Effect of the levels of Azadirachta indica dried leaf meal as phytogenic feed additive on the growth performance and haemato-biochemical parameters in broiler chicks

Abstract

A study was conducted to evaluate three different levels of Azadirachta indica dried leaf meal, using diets supplemented with 0 (negative control), 1.25 g antibiotic/kg (positive control), 1.25, 2.5 and 5.0 g leaf meal/kg of feed. The chicks were randomly divided into 15 separate floor pens (each 10×15 feet) each comprising 20 chicks and three pens (replicates) per treatment group following completely randomised design. At 28 and 42 d BW and feed conversion ratio (FCR) were determined. At 42 d, five birds per pen were slaughtered to measure dressing percentage and lymphoid organs weight was also determined. Serum protein, serum glutamic pyruvic transaminase (SGPT), serum glutamic oxaloacetic transaminase (SGOT), alkaline phosphatase (ALP), serum cholesterol, tissue cholesterol, growth hormone, thyroxine and haematological indices were also determined. At both 28 and 42 d, birds fed diets supplemented with 2.5 g/kg of leaf meal had significantly greater BW and better FCR than those fed diets with 1.25, 5.0 g/kg of leaf meal and controls. There was no significant (p>0.05) difference between BW and FCR when birds fed diets 1.25, 5.0 g/kg of leaf meal and antibiotic (positive control) at any age. There was no significant (p>0.05) effect on mortality at any time during the study. There was no marked variation in dressing percentage between leaf meal supplementation and antibiotic at 28 d of age. However, birds fed diet with 2.5 g/kg of leaf meal had significantly (p<0.05) highest dressing percentage than birds fed diets of 1.25 or 5.0 g/kg of leaf meal and control groups at 42 d of age. The mean lymphoid organs weight of control birds was significantly lower than the birds fed on diets containing leaf meal and antibiotics. Serum total protein was increased (p<0.05) in birds fed leaf meal up to 2.5 g/kg as compared to controls, antibiotic and higher level of leaf meal. The activities of blood enzymes (SGPT, SGOT and ALP) in groups of all broilers supplemented with leaf meal and antibiotic was lower (p<0.05) than negative control group. Serum and tissue cholesterol significantly decreased (p<0.05) with increasing the levels of leaf meal. Non-significant difference was observed amongst controls (positive and negative) and low level of leaf meal. Birds fed on diets containing antibiotic and leaf meal up to 2.5 g/kg had higher (p<0.05) packed cell volume values, red blood cell counts, white blood cell counts, haemoglobin and erythrocyte indices than birds fed diets without leaf meal (negative control) and third dose of leaf meal. It may be concluded that A. indica leaf meal can be included in the diets of broiler chicks up to 2.5 g/kg without any deleterious effects on their performance, serum biochemical constituents and haematological indices. Moreover, the dietary supplementation of A. indica leaf meal may lead to the development of low-cholesterol chicken meat as demanded by health-conscious consumers.

Keywords:

• Azadirachta indica leaf meal
• performance
• broiler chicken
• haemato-biochemical indices

Introduction

Only quality feed together with proper hygiene, potable water and management can ensure the production of nutritious animal products with desired organoleptic properties (Saxena 2008). Phytogenic feed additives are plant-derived products (often also called phytobiotics or botanicals) used in animal feeding to improve the performance of agricultural livestock through amelioration of feed properties, promotion of the animals’ production performance and improving the quality of food derived from those animals. This class of feed additives has recently gained increasing interest, especially for use in swine and poultry, as can be derived from a significant increase in the number of scientific publications since 2000 (Windisch et al. 2008). This appears to be strongly driven by the ban on most of the antibiotic feed additives within the European Union in 1999 (European Commission 1999), a complete ban enforced in 2006, and ongoing discussions to restrict their use outside the European Union because of speculated risk for generating antibiotic resistance in pathogenic microbiota (Collignon et al. 2005). In this context, phytogenic feed additives are discussed possibly to add to the set of non-antibiotic growth promoters, such as organic acids and probiotics, which are already well-established in animal nutrition. Phytogenics, however, are a relatively new class of feed additives and our knowledge is still rather limited regarding their modes of action and aspects of their application. Phytogenic feed additives include medicinal plants/herbs, which are non-woody flowering plants known to have medicinal properties; spices, which are herbs with intensive smell or taste, commonly added to human food; essential oils, which are aromatic oily liquids derived from plant materials such as flowers, leaves, fruits and roots; and oleoresins, which are extracts derived by non-aqueous solvents from plant material (Jacela et al. 2010). The consumption and demand for medicinal plants as phytogenic substances have been adopted in many countries because of low cost, easy availability, affordability for a common farmer, good anti-microbial natured, reduced diseases associated risks, lowering blood cholesterol level and diversified functions in improving performance, growth rate, feed conversion rate and weight gain in birds (Lewis et al. 2003). Medicinal plants are used in pharmaceuticals, neutraceuticals, cosmetics and food supplements and even as traditional source of medicines because of their anti-tumer, anti-arthritic and anti-thrombotic functions (Thomson and Ali 2003). Furthermore, scientists and researchers are trying to combat against fatal diseases in poultry through the use of medicinal plants, containing the most active ingredients to promote growth, weight gain and immune-stimulant.

Many plants have beneficial multifunctional aspects which are derived from their specific bio-active components (Kamel 2000). The Azadirachta indica is a tropical evergreen tree native to Indo-Pak sub-continent (Anonymous 1985). Presently these trees can be seen growing successfully in about 72 countries worldwide, in Asia, Africa, Australia, North, Central and South America (Girish and Shankara 2008). Biologically active principles isolated from different parts of the plant include: azadirachtin, meliacin, gedunin, salanin, nimbin, valassin and many other derivatives of these principles. Miliacin forms the bitter principles of its leaves (National Research Council [NRC] 1992). These compounds belong to natural products called triterpenoids (Limonoids). The active principles are slightly hydrophilic, but freely lipophilic and highly soluble in organic solvents like, hydrocarbon, alcohols, ketones and esters (NRC 1992). Various parts of the tree have medicinal value (Chakraborty et al. 1989). The A. indica leaf exhibits a wide range of pharmacological activities viz., anti-inflammatory, anti-hyperglycaemic, anti-ulcer, anti-malarial, anti-fungal, anti-bacterial, anti-viral, anti-oxidant, anti-mutagenic anti-carcinogenic, immunomodulatory and various other properties without showing any adverse affects (Subapriya and Nagini 2005). However, this leaf meal like most leaf meals contains anti-nutritional factors (Opender et al. 2004) which may affect nutrient utilisation. These anti-nutritional factors may alter the blood profiles and also affect the linear growth of animals fed this leaf meal.

Ogbuewu et al. (2010, 2011) investigated the effect of dietary A. indica leaf meal on body weight gain, linear body measurements and blood chemistry of pre-puberal buck rabbits. They suggested that buck rabbits could tolerate up to 15% dietary inclusion of meal without deleterious effects on body weight gain, linear body measurements, reproductive tract morphometry and some haematological parameters. Recent studies by Esonu et al. (2006) have shown that A. indica leaf meal could be of some value in the diet of laying hens both as feed ingredient and egg yolk pigmenter. Despite these findings, there is a dearth of information on growth performance, haematological, serum biochemical and immunity constituents in broiler chicks. The present study was therefore designed to determine the effects of various levels of A. indica leaf meal fed to broiler on the performance and haemato-biochemical indices.

Materials and methods

Experimental birds

A total of 300-day-old chicks of a Hubbard strain, having 47.0 g average body weight, were obtained from a local hatchery where they had been vaccinated for Marek's disease and had received vaccinations for Newcastle disease and infectious bronchitis post-hatch via a coarse spray. The chicks were randomly divided into 15 separate floor pens (each 10×15 feet) each comprising 20 chicks and three pens (replicates) per treatment group following completely randomised design. Each pen contained one tube-type feeder and one bell-type automatic water fount. Birds were provided with ad libitum access to feed and water with 23L:1D. Care and management of the birds followed accepted guidelines (FASS 1999). The brooder temperature was maintained at about 95°F up to 7 days of age and gradually decreased to75°F by 21st day of age, after which the chickens were kept at room temperature.

Experimental diets

All diets were formulated to meet or exceed NRC (1994) recommendations for essential amino acids in starter (0–28 d) and finisher (29–42 d) feeding periods. The leaves of A. indica were collected from and around the University of Agriculture, Faisalabad, Pakistan. The leaves were spread evenly and sundried for four days until they become crispy while still retaining the greenish colouration. The leaves thereafter were milled using a hammer mill to produce a leaf meal. Sample of the leaf meal was then analysed to determine the proximate composition (AOAC 2011) and the minerals (the macro and the microelements) profile was analysed using atomic absorption spectrometry (Atomic Absorption Spectrophotometer, Perkin Elmer, Analyst 100, Operation Manual) and flame emission spectrophotometry (Jenway-PFP7/C) after digesting the sample with nitric acid-perchloric acid mixture.

The antibiotic (TM-200; Pfizer Co.) used in this study as positive control that contained Terramycin 200 g/kg. Diets of broiler starter and finisher were supplemented with 0 (negative control), 1.25 g antibiotic/kg of feed (positive control), 1.25, 2.5 and 5.0 g leaf meal/kg of feed. The leaf meal and antibiotic were mixed in corn gluten meal prior to addition to the diets to enhance mixing. Each diet was analysed as described methods in AOAC (2011) for proximate composition at feed testing laboratory of Poultry Research Institute, Rawalpindi. All analyses and determinations were done in triplicate. The composition of experimental diets for broilers was given in Table 1.

Table 1. Composition (g/kg) and calculated nutrient content of basal diets.

Ingredients	0–4 week	5–6 week
Corn	500.00	600.00
Rice broken	50.00	–
Corn gluten meal (60%)	20.00	20.00
Canola meal	80.00	64.00
Soyabean meal (47.5%)	300.00	240.00
Vegetable oil	–	30.00
Molasses	30.00	30.00
Marble chips	5.00	5.00
Dicalcium phosphate	10.00	5.00
Vitamin premix^1	2.00	2.00
Trace mineral mix^2	1.00	1.00
Choline Cl (60%)^3	1.00	1.00
L-Lys HCl (98%)	1.00	2.00
Total	1,000.00	1,000.00
Calculated analysis
ME (kcal/kg)	2895.65	3155.90
CP (%)	22.80	20.13
CF (%)	3.75	3.39
Ash (%)	7.17	6.39
Available phosphorus (%)	0.40	0.40
Lysine (%)	1.27	1.08
Methionine (%)	0.50	0.42
Met + Cys (%)	0.84	0.72
Sodium (%)	0.21	0.21
Chloride (%)	0.28	0.29
Lino (%)	1.16	3.04
^1Provides per kilogram of diet: vitamin A, 7,714 IU; vitamin D3 3000 IU; vitamin E, 16.53 IU; vitamin B12, 0.013 mg; riboflavin, 6.6 mg; niacin, 39 mg; pantothenic acid, 10 mg; menadione, 1.5 mg; folic acid, 0.9 mg; thiamin, 1.54 mg; pyridoxine, 2.76 mg; D-biotin, 0.066 mg; ethoxyquin, 125 mg; Se, 0.1 mg.
^2Provides per kilogram of diet: Mn (from MnSO4 to H2O) 100 mg; Zn (from ZnSO4 to 7H2O) 100 mg; Fe (from FeSO4 to 7H2O) 50 mg; Cu (from CuSO4 to 5H2O) 10 mg; I from Ca (IO3)2 to H2O), 1 mg.
^3Provides 1040 mg choline per kilogram of diet.

Parameter measured

Pen body weights were obtained at 7, 14, 21, 28, 35 and 42 d of age. Feed consumption was determined for the same time periods. Birds were checked twice daily; weight of dead birds was used to adjust for feed consumption. At 42 d, five birds per pen were randomly selected, slaughtered and eviscerated to evaluate to record carcass weights. Carcass weight was recorded after removing skin, head, feathers, lungs, feet and gastro-intestinal tract. Dressing percentage was calculated by dividing the warm carcass weight by live body weight of the bird and expressing the result as a percentage. The lymphoid organs (thymus and bursa) were weighed separately. Total protein was quantitatively measured based on colorimetric determination as described by Cannon (1974). Serum glutamic pyruvic transaminase (SGPT) and Serum Glutamic oxaloacetic transaminase (SGOT) were measured using UV visible spectrophotometer (Shimadzu Corp., Tokyo, Japan). SGOT and SGPT were estimated using the nicotinamide adenine dinucleotide dehydrogenase (NADH) oxidation reaction method (Neeley et al. 1985). The serum (0.1 mL) was added to 1.0 mL of auto-reagent and incubated at 37°C for 1 min. The absorbance was measured at 340 nm, and the values were expressed as unit L⁻¹. The activity of alkaline phosphatase (ALP) was determined by method described by Bergmeyer and Wanlefeld (1980).

Serum cholesterol was measured by using diagnostic kits (Sigma Diagnostics, Catalog No. 352, Sigma Chemical Co., St Louis, MO, USA) and spectrophotometer apparatus. The total cholesterol content of the tissues was determined by the method of Salé et al. (1984). For this purpose, small equal portions of meat from breast and thigh muscles were taken, weighed and homogenised with chloroform+ methanol (2:1) to a final 20-fold volume and it was filtered for cholesterol analysis. One millilitre of extracted mixture was equal to 0.05 g of meat. The other procedure was like serum cholesterol analysis. The thyroxine (T4) concentration in serum was determined by the radioimmunoassay (RIA) technique by using hormonal assay kits (ImmuChem^TM Thyroxine, ICN Biomedicals, Irvine, CA). The growth hormone (GH) was determined by OMNIA^® GH kit. The anti-coagulated blood was also used to determine red blood cell (RBC) count, packed cell volume (PCV), haemoglobin (Hb) concentration and white blood cell (WBC) count. Differential WBC counts were made on monolayer blood films, fixed and stained with Giemsa-Wright's stain. Total RBC and WBC count were determined manually by using haemacytometer (Campbell 1995). PCV was measured by a standard manual technique using microhematocrit capillary tubes centrifuged at 2500 rpm for 5 min. Haemoglobin concentration was measured by Cyanmethemoglobin method. Erythrocyte indices, that is, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentrations (MCHC) were calculated from total RBC, PCV and Hb (Ritchie et al. 1994), respectively.

Statistical analysis

All data were determined by using the SPSS version 16 (SPSS, Cary, NC, USA) statistical analysis program. A p-value of <0.05 was considered a significant difference among groups and the comparison of means was made using Duncan's Multiple Range Test (Steel and Torrie 1984).

Results

Composition of meal

The A. indica leaf meal had the nutrient composition of 9% moisture, 20.52% crude protein (CP); 16.45% crude fibre (CF); 4.25% ether extract (EE); 7.00% total ash and 42.78% nitrogen free extract (NFE). Leaf meal contained macro minerals (%) that is Ca (0.71), P (0.28), Mg (0.75), Na (0.58) and K (2.00) and micro minerals (ppm) that is Cu (34), Zn (18), Fe (745), Co (10), Mn (60), Cr (0.8) and Pb (27). The calculated nutrient contents in the experimental diets are shown in Table 1. The results of the present study can be used with confidence in evaluating possible effects of A. indica leaf meal on performance of the broiler.

Bird's performance

The effect of feeding broiler chicks with leaf meal at three supplementary levels is shown in Table 2. The level of leaf meal had significant (p<0.05) effect at both 28 and 42 d of age. At 28 and 42 d of age, birds fed diets supplemented with 2.5 g/kg of leaf meal had significantly greater BW than those fed diets with 1.25, 5.0 g/kg of leaf meal and controls. There was no significant (p>0.05) difference between BW when birds fed diets 1.25, 5.0 g/kg of leaf meal and antibiotic (positive control) at any age. There was no significant (p>0.05) effect on mortality at any time during the study. The mortality of the birds in the present trial was in the expected range and was not influenced by the dietary level of leaf meal. Feed efficiency (weight gain/feed intake) was significantly influenced by the treatments used at both 28 and 42 d of age. At both age, the feed efficiency improved significantly (p<0.05) in broilers fed diet had 2.5 g/kg of leaf meal compared with fed the diets of other levels of leaf meal and controls (Table 2). However, the feed efficiency of broilers fed diets with 1.25 or 5.00 g/kg of leaf meal and antibiotic (positive control) was similar. Values of dressing percentage in different groups indicated that there was no marked variation between leaf meal supplementation and antibiotic as positive control at 28 d of age. Statistical analysis showed insignificant (p>0.05) results in above parameter of broiler amongst different levels of leaf meal and antibiotic treatments at 28 d of age (Table 2). However, birds fed diet with 2.5 g/kg of leaf meal had significantly (p<0.05) highest dressing percentage than birds fed diets of 1.25 or 5.0 g/kg of leaf meal and control groups at 42 d of age. The mean lymphoid organs weight of control birds was significantly lower than the birds fed on diets containing leaf meal and antibiotics (Table 2).

Table 2. Effect of different dosage levels of Azadirachta indica leaf meal on BW, feed conversion, mortality, dressing percentage, thymus and bursa weight of broilers.

	BW (g)		Feed:gain ratio		Mortality (%)		Dressing (%)		Thymus weight (g/100 g)		Bursa of fabricius Weight (g/100 g)
Treatments	28 d	42 d	28 d	42 d	28 d	42 d	28 d	42 d	28 d	42 d	28 d	42 d
Negative control	945^c	1537^c	1.68^a	2.24^a	1.50	1.50	46.90^b	55.10^c	0.18^b	0.16^b	0.18^b	0.16^b
Positive control	987^b	1707^b	1.54^b	2.09^b	1.00	1.00	52.81^a	62.17^b	0.24^a	0.22^a	0.24^b	0.22^a
Leaf meal (g/kg)
1.25	967^b	1771^b	1.57^b	2.10^b	1.00	0.50	51.50^a	61.20^b	0.25^a	0.23^a	0.25^a	0.22^a
2.50	1012^a	1875^a	1.49^c	1.99^c	1.00	1.00	53.85^a	68.30^a	0.26^a	0.24^a	0.26^a	0.23^a
5.00	965^b	1770^b	1.59^b	2.10^b	1.50	1.00	53.28^a	62.10^b	0.24^a	0.22^a	0.24^a	0.22^a
^a–cMeans with different letters in column differ significantly (p ≤ 0.05).

Blood biochemical parameters

Serum total protein was increased (p<0.05) in birds fed leaf meal up to 2.5 g/kg as compared to controls, antibiotic and higher level of leaf meal (Table 3). The activities of blood enzymes (SGPT, SGOT and ALP) in groups of all broilers supplemented with leaf meal and antibiotic was lower (p<0.05) than negative control group (Table 3). Serum and tissue cholesterol significantly decreased (p<0.05) with increasing the levels of leaf meal (Table 3). Non-significant difference was observed amongst controls (positive and negative) and low level of leaf meal. The birds fed diet with 2.5 g/kg of leaf meal had significantly (p<0.05) higher concentration of GH and T4 than birds fed diets of 1.25 or 5.0 g/kg of leaf meal and control groups (Table 3). However, non-significant difference (p>0.05) was found between antibiotic and 1.25 or 5.0 g/kg of leaf meal groups.

Table 3. Effect of different dosage levels of Azadirachta indica leaf meal on serum protein, serum glutamic pyruvic transaminase (SGPT), serum glutamic oxaloacetic transaminase (SGOT), alkaline phosphatase (ALP), serum cholesterol, tissue cholesterol, growth hormone and thyroxine in broilers.

Treatments	Serum protein (g/dl)	SGPT (IU/l)	SGOT (IU/l)	ALP (IU/l)	Serum cholesterol (mg/dl)	Tissue cholesterol (mg/100 g)	Growth hormone (ng/ml)	Thyroxine (ng/ml)
Negative control	2.89^b	33.00^a	161.33^a	7190^a	187.00^a	82.13^a	0.25^c	0.15^c
Positive control	2.95^b	30.00^a	121.00^b	6205^b	186.70^a	81.80^a	0.29^b	0.18^b
Leaf meal (g/kg)
1.25	3.30^a	26.25^b	125.33^b	6195^b	180.00^ab	80.78^ab	0.30^b	0.20^b
2.50	3.46^a	25.00^b	118.00^b	6065^b	178.69^b	79.65^b	0.35^a	0.26^a
5.00	3.05^b	23.00^b	115.00^b	6000^b	171.00^c	76.73^c	0.31^b	0.20^b
^a–cMeans with different letters in column differ significantly (p ≤ 0.05).

Blood haematological parameters

Birds fed on diets containing antibiotic and leaf meal up to 2.5 g/kg had higher (p<0.05) PCV values, RBC counts, WBC counts, Hb, MCV, MCH and MCHC concentrations than birds fed diets without leaf meal (negative control) and third dose of leaf meal (Table 4).

Discussions

The results of proximate analysis of leaf meal in the present study were almost similar to findings of Esonu et al. (2006), who reported that the leaf meal contained 20.68% CP; 16.60% CF; 4.13% EE; 7.10% ash and 43.91% NFE. However, Obikaonu et al. (2011) reported lower values of proximate analysis of leaf meal (18.10% CP; 15.56% CF; 2.50% EE; 5.62% ash and 58.22% NFE) than the present study. Overall, the A. indica leaf meal displayed same characteristics as leaf meals from other tropical browse plants – high crude fibre and moderate CP content as reported for Jacaranda mimosifolia (Okorie 2006) and for Microdesmis puberula (Esonu et al. 2002). Mineral composition profile was balanced in Ca and P but exceptionally high in K and Fe. These results are in accordance with those of Singhal and Mudgal (1984).

Table 4. Effect of different dosage levels of Azadirachta indica on blood haematological values in broilers.

Dosage level (g/kg)	PCV (%)	RBC (mil/mm^3)	WBC (thousands/mm^3)	Hb (g/dl)	MCV (cubic micron)	MCH (micro-micro gram)	MCHC (%)
Negative control	22.00^c	1.29^b	50.45^b	8.00^b	139.50^b	47.00^b	23.89^b
Positive control	32.50^b	1.45^a	75.00^a	8.38^a	197.80^a	65.54^a	35.70^a
Leaf meal (g/kg)
1.25	33.00^b	1.47^a	70.67^a	8.40^a	199.63^a	63.90^a	34.31^a
2.50	40.00^a	1.56^a	80.00^a	8.50^a	211.66^a	69.72^a	39.36^a
5.00	30.67^b	1.30^b	57.00^b	8.20^ab	148.95^b	50.96^b	25.47^b
PCV, packed cell volume; RBC, red blood cell; WBC, white blood cell; Hb haemoglobin; MCV, mean corpuscular volume; MCH, mean corpuscular haemoglobin; MCHC, mean corpuscular haemoglobin concentrations.
^a–bMeans with different letters in column differ significantly (p ≤ 0.05).

Azadirachta indica leaf meal fed to broilers gave live performance levels similar to those of the antibiotic growth promoter, results that agree with Jamroz and Kamel (2002), who observed improvements of 8.1% in daily gain and 7.7% in feed conversion ratios in 17-day-old poults fed a diet supplemented with a plant extract. In the current study, daily weight gain at 42 d of age was improved from 3.61 to 8.96% in broilers fed diet supplemented with leaf meal than antibiotic (positive control). An increasing trend was found in body weight gain with better feed efficiency with increased levels of leaf meal infusion up to 2.5 g/kg of diet. The results of the present study are in agreement with the study of Manwar et al. (2007), who supplemented leaf powder @ 1–2 gm/kg feed and reported significant increase in the live body weight of broilers with improvement of feed efficiency in the leaf powder fed groups when compared with control group. Similar findings have been reported by Tipu et al. (2002), who used salinomycine and A. indica fruit as feed additive and anti-coccidial in broilers and reported better results in terms of weight gain. The increase in weight gain could be possibly due to the presence of macro and micro minerals in A. indica leaf meal. The deficiency of these macro- and micro-minerals results in anorexia, osteoporosis and retarded growth in birds (Ensminger 1980). The higher body weight gain in broilers consuming leaf meal infusion could also be due to its appetite- and digestion-stimulating, anti-bacterial and hepatoprotective properties (Wankar et al. 2009), which help to reduce the microbial load of birds and improved the feed consumption and feed efficiency of the birds. Thus, this plant could substitute the existing antibiotic growth promoter. The high level of leaf meal (5.00 g/kg) implied a reduction in growth rate in broilers could be attributed to the presence of anti-nutritional factor contained in leaf meals. Similar adverse effect on growth and feed efficiency was shown in White Leghorn chicks fed de-oiled neem seed meal at or above 5% level for 8 weeks (Subbarayudu and Reddy 1975). Salawut et al. (1994) also found that lowest dose level of neem leaf meal in the diet of rabbits was better for growth performance than higher levels. Ogbuewu et al. (2010) reported that different bio-active components of leaf meal may be responsible for depression in nutrient utilisation and growth in rabbits at higher level.

The mortality of the birds in the present trial was in the expected range and was not influenced by the dietary level of leaf meal. Anti-bacterial activity of A. indica leaf meal suppresses pathogenic bacteria including Staphylococcus aures, Mycobacteria, Salmonella paratyphi and Klebsiella pneumonia (Koul et al.1990). The virus inhibiting activities of A. indica leaf in Newcastle disease both in vitro and in vivo (Koul et al. 1990), Herpes virus (NRC 1992) and Pox virus (Rai and Sethi 1972) might have also helped in survivability and the better performance of leaf meal fed birds.

The dressing percentage was improved in birds fed on the leaf meals in the current study. The results are in line with findings of Singh et al. (2009), who used poly-herbal growth promoter in broiler chicken and found improved dressing percentages in birds fed herbs diets than control. In contrast of the above study, Elangovan et al. (2000) reported that neem kernel meal treated groups neither affected the carcass characteristics nor acceptability of meat of quails. This indicated that the bitter taste of triterpenoids was not imparted to the meat. The better growth caused improvement in dressing percentage of birds fed diet with supplementation of leaf meal.

The greater bursa and thymus weight in birds supplemented leaf meal as compared to the negative control suggests that leaf meal supported these lymphoid organs. The two lymphoid organs are responsible for recruiting B and T lymphocytes and make up the vital and basic components of humoral and cellular immunity. The present study showed that leaf meal potentiated immune response in the experimental broilers. Similar results obtained by Toghyani et al. (2010) found that black seed supplementation caused a marked (p<0.05) increase in the weight of lymphoid organs. However, further investigation is required for the elucidation of the mechanism through which leaf meal produced systemic increase in the immune response.

Serum protein elevated in birds fed leaf meal at 1.25–2.5% levels than controls. Serum protein actually depends on availability of dietary protein. This means that the proteins of the leaf meal diets were more available to the birds confirming the recently observation by Obikaonu et al. (2011). The results are in line with findings of Samarth et al. (2003), who reported that herbs increased the serum proteins as compared to control. However, in the third dose level of leaf meal, total proteins were lowest as compared to other levels. This might be due to toxic effect of this very high dose level of A. indica as reported by Ibrahim et al. (1992).

Serum cholesterol levels were observed to decrease progressively with increasing dietary levels of A. indica leaf meal in this present study. This fall in serum cholesterol level of broilers fed leaf meal diets probably suggest a general decrease in lipid mobilisation. This could be that leaf meal has indirect inhibitory effects exerted at the levels of HMG-CoA reductase, a key enzyme in cholesterol biosynthesis. This suggests that leaf meal diets were capable of reducing serum cholesterol, thereby helping to reduce the deposition of cholesterol in the skin and muscles. This equally implies that A. indica leaf meal should be used to produce animal product with reduced cholesterol content. The reduced serum content of total cholesterol may reflect the hypocholesterolemic properties attributed to the defatted part of the leaves which are rich in fibrous content and may block intestinal cholesterol absorption (Ghazalah and Ali 2008). The reduction in the cholesterol level of broilers are in agreement with the earlier findings of Upadhyay (1990) and Ogbuewu et al. (2010), who reported that neem leaf meal in the diets of broiler birds, rats and rabbits resulted to a decrease in the cholesterol and liver lipid levels. Chattopadhyay (1996) reported that neem leaf meal contained different compounds that is Quercetin-3-O-β-D-glucoside, Myricetin-3-O-rutinoside, Kaempferol-3-O-β-D-glucoside, Quercetin-3-O-glucose and L-rhamnoside. It is presumed that these compounds either partially or wholly may be responsible for anti-hyperlipidermic activity of A. indica leaves. Similar trend was observed in cholesterol concentration in bird's tissue. Nagaraja Kumari et al. (2006) reported significant reduction in thigh muscle cholesterol by Amaranthus leaf meal (A. tricolor) in broiler. Lanjewar et al. (2009) also found that supplementation of tulsi leaf powder at the rate of 1% in broiler diet for 42 days reduced meat and blood cholesterol levels of broiler.

In the assessment of liver functioning after supplementation of leaf meal, SGOT, SGPT and ALP were determined. In this study, decreased in the activities of SGOT, SGPT and ALP in serum evidenced the positive effect of leaf meal on liver parenchyma of the birds. The SGOT is a cytoplasmic enzyme while SGPT is found in both cytoplasmic and mitochondria. According to Bhatti and Dil (2005), alteration in serum enzymes activity under stress conditions occur due to malfunctioning of liver, as degenerating and necrotic cells leak enzymes from cytoplasm. In this study, birds were quite healthy as shown low concentration of liver enzymes and low mortality occurred. The non-hepatotoxic nature of A. indica was proved in the study performed by Haque et al. (2006), who found unaltered and normal activities of serum SGOT, SGPT, ALP as well as retained architecture of liver after A. indica treatment. The aqueous extract of A. indica leaves was found to offer protection against paracetamol induced liver necrosis in rats (Bhanwra et al. 2000). The elevated levels of serum SGOT and SGPT indicative of liver damage were found to be significantly reduced on administration of the leaf aqueous extract. A. indica leaf meal is a promising hepatoprotective agent and this protective activity of leaf meal may be due to its anti-oxidant and normalisation of impaired membrane function activity (Mohamed et al. 2010). Moreover, the A. indica leaves contained quercetin and rutin compounds which are the most frequently studied bioflavonoid in the class of flavonols (Chattopadhyay 1998). It is well-established that quercetin, one of the most abundant flavonoids, is a more potent anti-oxidant than other anti-oxidant nutrients such as vitamin C, vitamin E and β-carotene (Rice-Evans et al. 1995).

The concentration of GH and T4 is significant in the birds fed second dose (2.50 g/kg) level of A. indica leaf meal. The circulating concentration of GH has been positively correlated with growth rate which is required for the normal development of chicken (Kikuchi et al. 1991) and high growth rate was recorded in birds fed leaf meal at 2.50 g/kg level. The increase in growth hormone level might be due to the presence of amino acids particularly arginine present in A. indica leaves (8.5%; Rao 1987); that provides a regularity system which results in secretion of growth hormone and ultimately facilitate uptake of amino acids in proteins (Muller et al. 1999). Sunanda and Anand (1998), who studied the effect of root extract of Withania somnifera on functions of thyroid in cockerel and observed increased serum T4 concentration suggesting its prothyroidic action. Similarly, Ghazalah and Ali (2008) reported that rosemary leaf meal stimulated thyroid function, as evidenced by increased plasma levels of T4 as compared to control in broilers. Increased T4 concentration in the treated group clearly indicates that leaf meal stimulates and/or release of T4 in the gland. Another possibility is that the leaf meal may be stimulating hypothalmohypophyseal system to secrete of thyrotropin releasing factor and or thyroid stimulating hormone ultimately stimulating the thyroid gland.

Haematological parameters are good indicators of the physiological status of birds and its changes are of value in assessing the response of birds to various physiological situations (Khan and Zafar 2005). In the current study, all values of haematological parameters fall in between the levels described by Aiello and Mays (1998). The higher (p<0.05) significant value of PCV, Hb and RBC indices (MCV, MCH and MCHC) of the birds on leaf meal diets relative to the control group is an indication that the birds were not anaemic. The PCV of birds across all the treatment levels were within the normal range (30–40%). The Hb values at all treatments levels were within the reported range of 9–13 g/dl. The high concentration of Hb in birds fed diets leaf meal might be due to hepato-stimulatory and hepatoprotective effects of leaf meal resulting in the synthesis of more Hb in the bone marrow which is under the control of erythropoietic factors released by hepatic cells (Browman et al. 1976). The values of WBC in birds possess phagocytic function and used as an indicator of stress response and sensitive biomarkers crucial to immune function. The results of the WBC in the present study clearly points to the fact that the birds on 5 g/kg leaf meal were stressed hence the significant (p>0.05) reduction in WBC. This tends to confirm the report of Talebi et al. (2005) that nutrition affects the blood profiles of birds and this implies that up to 2.5% inclusion of leaf meal had a positive effect on the relative quantity of blood cell as well as total volume of blood.

Based on the results, it may be concluded that A. indica leaf meal can be included in the diets of broiler chicks up to 2.5 g/kg without any deleterious effects on their performance, serum biochemical constituents and haematological indices. The use of antibiotics in boilers should be discouraged this can be replaced with A. indica leaf meal. Moreover, the dietary supplementation of A. indica leaf meal may lead to the development of low-cholesterol chicken meat as demanded by health-conscious consumers.