Introduction

There is an increasing concern among poultry consumers regarding the irrational use of synthetic medicines in the poultry feed for prophylactic and growth promoting effects. Moreover, widespread use of antibiotics in poultry feed has resulted in rapid development of resistance against several pathogens (Cowieson and Kluenter 2019). These undesirable effects led to ban on the use of antibiotic growth promoters in the poultry feed in Europe in January 2006 (Salim et al. 2018). These restrictions caused the enteric pathogens to grow more to affect the health and production of the poultry birds. Therefore, it is needed to substitute substances from natural sources to maintain and increase production in the poultry industry. Intensified research proceeded in the last 2‒3 decades for the development of certain substances that can be alternated to antibiotics for the improvement of health and production in poultry industry. Among these substances, probiotics, prebiotics, nutraceuticals, plant extracts and acidifiers were investigated (Dharma and Tomar 2007; Adhikari and Kim 2017; Abbas et al. 2020; Hazrati et al. 2020).

Coccidiosis is a major protozoal infection causing huge economic losses to the poultry producers in terms of compromised usage of feed and reduced growth rates of birds (Khater et al. 2020). Prophylactic medication to avian coccidiosis adds considerable increase in the prices of poultry feed (Lillehoj et al. 2004). Additionally, growth promoters are also commonly used in the poultry feed to exploit the production potential of genetically engineered birds. Different types of growth promoters including probiotics, prebiotics, enzymes, antioxidants and coccidiostats are being used in poultry industry (Angelakis et al. 2013). In case of withdrawal of these agents from the poultry feed, productive performance of the poultry birds severely affects the production parameters along with increasing the vulnerability of the birds towards different diseases (Yasmin et al. 2020).

Different mushrooms have been reported to possess growth promoting and therapeutic activities against several ailments (Willis et al. 2012, 2013; Gargano et al. 2017). Mushrooms have dietary priority over other sources from plant origin because of higher contents of good quality protein, dietary fibres and vitamin B with less fat and no cholesterol (Ghorai et al. 2009). Exudate secreted from the cell surface of mycelia of mushrooms contains natural antibiotics and some of these antibiotics target specific pathogens (Willis et al. 2009).

Keeping in view the beneficial effects of mushrooms, present study was designed to investigate the effects of extracts obtained from different culinary mushroom species on the production performance of healthy as well as commercially grown broiler birds infected experimentally with Eimeria.

Materials and Methods

Preparation of methanolic and polysaccharide extracts

Mushroom species including Pleurotus ostreatus (PO), P. sajor-caju (PSC) and Lentinus edodes (LE) procured from the local grower in Millat Town Faisalabad were identified (Voucher no. 173 (LE), 174 (PO) & 175 (PSC), dried, pulverized and sieved. All three mushroom species were processed for hot water extract (HWE), methanolic extract (ME) and polysaccharide extract (PSE). For HWE, mushroom powder (500 g) was added in 1500 mL of distilled water and stirred followed by boiling (4 h), centrifugation (3000 rpm for 30 min) and lyophilization. For ME, mushroom powder (500 g) was added in 1000 mL of 80% methanol and vortexed for 24 h followed by rotary evaporation and lyophilization. For PSE, mushroom powder (200 g) added in 95% ethanol for 24 h followed by ultrasonication, centrifugation (4000 rpm/30 min) and washing with ethanol, acetone and ether. Crude extract thus obtained was fractionated through diethylaminoethyl (DEAE‒52) cellulose column followed by Sephadex G-100 columns to obtain polysaccharide extracts (Ullah et al. 2014).

Preparation of mixed species Eimeria infection

Intestines of the broiler birds were collected from the butcher shops and poultry farms around Faisalabad. After direct microscopic examination of the intestinal contents, positive samples were separated and contents were squeezed to collect the infected material. Equal quantity of 2.5 per cent K₂Cr₂O₇ added to the contents having mixed species of Eimeria (for 48‒72 h). The mixture was centrifuged at 1500 rpm for 10 min. Sediment thus obtained was processed for ZnSO₄ floatation technique. Supernatant thus collected was washed with PBS three times and challenged dose was adjusted at 6.5–7.0 × 10⁴sporulated oocyst (Mumtaz et al. 2021).

Experimental design

Present study was divided into two experiments. In the experiment 1, HWE and ME, of LE, PO and PSC mushrooms were investigated. Briefly, a total of 350 birds were kept at experiment station of Parasitology Department, University of Agriculture-Faisalabad Pakistan. After 5 days, acclimatized birds were divided randomly into 7 (n=50) groups and given mushroom extracts on day 7^th, 8^th and 9^thof the experiment. In the experiment 2, PSE of LE, PO and PSC were investigated for production performance of broiler birds. A total of 200-day old birds were kept in the experiment station of Microbiology Department Veterinary Sciences Faculty University of Agriculture Faisalabad. On day 5^th, birds were divided into 4 (n=50) groups and given PSE on day 7^th, 8^th 9^th (Table 1) Experimental groups assigned were given different mushroom extracts dissolved in PBS (Phosphate Buffer Saline) as solvent. However, control groups of both experiments were given 2 mL of PBS.

In both experiments, following routine vaccination schedule for Newcastle Disease, Infectious Bursal Disease and Hydropericardium Syndrome was done and birds were offered withdrawal feed and water ad-libitum. All the experimental groups including controls were given mixed specie Eimeria infection orally, on day 21^st of the experiment at the dose rate of 6.5–7.0 × 10⁴oocysts/bird.

Weight gains and feed conversion ratios (FCR) of healthy birds

Birds belonging to all groups from 1^st and 2^nd experiments were kept in separate cages and monitored for feed consumption and weight gains weekly for up to 6 weeks of age and similarly, feed conversion ratios (FCR) were also calculated by using the following formula:

Weight gains post experimentally induced Eimeria infected birds

Half of birds from all experimental and control groups were separated in both 1^st & 2^nd experiments and were given mixed specie infection of Eimeria at the dose rate of (6.5‒7.0 × 10⁴) sporulated oocysts on day 21^st of the experiment and weights were recorded from day 4^th to day 12^th post Eimeria challenge. Oocysts per gram of feces, lesion scoring, and mortality percentages were also calculated (Ullah et al. 2014. 2015, 2018a, b).

Statistical analysis

Data obtained in the experiments was subjected to S.P.S.S. v. 21 and Tuckey’s range test was used for one-way ANOVA to determine the differences among various groups at P < 0.05.

Results

Experiment 1

Weekly weight gains: In the experiment 1, birds from all groups were weighed and feed consumed by each group was noted during 1^st to 6^thweeks of the experiment. Groups given Hot Water Extracts (HWE) and Methanolic Extracts (ME) of P. ostreatus (PO), L. edodes (LE) and P. sajor-caju (PSC), respectively showed significantly higher (P < 0.05) body weights as compared to control during the last three weeks (4^th, 5^th and 6^th) of the experiment. In the 4^th week of the experiment, among different groups highest weights (g) were observed in group given ME of LE (1016) followed by HWE of LE (967), ME of PSC (950), ME of PO (910), HWE of PSC (892) and HWE of PO (858). In the 5^th week, highest weights were observed in the ME of LE (1396) followed by ME of PSC (1295), HWE of LO (1253), ME of PO (1238), HWE of PSC (1218) and HWE of PO (1140). In the last week of the experiment, Maximum weights were observed in HWE and ME of LE (1722, 1669) followed by PSC (1631, 1608) and PO (1610, 1555), respectively (Table 2).

Feed conversion ratios (FCR): In control group, FCR observed during 6^th weeks of the experiment was significantly higher (P < 0.05) as compared to groups treated with HWE of LE, PSC and ME of PO, LE and PSC. In 5^th week of the experiment, groups given ME of LE and PSC showed significantly higher (P < 0.05) performance in terms of FCR as compared to control. In 4^thand 2^nd weeks of experiment, groups given ME of all three mushroom species showed significantly better performance in FCR. Among different experimental groups, groups administered with ME of LE showed better performance followed by ME of PSC and PO (Fig. 1).

Weight gains post Eimeria infection: The experimental and control groups were subjected to infective dose of Eimeria and weight gains were monitored from day 4^th to day 12^th post Eimeria infection. In the experiment 1, on day 6^th, 10^th and 11^th weight gains observed in all experiment groups were statistically higher (P < 0.05) as compared to control. Among different groups, in most of the days highest weights post infection were observed in ME and HWE of LE followed by ME and HWE of PSC and PO, respectively (Fig. 2).

Experiment 2

Table 1: Different treatment groups assigned in Experiment 1 and 2

Experiment 1:	Experiment 2:
G1: HWE of PO @ 200 mg/kg B.Wt.	G1a: PSE of PO @ 25 mg/kg B. Wt.
G2: HWE of LE @ 200 mg/kg B.Wt.	G2b: PSE of LE @ 25 mg/kg B. Wt.
G3: HWE of PSC @ 200mg/kg B.wt.	G3c: PSE of PSC @ 25 mg/kg B. Wt.
G4: ME of PO @ @ 200mg/kg B.wt.	G4d: Control: 2 mL PBS saline
G5: ME of LE @ 200 mg/kg B. wt.
G6: ME of PSC@ 200 mg/kg B. wt.
G7: Control: 2 mL PBS saline

HWE=Hot water Extract, ME=Methanol extract, PSE= Polysaccharide extract, PO= Pleurotus ostreatus, LE=Lentinus edodes, PSC=Pleurotus sajor-caju, B. Wt.=Body Weight

Fig. 1: Weekly feed conversion ratios (FCR) of experimental and control groups in experiment 1.

PO=Pleurotus ostreatus, LE= Lentinus edodes, PSC=Pleurotus sajor-caju, HWE= Hot Water Extract, ME= Methanol Extract. G1: Hot water extract of Pleurotus ostreatus administered @ 200 mg /kg body weight. G2: Hot water extract of Lentinus edodes administered @ 200 mg /kg body weight. G3: Hot water extract of Pleurotus sajor-caju administered @ 200 mg /kg body weight. G4: Methanolic extract of Pleurotus ostreatus administered @ 200 mg /kg body weight. G5: Methanolic extract of Lentinus edodes administered @ 200 mg /kg body weight. G6: Methanolic extract of Pleurotus sajor-caju administered @ 200 mg /kg body weight. G7: Control

Fig. 2: Weight gains of experimental and control groups post Eimeria infection in experiment 1.

Weekly weight gains: Polysaccharide extracts (PSE) of PO, LE and PSC were evaluated in term of weekly weight gains. Weights (g) observed during 5^th and 6^th weeks of the experiment were significantly higher (P < 0.05) in PSE of PO, LE and PSC. Among the mushroom treated groups in the 5^th week of the experiment, highest weights (g) were observed in PSE of LE (1352) followed by PO (1335) and PSC (1272). During 6^th week of the experiment, highest weights were observed in PSE of LE (1851) followed by PO (1820) and PSC (1580), respectively (Table 3).

Feed conversion ratios: FCR observed in control groups were statistically non-significant (P > 0.05) as compared to all experimental groups during 6^th, 5^th and 4^th week of the experiment. However, during 3^rd week, FCR observed in group given PSE of PO and in the 2^nd week, groups given PSE of PO and PSC showed significant improvement in FCR as compared to control (Fig. 3).

Table 2: Weekly weight gains (g) in experimental and control groups (Experiment 1)

Weeks	Groups
	G1 (mean ± SE)	G 2 (mean ± SE)	G 3 (mean ± SE)	G 4 (mean ± SE)	G 5 (mean ± SE)	G 6 (mean ± SE)	G 7 (mean ± SE)
1^st	103 ± 5.41^b	120 ± 8.37^ab	108 ± 4.32^b	110 ± 4.42^b	134 ± 2.99^a	124 ± 3.85^ab	110 ± 4.76^b
2^nd	241 ± 3.82^cd	276 ± 6.51^ab	251 ± 3.91^bcd	265 ± 6.24^abc	293 ± 6.73^a	241 ± 3.82^cd	232 ± 11.62^d
3^rd	367 ± 10.86^abc	408 ± 6.10^ab	397 ± 8.15^abc	394 ± 5.29^abc	435 ± 5.63^a	403 ± 7.82^abc	345 ± 31.58^c
4^th	858 ± 8.14^d	967 ± 7.93^b	892 ± 12.0^cd	910 ± 4.70^c	1016 ± 8.15^a	950 ± 8.4^b	802 ± 8.03^e
5^th	1140 ± 9.39^d	1253 ± 14.39^bc	1218 ± 7.75^c	1238 ± 11.39^c	1396 ± 10.62^a	1295 ± 7.88^b	1023 ± 17.97^e
6^th	1555 ± 9.09^d	1669 ± 12.93^ab	1608 ± 14.27^cd	1610 ± 13.09^cd	1722 ± 9.00^a	1631 ± 10.31^bc	1363 ± 18.71^e

Means sharing similar letters in a row are statistically non-significant (P > 0.05).

G1: Hot water extract of Pleurotus ostreatus administered @ 200 mg /kg body weight. G2: Hot water extract of Lentinus edodes administered @ 200 mg /kg body weight. G3: Hot water extract of Pleurotus sajor-caju administered @ 200 mg/kg body weight. G4: Methanolic extract of Pleurotus ostreatus administered @ 200 mg/kg body weight. G5: Methanolic extract of Lentinus edodes administered @ 200 mg /kg body weight. G6: Methanolic extract of Pleurotus sajor-caju administered @ 200 mg /kg body weight. G7: Control

Table 3: Weekly weight gains (g) in experimental and control groups (Experiment 2)

Weeks	Groups
	G1a (mean ± SE)	G1b (mean ± SE)	G1c (mean ± SE)	G1d (mean ± SE)
1^st	104 ± 3.15^b	124 ± 2.95^a	97 ± 2.197^b	85 ± 2.07^c
2^nd	212 ± 5.72^b	253 ± 3.45^a	209 ± 7.03^bc	187 ± 5.71^c
3^rd	320 ± 4.04^bc	359 ± 11.62^a	339 ± 3.94^ab	298 ± 5.26^c
4^th	920 ± 6.85^bc	1001 ± 7.07^a	943 ± 16.76^b	884 ± 18.87^c
5^th	1335 ± 12.35^ab	1352 ± 10.38^a	1272 ± 16.10^b	1059 ± 24.09^c
6^th	1820 ± 16.15^a	1851 ± 10.41^a	1580 ± 11.40^b	1411 ± 13.04^c

Means sharing similar letters in a row are statistically non-significant (P > 0.05)

G1a: Polysaccharide Extract of Pleurotus ostreatus administered @ 25 mg /kg body weight. G1b: Polysaccharide Extract of Lentinus edodes administered @ 25 mg /kg body weight. G1c: Polysaccharide Extract of Pleurotus sajor-caju administered @ 25 mg /kg body weight. G1d: Control

Fig. 3: Weekly Feed Conversion Ratios (FCR) of experimental and control groups in experiment 2

PO=Pleurotus ostreatus, LE= Lentinus edodes, PSC=Pleurotus sajor-caju, PSE= Polysaccharide Extract. G1a: Polysaccharide Extract of Pleurotus ostreatus administered @ 25 mg /kg body weight. G1b: Polysaccharide Extract of Lentinus edodes administered @ 25 mg /kg body weight. G1c: Polysaccharide Extract of Pleurotus sajor-caju administered @ 25 mg /kg body weight. G1d: Control

Fig. 4: Weight gains of experimental and control groups post Eimeria infection in experiment 2.

Weight gains post Eimeria infection: On day 7^th, 11^th and 12^th weight gains observed in the experimental groups were statistically higher (P < 0.05) as compared to control. Among different experimental groups, in most of the days highest weight gains were observed in PSE of LE followed by PO and PSC (Fig. 4).

Discussion

In production animals, growth augmentation due to the feeding of nondigestible carbohydrates, terpenes, tannins, flavonoids, saponins, alkaloids, phenolics, resins and minerals from plant source is well reported (Stanley et al. 1997; Grizard and Barthomeuf 1999; Toghyani et al. 2012; Ashraf et al. 2019; Elghobashy et al. 2020). In the present study, growth performance of commercial broiler birds was evaluated by monitoring the weekly weight gains and feed consumption ratios. In comparison to control, all experimental groups which were given mushroom extracts revealed significantly higher (P < 0.05) weight gains. However, in birds of groups given polysaccharide extracts (PSE), highest gains in the weight and FCR were observed in comparison to hot water extracts (HWE) and methanolic extracts (ME). Additionally, during comparison of different species of mushrooms, L. edodes (LO) showed highest values of gains in the weights and FCR followed by P. sajor-caju (PSC) and P. ostreatus (PO), respectively. These encouraging results of weight gains and FCR in mushrooms extracts administered chickens are pinpointing that feed utilization in these groups was relatively better as compared to control group. These results agree with the findings of another study in which broilers when supplemented with L. edodes and Tremella fuciformis revealed statistically significant values of weight gains and lesser conversion ratios of feed as compared to those experimental groups which were not supplemented with mushroom extracts. Moreover, values of feed conversions were lower in groups given L. edodes supplementation showed lower FCR than Tremella fuciformis administered birds (Guo et al. 2004). Similar findings were reported in other studies in which Agaricus bisporous, L. edodes and Fomitella fraxinea were supplemented (Guo et al. 2003; Dalloul et al. 2006). However, Daneshmand and his co workers in 2011 reported that inclusions of mushrooms alone in the feed had no promotional effects on the production performance, nevertheless if given with probiotics better growth performance can be achieved. Further, it was suggested that the differences observed may be due to the differences of structure, physical characteristics, sugar composition, molar ratios and geographical distribution of mushrooms belonging to different parts of the world. Nevertheless, current study results may suggest that HWE, ME and PSE of PO, PSC and LE can be used as production enhancer in commercial poultry farming.

In this study, post experimental Eimeria infection of all the groups, gains in the weight of birds from day 4^th to 12^th was recorded and found significantly higher (P < 0.05) in groups given different mushroom extracts than the birds of control groups. Among the different extracts, ME revealed highest gains in weights followed by HWE and PSE. Among the different mushrooms (LE) showed highest gains in weights after Eimeria infection followed by PSC and PO. During the experiment, all groups of chickens were also observed for general behaviour, attitude and health status. Birds belonging from control groups were emaciated, weak and lethargic with ruffled feathers and were slow to choose and pick feed particles and water. However, birds which were given different extracts of mushroom species were active and approach the feed anxiously and similar trend was also observed in the consumption of drinking water among different experimental groups. These changes in the behaviour of different groups may be due to variation in the intestinal homeostasis and ultimately causing variations in bird metabolism followed by lowered weight gains and reduced feed intake (Fernando and McCraw 1973; Adams et al. 1996; Pan and Yu 2014).

Conclusion

The study concludes that mushroom species including P. ostreatus, L. edodes and P. sajor-caju have potential to improve the production parameters of broiler birds in healthy animals. Moreover, above mentioned mushrooms can also be used with excellent production results in birds suffering from Eimeria infection.

Acknowledgements

Funds for this project were sponsored by Higher Education Commission (HEC), Islamabad, Pakistan under the scheme of PhD indigenous scholarship Batch V already enrolled PhDs Pin No. 085-12327-Av5-22.

Author Contributions

MIU, MA and MMA planned the experiment, MIU & MIA contributed equally in experimentation and interpreted the results, KK and NI finalized the write up.

Conflict of Interest

The authors declares no conflict of interest

Data Availability

Data is available

Ethical Approval

All procedures performed in studies were in accordance with the ethical committee of University of Agriculture.

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