Research Article

Promising Immunomodulation Potential of Ganoderma lucidium and Oregano as Mixture on Broiler Chicken

Fateh A. Badi*, Anwar A. G. Al-Kubati, Naif Al-Gabri

Department of Veterinary Medicine, Faculty of Agriculture and Veterinary Medicine Thamar University, Dhamar, Yemen.

Received | October 03, 2022; Accepted | December 20, 2022; Published | November 25, 2023

*Correspondence | Badi A Fateh, Department of Veterinary Medicine, Faculty of Agriculture and Veterinary Medicine Thamar University, Dhamar, Yemen; Email: [email protected], [email protected].

Citation | Badi FA, Al-Kubati AAG, Al-Gabri N (2023). Promising immunomodulation potential of Ganoderma lucidium and Oregano as mixture on broiler chicken. Adv. Anim. Vet. Sci. 11(12): 1968-1977.

DOI | http://dx.doi.org/10.17582/journal.aavs/2023/11.12.1968.1977

ISSN (Online) | 2307-8316

 

 

Copyright: 2023 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

The increment in poultry protein demand to cover the human requirements in the past few decades lead to using intensive rearing with different growth and production enhancement tools. This leads to defensive adverse effects on immunological parameters of poultry and increasing in the number of highly resistant microorganism (RU et al., 2015). In addition, antibiotic residues in poultry meat are dangerous to human health, therefore the researcher are continuously looking for safety and low cost natural alternatives. The first step in this issue was performed by European Commission which support the scientific community to focus on finding antibiotic alternative from the aromatic plants and extracts which day after day many scientific confirmation has been emphasis their beneficial productivity and health effects on animal (Franciosini et al., 2016).

Among many different natural resources that may benefit in this issue are the botanical and Mushroom, Although the different methods of plant and mushroom extraction effects the complex compounds in extracts and consequently the mode of action, but generally, the complex mixtures targets different physiological pathways. The using of single compound purified from plant or mushroom represent by modern pharmacological point of view, magic bullet for targeting certain cellular pathway. Several studies prove that the synergistic interaction of compounds in herbal plants and mushroom may present enhancing by that the action of one or both of them. This synergistic mode may attributes in some cases to the enhancement of absorption and bioavailability or to reduction of some mediators that effect the target pathway of another extracts (Martel et al., 2017). Despite of that, there are growing attention, devoted to environmental issues, led to new techniques capable of extracting plant bioactive compounds without using non-renewable resources (i.e. solvents) with a consequent reduction in the environmental impact of wastes because the plant material, remaining after solvent extraction, is considered hazardous waste (Setti and Zanichelli, 2009).

In poultry production, it is vital to enhance immunity to prevent infectious diseases. Minimizing immunosuppression and its impact is additionally crucial strategy for success in the broiler industry. However, strategies to control immunosuppression are largely based on vaccination programs for poultry and management to minimize stress during rearing (Hashemi and Davoodi, 2012). Utilization of natural immunostimulants is one solution to improve the immunity of animals and to decrease their susceptibility to infectious diseases (Liu, 1999).

Increasing number of researches reported the immunostimulation effects of Oregano (Hernandez-Coronado et al., 2019; Avila-Ramos et al., 2012; Mathlouthi et al., 2012; Giannenas et al., 2003; Botsoglou et al., 2002) or Ganoderma (Ahad et al., 2016; Ogbe et al., 2009; Liu et al., 2015; Pi et al., 2014; Ojiezeh and Eghafona, 2015). , so the aim of this study was:to study the effects of Oregano and Ganoderma mixture on some immunological parameters in broiler chicken and to assist potential synergistic and/or immunomodulation effects of combination of this natural immunostimulants.

Materials and Methods

Feed additives and preparation of ration

The effects of two feed additives, Ganoderma lucidium and Origanum Vulgare, were investigated in the present study. The Ganoderma lucidium were obtained from Dxn company branch at Dhamar city, while the Oregano was obtained from local herbal shop. Ganoderma lucidium and Origanum Vulgare grinded finally and were added into feeds in a ratio of 0.2% (2g/Kg) (Ogbe et al., 2008; Sofyan et al., 2012; Ri et al., 2017; Rahman et al., 2018). a combination of the additives were added into feeds in a ratio of 0.1% (1g/Kg) for each additive. The chicks were received on sugar solution of 5% for 6 hours. The feeding program were, ad libdo feeding, starter feed from one day to 14 days, grower feed from 15 day to 28 day and finisher from 29 to 42 day. The broilers’ feeders were from commercial source (Alfalah Corporation, Dhamar, Yemen). The lighting program were 23 hours per day at one day old and reduced 10 minutes per day. Initial brooding temperature was 33 ºC in the first week of age and reduced gradually 2 ºC per wk and reached 22 ºC at the end of experimental period.

Chickens and experimental design

Forty broiler chicks of the Ross breed were obtained from local provider (AL-Flah Hatchery, Ibn Al-Haj poultry incorporation) at age of one-day old. Birds were kept on deep beds and allowed to feed and drink ad libitum. At arrival, chicks were randomly allocated into four experimental groups, 10 chicks in each group. Three experimental groups were used to investigate the effects of Ganoderma lucidium, Origanum Vulgare and combination of them on immune status of birds (G, O and GO groups respectively). The fourth group was used as a control (C group).

Immunological performance of feed additives was evaluated using total and differential count of WBC, heterophil/lymphocyte ratio (Gross and Siegel, 1983), indices of immune organs, carbon clearance and phagocytosis index(Abbas et al., 2017, Dorhoi et al., 2006, Zheng et al., 2013). At age of 45 days, blood samples were taken.

Sample collection

At age of 42 days, blood samples form 24 chickens (six chicken per group) were collected from wing vein into EDTA containing vacutainer. Blood parameters measurements were performed according to the procedures recommended by Samour (2015). Accordingly, heterophil to lymphocyte (H/L) ratio was calculated (Gross and Siegel 1983). Heterophil to lymphocyte ratio was used as indication of stress (Lentfer et al., 2015, Jahanian and Rasouli, 2015, Gross and Siegel, 1983, Arczewska-Włosek et al., 2018).

Immunological parameters

Phagocytosis index: The phagocytic function of the mononuclear phagocytes system in chickens was evaluated with carbon clearance test. Six broilers from each treatment group were randomly selected at day 42 and used to measure carbon clearance. Indian ink was diluted in normal saline at rate of 1:3 and injected in right wing vein of broiler chickens at a rate of 2 ml/Kg. Three and ten minutes post injection, 100 µl of blood was collected from left wing vein into tubes containing 10 ml of 0.1% sodium carbonate. Optical density was measured at wavelength of 640 nm using PD-303UV (APEL, JAPAN) (Shahzad et al., 2015, Zheng et al., 2013). The carbon clearance index (K) were calculated as follows:

k = log OD­1 – log OD­­2)/(t2 –t1)

Where OD1 is the optical density at 3 min (t1) and OD2 is the optical density at 10 min (t2)

The phagocytosis indices were calculated from the carbon clearance (K), total weights of carcass (W) and weight of both spleen and liver (Wls) as following:-

α = W/Wls × K1/3, (Zheng et al., 2013)

Immunological organs indices

At age of 45 day, chickens were weighed and humanly sacrificed. Internal organs including bursa of Fabricius, spleen and liver, were removed surgically and weighed. Organ weight was presented as percentage of the live body weight.

Statistical analysis

Data were analyzed using SPSS (V22.0; IBM SPSS Statistics Inc., Chigaco, IL, US). All study groups were subjected to Analysis Of Variance (ANOVA). Least Significant Different (LSD) was used to separate the means were significant difference exist. Differences were considered significant at p < .05

Results

Haematological parameters (haematocrit, Hb, RBC, MCV, MHC and MHCH) were all within the normal range values in all the groups. Although were no significant differences among study groups in RBCs, MCV, MCH and MCHC, the addition of O.licidium induced significant decreased both hematocrite (Ht) (P=.025) and hemoglobin (P=.034) in comparison to control, (Table 1).

There was insignificant increase in eosinophil count due to addition of O. vulgare, while there was insignificant decrease in eosinophil count due to addition of G. lucidium as Table 2 The GO combined additives induced further increase in eosinophil count which was significant when compared with control (P<.05) and G.lucidium (P=.04) groups. In contrast to eosinophil count, there was significant increase in lymphocyte count due to addition of G. lucidium (P=.002) and significant decrease due to addition of O. vulgare (P=.01). The GO combined additives showed insignificant decrease in lymphocyte count (P=.518) when compared to control but significantly increased (P=.043) when compared to O. vulgare group.

There was significant decrease in heterophil count due to addition of G. lucidium (p=.039). The GO combined additives induced further decrease in heterophil count which was significant when compared with control group (p=.0004) while the addition of O. vulgare induced insignificant reduction in heterophil when compared with control (p=.053). Regarding heterophil/lymphocyte ratio, there was significant decrease due to addition of G. lucidium (p=.029) and the GO combined additives (p=.004). Decrease was insignificant due to addition of O. vulgare (p=.140).

There was significant increase in monocyte count due to addition of O. vulgare (p<.05) compared to other groups. Addition of G. lucidium alone or in combination with O. vulgare showed no effects on monocyte count. Overall, G. lucidium and O. vulgare showed insignificant decrease in TWB, while the GO combined additives showed insignificant increase in TWBC (p=.08) (Table 2).

G. lucidium and O. vulgare induced significant increase in bursa index when compared with control group (p= .0003 and .0004 respectively) and the GO combined additives decrease the bursa index in comparing to G. lucidium and O. vulgare (p=.004 and .0004 respectively) as shown table3. On the other hand, the spleen index was significant decrease in G. lucidium , O. vulgare and GO groups when compared with control (p=.002, .014 and .016 respectively). Significant increase in bursa to spleen (B/S) ratio were induced by G. lucidium and O. vulgare when compared with control group (p= .004 for both) and the GO combined additives significantly decrease the ratio in comparing to G. lucidium and O. vulgare (p= .006 and .004 respectively). The GO combined additives was significantly superior in increasing phagocytosis index when compared

 

Table 1: The Effects of G. lucidum and/or O. vulgare on hematological parameters of broiler chicken.

Treatment	RBC [1012 l−1]	Ht [l l−1]	Hb [g l−1]	MCV (FI)	MCH (Pg)	MCHC (g/dL)
G	2.5	34.11 a	11.46a	135.4	45.5	33.48
O	2.6	35.01	11.83	135.5	45.8	33.73
GO	2.6	34.75	11.69	134.1	45.5	33.81
C	2.7	36.28 b	12.16 b	135	45.5	33.49
SEM	0.0366	0.33	0.11	1.3306	0.4351	0.1

a, b, c, d means in columns with different superscripts differ significantly at p ≤ 0.05 for main treatment; G––Ganoderma lucidium; O––Origanum vulgare; GO––Ganoderma & Oregano mixture; C––Control; RBC––red blood cells; Ht––haematocrit; Hb––haemoglobin;MCV––mean corpuscular volume; MCH––mean corpuscular hemoglobin; MCHC––mean corpuscular haemoglobin concentration; SEM––standard errors mean.

 

Table 2: The effects of f G. lucidum and/or O. vulgare feed additive on leukogram of broiler chicken

Treatment	TWBC [109 l−1]	H [%]	L [%]	MONO [%]	EOS [%]	BASO [%]	H/L
G	5.50	12.55 a	65.88 a	11.22 a	10.33 a	0	0.19 a
O	6.01	12.70	53.37b	14.50 b	19.38 a	0	0.25
GO	6.33	8.60 a	57.7 c	11.10 a	22.60 b	0	0.15 a
C	6.02	17.66 b	59 cd	11.44 a	11.89 a	0	0.32 b
SEM	0.17	0.97	1.0	0.5	0.97	0	0.21

a, b, c, d means in columns with different superscripts differ significantly at p ≤ 0.05 for main treatment; G––Ganoderma lucidium; O––Origanum vulgare; GO––Mixture of Ganoderma and Oregano; C––Control;; WBC––white blood cells; ; H––heterophils; L––lymphocytes; MONO––monocytes; EOS––eosinophils; BASO––basophils; H/L––heterophils/lymphocytes; SEM––standard errors mean.

 

to control group (p=.003). Both G. lucidium and O. vulgare induced increase in phagocytosis index, though it was not significant when compared with control group (p=.06 and .051 respectively) (Table 3).

 

Table 3: The immunological indices of broiler chicken fed on additive of G. lucidum and/or O. vulgare

Treatment	SI	BI	B/S	PI
G	0.077 a	0.258 a	3.530	38.60
O	0.090 a	0.321 a	3.745	39.55
GO	0.087 a	0.114bc	1.349	52.81a
C	0.147 b	0.043 bc	0.309	17.94b
SEM	0.010	0.031	0.419	4.30

a, b, c, d means in columns with different superscripts differ significantly at p ≤ 0.05 for main treatment; G––Ganoderma lucidium; O––Origanum vulgare; GO––Ganoderma lucidium & Oregano vulgare mixture; C––Control; SI––spleen index; BI––bursa of Fabricus index; B/S––bursa to spleen ratio; PI––Phagocytosis Index,: SEM––standard errors mean.

Discussion

Immunomodulation. Immune-based therapies are gaining more importance than monovalent approaches which having limited benefits. The herbal products have privileged and high structural diversity with numerous biological activities (Ezzat Abd El-Hack et al., 2016, Christaki et al., 2020). In this study the immunomodulation effects of G.lucidium and O.vulgare with their mixture were evaluated.

Results showed that the hematological values of broilers were inconsistent and seemed to bear no direct relationship. It should be taken in consideration that the fluctuation and wide range in normal hematological values in poultry as normal phenomonia which have been reported in many research (Reece et al., 2015, Orawan and Aengwanich, 2007, Onibi et al., 2011, Ogbe et al., 2010, Ogbe et al., 2003, Islam et al., 2004).

This study revealed that the mixture of Ganoderma and Oregano insignificantly increase the total WBCs which may be indicative of hematopoietic stem cell stimulation (Ganeshpurkar and Saluja, 2017). Overall, G. lucidium and O. vulgare showed insignificant decrease in TWBC. The GO combined additives showed insignificant increase in TWBC. Similar findings were also reported. Extract of wild Ganoderma species was used to treat Eimeria tenella-infected broilers, result showed no significant effects on total WBC count (Ogbe et al., 2010). Similarly, wild Ganoderma lucidum together with other herbs were administered into broilers and showed no significant effects on TWBC count (Ogbe and Affiku, 2012). Likewise, addition of oyster mushroom to broiler feed at rates of 1, 10, 20 gm/kg showed no significant effects on total WBC (Toghyani et al., 2012). Feeding broilers on Oregano oil at rates of 60, 100 and 200, 300, 500 ppm induce no change in TWBC count (Mohiti-Asli and Ghanaatparast-Rashti, 2017; Hashemipour et al., 2013). Similarly, broilers supplied with Mexican Oregano oil at a rate of 0.4gm/kg of diet showed no change in total WBC count (Alagawany et al., 2018). In the present study, there was highly significant decrease in heterophil count due to addition of the combined additives, while the lymphocyte count was significant increased and decreased due to addition of G. lucidum and O. vulgare , respectively. This suggest that the G. lucidium is more effective as immunostimulant than O. vulgare. Although the GO combined reveal significant increase in lymphocytes when compare with O. vulgare . This reflected on the H/L ratio that were significantly decreased due to addition of G. lucidium and to lesser extent on the GO combined additives group.

Unfortunately, effects of G. lucidum on H/L ratio was not fully covered in previous studies. Willis et al. (2013) showed that addition of G. lucidum to chicken feeds at rate of at 5% and 10% induce increase of heterophil and decrease of lymphocyte and increase in H/L ratio Studies on oyster mushroom showed that its addition to broiler feed at a rate of 2% result in significant decrease of heterophil to lymphocyte ratio (Fard et al., 2014). While similar experiment on broiler showed that use of 10 and 20 g/kg of the oyster mushroom showed no effect on H/L ratio (Toghyani et al., 2012). Regarding effect of Oregano on H/L ratio, our results are in contrast to previous investigations where addition of ≤300 ppm to chicken feed induced significant decrease in H/L ratio while addition of higher rates induce no change in H/L ratio. Similarly, provision of broilers feeds with 60, 100 and 200 ppm of the Oregano essential oil was reported to significantly (P=.005) decrease H/L ratio (Hashemipour et al., 2013). Addition of 300 of the Oregano essential oil to broiler feeds result in significant decrease the heterophil and H/L ratio at 300 ppm but not at 500 ppm (Mohiti-Asli and Ghanaatparast-Rashti, 2017). While addition of Mexican Oregano oil to feeds of broilers at rate of 0.4gm/kg (=400ppm) induced slightly lower lymphocyte and monocyte count (Alagawany et al., 2018). Increase in H/L ratio may result from increase in circulating heterophil and decrease in circulating lymphocyte counts in stress condition (Maxwell and Robertson; 1995, Post et al., 2003).

There was significant increase (p=.00001) in eosinophil count due to addition of GO combined additives. Addition of G. lucidum to chicken feeds at rate 5% and 10% induce no change in eosinophil count (Willis et al., 2013). On the other, administration of Mexico Oregano oil to broiler feeds at rate of 0.4gm/kg induce insignificant increase in eosinophil count (Méndez Zamora et al., 2017). This may result from the higher dose of Oregano administered in the present study. Eosinophils and basophils have been described in avian systems and in mammals play an important role in defense against helminthes and in allergy responses. However, compared to other leukocytes such as heterophils and macrophages the functionality of eosinophils and basophils in chickens and other avian species has not been well determined. Using a range of intravenous injected immunostimulatory materials (e.g. horse serum, material from spontaneously occurring eosinophilia chicken, etc.), other researchers was unable to increase the levels of eosinophils in chickens. However intra-dermal injection of chickens challenged with dinitrochlorobenzene (DNCB) or citraconic anhydride (CA) increased eosinophil and heterophil levels in the skin indicating a potential role of eosinophils in early inflammatory responses (Maxwell, 1987).

There was significant increase in monocyte count due to addition of O. vulgare (p=0.030) while there was no change due to addition of G. lucidum. This is in agreement with result shown by Willis and collegues where addition of G. lucidum to chicken feeds at rate 5% and 5% result in no change in monocyte count (Willis et al., 2013) Similarly the addition of 600 and 900 ppm of Oregano cause significant increase in monocyte count (Eler et al., 2019). On the other hand, addition of 300 and 500ppm of the Oregano essential oil to broiler feeds result in no effect on monocyte count (Mohiti-Asli and Ghanaatparast-Rashti, 2017). addition of 0.4gm/kg of feed induced no significant change in relative monocyte (Méndez Zamora et al., 2017). The Oregano essential oil (OEO) 400 mg/ kg as natural antioxidant improved AFs induced oxidative stress, TWBCs, heterophil%, lymphocyte%, monocyte% and eosinophil % toward control values.

It’s known that oxidative stress influence hypothalamicpituitary-adrenal axis as well as the increased NO level that catalysed cortisol synthesis (Minka et al., 2012). Moreover, oxidative stress could affect differentiation and phagocytic ability of leucocytes especially monocytes against infection (Ponnappan and Ponnappan, 2011). Daily administration of corticosterone to chicken result in decrease of lymphocyte and eosinophil and increase in heterophil count (Glick B, 1958).

It has been reported that stress in chickens induce increase in plasma corticosterone concentration, decrease in total leukocyte count and increase in H/L ratio (Post et al., 2003, Mehaisen et al., 2017, Shini et al., 2009, Elnesr et al., 2019, Babacanoğlu et al., 2013). Decrease in lymphocyte and increase in heterophil and H/L ratio in response to stress and cortcosterone in chicken were previously documented (Lentfer et al., 2015, Jahanian and Rasouli, 2015, Gross and Siegel, 1983, Glick, 1961, Glick, 1958, Arczewska-Włosek et al., 2018). This give an evidence that the increasing in monocytes at peripheral blood indicate immune stimulation at non-infection-normal condition. GO combined additive antagoise the effect of stress as shown in H/L ratio under non-infection status.

In contrast to many researches, our results showed significant increase in bursa index and bursa to spleen ratio as result of G. lucidum and O. vulgare, while GO significantly decrease bursa index and bursa to spleen ratio. The spleen index was significant decrease in three treatments. Addition of 5% and 10% G. lucidum to feed of broiler did not induce significant change in bursa weight (Willis et al., 2013), Aqueous extracts of wild G. lucidum and other herbs were mixed at equal volume and administered in broiler feed at rates of 5% w/v at age of 2 weeks, 10% w/v at age of 4 weeks and 20% w/v at age of 6 weeks. Result showed no significant difference in weights of visceral organs, including spleen (Ogbe and Affiku, 2012). Similarly, addition of 1 or 2% of oyster mushroom to broiler feeds induce no change in weight of bursa, spleen or bursa to spleen ratio (Toghyani et al., 2012, Kavyani et al., 2012, Fard et al., 2014).

Similarly, several studies were performed on the effects of Oregano active ingredients, Oregano oil or Oregano powder on weight of lymphoid organ in broiler chicken or bursa: spleen ratio. No change in weight of bursa or spleen were induced by addition of Oregano active ingredient thymol and carnvacrol at rate of 60, 100, 200 ppm (Hashemipour et al., 2013); 150 mg of the Oregano powder/kg diet . (Ri et al., 2017); 250 and 500 mg of the Oregano oil/kg diet (Basmacioğlu Malayoğlu et al., 2010); 300 mg of Oregano essential oil /kg (OEO) diet (Kırkpınar et al., 2011). Combination of OEO with garlic oil at rates of 150mg of both/kg diet induce no change in weight of bursa and spleen (Kırkpınar et al., 2011). On the other hand, combination of OEO with garlic and thyme oils at rates of 10mg or 20mg of each/kg diet for 15 days induce no change in weight of bursa but significant increase in weight of spleen. Extending feeding period for 35 days abolish the effect on spleen weight (Abou-Elkhair et al., 2014).

Thymus gland, bursa of Fabricius and spleen are important immune organs of broilers which are involved in humoral and cell-mediated immunities and as generative organ for T and B cells. Generally, cell growth, development and division caused animal immune organ weight increase, and the immune organ weights reflected the immune functions. The greater bursa weight commonly reflects better health status and also the better anatomical response to immune system changes due to stress (Willis et al., 2013). Beside that There is a strong correlation between the relative size of bursa and the average levels of IgG antibody expression (Yonash et al., 2002; Glick et al., 1956). The Bursa of Fabricius is an essential lymphoid organ in chickens that plays an important role in adaptive immunity. Bursa size can reflect the immunological health status of birds (Glick, 1979). Healthy broilers produce larger bursas than broilers whose immune systems have been compromised (Glick, 1979). Glick et al. (1956), also reported that the Bursa of Fabricius plays an important role in antibody production.

Therefore, it may be perceived that unchallenged broilers in treatment groups with higher bursa have superior immune systems compared to broilers whose bursa weights were smaller. Although, there is no apparent evidence that bursa size had any significant influence on the lymphocyte values or antibody titers of unchallenged broilers in this study, there has been a noticeable immunological response to Eimeria infections in both challenged and unchallenged broilers. In addition, there have been reports that have indicated positive effects on the integrity of the immune system of chickens given medicinal mushrooms in their diets (Guo et al., 2004; Willis et al., 2012; Willis et al., 2013).

Therefore, it may be suggested that broiler diets supplemented with certain mushrooms can exert positive health attributes via bursa performance. Immune stimulation by using herbal extracts may reduce the animal’s susceptibility to infectious diseases (Alagawany et al., 2015). Not all trials have been positive, for example Bozkurt et al. (2009) studied the effect of 1.0 g/kg diet of Oregano on the performance of broilers and found that bursa was not affected by supplementation.

The primary task of ‘phagocytosis’ is amputation of foreign bodies and microorganisms along with the eradication of injured and dead cells, malignant cells, and inorganic particles (White and Gallin, 1986). Carbon clearance test is one of the necessary methods to assess phagocytosis immunomodulatory effects of drugs and phytoconstituents in different animal model including chicken (Nudo and Catap, 2011; Kundu et al., 2015). increasing in phagocytosis is index is indicative of stronger immune response (Sun et al., 2008).

Separated additives of G. lucidum and O. vulgare or combination of them induce insignificant increase in carbon clearance and phagocytosis index in comparing to Control Group. Unfortunately, phagocytosis and carbon clearance were not tested in previous studies on G. lucidum and O. vulgare. Investigating the effects of oral administration of aqueous methanol extracts of roots of Glycyrrhiza glabra, leaves of Artemisia brevifolia and Ageratum conyzoides at doses of 100, 200 and 300 mg/kg body weight of broilers. carbon clearance was low at low doses and increase with higher doses to a level similar to those result from treatment with vitamin E (Hussain et al., 2017). Acamovic and Brooker (2005) reported an immune stimulatory effect with thymol and Oregano oil fed in broiler diets in terms of mononuclear phagocyte proliferation, as well as in cell mediated and humoral immunity. In a study by (Perez-Roses et al., 2015), it was reported that immunity partially participates in resistance against different infections. Phagocyte proliferation, cellular and humoral immune responses in broiler chickens could be improved by using herb oils, which elevated the ability of defence system to interact with infectious agents.

Although, there are many evidence on the phagocytosis activation ability of G. lucidium. Both the classical and alternative complement pathways were activated by an alkali extract obtained from G. lucidum cultivated mycelium. In the carbon clearance test, this fraction also activated the reticuloendothelial system of mice and enhanced haemolytic plaque producing cells in the spleen. In RAW 264.7 macrophages, Ganoderan, a D-glucan isolated from G. lucidum, increased NO generation. Raw 264.7 cell lines treated with GAN had their cell growth reduced. These findings suggest that the higher fungus’ -glucan-related polysaccharides activate macrophages and produce NO, a key chemical messenger for the generation of a variety of biological responses. GLB, a protein-polysaccharide fraction derived from the developing tips of G. lucidum, is a potent macrophage stimulant. GLB (100 g/ml) boosted the phagocytic activity of BALB/c mouse peritoneal macrophages and chicken macrophage BM2CL cells against FITC-labeled C. albicans by 55.2 percent and 21.2 percent, respectively, when examined using a flow cytometer. GLB boosted the spreading and expression of MHC class II molecules in BM2CL cells and mouse peritoneal macrophages as well (Sanodiya et al., 2009).

Reishi mushroom extracts boosted leukocyte respiratory burst activity, phagocytic activity, and lysozyme synthesis, all of which are antimicrobial enzymes. The phenotypic functions of macrophages such as phagocytic absorption, generation of NO and other reactive oxygen species, cytokine gene expression, and morphological modifications were all up-regulated by glucan. Many immune cells which have TLR 4 on their surface, can mediate the immunological response. Ganoderma polysaccharide appears to be a ligand for these receptors as well as an activator. Many of the effects identified in dendritic cells may be blocked by blocking the TLR4 receptor; this, together with the activation of TLR4 and the presence of certain polysaccharides, suggests that receptor activation is a fundamental mechanism of action. On macrophages, TLR4 activation has been seen, with downstream effects that boost macrophage phagocytic capacities. Polysaccharides and proteoglycans may be involved in these actions. It’s been proposed that a polysaccharide fraction F3 containing fucose binds to TLR4 on macrophages, activating proteins like ERK, which regulates meiosis, mitosis, and postmitotic functions, as well as JNK and p38, which regulate cell proliferation/differentiation, inflammation, and cytokine production (Bhardwaj et al., 2014).

Conclusion

In: conclusion, it is known that an unbalanced immunitory system provides the premise for a great number of diseases in both humans and animals the combination of Ganoderma and Oregano in broiler chicken may be promising immunomodulation and/or immunobalancer feed additive, although further studies are required to assess different biological effects and the best optimal synergistic concentration.

acknowledgements

The authors would like to thank the Faculty of Agriculture and Veterinary Medicine at Thamar University and Yemen-Agrivet for Agriculture and Veterinary Medicine for their support.

conflict of interest

The authors declare no conflict of interest.

novelty statement

Up to our knowledge, this is the first study to evaluate the effects of combined feed additives, which include G. lucidium and O. vulgare, on the haematological and immunological parameters of broiler chicks. Obtained findings showed these phytobiotic feed additives are promising

authors contribution

Fateh Badi: conceptualization and experimental design; performed the experiment; data curation. Fateh Badi, Anwar Al-Kubati, Naif Al-Gabri: validation, data analysis, writing, and editing the manuscript. All authors have read and agreed to publish this version of the manuscript.

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