Effect of White Button Mushroom (Agaricus bisporus) on Immunity and Haematological Parameters of Oreochromis niloticus

1Department of Zoology, University of Sargodha, Sargodha 2Institute of Molecular Biology and Biotechnology, University of Lahore Sargodha Campus 3Department of Biosciences, COMSATS University, Islamabad 4Department of Zoology, Kohat University of Science and Technology, Kohat-26000 5Department of Zoology, Government Post Graduate College, Bannu 6Department of Biotechnology, Fatima Jinnah Women University, Rawalpindi 7Department of Zoology, Government College University, Lahore Article Information Received 20 October 2020 Revised 23 November 2020 Accepted 15 December 2020 Available online 09 June 2021


INTRODUCTION
F ish is considered as one of the best economical sources of protein. Their meat is rich in omega-3 fatty acid (Sarvenaz and Sabine, 2017). Worldwide, different fish species have been cultured including Neil tilapia which is famous for fast growth and high tolerance range (environmental stress). In Pakistan it was introduced as a farm fish since 2016, and its demand is increasing day by day (Prabu et al., 2019). Fish farmers worldwide are facing many challenges regarding health, disease and mortality rate of fish, hence decreasing the output of the aquaculture * Corresponding author: Sikandarzoo00@yahoo.com 0030-9923/2021/0001-0001 $ 9.00/0 Copyright 2021 Zoological Society of Pakistan (Sahoo et al., 2020). To overcome these situations many antimicrobial and disinfectants are used but no satisfactory result is achieved so far. Besides this, over use of these drugs can result in development of multi drugs resistance strains of the pathogens, which can pass their resistance genes to the next generation. It not only promotes a drug resistant pathogen but even can harm the consumer health and causes serious environmental hazards (Lúcia and Fernando, 2018;Parrino et al., 2020). Therefore, demand for a better alternative to control or prevent fish disease is increased. One of the best method is to use ecofriendly and bio-compatible methods that is using natural immunestimulants (mostly medicinal herb derivatives). Many researchers successfully use the natural herbal products in aquaculture which can enhance the immune system or can improve the blood parameters for example, pomegranate O n l i n e F i r s t A r t i c l e seed oil (PSO) , hot pepper (Capsicum sp.) (Parrino et al., 2019), essential oil from leaves of the Cupressus macrocarpa (Kesbic et al., 2020) and essential oil from Thymus vulgaris (Zargar et al., 2019). Different other plants are also used in the aquaculture practices like garlic (Allium sativum), pomegranate (Punica granatum), bermuda grass (Cynodon dactylon), Indian ginseng (Whitania somnifera) and ginger (Zingiber officinale) (Reverter et al., 2017). Furthermore, fish hematology can be used as a tool to determine the health status of the species, but the blood profile of the fish can be greatly influence by the environment, sexual maturity, age and feed they consume Ahmed et al., 2020).
Mushrooms are considered one of the most favored healthy foods across the world. Among them Agaricus bisporus is well known for its delicious taste and health benefits, hence fetches high price and it ruled the international market. White button mushroom comprises 28-42.5% crude protein, 90-93% moisture, 8.3-16.2% crude fiber, 59.4% carbohydrates, 9.4-14.5% ash and 3.1% fat. It also contains a number of essential minerals and vitamins like 2850 mg potassium, 106 mg sodium, 8.8 mg iron, 912 mg phosphorous, 71 mg calcium, 8.9 mg thiamine (B1), 3.7 mg riboflavin (B2), 26.5 mg ascorbic acid (C), and 42.5 mg niacin (B3). Besides this, Agaricus bisporus also contains essential amino acids like lysine and tryptophan. Carbohydrate is stored in the form of glycogen in level 4.5-5.0% and fat is rich in essential fatty acid (linoleic acid) (Harikrishnan et al., 2018). Many other pharmaceutical properties are also associated with the white button mushroom like anti-bacterial, anti-fungal, stimulate non-specific immune system, lowering the blood cholesterol and glucose level. However, very limited work was carried out on button mushroom (Rajni et al., 2006). For the first time in aquaculture it was used by Harikrishnan et al. (2018) and conclude that 5% and 10% of its extract can be used to improve Clarias gariepinus hematology and enhance its immunity against Flavobacterium columnare. Current study focus to evaluate the effect of the white button mushroom on hematology and immune system of the Oreochromis niloticus which can in turn increase the output of the aquaculture.

Study site
The experiment was performed in the laboratory of University of Sargodha Sub campus Mianwali located in District Mianwali Punjab, Pakistan and the fish were fed for six weeks.

Feed formulation
The mushroom (Agaricus bisporus) commonly known as white button mushroom was purchased from the local super market. The supplemented (control) diet contains crude protein 38%, crude lipids 18%, crude cellulose 1.3%, ashes 8.7%, calcium 1.62%, total phosphorous 1.7% with mineral ash content and vitamin. The mushroom was ground into powder form and added into the basal diet with variant level of 0%, 2%, 4% and 6%. By using vacuum freeze drier the experimental diet was dried for 15 h and was filtered by using 2mm mesh. For further use the prepared feed was stored at -4ºC. The diet was formulated according to the Harikrishnan et al. (2018), in which A. bisporus replaced maize powder of 1%, 5% and 10% of control diet in groups 1, 2 and 3, respectively and the proximate composition was analyzed for its nutrient content by using the method of Horwitz and Latimer (2005).

Fish sampling and experimental design
A total number of 120 Nile tilapia fish (Oreochromis niloticus) of mean body weight 24.0±12.5g were purchased from the Tawakkal fish hatchery (located in district Muzaffar Garh Punjab, Pakistan). All the fishes were healthy and to prevent infection the fishes were treated with 0.2% of KMnO 4 solution. The fishes were acclimatized for a period of 14 days with normal L13/ D11 and were divided into four groups such as group 1 (control group) fed on control diet with 0% of white button mushroom while group 2, 3 and 4 fed on basal diet with 2%, 4% and 6% of mushroom extract, respectively. Ten fishes in each group were stocked in triplicates in 12 fiber glass tanks (water holding capacity 180 L). During that period basal feed was provided twice a day and water quality parameters were measured on daily basis. The mean water quality parameters including temperature 29±0.5 °C, DO 5.7±0.04 (Hanna Instrument HI9132) and pH 7.5 (Hanna Instrument HI221). The fresh water was added after every three days and the aerator was fixed in each tank. After adaptation the fishes were treated with experimental diet. The feed was provided twice a day of about 3-5% of their body weight.

Blood sampling and haematological parameters
At the end of the experimental diet the blood samples of 0.4 cc were collected from the caudal vein of the healthy randomly selected 4 fishes by using sterile syringe with needle (22 gauges) and stored at 4 °C in plastic tubes containing EDTA (anticoagulant) and transport it to the laboratory where they were immediately analyzed. During blood sampling fishes were not anesthetized because it may influence the blood indices (Torrecillas et al., 2011). Total red blood cell count and white blood cell count was measured with the help of the Neubauer hemocytometer O n l i n e F i r s t A r t i c l e as described by Goldenfarb et al. (1971). In this method 0.02cc of blood (non-clotted) was taken and diluted with Drabkin solution (5cc) and stored at room temperature for ten minutes. After that the Hb concentration was calculated by the absorbance at 540 nm. Besides this Wright-Giemsa stains was used for the determination of the differential leukocytes count while determination of percentage composition of leukocytes was based on the identification character (Ivanova, 1983). Besides this, on the basis of blood sedimentation the value of hematocrit (Hct) was estimated. In this process 50µL of heparinized blood in micro-heparinized capillary and spun at 12000 rpm for 5 minutes in micro-hematocrit centrifuge (RM-12C, REMI India). Hct values were obtained using hematocrit reader. Findings are presented in percentage (Natt and Herrick, 1952).

Lysozyme activity
To determine the lysozyme activity a slight modification was made in the lysoplate method described by the (Siwicki and Anderson, 1993). In this procedure, 2 ml of suspension comprises of Micrococcus lysodeikticus (Sigma, USA: 0.375 mg ml-1, 0.05 M sodium phosphate buffer, pH 6.2) and 50 ml of serum were mix thoroughly and then concentration was measured in spectrophotometer at 450 nm (Biochorm Libra S12, UK). Optical density was measured at different interval of time i.e. 15, 30 and 270s. Lysozyme activity was measured by the reduction in absorbance. Value obtain from the experiment was expressed in the unit of time (min -1 ).

Serum Ig level
To determine the Ig level, we have to precipitate it from the serum, for this 100 ml of 12% polyethylene glycol was mixed well with the 100 ml of plasma sample was incubate for 2h at 25°C. After incubation it was centrifuge at 5000rpm for 15 min. Then the supernatant was discarded and level of Ig was determined by difference between remaining and total concentration of the serum.

Alternative complement activity (ACH50)
For this, procedure proposed by the Ortuño et al. (2002) was followed. In this method the 500 µl of test sample was serially diluted with the Hank's buffer to achieve the final concentration of the 10-0·078%, then 500 µl of SRBS was added. And incubate for 1 hour at 22°C. After that it was centrifuge at 800rpm for five mints at 4°C and un-lysed RBC was removed. By using 450 nm optical density of spectrophotometer (Biochorm Libra S12, UK). The value of the Hb content in the supernatant was measured. The minimum and maximum value of hemolysis was determined by adding the 500 l HBSS to 500 l of SRBC. The hemolysis degree was obtained by plotting a curve (Y) against the total volume of the serum added on log-log scaled graph. The 50% hemolysis produce by the volume of the serum was determined and ACH50 % ml -1 was obtained for each set of experiment.

Statistical analysis
The data was analyzed statistically (SPSS Software version 17.0) by applying ANOVA (one way) with significant level 5%. Data are depicted in tables as mean ± standard deviation. Among the groups the significant difference was compare by applying Duncan test.

RESULTS
The immunological and hematological parameters of Oreochromis niloticus fed on variant level of Agaricus bisporus for six weeks are shown in Table I. The result showed that there was notable increase (P < 0.05) in lysozyme activity and ACH50 in treated groups than control group. But in the Ig level there was no significant difference (P > 0.05). There was also notable change in the hematological parameters of the fish when compared with those of control group.

DISCUSSION
The present study was conducted to investigate the effect of the basal diet with various level of white button mushroom (Agaricus bisporus) on immune response and haematological parameters of Oreochromis niloticus. The result revealed that the fish fed on 6% of white button mushroom is effective than other treatments of the diet especially lysozyme and ACH50. Lysozyme is involved in phagocytic activity, increases fish immune response and causes the destruction of bacteria cell wall. Chitsaz et al. (2018) carried out study on juvenile of great sturgeon and study the effect of Lentinula edodes on fish immune response and they observed that the lysozyme activity and SOD (superoxide dismutase) was significantly higher in fish fed on 2% of mushroom. Another study revealed that the Pleurotus eryngii and Lactobacillus plantarum enhance the Pangasius catfish lysozyme activity, respiratory burst activity and phagocytosis (Van et al., 2016). Besides this Akrami et al. (2015) reported that in beluga the basal diet with onion powder of 1% increased the lysozyme activity. According to Uluköy et al. (2016) the rainbow trout fed on extract of 1 and 2% of Pleurotus ostreatus which significantly increase the fish lysozyme activity, phagocytic activities, but no significant increase was observed in Ig (total serum immunoglobulin). Similar findings were observed in our study in which the Ig level was not significantly increased. Values are shown as mean ± SD. Values of the different superscripts in the same rows are significantly (P < 0.05) different. *Abbreviations: ACH50 (Alternative complement activity), Ig (serum total immunoglobulin); WBC (white blood cell), RBC (red blood cell), Hct (hematocrit), Hb (hemoglobin). Harikrishnan et al. (2011) reported the higher level of ACH50 when fish was treated with experimental diet contains prickly ash extract, pyrethrum and pomegranate. According to Bazari et al. (2017) the 1.5% of Aloe vera extract can boost the immunity of Siberian sturgeon such as lysozyme activity and ACH50 than control group. Awad et al. (2013) study the effect of Managifera indica, Lupinus perennis and Urtica dioiaca on rainbow trout and they reported that the extract of 1% and 2% of these plants are effective in fish complement activity (ACH50).

O n l i n e F i r s t A r t i c l e
In several studies the haematological indices have been used as they are important to identify the nutrients and pathogenic condition of the animal body (Jahanbakhshi et al., 2013). In the current study the haematological parameters such as WBC, RBC, Hct, Hb. Lymphocytes, monocytes and neutrophils are significantly higher in Oreochromis niloticus which fed on variant concentration of Agaricus bisporus. Chitsaz et al. (2018) reported that the effect of 2% of Lentinula edodes can enhance the RBC, Hct and Hb value in sturgeon juvenile which is similar to our study, but WBC and differential cells were significantly decrease than control group which is contrary to our study. According to Ndong and Fall (2007) the WBC counts was higher in hybrid tilapia which fed on basal diet with garlic at 0.5% and 1%. Another study reported that the G. cambogia extract can enhance the WBC counts (Dada and Ikuerowo, 2009). According to Bazari et al. (2017) who conducted study on Siberian sturgeon (Acipenser baerii) by introducing alovera extract to evaluate the haematological parameters and concluded that the WBC, RBC and MCV showed significant increased. According to Nwabueze (2012) the effect of garlic increases the WBC counts, Hb, packed cell volume (PCV) and RBC in the fingerlings of Clarias gariepinus fed on 0.5% of garlic extract. Sahu et al. (2007) described that the fingerlings of Labeo rohita which fed on Magnifera indica kermel, their RBC counts were increased and this intensification of RBC is sign of higher cellular immunity. Binaii et al. (2014) reported that the differential leukocytes were observed increased in beluga body on treatment with Urtica dioica.

CONCLUSION
Current study concluded that the 6% of white button mushroom (Agaricus bisporus) has shown progressive increase in fish immunity and hematology. Hence their use can be effective in order to reduce the mortality rate and to maintain the healthy aquaculture environment. Many mushrooms possess therapeutic properties. Therefore, these are the best alternative to the antibiotics used in aquaculture. So, result of current research open a new perspective of the mushrooms use in the aquaculture and further studies are needed to evaluate cost-benefits and molecular pathways.

Statement of conflict of interest
The authors have declared no conflict of interest.

O n l i n e F i r s t A r t i c l e
Effect of White Button Mushroom