Experimental animals
This research was performed in accordance with of the animal ethics rules of the Faculty of Veterinary Medicine, University of Sadat City. Research study was carried out in the animal facility of the previously mentioned faculty. Ninety, one-day old, Cobb chicks of both sexes have been obtained from El-Arabia Poultry Company. Water and feed were ad libitum provided. Light, naturally, and artificially, was kept 24 hours per day for 2 week, then one hour of darkness was applied daily.  Wood shaving was used as bedding material. Strict sanitation practices were applied throughout the experiment. Chicks of all the groups were immunized against infectious bronchitis (IB) strains HIB120 and Newcastle disease (ND) at day 9 of age and against infectious bursal disease (Gumboro) at day 13 of age. There was no vaccination applied during the experimental period (day 14 – 35).

Experimental design
Chicks were assigned into three groups, 30 birds each, at day 14 of age. The first group received a standard basal diet (Starter 23% CP 2900 kcal, Grower 20% Crude Protein 3000 kcal, and Finisher 18% CP 31000 kcal)²¹ without any treatment and was used as control group (CON). The second group received a diet mixed with methanolic Moringa oleifera leaf extract (MOLE, 250mg/kg feed, MOLE250) from day 14 to day 35 of age. The third group received a diet mixed with MOLE 500mg/kg feed (MOLE500) from day 14 to day 35 of age. All the chicken groups were reared in the same building with identical managing and environmental conditions.

Preparation of Moringa oleifera leaf extract
Fresh Moringa oleifera (MO) leaves were purchased from a farm at Sadat City, Menoufia governorate, Egypt. For 7 days, fresh MO leaves were collected and shed dried at room temperature (22°C) with relative humidity of 65 %. The dry leaves have been ground, filtered, and drenched in 70 % methanol (50g/L). The extract remained for 24 h with soft shaking at ambient temperature (22°C). Through Whatman filter paper size No.1, extract was then filtered, and the solvent was evaporated by air current for 24 h. Finally, it was entirely dried out in a hot air oven at 40°C for 24 h. The extract was kept in a sealed bottle at 4°C until use²².

Sample collection
At the end of the experiment (day 35), chickens were overnightly fasted and euthanized according to the Islamic method of slaughtering. Bleeding of chickens were for 2 min, scalding at 54°C for 2 min, defeathering manually, and evisceration. Six animals per group were randomly selected and sampled. Breast was dissected from the left half of the carcasses for further analyses by collecting the part from the caudal ending of the sternum headed for a shoulder joint on either side of every carcass and by dragging the breast aside from the back and shattering the link at the shoulder joints. Each breast sample was divided into four equal parts and kept stored in plastic storage bags at 4°C for further analyses at days 0, 2, 4, and 6 post-mortem.

Determination of sensory parameters
Meat samples were evaluated at day 0, 2, 4, and 6 post-mortem. Sensory attributes and organoleptic characterization of raw meat of broiler breast were determined by a rating scale from 1 to 3 presented in Table 1²³. The examined sensory characteristics were visual appearance (meat and skin color), meat texture, odor, and elasticity. Five qualified judges were trained in the method of tests on raw chicken meat. Also, another five trained judges re-examined the suggested organoleptical method of raw meat assessment. To acquaint the panel with various intensities for the various sensory characteristics of raw chicken meat, and to adapt a scoring scale that should be used, two samplings of 1.5h each have been carried out. The sensory profile utilized was received from the previous two sessions opened. Assessments were carried out in a sensual testing room equipped with the positive air pressure, personal stations, and appropriate illumination²⁴.

Table (1):
Sensory attributes and quality of raw, refrigerated chicken breast.

Meat composition
Crude fiber, protein, fat, water, and ash of broiler breast muscle were determined in accordance with AOAC methods²⁵. Moisture has been determined by dry up 1 g of meat in a furnace at 100-105°C till a steady weight has been obtained. Raw protein was determined in accordance with the Kjeldahl method. The raw protein had been obtained as 6.25×N%. Lipid content of the meat was determined in accordance with the Soxhlet extraction method utilizing petroleum ether. Ash content of the meat was determined by burning the sample in a muffle furnace at 550°C for 3h. Crude fiber was determined in accordance with boiling sample with 1.25% diluted H₂SO₄, rinsed with water, further boiled with 1.25% diluted sodium hydroxide and the residual deposit after the digestive process had been taken as crude fiber.

Determination of meat quality
Determination of pH of the breast muscle
Determination of pH values was performed using a pH Measuring Device (Germany, Testo 205) by penetrating breast muscle.

Determination of Total Volatile Nitrogen (TVN) of the breast muscle
The TVN of the breast muscle was determined according to Conway and  Byrne²⁶. In a clean dry beaker, 10g of the sample were added to 30 ml of distilled water and thoroughly mixed by a blender for 2 min. Thus, 2 drops of 0.02 M HCl were added to bring the pH value to 5.2. The homogenate was slowly heated to 70°C and then cooled to room temperature and filtered into the inner compartment of Conway dish and then 2ml of 0.01M HC1 were added. The outer ring was filled with 2ml of the sample extract and 1ml of saturated potassium carbonate (KCO3). The Conway unit was rotated as gently as possible and the dish was covered and incubated at 36°C for two hours, HCl in the inner ring was titrated against 0.01M NaOH by using methyl red indicator (T₁ ml).  TVN/l00g = 26.88 x (2-T₁), Where T₁ = volume of NaOH expended in the titration.

Determination of Thiobarbituric Acid Number (TBA)²⁷
TBA test is based on determination of malonaldehyde (MDA) values as a product of lipid peroxidation. The degree of oxidative rancidity is usually stated as TBA number and stated as milligrams of malonaldehyde counterparts per kilogram of samples. A sample of ten grams was mixed with 50ml of distilled water in warring blender for 2 min. The mix was quantitatively transported into Kjeldahl flask by wash with an extra 47.5ml of distilled water. Though, 2.5ml of HCL were added to 125ug/g fat and then a small number of stones was sited to stop pumping. The flask was heated at the maximum heat reachable in the Kjeldahl distillation device. Approximately, 10 min after the boiling were essential to gather 50ml of the concentrate.  Therefore, 5ml of the concentrate were mixed to 5ml of TBA reagent in 50ml glass stoppered tube. The contents were immersed and mixed in a boiling water bath for 35 min. Like samples, a distilled water TBA reagent blank was intended and handled.  After boiling, cooling under tape water for 10 min was applied. The sample optical density alongside the reference liquid (blank) was measured by using Spectrophotometer (UNICAM969AA Spectronic, USA) at a wavelength of 538 nm. Concentration of malonaldehyde (mg/kg) = Rx7.8, Where R = the absorbance.

Determination of Peroxide Value (PV)
The peroxide value has been determined according to Asakawa et al.²⁸. The titre of the sodium thiosulfate solution was determined, where the precise normality of Na₂S₂O₃ realizing that in this chemical reaction 1gram-equivalent of Na₂S₂O₃ react with 1gram-equivalent of K₂Cr₂O₇ (6moles Na2S2O3 react with 1mole K₂Cr₂O₇) was determined. Peroxide value determination by titration with sodium thiosulfate until blue colour disappeared. The titration procedure was repeated as a minimum 3 times and the single findings should not differ greater than 0.3ml.  For all samples, PV was evaluated by the formula: PV = (V₁–V₀) x T x 1000/m (ml equivalent available oxygen/kg). Where, V₁= volume of thiosulfate solution needed to titrate the sample (ml), V₀= volume of thiosulfate solution essential to titrate the blank, T= sodium thiosulfate solution titre (normality) and m= sample weight (g).

Determination of Free Fatty Acids (FFA)
Lipid was extracted by using 1 part of the inspected sample to the original extract ratio of 20 parts of 2:1 dichloromethane/methanol²⁹. A final ratio of 8:4:3 dichloromethane/ methanol/ water was achieved, by adding a weak salt solution (0.66% NaCl), containing the water included inside the tissue. Titration procedure³⁰ was modified to the sample nature and quantity. The sample fat extract was vaporized with nitrogen and dissolved in previously neutralized ethanol with cresol purple and 60°C preheated. Three repeats of sample were used titrated with N NaOH to a violet end point Cresol purple has been planned to enhance the sensitivity of the technique and has been used to a titration of lipid fish extracts³¹. In this case, 0.05N NaOH had been used for the titration of an extract to a violet end point cresol purple. Consequently, FFA values were registered as a proportion of oleic acid which equivalent (ml NaOH X NaOH normality X 28.2) / sample weight.

Determination of heavy metals
The standard, blank and digest solutions were aspirated by Atomic Absorption Spectrometer (AAS, VARIAN, Australia, model AA240 FS) and analyzed for content of heavy metal. Investigation was carried out by air/acetylene flow (5.5/1.11/m) flame for Cd, Cu and Pb concentrations (mg/kg wet weight)²⁵. Detection limit (ppb) 8-40 for Pb, 0.2-0.8 for Cd and 11-25 for Cu. The three heavy metal levels were verified from the digital scale of AAS and intended rendering to the equation: Metal concentration (ppm) C=Rx(D/W), where C= heavy metal concentration (wet weight), R= digital scale reading of AAS, D= prepared sample dilution and W= sample weight. Each heavy metal concentration in the blank solution was also determined and subtracted from each examined sample³².

Bacteriological examination
Samples were prepared for bacteriological examination according to ICMSF³³. In brief, 225ml of sterile peptone water were added to 25g of the sample, and completely blended utilizing sterile stomacher for 1–1.5 min.

Bacterial screening
E. coli screening
Immediately after slaughter, samples were screened for E. coli according to ISO³⁴. Concisely, 1 milliliter from the initial dilution, was injected into MacConkey broth tubes supplemented by inverted Durham’s tubes. The injected tubes remained at 37°C for 24 hours. From positive MacConkey tube, 1 milliliter was injected into one more MacConkey broth tube and developed at 44°C for 24 hours. Loopfuls from the positive MacConkey broth tubes, were individually streaked onto Eosin Methylene Blue agar medium (EMB), which was then developed at 37°C for 24 hours. The presumed colonies were seemed like metallic green in color. Into slope nutrient agar tubes, the presumed colonies were then inoculated and purified for further recognition.

Salmonella screening
Salmonella screening was carried out according to the method described by Harvey and Price³⁵. In brief, one milliliter of the initial dilution was injected into nine milliliter Rappaport Vassilidis broth tube, and then the tube was stored at 43°C for 24 hours. On Xylose lysine desoxychoclate (XLD) agar medium, loopfuls from the inoculated tubes were individually streaked and developed at 37°C for 24 hours. The presumed colonies were looked as red with or without black centers. On nutrient agar plates, the presumed colonies were then reared and sub-cultivated at 37°C for 24 hours. On slope nutrient agar, the purified suspected colonies were selected and streaked for further recognition.

Staphylococcus aureus screening
Staphylococcus aureus screening was performed according to ICMSF³³. On duplicated plates of Baired Parker agar (BPA) and using a sterile glass spreader, 0.1ml from formerly ready dilution was spread. At 37°C for 48 hours, the injected and control plates were incubated. On nutrient agar slopes, the black colonies surrounded with clear zone have been chosen up and purified for further proof of identity.

Morphological, biochemical, and serological identification
According to Cruickshank et al.³⁶, morphological examination has been conducted, while biochemical identification of E. coli was conducted according to the Krieg and Holt³⁷. According to Kok et al.³⁸, isolates were serologically recognized by using rapid diagnostic E. coli antisera sets (DENKA SEIKEN Co., Japan). The presumptive colonies of Salmonella were further subjected to biochemical tests; indole, methyl red, Voges-Proskauer and Citrate test (IMViC) as per the standard test protocol described in Bacteriological Analytical Manual FDA³⁹. Salmonellae were serologically identified according to Kauffman-White scheme⁴⁰ for the determination of flagellar (H) and Somatic (O) antigens using Salmonella antiserum (DENKA SEIKEN Co., Japan). Staphylococcus aureus was tested for production of catalase, free coagulase, yellow pigment, and thermonuclease (TNase) according to Lancette et al.⁴¹.

Statistical analysis
Data obtained in this research were analyzed by using SAS statistical software (Version 9.3, SAS Inst., Cary, USA), data obtained in this research were investigated. Heavy metal concentration and meat composition data were analyzed with simple ANOVA and Tukey’s post-hoc test. Quality traits and Sensory data were analyzed by ANOVA using the MIXED procedure with fixed factor group and repeated factor storage day (with autoregressive covariance structure) and group x storage day interaction.  The Tukey-Kramer correction was used to manage the Family-wise Error Rate (referred to as Padj). The results are presented as LSmean ± standard error (SE). Differences were deemed to be significant if Padj≤0.05.