Experimental site
The experiment was carried out at the Poultry unit of the Directorate of University Farms (DUFARMS) Federal University of Agriculture, Abeokuta, Alabata, Ogun State, Nigeria following the approved guidelines for Animal Research by Nigeria Institute of Animal Science in Nigeria (NIAS). The site is situated in the derived savanna zone of South-western Nigeria on Latitude 7o9' 39N and 3o20' 54E and 76m above sea level. The Mean Annual Rainfall is 1040mm and occurs from March to October, the temperature average is 34oC throughout the year.
Test Ingredients And Processing
The test ingredients which are dried Ethiopian pepper (Xylopia aethiopica) and clove (Syzgium aromaticum) were purchased from Kuto market in Abeokuta Ogun State, Nigeria. The properly dried fruit of clove and Ethiopian pepper were milled to particle size 0.2mm using a Kenwood blender and stored in plastic containers before diet formulation and commencement of feeding trial.
Chemical Composition Of Test Ingredients
3g ground samples of the test ingredients were taken and analysed for proximate composition (AOAC, 2005). Gross energy was estimated using the adiabatic bomb calorimeter (Model 1261; Parr Instrument Co., Moline, IL, USA). Analysis of mineral nutrients was determined according to the method by Kumari et al. (2017). The quantitative analysis was carried out by using inductively coupled plasma atomic absorption emission spectroscopy (ICP-AES). Samples were also analysed for mineral contents using atomic absorption spectrophotometer (Perkin Elmer Optima 4300DV ICP spectrophotometer, UK). The phytochemical constituents were analysed following standard procedures; total polyphenol (Wright et al., 2000), flavonoid (Arvouet-Grand et al., 1994), extractable tannin (Hoff and Singleton, 1977) and saponin content (Edeoga et al., 2005).
Experimental Birds And Management
Three hundred and sixty (360) unsexed Ross broilers were purchased from Agric International Technology and Trade Limited (AGRITED) hatchery in Ibadan, Nigeria. The necessary biosecurity measures were observed before the arrival of the broiler chicks. The birds were reared on a deep litter system, with wood shavings provided as bedding for the birds. Feeding trays and water troughs were made available for the birds. External heat (electric bulb and charcoal) was provided in the poultry pen.
Experimental Design And Dietary Treatment
The broilers were allotted on a weight equalisation basis to four dietary treatments using a completely randomised design. Each treatment has six replicates with 15 birds per replicate. The formulated diet consists of a control (Diet 1) (without Phytogenic supplementation), Diet 2 (supplemented with 1% Ethiopian pepper (EP)), Diet 3 (supplemented with 1% Clove (CL)) and Diet 4 (supplemented with 1% mix of equal quantity (0.5% each) of Ethiopian pepper and clove (EPCL)). The phytogenic additive was added to the basal diet and thoroughly mixed to ensure uniform distribution. The diets were formulated (Table 1) for starter (0–28 days) and finisher phases (29-56days) based on the nutrient requirement guide for Ross 308 (Ross Broiler Nutrition Specifications 2016). The experiment lasted for eight weeks and all birds were raised intensively on a deep litter system with a spacing of 1m2 per bird while feed and water were supplied ad-libitum during the experimental period. No in-feed antimicrobials and anti-coccidial drugs or antibiotics were fed to the birds throughout the duration of the study.
Table 1
Composition of experimental diet
Ingredients (%) | Starter (0–28 days) | Finisher (29–56 days) |
Maize | 54.00 | 63.00 |
Soya meal | 24.00 | 19.00 |
Wheat offal | 13.00 | 10.00 |
Fish meal | 3.00 | 2.00 |
Bone meal | 3.00 | 3.00 |
Oyster shell | 2.00 | 2.00 |
*Vitamin/Mineral Premix | 0.25 | 0.25 |
Salt | 0.25 | 0.25 |
Lysine | 0.25 | 0.25 |
Methionine | 0.25 | 0.25 |
Total | 100 | 100 |
Determined nutrients (%) | | |
**Metabolised energy (kcal/kg) | 2,827.80 | 3,078.80 |
Crude protein | 23.41 | 20.41 |
Ether extract | 3.62 | 3.32 |
Crude fibre | 3.33 | 3.21 |
Calcium | 1.34 | 1.25 |
Phosphorus | 0.66 | 0.61 |
*Starter Premix: Vit. A 10,000,000 (iu), Vit D3, 2,000,000 (iv), Vit. 23,000(mg), Vit.k3 2000 (mg), Vit. B1, 1800 (mg), Vit. B2 5,500 (mg), Niacin 27, 500mg, Pantothenic acid 750mg Vit B6 3,000mg, Vit B12 15mg, folic acid 750mg, Biotin H2 60mg, chlorine chloride 300,000 mg, Cobalt 200mg, Copper 300mg, Iodine 1000mg, Iron 20,000mg, Manganese 40,000 (mg), Selenium 200mg, Zinc 30,000mg, Anti-oxidant 1,250mg.
*Finisher premix-Vit. A. 5,500,000 (iu), Vit D3. 1500,000 (iu), Vit E. 10,000 (mg), Vit.k3 1,500 (mg), Vit. B1, 1,600 (mg), Vit. B2 24,000 (mg), niacin 20,000mg, pantothenic acid 5,000mg vit B6 1,500mg, Vit. B12 10mg, folic acid 500mg, Biotin H2 750mg, chlorine chloride 175,500 mg, cobalt 200mg, copper 300mg, iodine 1,000mg, iron 20,000mg, manganese 40,000 (mg), selenium 200mg, zinc 30,000mg, anti- oxidant 1,250mg.
**Estimated using the Nutrient Requirement of Poultry, NRC (1994) formulae, ME = 26.7 (%Dry matter) + 77 (%Ether extract) − 51.22 (%Crude fibre)
Apparent Total Tract Nutrient Digestibility
A digestibility trial was conducted on days 28 and 56 of the study to determine the apparent total tract nutrient digestibility. A bird per replicate (6 birds /treatment) was randomly selected and housed separately in appropriate metabolism cages fitted with individual feed troughs, water troughs and facilities for separate excreta collection. The birds were acclimatized for 2 days prior to the commencement of 5 days collection period. A known weight of feed (slightly above the respective daily requirements) with the addition of 0.5% titanium dioxide as an indigestible marker was fed to the birds housed in individual metabolic cages. Excreta collected per bird per day (collected twice daily) were stored in air-tight bags and stored at -180C till analysed. The frozen excreta per bird were thawed, pooled for each treatment, oven dried (60oC) for 48h, and then ground to 0.5 mm size. The concentration of titanium dioxide in diets and excreta was measured according to short et al. (1999). The proximate composition of feed and dried excreta samples was determined using the standard method of AOAC (2005). The apparent nutrients digestibility was calculated according to Maynard et al. (1979) using the equation
Gut Microflora
At day 56, 1 bird per replicate was selected and slaughtered for the collection of two sets of intestinal contents. Fresh digesta from the small intestine (from the distal end of the duodenum to the ileo-cecal junction) were collected and emptied into labeled sterile bottles. Fresh cecal content collected from a pair of ceca of the selected broiler was also collected in a different labeled sterile bottle. Samples collected were analysed for the estimation of gut microbiota according to the methods of Xia et al. (2004). One gram of the sample was dispersed in a 9 mL phosphate buffered saline solution with 0.5 g/L of Cys. HCl, and further diluted to a factor of 10 − 8. 0.1 mL of diluted sample was spread onto a Petri dish containing selective media for the enumeration of bacteria. The ileum content samples were incubated with Wilkins-Chalgren agar (Merck GmbH, Darmstadt, Germany) + novobiocin (8mg/L) + colistin sulphate (8mg/L) at 37°Cfor72h for estimation of clostridium counts, ES agar (MerckGmbH, Darmstadt, Germany) at 37°C for 24 h for estimation of coliform counts, de Man Rogosa Sharpe agar (Merck KGaA, Foster City, California, United States) at 37°C for 72 h for estimation of lactobacilli count, and brilliant green agar (Merck Ltd, Mumbai, India) at 37°C for 24 h for estimation of salmonella count. Microbial counts were expressed as colony-forming units (CFU) of microorganisms per gram of fresh sample.
Carcass Yield
At d 56, 1 bird per pen whose weight is representative of the average weight of broilers in each pen was selected, slaughtered, de-feathered, and eviscerated using standard procedures (Jensen, 1984). The body weight and dressed weights were measured, while the dressing percentage was calculated. The cut parts, which are the head, neck, breast, back, thighs, drumsticks, and shanks, were weighed and recorded as relative weights (percentage of body weight). The organs which are the kidney, lungs, gizzard, Pancreas, gastrointestinal tract (GIT), liver, heart, thymus, bursa, and spleen, were collected, weighed, and expressed as percentages of body weight.
Meat Microbiology
At d 56, 1 bird per replicate was selected and slaughtered for the collection of breast meat samples. The meat samples were stored in the refrigerator at 4 to 6oC for 3 days. After 3 days, the meat samples were removed and allowed to thaw and meat samples were analyzed using methods by Domanska and Rozanska (2003). Using sterile spoons, 10g of breast meat was weighed aseptically into a sterile blender jar. About 90ml of sterile Butterfield's phosphate diluent or Buffered Peptone Water (BPW) was added and blended for two minutes. From the prepared homogenate about 10 serial dilutions were prepared by diluting 1 ml of homogenate with 9 ml of 0.1% peptone water and spread plated (0.1 ml) on a range of enumeration agar plates: Wilkins-Chalgren agar (Merck GmbH, Darmstadt, Germany) for estimation of clostridium counts, MacConkey agar (MAC) for coliforms; plate count agar (PCA) for heterotrophic bacteria (total aerobic plate counts); De Man Rogosa Sharpe agar (Merck KGaA, Foster City, California, United States) for estimation of lactobacilli count, and brilliant green agar (Merck Ltd, Mumbai, India) for estimation of salmonella count. The agar plates were incubated (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 370C for 24 hours. Microbial counts were expressed as colony-forming units (CFU) of microorganisms per gram of fresh sample.
Statistical analysis
Data obtained were subjected to one-way analysis of variance using a completely randomised design. The pen was used as the experimental unit for the statistical analysis. The data were analysed using the ANOVA procedure of SAS (2000). Mean separation was done using the Tukey test of SAS to determine the effect of phytogenic supplementation. Significant differences were considered at P < 0.05