotoxicity in Bovans chicks. In chicks fed R. graveolens leaves, the decrease in Hb, RBC, PCV and the increase in MCV without significant effect on MCHC indicate macrocytic normochromic anemia. Previous studies showed macrocytic anemia in Bovans chicks which had been fed 10% Cassia italica or normocytic normochromic anemia in chicks which had been fed Ambrosia maritima (Bakhiet and Adam, 1996a, b).

Investigations into the isolation and characterization of the active constituents in the plants are necessary for elucidating their modes of actions and interactions.