Estimation of Protein and Productive Eciency Prole of Locally Produced Oyster Mushroom (Pleurotus Ostreatus) in Broiler

Background: Pleurotus ostreatus (Oyster) is a worldwide used mushroom, known to have a wide range of nutrients. Effects of the ethanolic and aqueous extracts of Oyster and its isolated proteins on the production proles and bio health markers in broiler birds were evaluated in the present research. Methods: Four hundred and twenty, day-old Ross-308 broiler birds were distributed into seven groups with sixty number of birds in each group (N=60). Each group was divided into three subgroups with twenty number of birds in each subgroup (N=20). The rst group was selected as control (CONT) with no treatment and other six groups were supplemented orally with the mushroom protein at a dose of 200 (Pr200) and 400mg/Kg BW (Pr400), an aqueous extract of mushroom at a dose of 200 (Aq200) and 400mg/Kg BW (Aq400) and ethanolic extract of mushroom at a dose of 200 (Eth200) and 400mg/Kg BW (Eth400) respectively. On day 28 th of the experiment, 1 st sub-group of each group was evaluated for cell-mediated immunity by assessing the lymphoproliferative reaction to Phytohemagglutinin-P (PHA-P; Toe web assay) and the humoral immune reaction was evaluated in the 2 nd subgroup of each group by antibody reaction to sheep red blood cells (sRBCs; Hemagglutination assay). On day 42, serum was separated by slaughtering (decapitation) all the birds and evaluated for the oxidative stress, lipid prole, blood glucose level, and immune parameters. One-way analysis of variance (ANOVA), Tuckey range tests, and geometric mean titer (GMT) was used to statistically analyze the results. Results: The Eth400 birds showed signicantly (P<0.05) higher cell-mediated immune response (1.1±0.04 mm) and humoral immune response (49.31±0.03 mm) in comparison with the control group. Birds supplemented with mushroom extracts and protein revealed signicantly greater growth performance, maximum in Eth400, in terms of feed conversion ratios (FCR;


Background
Poultry diet with speci c inclusion of mushroom is one of the substitutes attaining much importance among the poultry farmers and nutritionists, because many of the biologically active compounds like proteins, polysaccharides and polysaccharide-protein complexes have been separated from the mushrooms and analyzed for their antimicrobial, antiparasitic, immune enhancing and antiviral activities (1). Polysaccharides and many other bioactive compounds in the mushrooms have been evaluated and considered to have useful effects on growth performance and immune responses (2). One of the active compound, A xylose lectin which is isolated from Xylaria hypoxylon showed remarkable anti tumour activities and antimitogenic (3). Similarly, Zhang et al., 2009 separated lectin from the Pholiota adiposa through DEAE cellulose ltration method followed by carboxy methyl cellulose and a fast protein by using liquid chromatography gel ltration on the superdex 75 resulted in excellent antitumor effect against mammary hepatomas and tumor (4). Various studies have been performed in the past decade to isolate proteins responsible for the inactivation of ribosomes, from Flammulina velutipes, and Calvatia caelata Hypsizigus marmoreus and these proteins work by removing one or more than one of adenosine residue from rRNA. Various therapeutic effects have been observed by these proteins including antitumour, immunomodulatory and antifungal, activities (5). An antiviral substance laccase has been separated from Pleurotus ostreatus (6). In all the therapeutically active compounds isolated so far, protein related compounds like antifungal, immunomodulatory proteins, ribosomal inhibiting proteins, ribonulcleases, proteins for antimicrobial effect, laccases and lectins are of important concerns (7); (8). Proteins separated from the mushrooms might be a unique source of nutrition for the poultry industry of Pakistan.
Mushrooms may also possess many therapeutically effective moieties i.e protocatechuic acid, gallic acid, naringenin, chlorogenic acid, biochanin-A and hesperidin (10); (11). Due to the presence of crude ber which are indigestible, fraction amount of the carbohydrates or polysaccharides, low fat and sugars have an important function in the digestive system of broilers, thereby potentiating the growth rate of nonpathogenic bacteria i.e one of the specie Bi dobacterium (12). Polysaccharides from herbs and mushrooms have been utilized as immunomodulator or immune enhancers and resulted in antiparasitic, antiviral, antibacterial and anticarcinogenic bioactivities (13). Along with the polymers (β-glucans) obtained from mushroom can enhance the response of immune cells in human and animals by binding to other receptors like Dectin-1 (14). One of the mushroom which is oyster mushrooms (Pleurotus ostreatus) found to possess immunomodulatory effects and having antioxidant potential (15) has also been proved to improve the intestinal health, immunity and growth in poultry (16); (17); (18). In an experiment on layer, Cho et al. (2010) (19) found that fermented spent mushroom compound may be utilized as an important resource in laying hen which fed at 5-15% without negatively affecting the egg quality and their effective performance related to laying eggs.
Several studies demonstrated the isolated proteins as antitumor, antioxidative, and immunomodulatory response in various species (20). Supplementation of one of the most important components from the locally available mushroom (Pleurotus ostreatus) like FIPs (Fungal Immunomodulatory Proteins) added in the broiler feed can potentiate immunity and growth of broilers. This recent literature may be a food for thought for the farmers to utilize this natural protein which are the best source for the chickens in having biological effect of good immunomodulator which may be cost effective for the farmers by rstly enhancing the production pro les and secondly by decreasing the economy of medication. So, Current study is designed to separate the proteins from Pleurotus ostreatus and evaluation of these proteins and ethanolic and aqueous extracts of the oyster mushroom on immunity, oxidative stress and production pro les of broiler poultry.

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 identi ed 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 ne 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). Brie y, 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 quanti cation 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 quanti ed 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 ltered through Whatman lter paper No. 4. Both the ethanolic and aqueous residues were re extracted twice and the ltrates 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 quanti ed 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). Brie y, 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 NH 4 ) 2 SO 4 was removed by dialyzing twice with PBS. The extracted proteins were nally puri ed by ion exchange chromatography by using DEAE-cellulose column which was equilibrated with PBS with N-acetyl, D-Galactosamine (12.5mM). The quanti cation 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 penbased 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 nisher (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 3 rd , 5 th and 7 th week interval to assess the performance pro le and FCR of the birds in all the groups.
On day 28 th of the experiment, 1 st 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 2 nd 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 pro le and immune parameters.

Toe web Assay
At day 28 th of the experiment, 1 st replicate of each group was injected 0.1ml of PHA-P (Sigma®, USA; 100μg/100μl) and PBS in the toe web between 3 rd and 4 th 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 28 th of the experiment, 2 nd replicate of each group was injected intramuscular with 5% suspension of sheep RBCs (1ml). At 7 th day of injection, a 2 nd dose of sheep RBCs was injected. Anti-sheep RBCs immunoglobulin titers were identi ed by hemagglutination assay in the serum of experimental birds (26); (27).
Total Antioxidant Capacity, (TAC, mmol Trolox equiv. L -1 ) Nisar et al. (28) described the method to evaluate the total antioxidant capacity (TAC) in the serum samples. Brie y 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 coe cient of variance (CV) below 3% (29) 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 signi cance of results of weekly weight gain, Feed conversion ratio (FCR) and other biochemical analysis related to oxidative stress and lipid pro le, 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 signi cant.

Results
Proximate Analysis of oyster mushroom Proximate composition of the dried mushroom powder is given in the table 3. The results are given in mean values of three replicates of the mushroom powder. Results show high carbohydrate (48.36g/100g) and protein contents (33.24g/100g) and low-fat content (1.2g/100g). Moisture content is 3.84 and ash is 14.34g/100g whereas the mushroom contains 405 kcal/100g of energy.
Amino acid composition of oyster mushroom Crude powder of the mushroom was evaluated for the presence or absence of different amino acids and the results are given in table 4. Ethanolic and aqueous extracts of the mushroom were evaluated the quantity of different amino acids and the results are given in the table 5. Glycine (210.53 mg/g) was found maximum in aqueous extract whereas B-alanine was higher in aqueous extract (31.56 mg/g).
Lowest concentration of Proline amino acid was seen in both the extracts.
Protein quanti cation in isolated protein of Oyster mushroom The mean value of 3 replicates of protein samples was 27.42 µg/ml.

Production Pro le
Results of body weights and feed conversion ratio (FCR) of different groups of broiler birds at day 14, 28 and 42 are given as mean ± SE is given in the gure 1, after applying one-way ANOVA. Our results showed that there is a signi cant increase in body weight of broiler chickens in all the treatment groups as compared with the control group, with the passage of experimental trial. Ethanolic extract of the mushroom at dose 400 mg/kg body weight has greater values of body weight as compared to other treatment groups and lower FCR values.

Oxidative Stress
Oxidative stress was measured in all the groups of broiler birds in terms of Total antioxidant capacity (TAC), Total oxidant status (TOS), Paraoxonase enzyme (PON) and Arylesterase enzyme (ARY) activity. The results, after analyzing with one-way ANOVA are shown in the gure 2. Oyster mushroom protein showed higher TAC levels in Pr400, Eth200 and Eth400 groups with signi cant difference as compared to control group. Mushroom proteins also increased antioxidant values in Pr200 (2.53 mmol/l) as compared to control group. Whereas, TAC results of Aq200 (2.25 mmol/l) and Aq400 (2.2 mmol/l) are nonsigni cantly different from the control group. Signi cantly lower values of TOS in the Eth400 group shows that ethanolic extract of the oyster mushroom reduces oxidative stress at the dose of 400 mg/kg body weight. Moreover, the paraoxonase activity is also signi cantly higher in the same ethanolic extract

Cell Mediated Immune Response
Lymphoproliferative reaction to PHA-P was calculated and the results are shown in gure 6 in terms of differences (mm) in the thickness of toe web at 24, 48 and 72 hours post administration to PHA-P (means ± standard error). All the treatment groups showed signi cantly higher (P<0.05) lymphoproliferative reaction when compared with control after 72 hours of PHA-P injection. Maximum response was observed in ethanolic extract treated groups and that is in a dose dependent manner.

Humoral Immunity
For the evaluation of humoral immune response, antibody reaction to sheep RBCs is in the form of total antibody, IgM and IgG titers were determined. Results were shown in the form of geomean titers (GMT). Increased titers are seen in the ethanolic extract of oyster mushroom treated groups in a dose dependent manner ( gure 7).

Discussion
Therapeutic use of mushrooms have been reported since centuries to be used in the treatment of different diseases and various efforts have been made since the second half of the last century to separate different biologically active substances from the mushrooms which are responsible for various pharmacological effects (34). Various substances with their immunodecorated effects have been isolated from different types of mushrooms and also have their subsequent factors against several ailments like in ammation (35), (36), food allergy (37) Considering such a broad spectrum of action, and based on the recent literature data, the use of mushrooms is justi ed in the feeding of farm animals, including poultry (56).
Boletus badius was evaluated for the presence of phenolic compounds (protocatechuic acid, phydroxybenzoic acid, p-coumaric acid and cinnamic acid). Elmastas et al. (2007) (15) in their study showed that the total antioxidant activity of the extracts obtained from dried fruiting bodies of B. badius at a concentration of 100 µg/mL was 99.2%. Another edible mushroom, Fistulina hepatica which is a very rich source of phenolic compounds specially protocatechuic acid and phydroxybenzoic acid contents in this species have been studied for tits antioxidant properties (57 Such kind of versatile effects of mushrooms against several disorders need detailed work and, in this context, the current study was done to nd out antioxidant activities and immunomodulatory effects of separated protein, Ethanolic and Aquous extracts of a locally available mushroom Pleurotus ostreatus in broiler chicken. Results of proximate analysis are described in table 2, which shows high carbohydrates and protein contents and low fat contents which are similar to another study on the oyster mushroom (59). Another study of Tolera et al, (60) also showed high content of carbohydrates, proteins and bres in the fresh oyster mushroom sample as compared to dried mushroom sample. Mushrooms have a variable amount of Moisture content depending upon the location, environmental factors, harvesting season, and preservation conditions (61). High contents of ber represent the mushroom as an e cient source of dietary bers which can help to reduce high cholesterol levels and to decrease the risk of tumors (62).
Amino acids are important with respect to involving biological activities relative to human body as they are well known for building blocks of the body. Amino acids also serve in repairing of tissues as well as building block of cells and in formation of antibodies to kill viruses and bacteria. They are also important part of the enzymes that produced in body endocrine system (63). Results of amino acid analysis of our mushroom samples given in table 5 shows the concentration of different evaluated amino acids that varied signi cantly (p < 0.05) in all respective different extracts of mushrooms. Total sixteen different types of amino acids were detected in both the ethanolic and aqueous extracts that includes essential and non-essential amino acids.
Glycine (210.53 ± 6.32 mg/g) was found high in aqueous extract. In ethanolic extract maximum amount of amino acid was B-ala i.e. (31.56 ± 1.58 mg/g). Proline amino acid was observed in minimum amount among all extracts as discussed by (64) (65). Their occurrence in low concentration is might be due to their non-essential nature. Among specimen lowest concentration of proline i.e. (0.03 ± 0.00 mg/g) was detected in ethanolic extract.
Mean values of body weights and feed conversion ratio of different groups of broiler birds at day 14, 28 and 42 are given in the table 5. Our results showed that there is a signi cant increase in weight of the broiler in all the treated groups when compared with control group. Ethanolic extract of the mushroom at dose 400 mg/kg body weight has greater values of body weight as compared to other treatment groups and lower FCR values. In a previous study an extract of a common edible mushroom. Lentinus edodes, had a similar effect on the body weight (66). The signi cant increase in body weight and a lower ratio of FCR in the oyster mushroom protein and extracts treated groups may be due to its high nutrition value (67) which makes it the most widely cultivated mushroom in the world (68).
The results of oxidative stress in all the groups are given in the Fig. 2. Oyster mushroom protein at dose rate of 400 mg/kg of the body weight and its ethanolic extract at both lower (200 mg/kg body weight) and higher doses (400 mg/kg body weight) produced highest antioxidant levels with signi cant difference as compared with the control group. Mushroom proteins at lower dose (200 mg/kg body weight) also increased antioxidant values as compared with the control group. Values of TOS signi cantly lower level in the Eth400 group shows that ethanolic extract of the oyster mushroom reduces oxidative stress at the dose rate of 400 mg/kg of the weight of body. Moreover, in the group treated with ethanolic extract showed signi cantly higher level of the paraoxonase activity. In ethanolic extract treated group, higher antioxidant levels and lower TOS values may be due to the higher phenolic contents in the ethanolic extract because phenolics are directly involved in the antioxidant activity (69). Khatun et al. 2015 (68) reported the antioxidant activities of some pleurotus species mushrooms. They reported that pleurotus species are the best mushrooms in reducing the oxidative stress due to their higher phenolic and protein contents.
Results of all the lipid pro le parameters and serum glucose levels, presented in Fig. 4 and Fig. 5 respectively, are non-signi cant among all the groups. A very limited data is available to study the effects of the mushroom on the lipid pro le of the broiler birds with normal serum levels of glucose and lipid pro le parameters. Although, various mushrooms including oyster mushroom have been studied by many researchers for their good anti hyper cholesterolemic and hypoglycemic effects in hypercholesterolemic or hyperglycemic animal models (70) (71). But in the present study the broiler birds are not induced any condition with associated abnormal levels of lipid pro le so we could not see any signi cant difference in the serum glucose level and serum levels of lipid pro le parameters among the control and treated birds.
Lymphoproliferative reaction to PHA-P was observed to elaborate the cell mediated immune response, and their results were shown in terms of variation (mm) in the amplitude of swelling in toe web at 24, 48 and 72 hours after giving PHA-P (means ± standard error). Lymphoproliferative response was signi cantly greater (P < 0.05) in the treatment groups when compared with control. In ethanolic treated group, maximum response was found in a dose dependent manner (Fig. 6).
Immune system and oxidant status play an important role in body homeostasis and wellbeing. Any interruption in oxidative balance or in immune system may lead to different diseases. Precautions and preventions may signi cantly improve health status and protect from developing the several diseases.
Supplementation of different types of immune boosters is generally recommended to prevent infectious diseases. The present study also evaluated the immune response of Oyster mushroom which shows that the mean value of cell mediated immune response was higher in the Eth400 group compared to the control groups. A study on partially puri ed polysaccharides from four medicinal mushroom species, has shown that the polysaccharides in the mushroom's extracts express an immunostimulating effect (72). In another study, the researchers puri ed the (1 → 6)-β-d-glucans from an edible Agaricus bisporus and evaluated its effect on innate immunity. They concluded that (1 → 6)-β-d-glucans present in Agaricus bisporus has the immunostimulatory effect (73). The mode of immunomodulatory effect of mushroom can be attributed to its stimulatory effect on different immune component cells, like, B lymphocytes, T lymphocytes and granulocytes, macrophages and natural killer cells, monocytes and dendritic cells (74). These mushroom contents also have inhibitory effect on the pro-in ammatory genes expression and ultimately reduce the in ammation by inhibiting IL-1 and COX-2 proteins (73).
Previous studies showed signi cant increase in T cell proliferation (18%) as reported by Liu et al., (1999) (75) in Marek's disease affected chickens and found with a signi cantly improved production of lymphocyte IL-2 (37-52%). Another study in which polysaccharides present in Tremella fuciformis mushroom signi cantly improves the lymphocyte production and in cholera vaccine these polysaccharides were being used as adjuvants (76); (77); (78); (79). Present study has consistent results to a previous research in which greater humoral reaction was seen after supplementation of a mushroom Fomitella foraxina (80). Similarly, one more study was done on Tremella fuciformis and Lentinus edodes supplementation in broiler birds resulted in signi cant production of antibodies in the sera, identi ed after binding with the antigens, secreted from Eimeria tenella infection (81). Girard et al., (1997) also stated notable synthesis of particular antibodies including IgA, IgM and IgG in the intestinal mucosa of birds infected with Eimeria (82). Various factors are involved in the cells to produce different types of cytokines when some particular mushroom is supplemented as a part of diet to an animal. These differences in immune responses in the cells may be due to the presence of different physicochemical properties of mushrooms, such as, purity of polysaccharides and methods of preparation of the extracts (83); (84); (85).

Conclusion
The oyster mushroom showed good physiochemical properties in terms of its proximate composition and presence of a good amount of all the essential and non-essential amino acids. This shows the nutritious value of the mushroom along with its health promoting effects. The broiler birds when treated with isolated protein of the mushroom and its aqueous and ethanolic extract in two different doses, the treated birds showed signi cantly higher cell mediated immune response in terms of lymphoproliferative response to PHA-P (P < 0.05) as compared to control group being maximum in the ethanolic extract treated group. Humoral immune response in terms of antibody (total immunoglobulins Ig, IgM, IgG) titers to sheep RBCs was observed signi cantly (P < 0.05) higher in treatment groups as compared to control.
The highest antibody titers were seen in ethanolic extract treated group. Birds supplemented with mushroom extracts and protein showed signi cantly higher growth performance in terms of weight gain and feed conversion ratios (FCR) in comparison to the control. Different parameters to evaluate oxidative stress also showed that the mushroom extracts and its protein possess antioxidant properties, hence can be used to reduce the oxidative stress in broiler chicken. The results of Hematology, Lipid pro le and blood glucose level showed no signi cant changes in the treated groups from the control group, but the result of these parameters were in normal physiological values which can be concluded as the mushroom has no adverse effects on these parameters. So, the mushroom has health promoting effects in term of immunomodulator and antioxidant with a considerable amount of essential and non-essential amino acids.

Declarations
Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
The study has been approved by the Directorate of Advance studies, Government College University, Faisalabad, Pakistan.

Consent for publication
Not applicable.

Competing interests
All the authors declare that they have no con ict of interest.

Funding
The funding for the current experiment was provided by Pakistan Agricultural Research Council under Agricultural linkages program (ALP; AS014). Eth400-1 Eth400-2 Eth400-3 Ethanolic extract of the mushroom at dose 400 mg/kg body weight          Haemagglutination Assay in all groups of broiler birds Total anti-SRBC antibody, IgG, IgM titres and total (IgG+IgM))

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