MATERIALS AND METHODS

Plant Material
Cuminum cyminum seeds and H. sabdariffa calyces were purchased from a local market, ground separately to a fine powder and then mixed into a basal diet (Table 1).

Experimental Design
Sixty one day-old Bovans cockerels were obtained from Coral Company Ltd. Khartoum and housed within the premises of the Faculty of Veterinary Medicine, University of Khartoum, under light/dark cycle with diet and water provided ad libitum. This experiment was carried out in the year 2006. After 7 days, the chicks were randomly allotted to 5 groups of 12 chicks each. Chicks in group 1 were the controls and fed normal basic diet. Chicks of groups 2 and 3 were fed diets containing 2% (w/w) and 10% (w/w) of ground C. cyminum seeds, respectively. Chicks in groups 4 and 5 received diets containing 2% (w/w) and 10% (w/w) of ground H. sabdariffa calyces, respectively. All chicks were fed their designated experimental diets for 9 weeks.

Average body weights, body weight gains and feed conversion ratios (kg feed kg^-1 gain) for each group were estimated weekly. Batches of 4 birds from each group were slaughtered at weeks 3, 6 and 9 for pathological examinations. Blood samples were collected from each of the killed birds for haematology and serum analysis.

Blood Analyses
Haemoglobin (Hb) Concentration, Packed Cell Volume (PCV), Red Blood Cell (RBC) Counts, Mean Corpuscular Volume (MCV), Mean Corpuscular Haemoglobin (MCH) and Mean Corpuscular Haemoglobin Concentration (MCHC) were estimated by standard methods (Schalm et al., 1975). Sera were analyzed for the activities of aspartate aminotransferase (AST), acid phosphatase (ACP) and Creatine Kinase (CK) and for concentrations of total lipid, cholesterol and uric acid using commercial kits (Stanbio Laboratory Inc., San Antonio, TX).

Table 1:	Percent composition of basal diet fed

Pathological Examinations
Necropsies were made on all birds to identify gross lesions and specimens of liver, kidneys, spleen, heart, proventriculus and intestines were fixed in 10% neutral buffered formalin, embedded in paraffin wax, sectioned at 5 μm and stained with haematoxylin and eosin (H and E) for histopathological examinations.

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).

RESULTS

Effect on Growth
The effects of treatment with diets containing 2 and 10% C. cyminum seeds or 2 and 10% H. sabdariffa calyces on body weight, body weight gain and feed efficiency of the chicks are presented in Table 2. The chicks fed a diet consisting of 10% H. sabdariffa calyces (group 5) for 3, 6 and 9 weeks had the lowest growth rate, but none of the birds died during the 9-week period.

Haematological Changes
In the chicks fed a diet containing 10% H. sabdariffa calyces (group 5) for 3 weeks, there were decreases in Hb, RBC, PCV and MCV without effect on MCHC Table 3. After 6 and 9 weeks, changes in erythrocytic series of this group persisted. In chicks fed a diet consisting of 10% C. cyminum seeds (group 3) for 6 or 9 weeks, the values of Hb, RBC, PCV and MCV were lower (p<0.05) than those in the control and other groups.

Serobiochemical Changes
In the chicks fed 10% H. sabdariffa (group 5) for 3, 6 and 9 weeks Table 4, there were increases in the activity of AST and CK and concentration of cholesterol, total lipid and uric acid.

Table 2:	Growth changes in Bovans chicks fed diets containing C. cyminum or H. sabdariffa for 9 weeks
Values are means±SE. Means within column with no common letters are significantly different (p<0.05)

Table 3:	Haematological changes in chicks fed C. cyminum or H. sabdariffa for 9 weeks
Values are means±SE, Means within rows with no common letter (s) are significantly different (p<0.05)

Table 4:	Serobiochemical changes in chicks fed C. cyminum or H. sabdariffa for 9 weeks
Values are means±SE. Means within row with no common letter (s) are significantly different (p<0.05)

Serum enzyme activity and concentrations of cholesterol, total lipid and uric acid were also higher (p<0.05) in the chicks fed a diet containing 10% C. cyminum (group 3) or 2% H. sabdariffa (group 4) for 6 or 9 weeks than those in the control and chicks on 2% C. cyminum diet (group 2) no significant changes in ACP activity were observed in the serum of test chicks.

Pathological Changes
After 3 weeks of treatment, no significant macroscopical or microscopical changes were observed in the vital organs of the chicks on the 2% C. cyminum (group 2), 10% C. cyminum (group 3) or 2% H. sabdariffa diets (group 4). In the chicks fed a diet containing 10% H. sabdariffa (group 5), the microscopical examinations showed that the centrilobular hepatocytes and cells of renal proximal convoluted tubules contained fatty vacuoles. In this group, the lesions observed at weeks 6 and 9 of treatment included catarrhal enteritis, fatty cytoplasmic vacuolation or necrosis of the centrilobular hepatocytes and of the cells of renal tubules with accumulation of lymphocytes. These changes were also observed in the chicks of groups 3 and 4, but were less pronounced. No lesions were observed in the proventriculus, spleen or heart of the chicks of groups 3, 4 and 5 or in the vital organs of the chicks of group 2 or of the control group 1 throughout the 9-week feeding period.

DISCUSSION

In spite of the use of C. cyminum seed and H. sabdariffa calyx in Sudanese traditional medicine for the treatment of various ailments toxicological information is unavailable. The results of the present study indicated that feeding Bovans chicks with 2 and 10% H. sabdariffa or 10% C. cyminum of the normal diet is toxic as evidenced by growth impairment, of lesions in the vital organs and of haematological and serobiochemical alterations.

It is well known that susceptibility of animals to feeding plant material is dependent on the type of the active constituents and concentrations in the amount added to the diet as well as the rate of their metabolic conversion in the liver to metabolites and consequent excretion. For chicks, the dietary levels (2 and 10%) represented non-toxic concentrations of some plants and are exemplified by Nigella sativa (Al-Homidan et al., 2002). On the other hand, levels of 2% or more of dietary Abrus precatorius, Rhazya stricta and Ammi visnaga have been toxic to chickens and rodents (Omer et al., 1992; Adam 1999; Ibrahim et al., 2004).

No research has been done to investigate the safety of C. cyminum seed or H. sabdariffa calyx fed to chicks. The present study suggests the hazard associated with feeding of the calyx of H. sabdariffa may be related to the concentration and properties of the compounds in H. sabdariffa calyx as compared with those in C. cyminum seed. Phytochemical investigations of H. sabdariffa calyces have so far demonstrated the presence of flavone glycosides, alkaloids, anthocyanin pigments, amino acids, pectins, acids and probably other constituents (Chen et al., 2003; Odigie et al., 2003). Cuminum cyminum aerial parts contain flavonoid gycosides, essential oil rich in cuminaldehyde, α-β pinene, δ-3-carene, 1, 8 lineole, terpenes and sesquiterpenes (Ageel et al., 1987; Banerjee et al., 2002).

In the chicks fed diets consisting of 2 and 10% H. sabdariffa calyces or 10% C. cyminum seeds, the damage to the intestines, liver and kidneys could explain depression in growth. However, the mechanism whereby the plant constituents damage body tissues can not be derived from the present study, but the damage to these organs probably contributed to the increased serum AST and CK activity and cholesterol, total lipid and uric acid concentrations.

The anaemia is microcytic normochromic as indicated by the low MCV and normal MCHC values. These findings suggest that the plants constituent (s) may be involved in a derangement of the haemopoietic process.

Further investigations into the appropriate isolation, characterization and concentration of the active constituents in H. sabdariffa calyces and C. cyminum seeds are deemed necessary for elucidating their respective modes of action.