This study was conducted to determine the effect of dietary Moringa oleifera leaf (MOL) supplementation of feed on performance, antibody response, haematological and biochemical parameters of broilers challenged with a very virulent infectious bursal disease virus (vvIBDV). Fresh MOL was collected from Potiskum, Yobe State and air dried for five days, ground into powder using a milling machine and analyzed for nutrients and elements using standard method of the Association of Official Analytical Chemists. The phytochemical constituents analysis of MOL was done using the method described by Sofowora. Two hundred and forty day-old Ross 308 hybrid broiler chicks were randomly assigned into four groups (A, B, C and D) of 60 chicks each and raised in a deep litter house consisting of 4 separate compartments. All birds were fed with broiler starter (BS) from 0 to 28 days of age and broiler finisher (BF) from 29 to 49 days of age. Broiler starter (22% crude protein CP) and BF (20% CP) mash were formulated each with 5% MOL included as part of the feed ingredient for broilers in groups A and B while BS and BF for broilers in groups C and D were formulated without MOL. Only birds in groups A and C were vaccinated intramuscularly with 0.5 ml of an inactivated intermediate strain infectious bursal disease (IBD) vaccine at 14 and 21 days of age. Inactivated Newcastle disease virus (NDV) vaccine (Komorov strain) was also administered (0.5 ml) intramuscularly at 18 days of age to groups A and C. Birds in groups A, B and C were challenged intraocularly at 35 days of age with vvIBDV while those in group D served as control. Daily feed intake (DFI), average daily weight gain (ADWG), feed conversion ratio (FCR) and selling price (SP) per bird were determined for each group. Five birds were randomly selected and euthanized from each group on 35, 38 and 42 days of age and the bursa of Fabricius, thymus, spleen and Harderian glands were removed for evaluation of organ body weight index. Blood was collected from ten broilers in each group via the wing vein on 14, 21, 35, 38 and 42 days of age to determine the IBD antibody titre level, haematological and biochemical parameters. At 49 days of age, five birds from each group were slaughtered and their carcasses weighed to assess the performance of birds fed with MOL supplemented diets. The results of the nutrients analysis showed that MOL contained carbohydrate (55.14%), CP (25.9%), crude fibre (13.91%), moisture (7.94%), fat (5.85%), ash (3.72%) and energy (2930.63 Kcal/Kg). The phytochemical analysis of MOL revealed phytates (2.57%), tannins (2.19%), saponins (1.06%), oxalates (0.45%) and cyanides (0.1%). The elemental analysis on MOL revealed Ca (2.26%), P (0.35%), Mg (0.45%), K (1.9%), Na (0.11%), Zn (34 ppm), Cu (7.5 ppm), Mn (40.5 ppm), Fe (116.5 ppm) and Se (0.85 ppm). Broilers in group D correlated more strongly (Pearson correlation = 0.921; P = 0.000) in their feed intake than those in groups B (Pearson correlation = 0.875; P = 0.000), C (Pearson correlation = 0.863; P = 0.000) and A (Pearson correlation = 0.862; P = 0.000). Broilers in groups A and B had weaker correlation (Pearson 0.379 108; P = 0.273). Broilers from groups D, A and B had a higher selling price/bird (N 1,151 ± 82.82, N1,093 ± 54.11 and N935.9 ± 70.69, respectively) than those in group C (N908.3 ± 63.97). There was an increase in the enzyme linked immunosorbent assay (ELISA) IBD antibody titre level of broilers between 21 and 35 days of age in groups A (1,379.89 ± 829.98 to 2,836.83 ± 463.58), C (1,576.94 ± 566.51 to 3,290.51 ± 848.87) and D (1,542.43 ± 106.80 to 2,953.49 ± 561.88), and up to 42 days of age in broilers of group B (1,205.94 ± 612.32 to 3,193.10 ± 478.52). Moringa oleifera leaf feed supplementation improved bursa (1.4, 1.4), spleen (1.3, 1.1) and Harderian gland (2.0, 2.0) organ to body weight index of broilers of groups A and B. Feeding broilers with 5% MOL supplemented diet without vaccination (group B) did not prevent vvIBDV from causing a decrease in lymphocyte count from 3.87 ± 1.52 to 2.67 ± 1.26 on day 3 pi. Supplementation of broiler feed with 5% MOL and vaccination with inactivated IBD vaccine did not prevent a decrease in PCV (28.60 ± 1.77 % to 21.80 ± 3.46 % and 25.22 ± 2.28 % to 21.44 ± 1.33 %) and RBC (2.44 ± 0.44 x 1012/L to 2.01 ± 0.42 x 1012/L and 2.25 ± 0.26 x 1012/L to 1.79 ± 0.52 x 1012/L), but caused an increase in Hb (10.55 ± 2.21 g/dl to 10.78 ± 1.95 g/dl and 9.81 ± 0.97 g/dl to 11.39 ± 1.17 g/dl) concentration on day 3 pi with vvIBDV in groups A and C. There were respective increase in aspartate aminotransferase and alanine aminotransferase levels in groups A (39.90 ± 3.96 IU L-1 to 45.10 ± 5.70 IU L-1and 42.90 ± 3.21 IU L-1 to 49.60 ± 3.56 IU L-1), B (39.80 ± 3.68 IU L-1 to 48.50 ± 4.22 IU L-1 and 43.30 ± 3.83 IU L-1 to 54.20 ± 5.53 IU L-1) and C (38.40 ± 3.20 IU L-1 to 42.80 ± 4.02 IU L-1 and 42.70 ± 4.80 IU L-1 to 48.60 ± 4.45 IU L-1). However, similar increase were observed in group D following challenge with vvIBDV (41.80 ± 3.85 IU L-1 to 45.30 ± 5.64 IU L-1 and 44.20 ± 4.52 IU L-1 to 51.60 ± 3.69 IU L-1). Supplementing broilers feed with MOL without vaccination against vvIBDV could not prevent lipid peroxidation (from 1.37 ± 0.23 IU-1 to 1.51 ± 0.30 IU-1) in broilers of group B following inoculation with vvIBDV. Supplementing broilers feed with MOL maintained the level of Na+ concentration in broilers of group A (140.00 ± 2.79 mg/dl to 139.90 ± 2.69 mg/dl) and B (140.10 ± 2.51 mg/dl to 138.30 ± 2.50 mg/dl) following inoculation with vvIBDV. Feed millers are encouraged to create awareness among poultry farmers on the nutritional and health benefits of MOL inclusion in the diets of broilers. Broilers fed with MOL supplemented diets can be vaccinated by farmers since it has no adverse effect against immune response to IBD.



1.1 Background Information

Moringa oleifera belongs to the single genus monogeneric family Moringaceae and is indigenous to Northwest India, but at present, it is widely distributed in the tropics throughout the Pacific region, Central America, the Caribbean, as well as West Africa (Ramachandran et al., 1980; Freiberger et al., 1998; Foidl et al., 1999; Makkar and Becker, 1999; Lockett et al., 2000; Aregheore, 2002). In Nigeria, this plant is commonly and widely cultivated in the Northern region of the country and is known by most ethnic groups in the country (Anjorin et al., 2010). Moringa Oleifera is also referred to as Horseradish tree, Radish tree, Drumstick tree, Mother‘s best friend (English); Gawara, Konamarade, Rini maka (Fulfulde); Zogale, Bagaruwa maka (Hausa); Ikwe oyibo (Igbo); Ewe ile, Ewe Igbale, and Idagbo monoye (―the tree which grows crazily‖) (Yoruba) (Lowell and Sreeja, 2001).

The demand for Moringa oleifera products is on the increase due to the consideration of the tree as one of the world most useful plants known for its nutritional, medicinal and significant economic importance. The leaves, fruits, flowers and immature pods of the tree are edible and they form part of traditional diets in many countries and posses several nutrients, including; calcium, magnesium, potassium, iron, vitamin A, vitamin C and a crude protein content that varies from 16 to 40% (Foidl et al., 2001; Marcu and Pharm, 2005; Rweyemamu, 2006). The Moringa oleifera leaves (MOL) have been reported to be a valuable source of both macro- and micronutrients, rich source of β-carotene, protein, vitamin C, calcium and potassium and act as a good source of natural antioxidants; and thus enhance the shelf-life of fat-containing foods (Dillard and Bruce German, 2000; Siddhuraju and Becker, 2003).

Moringa oleifera leaf has been reported to contain a high pepsin and total soluble protein, which makes it more suitable for monogastric animals such as poultry (Kakengi et al., 2007). The use of MOL as a supplement can improve voluntary intake, digestability and performance (Aregheore, 2001). Moringa oleifera leaves are known to be very poor in anti-nutritional factors and have been used in poultry feeding with various performance results depending on their nutritional value and inclusion level in the diet (Foidl et al., 2001; Sarwatt et al., 2002; Kaijage et al., 2003; Soliva et al., 2005; Kakengi et al., 2007; Nuhu, 2010; Olugbemi et al., 2010a; 2010b).

In Nigeria, the state of nutrition of the populace is predominantly marked by inadequate protein intake both in quantity and quality (Taiwo et al., 2005). Food and agriculture organization (FAO, 1992) recommended 27 g as the animal protein daily requirement for human beings. However, the intake per average Nigerian is grossly inadequate (3.24 g animal protein/day) (ILCA, 1980; FAO, 1986; FAO, 1992). Broiler production has been suggested as a means of massively producing animal protein because of its short generation interval and high growth rate (Larry, 1993; Dipeolu et al., 1996; Nworgu et al., 2000; Essen et al., 2005), since they are prolific, possess a high feed conversion ratio and are accepted by all, irrespective of religion. Broilers are chickens reared from chick to slaughter weight in eight weeks (Smith, 2001). Broilers have several desirable nutritional characteristics such as high protein, low lipid content and high polyunsaturated fatty acids; these make them preferable, health wise, when compared to red meat (Mothershaw et al., 2009).

Infectious bursal disease (IBD) has been considered as one of the important naturally occurring viral diseases of commercial chicken threatening the poultry industry worldwide (Müller et al., 2003). In Nigeria, IBD was reported by Ojo et al. (1973), Onunkwo (1975) and Okoye (1984). The dreaded nature of IBD has rendered investment in poultry to be fearful and unrealistic to both organizations and individuals (Okoye, 1983; Abdu, 1986; Lukert and Saif, 1997; Shane, 1997). Shane (1997) and Sainsbury (2000) reported the disease as a setback to productivity and profitability in the poultry industries of both developing and industrialized nations. Infectious bursal disease is caused by an infectious bursal disease virus (IBDV) (Dobos, 1979; Kibenge et al., 1988; Lukert and Saif, 2003) that is non-enveloped and has a bisegmented double-stranded RNA (dsRNA) genome (Kibenge et al., 1988; Da Costa et al., 2003). Infectious bursal disease virus is a member of the Birnaviridae family (Dobos, 1979).

The diagnostic application of haematology and biochemistry in human and veterinary medicine is a well established procedure (Ross et al., 1976). The present organisation within the poultry industry, where large numbers of individuals of low genetic variance are maintained in controlled environments, present an ideal situation for the use of clinical chemistry. The circumstances are unique in that clinical examination and clinical chemistry estimates can readily be combined with postmortem examination on individuals sacrificed to complement disease investigation (Ross et al., 1976). The introduction of rapid micro methods and automated line analyses to haematology and biochemistry has helped in the development of clinical chemistry and ―profile‖ studies in poultry (Ross et al., 1976).

Lipid peroxidation is a complex process occurring in aerobic cells and reflects the interaction between molecular oxygen and polyunsaturated fatty acids via a free radical chain mechanism, forming fatty acyl hydroperoxides, generally called peroxides or primary products of oxidation (Rasooli, 2007). The primary auto-oxidation is followed by a number of secondary reactions which lead to degradation of lipids and the development of oxidative rancidity (Ladikos and Lougovois, 1990). Lipid peroxidation is one of the primary causes of quality deterioration in meat and meat products, as it largely contributes to colour and flavour deterioration, loss of nutritional value and safety and generates compounds that may be detrimental to consumers (Min et al., 2008). The degree of lipid peroxidation is often used as an indicator of reactive oxygen species (ROS) mediated damage (Kuun and Borchert, 2002) and the concentration of MDA in blood and tissues are generally used as biomarkers of lipid peroxidation (Sehirli et al., 2008; Yousef et al., 2009).