Rodolphe Sonnier
email: rodolphe.sonnier@mines-ales.fr

Amandine Viretto

Belkacem Otazaghine

Loïc Dumazert

Alexei Evstratov

Jean-Claude Roux

Cai Heruijing

Charlene Presti

Johan G Alauzun

P Hubert Mutin

Henri Vahabi

Studying the thermo-oxidative stability of chars using pyrolysis-combustion flow calorimetry

Keywords:

Introduction

During burning, many polymeric materials form a carbonaceous or partly carbonaceous residue. This residue can act as a protective insulating layer. Except in the case of porous materials [START_REF] Kramer | Heat release and structure collapse of flexible polyurethane foam[END_REF], pyrolysis is believed to be anaerobic as long as the flame is sustained. But when the flame vanishes, the residue can undergo aerobic pyrolysis (or thermo oxidation) at the end of cone calorimeter test for example. Chars from textiles also undergo thermo oxidation when a small flame propagates, as evidenced in a previous article [START_REF] Sonnier | Improving the flame retardancy of flax fabrics by radiation grafting of phosphorus compounds[END_REF]. A critical situation is probably the use of intumescent coating to protect structural materials. The expanded carbonaceous layer must protect the underlying material for several hours. The char layer must fulfil some requirements to be efficient. Cohesion, thickness, porosity are important properties influencing the barrier effect of this char layer. Above all, the thermal stability of the char is a critical parameter.

Aerobic pyrolysis (thermo oxidation) of carbonaceous materials is a heterogeneous catalysis which has been extensively studied, in particular by scientists working on the coal boiler efficiency [START_REF] Walker | An update on the carbon-oxygen reaction[END_REF]. Indeed, the rate of CeO 2 reaction has a strong effect on efficiency of combustion, heat transfer and particulate removal processes. The intrinsic reactivity of carbon to oxygen depends on the temperature and activation energies were found in the range 126e290 kJ/mol [START_REF] Smith | The intrinsic reactivity of carbons to oxygen[END_REF]. But the reactivity of true coals and chars may deviate from the intrinsic reactivity due to a couple of parameters. Many studies have been carried out for several decades on coals or chars pre pared from coals.

One main result from these studies is that the reactivity of carbon to oxygen decreases when carbon ordering increases. Several methods were developed to characterize the carbonaceous structure of coals and chars. X ray diffraction is an appropriate method to assess the structural changes of a carbonaceous material during heat treatment, including fraction of amorphous carbon, aromaticity and crystallite size [START_REF] Schoening | X-ray structural parameter for coal[END_REF][START_REF] Lu | Quantitative X-ray diffraction analysis and its application to various coals[END_REF]. Other methods include Raman spectroscopy [START_REF] Rao | Diameter-selective Raman scattering from vibrational modes in carbon nanotubes[END_REF][START_REF] Sun | Effects of oxygen on the structure and reactivity of char during steam gasification of Shengli brown coal[END_REF], nuclear magnetic resonance or high resolution transmission electron microscopy [START_REF] Russell | Structural ordering in high temperature coal chars and the effect on reactivity[END_REF]. The H/C ratio is indicative of aromatization and the reactivity was found to decrease when the ratio decreases [START_REF] Mcdonald | Low temperature char oxidation kinetic: effect of preparation method[END_REF]. Reactivity of oxygen was found to be well correlated with a disorder index taking into account amorphous fraction and aromaticity [START_REF] Lu | Char structural ordering during pyrolysis and combustion and its influence on char reactivity[END_REF].

Other fuel properties have obviously a strong influence on reactivity of carbon to oxygen. Size and porosity of particles influ ence the diffusion of oxygen [START_REF] Smith | Internal burning of pulverized semi-anthracite: the relation between particle structure and reactivity[END_REF]. The presence of catalytic ele ments (magnesium, calcium…) has been pointed out as one of the most influent parameters on reactivity to oxygen [START_REF] Wells | Relation between reactivity and structure for coals and chars[END_REF].

The ordering of carbonaceous material as well as the morphology of coal/char particles influences its reactivity to oxy gen but is also evolutive upon heat treatment. Since the combus tion conditions (temperature, heating rate, residence time) change from one boiler to another one, the effect of these conditions on the reactivity has been studied [START_REF] Mcdonald | Low temperature char oxidation kinetic: effect of preparation method[END_REF][START_REF] Radovic | Reactivities of chars obtained as residues in selected coal conversion processes[END_REF][START_REF] Cai | Combustion reactivity and morphological change in coal chars: effect of pyrolysis temperature, heating rate and pressure[END_REF]. Temperature reached during char preparation and heating rate are two parameters influencing the organization of char. Davis et al. explain that the typical com bustion conditions are insufficient to lead to true graphitization but allow the growth of regions of turbostratic order [START_REF] Davis | Evolution of char chemistry, crystallinity, and ultrafine structure during pulverized-coal combustion[END_REF]. As an example, char deactivation was observed by Russell et al. [START_REF] Russell | Structural ordering in high temperature coal chars and the effect on reactivity[END_REF] after treatment at high temperature. The presence of 1% of oxygen dur ing steam gasification of brown coal leads to a lower reactivity of char because oxygen enhances the generation of H radicals which promote the condensation of aromatic ring systems and a more ordered structure [START_REF] Sun | Effects of oxygen on the structure and reactivity of char during steam gasification of Shengli brown coal[END_REF].

There is a real need to understand the influence of the compo sition and the structure of chars on their thermo oxidative stability. Nevertheless, the relations between carbonaceous structure and reactivity to oxygen are out of the scope of this article. The main goal of this work is to propose a convenient method to study the reactivity of chars to oxygen using a henceforth well known apparatus available in many fire laboratories. The thermo oxidative stability is studied using a pyrolysis combustion flow calorimeter (PCFC) upon a wide range of oxygen fraction in pyrol ysis chamber (0e0.4, i.e. from 0 to 40%). The PCFC analysis is most generally used in standard conditions [17] (i.e. anaerobic pyrolysis followed by a complete combustion of gases). Nevertheless, other conditions (incomplete combustion, aerobic pyrolysis) can be applied to obtain new insights on materials properties.

This new method may allow identifying the influence of various charring promoters on the thermo oxidative stability of the char. Among charring promoters, phosphorus based compounds are nowadays an important class of flame retardants (FRs) [START_REF] Perret | Fire retardancy mechanisms of arylphosphates in polycarbonate (PC) and PC/acrylonitrile-butadiene-styrene: the key role of decomposition temperature[END_REF][START_REF] Schartel | Flame retardancy of polymers: the role of specific reactions in the condensed phase[END_REF]. Other flame retardants, as some metal hydroxides [START_REF] Viretto | Thermal degradation of polyesters filled with magnesium dihydroxide and magnesium oxide[END_REF], can also promote charring of polymers. Some materials are intrinsically charring as polycarbonate, for example. It is also the case of ligno cellulosic fibers which are considered to be promising alternatives to replace glass fibers due to their excellent mechanical properties [START_REF] Kozłowski | Flammability and fire resistance of composites reinforced by natural fibers[END_REF]. In the present article, we have studied the thermo oxidative stability of chars obtained from the degradation of various mate rials: charring polymers, flax fibers grafted with phosphonate based molecules, epoxy resins flame retarded with phosphorus containing additives, polybutylene terephthalate containing aluminum based flame retardants and phosphorus containing nanodiamonds. The denomination of char is used in this article for various residues obtained from organic materials after a first degradation step whichever the heating conditions. The residues can be partly carbonaceous and partly inorganic.

Experimental

Materials

Poly(ether ether ketone) (PEEK 151G Natural) was provided by Victrex. Polycarbonate (PC Makrolon 2647) was provided by Bayer SA. Poly(butylene terephthalate) (PBT Vestodur 3000) was pro vided by from Evonik.

Dried flax fibers used in this study were extracted from a fabric kindly provided by Hexcel. The fibers had the following composi tion: cellulose (81 wt%), hemicellulose (13 wt%), lignin (2.7 wt%). This composition was in a good agreement with literature data [22e24]. Dimethylvinyl phosphonate (MVP) from Acetolac was grafted onto flax fibers. While MVP was grafted in the bulk, its content can reach high level (up to 17 wt%). The grafting method (e beam irradiation) and the characterization of the modified flax fi bers have been described elsewhere [START_REF] Sonnier | Improving the flame retardancy of flax fabrics by radiation grafting of phosphorus compounds[END_REF].

Epoxy resin was flame retarded with phloroglucinole tris(diethyl phosphate) (P3P(OEt)). Flame retardant was prepared as described by Menard et al. [START_REF] Negrell-Guirao | Synthesis of biobased phosphate flame retardants -characterization of flame retardancy on epoxy thermosets[END_REF]. Ammonium polyphosphate (APP Exolit APP423 provided by Clariant) was also incorporated into epoxy resin according to the same procedure. APP content was fixed to 9.4 wt% in order to maintain a phosphorus content equal to 3 wt%.

Polyamides were flame retarded with two organophosphorus silica generated in situ from alkoxysilanes called SiP and SiDOPO. The preparation methods of SiP and SiDOPO as well as the char acterization of the composites can be found elsewhere [START_REF] Sahyoun | Influence of organophosphorus silica precursor on the thermal and fire behaviour of a PA66/PA6 copolymer[END_REF][START_REF] Sahyoun | Synthesis of a new organophosphorous alkoxysilane precursor and its effect on the thermal and fire behavior of a PA66/PA6 copolymer[END_REF].

Magnesium hydroxide (MDH Magnifin H10) was provided by Albermarle. Particles median size was 0.8 mm. Its efficiency as char promoter in polyesters, and particularly PBT, has been extensively studied in Viretto et al. [START_REF] Viretto | Thermal degradation of polyesters filled with magnesium dihydroxide and magnesium oxide[END_REF].

Poly(butylene terephthalate) (PBT) composites were prepared by incorporating 10 wt% of pure g alumina and modified g alumina. The alumina from Prolabo was crushed by grinder (Retsch S1000) into 160e315 mm particles. Nickel sulfate was dissolved in water and dripped slowly on the alumina surface. The mixture obtained was dried at 150 C for 24 h, and then oxidized at 550 C for 6 h, followed by the H 2 /N 2 flow reduction for 3 h in order to form metallic nickel (Ni 0 ) on alumina. The H 2 and N 2 flows were 75 ml/min and 150 ml/min respectively, the reduction temperature was 750 C. The procedure has already been described by Majoli et al. [START_REF] Majoli | Composite carbon nanostructures as promising carriers for gaz analysis[END_REF]. PBT was compounded with additives in the micro compounder (DSM). Processing was operated at 247 C at 80 rpm during 10 min. The composites were prepared under nitrogen at mosphere to avoid the degradation of PBT. Nanodiamonds (International Technology Center, Raleigh, NC, U.S.A.) were also studied as model materials. Such materials were modified through surface phosphorylation [START_REF] Presti | Improvement of the Oxidative Stability of Nanodiamonds by Surface Phosphorylation[END_REF]. Phosphorylation leads to a strong improvement of the thermo oxidative stability measured using dynamic and isothermal thermogravimetric ana lyses in air, as already reported by Presti et al. [START_REF] Presti | Improvement of the Oxidative Stability of Nanodiamonds by Surface Phosphorylation[END_REF]. Details about all these materials are given in Table 1.

Methods

The flammability was studied using a Pyrolysis Combustion Flow Calorimeter (PCFC) from Fire Testing Technology (FTT). Heating rate, maximum pyrolysis temperature, combustion tem perature and gas flow rate were respectively 1 C s À1 , 750 C, 900 C (corresponding to complete combustion) and 100 cm 3 min À1 . Ox ygen was introduced at various fractions (up to 0.4) into the py rolyzer to measure the thermo oxidative stability of the char. When the method is applied, the oxygen fractions in pyrolyzer and in combustor are not independent. Therefore, if low oxygen fractions are chosen for aerobic pyrolysis, oxygen may be fully consumed and the combustion may become incomplete. To avoid this issue, the weights of samples were carefully chosen for each test. For example, in most cases (low to medium peak of heat release rate), a 1 mg sample can be successfully tested even with a very low oxy gen fraction (0.01) in pyrolyzer.

In order to highlight the influence of preparation method of chars as already pointed out in literature [START_REF] Mcdonald | Low temperature char oxidation kinetic: effect of preparation method[END_REF], some chars were obtained from cone calorimeter (Fire Testing Technology, FTT) tests according to ISO5660 standard. Heat flux was fixed at 50 kW/m 2 . Chars were removed arbitrarily two minutes after flaming out.

The solid residues were observed using a FEI Quanta 200 scanning electron microscope and their elemental compositions (C, O, P, Si atoms) were determined by Energy Dispersive X ray spec troscopy (EDX) using Oxford INCA Energy 300 system and a de tector of 133 eV (at Mn). Note that the hydrogen fraction cannot be measured using this method. The P content in phosphorylated nanodiamonds was determined by ICP OES [START_REF] Kozłowski | Flammability and fire resistance of composites reinforced by natural fibers[END_REF].

Description of the test method

In the following, "char" is referred to the carbonaceous part of residue undergoing the degradation during aerobic pyrolysis. Keep in mind that in many cases the residue is not only constituted by a carbonaceous part but also by an inorganic part.

Nanodiamonds were studied according to a specific procedure as discussed below. All other materials were subjected to a first pyrolysis step in anaerobic atmosphere up to 500e600 C (when the heat release rate is stable and equal to 0) in PCFC. The obtained residues were cooled down to room temperature and a second degradation step was carried out under aerobic atmosphere up to 800 C in order to degrade the char, still using PCFC. This procedure is considered as the standard one.

In all cases, the temperature of peak of Heat Release Rate (pHRR) (called T fO 2 , i.e. char degradation temperature when oxygen fraction f O2 ) decreases when the oxygen fraction increases. The pHRR temperature changes according to Equation (1):

T fO 2 ðKÞ T 1 ðKÞ Â f Àa O2 (1)
Equation ( 1) can be easily linearized leading to Equation (2).

Ln T fO

2 LnðT 1 Þ a  Ln À f O2 Á ; ( 2
)
where f O2 is the oxygen fraction in pyrolysis chamber, T 1 is the temperature of pHRR corresponding to the char degradation in pure oxygen atmosphere (f O2 1), and a is a parameter (>0) corre sponding to the sensitivity of char degradation temperature to the oxygen fraction (the higher a is, the higher the sensitivity of char degradation temperature to the oxygen fraction is). ) versus Ln(f O2 ) and shows that the experimental points are well fitted with equation (2) (R 2 0.9981). For some materials, the temperature of pHRR at low oxygen fraction (<0.02) is too high and only four or five oxygen fractions were used.

Equation ( 1) means that the temperature of char degradation increases when the oxygen fraction decreases and this change is faster and faster when the oxygen fraction becomes very low. On the contrary, when the oxygen fraction is high, the temperature of char degradation is almost constant.

The thermo oxidative stability of nanodiamonds has been studied directly. The temperature of pHRR is not observed due to a very good stability of these materials (see Fig. 3). In this case, Equation (1) was used considering the temperature when Heat Release Rate (HRR) reaches 50 W/g (i.e. when the degradation starts). Of course, the parameters T 1 and a determined by means of this modified method cannot be compared to those measured using the previous one.

A series of chars was prepared according to other procedures in order to assess the influence of particular conditions of char preparation. Two of these chars were sampled from the cone calorimeter tests and their properties were compared to those of the chars prepared according to the standard procedure from the same materials. Some flax fibers were also subjected directly to an aerobic degradation step. In this case, the heat release rate curve exhibits two steps. The first one corresponds to the degradation of flax fibers (especially cellulose component). The second one is assigned to the degradation of the char resulting from the first degradation step. Hence, in this case, the char is formed under aerobic conditions. The stability of the char was obviously evalu ated from the temperature of the second peak (see Fig. 4).

The method proposed here also allows studying the influence of oxygen fraction on heat release rate. Although such a study is out of the scope of the present article, an example about polycarbonate is given in supporting information 1. A brief comparison between PCFC results and thermogravimetric analyses is shown in supporting information 2.

Results and discussion

Preliminary comments

According to several authors, the decomposition rate _ u of a material in aerobic pyrolysis can be written as Equation ( 3)

[30e32]. _ u Ae E RT f ðaÞf d O2 (3)
where f O2 is the oxygen fraction, A is the pre exponential factor, E is the energy of activation, T the temperature, R the universal con stant of gases, d the reaction order for oxygen and f(a) the differ ential conversion function.

Note that this equation is only proposed to predict the kinetics of a complex process involving several heterogeneous solid state reactions but does not represent a "real" reaction. The set of ki netic parameters properly fitting the data is not unique and these parameters must be considered only as "numerical parameters" [START_REF] Marquis | Influence of reaction mechanism accuracy on the chemical reactivity prediction of complex charring materials in fire condition[END_REF].

A very common result evidenced by this equation is that the decomposition rate increases when the oxygen fraction increases, which is obviously observed in the present work. Nevertheless, most of thermogravimetric analyses are carried out in nitrogen or in air and the data about the influence of oxygen on decomposition are scarce. Hu and You have studied the effect of oxygen into the pyrolysis of polyurethane. The oxygen fraction ranges from 0 to 0.5. Even if the oxygen fraction affects the two decomposition steps at different extents, the authors show that oxygen promotes a faster decomposition rate occurring at lower temperature [START_REF] Hu | The effects of oxygen contents and heating rates on characteristics of pyrolysis prior to smoldering of flexible polyurethane foam[END_REF]. This is in agreement with Equation [START_REF] Kramer | Heat release and structure collapse of flexible polyurethane foam[END_REF] indicating that the temperature of char decomposition decreases with higher oxygen fraction.

General results

Fig. 5 and Table 2 summarize the main results obtained for all chars prepared according to the standard procedure. It can be observed that the temperature T 1 ranges from 550 to 900 K (i.e. 277e627 C) and a ranges from 0.02 to 0.14. As a rule, a decreases when T 1 increases. In other words, a low char stability in pure ox ygen (low T 1 ) is correlated with a high sensitivity of the char degradation temperature to the oxygen fraction (high a, i.e. strong increase in thermo oxidative stability when the oxygen fraction decreases).

Untreated flax fibers were tested in duplicate. It appears that both series of measurements are consistent: T 1 is almost the same (554e560 K) and a is in the range 0.087e0.097, which is relatively narrow in comparison to the range obtained for all materials tested (0.021e0.135).

Fig. 6 compares the chars exhibiting the worst and the best thermo oxidative stabilities: these two chars are respectively un treated flax fibers (standard procedure) and epoxy containing ammonium polyphosphate (sampled from the cone calorimeter test). The difference in the thermo oxidative stability is very important: 242 C at the oxygen fraction equal to 0.02. It increases when oxygen fraction increases up to a plateau around 317e332 C above 10% of oxygen content.

Finally, it is interesting to note that the results obtained for similar chars are quite close. Then it become possible to identify and to distinguish the main groups of materials studied in this work: charring polymers, flax (i.e. lignocellulosic) fibers, poly butylene terephthalate containing inorganic additives and phosphorus containing polymers (both polyamide and epoxy). We discuss these results in the following section according to the materials group.

Charring polymers

Three charring polymers were tested: polycarbonate (char content in anaerobic pyrolysis equal to 24%), poly( etheretherketone) (char content: 40%) and polybutylene tere phthalate (char content: 6%). The chars exhibit very close properties: T 1 ranges from 707 to 728 K (i.e. from 434 to 455 C) and a ranges from 0.063 to 0.081 (Fig. 5 and Table 2). This is not surprising because these polymers contain only C, O and H atoms and the corresponding chars must exhibit similar chemical com positions. Lyon et al. have shown that chars resulting from many charring polymers have elemental compositions close to C 5 H 2 [START_REF] Walters | A microscale combustion calorimeter study of gas phase combustion of polymers[END_REF].

The effective heat of combustion (EHC) of these chars was calculated as the ratio between the total heat release (THR) and the mass loss (ML). The effective heat of combustion depends on the elemental composition according to Huggett's relation [START_REF] Huggett | Estimation of rate of heat release by means of oxygen consumption measurements[END_REF]: 1 kg of oxygen consumed during combustion corresponds to 13.1 MJ of energy released. To ensure a complete degradation of the char, its pyrolysis was carried out at an oxygen fraction of oxygen equal to 40%. The EHC of PEEK and PC were found to be respectively equal to 30.6 and 30.8 kJ/g. Unfortunately, the char content of PBT was too low (<10 wt%) to calculate accurately its EHC. Note that the calculated EHC for PEEK and PC are slightly lower than expected from literature [START_REF] Factor | Char formation in aromatic engineering polymers[END_REF]. The reasons may be twofold. First Lyon et al. [START_REF] Walters | A microscale combustion calorimeter study of gas phase combustion of polymers[END_REF] have shown that the heat of combustion in PCFC slightly de creases when the oxygen fraction increases. Second, the heat release would be slightly underestimated by our apparatus.

Epoxy and polyamide flame retarded with phosphorus additives

The chars prepared from flame retarded polymers according to the standard procedure exhibit higher stabilities in pure oxygen than charring polymers studied just above (Fig. 7). The temperature T 1 is high: 747 K for the epoxy flame retarded with P3P(OEt) and 795e800 K for the polyamides flame retarded with SiP and SiDOPO and the parameter a is very low for the two polyamide based materials (around 0.04) but much higher for the flame retarded epoxy (0.0825). It is interesting to note that the chars resulting from polyamide based materials exhibit higher temperature T 1 despite a lower phosphorus content (3.2, 8.2 and 10.3 wt% respectively for the polyamides flame retarded by SiDOPO, SiP and flame retarded epoxy). Moreover, SiDOPO seems to promote a more stable char in comparison to SiP: the char properties are similar but the phos phorus content is much lower in the first case. This fact may be not related to the presence of silicon in the char. The silicon content is 19.5 wt% for the char from the polyamide flame retarded with SiP and only 16.4 wt% for the char prepared from the polyamide flame retarded with SiDOPO. The chars obtained from the cone calorimeter tests exhibit significantly better thermal stability. The parameters T 1 and a are in the range 860e900 K and 0.35e0.53, respectively. In particular, the char arising from the epoxy flame retarded with P3P(OEt) exhibits higher T 1 and lower a after the cone calorimeter test than according to the standard procedure, despite the same phosphorus content (10.3 wt%). This observation may be explained by a deeper thermo oxidation of a char fraction at the end of the cone calorimeter test. This point needs further investigation to be confirmed.

Flame retarded flax fibers

The chars obtained from flax fibers using standard procedure exhibit low T 1 (Fig. 8). T 1 was in the range 554e560 K (i.e. 281e287 C) and a was found to be in the range 0.087e0.097 (high sensitivity to the oxygen fraction). The grafting of phosphorus compound (dimethylvinyl phosphonate MVP) on flax fibers allows an increase of T 1 up to 643e652 K. This value was the same whichever the phosphorus content in the char (0.9e9.3 wt%).

The changes in a were observed to be more complex. The lowest a (0.068) was obtained for the char containing 0.9 wt% of phos phorus. However, the a value increased up to 0.13 when the phosphorus content increases up to 9.3 wt%. Such a value is very high and indicates a strong sensitivity to the oxygen fraction in air. It also means that the char becomes the most thermally stable when the oxygen fraction is the lowest. Its degradation tempera ture for an oxygen fraction of 0.01 is found equal to 1200 K, higher than the degradation temperature of the char prepared from epoxy composite flame retarded with APP.

A comparison of the chars prepared using the standard pro cedure or using a direct aerobic pyrolysis of the material provides a consistent tendency: T 1 increases from 554 to 600 K for untreated flax fibers and from 643 e 652 to 660e679 K for treated fibers. a seems to decrease when a direct aerobic pyrolysis is applied. a is stable around 0.068e0.072 according to both methods only in the case of flax fibers containing 0.4 wt% of MVP. This seems to be consistent with the observations of Sun et al. who found that the chars prepared in presence of a small amount of oxygen (1%) has lower reactivity than the chars prepared from anaerobic pyrolysis [START_REF] Sun | Effects of oxygen on the structure and reactivity of char during steam gasification of Shengli brown coal[END_REF].

The same conclusions can be drawn on the influence of phos phorus content: T 1 does not change appreciably and a increases when the phosphorus content increases whichever the procedure used (standard procedure or direct aerobic pyrolysis).

In the whole, the influence of phosphorus on the thermo oxidative stability is complex. It seems that only a small content of phosphorus is needed to improve slightly the thermo oxidative stability. Higher phosphorus content does not improve anymore the thermo oxidative stability but increases the char content. The chars resulting from flax fibers modified with high content of dimethylvinyl phosphonate (MVP) do not reach the same thermo oxidative stability as charring polymers. Nevertheless, even a slight increase in thermo oxidative stability improves significantly the behavior of a fabric prepared from flax fibers during a fire test, as proved in a recent publication [START_REF] Sonnier | Improving the flame retardancy of flax fabrics by radiation grafting of phosphorus compounds[END_REF].

Polybutylene terephthalate containing mineral additives

The use of phosphorus containing flame retardants allows modifying the thermo oxidative stability of the char but also increasing significantly the char content. It is a well known effect of these additives. However, other additives can also act as efficient char promoters. Viretto et al. [START_REF] Viretto | Thermal degradation of polyesters filled with magnesium dihydroxide and magnesium oxide[END_REF] had shown that only 10 wt% of magnesium hydroxide and magnesium oxide can increase signifi cantly the char amount from PBT degradation and thus modify the char properties.

Recently, we have observed that alumina and alumina modified with nickel elaborated following the procedure described by Majoli et al. [START_REF] Majoli | Composite carbon nanostructures as promising carriers for gaz analysis[END_REF] do not only lead to an enhancement of the char amount from PBT pyrolysis but also changes its properties in a certain extent.

Pure alumina and magnesium hydroxide (MDH) change the thermo oxidative stability of the char. T 1 increases up to 785 K in presence of MDH and a decreases. The presence of nickel at the surface of alumina particles leads to a slight decrease in thermo oxidative stability in comparison of pure alumina. Nevertheless, the change in char properties does not follow a clear tendency according to nickel content and further research will focus on this issue.

Interestingly, it can be observed that all the data points corre sponding to this series are aligned. Higher char stability under pure oxygen is closely related to lower sensitivity to oxygen.

Influence of elemental composition of the char on its thermo oxidative stability

The elemental compositions of all chars were investigated by Energy Dispersive X ray spectroscopy (EDX) coupled to scanning electron microscopy (Table 3). It appears that there is no simple relation between the elemental composition and the thermo oxidative stability of char. Indeed, some chars exhibiting similar phosphorus content (around 9e12 wt%, both for chars prepared from flax MVP17 and from epoxy P3POEt) exhibit very different T 1 (920e1173 K, i.e. 647e900 C). In the whole, contrarily to the effective heat of combustion which can be calculated from the elemental composition, the char thermo oxidative stability during an aerobic pyrolysis depends on a couple of parameters, as already explained in the introduction part.

Concerning more specifically the chars prepared from fire tests, several researchers have assumed the influence of various param eters. Hence a higher degree of graphitization measured from the ratio between D and G bands intensities arising from Raman spectroscopy data corresponds to more thermally stable char [START_REF] Zhou | MoS2 nanolayers grown on carbon nanotubes: an advanced reinforcement for epoxy composites[END_REF][START_REF] Rajarao | Study of structural evolution of chars during rapid pyrolysis of waste CDs at different temperatures[END_REF]. Shao et al. considered that the presence of PeNeC bonds improves the char thermal stability [START_REF] Shao | Flame retardation of polypropylene via a novel intumescent flame retardant: ethylenediaminemodified ammonium polyphosphate[END_REF][START_REF] Shao | An efficient monocomponent polymeric intumescent flame retardant for polypropylene : preparation and application[END_REF]. Quach et al. observed that the decomposition of PMMA flame retarded with ammonium polyphosphate (APP) and silica leads to a more insulating char when SiP 2 O 7 crystals are formed by a chemical reaction between APP and silica [START_REF] Quach | Barrier effect of flame retardant systems in poly(methyl methacrylate): Study of the efficiency of the surface treatment by octylsilane of silica nanoparticles in combination with phosphorous fire retardant additives[END_REF]. Unfortunately, no quantitative relation was drawn between the composition and the structure of the chars and their thermo oxidative stability. Further research is needed to ascertain such relations.

At the same time, it may be noted that there is just a rough relation between the carbon weight fraction, T 1 and a. A low carbon fraction allows reaching higher T 1 and lower a.

It can also be confirmed (Table 3) that the char sampled from the cone calorimeter test exhibits a better thermo oxidative stability (higher T 1 ) than their counterparts prepared using the standard procedure. Indeed, the carbon contents of the chars sampled from the cone calorimeter test are much lower. For example, the carbon content of the char prepared from epoxy material flame retarded with P3P(OEt) is 56.8 wt% after the cone calorimeter test against 77.4 wt% when the standard procedure was applied. This fact is probably due to an extended thermo oxidation step at the end of the cone calorimeter test when the flame is vanished.

During a fire test, a char can act according to two different modes. Firstly, it reduces the heat release by trapping carbon into the condensed phase. Higher is the carbon content in the char, higher is its "potential" effective heat of combustion and lower is the heat actually released during the thermal degradation of a material. However, it can also act as a layer insulating the under lying polymer. From this point of view, higher is its thermal sta bility, better is its efficiency to slowdown the thermal degradation.

Combining these two modes of action is challenging because a high carbon content corresponds to a lower thermal stability. It appears quite difficult to obtain a char storing a lot of energy with a high thermal stability as shown in Fig. 9. Effective heat of com bustion was calculated considering a complete oxidation and the composition based only on C, O, P and Si atoms (representing more than 96 wt% of the char composition).

Phosphorylated nanodiamonds

Due to the very high thermo oxidative stability of nano diamonds, the procedure was modified to study these materials. The temperature considered to assess their stability is the tem perature for which the heat release rate reaches 50 W/g (and not the temperature of the peak of heat release rate). Obviously the parameters T 1 and a calculated according to this method cannot be compared to the parameters calculated using the standard method. Table 4 and Fig. 10 summarize the results obtained with un modified and phosphorylated nanodiamonds. It appears that phosphorus leads to a strong improvement of thermo oxidative stability in good agreement with previous findings [START_REF] Presti | Improvement of the Oxidative Stability of Nanodiamonds by Surface Phosphorylation[END_REF] (see supporting information S2). For unmodified nanodiamonds, the temperatures allowing to reach a heat release rate equal to 50 W/g in pure oxygen and in air (f O2 0.2) are 691 K and 726 K respec tively. In the case of nanodiamonds containing 5.15 wt% of phos phorus, these temperatures rise correspondingly, up to 938 K and 965 K. The increase of temperature is thus about 250 K. Once again, the influence of phosphorus appears nonlinear. Especially, a does not change when phosphorus content increases up to 4 wt% but starts decreasing with a further increase in phosphorus content (ND5.15).

Conclusions

A simple method for an evaluation of the thermo oxidative stability of chars collected from burnt polymers is proposed. The method is based on the application of the pyrolysis combustion flow calorimetry in a broad range of the oxygen contents in py rolysis chambere from 0% vol. (anaerobic conditions) up to 40% vol. The method allows defining two main parameters which charac terize the thermo oxidative properties of the char, namely the temperature T 1 of char degradation in pure oxygen and the value a representing the dependence of the char degradation temperature on the oxygen fraction.

a usually decreases when T 1 increases. This is likely to mean that a low thermo oxidative stability of a char in pure oxygen (low T 1 ) causes it to become highly sensitive to the oxygen content. How ever, the best thermo oxidative profile should correspond both to high T 1 and a values. Nevertheless, it depends also on the burning conditions. For an aerobic pyrolysis in an atmosphere relatively rich in oxygen (the oxygen fraction arises to 0.1 and more), a high T 1 value must be targeted. On the contrary, if pyrolysis is carried out in an atmosphere poor in oxygen, a high a value can compensate a low temperature T 1 .

Due to a broad range of materials studied in this work (ligno cellulosic fibers, synthetic polymers, carbon based composite ma terials, polymers containing phosphorus flame retardants, organ ophosphorus silica and metal based fillers), it appears quite difficult to point out any general relationships between the char elemental composition and its thermo oxidative stability. However, some particular observations can be already made.

First of all, high carbon content appears to be harmful for the thermo oxidative stability of a material. Combining high thermal stability and great energy storage capacity is a challenging task. Further work is needed to link the thermo oxidative stability and the structure of the char.

One of the main expected results concerns the influence of phosphorus on the thermo oxidative stability. Phosphorus im proves the temperature of char degradation and allows chars to be thermally stable during fire tests even in aerobic conditions. Nevertheless, the effect of phosphorus also depends strongly on the materials composition. Even with high content of phosphorus, chars from ligno cellulosic fibers exhibit low thermo oxidative stability. On the contrary, in the case of epoxy, polyamide or nanodiamonds, the char degradation temperature can reach or exceed 800 K. The sensitivity of char degradation to the oxygen fraction also changes with the phosphorus content but no clear tendency can be noted.

It must be also mentioned that the conditions of char elabora tion are highly influent on the char properties. In particular, the chars obtained from the cone calorimeter test or from direct aer obic pyrolysis usually manifest better thermo oxidative stabilities than those prepared from anaerobic pyrolysis in PCFC.

Figs. 1

 1 and 2 show the change of the pHRR temperature versus the oxygen fraction in the case of a char prepared from poly carbonate (PC) anaerobic pyrolysis. The analyses were carried out for six oxygen fractions: 0.01, 0.02, 0.05, 0.1, 0.2, 0.4. Fig. 2 repre sents Ln(T fO 2

Fig. 1 .

 1 Fig. 1. Heat release rate curves for char prepared from polycarbonate (PC) anaerobic pyrolysis and degraded using various oxygen fractions.

Fig. 2 .Fig. 3 .

 23 Fig. 2. Ln(T fO 2) versus Ln(f O2 ) for char prepared from polycarbonate (PC) anaerobic pyrolysis.

Fig. 4 .

 4 Fig. 4. HRR curves for flax fibers during direct aerobic pyrolysis.

Fig. 5 .

 5 Fig.5. a parameter versus T 1 for all studied chars prepared according to the standard procedure (labels in italic correspond to the phosphorus content in the char).

Fig. 6 .

 6 Fig. 6. Temperature of char degradation versus the oxygen fraction for a char prepared from untreated flax fiber (standard procedure, open circles) and for a char prepared from epoxy containing ammonium polyphosphate (sampled from the cone calorimeter test, black circles).

Fig. 7 .

 7 Fig. 7. Thermo-oxidative stability for epoxy and polyamide-based materials flame retarded with phosphorus-containing additives according to two procedures for char preparation (only the chars discussed in this section are labelled).

Fig. 8 .

 8 Fig. 8. Thermo-oxidative stability of flax fibers grafted with MVP according to the standard procedure or determined during their direct aerobic pyrolysis (only the chars discussed in this section are labelled).

Fig. 9 .

 9 Fig. 9. Effective heat of combustion versus the thermal stability in pure oxygen (T 1 ) for the chars listed in Table3(EHC of PEEK and PC experimentally measured was also added).

Fig. 10 .

 10 Fig.10. Evolution of the nanodiamonds properties versus the phosphorus content on nanodiamonds (white circles: calculated temperature for a heat release rate (HRR) equal to 50 W/g in pure oxygen; black circles: alpha parameter).

Table 1

 1 Materials studied in this work.

	Char from	Test conditions	Additive	Initial content (wt%)	Phosphorus content in the char (wt%)
	PEEK	Standard procedure	/	/	0
	PC	Standard procedure	/	/	0
	PBT	Standard procedure	/	/	0
	Epoxy-P3P(OEt)	Standard procedure	P3P(OEt)	17.2	10.3
	Epoxy-P3P(OEt)	Cone residue	P3P(OEt)	17.2	10.3
	Epoxy-APP	Cone residue	APP	9.4	11.6
	PA-SiP	Standard procedure	SiP	11	8.2
	PA-SiDOPO	Standard procedure	SiDOPO	10	3.2
	Flax fibers	Standard procedure	/	/	0
	Flax fibers	Direct aerobic pyrolysis	/	/	0
	Flax-MVP 0.4	Standard procedure	MVP	0.4	0.9
	Flax-MVP 0.4	Direct aerobic pyrolysis	MVP	0.4	Unknown
	Flax-MVP 2.1	Standard procedure	MVP	2.1	3.5
	Flax-MVP 2.1	Direct aerobic pyrolysis	MVP	2.1	Unknown
	Flax MVP 17	Standard procedure	MVP	17	9.3
	Flax-MVP 17	Direct aerobic pyrolysis	MVP	17	Unknown
	PBT-MDH	Standard procedure	MDH	10	0
	PBT-alumina	Standard procedure	Alumina	10	0
	PBT-alumina Ni1	Standard procedure	Modified alumina	10	0
	PBT-alumina Ni2.5	Standard procedure	Modified alumina	10	0
	PBT-alumina Ni5	Standard procedure	Modified alumina	10	0
	Nanodiamond ND	Direct aerobic pyrolysis	/	/	0 a
	ND3.85	Direct aerobic pyrolysis	Phosphate	/	3.85 a
	ND3.99	Direct aerobic pyrolysis	Phosphate	/	3.99 a
	ND5.15	Direct aerobic pyrolysis	Phosphate	/	5.15 a
	a Phosphorus content in nanodiamonds.

Table 2

 2 Main data on thermo-oxidative stability for the studied chars.

	Char from	Test conditions	T 0.01 f O2	0.01	T 0.2 f O2	0.2	T 1 (K) f O2	1	a	R2
	PEEK	Standard procedure	1059		833		728		0.081	0.9939
	PC	Standard procedure	1034		823		721		0.078	0.9981
	PBT	Standard procedure	932		792		706		0.060	0.9617
	Epoxy-P3P(OEt)	Standard procedure	1092		842		747		0.083	0.9822
	Epoxy-P3P(OEt)	Cone residue	1094		946		859		0.053	0.9457
	Epoxy-APP	Cone residue	1060		955		900		0.036	0.9572
	PA-SiP	Standard procedure	962		858		794		0.042	0.9553
	PA-SiDOPO	Standard procedure	956		845		799		0.039	0.9433
	Flax fibers	Standard procedure	865		624		554		0.097	0.9607
			836		626		560		0.087	0.9741
	Flax fibers	Direct aerobic pyrolysis	871		668		600		0.081	0.957
	Flax-MVP 0.4	Standard procedure	878		718		643		0.068	0.9815
	Flax-MVP 0.4	Direct aerobic pyrolysis	919		735		660		0.072	0.9939
	Flax-MVP 2.1	Standard procedure	997		749		652		0.092	0.9796
	Flax-MVP 2.1	Direct aerobic pyrolysis	959		769		679		0.075	0.9962
	Flax MVP 17	Standard procedure	1201		780		647		0.135	0.9523
	Flax-MVP 17	Direct aerobic pyrolysis	1157		788		669		0.119	0.9621
	PBT-MDH	Standard procedure	866		811		785		0.021	0.9847
	PBT-alumina	Standard procedure	925		827		769		0.040	0.9805
	PBT-alumina Ni1	Standard procedure	944		794		723		0.058	0.9761
	PBT-alumina Ni2.5	Standard procedure	963		793		694		0.071	0.9707
	PBT-alumina Ni5	Standard procedure	904		801		759		0.038	0.9891

Table 3

 3 Elemental compositions of several studied chars.

	Char obtained from	Test conditions	T 1 (K) f O2	1	a	Weight fraction (%)
						C	O	P	S i
	Epoxy-P3P(OEt)	Standard procedure	747		0.083	77.4	12.3	10.3	0
	Epoxy-P3P(OEt)	Cone residue	859		0.053	56.8	29.5	10.3	0
	Epoxy-APP	Cone residue	900		0.036	51.0	33.7	11.6	0
	PA-SiP	Standard procedure	794		0.042	31.5	40.0	8.3	19.5
	PA-SiDOPO	Standard procedure	799		0.039	48.7	31.7	3.2	16.4
	Flax fibers	Standard procedure	554		0.097	81.7	14.8	0	0
			560		0.087
	Flax-MVP 0.4	Standard procedure	643		0.068	87.9	9.5	0.9	0
	Flax-MVP 2.1	Standard procedure	652		0.092	84.3	11.5	3.5	0
	Flax MVP 17	Standard procedure	647		0.135	76.9	13.5	9.3	0

Table 4

 4 Main data on thermo-oxidative stability of nanodiamonds.

	Char from	Test conditions	T 0.2 (K) f O2	0.2	T 1 (K) f O2	1	a	R2
	ND	Direct aerobic pyrolysis	726		691		0.0325	0.9925
	ND3.85	Direct aerobic pyrolysis	917		875		0.032	0.9922
	ND3.99	Direct aerobic pyrolysis	951		906		0.0309	0.9802
	ND5.15	Direct aerobic pyrolysis	965		938		0.0204	0.9673

Acknowledgments

The authors thank the french "Fonds Unique Interminist eriel" the french "Pôles de Comp etitivit e" for supporting the BIONICOMP project. J. Sahyoun and R. M enard are acknowledged to have pre pared some materials studied in this article.

Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.polymdegradstab.2016.11.007.


Louise Chantepie

Loys Bodin

Julien Sarry

Florent Woloszyn

Florence Plisson-Petit

Julien Ruesche

Laurence Drouilhet

Stéphane Fabre
email: stephane.fabre@inra.fr

Genome-wide identification of a regulatory mutation in BMP15 controlling prolificacy in sheep

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

Introduction

There is now an accumulation of evidence that oocyte plays a central role in controlling the ovarian folliculogenesis, from the early stages up to ovulation. Among the local factors produced by the oocyte itself, members of the bone morphogenetic protein/growth and differentiation factor (BMP/GDF) family play an integral role in this control (Persani et al., 2015 [START_REF] Persani | The fundamental role of bone morphogenetic protein 15 in ovarian function and its involvement in female fertility disorders[END_REF]). Among them, the most important are surely BMP15 and GDF9. Knock-out mice models gave the first evidence of the importance of these two oocyte-derived factors acting individually as homodimers and/or through a synergistic co-operation to control the ovarian function [START_REF] Elvin | Paracrine Actions Of Growth Differentiation Factor-9 in the Mammalian Ovary[END_REF], Yan et al., 2001[2,3]).

In human also, a focus was done on BMP15 and GDF9 about their implication in various ovarian dysfunctions. Indeed, numerous heterozygous missense mutations have been identified in both genes associated with primary or secondary amenorrhea in different cohorts of women affected by primary ovarian insufficiency (POI) all over the world. Particularly, the 10-fold higher prevalence of BMP15 variants among patients with POI compared with the control population supports the causative role of these mutations (Persani et al., 2015 [START_REF] Persani | The fundamental role of bone morphogenetic protein 15 in ovarian function and its involvement in female fertility disorders[END_REF]). Alteration of BMP15 and GDF9 were also searched in association with the polycystic ovary syndrome (PCOS). Here again several missense variants were discovered in both genes, but the pathogenic role of these mutations remains controversial in the etiology of this syndrome. However, several studies have reported an aberrant expression of BMP15 and GDF9 in the ovary of PCOS patients (Teixera [START_REF] Filho | Aberrant expression of growth differentiation factor-9 in oocytes of women with polycystic ovary syndrome[END_REF][START_REF] Wei | Reduced and delayed expression of GDF9 and BMP15 in ovarian tissues from women with polycystic ovary syndrome[END_REF] [START_REF] Filho | Aberrant expression of growth differentiation factor-9 in oocytes of women with polycystic ovary syndrome[END_REF][START_REF] Wei | Reduced and delayed expression of GDF9 and BMP15 in ovarian tissues from women with polycystic ovary syndrome[END_REF]). Interestingly, some BMP15 polymorphisms situated in the 5'UTR are significantly associated with the over response to recombinant FSH applied during assisted reproductive treatment and with the risk to develop an ovarian hyperstimulation syndrome (OHSS, Moron et al. 2006;[START_REF] Hanevik | A single nucleotide polymorphism in BMP15 is associated with high response to ovarian stimulation[END_REF] [START_REF] Morón | Bone morphogenetic protein 15 (BMP15) alleles predict over-response to recombinant follicle stimulation hormone and iatrogenic ovarian hyperstimulation syndrome (OHSS)[END_REF][START_REF] Hanevik | A single nucleotide polymorphism in BMP15 is associated with high response to ovarian stimulation[END_REF]). Finally, polymorphisms in BMP15 and GDF9 genes were also searched in association with dizygotic twinning in human. If no convincing results were obtained for BMP15, some lost-of-function variants of GDF9 were observed significantly more frequently in mothers of twins compared to the control population [START_REF] Palmer | Novel variants in growth differentiation factor 9 in mothers of dizygotic twins[END_REF][START_REF] Simpson | Aberrant GDF9 expression and activation are associated with common human ovarian disorders[END_REF] [START_REF] Palmer | Novel variants in growth differentiation factor 9 in mothers of dizygotic twins[END_REF][START_REF] Simpson | Aberrant GDF9 expression and activation are associated with common human ovarian disorders[END_REF]).

In parallel, the search for the genetic determinism of ovulation rate and prolificacy variability in sheep has also highlighted the crucial role of BMP15 and GDF9 by evidencing numerous independent loss-of-function mutations all altering the coding sequence of these two genes (Persani et al. 2015;[START_REF] Abdoli | A review on prolificacy genes in sheep[END_REF] [START_REF] Persani | The fundamental role of bone morphogenetic protein 15 in ovarian function and its involvement in female fertility disorders[END_REF][START_REF] Abdoli | A review on prolificacy genes in sheep[END_REF]). Depending on the mutation and its hetero-or homozygous state, the phenotype controlled by these mutations in BMP15 and GDF9 goes from the early blockade of the folliculogenesis, and subsequent sterility, to an extraordinary increase of the ovulation rate (OR) and thus litter size (LS) of carrier ewes [START_REF] Galloway | Mutations in an oocyte-derived growth factor gene (BMP15) cause increased ovulation rate and infertility in a dosage-sensitive manner[END_REF][START_REF] Hanrahan | Mutations in the Genes for Oocyte-Derived Growth Factors GDF9 and BMP15 Are Associated with Both Increased Ovulation Rate and Sterility in Cambridge and Belclare Sheep (Ovis aries)1[END_REF]Sylva et al., 2011;Demars et al., 2013 [11-14]). Thus, sheep exhibiting an extremely high prolificacy are of great interest for identifying genes and mutations involved in molecular pathways controlling the ovarian function. These animal models have a double interest, in agriculture for the genetic improvement of the prolificacy, and in human clinic for providing valuable candidate genes in the genetic determinism of female infertility or subfertility, as described above.

The Noire du Velay (NV) population is a French local sheep breed mainly reared in the Haute-Loire and Loire departments. Ewes present naturally out-of-season breeding ability, very good maternal characteristics and a quite high prolificacy (mean LS=1.62 lamb per lambing). Large variation in LS has been observed in this breed and a recent genetic study has evidenced the segregation of an autosomal mutation named FecL L controlling this trait (Chantepie et al. 2018[15]). This variant located in the intron 7 of the B4GALNT2 gene and associated with its ectopic ovarian expression, was originally discovered in the Lacaune meat sheep breed, increasing OR and prolificacy (Drouilhet et al. 2013[16]). For the segregation study, more than 2700 NV ewes with LS records were genotyped at the FecL locus (Chantepie et al. 2018[15]). Surprisingly, the distribution of LS and the existence of high prolific ewes among the FecL L non-carrriers have suggested the possible segregation of a second prolificacy major mutation in this population as already observed in the Lacaune breed carrying both FecL L and FecX L [START_REF] Bodin | A novel mutation in the bone morphogenetic protein 15 gene causing defective protein secretion is associated with both increased ovulation rate and sterility in Lacaune sheep[END_REF], Drouilhet et al. 2013[16,17]). In order to validate this hypothesis, after specific genotyping excluding all other known mutations affecting OR and LS and segregating in French sheep populations, we have performed a genome-wide association study (GWAS) based on a case/control design. Completed by the whole genome sequencing of two finely chosen animals, we have identified a new regulatory variant called FecX N affecting the oocyte-dependent expression of BMP15 in association with increased prolificacy in sheep.

Results

Genetic association analyses

A first set of genomic DNA from 30 NV ewes without the FecL L prolific allele at the B4GALNT2 locus (LS records ranging from 2.00 to 3.00) was genotyped for already known mutations affecting sheep prolificacy at the 3 other loci, BMPR1B, GDF9 and BMP15. Using specific RFLP assay (BMPR1B, Wilson et al. 2001[18]) or Sanger sequencing of coding parts (GDF9 and BMP15, Talebi et al, 2018 [START_REF] Talebi | Transcriptome analysis of ovine granulosa cells reveals differences between small antral follicles collected during the follicular and luteal phases[END_REF]), none of the known mutations were evidenced (data not shown). Thus, to establish the genetic determinism of the remaining LS variation in this population, 80 ewes were genotyped by Illumina Ovine SNP50 Genotyping Beadchip. The allele frequencies of the most highly prolific ewes (cases, n=40, mean LS=2.47) and lowly prolific ewes (controls, n=40, mean LS=1.23) were compared to identify loci associated with LS using GWAS according to the procedures described in the Materials and Methods. Finally, genotype data were obtained from 79 animals (39 cases, 40 controls). Six markers located on OARX were significantly associated with LS variation at the genome-wide level after Bonferroni correction (Fig 1A, Table 1). Importantly, at the chromosome-wide level, a cluster of 26 significant markers encompassed the location of the BMP15 candidate gene (Fig 1B). In order to better characterize this locus on the X chromosome, we have determined for each individual the most likely linkage phase across 80 markers (10Mb) including the significant region. After haplotype clusterization, a specific segment of 3.5 Mb (50639087-54114793 bp, OARv3.1 genome assembly) was identified to be more frequent in highly prolific cases than in controls (f cases = 0.51 vs. f controls =0.37, P= 1.92E -11 , Chi-square test) (Fig 2). This identified segment contained the BMP15 gene (50970938-50977454 bp, OARv3.1) well-known to play a crucial role in the ovarian function and to be a target of numerous mutations in its coding region controlling 133 prolificacy (Persani et al, 2015 [START_REF] Persani | The fundamental role of bone morphogenetic protein 15 in ovarian function and its involvement in female fertility disorders[END_REF]). RFLP assay to specifically genotype for this polymorphism. Among the 79 animals of the GWAS, 31 ewes were heterozygous and 6 homozygous for the A variant allele. As shown in Table 2, most of the A carrier ewes were in the highly prolific Case group (34 among 39), while only 3 set in the Control group. When associating the LS performance of the 79 ewes to their genotype at the OARX: 50977717T>A SNP, the A non-carriers exhibited a mean LS of 1.36, heterozygous T/A a mean LS of 2.32 and homozygous A/A a mean LS of 2.73 indicating that the A allele of this polymorphism was strongly associated with increased LS in NV (T/A or A/A vs. T/T, P<1E -3 , one-way ANOVA).

Furthermore, this polymorphism appears in total linkage disequilibrium with the six more significant markers from the GWAS analysis (Fig 3). Genotype information at the OARX: 50977717T>A locus was introduced in the GWAS analysis. This SNP appeared as the most significant marker associated to the prolificacy phenotype (P unadjusted =1.93E -11 , P Chromosome-wide corrected =1.62E -14 and P Genome-wide corrected =9.13E -07 )

suggesting that it could be the causal mutation (S3 Figure). In accordance with the Fec gene nomenclature, the mutant allele identified upstream of the BMP15 gene in NV sheep was named FecX N . [START_REF] Chantepie | Presence of causative mutations affecting prolificacy in the Noire du Velay and Mouton Vendéen sheep breeds[END_REF][START_REF] Davis | DNA tests in prolific sheep from eight countries provide new evidence on origin of the Booroola (FecB) mutation[END_REF][START_REF] Mullen | Investigation of prolific sheep from UK and Ireland for evidence on origin of the mutations in BMP15 (FecX(G), FecX(B)) and GDF9 (FecG(H)) in Belclare and Cambridge sheep[END_REF][START_REF] Jemaa | The high prolificacy of D'man sheep is associated with the segregation of the FecLL mutation in the B4GALNT2 gene[END_REF]). We have tested the FecX N allele presence in a diversity of 26 sheep breeds representing 725 animals (Rochus et al. 2018[23]). Among the breeds tested, the FecX N genotyping has confirmed the segregation of this mutation in NV breed and revealed its presence in the Blanche du Massif Central (BMC) and Lacaune breeds (Table 3). Additionally, the

184

FecX N genotype frequency and effect on prolificacy 185 Large cohorts of ewes, chosen at random, were genotyped in order to accurately 186 estimate the allele frequencies in the NV and the BMC populations (Table 4). The Based on the raw mean LS observations, the FecX N carrier ewes clearly exhibited increased LS compared to non-carriers in both populations (Table 4). The L prolific allele at the FecL locus is also segregating in NV (Chantepie et al. 2018 [START_REF] Chantepie | Presence of causative mutations affecting prolificacy in the Noire du Velay and Mouton Vendéen sheep breeds[END_REF]). Results

of the linear mixed model showed that for the NV breed, one copy of the FecX N allele significantly increased LS by +0.22 and two copies increased LS by +0.65, while a single copy of the FecL L allele increased LS by +0.41 and two copies by +0.72. Based on the 80 ewes genotyped heterozygous at both loci it appeared that the effect of FecX N and FecL L on LS was not fully additive, the expected LS being significantly slightly reduced by -0.05 (0.58 instead of 0.63) (Fig. 4A). For the BMC population, compared to FecX + /FecX + ewes, FecX N /FecX + exhibited increased LS by +0.18 and

FecX N /FecX N by +0.30 under natural estrus (Fig. 4B). The use of PMSG for estrus synchronization increased LS significantly among FecX + /FecX + ewes (+0.23) and

FecX N /FecX + ewes (+0.18) while the effect on FecX N /FecX N ewes was negative (-0.13). The combined effect of the first copy of the FecX N allele and the use of PMSG treatment was not fully additive, the interaction being significant although low (0.35 instead of 0.41) (Fig. 4B).

Functional effects of the FecX N mutation

As described above, FecX N is located upstream of the coding region of the BMP15 gene when referencing to the ovine genome v3.1 (ensembl.org) or v4.0 (ncbi.nlm.nih.gov). In both versions of the ovine genome, the BMP15 gene annotation begins at the ATG start site and FecX N is located -290pb upstream, possibly in the 5'UTR and/or the proximal promoter region. As a first approach, we took advantage of RNA sequencing data from ovine oocytes publicly available at EMBL-EBI (Bonnet et al., 2013[24]). After reads mapping against the ovine genome (v3.

Discussion

The present study identified the g.50977717T>A variant on the ovine chromosome X upstream of the BMP15 gene as the most likely causative mutation for the increased prolificacy of the NV ewes. The highly significant genetic association with the extreme LS phenotype, the significant effect of the A variant on increasing prolificacy by +0. [START_REF] Wan | Identification and Analysis of Regulatory Elements in Porcine Bone Morphogenetic Protein 15 Gene Promoter[END_REF]). In human, a regulatory mutation in the 5'UTR of BMP15 (c.-9C>G) was associated to non-syndromic premature ovarian failure (Dixit et al. 2006 [29]), but also to iatrogenic ovarian hyperstimulation syndrome (Moron et al. 2006 [START_REF] Morón | Bone morphogenetic protein 15 (BMP15) alleles predict over-response to recombinant follicle stimulation hormone and iatrogenic ovarian hyperstimulation syndrome (OHSS)[END_REF]). This mutation was shown to enhance the fixation of the PITX1 factor transactivating the BMP15 promoter (Fonseca et al. 2014 [START_REF] Fonseca | BMP15 c.-9C>G promoter sequence variant may contribute to the cause of non-syndromic premature ovarian failure[END_REF]). However, the FecX N position does not fit with the syntenic location of porcine LHX8 and human PITX1 binding sites on the ovine BMP15

promoter. Using the MatInspector promoter analysis tool (Genomatix), we were only able to hypothesize an alteration by FecX N of a putative TATA-box like sequence (TTAAATA >TTATATA). Unfortunately, our electromobility shift assay attempts using CHO nuclear extracts failed to demonstrate the binding of any factor at the FecX N position, preventing us from defining the precise molecular mechanism by which FecX N decreases the BMP15 promoter activity.

The inhibition of the promoter activity combined with the apparent decreased of BMP15 mRNA accumulation in homozygous FecX N /FecX N oocytes seem to confirm the transcriptional regulatory role of FecX N . However, the moment we have chosen during the follicular phase of the late folliculogenesis for the comparative analysis between FecX + and FecX N oocytes from antral follicles could not be optimal to visualize a highly 2017 [START_REF] Galloway | Mutations in an oocyte-derived growth factor gene (BMP15) cause increased ovulation rate and infertility in a dosage-sensitive manner[END_REF][START_REF] Bodin | A novel mutation in the bone morphogenetic protein 15 gene causing defective protein secretion is associated with both increased ovulation rate and sterility in Lacaune sheep[END_REF][START_REF] Lassoued | FecX Bar a Novel BMP15 mutation responsible for prolificacy and female sterility in Tunisian Barbarine Sheep[END_REF]). Consequently, it would certainly be appropriate to follow the BMP15 expression in FecX N carrier ewes from these early stages of folliculogenesis to better decipher the mutation impact on ovarian physiology. Nevertheless, the fact that FecX N inhibits the BMP15 gene expression fits well with the physiological and molecular models associating BMP system loss-of-function and increased sheep prolificacy [START_REF] Fabre | Regulation of ovulation rate in mammals: contribution of sheep genetic models[END_REF], Demars et al. 2013[14,33]).

One copy of FecX N allele significantly increased by +0.30 to +0.50 the raw mean LS of NV ewes. When corrected for different environmental effects and more particularly for the genotype at the FecL locus, the estimated effect of FecX N on LS was +0.22 lamb per lambing for the first copy and +0.43 for the second copy. This effect was in the range of already known prolific alleles in various sheep breeds (Jansson, 2014 [34]).

The effect of FecX N on LS seems independent of the genetic background. Indeed, the estimated positive effect of FecX N on prolificacy was confirmed in BMC breed with +0.18 lamb per lambing based on natural estrus. Moreover, the same robust effect was observed even in the presence of PMSG for synchronizing the estrus cycles preceding the lambing. The same observation is made for other mutations controlling sheep prolificacy. For instance, the FecL L allele exhibited a similar effect on LS in NV (+0.41, present study; +0.42, Chantepie et al. 2018 [START_REF] Chantepie | Presence of causative mutations affecting prolificacy in the Noire du Velay and Mouton Vendéen sheep breeds[END_REF]), Lacaune (+0.47, Martin et al. 2014 [START_REF] Martin | Effects of the FecL major gene in the Lacaune meat sheep population[END_REF]) and D'man (+0.30, Ben Jemaa et al. 2018 [START_REF] Jemaa | The high prolificacy of D'man sheep is associated with the segregation of the FecLL mutation in the B4GALNT2 gene[END_REF]), and this was also observed for the FecB B allele introgressed in several populations (Kumar et al. 2008 [36]).

By genotyping a diversity panel, we also evidenced the presence of 2 FecX N carrier animals in the Lacaune meat strain which will require further genotyping of numerous animals. If this is confirmed, the Lacaune meat breed will be another population, as Belclare, where 3 different natural prolific mutations are segregating [START_REF] Hanrahan | Mutations in the Genes for Oocyte-Derived Growth Factors GDF9 and BMP15 Are Associated with Both Increased Ovulation Rate and Sterility in Cambridge and Belclare Sheep (Ovis aries)1[END_REF][START_REF] Bodin | A novel mutation in the bone morphogenetic protein 15 gene causing defective protein secretion is associated with both increased ovulation rate and sterility in Lacaune sheep[END_REF]Drouilhet et al. 2013 [12,16,17]). The presence of FecL L in both NV and Lacaune, and the presence of FecX N in NV, BMC and Lacaune, also raises the question of the origin of these mutations. From population structure analysis, it was shown that NV, BMC and Lacaune shared the same origin within the European southern sheep populations that may explain the segregation of the same mutations in these populations (Rochus et al., 2018 [23]).

In conclusion, through a case/control GWAS strategy and genome sequencing, we

Materials and Methods

Animals

Ewes (Ovis aries) from the NV breed (n=2266) were genotyped on blood DNA at the FecL locus as already described (Chantepie et al. 2018[15]). In order to test the hypothesis of the segregation of a second major mutation controlling LS in this breed, a first set of 80 ewes with at least 5 LS records (mean LS=1.84; ranging from 1.00 to 3.50) were selected among the FecL + homozygous genotype (n=2151, mean LS=1.58). Subsequently, for NV breed, the effect of the FecX N mutation on LS was estimated on 2252 ewes, considering the genotype at the FecL locus. The presence of the FecX N mutation in other breeds was checked on a diversity panel of 725 animals from 26 French sheep breeds (Rochus et al. 2018 [START_REF] Rochus | Revealing the selection history of adaptive loci using genome-wide scans for selection: an example from domestic sheep[END_REF]; Table 3). For the BMC population, the effect of the FecX N mutation on LS was estimated on 2456 ewes. For

Biological samples

All blood sampling from the numerous sheep breeds studied were collected from jugular vein (5 ml per animal) by Venoject system with EDTA and directly stored at -20°C for further use. Part of these blood samples (GWAS and diversity panel) was used for extraction of genomic DNA as described (Bodin et al. 2007[17]). All other samples were used for direct genotyping on whole blood without DNA purification (Chantepie et al. 2018[15]).

For ovary collection and oocyte isolation, the estrus cycles of all adult NV and BMC ewes were synchronized with intravaginal sponges impregnated with flugestone acetate (FGA, 30 mg, CEVA) for 14 days. Ovaries were collected at slaughtering during the follicular phase 36h after FGA sponge removal. Cumulus-oocyte complexes (COC)

were immediately recovered from all visible 1-3mm follicles by aspiration using a 1ml syringe with a 26G needle and placed in McCoy's 5A culture medium (Sigma-Aldrich).

COC were mechanically dissociated by several pipetting and washing cycles in 150µl drops of McCoy's 5A medium and finally, denuded oocytes devoid of granulosa cells were recovered in 1X PBS. Only intact oocytes with a good homogeneity of the cytoplasm were grouped to obtain two to three pools of 5 oocytes per animal and stored at -80°C before RNA extraction.

Genotyping analyses

The FecL L mutation (OAR11:36938224T>A, NC_019468) was genotyped directly on whole blood samples by the KAPA-KASP assay as already described (Chantepie et al. 2018 [START_REF] Chantepie | Presence of causative mutations affecting prolificacy in the Noire du Velay and Mouton Vendéen sheep breeds[END_REF]). As a prerequisite before GWAS, a set of 30 high prolific FecL + /FecL + ewes were controlled for the absence of other evidenced major mutations affecting sheep prolificacy in French populations. Using the same KAPA-KASP assay, FecX L and FecX Gr alleles in BMP15 were genotyped as described (Chantepie et al. 2018[15]).

FecB B in the exon 7 of the BMPR1B gene (OAR6:29382188A>G, NC_019463.1) was genotyped using forced restriction fragment length polymorphism (RFLP) as described by [START_REF] Wilson | Highly prolific Booroola sheep have a mutation in the intracellular kinase domain of bone morphogenetic protein IB receptor (ALK-6) that is expressed in both oocytes and granulosa cells[END_REF] [START_REF] Wilson | Highly prolific Booroola sheep have a mutation in the intracellular kinase domain of bone morphogenetic protein IB receptor (ALK-6) that is expressed in both oocytes and granulosa cells[END_REF].

The whole genome genotyping was performed on 80 ovine genomic DNA using the [START_REF] Depristo | A framework for variation discovery and genotyping using next-generation DNA sequencing data[END_REF][START_REF] Van Der Auwera | From FastQ data to high confidence variant calls: the Genome Analysis Toolkit best practices pipeline[END_REF]). Variants effect and annotation were realized by SNPEff version 4.1 and filtering of interesting variants was performed using the SNPSift tool.

In vitro transcription and translation of BMP15

The full-length cDNA of ovine BMP15 (1480bp [START_REF] Pfaffl | Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper--Excel-based tool using pair-wise correlations[END_REF].

Data analysis

Single-marker association analyses were conducted using a Fisher's exact test and a

Bonferroni correction has been applied to check for significance levels. The chromosome-wide and genome-wide values have been established as mentioned by Balding et al. 2006 [42] . Statistical analyses were done using PLINK1.9 software under a case/control design [START_REF] Chang | Second-generation PLINK: rising to the challenge of larger and richer datasets[END_REF]. Among the 79 datasets of 47446 SNPs analyzed, the LS trait was considered as case when mean LS ≥ 2.18 (n=39) and control when LS ≤ 1.45 (n=40). Haplotypic association analysis on X chromosome were performed using

FastPhase software [START_REF] Scheet | A fast and flexible statistical model for large-scale population genotype data: applications to inferring missing genotypes and haplotypic phase[END_REF]. Empirical significance levels were calculated using maximum statistic permutation approach (max (T), n=1000).

Allele effect on LS was estimated in NV and BMC breeds on data extracted from the follow by Newman-Keuls post-hoc test. For the large animal cohort analyses, the linear mixed models used were as similar as possible to those of the national genetic evaluation system (Poivey et al. 1995 [45]). In the present study, the following fixed effects were considered: i) the genotype at the FecX locus, ii) the month of birth (12 levels) iii) a physiological status effect combining parity, age at first lambing, rearing mode and postpartum interval (44 levels) and iv) a combination of the flock year and season effect. Two random effects were added to the model: a permanent environmental effect and an animal additive genetic effect. Moreover, an additional fixed effect of the reproduction type was considered for the BMC breed for which some hormonal treatments are used each year (87% and 13% after natural and induced estrus in the data set). For the NV breed, since the FecL L allele is also segregating in the population (Chantepie et al. 2018 [15]), the effect of the genotype at this locus (2252 known and 25502 unknown genotypes) as well as its interaction with the genotypes at the FecX locus were considered. All these models were fitted using the ASReml software (Gilmour et al. 2009 [START_REF] Glimour | R: ASReml User-Guide Release 3[END_REF]).

The comparison between FecX alleles for BMP15 protein quantification was analyzed using Student's t-test, using Welch's correction. For reporter luciferase assays, differences between constructs were analyzed by one-way ANOVA followed by Newman-Keuls post-hoc test. QPCR data for BMP15 and GDF9 expression in oocytes were analyzed by two-way ANOVA considering genotype and breed effects. P > 0.05 was considered as not significant. All these experimental data are presented as means ± SEM and were analyzed using Prism 6 (GraphPad Software Inc.).

  //www.ensembl.org) compiling information from i) dbSNP, ii) whole genome sequencing information from the NextGen project (180 animals from various Iranian and Moroccan breeds) and iii) the International Sheep Genome Consortium (551 animals from 39 breeds all over the world).

  187 frequency of the N prolific allele at the FecX locus was similar in both populations, 0.11 188 and 0.10, with a distribution of 19.4% and 17.6% heterozygous, 1.5 % and 1% 189 homozygous carriers in NV and BMC, respectively. The genotype frequencies were 190 consistent with the Hardy Weinberg equilibrium (HWE) in both breed (NV P= 0.28 and 191 BMC P= 0.76).

  1) using STAR2 aligner within the Galaxy pipeline and visualization with Integrative Genome Viewer (IGV), the Fig 5 shows the location of FecX N within the possible 5'UTR of the BMP15 gene when expressed in the oocyte. Consequently, we have first tested the potential functional impact of FecX N on the in vitro stability and translatability of the BMP15 mRNA. Thus, the reference (T, FecX + ) and variant (A, FecX N ) forms of the ovine BMP15 cDNA (-297, +1183 referring to ATG start codon) were cloned in a pGEM-T vector for subsequent in vitro T7 promoter-dependent transcription/translation experiment using reticulocyte lysate solution. As shown in Fig 6, the western blotting of the BMP15 proteins produced from both forms and their chemiluminescent quantification revealed that the FecX N mutation had no significant impact on the overall stability and translatability of the BMP15 mRNA in this condition. As a second hypothesis, we have tested the FecX N impact on the BMP15 promoter activity. Two promoter regions were tested ([-743,-11] bp and [-443,-102] bp referring to ATG start codon) cloned in front of the luciferase reporter gene and transiently expressed in CHO cells cultured in vitro. As shown by the luciferase assays (Fig 7), the FecX N variant was able to significantly reduce the luciferase activity in the context of both the long or the short BMP15 promoters, indicating the possible inhibitory impact of FecX N on BMP15 gene expression. To go further with this hypothesis, in vivo BMP15 gene expression was measured directly on isolated oocytes pools from NV and BMC homozygous ewe carriers and non-carriers of the FecX N allele. Real-time qPCR experiments revealed a tendency of the BMP15 expression to be decreased by 2-fold (P=0.17, genotype effect, two-way ANOVA) in the oocytes of FecX N carriers despite a large inter-animal variability. In contrast, the expression of the second oocyte-specific prolificacy major gene GDF9 seemed unaffected (Fig 8).

  significant differential expression of BMP15. The BMP15 gene expression in ovine oocytes begins during the primary stage of follicular development and its expression increases up to the antral stages (McNatty et al. 2005; Bonnet et al. 2011[START_REF] Mcnatty | Physiological effects of major genes affecting ovulation rate in sheep[END_REF][START_REF] Bonnet | Transcriptome profiling of sheep granulosa cells and oocytes during early follicular development obtained by laser capture microdissection[END_REF]).Moreover, the streak ovaries phenotype of infertile ewes carrying homozygous mutations in BMP15 have evidenced its crucial role in controlling the primary to secondary follicle transition[START_REF] Galloway | Mutations in an oocyte-derived growth factor gene (BMP15) cause increased ovulation rate and infertility in a dosage-sensitive manner[END_REF][START_REF] Bodin | A novel mutation in the bone morphogenetic protein 15 gene causing defective protein secretion is associated with both increased ovulation rate and sterility in Lacaune sheep[END_REF][START_REF] Lassoued | FecX Bar a Novel BMP15 mutation responsible for prolificacy and female sterility in Tunisian Barbarine Sheep[END_REF]

  have identified in the NV breed a second prolific mutation named FecX N affecting the expression of the BMP15 gene, a well-known candidate gene controlling OR and LS in sheep. This work confirms the relevance of the whole genome approaches to decipher the genetic determinism of the prolificacy trait. Homozygous FecX N /FecX N animals were still hyperprolific as already observed for FecX Gr and FecX O , but in contrast with sterile animals observed for the 7 other FecX homozygous variants in BMP15. As an upstream regulatory mutation, FecX N also contrasts with these 9 other prolific causal mutations all evidenced in the coding part of BMP15 and altering the protein function. Thanks to this new sheep model, the genetic etiology of ovarian pathologies in women could be improved by searching polymorphisms, not only in the coding region, but also in the regulatory parts driving the BMP15 expression within the oocyte.

  gene expression analysis, 10 homozygous ewes at the FecX N locus (5 carriers and 5 non-carriers of the N allele) were bought from private breeders (6 NV and 4 BMC) and reared at INRA experimental facility (agreement number: D3142901). All experimental procedures were approved (approval number 01171.02) by the French Ministry of Teaching and Scientific Research and local ethical committee C2EA-115 (Science and Animal Health) in accordance with the European Union Directive 2010/63/EU on the protection of animals used for scientific purposes.

French national database for

  genetic evaluation and research managed by the Institut de l'Elevage (French Livestock Production Institute) and the CTIG (Centre de Traitement de l'Information Génétique, Jouy-en-Josas, France). Only females born after 2000 were retained (27 754 NV ewes with 122 110 LS records and 110 848 BMC ewes with 461 405 LS records) with their pedigree over 5 generations. FecX N genotype effect on the subset of 79 case/control animals was assessed by one-way ANOVA,

134 Table 1. Markers significantly associated with litter size SNP Chromosome Position a MAF b PUnadj c PChrom d PGenome e

		Variant search analysis and annotations through GATK toolkit was limited to the
		OARX: 50639087-54114793 region. We detected 60 SNPs and 90 small insertions
		and deletions (INDELs) with quality score >30 (S1 Table). Among them, we particularly
		focused on the 85 variants located within annotated genes (upstream, exon, intron,
		OARX_51294776.1 splice acceptor or donor, and downstream localization). After filtering these 85 variants OARX 53756339 0.28 3.52E -11 4.18E -08 1.67E -06
		s27837.1	OARX	53825247	0.30	5.83E -10	6.92E -07	2.77E -05
		s73460.1 for allele sharing with other breeds based on SheepGenome DB OARX 53905939 0.44 9.59E -10 1.14E -06 4.55E -05
		s39212.1	OARX	53852735	0.44	3.46E -09	4.11E -06	1.64E -04
		OARX_52608221.1 s46003.1 (http://sheepgenomesdb.org/) and 68 publicly available domestic sheep genomes OARX 52367253 0.41 1.68E -08 2.00E -05 7.99E -04 OARX 80222479 0.44 2.56E -07 3.03E -04 1.21E -02
		OARX_55032299.1	OARX	48942926	0.30	1.57E -06	1.86E -03	NS
		OARX_72164491.1	OARX	74388397	0.23	1.71E -06	2.03E -03	NS
		OARX_49135019.1	OARX	42475099	0.35	1.80E -06	2.13E -03	NS
		OARX_111306030.1	OARX	92041520	0.27	2.25E -06	2.67E -03	NS
		OARX_102620828.1	OARX	82796975	0.41	3.02E -06	3.59E -03	NS
		s31917.1	OARX	58202482	0.44	3.12E -06	3.70E -03	NS
		OARX_72351736.1	OARX	74590448	0.19	5.63E -06	6.68E -03	NS
		OARX_72263548.1	OARX	74498463	0.17	6.45E -06	7.66E -03	NS
		OARX_49564109.1	OARX	42876169	0.49	8.16E -06	9.69E -03	NS
		DU400878_520.1	OARX	73847207	0.27	9.96E -06	1.18E -02	NS
		s54281.1	OARX	58993959	0.24	1.20E -05	1.42E -02	NS
		s05229.1	OARX	58346644	0.46	1.71E -05	2.03E -02	NS
		s27938.1	OARX	53275559	0.35	1.77E -05	2.10E -02	NS
		OARX_54104393.1	OARX	49870983	0.36	2.03E -05	2.41E -02	NS
		OARX_72236232.1	OARX	74464263	0.18	2.23E -05	2.64E -02	NS
		OARX_111349974.1	OARX	92085118	0.18	2.23E -05	2.65E -02	NS
		OARX_53703822.1	OARX	51193144	0.35	2.59E -05	3.08E -02	NS
		OARX_43227227.1	OARX	36235514	0.32	3.41E -05	4.04E -02	NS
		OARX_51842287.1	OARX	53162079	0.49	3.43E -05	4.08E -02	NS
		OARX_102654502.1	OARX	82837158	0.20	3.65E -05	4.33E -02	NS
		a Position of markers are based on the OARv3.1 assembly in bp.
		b MAF, minor allele frequency.
		c PUnadj corresponds to exact unadjusted p-value for the Fisher's test.
		d PChrom corresponds to p-value after chromosome-wide Bonferroni correction.
		e PGenome corresponds to p-value after genome-wide Bonferroni correction (NS, non-significant).
	135	Characterization of the mutation
	136	While the BMP15 gene could be considered as a positional and functional candidate
	137	gene, no mutation was evidenced by Sanger sequencing of the BMP15 coding regions
	138	of the most prolific ewes studied. In order to find the potential causal mutation, we
	139	sequenced the whole genome of two finely chosen ewes based on the shortest
	140	haplotype within the region (homozygous reference vs. homozygous variant) and their
	141	opposite extreme phenotypes (LS 1.1 vs. 2.8).

(International Sheep Genomics Consortium; http://www.sheephapmap.org/), none of them were removed, all being NV breed specific. Finally, and based on prolificacy gene knowledge, we were particularly interested in one SNP (T>A) identified in the upstream region of the BMP15 gene at position 50977717 on OARX v3.1. We then developed a

Table 2 . Distribution of OARX:50977717T>A SNP genotypes and associated LS in case and control groups

 2

	Group	TT	TA AA
	Low prolific control	n=	37	3
		Raw mean LS	1.22 1.39
	Highly prolific case	n=	5	28	6
		Raw mean LS	2.41 2.43 2.73
	Total		42	31	6

As described for other prolific alleles such as FecB B , FecX G , FecG H , FecX Gr and FecL L , a given mutation can segregate in several sheep populations

(Davis et

al., 2002; Mullen et al., 2013, Chantepie et al. 2018, Ben Jemaa et al. 2019

Table 3 . FecX N genotype distribution from a diversity panel of French ovine breeds

 3

			Genotype			Genotype
	Breed	Total +/+	N/+	Breed	Total	+/+	N/+
			(+/Y)	(N/Y) a			(+/Y)	(N/Y) a
	Berrichon du Cher	29	29		Mourerous	26	26
	Blanche Massif Central 31	27	4	Mouton Vendéen 30	30
	Causse du Lot	32	32		Noire du Velay	28	26	2
	Charmoise	31	31		Préalpes du sud	27	27
	Charollais	29	29		Rava	29	29
	Corse	30	30		Romane	29	29
	Ile de France	28	28		Romanov	26	26
	Lacaune (meat)	42	40	2	Rouge de l'Ouest	28	28
	Lacaune (dairy)	40	40		Roussin	30	30
	Limousine	30	30		Suffolk	20	20
	Manech tête rousse	29	29		Tarasconnaise	32	32
	Martinik	22	22		Texel	21	21
	Merinos d'Arles	26	26
					TOTAL	725	717	8

a : +/+ or N/+ females, N/Y or N/+ hemizygous males.

Table 4 . FecX N frequencies and effects on LS in NV and BMC breeds.

 4 increased LS compared to L and N non-carriers from the linear mixed models with interaction between genotypes at the FecX and FecL loci for the Noire du Velay (NV) breed and between FecX genotypes and estrus type (NE= natural estrus; IE = Induced estrus) for the Blanche du Massif Central (BMC) breed.

					Breed
			NV (n=2323)				BMC (n=2456)
	FecX genotype	+/+	N/+	N/N		+/+	N/+	N/N
	Number of animals	1839	450	34		1999	432	25
	Frequency ( % )	79.2	19.4	1.5		81.3	17.6	1
	Raw mean LS	1.66	1.93	2.45		1.56	1.79	1.87
	ANOVA solutions a
	+/+	0.00	0.22	0.65	NE	0.00	0.18	0.30
	FecL genotype L/+	0.41	0.58	0.56	IE	0.23	0.35	0.17
	L/L	0.72	/	/

a. estimated

  2 lamb per lambing in a large set of NV ewes, also found in the BMC genetic background, and the demonstrated action on BMP15 transcriptional activity all support the causality of this mutation named FecX N . The BMP15 gene is at the top of the list of candidate genes controlling the ovarian function, ovulation rate and thus prolificacy in the ovine species, with nine independent causal mutations identified out of the sixteen already known. Indeed, 7 SNPs and 2 small INDELs all within the open reading frame were evidenced affecting the BMP15 FecX G , Hanrahan et al. 2004 [11,12]) or by breaking the reading frame (FecX R , Martinez-Royo et al. 2008; FecX Bar , Lassoued et al. 2017[START_REF] Martinez-Royo | A deletion in the bone morphogenetic protein 15 gene causes sterility and increased prolificacy in Rasa Aragonesa sheep[END_REF][START_REF] Lassoued | FecX Bar a Novel BMP15 mutation responsible for prolificacy and female sterility in Tunisian Barbarine Sheep[END_REF]). The 5 other SNPs generate non-conservative amino acid substitutions all leading to a loss of function of BMP15 ranging from inhibited protein production (FecX L , Bodin et al. 2007[START_REF] Bodin | A novel mutation in the bone morphogenetic protein 15 gene causing defective protein secretion is associated with both increased ovulation rate and sterility in Lacaune sheep[END_REF]), impaired interaction with GDF9 (FecX I and FecX B ,Liao et al. 2004 [27]), to altered cell signalling activity (FecX Gr and FecX O , Demars et al. 2013[START_REF] Demars | Genome-wide association studies identify two novel BMP15 mutations responsible for an atypical hyperprolificacy phenotype in sheep[END_REF]). In contrast with the 9 mutations described above, the FecX N variant evidenced in the present study is not located in the open reading frame of BMP15 and does not alter the protein sequence.

	NOBOX and PITX1 transcription factors. However, only LHX8 was demonstrated as
	functionally activating the porcine BMP15 promoter activity (Wan et al. 2015
	Whatever the version of the ovine reference genome (Oar_v3.1, Oar_v4.0 or even the
	last Oar_rambouillet_v1.0) the annotation of the BMP15 gene always starts at the ATG
	initiating codon. Using publicly available transcriptome data from ovine oocytes
	RNAseq analysis, we were able to show that FecX N located 290bp upstream of BMP15
	could stand in its 5'UTR region. From our in vitro functional analyses, FecX N was not
	demonstrated to influence the translatability of the BMP15 mRNA, but in the contrary
	it was shown to decrease the BMP15 promoter activity. Little is known about
	transcription factors able to regulate BMP15 expression. Several regulatory elements
	were evidenced in the pig BMP15 promoter hosting consensus binding sites for LHX8,

function. Among these mutations, 2 SNPs and the 2 INDELS impaired the protein production either by generating premature stop codon (FecX H , Galloway et al. 2000; However, no other polymorphism genetically linked to FecX N was found in the BMP15 coding sequence when checked by whole genome or local Sanger sequencing of the BMP15 gene from FecX N carrier animals. Of course, this does not rule out the possibility of a polymorphism lying in another gene nearby with a still unknown role in the ovarian function and prolificacy. Nevertheless, we did not find any polymorphism (SNP and INDEL) altering the coding sequence of genes annotated in the significantly LS-associated genetic region of 3.5Mb on OARX (S1

Table), leaving BMP15 as the most obvious candidate.

  cycles of 30s at the specific melting temperature, 30s at 72 °C and 30s at 94 °C, followed by 5 min at 72 °C. The primers used in this study are listed in S2 Table.

	also discarded. Finally, from the available design of 54241 SNPs available on the and mapped against the ovine genome assembly (Oar_v3.1.86) using BWA-MEM
	Illumina OvineSNP50 Beadchip and 80 selected NV ewes, the final dataset was (Galaxy version 0.7.17.1). The resulting BAM files were sorted using Samtools_sort
	reduced to 47446 SNPs analyzed in 79 individuals. (Galaxy version 1.0.0). Sorted and indexed BAM files were visualized through
	The FecX N mutation (OARX: 50977717T>A, NC_019484) was genotyped by a RFLP Integrative Genome Viewer, IGV software version 2.4.10 (Robinson et al. 2011[38]). A
	analysis using the Mse1 restriction enzyme (New England Biolabs) after a first step of GFF3 annotation file was obtained from Ensembl (Ovis_aries.Oar_V3.1.78). We
	Terra PCR Direct Polymerase Mix amplification (Takara) using one μl sample of total applied GATK version 3.5-0 to performed SNP and InDel discovery and genotyping
	blood. The accuracy of the FecX N RFLP genotyping was controlled by Sanger across the two samples simultaneously using standard filtering parameters according
	sequencing on few samples using the same amplification primers. to GATK Best Practices recommendations (DePristo et al. 2011; Van der Auwera et
	al. 2013
	PCR amplifications were conducted independently for each locus studied on an
	ABI2400 thermocycler (Applied Biosystems) with the following conditions: 5min at
	94°C, 32 Whole genome sequencing (WGS) analysis
	DNA sequencing libraries were constructed from 1 µg of genomic DNA using TruSeq
	DNA PCR-free Library Prep kit (Illumina). Sequencing was run on an Illumina HiSeq
	2500 apparatus using a paired-end read length of 2x150 pb with the Illumina Reagent
	Kits as already described (Demars et al. 2017[37]). WGS was performed at the
	Genotoul-GeT core facility (INRA Toulouse, https://get.genotoul.fr). The raw reads of
	OvineSNP50 Genotyping Beadchip from Illumina according to the manufacturer's
	Illumina DNA sequencing were preprocessed by removing adapter sequences. After
	protocol at the Laboratoire d'Analyses Génétiques pour les Espèces Animales,
	quality control, the FastQ files and metadata were submitted to the European
	(LABOGENA, Jouy en Josas, France; www.labogena.fr). From the dataset, individuals
	Nucleotide Archive (ENA) at EMBL-EBI (accession number PRJEB35553). Reads
	with a call rate <0.98 were excluded. SNP exclusion thresholds were: call frequency
	mapping and variants calling were performed using the local instance of Galaxy
	<0.95, and minor allele frequency (MAF) <0.01; or a significant deviation from Hardy-
	(https://galaxyproject.org) at the Toulouse Midi-Pyrénées bioinformatics platform
	Weinberg equilibrium (HWE) in the controls (p<1.10 -6 ). Non-polymorphic SNP
	(http://sigenae-workbench.toulouse.inra.fr). The cleaned paired reads were combined
	positions and markers with no position on the OARv3.1 reference genome map were

  Chemiluminescent signal was capture by a ChemiDoc MP imaging system and images were analyzed with the Image Lab Software (Bio-Rad). Primer sequences, amplicon length and amplification efficiency are listed in S2 Table. RNA transcript abundance was quantified using the ∆Ct method with the mean expression of GAPDH and SDHA as internal references and following the formula R=[E ref Ct ref /E target Ct target ]. The two reference genes were validated by the Bestkeeper algorithm[START_REF] Pfaffl | Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper--Excel-based tool using pair-wise correlations[END_REF]

	equivalent of 1.25 oocyte was reverse-transcribed using SuperScript II reverse transcriptase (Invitrogen) and anchored oligo(dT)22 primer (1µl at 10 µM). Primer membrane. BMP15 promoter activity design using Beacon designer 8.20 (Premier Biosoft), SYBR green real-time PCR
	The promoter sequence of the ovine BMP15 gene was amplified by PCR on genomic cycling conditions using QuantStudio 6 Flex Real-Time PCR system (ThermoFisher
	DNA from both homozygous FecX + and homozygous FecX N ewes. Two sizes of Scientific) and amplification efficiency calculation (E=e (-1/slope) ) were as already
	fragments were generated for cloning the BMP15 promoter in front of the luciferase described in Talebi et al. (2018)[19].
	(Luc) reporter gene, a long (lg) form of 732 bp ([-743,-11] bp referring to ATG start
	codon) and a short (sh) form of 341bp ([-443,-102] bp). The PCR products were
	engineered for digestion using Kpn1 and Hind3 restriction enzymes (New England
	Biolabs) and inserted into the pGL4.23 vector (Promega). The four resulting constructs
	(lgBMP15 + -Luc; lgBMP15 Twenty-four hours after seeding (3.10 4 cells/well, 24 wells plate), CHO (Chinese
	Hamster Ovary) cells were transfected using Lipofectamine 3000 (Invitrogen) with
	500ng/well of pGL4.23 constructs either empty or containing BMP15 promoter
	fragment. Forty-eight hours after transfection, cells were lysed and assayed for
	luciferase activity (Luciferase reporter assay kit, Promega). Luminescence in relative
	light units (RLU) was measured by a Glomax microplate reader (Promega). Each
	In vitro transcription and translation were realized from 500ng of cDNA pGEM-T construct was assayed in triplicate in 6 independent transfection experiments.
	construct using the TnT T7 Quick Coupled Transcription/Translation kit (Promega) and
	Transcend Biotin-Lysine-tRNA following the manufacturer protocol. Reactions for each RNA extraction, reverse transcription and quantitative PCR
	construct were run in duplicate in 6 independent TnT experiments. The resulting Total RNA from pools of 5 oocytes were extracted using the Nucleospin RNA XS kit
	BMP15 protein was revealed using Transcend non-radioactive translation detection according to the manufacturer's protocol (Macherey-Nagel) and including a DNase1
	system with chemiluminescent method (Promega) after reducing SDS-PAGE on a treatment. The low quantity of RNA recovered did not allow quantification. So, the
	gradient (4-15%) polyacrylamide gel (Promega) and transfer onto nitrocellulose

[-297,+1183]

referring to ATG start codon) with or without the FecX N mutation was generated from oocyte-derived RNA after a reverse transcription (RT) step (described in RNA extraction and RT paragraph, primers are listed in S2 Table

)

. The resulting PCR products were inserted by TA cloning into pGEM-T Easy plasmid (Promega) possessing T7 and SP6 promoters. The orientation of insertion and exclusion of unexpected PCR-induced mutations were controlled by Sanger sequencing. N -Luc; shBMP15 + -Luc; shBMP15 N -Luc) were controlled by Sanger sequencing. Primers used to generate these constructs are listed in S2 Table.

Acknowledgments

We thank Claire Chantaduc, Didier Cathalan and Kévin Chile from ROM Sélection managing the NV and BMC populations, for their precious help in the planning of blood sampling. We are grateful to the breeders who made their animals available for this study. LC was supported by a PhD grant co-funded by APIS-GENE through the Proligen project and the European Funds for Regional Development (FEDER) through the Interreg POCTEFA programme in the framework of the PIRINNOVI project (EFA103/15). Part of the NV sampling was supported by the DEGERAM project cofunded by the FEDER Massif Central, the Régions: Aquitaine, Midi-Pyrénées, Limousin and Auvergne; and the French government.

Author Summary

In the genetic etiology of women infertility syndromes, a focus was done on the oocyteexpressed BMP15 and GDF9 genes harboring several mutations associated with

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