Elodie Merlot

Nathalie Le Floc'h

Caroline Leclercq

Armelle Prunier

Response of blood nutients to an LPS inflammatory challenge in pigs

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.


Serge Moukha

Cyril Ferandon

Theophile Mobio

Edmond E Creppy

SAFETY EVALUATION OF AGARICUS SUBRUFESCENS VARIETIES AND THEIR PRODUCTS OF THERARPEUTIC INTEREST OR FOR DISEASE PREVENTION

Keywords: Agaricus subrufescens, Agaricus bisporus, Safety, Prevention, Rodents

Mushrooms are food traditionally consumed in Europe, Asia and America. They are being studied for medicinal benefits. Extensive studies have shown that Agaricus subrufescens (A. blazei Murrill or A. brasiliensis) has anticancer properties. A comparative study of Agaricus subrufescens strains (from Brazil and France) is presented herein using Agaricus bisporus (champignon de Paris) as control. In vivo OCDE test were performed to evaluate either tolerance and/or acute and sub chronic toxicity in rats and mice. Our data reveal that all A. subrufescens strains are not toxic, either in vivo or in vitro, except some locomotor hypoactivity. All show a preventing effect against carcinogenesis, including A. bisporus. This is the first time that this mushroom is shown to be effective, even thought it is clearly less effective than A. subrufescens. However no anti tumour effect is found using Balb-c mice implanted with leukaemia cells. Furthermore they elicit slight cell growth stimulation at the concentrations tested in vitro, in Hep G2 (human hepatoma cells) and Neuro 2a (mouse neuroblastoma cells). The most active is A. subrufescens from Brazil.

These mushrooms do have many bioactive compounds, different from the polysaccharides that need to be isolated and characterised for their curative properties following accurate evaluation of toxicological effects. Indeed there is clearly a lake of information on toxicological assessment (acute and chronic toxicity) of compound such as agaritine, blazein among others and of the whole mushroom A. subrufescens itself, and overall on epidemiological data linking the consumption of Agaricus sp and eventual prevention and/or pathologies.

INTRODUCTION

Agaricus subrufescens Peck is also known as Agaricus blazei Murrill and/or Agaricus brasiliensis Wasser [START_REF] Wasser | Is a widely cultivated culinarymedicinal royal sun Agaricus (the Himematsutake Mushroom) indeed Agaricus blazei Murrill?[END_REF][START_REF] Kerrigan | Agaricus subrufescens, a cultivated edible and medicinal mushroom, and its synonyms[END_REF] and has other common names, such as "piedade mushroom" or "Cogumelo do sol" in Brazil, "Himematsutake" in Japan and "Almond mushroom" in United States of America. It is named as ABM in several articles. This Agaricus is rich in substances that are expected to modulate biological functions in mammals. Various active compounds that can potentially be used to treat or prevent different diseases have been reported [START_REF] Wasser | Medicinal mushrooms as source of antitumor and immunomodulating polysaccharides[END_REF][START_REF] Takaku | Isolation of an antitumor compound from Agaricus blazei Murrill and its mechanism of action[END_REF][START_REF] Firenzuoli | The Medicinal Mushroom Agaricus blazei Murrill: Review of Literature and Pharmaco-Toxicological Problems[END_REF]. This mushroom has been used as a medicinal food for the prevention of cancer, diabetes, hyperlipidemia, arteriosclerosis, and chronic hepatitis and is known to impact putatively the immune system [START_REF] Takaku | Isolation of an antitumor compound from Agaricus blazei Murrill and its mechanism of action[END_REF]. Potential effective compounds of this mushroom can be isolated either from their fruiting bodies, or even from pure culture of mycelia and culture broth filtrate.

Consumption of cultivated mushrooms or their extracts is suggested to have several benefits. Paradoxically no real safety study on wild A. subrefescens or A. bisporus has been performed. ABM is currently studied and commercialised as a potential functional food or beverage (tea). Several studies on many substances of this mushroom such as polysaccharides, sterol, sodium pyroglutamate, lectins and RNA-protein complex have been reported as potential bioactive substances [START_REF] Wasser | Is a widely cultivated culinarymedicinal royal sun Agaricus (the Himematsutake Mushroom) indeed Agaricus blazei Murrill?[END_REF][START_REF] Kerrigan | Agaricus subrufescens, a cultivated edible and medicinal mushroom, and its synonyms[END_REF][START_REF] Wasser | Medicinal mushrooms as source of antitumor and immunomodulating polysaccharides[END_REF][START_REF] Takaku | Isolation of an antitumor compound from Agaricus blazei Murrill and its mechanism of action[END_REF]. Among those molecules the most extensively studied are the polysaccharides for their anti tumour effect. One has to admit that polysaccharide structures from isolated fractions are poorly resolved and not forceful. These enriched fractions of polysaccharide could be contaminated by undetected compounds (possibly strongly cytotoxic ones). Many questions arise that fully justify the proposed studies aiming to evaluate the safe use of this mushroom and its eventual pure substances of interest.

What do we learn from the literature on A. subrufescens Peck, A. blazei Murrill and/or A. brasiliensis Wasser? Immuno-active involved in anti tumour effect and/or prevention; antimicrobial [START_REF] Wasser | Medicinal mushrooms as source of antitumor and immunomodulating polysaccharides[END_REF], [START_REF] Angeli | Protective effects of beta-glucan extracted from Agaricus brasiliensis against chemically induced DNA damage in human lymphocytes[END_REF], [START_REF] Yu | Inhibitory mechanisms of Agaricus blazei Murrill on the growth of prostate cancer in vitro and in vivo[END_REF], [START_REF] Jumes | Effects of Agaricus brasiliensis mushroom in Walker-256 tumor-bearing rats[END_REF] [6] [START_REF] Fujimiya | Selective tumoricidal effect of soluble proteoglucan extracted from the basidiomycete, Agaricus blazei Murrill, mediated via natural kill cell activation and apoptosis[END_REF], [START_REF] Gonzaga | In vivo growth-inhibition of Sarcoma 180 by an alpha-(1, 4)-glucan-beta-(1, 6)-glucan-protein complex polysaccharide obtained from Agaricus blazei Murill[END_REF] [11] [START_REF] Mizuno | Antitumor activity and some properties of water-soluble polysaccharides from "Himematsutake", the fruiting body of Agaricus blazei Murrill[END_REF] [11] [START_REF] Hikichi | Protein polysaccharide 0041[END_REF] [13], [START_REF] Mizuno | Antitumor activity and some properties of water-soluble polysaccharides from Himematsutake, the fruiting body of Agaricus blazei Murrill[END_REF], [START_REF] Mizuno | Anti-tumor polysaccharide from the mycelium of liquid-cultured Agaricus blazei mill[END_REF] Glucan-Protein complex Oncogenesis prevention [START_REF] Angeli | Protective effects of beta-glucan extracted from Agaricus brasiliensis against chemically induced DNA damage in human lymphocytes[END_REF], [START_REF] Fujimiya | Selective tumoricidal effect of soluble proteoglucan extracted from the basidiomycete, Agaricus blazei Murrill, mediated via natural kill cell activation and apoptosis[END_REF], [ The average composition of mushrooms is normally 90% water, 2-40% protein, 2-8% fat, 1-55% carbohydrate, 3-32% fibber, yielding 8-10% ash [START_REF] Firenzuoli | The Medicinal Mushroom Agaricus blazei Murrill: Review of Literature and Pharmaco-Toxicological Problems[END_REF]. Several categories of molecules are supposed to be involved in beneficial effects and most of the molecules categories found in A. subrufescens (A. blazei Murill) are common to the entire fungal kingdom. Active compounds found in A. blazei are listed in Table 1. The active compounds isolated from A. subrufescens are believed to be mainly polysaccharides [START_REF] Wasser | Medicinal mushrooms as source of antitumor and immunomodulating polysaccharides[END_REF], [START_REF] Angeli | Protective effects of beta-glucan extracted from Agaricus brasiliensis against chemically induced DNA damage in human lymphocytes[END_REF], [START_REF] Yu | Inhibitory mechanisms of Agaricus blazei Murrill on the growth of prostate cancer in vitro and in vivo[END_REF], [START_REF] Jumes | Effects of Agaricus brasiliensis mushroom in Walker-256 tumor-bearing rats[END_REF] β-glucan [START_REF] Fujimiya | Selective tumoricidal effect of soluble proteoglucan extracted from the basidiomycete, Agaricus blazei Murrill, mediated via natural kill cell activation and apoptosis[END_REF], [START_REF] Gonzaga | In vivo growth-inhibition of Sarcoma 180 by an alpha-(1, 4)-glucan-beta-(1, 6)-glucan-protein complex polysaccharide obtained from Agaricus blazei Murill[END_REF], [START_REF] Mizuno | Antitumor activity and some properties of water-soluble polysaccharides from "Himematsutake", the fruiting body of Agaricus blazei Murrill[END_REF], [START_REF] Mizuno | Antitumor activity and some properties of water-soluble polysaccharides from Himematsutake, the fruiting body of Agaricus blazei Murrill[END_REF] and glucomannan [START_REF] Hikichi | Protein polysaccharide 0041[END_REF], [START_REF] Tsuchida | Glucomannan separated from Agaricus blazei mushroom culture and antitumor agent containing as active ingredient[END_REF], [START_REF] Mizuno | Antitumor activity and some properties of water-soluble polysaccharides from Himematsutake, the fruiting body of Agaricus blazei Murrill[END_REF] or riboglucan [START_REF] Cho | Chemical features and purification of immunostimulating polysaccharides from the fruiting bodies of Agaricus blazei[END_REF] (Table 1). Additionally, in A. blazei, a protein (or glycoprotein), Glucan protein complex, a lectin [START_REF] Kawagishi | Formolysis of a potent antitumor (1-6)-b-D-glucan-protein complex from Agaricus blazei fruiting bodies and antitumor activity of the resulting products[END_REF], [START_REF] Cho | Chemical features and purification of immunostimulating polysaccharides from the fruiting bodies of Agaricus blazei[END_REF], originally found associated to β (1, 6) -glucan, were characterized and claimed to be anti-tumour (Table 1). Recently, it has been shown that A. blazei sp do contain aromatic hydrazines, especially agaritine [START_REF] Stijve | Potential toxic constituents of Agaricus brasiliensis (A. Blazei ss. Heinem.), as compared to other cultivated and wild-growing edible mushrooms[END_REF] (Table 1) and its derivatives [START_REF] Nagaoka | Measurement of a genotoxic hydrazine, agaritine, and its derivatives by HPLC with fluorescence derivatization in the Agaricus mushroom and its products[END_REF]. More recently, blazein a steroid derivative found in A. blazei (A. subrufescens), was shown to kill Human lung cancer LU99 cells but not normal Human lymphocyte [START_REF] Itoh | Blazein of a new steroid isolated from Agaricus blazei murrill (himematsutake) induces cell dealth and morphological change indicative of apoptotic chromatin condensation in human lung cancer LU99 and stomach cancer KATO III cells[END_REF]. Agaritine extracted from A. blazei was also showed to kill leukemic cell in vitro. Its cytotoxicity is triggered cell apoptosis with an IC50 in the range of 2.7 to 16 µg /ml depending on the cell line [START_REF] Akiyama | Agaritine from Agaricus blazei Murrill induces apoptosis in the leukemic cell line U937[END_REF], [START_REF] Endo | Agaritine purified from Agaricus blazei Murrill exerts anti-tumor activity against leukemic cells[END_REF]. Agartine is found at similar concentration in both A. blazei and A. bisporus (approximately 1.8 mg/g dry weight) [START_REF] Stijve | Potential toxic constituents of Agaricus brasiliensis (A. Blazei ss. Heinem.), as compared to other cultivated and wild-growing edible mushrooms[END_REF].

Several authors found antibacterial properties of glucan against Staphylococcus aureus [START_REF] Endo | Agaritine purified from Agaricus blazei Murrill exerts anti-tumor activity against leukemic cells[END_REF] rendering this compound an attractive immunotherapeutic agent even against diverse virus [START_REF] Williams | Glucan-induced modification of murine viral hepatitis[END_REF], [START_REF] Taniguchi | Depression of protective mechanisms against ectromelia virus infection in tumor-bearing mice and its prevention by PSK[END_REF], [START_REF] Ooi | Immunomodulation and anti-cancer activity of polysaccharideprotein complexes[END_REF]. [START_REF] Jung | Antiviral effect of Saccharomyces cerevisiae betaglucan to swine influenza virus by increased production of interferon-gamma and nitric oxide[END_REF].

Further, these immune modulators properties were also described for several diverse mushrooms polysaccharides extracts [START_REF] Ooi | Immunomodulation and anti-cancer activity of polysaccharideprotein complexes[END_REF]. This potential effect was also clearly reported for Agaricus blazei extracts by several authors such as for example Mizuno et al. [START_REF] Mizuno | Polysaccharides from Agaricus blazei stimulate lymphocyte T-cell subsets in mice[END_REF]. Therefore it is believed that mushrooms glucans or protein-polysaccharides complex have host mediated immune-modulator properties that trigger anti-cancer and anti-microbial effects. Furthermore, the water-soluble extracts (proteo-glucans) and AndoSan from crude A. blazei up regulate the in vitro maturation of dentritic cells [START_REF] Kim | Effect of water-soluble proteoglycan isolated from Agaricus blazei on the maturation of murine bone marrow-derived dendritic cells[END_REF], [START_REF] Førland | Effect of an extract based on the medicinal mushroom Agaricus blazei Murill on expression of cytokines and calprotectin in patients with ulcerative colitis and Crohn's disease[END_REF]. Ex vivo experiment on total heparinised blood of volunteers and in vitro experiment show that the monocytes-derived dendritic cells from peripheral blood mononuclear cells produce an increase level of cytokine and chemokines. The most abundant cytokines after A. blazei Murill stimulation were mainly pro-inflammatory cytokine and chemocytokines IL-8, G-CSF, TNF-α, IL-1β, IL-6, IL 17 and MIP-1β. The synthesis of IL-2 and IFN γ is also reported but not of IL-12 as previously said. Therefore only some Th1-type and Th17 type cytokines were up regulated whereas no anti-inflammatory or Th2 cytokines were stimulated in vitro. Whereas ex vivo there is a release of different cytokines including Th-1, TH-2, pro inflammatory or anti-inflammatory as well as the chemokines and leukocyte growth factor. Therefore this A. blazei immunostimulatory extract proceed mainly by pro inflammatory chemocytokine and cytokines in vitro or ex vivo.

The inhibition of pro-inflammatory cytokines appears to be dose dependant [START_REF] Johnson | Effect of an extract based on the medicinal mushroom Agaricus blazei murill on release of cytokines, chemokines and leukocyte growth factors in human blood ex vivo and in vivo[END_REF]. This is in contradiction with most of the tests made in murine models with different A. blazei extracts. This contradiction can be explained only by the capacity of certain glucans to cross the murine intestinal barriers. Ex vivo experiment with healthy volunteers heparinised blood controversially showed stimulation of all pro-inflammatory cytokines tested as expected. More recently, a clinical test with patients suffering of inflammatory bowel disease (IBD) inflammatory disease of colon and intestine has been conducted [START_REF] Førland | Effect of an extract based on the medicinal mushroom Agaricus blazei Murill on expression of cytokines and calprotectin in patients with ulcerative colitis and Crohn's disease[END_REF]. Patients with Chron's disease (CD) widely regarded as an autoimmune disease, and ulcerative colitis (UC) were exposed orally to A. blazei enriched mixture (AndoSan). After 12 days ingestion, the A blazei extract promotes in these patients anti-inflammatory effects with no side effect. There are opposite effects between in vivo and ex vivo assays with normal patients, proinflammatory cytokines and chemokines are controversially down regulated and up regulated respectively. This discrepancy are tentatively explained by the antioxidant potential of A. blazei [START_REF] Ker | Antioxidant capability of polysaccharides fractionated from submerge-cultured Agaricus blazei mycelia[END_REF], [START_REF] Oliveira | Antioxidant activity of Agaricus blazei[END_REF] and the limited absorption of large macromolecules complex or bioactive molecule such β-glucans across the intestinal mucosa to the reticulo-endothelial system and blood through the gastrointestinal tract. With CD and UC patients the same conclusive explanation is proposed, but the intestinal barrier is hypothesized to be presumably selectively permeable to certain βglucans bioactive fragments to body fluids (blood and lymphoid system) as in murine [START_REF] Førland | Effect of an extract based on the medicinal mushroom Agaricus blazei Murill on expression of cytokines and calprotectin in patients with ulcerative colitis and Crohn's disease[END_REF]. This is yet too speculative. Does the presence of molecules that differ from polysaccharides or PAMPS (Pathogen Associated Molecular Patterns) exert immunosuppressive effect on the innate immune system?

It appears clearly that most of the studies were performed on mixtures of substances (even through purified) allowing these contradictory data in which the same substance does something and the contrary. Thus purification and specific studies are absolutely needed in each of the structural groups of substance.

All together it appears that A. blazei extracts do exert both immunostimulatory and immunosuppressive effects. Globally glucan, proteoglucan complex peptide-glucan appeared to be rather immunostimulating and promoting indirectly anti cancer benefits. Whether immunosuppression is triggered by the same categories of molecules such as more or less branched polysaccharides or proteoglucans remains unclear and too speculative. However, because of the immunosuppressive potential, safety of chronic exposure to dietary edible commercial A. blazei should be evaluated.

The biological material to be studied consists of original Brazilian and European A. subrufescens cultivated on conventional compost using A. bisporus as reference material for comparison. The whole study has then been designed to investigate on mushrooms safety and new products of therapeutic interest or for disease prevention: fruitbody composition, nature of the metabolites produced by this species and their toxicological or pharmaceutical effects. Preliminary data are included herein.

MATERIALS AND METHODS

Production of mushrooms extracts. A. subrufescens CA454 (CGAB collection, INRA, France)

formerly A. blazei (strain ATCC 76739) from Brazil and at the origin of commercial strains, A. subrufescens CA 487, European strain of French origin (CGAB collection, INRA, France) and Agaricus bisporus 30A (commercial strain, France Mycelium), were grown in the facilities of the research group MycSA, INRA France. Fungal preparation for animals' treatment: A mass of 100 g of freeze-dried mushrooms was ground with a blender; powder was added to 100 mL of ultra-pure sterile water (Milli-Q plus, Millipore, France) at room temperature for 30 min with stirring to yield a suspension of mushroom (mother suspension) which was subsequently diluted to 0.5g and 0.25g/ml, used for gavages. When needed suspensions were centrifuged at 2000 g for 20 min to yield supernatants used for intra-peritoneal injections. Fungal water extraction methods for in vitro alternative assays: Hot water hydrophilic extracts: A mass of 10 g of freeze-dried mushrooms was ground with a blender; powder was added to 100 mL of ultra-pure boiling water (100 °C) for 6 hours with stirring [START_REF] Nakamura | Fractionation and anti-tumor activity of the mycelia of liquidcultured Phellinus linteus[END_REF]. The suspension was then centrifuged at 2000g for 10 min at 20 °C. The supernatant was freeze dried and the resulting powder weighted. For in vitro alternative assay, a stock solution was prepared by re-suspending the powder directly in cell culture medium without foetal calf serum and dilution was made with the same medium. Cold water hydrophilic extracts: A mass of 10 g freeze dried mushroom was ground and lyophilized. The matrix was put into a flask by adding 100 mL of sterile ultrapure water. The vial was placed 1 h at room temperature and then at 4 °C for 48 h with agitation. The suspension was then centrifuged at 2000xg for 10 min at 4 °C. The supernatant was recovered, lyophilized and the resulting powder weighted. For in vitro alternative assay, stock solution and diluted solutions were made as described above.

Animal testing. Male and female Swiss mice (22 ± 2 g) and male and female Wistar rats (200 ± 20g) were weighed and stall feeding one week before the test. They were divided into two groups consisting of four subgroups of five animals.

Group 1, "Evaluation of tolerance and safety": Groups of 10 males and females Wistar rats separately caged received each day for a week, 1 gram of mushroom suspension per kilogram of body weight. They were kept under observation for an additional 15 days after the end of treatment for any signs of toxicity or behavioural modification. They were weighed daily and at the end of the experiment, 21 days (n = 10). Similarly, mice have been treated to evaluate the tolerance. Group 2, Evaluation of acute oral dose "(limit test)": Animals were divided into groups of 5 males and females separately caged and received mushroom suspension either 1 g / kg of body weight or 2 g / kg of body weight in a single intra gastric tube. "Evaluation of acute oral dose (limit test)": The animals received by gavages a dose of either 2g/kg or 5g/kg body weight and kept under observation for two weeks. The OECD test 423 was conducted with female rats of the Sprague Dawley strain F344 (SPF Caw) of about 8 to 12 weeks at the beginning of the test. These tests were performed in GLP conditions at the Phycher-Biodevelopment Laboratory in Cestas (France). In vivo Model to assess anti carcinogenesis effect: The rat gut carcinogenesis model of methylimidazol and phenylimidazol which are known to promote cancer has been used. F344 rats were treated with aromatic amine derivatives (methylimidazol and phenylimidazol) for one week. Then they received crude extracts of Agaricus sp or suspension of mushrooms powder, or extracts daily by oral route (0.25, 0.5 and 1g/kg body weight) for three months. Intestines from all animals were examined following staining with methylene blue (1% in NaCl 0.9%) to assess the pre-neoplasic changes (different foci following histological observation under microscope; magnification 40). Preliminary test in cancer model mice: Animals, Balb-c male and female used to develop the mouse model of cancer were treated (n = 5) orally and / or by the intra peritoneal route (supernatant of mushroom suspension following centrifugation at 2000g for 20min) with a dose of extract corresponding to 0.5, 1 and 2 g of fresh mushrooms each day for 15 days. They were observed daily before treatment and kept under observation for 15 days after the end of treatment. Then 4 groups of 10 mice were treated following transplant of L1210 and/or SP2-O leukaemia cells in the peritoneal cavity. After 10 days of incubation, animals were treated daily with either pure sterile saline (NaCl, 0.9%) or extracts of A. subrufescens CA454, A. subrufescens CA487, and/or A. bisporus 30A. They were kept under observation until death.

In vitro assays. Cell culture: The HepG2 human hepatocarcinoma cell line (ATCC HB8065) was grown in MEM-Glutamax containing 1 g/l glucose supplemented with 10% foetal calf serum (FCS), 50 U/ml penicillin and 50 µg/ml streptomycin and 1% non-essential amino acids. The Neuro2a mouse neuroblastoma cell line was cultured in RPMI 1640 containing 2g/l glucose, 2% L-glutamine and supplemented with 10% foetal calf serum, 1 mM sodium pyruvate, 50 U/ml penicillin and 50 µg/ml streptomycin. All cells were routinely grown in 75cm 2 and incubated in humidified atmosphere consisting of 5% CO2, 95% air mixture at 37° C. Cytotoxicity assay by MTT test: Before treatment, cells (10 5 cells/ml) were cultured (100 µl) in 96-well plates for 24 h. The medium was removed from the wells and cells were exposed (triplicates) during 48 h to different concentrations of fungal extracts amended into the medium without foetal calf serum (100µl/well). Plate design omitted marginal rows and columns and 6 wells were used for cell viability control. The cell viability was measured using the 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. After treatment, the medium was replaced by a FCS-free medium containing 0.5 mg/ml MTT (Sigma) for 2 h at 37°C. The medium was discarded prior the addition of DMSO to dissolve the formazan. The absorbance was read at 495 nm with a microplate reader (LT4000 Labtech, France). Viability was expressed as the percentage of mean absorbance of the cell viability in control (100% viability).

Statistical analysis. Data were expressed as mean ± SEM or absolute value where needed and always compared to controls by using Analysis of Variance and Tukey Honestly Significant Difference tests. Statistical analysis was done by using the R software (v2. 3.1, 2006) and P values less than 0.05 was considered to be significant.

RESULTS AND DISCUSSION

First we found during the grinding and preparation of diverse solutions of the mushrooms that the strains of A. subrufescens showed different colours: CA487 yielded a yellowish white suspension in cold extracts while those of CA 454 were yellow curry. Comparatively suspensions of A. bisporus 30A were light brown. The colour differences between the suspension of strains of A. subrufescens (CA 454 and CA 487) and A. bisporus A30 might be related to a difference in composition including that of the active substances.

Evaluation of Tolerance and Acute Oral Dose "(Limit Test)". Animals, rats and mice (group 1) showed no toxic effect during treatment or within the 15 days of surveillance following treatment, excepted, those treated with the suspension of powder of A. subrufescens strains at a dose of 2 g / kg. In these animals a decrease in locomotor activity and a soothing effect were observed. The gains in body weight did not change significantly compared with controls who received only water. The relative weights of major organs (liver, kidney, heart, brain, genitals) have not changed either.

Group 2 animals (rats and mice) showed no acute toxic effects with the exception of animals receiving 2 g / kg body weight of A. subrufescens CA454. Similarly, decrease in locomotor activity and a soothing effect were observed within 24 to 72 hours. As before there were no other changes.

The OECD 423 test conducted under the conditions of COFRAC GLP accreditation revealed in the rat a LD50> 5g/kg of body weight.

Treatment resulted in no significant effect either by the intra peritoneal route (supernatant of suspension) or by the oral route. The product is well tolerated and does not lead to toxic effect except locomotor hypoactivity with all the suspensions of A. subrufescens CA454 and CA487. Neither weight gain nor the relative mass of the organs were affected. Safety testing (12 weeks) and oral acute toxicity tests show a difference between CA454 and CA487. The first one involved an unexpected and currently unexplained locomotor hypoactivity. We were unable to connect this hypoactivity (during the first 2-3days) with the swelling of foot pads as described by Chan et al. [START_REF] Chan | Immunomodulatory effects of Agaricus blazei Murill in Balb / cByJ mice[END_REF] in connection with a pro-inflammatory (TNF-alpha) and hypersensitivity phenomena. Altogether these data suggest that the extraction protocol using water (room temperature, and centrifugation) is not sufficient to extract all substances of the mushrooms. Hence extraction following boiling is subsequently proposed. Indeed, Chan et al. [START_REF] Chan | Immunomodulatory effects of Agaricus blazei Murill in Balb / cByJ mice[END_REF] have performed their extract preparation in hot water (80-85°C) and finished by sterilization at 105°C for 10 min. They have certainly extracted additional substances as compared to our extracts. The confirmation of this hypothesis is underway with further solid state NMR studies of our recent extracts at high temperature. The dose of 5 g of fresh mushrooms per kilogram body weight would make 300 g of fresh mushrooms for a person of 60 kg, without acute toxic effects. However, one cannot exclude chronic or sub chronic effects, following daily or frequent ingestion of such a dose of mushrooms or the corresponding extract, for several weeks or months.

Such a chronic toxic effect (tumour-promoting activities) has been revealed in rodents fed with feeds containing A. blazei Murill in Japan [START_REF] Itoh | Blazein of a new steroid isolated from Agaricus blazei murrill (himematsutake) induces cell dealth and morphological change indicative of apoptotic chromatin condensation in human lung cancer LU99 and stomach cancer KATO III cells[END_REF]. This finding led to a severe restriction of commercialisation of A. blazei Murill, whatever the form, for human consumption by the Ministry of Health, Labour and Welfare of Japan in 2006.

This apparent lack of toxicity does not exclude the presence of active substances. Indeed many situations can be considered. It can there be a substance having an effect on immunity as described in the literature, immuno stimulant [START_REF] Chan | Immunomodulatory effects of Agaricus blazei Murill in Balb / cByJ mice[END_REF] or immunosuppressant [START_REF] Førland | Effect of an extract based on the medicinal mushroom Agaricus blazei Murill on expression of cytokines and calprotectin in patients with ulcerative colitis and Crohn's disease[END_REF][START_REF] Johnson | Effect of an extract based on the medicinal mushroom Agaricus blazei murill on release of cytokines, chemokines and leukocyte growth factors in human blood ex vivo and in vivo[END_REF].

In Vivo Model to Assess Anti Carcinogenesis Effect and Preliminary Testing of Anti

Cancer Properties. The administration of the Agaricus powder suspension or fungal extracts (0.25, 0.5 and 1g/kg Body Weight) elicits positive effect on intestinal carcinogenesis. All mushrooms were effective in preventing gut-induced carcinogenesis (Tables 234). The relative preventions were respectively of 72%, 64% and 56% for CA454, CA487 and A30 (Table 5). For the later this preventive effect is unexpected and indeed demonstrated for the first time. This justifies the use of Agaricus sp in cancer prevention by millions of people all over the world. The doses used in our experiments were fixed at respectively 1/20 to 1/5 of the maximum dose experimentally ingested since LD50≥ 5g/kg BW (as obtained in a previous work, BRG national project in 2007) that is 0.25, 0.5 and 1 g/kg BW. From the lower dose of 0.25 g/kg BW to the rat, we already observed almost the maximum protective effect. It appears that there is no dose-effect relationship in this protective effect. Interestingly Johnson et al. [START_REF] Førland | Effect of an extract based on the medicinal mushroom Agaricus blazei Murill on expression of cytokines and calprotectin in patients with ulcerative colitis and Crohn's disease[END_REF] and Forland et al. [START_REF] Johnson | Effect of an extract based on the medicinal mushroom Agaricus blazei murill on release of cytokines, chemokines and leukocyte growth factors in human blood ex vivo and in vivo[END_REF] have given to human a dose of 60 ml per day of a solution containing 340g/L that makes 0.34 g/kg BW for a person of 60 kg. These doses are quite similar to those given to the rats in the present experiments, however in our case and having the end-point of pre neoplasic changes, we found a very significant protective effect in the rats whereas in [START_REF] Førland | Effect of an extract based on the medicinal mushroom Agaricus blazei Murill on expression of cytokines and calprotectin in patients with ulcerative colitis and Crohn's disease[END_REF] and [START_REF] Johnson | Effect of an extract based on the medicinal mushroom Agaricus blazei murill on release of cytokines, chemokines and leukocyte growth factors in human blood ex vivo and in vivo[END_REF] immunosuppressant effects in humans were observed. If the preventive effect observed is triggered by immune stimulation as forecasted or hypothesised by several authors [START_REF] Endo | Agaritine purified from Agaricus blazei Murrill exerts anti-tumor activity against leukemic cells[END_REF][START_REF] Chan | Immunomodulatory effects of Agaricus blazei Murill in Balb / cByJ mice[END_REF], our data appear incongruent with those of Johnson et al. and Forland et al. [33,[START_REF] Johnson | Effect of an extract based on the medicinal mushroom Agaricus blazei murill on release of cytokines, chemokines and leukocyte growth factors in human blood ex vivo and in vivo[END_REF]. But, if the tumour preventing effects are triggered by cytotoxicity against modified cells bearing rapid growth rate [START_REF] Jin | Bcl-2 and caspase-3 are major regulators in Agaricus blazei-induced human leukemic U937 cell apoptosis through dephoshorylation of Akt[END_REF][START_REF] Kim | Inhibitory effect of Agaricus blazei extracts on human myelomia cells[END_REF], then our data are congruent with the above studies [START_REF] Førland | Effect of an extract based on the medicinal mushroom Agaricus blazei Murill on expression of cytokines and calprotectin in patients with ulcerative colitis and Crohn's disease[END_REF][START_REF] Johnson | Effect of an extract based on the medicinal mushroom Agaricus blazei murill on release of cytokines, chemokines and leukocyte growth factors in human blood ex vivo and in vivo[END_REF], since the immunosuppressant effect observed could be triggered by cytotoxicity.

Based on the present data our preliminary conclusion is that A. subrufescens CA454, CA 487 and A. bisporus 30A might have tumour preventing properties. Then further step was investigation on anti tumour properties. This has been done using the mouse transplanted-tumour model. Balb/c mice have been treated with similar doses of mushroom extracts by both intra peritoneal and oral routes after having been injected with L1210-SP2O leukaemia cells. We were unable to prevent death of animals. Thus no anti-tumour effect can be claimed for A. subrufescens CA 454, CA 487 and A. bisporus A30, at least with these extracts.

In Vitro Assays. After obtaining cold and hot hydrophilic extracts they were tested in vitro on two cell lines Hep-G2 and Neuro-2A. The concentrations used in vitro were chosen from our in vivo data, considering that we should not put more than 1000 µg/ml when 1 g/kg BW is effective in vivo. Further we have seen that most of the research groups never exceeded 600 µg/ml of extracts or polysaccharides in their culture medium. Our in vitro data revealed no effect until concentrations of 200 µg/ml, then slight mitogenic effect, when cell growth was stimulated somehow between 200 and 500 µg/ml and then no clear inhibitory effect was observed above these concentrations (Fig. 1, a,b,c andd). Hep-G2 cells were more sensitive to these extract than Neuro-2a cells. Since Hep-G2 cells originate from the liver, it could be that liver cells are more sensitive to A. subrufescens than other cells. Indeed several authors found some liver damages (fulminant hepatitis) with extract of A. subrufescens sp [START_REF] Firenzuoli | The Medicinal Mushroom Agaricus blazei Murrill: Review of Literature and Pharmaco-Toxicological Problems[END_REF][START_REF] Mukai | An alternative medicine, Agaricus blazei, may have induced severe hepatic dysfunction in cancer patients[END_REF], A. subrufescens strains were more active than A. bisporus, with CA454 being the more active.

These data are congruent with the stimulatory effects observed on immune cells both in vitro and in vivo. Purification and characterization of active substances could help finding in the future any curative effect borne by these mushrooms. At present our in vitro data show some growth stimulatory effects at the tested concentrations. It is noticeable that the cold extracts are more effective than the hot extracts (Fig. 1 a, b, c andd). This observation could be due to the partial destruction of some active mitogenic substances by heating or differential extraction.

CONCLUSION

The final goal of the toxicological and chemical studies is to give safety information and to promote any of these mushroom strains which will prove to be of interest either as food or as drug to be used for curative or preventive purposes so that implementation of its culture at a large scale will proceed of sustainable and profitable agriculture. Agaricus subrufescens or A. blazei, A. brasiliensis has been consumed by human for decades. Due to the popularity, it is increasingly cultivated in many countries for its putative medicinal properties. Many scientific studies reported various medicinal benefits i.e. anti-cancer, anti-microbial anti oxidant and anti inflammatory activities. Although these mushrooms have many bioactive compounds that could be developed for curative properties toxicological problems are still discussed. There is clearly a lake of information on toxicological assessment (acute and chronic toxicity) of compounds such as agaritine, blazein and of the whole mushroom A. subrufescens itself, and overall on epidemiological data linking the consumption of Agaricus sp and eventual prevention and/or pathologies.

Our data reveal that A. subrufescens strains of different origins are not toxic either in vivo or in vitro except some locomotor hypoactivity. Additionally they elicit slight cell growth stimulation at the concentrations tested in vitro. The most active is A. subrufescens (A. blazei Murrill) from Brazil whereas the most productive is the European species [START_REF] Hernández | Phenotypic variability of Agaricus subrufescens[END_REF].

Figure 1 :

 1 Figure 1: Effects of hydrophilic extracts of A. subrufescens sp (Asub) and A bisporus (Abi) on the viability of Hep G2 and Neuro-2a cells; (a) and (c) = cold water extracts of total mushrooms; (b) and (d) = hot water extracts of total mushrooms.

Table 1 :

 1 Bioactive compounds from Agaricus subrufescens

	Bioactive Compounds	Main Potential Effects	References
	Polysaccharides
	β-(1,6)-glucan

α-(1,4)-; β-(1,6)-glucan α-(1,6)-; α-(1,4)-glucan β-(1,6)-; β-(1,3)-glucan β-(1,6)-; α-(1,3)-glucan Glucomannan β-(1,2)-β-(1,3)-glucomannan

  [START_REF] Gonzaga | In vivo growth-inhibition of Sarcoma 180 by an alpha-(1, 4)-glucan-beta-(1, 6)-glucan-protein complex polysaccharide obtained from Agaricus blazei Murill[END_REF]

	Lectin	Anti-tumour	[17]
	Riboglucan	Anti-tumour	[18]
	Ergosterol	Anti-tumour	[4]
	Sodium pyroglutamate	Anti-tumour	[19]
	RNA-protein complex	Anti-tumour	[20]
	Agaritine	Anti-tumour	[21]
	Blazein	Anti-tumour	[23]

Table 2 :

 2 Number of histological foci observed in intestinal tissues of rats treated by A. subrufescens suspension and/or abstracts (0.25g/kg BW) following induction carcinogenesis process with phenylimidazolic compounds. Each number represents the sum of foci counted in 4 different fields by slice.

	Treatments		Pre neoplastic lesions observed by slices of intestinal tissue
		Type 1 lesions	Type 2 lesions	Type 3 lesions	Type 4 lesions
		(Big size foci)	(Foci with thick	(Extended Foci)	(Foci with peripheral
			margin)		zones)
	Untreated Controls
	(n=5)	1	0	0	0
	A. subrufescens CA 487 (n=5)	3	3	2	3
	A. subrufescens CA 454 (n=5)	1	2	2	4
	A. bisporus A30 (n=5)	4	4	2	4
	Imidazol-Treated Controls (n=5)	6	9	5	8

Table 3 :

 3 Number of histological foci observed in intestinal tissues of rats treated by A. subrufescens suspension and/or abstracts (0.5g/kg BW) following induction of carcinogenesis process with phenylimidazolic compounds. Each number represents the sum of foci counted in 4 different fields by slice.

	Treatments

Pre neoplastic lesions observed by slices of intestinal tissue Type 1 lesions (Big size foci) Type 2 lesions (Foci with thick margin) Type 3 lesions (Extended Foci) Type 4 lesions (Foci with peripheral zones)

	Untreated (n=5)	Controls	1	0	0	1
	A. subrufescens CA 487 (n=5)	2	3	1	5
	A. subrufescens CA 454 (n=5)	2	2	1	3
	A. bisporus A30 (n=5)	3	5	1	3
	Imidazol-Treated Controls (n=5)	4	6	4	10

Table 4 :

 4 Number of histological foci observed in intestinal tissues of rats treated by A. subrufescens suspension and/or abstracts (1g/kg BW) following induction of carcinogenesis process with phenylimidazolic compounds. Each number represents the sum of foci counted in 4 different fields by slice.

	Treatments	Pre neoplastic lesions observed by slices of intestinal tissue
		Type 1 lesions	Type 2 lesions	Type 3 lesions	Type 4 lesions
		(Big size foci)	(Foci with thick	(Extended Foci)	(Foci with
			margin)		peripheral zones)
	Untreated Controls (n=5)	0	1	0	1
	A. subrufescens CA 487 (n=5)	4	3	2	2
	A. subrufescens CA 454 (n=5)	3	2	3	2
	A. bisporus A30 (n=5)	4	5	3	1
	Imidazol-Treated Controls (n=5)	8	10	6	7

Table 5 :

 5 Sum of all types of foci for each mushroom and percent of prevention following normalisation of values, taking into account that 5 foci/group of 5 animals is not significant.

	Treatment	Total pre-neoplasic foci	% of prevention
	Untreated Controls (n=15)	5	100
	A. subrufescens CA 487 (n=15) (0.25-1g/kg BW)	33	64
	A. subrufescens CA 454 (n=15) (0.25-1g/kg BW)	27	72
	A. bisporus A30 (n=15) (0.25-1g/kg BW)	39	56
	Imidazol-Trémate Controls (n=15)	83	0

ACKNOWLEDGEMENTS

We acknowledge research funding from the Bureau des Ressources Génétiques (BRG), France, project 2007-2008 n°51, and from the Agence Nationale de la Recherche, project ANR-09-BLAN-0391-01.


Genetic variability of FCR with suboptimal food and prospects for selection F eed cost represents a high part of production cost in chickens. So, feed efficiency is a key factor of poultry production profitability. Genetic selection of standard broiler chickens has resulted in a huge decrease in feed conversion ratio. Nowadays, as high quality feedstuffs price and human population both increase, there is a growing interest in improving efficiency of birds on alternative diets, for economical and ethical reasons. These diets could include low cost feedstuffs or by products of agriculture that are not consumed by humans. However, these alternative diets often show low and variable quality. Moreover, this approach is a kind of challenge since selection is usually made on high quality diets in order to express the growth genetic potential of modern genotypes. This review aims at evaluating the potential interest in using alternative and lower quality diets in a selection for feed efficiency, keeping in mind that the diet quality may interact with the selection criteria used to reach this goal.

Introduction

Feed cost represents the highest part of the production cost of meat-type chickens, from 55 to 65 % depending on the production type [START_REF] Riffard | Performances techniques et coûts de production en volailles de chair, poulettes et pondeuses[END_REF]. Therefore, feed efficiency is a key factor in the profitability of poultry rearing. The selection of rapid growing broilers for 3 decades resulted in a continuous decrease of feed conversion ratio in these birds, as shown in Figure 1. The "certified chickens" that result from a cross between a standard broiler sire and a slow-growing dam also show such a tendency (Figure 1). However, no decrease in feed conversion ratio is observed for slow growing "label rouge" chickens (Figure 1) [START_REF] Riffard | Performances techniques et coûts de production en volailles de chair, poulettes et pondeuses[END_REF].

Nowadays, the importance of feed efficiency has been reinforced for economic and ethical reasons. Feed price, especially for the high quality diets required for modern genotypes, is still increasing. For example, in France in 2010, feed price was 9.1% and 30.9% than in 2009 and 2006, respectively [START_REF] Riffard | Performances techniques et coûts de production en volailles de chair, poulettes et pondeuses[END_REF]. These prices become limiting for the development of production in developing countries which have to import these feedstuffs and cannot achieve maximum performances due to environmental constraints as hot and humid climate [START_REF] Farrell | Matching poultry production with available feed resources: issues and constraints[END_REF]. Simultaneously, the increase of human population leads to an increased demand for poultry products, which in turn increases the demand for the high quality feedstuffs used in poultry diets. From an ethical point of view, it is important to be able to reduce the pressure of [START_REF] Riffard | Performances techniques et coûts de production en volailles de chair, poulettes et pondeuses[END_REF].

World´s Poultry Congress 5 -9 August -2012 • Salvador -Bahia -Brazil XXIV animals on cultivated surfaces, as 80% of cultivated surfaces are used to provide resources to animals and only 20% to humans [START_REF] Steinfeld | Livestock's Long Shadow: environmental issues and options. Food and Agriculture Organization of the United Nations[END_REF].

To reach this goal, it would be interesting to replace part of the high quality feedstuffs used in poultry diets by other feedstuffs, especially those that cannot be consumed directly by humans. However, these products often show a poor and variable quality, and may not be adapted to modern genotypes. Thus, it would be needed to select for chickens that would be adapted to such suboptimal diets.

Composition of feed effi ciency traits

Feed conversion ratio (FCR) (feed intake / weight gain) is a composite trait depending on compositions of feed and body growth. It can be modeled as follows (Carré et al., 2008a;[START_REF] Carré | Understanding and breeding for improved poultry feed efficiency[END_REF]

:
Where D is the duration of production, MBW the mean metabolic weight (i.e. mean body weight raised to the power 0.70), PW the production weight, C L and C P the lipid and protein concentrations of production, and AMEn the apparent metabolizable energy value of diet, corrected to zero nitrogen retention.

This equation illustrates why rapid growth and body composition have often been used as selection criteria to improve feed efficiency. Direct selection has also been performed but more scarcely, as it implies to measure individual feed intake, i.e. to place animals in individual cages. [START_REF] Fairfull | Breeding for feed-efficiency -poultry[END_REF] estimated that 65% of the differences in FCR originated from differences in weight, growth rate and body composition.

Selection for improved FCR

Direct selection for FCR Direct selection on FCR is possible as this trait present a moderate to high heritability [START_REF] Pym | Nutritional genetics[END_REF]. It has been confirmed by selection experiments on this trait. [START_REF] Leenstra | Fat deposition in a broiler sire strain. 2. Comparisons among lines selected for less abdominal fat, lower feed conversion ratio, and higher body weight after restricted or ad libitum feeding[END_REF] and [START_REF] Sutedjo | Selection for the components of lean tissue growth in Japanese quail (Coturnix coturnix japonica)[END_REF] showed that direct selection on FCR was more efficient to reduce FCR than selection on fatness or growth and that this selection also led to leaner birds. However, all these experiments have been done using standard diets in order to allow birds to express their genetic potential for growth, and did not try to show adaptability to poorer quality diets.

Indirect selection on growth under suboptimal environment [START_REF] Godfrey | Ten generations of selection for lysine utilization in Japanese quail[END_REF], [START_REF] Neisheim | Genetic variation in nutritional requirements of poultry. The Effect of Genetic Variance on Nutritional Requirements of Animals: Proceedings of a Symposium[END_REF], [START_REF] Sorensen | Selection for growth rate in broilers fed on diets with different protein levels[END_REF] and [START_REF] Marks | Long term selection for four week body weight in Japanese quail under different nutritional environments[END_REF] selected broilers or quails for growth under normal or 30% reduced protein rate, or low arginine or lysine content. [START_REF] Marks | Selection for 8-week body weight in normal and dwarf chickens under different water/ feed environments[END_REF] selected quails for growth under normal or high salt diet (0.4 vs 1.6%). Finally, [START_REF] Leenstra | Fat deposition in a broiler sire strain. 2. Comparisons among lines selected for less abdominal fat, lower feed conversion ratio, and higher body weight after restricted or ad libitum feeding[END_REF] selected chickens for growth under ad libitum or feed restricted conditions. All these studies found that the response to selection for growth was lower in the suboptimal environment than in the normal environment. In the feed restricted environment, [START_REF] Leenstra | Fat deposition in a broiler sire strain. 2. Comparisons among lines selected for less abdominal fat, lower feed conversion ratio, and higher body weight after restricted or ad libitum feeding[END_REF] had to apply a selection pressure twice as high as in the ad libitum environment to obtain similar responses in both environments. Without no difference in selection intensity, [START_REF] Sorensen | Selection for growth rate in broilers fed on diets with different protein levels[END_REF] found lower response to selection for body weight at 40 d in the line selected on low protein diet (LP) than in the line selected on normal protein diet (NP) after 7 generations of selection (+ 80 g vs +250 g). When both lines were fed the normal protein diet, the NP line was 75 g heavier than the LP line [START_REF] Sorensen | Influence of diet on response to selection for growth and efficiency[END_REF]. At the opposite, when fed with reduced protein content, the LP line was 180 g heavier than the NP line. So, this strategy did not improve adaptability to suboptimal diets, but adaptation to a special diet. Moreover, when the protein rate decreased too much, the line selected on low protein content became extremely variable.

These results are consistent with the fact that heritability of growth traits is 0.05 to 0.10 points lower in the unfavorable environments than in the favorable environments [START_REF] Godfrey | Ten generations of selection for lysine utilization in Japanese quail[END_REF]Marks, 1986[START_REF] Marks | Selection for 8-week body weight in normal and dwarf chickens under different water/ feed environments[END_REF].

Finally, this selection on growth in unfavorable environment had a negative effect on body composition and on efficiency. Indeed, as protein but not energy content was modified, birds increased their feed consumption to meet their protein requirements. For example, the quail line selected on growth with a low lysine diet consumed 11 to 13% more than the line selected on adequate XXIV lysine content when fed a diet with low lysine content [START_REF] Godfrey | Ten generations of selection for lysine utilization in Japanese quail[END_REF], but the difference of feed intake vanished when they were both fed an adequate diet. The excess of energy consumption is deposited as fat, which could be deduced from equation [1] (Carré et al., 2008a). So, the LP line had 17% more total fat and 37% more abdominal fat than the NP line when both lines were fed a standard commercial diet, 25% more total fat and 60% more abdominal fat when they were fed a restricted protein diet [START_REF] Sorensen | Selection for growth rate in broilers fed on diets with different protein levels[END_REF]. When fed a normal diet, the LP line had a higher FCR (+0.05), lower digestibility of dry matter (-1.3%) and a lower proportion of nitrogen retention (-6.4%, [START_REF] Sorensen | Protein and energy metabolism in two lines of chickens selected for growth on high or low protein diets[END_REF].

Indirect selection on digestive effi ciency

It has long been argued that digestive efficiency was not controlled by genetics as differences between breeds were low and as birds were already very efficient (Neishem, 1975;[START_REF] Pym | Nutritional genetics[END_REF]. However, [START_REF] Carré | Relationships between digestibilities of feed components and characteristics of wheat (Triticum aestivum) introduced as the only cereal source in a broiler chicken diet[END_REF] compared different varieties of wheat used in poultry diets, and observed that the variability of metabolizable energy of diets was much lower between wheat varieties (around 300 kcal.kg -1 DM) than between animals within a variety (around 900 kcal.kg -1 DM) (cf. Figure 2). More recently, Bandengan et al. (2009) also stated that the variability of ileal digestibility of proteins and amino acids was much higher between animals than between diets, even when using highly variable feedstuffs such as DDGS. For instance, the proportion of total variability of amino acids digestibility explained by the diet was ranging between 4.9% for phenylalanine to 24.1% for lysine (9.2% for total crude proteins), whereas variability due to animals within a diet represented respectively 95.1%, 75.9%, and 90.8% for phenylalanine, lysine and total crude proteins. [START_REF] Muley | Heritability of digestibilities and divergent selection for digestion ability in growing chicks fed a wheat diet[END_REF] showed that digestive efficiency of broilers was heritable when birds were fed a challenging diet containing 50% wheat from the Rialto variety, which is especially difficult to be digested because of its hardness and its high viscosity [START_REF] Carré | Relationships between digestibilities of feed components and characteristics of wheat (Triticum aestivum) introduced as the only cereal source in a broiler chicken diet[END_REF]. The genetic basis of digestibility was confirmed by a divergent selection experiment on AMEn using this challenging diet. After 8 generations of selection, AMEn of the Rialto wheat diet differed by 35 to 40% between the high (D+) and low (D-) line [START_REF] Juin | Genetic parameters of digestibility of wheat-or cornbased diets in chickens[END_REF][START_REF] De | Digestive tract measurements and histological adaptation in broiler lines selected for digestive efficiciency[END_REF]de Verdal et al., , 2011a)). This AMEn divergence was associated with a much lower excretion of nitrogen and phosphorus in D+ than in D-birds (de Verdal et al., 2011b). On the 7 th generation with the same diet, digestibilities of starch, proteins and lipids were also 42%, 15%, and 50 % higher in D+ than in D-line (Carré et al., 2008b). D+ birds also had 6.2 to 8.7 % higher AMEn and a 17.6% lower excretion rate of nitrogen and phosphorus when compared to commercial broiler genotypes on wheat (Carré et al., 2008a;[START_REF] De | Possibility of reduction of excretion in chicken by genetic selection[END_REF]. When birds were fed diets easier to digest (i.e. based on corn and soybean), differences were still observed between D+ and either Dor commercial broiler birds, but were largely reduced, ranging between 2.5 and 5 % for AMEn (Carré et al., 2008a;[START_REF] Juin | Genetic parameters of digestibility of wheat-or cornbased diets in chickens[END_REF]. These differences in digestive efficiency were associated with a smaller intestine, a bigger gizzard (Garcia et al., 2007;Rougière et al., 2009;[START_REF] De | Digestive tract measurements and histological adaptation in broiler lines selected for digestive efficiciency[END_REF]de Verdal et al., , 2011a)), and a longer gizzard retention time [START_REF] Rougière | Comparison of gastrointestinal transit times between chickens from D+ and D-genetic lines selected for divergent digestion efficiency[END_REF] in D+ than in D-birds. So, gizzard physiology seems to be involved in the differences between D+ and D-lines, as reported by a study on gizzard motility showing a higher responsiveness of gizzard to environment variations in D+ than in D-birds [START_REF] Rougière | Comparison of gizzard activity between chickens from genetic D+ and D-lines selected for divergent digestion efficiency[END_REF].

In agreement with this statement, genetic correlations between AMEn

Figure 2 -Variability of AMEn of wheat diets between wheat varieties and between animals within a wheat variety (after Carré et al., 2002).

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World´s Poultry Congress 5 -9 August -2012 • Salvador -Bahia -Brazil XXIV and relative weights of gizzard and proventriculus were observed to be rather high (0.43 and 0.59, respectively) (de Verdal et al., 2011a).

Various studies performed on these birds indicated that performances of D+ birds depended less on characteristics of diets than D-birds (Carré et al., 2008a;Rougière et al., 2009) and that, in contrast with a selection on growth in suboptimal environment, selection on AMEn led to more adaptable birds. These results are consistent with the strong and positive genetic correlations obtained between digestibility traits on corn or on wheat diets, which ranged from 0.67 for digestibility of starch to 0.88 for digestibility of proteins. This implies that selection on digestive efficiency on one diet would also improve performances on the other diet. However, progress would be more efficient if selection was made on wheat, as, except for starch, heritability of digestibility is much higher with wheat that with corn diet: respectively 0.32 and 0.15 for AMEn, 0.28 and 0.26 for starch, 0.25 and 0.04 for lipids, 0.29 and 0.09 for proteins [START_REF] Juin | Genetic parameters of digestibility of wheat-or cornbased diets in chickens[END_REF]. Again, at the opposite to selection on growth in suboptimal environment, the D+ and Dlines did not show significant differences in fatness at market age [START_REF] De | Possibility of reduction of excretion in chicken by genetic selection[END_REF].

Genomic data on FCR

QTL detection

Despite the importance of FCR for poultry production, very few QTLs have been detected on FCR. In pigs for example, Rotschild et al. (2007) stated that on 1675 QTL that had been detected, only 8 were detected on feed efficiency, as compared to 16, 224, and 404 for feed intake, growth and fatness. Similarly, on 2736 QTL detected until now in poultry (laying hens and growing chickens), only 25 are related to feed efficiency traits (cf. Figure 3), coming from 5 studies, among which none detailed the diet used in the experiment (de [START_REF] De Koning | Quantitative trait locus detection in commercial broiler lines using candidate regions[END_REF][START_REF] De Koning | Segregation of QTL for production traits in commercial meat-type chickens[END_REF][START_REF] Parsanejad | Ornithine decarboxylase: haplotype structure and trait associations in White Leghorn chickens[END_REF][START_REF] Hansen | Identification of QTL for production traits in chickens[END_REF][START_REF] Ewald | Association of BF2 alleles with antibody titres and production traits in commercial pure line broiler chickens[END_REF]. Most studies that were looking for QTL on populations differing by their feed efficiency finally found QTL on components of feed efficiency, mainly feed intake and body composition [START_REF] Hocking | Review of QTL mapping results in chickens[END_REF][START_REF] Abasht | Review of quantitative trait loci identified in the chicken[END_REF]. None of them were performed on a challenging diet.

Regarding performances on low quality diet, a QTL detection program has been initiated using a F2 cross between the D+ and D-lines fed a diet including 55% of Rialto wheat. A total of 945 animals have been genotyped on 6000 SNPs and the 864 F2 birds were measured for digestibility, performances and anatomy. The first analyses show the existence of several QTLs, which in most cases co-localized for AMEn, digestibility of dry matter, starch and proteins.

Candidates genes and expression data [START_REF] Parsanejad | Genetic variability of the cytosolic phosphoenolpyruvate carboxykinase gene in white leghorn chickens[END_REF][START_REF] Parsanejad | Ornithine decarboxylase: haplotype structure and trait associations in White Leghorn chickens[END_REF]) ) proposed that PEPCK-C and ornithine decarboxylase 1 may be involved in residual feed intake of adult laying hens. The former is part of the process of glucose generation from non-sugar carbon substrates. The latter is implied on efficiency of nutrient absorption including calcium. More recently, studies found in broilers that FCR was correlated with SNP variations in genes regulating metabolism and energy partitioning (growth hormone receptor or neuropeptide Y, [START_REF] Sherman | Identification of polymorphisms influencing feed intake and efficiency in beef cattle[END_REF], genes implied in thermogenesis and proton leak (avian uncoupling protein, [START_REF] Sharma | Polymorphisms in uncoupling protein, melanocortin 3 receptor, melanocortin 4 receptor, and pro-opiomelanocortin genes and association with production traits in a commercial broiler line[END_REF][START_REF] Bottje | Mitochondrial proton leak kinetics and relationship with feed efficiency within a single genetic line of male broilers[END_REF] or in energy homeostasis and feeding behaviour (melanocortin 3 receptor, Sharma et all., 2008). By comparing expression of genes between high and low FCR chickens, differences have been detected in genes implied in the regulation of mitochondrial energy metabolism (Ojano-Dirain et World´s Poultry Congress 5 -9 August -2012 • Salvador -Bahia -Brazil Area: Genetics and Breeding • August 07 XXIV environment would also improve performances in a more favorable (corn) environment. [START_REF] Sorensen | Selection for growth rate in broilers fed on diets with different protein levels[END_REF] found a much lower genetic correlation (0.30) between broiler growth obtained on a normal or a low protein diet, but in their case, the low protein diet was really out of requirement, which could partly explain this very low correlation.

Criterion of selection

Criterion of selection has to be chosen carefully when selecting on improved feed efficiency on low quality diets. Results from [START_REF] Sorensen | Selection for growth rate in broilers fed on diets with different protein levels[END_REF][START_REF] Sorensen | Protein and energy metabolism in two lines of chickens selected for growth on high or low protein diets[END_REF] showed that selection for growth of birds fed diets with low protein contents would lead to increased feed consumption and fatness, whereas selection on digestive efficiency with a wheat diet does not increase neither feed consumption nor fatness in the high line [START_REF] Muley | Heritability of digestibilities and divergent selection for digestion ability in growing chicks fed a wheat diet[END_REF], 2010). Thus, when suboptimal diets are considered, selection criteria such as digestive efficiency (AMEn or digestibility of dry matter) seems to be more appropriate than the growth criteria. Attention has also to be given to age at selection. Indeed, the development of gastrointestinal tract is strong in the first weeks of age, which increases the chances of success when selecting at this age. Birds of D+ and D-lines were selected at 3 weeks of age. The differences between both lines for development of gastrointestinal tract and for digestive efficiency appear after one week, are maximal at 3 weeks and decrease or disappear at 8 weeks (de Verdal, 2011).

Homogeneity of results

When selection is performed in a difficult environment, attention has also to be given to homogeneity of performances in addition to the mean value. Data collected on several generations of D+ and D-lines showed that the coefficients of variation of AMEn, coefficients of digestibility of starch and proteins were diet dependent in D-line but not in D+ line. Coefficient of variation of lipid digestibility was diet dependent in both lines (cf. Table 1, [START_REF] Juin | Genetic parameters of digestibility of wheat-or cornbased diets in chickens[END_REF]). al., 2007), and in heat shock protein involved in oxidative stress [START_REF] Kong | Gene expression in breast muscle associated with the efficiency in a single male broiler line using a 44K oligo microarray. I. Top differentially expressed genes[END_REF].

Perspectives for selection with suboptimal diets

Variability of feedstuffs

Variability in the quality of feedstuffs may be a problem in estimating the potential of genetic improvement of feed efficiency on alternative diets as it does not allow a reproducible selection environment. It is especially the case of byproducts such as bran or DDGS, that highly depend on the raw material composition and technological process (drying, milling, addition of solubles). For instance, [START_REF] Ravindran | Feed resources for poultry production in Asia and the Pacific region. I. Energy sourcesµ[END_REF] highlighted that the machinery used for milling rice affects the proportion of hulls in the rice bran, which fiber and fat contents can vary respectively from 12.6% to 30.9% and from 6.6 to 31.6%. [START_REF] Bandegan | Standardized ileal amino acid digestibility in wheat distillers dried grains with solubles for broilers[END_REF] also showed that depending on feedstuff sample, apparent ileal digestibility of essential amino acids as lysine and threonine varied from 24.4 to 45.7% and from 48.2 to 60.9%, respectively.

It is often claimed that selection efficiency is decreased when environment, including diet, is suboptimal, because variance due to the environment increases and heritability, and thus possibilities of selection, decreases. Indeed, if selection is done on maximum growth, the animal needs an optimized environment to fully express its genetic potential for growth. This is especially true for modern genotypes, since their rapid growth and high protein deposition rate require a high quality diet. However, if the criterion of interest is the robustness of animals in a difficult environment, it is precisely in a difficult environment that the capacity of animals to cope with difficulty will be expressed. The high positive genetic correlations found between capacities to digest wheat and corn diets [START_REF] Juin | Genetic parameters of digestibility of wheat-or cornbased diets in chickens[END_REF] show that improving performances in a difficult (wheat) World´s Poultry Congress 5 -9 August -2012 • Salvador -Bahia -Brazil XXIV However, it is worthy to note that, in most studies on digestibility or feed efficiency using suboptimal or alternative diets, only the global standard error of the mean is given in studies, which does not allow to see whether variance is modified in each treatment.

Conclusions

This review shows that selection for improved feed gain ratio using challenging or alternative diets is efficient, provided that conditions, age, and especially criterion of selection are appropriate. It is especially obvious that, in suboptimal conditions, a selection on growth is less suitable than a selection on digestive efficiency. Finally, in addition to an improved average value in selected populations, another goal that has to be reached for selection in such environments is homogeneity of population, which is a major factor of profitability of poultry breeding.

Figure 3 -

 3 Figure 3 -QTL detected on feed efficiency (FE), feed conversion ratio (FCR) and residual feed intake(RFI) in chickens and laying hens (www.animalgenome.org/cgi-bin/QTLdb, 02/25/2012).

Table 1 -

 1 Effect of the diet on mean value and on homogeneity of digestive performances in D+ and D-lines.

			Corn diets			Wheat diets
		D+		D-		D+		D-
		mean CV (%)	mean CV (%)	mean CV (%)	mean CV (%)
	AMEn (kcal.kg -1 DM)	3298	6.6	3063	7.3	3247	8.6	2411	26.5
	Digestibility of lipids (%)	90.9	3.1	84.6	11.2	83.0	12.0	68.6	26.2
	Digestibility of starch (%)	97.9	0.9	96.8	2.0	96.1	3.6	87.4	14.2
	Digestibility of proteins (%) 83.1	4.8	78.6	6.7	77.4	6.0	68.1	11.7

World´s Poultry Congress 5 -9 August -2012 • Salvador -Bahia -Brazil Area: Genetics and Breeding • August 07 XXIV


Marc Bardin

Claire Troulet

Philippe C Nicot

The efficacy of biocontrol against Botrytis cinerea on tomato depends on the strain of the pathogen

The objective of the study was to estimate the risk of loss of biocontrol efficacy towards plant pathogens. To this end, the protective efficacy of both biocontrol agents Microdochium dimerum and Bacillus subtilis (Serenade Max®) was evaluated on tomato against 41 strains of Botrytis cinerea differing in their geographic origin, host of isolation and level of aggressiveness. The protective efficacy of M dimerum was evaluated on whole tomato plants after co-inoculation of pruning wounds with B. cinerea and M dimerum at the desired dose. Lesion expansion was measured on the stem from the 3 rd to the 7 th day after inoculation. A very high level of efficacy against all strains was obtained when the biocontrol agent was used at the recommended dose. At a 10-fold reduced application dose, a wide range of sensitivity was observed and a significant correlation was observed between the level of aggressiveness of a strain to tomato and its sensitivity to the biocontrol agent. For B. subtilis, tomato leaves were first treated with the biocontrol agent at the desired dose, two days before inoculation with a mycelial agar plug of B. cinerea. The resulting lesions were assessed from the 2 nd to the 4 th day after inoculation. The efficiency of this product was also significantly influenced by the strain of B. cinerea but no correlation was observed between the aggressiveness of B. cinerea and the protection provided by the biocontrol agent. This study reveals the importance of considering several strains of the pathogen when evaluating the efficacy of biocontrol agents, to obtain a good representation of the pathogen population.

P03. 046

 046 The efficacy of biocontrol against Botrytis cinerea on tomato depends on the strain of the pathogen M Bardin, C. Troulet and P.C. Nicot INRA, UR407 Pathologie végétale, Domaine Saint Maurice, BP94, F-84143 Montfavet Cedex, France Email: marc.bardin@avignon.inra.fr