3.2 Materials and methods
3.2.3 Birds, treatments and their management
A total of 200 unsexed Ovambo chicks that were hatched from parent stock by the Agricultural Research Council (ARC), Irene, Pretoria, were raised together in a well-ventilated floor area under a deep litter management system at Cedara College of Agriculture where the research was conducted. Floors were adequately covered with wood shavings. A broiler starter meal was given ad libitum to the chickens from Day 1 to Day 49 and grower diet was given from Day 50 to 91.
The chemical compositions of the starter and grower diets are given in Table 3.2. Water was offered ad libitum in 4L plastic founts and feed was provided in tube feeder made of standard gutter materials. Light and heat using infra-red were provided continuously. The birds were vaccinated against Newcastle disease at 14 days of age. Gumboro vaccine was also given at 6 weeks of age. The vaccinations were administered orally by putting the vaccines in the drinking water of the chickens.
Two dietary treatments were used. The control, with low level of vitamin A (WM), was formulated with 100 % white maize. The high vitamin A (PABM) was formulated with 100 % PABM maize variety HP326-6 (Table 3.3). The PABM maize was obtained from the Makhathini Research Station, Jozini, KwaZulu-Natal, where it was planted in April, 2014. The aim of biofortification of maize with provitamin A was to increase the concentration of β carotene in the endosperm of the maize. Temperate maize has more β- carotene than the tropical maize (Yan et al., 2010). Therefore, the high provitamin A temperate maize sources was tropicalized to convert it to the popular African open pollinated varieties and inbred lines. The inbred line was planted at a spacing of 0.75 m between rows and 0.25 m between plants within a row
61 Table 3.2: Chemical composition of basal starter and grower diet
Composition Starter Grower
Crude protein (g/kg) 200 180
Metabolisable energy (MJ) 12.8 13.0
Fat (g/kg) 25.0 25.0
Crude fibre (g/kg) 50.0 60.0
Dry matter (g/kg) 880.0 880.0
Calcium (g/kg) 12.0 12.0
Lysine (g/kg) 12.0 10.0
Supplied by Smith Animal Feeds. Pietermaritzburg, South Africa ME/CP= Metabolisable energy/ crude protein
62 Table 3.3: Feed composition of experimental diets
Ingredients (Control-WM) kg PABM
Provitamin A biofortified maize 0.0 417.7
White maize 417.7 0
Soyameal 175.4 175.4
Vegetable oil 23.8 23.8
Limestone 12.3 12.3
Dicalcium phosphate 6.9 6.9
Salt 1.9 1.9
DL-Methionine 1.2 1.2
L-Lysine 0.1 0.1
Vit.-min. premix (excluding vit A) 3.2 3.2 Nutrient composition
Metabolisable energy (MJ/kg) 12.56 13.01 Crude protein (g/kg) 199 198
Fat (%) 3.52 5.09
Ash (%) 11.04 9.73
Calcium (%) 1 1
Phosphorus (%) 0.74 0.81
Provitamin A carotenoids(mg/kg) 0.1 0.5
One kg of feed contained the following:; cholecalciferol,60 mg; all-rac-_ tocopheryl acetate, 30 mg; menadione, 3 mg; thiamine, 22 mg; riboflavin, 8 mg; pyridoxine, 5 mg; cyanocobalamin, 11 mg; folic acid, 1.5mg; biotin, 150 mg; calcium pantothenate, 25 mg; nicotinic acid, 65 mg; Mn, 60 mg; Zn, 40 mg; I, 0.33 mg; Fe, 80 mg; Cu, 8 mg; Se, 0.15 mg; ethoxyquin, 150mg.
63 Fertilizer and field management practices recommended for optimum maize production were used (Azmach et al., 2013). The maize was grown in a secluded area to prevent cross pollination from other maize varieties. After the maize were harvested, it was dried sufficiently, shelled, bagged in vacuum sealed bags and stored in a cool, pest free and dark place to preserve the quantity and quality of the pro-vitamin A carotenoids.
The PABM and WM maize were milled separately into flour using a hammer mill (Glen Creston, Stanmore, England) fitted with a 1.0 mm sieve. The fat content of dry milled maize flour was determined using Soxhlet extraction method (Association of Official Analytical Chemists (AOAC), 1984) About 2 g maize flour was weighed into the Soxhlet cellulose extraction thimbles. The thimbles were plugged with a cotton wool to avoid loss of sample. The thimbles were transferred into the Soxhlet extractor. Sufficient petroleum ether was poured into the Soxhlet extractor beakers. The beakers were clamped into the Soxhlet machine and the electric heating plates were lifted to the base of the beakers. The samples were subjected to extraction for 3 hours. Recovered solvent was transferred into an air oven (100oC) for 1 hour and then cooled in desiccators and weighed. The amount of oil extracted was calculated and expressed as a percentage of the original sample.
To determine the crude protein, the total nitrogen content was determined by Kjeldahl nitrogen analysis, according to AOAC (1995). The percentage of crude protein was estimated by multiplying the total nitrogen content by a factor of 6.25. To determine the ash/ mineral content, dry samples of milled maize was weighed in to a dry porcelain dish and heated in a muffle furnace at 6000C for 12 hours after which it was placed in desiccators for cooling before it was
64 weighed. The percentage ash content was calculated as: % ash = weight of ash x 100/ weight of sample (AOAC, 1980). The gross energy values were estimated by multiplying the crude protein, fat and carbohydrate by their water values of 4, 9 and 4 kcal/g, respectively. Each sample was analyzed in triplicate. Calcium and phosphorus were determined by atomic absorption spectrophotometer method, and colorimetrically respectively according to AOAC (1984).
Carotenoid analysis was carried out using a HewlettPackard 1100 HPLC (Agilent Technologies Incorporated, Loveland, CO, USA) consisting of a binary pump, autosampler, column thermostat, diode array detector and ChemStation software (Revision B.03 02, Agilent Technologies Incorporated, Loveland, CO, USA).
The birds were acclimatised to their experimental pen environment for 14 days prior to the commencement of the trial. During this period, the birds were fed a common proprietary grower diet (Table 3.2). The pens were placed in open sided houses with cement floor on a deep litter system. The wood shavings were changed fortnightly or whenever there was water spillage.
At 13 weeks of age, 44 male and 58 female birds of similar body weight were selected from the 200 birds raised from day one, weighed and randomly placed in 16 pens (eight pens were allocated for each diet, four pens for males and four pens for the females). The initial individual weights of the birds were 1.5 ± 0.5 and 1.0 ± 0.5 kg for males and females, respectively. The pens were 230 cm long, 13cm wide and 120 cm high. Each experimental unit, represented by a pen, contained a minimum of five birds. A minimum of 15h light was provided daily throughout the experimental period. No antibiotic or growth promotant was administered. Water and feed
65 were given ad libitum. Water was provided in 4L plastic founts and the feed was given in a 10L plastic hanging feeders.