Procurement and processing of Pleurotus ostreatus
Oyster mushroom used in the present study was procured from Department of Horticulture, University of Agriculture, Faisalabad, Pakistan. The mushroom specimen was identified by an expert botanist from Department of Botany, Government College University, Faisalabad. Six hundred grams of the fresh mushroom was used for proximate analysis. The remaining mushroom after washing with distilled water, was shade dried under sun light and grinded to form fine powder. After the qualitative and quantitative analysis of amino acids, the mushroom powder was used for the isolation of proteins and ethanolic and aqueous extracts preparation to be used in the experimental trial in broiler poultry feed.
Proximate analysis
Proximate analysis was done according to the procedure described by Agrahar-Murugkar and Subbulakshmi 2004 (21). Briefly, 5g of the mushroom sample was placed in oven at 105 ºC for 5 hours, the loss of moister was calculated and expressed as percent moisture. Soxhlet extraction method was used for quantification of fat content using the solvent petroleum ether. Ash was estimated by incineration of known quantity of powdered mushroom sample at 550 ºC in electric furnace. Carbohydrates were quantified by using anthrone reagent (21).
Aqueous and Ethanolic Extract Preparation
The dried mushroom powder was subjected to ethanolic and aqueous extraction. The dried sample (500 g) was extracted with 2000 ml solvent, distilled water and absolute (95%) Ethanol at room temperature for 24 h and filtered through Whatman filter paper No. 4. Both the ethanolic and aqueous residues were re extracted twice and the filtrates were collected to evaporate almost to dryness in a rotary evaporator (Scilogex RE-100 pro; USA) at 40 °C. The dried extracts were stored at 4 °C to be used for clinical trial (22).
Amino acid analysis of Aqueous and Ethanolic Extracts
Amino acids present in the mushroom extracts were quantified by using the Amino Acid Analyzer (TSM–1 Technicon Instrument Basingstoke, UK). Norleucine was used as standard.
Protein Isolation
Proteins were isolated from the dried fruiting bodies of the mushroom Pleurotus ostreatus by following the previously described method (8). Briefly, 100g of mushroom powder was dissolved in 0.15M sodium chloride at and soaked overnight at 4°C. After centrifugation for 15 min at 8000g, 1M ammonium sulfate was added to almost 80% saturation. After a second centrifugation, extra NH4)2SO4 was removed by dialyzing twice with PBS. The extracted proteins were finally purified by ion exchange chromatography by using DEAE-cellulose column which was equilibrated with PBS with N-acetyl, D-Galactosamine (12.5mM). The quantification of the extracted proteins was done by using Bradford’s method (23) with bovine serum Albumin (BSA) as standard protein with six different concentrations (0-30 μg/ml).
Clinical Trial
Four hundred and twenty, day old broiler birds (Ross-308) were procured from a commercial hatchery, Faisalabad and reared at the Animals Experiment station, Department of Physiology, Government College University Faisalabad, Pakistan. Chicks were reared under standard management practices. All the chicks were kept on mash feed ration. The birds were randomly distributed into seven groups (N=60) with three replicates in each group with 20 number of birds in each replicate (n=20) (table. 1). First group was selected as control and other six groups Pr200, Pr400, Aq200, Aq400, Eth200, and Eth400 were supplemented orally with Mushroom protein at dose of 200mg/kg BW, 400mg/Kg BW, Aqueous extract of mushroom at dose of 200mg/Kg BW, 400mg/Kg BW and ethanolic extract of mushroom at dose of 200mg/Kg BW, 400mg/Kg BW respectively. The dose of different treatments was measured daily on pen-based weight measurement to cope with the daily weight gain of the birds. A complete basal diet was formulated for each of the 3 stages of growth: starter, grower, and finisher (table 2). Water was provided ad libitum to all the birds in each of the 7 groups. Weight of birds in all groups along with food consumption were recorded at 3rd, 5th and 7th week interval to assess the performance profile and FCR of the birds in all the groups.
On day 28th of the experiment, 1st replicate of each group was assessed for cell mediated immunity by assessing the lymphoproliferative reaction to Phytohemagglutinin-P (PHA-P; Toe web assay) (24) and humoral immune reaction was evaluated in the 2nd replicate of each group by antibody reaction to sheep red blood cells (RBCs; Hemagglutination assay) (25); (26); (27).
Sampling
At the end of the experiment at day 42 a total of 6 birds from control group and each sub-group of all the treatment groups were slaughtered (decapitation) for blood collection. During the experiment, the blood was taken from jugular vein in EDTA tubes for hematology and clot activator tubes for serum separation. Serum was separated and stored at -20 ºC for biochemical analysis related to oxidative stress, lipid profile and immune parameters.
Toe web Assay
At day 28th of the experiment, 1st replicate of each group was injected 0.1ml of PHA-P (Sigma®, USA; 100μg/100μl) and PBS in the toe web between 3rd and 4th digit of right and left foot respectively. Thickness of toe web skin of injected feet of birds was measured before the injections and after the 24, 48 and 72 hours of injections, using micrometer screw gauge. Final values of lymphoproliferative reaction to PHA-P was measured by means of following equation:
Lymphoproliferative reaction = (PHA-P reaction, right foot) – (PBS reaction, left foot).
Hemagglutination assay
Sheep RBCs were used as T-dependent antigen to evaluate the antibody reaction. Antibodies IgG (mercaptoethanol-resistant) and IgM (mercaptoethanol-sensitive) were estimated in the treatment groups compared with the control group (25). At day 28th of the experiment, 2nd replicate of each group was injected intramuscular with 5% suspension of sheep RBCs (1ml). At 7th day of injection, a 2nd dose of sheep RBCs was injected. Anti-sheep RBCs immunoglobulin titers were identified by hemagglutination assay in the serum of experimental birds (26); (27).
Total Antioxidant Capacity, (TAC, mmol Troloxequiv. L-1)
Nisar et al. (28) described the method to evaluate the total antioxidant capacity (TAC) in the serum samples. Briefly it’s a colorimetric assay based on bleaching of ortho dianisidine color in the assay reagent by the presence of antioxidants in the sample. Increased level of antioxidants in the sample leads to the increased bleaching and decreased absorbance which shows an inverse standard curve. Semi auto analyser Biolab® 320 was used adjusted on bichromatic wavelength (660 and 870 nm) by calibrating vitamin C standards at concentrations of 1, 3, 5, and 7 mmol/L. The minimum measurable value of this assay was 0.18 mmol · L-1 and the linearity was up to 7 mmol vitamin C equivalent · L-1, with an intra assay coefficient of variance (CV) below 3% (29); (30).
Total Oxidant Status (TOS; µmol H2O2 equiv. L-1)
Methodology for measuring the serum TOS was adopted as described by Nisar et al. Calibration curve was prepared from different concentrations of hydrogen peroxide (H2O2) and TOS was expressed as µmol of H2O2 equivalent/l. The precision value of the assay was up to < 3% and Intra assay coefficient of variance (CV) was kept under 10 %. The linearity of the assay was up to 200 μmol H2O2 equivalent /l (31); (30).
Paraoxonase activity (U/L)
The method described by Juretic (2006) was used for the estimation of paraoxonase activity in the serum samples. The activity of the enzyme paraoxonase was calculated by using the formula described in the reference methodology and expressed in Unit/min/l. Intra assay CV was below 10 % and a reaction rate/hydrolysis rate of paraoxon was stable up to 5 min. The minimum level of activity for this assay ranged from 80 to 100 U/min/l (32); (30); (33).
Arylesterase Activity (KU/L)
The enzymatic activity of arylesterase was evaluated by following the procedure as previously adopted by Juretic, 2006. The enzymatic activity of the arylesterase was calculated by using the prescribed formula given in reference method. Intra assay CV was below 7% and initial rate of hydrolysis of paraoxon was stable up to 5 min.(32); (30); (33).
Statistical Analysis
One-way analysis of variance (ANOVA) and Tuckey range tests were adopted to analyze the statistical significance of results of weekly weight gain, Feed conversion ratio (FCR) and other biochemical analysis related to oxidative stress and lipid profile, using SPSS®, 23 software. Data on antibody reaction to sheep RBCs was evaluated by geometric mean titer (GMT). Value of p<0.05 was considered statistically significant.