The experimental protocol was approved by the Animal Care and Use Committee of Dankook University.
Experimental design, animals and diets
Bacteriophages used in our study contain
Salmonella (
S.)
gallinarum,
S. typhimurium, and
S. Enteritidis at the ratio of 3:3:4. The concentration of bacteriophages is 10
8 plaque forming unit per gram. A total of 720 1-d-old male broilers (Arbor Acres) were used in a 32-d experiment. Birds were randomly allotted to 1 of 4 treatments in a completely random block design. The bacteriophage was administrated by replacing the same amount of corn. Each dietary treatment consisted of 12 replicate cages, with 15 broilers per replicate. Dietary treatments included: i) CON (basal diet), ii) ANT (CON+0.5 g antibiotics/kg feed (bacitracin methylene disalicylate)), iii) BP1 (CON+0.25 bacteriophage/kg feed), and iv) BP2 (CON+0.5 g bacteriophage/kg feed). All the chicks were kept in a battery brooder with three levels of stainless steel cages (124 cm-width×64-cm length×40-cm height), with 8 adjacent cages per level. Each cage was equipped with 2 drinker nipples and 2 open trough feeders. The basal diet was formulated to meet or exceed all the nutrient requirements of broilers (
NRC, 1994), and supplied in mash form. There were two nutritional phases, including starter (1 to 14 d) and finisher phase (15 to 32 d), and the ingredients and calculated nutrient composition of the basal diet are shown in
Table 1. The temperature of the battery brooder was maintained at 33±1°C during the first week and decreased by 3°C per week until reaching 24°C. Artificial light was provided 24 h/d by the use of fluorescent lights. All diets were fed in mash form with feed and water being provided
ad libitum throughout the experimental period.
Sampling and measurements
Broilers were weighed by pen and feed intake (FI) was recorded weekly. This information was then used to calculate BW gain (BWG) and feed conversion ratio after correcting mortality (FCR).
On day 24 d, broilers were fed the respective diets containing 0.20% chromic oxide (Cr
2O
3) as an indigestible marker for 4 d prior to the collection period on 28 d. The total excreta of the broilers were collected for 3 d. All feed and fecal samples were immediately stored at −20°C until analysis. The fecal samples were dried for 72 h at 70°C and finely ground to allow for passage through a 1-mm screen. The procedures utilized for the determination of coefficient of total tract apparent digestibility for dry matter (DM), gross energy (GE) and nitrogen (N), in accordance with the methods established by the
AOAC (2000). Chromium levels were determined via UV absorption spectrophotometry (UV-1201, Shimadzu, Kyoto, Japan) and the apparent total tract digestibility (ATTD) of DM, N, were calculated using indirect methods described by
Williams et al. (1962). The gross energy was determined by measuring the heat of combustion in the samples using a Parr 6100 oxygen bomb calorimeter (Parr instrument Co., Moline, IL, USA).
On d 32, 24 broilers were randomly selected from each treatment (2 birds per cage) and blood samples were collected from the brachial vein into a sterile syringe. After collection, sample was aliquoted into both a vacuum (clot activator with gel) and K3EDTA vacuum tube separately (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ, USA) and stored at −4°C. Samples for serum analysis were then centrifuged at 3,000×g for 15 min, and then separated and stored at −4°C. The white blood cells (WBC), red blood cells (RBC), and lymphocyte percentage were analyzed using an automatic blood analyser (ADVIA 120, Bayer, NY, USA).
After blood collection, the same broilers were weighed individually and killed by cervical dislocation and exsanguinated. The breast muscle (pectoralis major), bursa of Fabricius, liver, spleen, and abdominal fat were then removed by trained personnel and weighed. Organ weights were expressed as a relative percentage to the whole body weight. The breast muscle Hunter lightness (L*), redness (a*), and yellowness (b*) values were determined using a Minolta CR410 chromameter (Konica Minolta Sensing Inc., Osaka, Japan). The pH of the breast muscle sample was measured by a calibrated, glass-electrode pH meter (WTW pH 340-A, WTH Measurement Systems Inc., Ft. Myers, FL, USA).
At d 32, excreta samples were collected from each pen and pooled and placed on ice for transportation to the laboratory, where analysis was immediately carried out. The composite fecal sample (1 g) from each pen was diluted with 9 ml of 1% peptone broth (Becton, Dickinson and Co.) and homogenized. Viable counts of bacteria in the fecal samples were then determined by plating serial 10-fold dilutions (in 1% peptone solution) onto MacConkey agar plates (Difco Laboratories, Detroit, MI, USA) and lactobacilli medium III agar plates (Medium 638, DSMZ, Braunschweig, Germany) to isolate Escherichia coli (E. coli) and Lactobacillus, respectively. The lactobacilli medium III agar plates were then incubated for 48 h at 39°C under anaerobic conditions. The MacConkey agar plates were incubated for 24 h at 37°C. Escherichia coli and Lactobacillus colonies were counted immediately after removal from the incubator. For Salmonella, the serially diluted peptone broth tubes were incubated overnight at 37°C, after which 1 ml was transferred to 9 ml of tetratinate broth (Neogen Corporation, Lansing, MI, USA) and then incubated for 48 h at 42°C. From these tubes, 1 ml was used to inoculate 9 ml of Rappaport Vassiliadis broth (Neogen Corporation, Lansing, MI, USA) and incubated for 48 h at 42°C. The Rappaport was used to inoculate XLT4 plates for Salmonella isolation, and the Salmonella was, then, presumably identified using LIS and TSI agar tubes (Difco Laboratories, Detroit, MI, USA).