Experimental Site: The experiment was carried out at the Poultry unit of the Teaching and Research Unit of Directorate of University farms, Federal University of Agriculture, Abeokuta, Ogun State, Nigeria. The site is located in the rain forest vegetation zone of south – western Nigeria on Latitude 70 10’ N and Longitude 30 2’E and altitude of 76m above the sea level (Google Earth, 2020). The climate is humid, with a mean annual rainfall of about 1037mm and mean temperature and humidity of 34.70C and 83%, respectively.
Preparation of the extracts: the extraction methods were two: the fresh and air-dried leave extracts. The fresh leaf extraction was prepared as follow; 200 and 400 g of fresh leaves was blended with six litre of water respectively. The procedure for the extraction of air-dried leaf extract is as follows; fresh leaves of the plant were harvested and spread in a room for air- drying. Air-drying method was adopted to prevent loss of volatile oils when spread under direct sunlight. The air-dried leaves were milled and dissolved in water at 40 and 80g (an equivalent of 200 and 400g fresh leaves, respectively) per six litres of water respectively. The solutions were stirred every 30 minutes for 3 hours and allowed to stand 24 hours. After the 24 hours, the solution was sieved to get extract according to the procedure of Jesuwenu and Okozi (2017).
Management of Experimental Birds
A total of one hundred and ninety-five (195) day old chicks of Cobb 500 strain of broiler chickens was purchase from a reputable hatchery. The birds were randomly divided into five experimental groups. Each group was further sub-divided to three replicates of thirteen chicks per each. The groups were: control (use of synthetic antibiotics), extracts from 200 g fresh leaf, 400 g of fresh leaf, 40 g of air-dried leaf and 80 g of aid-dried leaf respectively. The extract was served to the birds at the rate of one-third of daily water intake throughout the experimental period expect a day prior and the day of vaccination. Water was served immediately the birds in each replicate finished the served extract. Brooding of chicks lasted for two weeks in individual pen with the use of charcoal pots and electrical bulbs as heat source and lightings. Synthetic antibiotic (enrofloxacin) was used for the birds in the control group. Commercial broiler starter diet was supplied for the first four weeks of the study while commercial broiler finisher diet was used for the last three week. Feed and water were supplied ad-libitum. The experiment lasted for seven weeks.
Data collection: Growth performance indices (feed intake, weight gain) were taken weekly while feed conversion ratio was calculated by dividing feed intake by weight gain. Blood samples were collected on the 28th (end of starter phase) and 49th (finisher phase) days of the experiment from each replicate. Blood sample of 2.0ml was drawn via wing vein puncture with hypodermic syringes into bottles containing an anticoagulant; Ethylene diamine tetra acetate (EDTA) for the determination of haematological parameters. Packed cell volume (PCV), haemoglobin (Hb) concentration, red blood cells (RBC) and white blood cells (WBC) and differential counts were determined according to the procedure of Schalm et al. (1975); mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) were derived by calculation from the RBC, Hb, PCV values as described by Jain (1986). Another 2.0ml of blood was drawn into labelled sample bottles without EDTA for serum biochemical indices determination. Serum was separated from the clot blood by centrifuge. Serum indices; Total protein (TP) and albumin values were determined by Biuret method. Globulin was calculated as the difference between total protein and albumin. Cholesterol was determined according to the procedures of Thu et al. (2011), urea and creatinine were determined by the method Harr (2006), serum enzymes (ALT, ALP and AST) actions were determined spectrophotometrically (McComb et al., 1983).
Collection of faecal and gut samples: On the 28th day of the experiment, black nylon was spread on the litter in each pen. Each pen was observed and the birds were allowed to roam freely over the nylon. The nylon was then withdrawn from the pen as soon as the feacal sample were collected on the nylon. Each sample collected was kept in a universal bottle. On the 49th day of the experiment a chicken of average weight of each replicate was picked, fasted overnight and slaughtered. The gut sample was aseptically collected from the slaughtered bird for all replicates. All samples were taken to the laboratory for analysis to determine bacterial identification and count following procedure outlined by Adhikari and Kwon, (2017).
Determination of phytochemicals, microbial count and identification
Gram-positive and negative bacteria species were examined as caecal contents were serially diluted and plated, then incubated at 37°C under a microaerophilic condition for 15-18 hrs. Diluted samples were cultured on nutrient agar. Subsequently, adapted Mueller Hinton Agar (MHA) was modified according to Al-blooshi et al. (2021) to develop MHA-C15 (MHA containing 15% volume/volume water extract of clove). Gram-negative bacterial pathogens such as Escherichia coli, Proteus mirabilis and Pseudomonas aeruginosa grew on MHA-C15. However, none of the major Gram-positive bacterial pathogens such as Streptococcus faecalis and Clostridium perfrigens grew on it. To isolate bacteria in caecum-ilium mucosa, as well as the faeces, samples were rinsed in sterile Phosphate Buffered Saline (PBS) of pH 7.4 after luminal contents were separated three times, and homogenized in 20 ml of PBS. Samples extracted were kept in cold storage and transported to the laboratory prior to subsequent analysis. Thereafter, Isolation and identification was carried out by observing colony morphology, grams staining and standard cultural and biochemical test.
Statistical Analysis: Data collected were subjected to One-way Analysis of variance using SAS (2010). The significant means were separated at p<0.05 using Duncan’s Multiple Range Test in the software package.