Evaluation of the effect of the supplementation of Agaricus brasiliensis mushroom in cytohematological, growth and stress parameters in Nile tilapias (Oreochromis iloticus)

Currently, in fi sh farming, Nile tilapia farming is one of the most widespread types worldwide and is present on all continents. However, stressors are normal in intensive rearing systems, which triggers immune suppression leading to a higher chance of infection, with Aeromonas hydrophila being one of the main responsible for the frequent diseases. Inhibition of reproductive and growth performance are also frequently observed in these situations. Thus, it is more than necessary to develop effi cient treatments not only against the immune damage generated by stress in fi sh but also to prevent oxidative stress itself as well as against its side effects related to physiology and infections. The mushroom Agaricus brasiliensis is widely recognized in the literature for its immunomodulatory and antioxidant effects, which makes it a potential supplement to counteract the effects of stress on intensive fi sh farming. In this study, we evaluated the effects of supplementation with the mushroom Agaricus brasiliensis through a randomized clinical trial regarding the immunological hematological variables in face of a normal situation and an induced stress situation as well as in the face of an immunological challenge by Aeromonas hydrophila. The mushroom proved to have a positive effect under the tested conditions, however, further studies will be necessary to determine its defi nitive effects. Research Article Evaluation of the effect of the supplementation of Agaricus brasiliensis mushroom in cytohematological, growth and stress parameters in Nile tilapias (Oreochromis iloticus) Flávio F Silva1,2, Alessandra Hugo de Souza2 and Wiliam CB Regis1-3* 1Nutrition Science Department, Pontifi cal Catholic University of Minas Gerais, Belo Horizonte, Brazil 2Graduate Program in Vertebrate Biology, 3Biological and Health Institute, Pontifi cal Catholic University of Minas Gerais, Belo Horizonte, Brazil 3Graduate Program in Nutrition and Health, Federal University of Minas Gerais, Belo Horizonte, Brazil Received: 31 August, 2020 Accepted: 05 November, 2020 Published: 07 November, 2020 *Corresponding author: Wiliam CB Regis, Graduate Program in Nutrition and Health, Federal University of Minas Gerais, Belo Horizonte, Av. Dom José Gaspar, 500 Coração Eucarístico, Belo Horizonte, Brazil, Tel: +55 31992417276; E-mail:


Introduction
Nile tilapia farming is one of the most widespread types of fi sh farming in the world and is present on all continents [1]. In this type of farming, cropping systems can be classifi ed as extensive, semi-intensive and intensive systems such as Raceway's, which are characterized by high risk and productivity environments due to the stress caused by the conditions of creation [2]. The stress factors in fi sh that are commonly found in these breeding systems [3] may be physical, environmental or social [4], with plasma cortisol being the predominant factor in the stress mechanism [5].
The main biochemical effects triggered by stress and release of cortisol in fi sh are the alteration of osmotic and ionic regulation Citation: Silva [6], reduction of mobilization of circulating leukocytes and lymphocytes [5] and reduction of the rate of growth and suppression of immune and reproductive functions [7]. Given this fact, a frequent problem in aquatic animal populations is the occurrence of diseases, since water acts as a facilitator of transmission of pathogens in a population of the same species [8]. Many natural products have been described to be safe to use on acquacuture like extracts from clove seed used to sedate fi sh for different farm operations [9]. This study further revealed that the extracts could be safely applied at 100.0 mg/l, which is suffi cient to anaesthetize the fi sh with little or no changes on its heamatological parameters.
Among the diseases observed in fi sh, those caused by bacteria are the ones with the greatest risk potential, since bacteria of economic importance for fi sh farming are present in fi sh water and microbiota [10], in captivity annual losses associated with streptococcus are approximately 150 million dollars [11].
The Mushroom Agaricus brasiliensis has always been reported in the literature as a potent antioxidant and immunomodulator, and currently arouses great scientifi c and economic interest due to its well documented properties [12], and can be used in fi sh farming and aquaculture to avoid sanitary problems and excessive use of antibiotics [13]. In the case of direct cytohematological parameters, it is not new that the supplementation of the A. brasiliensis mushroom can stimulate responses. In 1998 its potential benefi ts were already elucidated by stimulating the production of lymphocytes [14]. Supplementation of this mushroom under normal conditions may also increase the number of circulating eosinophils and macrophages [15]. However, in the case of the immunomodulation promoted by this mushroom, the stimulatory effect on monocytes is also described be the activity of isolated fractions of A. brasiliensis [12]. A single study evaluated the supplementation of this mushroom in tilapia, however, in a short experimental period, but still, are be observed an increase in immature leukocytes [16].
The objective of this work was to evaluate the cytohematological, growth and weight effects associated with the supplementation with the mushroom Agaricus brasiliensis, juveniles of Nile tilapia in normal conditions and the situation of stress and in front of immunological challenge by Aeromonas Hydrophila.

Methodology
The present study was carried out at the Pontifi cal Catholic during 90 days prior to stress induced by starvation [17]. Feeding was done at will once a day (4:30 p.m.), so that there was not too much leftover in the tanks. A commercial feed with 32.0% digestible protein was used, both for mushroom enrichment and control diet. To perform the enrichment, the feed was ground and the mushroom Agaricus brasiliensis was added in 1% dry weight, 60% humidifi ed, extruded and dried by exposure to the sun for 24 hours. For evaluation of hematological parameters, four fi sh were randomly removed per treatment and anesthetized with 1.0 ml / L Eugenol. After anesthesia, blood was collected utilizing a puncture of the caudal vessel with 1.0 ml syringe, bathed with anticoagulant (EDTA 5.0%) and blood (0.5 ml) collected was stored in heparin tubes and taken immediately for analysis in the laboratory DMVET. The fi sh submitted to blood collection were separated in another tank with the same environmental conditions and after the effect of the anesthesia were reallocated in the tank of the experiment/control.

Immunological challenge
For this experiment, 100 Nile tilapia (Oreochromis niloticus) fi ngerlings were used and maintained in two circular tanks of 300 l in the density of 50 fi ngerlings per tank, at a mean temperature of 27 ± 1.2 in a photo-lighting period of 12 h: 12 h light / dark and after the acclimation period, the experimental treatments were different for each tank, consisting of a control diet composed of commercial feed and an experimental diet composed of commercial feed enriched with 1% with Agaricus brasiliensis for 30 days. Shortly after the stress period the fi sh were challenged by Aeromonas hydrophila (ATCC7966) via intraperitoneal injection in low doses (1 x 10-4 CFU) or saline solution to 0.9%. The appearance of clinical signs was observed for 10 days. After this period, the fi sh were challenged again with a dose of 7.1 x 10-2 CFU of the same bacterium via intraperitoneal injection and were then observed for the next 10 days or until death.
Statistical analyses were performed in the Bioestat 5.0 program, the t-tests of Student are used.

Biometric analysis between groups (90 days)
The tables below (Tables 1,2) present the mean and initial and fi nal standard deviation for the size of the groups tested.
Although not statistically signifi cant, the A. brasiliensis mushroom showed a tendency to retard fi sh weight gain and growth rate. Regarding weight a slight retard was noted at the end of the experiment, but also did not present a signifi cant difference (P> 0.05) (Tables 3,4). When the post-stress variables between groups were assessed, no signifi cant differences were observed (Tables 13-16).

Biometric analysis of supplementation (30 days)
Below (Tables 17,18), we compared fi sh weight at the  Experiment starts with -33.14 g compared to control         showing that the mushroom had no negative effect on growth.
The same result can be observed with respect to size (Tables   19,20).

Survival challenge
In the challenge of survival against infection we can observe a higher mortality related to the supplementation with the mushroom Agaricus blazei (Table 21).

Discussion
In this study, even slightly, the A. brasiliensis mushroom proved to be able to modulate the immune response of Nile tilapia concerning the number of circulating monocytes, and in fi sh, in general, the innate defense is mainly composed of monocytes, macrophages and granulocytes (lymphocytes) [18] and just like in mammals, circulating monocytes give macrophage origin [19]. During an infl ammation or an infectious process, monocyte levels can be increased, being one of the major cells involved in acute infl ammation as part of the innate immune response [20], thus, the reduction of circulating monocytes observed in the results presented, may indicate that there was a reduction in acute infl ammation, which is possibly generated by the capture management itself [21], this idea are reinforced by the confi rmation that monocytes are the main producers of cytokines and secrete free radicals [22,23].
In immunostimulatory-based diets containing PAMPs, recognition and activation pathways are unclear [22], but TLRs play a crucial role in both innate and adaptive immunity. Its ability to detect PAMPs is related to the infl ammatory response [24] and although there are more than 17 types of TLRs identifi ed in fi sh, it is possible that the function of TLR4, one of the main monocyte modulation sites of the mushroom A.
Another fact that should be taken into account is that for immunostimulants, such as -glucan, the route of administration is a determinant factor in the immune response, especially in the case of tilapia, since the supply of immunostimulants via feed produces an effect attenuation in the cells of the immune system when compared to the supply via immersion and intraperitoneal injection, in regard to monocyte, leukocyte, and lymphocyte counts. This result is          also observed when we compared the oral immunostimulant intake against a control diet [27].
The results of our study corroborate with another results presented [16], thus bringing the concept that low doses of this mushroom did not present a strong immunomodulatory activity in tilapias, similar results were observed in FOS supplementation studies [28].
Recently, it was evidenced that hypoxia stress can generate alterations in the immune system of fi sh [29], but to date, the effect of starvation on the count of circulating leukocytes in fi sh has not been investigated. Thus, based on the results presented in this paper, we suggest that the response to starvation may not trigger signifi cant immunological changes in adult O. niloticus, or that fi sh may present different mechanisms of non-cortisol-dependent immune response.
Ichthyologists consider changes in leukocyte differential counts as one of the most sensitive indicators of acute stress in fi sh because there is a close link between leukocyte profi les and glucocorticoid levels. High neutrophil rates for lymphocytes in blood samples strongly indicate high levels of glucocorticoids as acute responses to stress [30]. In this work we can observe a higher neutrophil: lymphocyte ratio for both groups post stress period when compared to the non-stress period, we observed that the treatment with A. brasiliensis was also able to maintain this score calculated by the neutrophil: lymphocyte ratio at 0.7 after the stress period, whereas in the control group the score was approximately 0.9 which may indicate a lower stress in the supplemented group.
It was elucidated that there was an interesting difference, although not statistically signifi cant among the means of the weight of the groups, where A. brasiliensis tended to delay the weight gain in approximately 20% in the supplemented group, also, we observed a higher mortality rate for the experimental group.
This mushroom has 33.14 to 54.22% of carbohydrates [31], which represents a considerable increase (0.3g to 0.54g / kg) of carbohydrates in the supplementation since the diet control is carbohydrate-free. The low capacity of fi sh to use dietary carbohydrates is reported by inadequate regulation in the use (glycolysis) and production (gluconeogenesis) of hepatic glucose [32], which can also lead to a higher mortality or lead to losses in fi sh growth and weight gain [33]. In fi sh fed a highcarbohydrate diet compared to a high-protein diet, a lower rate of weight gain and a specifi c growth rate can be observed [34]. The studies are not conclusive as to the carbohydrate content; however, apparently Nile tilapia supports high carbohydrate dosages [35]. In our second attempt we observed that the mushroom had no negative effects on the physiological aspects of tilapia growth and weight. However, the effects on growth and weight may be dependent on the amount of ß-glucan incorporated into the diet, duration of feeding, room temperature and the species under study [36].

Infection challenge
Although knowledge about Aeromonas hydrophila is limited, its virulence has already been elucidated against aquatic populations. Among the virulence factors already documented for A. hydrophila, hemolysins, arabinasins, proteases, adhesins, enterotoxins, phospholipase and lipase, enzymes leading mainly to hemorrhagic septicemia in stressed fi shes [37].
We observed a systemic response to the second challenge of bacteria, where the fi sh supplemented died soon after the application. One of our hypotheses is the hyper reaction caused by supplementation, however, glucans alone or in combination with PAMPs or bacterial injection do not cause apoptosis in organs related to the immune system [38]. Another plausible biochemical pathway is the interaction between the biochemical mechanisms of the pathway of Agaricus brasiliensis and Eugenol.
It has recently been shown that Eugenol can act synergistically with cisplatin increasing its chemotherapeutic potential by mainly inhibiting the transcription factor pathway (NF-kB) [39]. The mushroom, on the other hand, modulates the immune response of enterocytes, reducing the translocation of NF-kB in Caco-2 cells [40].
In addition to acting as a stress mediator, NF-B is constitutively present in neurons participating in physiological functions of the CNS, such as synapse, development and neural plasticity, therefore, an exacerbated physiological response to a stressor agent, resulting in a drastic reduction of the level of this neurotransmitter, could result in death.

Highlights
This fi rst clinical trial randomized presents a new supplementation with potential benefi ts; this is the fi rst study of Agaricus brasiliensis mushroom supplementation in fi sh; this study brings new possibilities for study.

Conclusion
Based on our fi ndings, we can conclude that the supplementation with the mushroom Agaricus brasiliensis is justifi ed in the pisciculture by the potential immunomodulatory activity and, possibly, antioxidant effect, without bringing any physiological losses in terms of yield and biomass conversion in short periods of supplementation.