Nathalie Le Floc'h

Ce ´line Deblanc
email: celine.deblanc@anses.fr.

Roland Cariolet

Anne V Gautier-Bouchardon

Elodie Merlot

Gae ¨lle Simon

Effect of Feed Restriction on Performance and Postprandial Nutrient Metabolism in Pigs Co-Infected with Mycoplasma hyopneumoniae and Swine Influenza Virus

As nutritional status and inflammation are strongly connected, feeding and nutritional strategies could be effective to improve the ability of pigs to cope with disease. The aims of this study were to investigate the impact of a feed restriction on the ability of pigs to resist and be tolerant to a coinfection with Mycoplasma hyopneumoniae (Mhp) and the European H1N1 swine influenza virus, and the consequences for nutrient metabolism, with a focus on amino acids. Two groups of specific pathogen-free pigs were inoculated with Mhp and H1N1 21 days apart. One group was fed ad libitum, the other group was subjected to a two-week 40% feed restriction starting one week before H1N1 infection. The two respective mock control groups were included. Three days post-H1N1 infection, 200 g of feed was given to pigs previously fasted overnight and serial blood samples were taken over 4 hours to measure plasma nutrient concentrations. Throughout the study, clinical signs were observed and pathogens were detected in nasal swabs and lung tissues. Feed-restricted pigs presented shorter hyperthermia and a positive mean weight gain over the 3 days post-H1N1 infection whereas animals fed ad libitum lost weight. Both infection and feed restriction reduced postprandial glucose concentrations, indicating changes in glucose metabolism. Post-prandial plasma concentrations of the essential amino acids histidine, arginine and threonine were lower in co-infected pigs suggesting a greater use of those amino acids for metabolic purposes associated with the immune response. Altogether, these results indicate that modifying feeding practices could help to prepare animals to overcome an influenza infection. Connections with metabolism changes are discussed.

Introduction

In pigs, infectious diseases and inflammatory states induce metabolic alterations and changes in nutrient partitioning between tissues associated with growth (e.g. muscle) and those involved in body defence (liver, lymphoid organs, etc.) [START_REF] Floc | Modifications of protein and amino acid metabolism during inflammation and immune system activation[END_REF][START_REF] Melchior | Chronic lung inflammation affects plasma amino acid concentrations in pigs[END_REF]. Metabolism disruption during disease or inflammation has also been demonstrated and is well-known in humans [START_REF] Wannemacher | Key role of various individual amino acids in host response to infection[END_REF][START_REF] Reeds | The amino acid requirements of disease[END_REF][START_REF] Obled | Amino acid requirements in inflammatory states[END_REF]. Conversely, many nutrients are directly or indirectly involved in the regulation of inflammatory and immune responses [START_REF] Grimble | Nutritional modulation of cytokine biology[END_REF][START_REF] Mora | Vitamin effects on the immune system: vitamins A and D take centre stage[END_REF][START_REF] Floc | Tryptophan metabolism, from nutrition to potential therapeutic applications[END_REF]. Particularly, amino acids (AA) are redistributed away from muscle protein synthesis towards tissues and cells involved in inflammatory and immune responses either to be used for the synthesis of specific compounds or to be degraded for use as energetic precursors [START_REF] Floc | Modifications of protein and amino acid metabolism during inflammation and immune system activation[END_REF]. Consequently, both utilization of dietary AA and intensity of AA mobilization may influence the ability of pigs to resist to an infectious agent and limit the detrimental consequences of the inflammation caused by the response to infection.

Infectious diseases and inflammatory states decrease feed consumption [START_REF] Konsman | How the immune and nervous systems interact during disease-associated anorexia[END_REF] and may induce a nutrient deficiency that can, in turn, reduce the ability of animals to mount an efficient immune response. Conversely, it has been shown that a feed restriction can have a beneficial effect on animal health [START_REF] Matsuzaki | Inflammatory responses to lipopolysaccharide are suppressed in 40% energy-restricted mice[END_REF][START_REF] Rantzer | Effects of a strategic feed restriction on pig performance and health during the post-weaning period[END_REF]. Indeed, in mice, a 40% feed restriction decreased the acute release of proinflammatory cytokines and induction of inducible nitric oxide synthase expression in response to an LPS challenge, thereby avoiding extreme inflammatory responses which could damage organs [START_REF] Matsuzaki | Inflammatory responses to lipopolysaccharide are suppressed in 40% energy-restricted mice[END_REF]. In growing pigs, caloric restriction modified the expression of immune genes [START_REF] Lkhagvadorj | Gene expression profiling of the short-term adaptive response to acute caloric restriction in liver and adipose tissues of pigs differing in feed efficiency[END_REF] suggesting that a short-term feed restriction may modify the immune response. However, the beneficial effect of feed restriction on pig health remains to be established for diseases other than digestive diseases [START_REF] Rantzer | Effects of a strategic feed restriction on pig performance and health during the post-weaning period[END_REF]. The feed cost represents more than 60% of the pig productive cost. Moreover, after a period of feed restriction, pigs usually exhibite a compensatory growth that limits the impact of a transient limitation of feed intake [START_REF] Heyer | Compensatory growth response in pigs: effects on growth performance, composition of weight gain at carcass and muscle levels, and meat quality[END_REF]. Thus, strategies based on a moderate and transient feed restriction deserve to be considered to reduce the impact of health problems and to maintain the productivity of farm animals without increasing feed cost.

In modern pig production, respiratory diseases are common and one responsible pathogen is the swine influenza virus (SIV) [START_REF] Opriessnig | Polymicrobial respiratory disease in pigs[END_REF]. Three SIV subtypes (H1N1, H1N2 and H3N2) are simultaneously circulating among pigs worldwide, but genetic lineages may vary within each subtype depending on the region (i.e. Europe, Asia and North America) [START_REF] Kuntz-Simon | Genetic and antigenic evolution of swine influenza viruses in Europe and evaluation of their zoonotic potential[END_REF]. Swine influenza is characterized by hyperthermia, loss of appetite, lethargy, respiratory problems and coughing, similarly as human flu. The severity of the disease is highly variable and influenced by many factors such as the virus strain, the age and immune status of the infected pig, the housing environment, and the presence of other respiratory pathogens [START_REF] Kuntz-Simon | Swine influenza In[END_REF][START_REF] Deblanc | Pre-infection of pigs with Mycoplasma hyopneumoniae modifies outcomes of infection with European swine influenza virus of H1N1, but not H1N2, subtype[END_REF][START_REF] Deblanc | Preinfection of pigs with Mycoplasma hyopneumoniae induces oxidative stress that influences outcomes of a subsequent infection with a swine influenza virus of H1N1 subtype[END_REF][START_REF] Rose | Dynamics of influenza A virus infections in permanently infected pig farms: evidence of recurrent infections, circulation of several swine influenza viruses and reassortment events[END_REF]. The infection is restricted to the respiratory tract, without viremia [START_REF] Van Reeth | Swine influenza[END_REF]. It has an impact on animal health and welfare but also generates important economic losses because of the decline in animal performance and the medication costs in the case of complications. Recently, the European avian-like swine H1N1 virus subtype has been identified in pig herds as a major pathogen involved in the Porcine Respiratory Disease Complex, together with Mycoplasma hyopneumoniae (Mhp) [START_REF] Fablet | Infectious agents associated with respiratory diseases in 125 farrow-to-finish pig herds: a cross-sectional study[END_REF], another respiratory pathogen widespread in French farms. Alone, Mhp induces chronic pneumonia with dry, non productive cough beginning 7 to 14 days after infection. Under experimental conditions, the diseases induced by intra-tracheal inoculation of Mhp or H1N1 mimicked respiratory outbreaks observed in the herds of origin [START_REF] Deblanc | Pre-infection of pigs with Mycoplasma hyopneumoniae modifies outcomes of infection with European swine influenza virus of H1N1, but not H1N2, subtype[END_REF][START_REF] Marois | Development of a quantitative Real-Time TaqMan PCR assay for determination of the minimal dose of Mycoplasma hyopneumoniae strain 116 required to induce pneumonia in SPF pigs[END_REF] and it was shown that pre-infection of pigs with Mhp increased the severity of clinical symptoms induced by H1N1 [START_REF] Deblanc | Pre-infection of pigs with Mycoplasma hyopneumoniae modifies outcomes of infection with European swine influenza virus of H1N1, but not H1N2, subtype[END_REF], inducing a strong inflammatory response [START_REF] Deblanc | Preinfection of pigs with Mycoplasma hyopneumoniae induces oxidative stress that influences outcomes of a subsequent infection with a swine influenza virus of H1N1 subtype[END_REF].

The aim of the present study was to evaluate the impact of 1) a Mhp/H1N1 co-infection on nutrient metabolism and 2) a 40% feed restriction on the ability of Mhp/H1N1 co-infected pigs to resist and to cope with the metabolic consequences of inflammation caused by infection (refered as tolerance) [START_REF] Detilleux | Effectiveness analysis of resistance and tolerance to infection[END_REF]. Resistance and tolerance to the infection were assessed by measuring pathogen excretions, clinical signs, and changes in nutrient metabolism, with a special focus on AA. A meal standardized for size, duration and composition was used to study post-prandial AA utilization and to identify which AA exhibited modified metabolism.

Materials and Methods

Pathogens and animals

The two pathogens, M. hyopneumoniae strain 116 and the avian-like swine H1N1 virus strain A/Sw/Cotes d'Armor/0231/ 06, were isolated from French farms. Strain isolations, as well as preparation and titration of inocula were performed as previously described [START_REF] Deblanc | Pre-infection of pigs with Mycoplasma hyopneumoniae modifies outcomes of infection with European swine influenza virus of H1N1, but not H1N2, subtype[END_REF].

Twenty-four specific pathogen-free pure-bred 6-week-old Large White pigs were randomly assigned to this study, taking into account litter origin, weight and sex. Young males were castrated and had similar metabolism and growth rate to those of females. These animals were obtained from the experimental pig herd of the French Agency for Food, Environmental and Occupational Health and Safety (Anses) at Ploufragan. These animals were free from Mhp and SIV at the beginning of the study.

Experimental design and sample collection

Experiments were performed in the Anses facilities in accordance with the animal welfare experimentation recommendations drawn up by the Directions De ´partementales de la Protection des Populations des Co ˆtes d'Armor (Anses registration number B-22-745-1), under the responsibility of G. Simon (authorization number [START_REF] Marois | Development of a quantitative Real-Time TaqMan PCR assay for determination of the minimal dose of Mycoplasma hyopneumoniae strain 116 required to induce pneumonia in SPF pigs[END_REF][START_REF] Detilleux | Effectiveness analysis of resistance and tolerance to infection[END_REF][START_REF] Madec | Bilan le ´sionnel des poumons de porcs charcutiers a l'abattoir[END_REF][START_REF] Pol | Validation of commercial real-time RT-PCR kits for detection of influenza A viruses in porcine samples and differentiation of pandemic (H1N1) 2009 virus in pigs[END_REF][START_REF] Koong | Effects of plane of nutrition on organ size and fasting heat production in pigs[END_REF]. This animal experiment protocol was approved by the French national ethics committee ComEth Anses/ENVA/ UPEC (approval nu15/02/11-7). Infected and Control pigs were housed in separate rooms. Very strict biosecurity measures, i.e air filtration system and airlocks for each room, room-specific clothes and compulsory showering before and after visiting the pigs were implemented to avoid undesirable contamination of the pigs (BSL3).

Two groups of 8 pigs were intra-tracheally inoculated on two consecutive days with 5 ml of 10 8 Colour Changing Unit (CCU)/ ml of Mhp 116 (at Day (D) 0 and D1) and, 3 weeks later (at D21), with 10 5 Embryonic Lethal Dose (ELD 50 ) of H1N1 in a total of 5 ml (MH1N1 groups). Simultaneously, two control groups of 4 pigs each received intra-tracheally pathogen culture media, i.e. 5 ml of Friis broth medium at D0 and D1, and 5 ml of allantoic fluid at D21 (C groups). All pigs were transferred to individual pens one week before H1N1 infection (D15). Drinking water was provided ad libitum to all groups throughout the study. One infected group and one control group were fed ad libitum (MH1N1-AL and C-AL groups) whereas the other two groups were subjected to a 40% feed restriction (MH1N1-FR and C-FR groups) from D15 until the end of the experiment (Table 1). Pigs were fed a standard starter diet until D14 then a standard growing diet until the end of the experiment. These diets were formulated to cover the nutritional requirements of pigs (see Table S1). The level of 40% feed restriction was determined in a preliminary experiment as the highest level of restriction that did not induce body weight loss (unpublished data).

After an overnight fasting, the pigs were surgically fitted with a jugular catheter at D14 (control groups) or at D16 (infected groups) under general anaesthesia (premedication with ketamine, 400 mg/pig IM, and anaesthesia with propofol, 40 mg/pig IV).

Three days post-infection (DPI) with H1N1 (i.e. at D24), after an overnight fasting, 200 g of feed were given to all animals. The meal size was calculated to ensure that all pigs ate their meal in less than 10 minutes. Blood samples (4 ml) were sampled in heparinized tubes just before the distribution of the test meal, then every 15 min during the first 2 hours and every 30 min during the next 2 hours, for measurement of plasma postprandial glucose, urea and AA concentrations.

Additional blood samples were taken in heparinized tubes 4 days before Mhp inoculation (D0), on D17 (i.e., 4 days before H1N1 inoculation) and on D28 for determination of total IgG levels. All plasmas were separated by centrifugation and stored at 220uC until use.

Pigs were examined daily for clinical signs (rectal temperature, coughing, respiratory rates, and feed consumption) throughout the study and weighed on days 0, 7, 16, 21, 24 and 28. Nasal swabs were taken on days 23, 24 and 28 (i.e., 2, 3 and 7 DPI with H1N1) for determination of viral excretion. After collection, swabs were suspended in 2 ml of Eagle's minimum essential medium (EMEM, LONZA, Levallois-Perret, France) containing penicillin (100 UI/ ml) and streptomycin (100 mg/ml) (Sigma, Saint-Quentin Fallavier, France), vigorously vortexed and then the supernatants were stored at 270uC. One week post-H1N1 infection (on D28), the pigs were euthanized by intravenous injection of sodium pentobarbital followed by exsanguination. Post-mortem examinations were immediately carried out and macroscopic pulmonary lesions were scored visually as previously described [START_REF] Madec | Bilan le ´sionnel des poumons de porcs charcutiers a l'abattoir[END_REF]. A sample of each (apical, cardiac and diaphragmatic) left lobe of the lungs was collected for detection of Mhp and SIV and stored at 270uC until use. A sample of the left diaphragmatic lobe was also fixed in 10% neutral buffered formalin for histopathological examination, as previously described [START_REF] Deblanc | Pre-infection of pigs with Mycoplasma hyopneumoniae modifies outcomes of infection with European swine influenza virus of H1N1, but not H1N2, subtype[END_REF]. The severities of hyperplasia of the bronchial epithelium, hyperplasia of the bronchiolar-associated lymphoid tissue, interstitial pneumonia and bronchiolitis were scored between 0 ( = no lesion) and 4 (lesion very marked) in order to obtain a total score of 16.

Detection and quantification of pathogens

For Mhp detection and quantification, DNA was extracted from 25 mg of lung samples with DNeasy Blood and Tissue kit (Qiagen, Courtaboeuf, France) and analyzed by specific PCR assay as previously described [START_REF] Marois | Development of a quantitative Real-Time TaqMan PCR assay for determination of the minimal dose of Mycoplasma hyopneumoniae strain 116 required to induce pneumonia in SPF pigs[END_REF].

H1N1 virus genome was detected in nasal swab supernatants and lung samples by matrix (M) gene real-time RT-PCR. RNA was extracted from 200 ml of nasal swab supernatants and from 30 mg of lung samples with the RNeasy Mini kit (Qiagen, Courtaboeuf, France) and tested using the TaqVet Influenza A, INFAP-Swine kit (LSI, Lissieux, France) [START_REF] Pol | Validation of commercial real-time RT-PCR kits for detection of influenza A viruses in porcine samples and differentiation of pandemic (H1N1) 2009 virus in pigs[END_REF] according to the manufacturer's instructions. M gene positive nasal swab supernatants were then titrated in MDCK cells as previously described [START_REF] Deblanc | Pre-infection of pigs with Mycoplasma hyopneumoniae modifies outcomes of infection with European swine influenza virus of H1N1, but not H1N2, subtype[END_REF] to determine the H1N1 particle content.

Total IgG assay

Total IgG concentration was determined by quantitative sandwich ELISA, using goat antibodies directed against the swine IgG Fc fragment and a pig immunoglobulin reference serum for the standard curve (A100-104A, A100-104P and RS10-107 respectively, Bethyl, Interchim, Montluc ¸on, France). The colorimetric reaction following the addition of 3,39,5,59-Tetramethylbenzidine (TMB) substrate (Sigma-Aldrich, Saint Quentin Fallavier, France) was halted by adding H 2 SO 4 2.5N and absorbance was read with a microplate reader at a test wavelength of 450 nm.

Plasma postprandial nutrient measurement

Plasma concentrations of glucose, urea and AA were determined from blood collected during the test meal. Concentrations of glucose and urea were determined using the Glucose RTU kit (Biome ´rieux, Marcy l'Etoile, France) and UREA UV 250 kit (Biome ´rieux, Marcy l'Etoile, France), respectively, with a Konelab 20i analyzer (ThermoFisher Scientific). The intra-assay CV was less than 5%. Plasma AA concentrations were determined by an ultra performance liquid chromatography (UPLC) system (Waters Acquity Ultra Performance LC, Waters, Milford, MA, USA) coupled to two detectors (Acquity Tunable UV detector and Mass SQD detector; Waters, Milford, MA, USA) after derivatization of samples using the AccQ?Tag Ultra method (MassTrak AAA; Waters, Milford, MA, USA). Norvaline (Sigma-Aldrich, Saint Quentin Fallavier, France) was used as an internal standard. This method allowed separation and identification of 33 AA in plasma, 21 of which were analysed statistically: 20 are proteinogenic AA (cysteine was not analysed because unstable during sample conservation) plus citrulline because of its involvement in arginine metabolism. The other AA were either detected at very low level in the plasma, or not directly related to dietary AA.

For each individual kinetic of AA, glucose and urea, the area under the curve (AUC) was calculated as the sum of trapeze areas. For that purpose, it is assumed that the shape of nutrient change between two consecutive sampling times was linear and the area of each consecutive trapeze from x-axis was calculated.

Statistical analyses

The clinical data, virus titrations and Mhp DNA quantifications obtained in the different groups were compared by Kruskal-Wallis test using Systat 9 software. Plasma IgG and postprandial nutrient concentrations were subjected to a variance analysis, using the MIXED procedure of SAS (SAS Inst. Inc., Cary, NC). Plasma total IgG concentrations were analyzed using the repeated statement and taking into account the day of sampling, feed restriction (FR), infection (I) and the corresponding interactions. Because of non-normal distribution and non-homogenate variances for glucose, urea and AA plasma concentrations due to the major effect of time, statistical analyses of these data was performed separately at each sampling time and data were logtransformed to be normalized when necessary. For nutrient concentrations and AUC, the model included the effects of feed restriction (FR), infection (I) and the corresponding interactions. For simplification, AA average concentrations measured at basal state, 60 and 240 min after the meal are presented as Supporting information (Tables S2, S3 and S4), whereas AUC values for each AA and the postprandial kinetics of glucose, urea, threonine and arginine were presented and commented in the Result section. The results are expressed as least square means calculated for each time and each experimental treatment.

For all statistical analyses, probabilities less than 0.05 were considered significant. Probability values for the effect of infection and feed restriction comprised between 0.05 and 0.10 were considered as a trend.

Results

Clinical disease

All pigs inoculated with Mhp were coughing 2 weeks later, demonstrating they were infected (data not shown). The week before H1N1inoculation, the average frequencies of coughs per pig per 15 min were similar in MH1N1-FR and MH1N1-AL groups (0.360.3 and 0.660.3, respectively; P = 0.07). At 1 DPI H1N1, they were higher but still similar (1.4 and 1.3, respectively). No coughing was observed in the two control groups.

During the first two weeks of the study, rectal temperatures were normal in all pigs, ranging from 39.2uC to 39.9uC (Figure 1). After feed restriction was applied, they decreased to 38.660.4uC and 39.060.4uC in C-FR and MH1N1-FR groups, respectively, leading to a significant difference (P,0.05) between FR and AL groups during the week before H1N1 inoculation. At 1 DPI H1N1, hyperthermia (temperature .40.0uC) was observed in both co-infected groups. A second peak of hyperthermia appeared in MH1N1-AL group between 3 and 5 DPI H1N1 and temperatures returned to normal at 6 DPI H1N1. In MH1N1-FR group, hyperthermia was of shorter duration, as temperature returned to normal from 2 DPI H1N1, leading to a significant difference between both co-infected groups at 5 DPI H1N1 (P = 0.02). No hyperthermia was observed in C groups and a significant difference due to the feed restriction was maintained between both C groups during the 4 th week of the assay (P,0.05).

The day after H1N1 inoculation, the mean respiratory rate increased similarly in both infected groups. It reached 65623 and 62616 breaths per min in MH1N1-AL and MH1N1-FR groups respectively, while it was maintained to 35 in both control groups.

Viral infection led to a reduction in feed consumption in the MH1N1-AL group. It was strongly marked during the 3 first days, with pigs eating 30 g of feed per kg of body weight on average against 60 g before H1N1 infection (P = 0.001). This resulted in a reduction of weight and mean weight gain (MWG) as compared to other groups (P = 0.006) (Figure 2). In contrast, H1N1 infection had no impact on feed consumption in the MH1N1-FR group in which pigs were eating on average 30 g of feed per kg of body weight both before and after inoculation. In this group, MWG was identical to MWG measured in C-FR.

At necropsy, all co-infected pigs had macroscopic lesions of pneumonia (data not shown). They extended into apical, cardiac and diaphragmatic lobes with no difference in the mean score severity between MH1N1-AL and MH1N1-FR groups (17.463.3 and 16.563.5, respectively). In C-AL and C-FR groups, the scores were 060 and 262.8, respectively. Histological examinations showed that all co-infected animals developed bronchiolitis, broncho-interstitial and interstitial pneumonia (data not shown). The mean microscopic lesion scores were identical for both MH1N1-AL and MH1N1-FR groups (8.861.5 and 7.162.4, respectively). Microscopic lesions were not observed in pigs from the control groups.

Detection and quantification of Mhp and H1N1 virus

Mhp genome was detected in all lung lobes from infected pigs without significant difference in DNA quantities between MH1N1-AL and MH1N1-FR groups (Table 2).

SIV RNA detection in nasal swabs showed that 6/8 pigs in the MH1N1-AL group and 5/8 pigs in the MH1N1-FR group shed H1N1 virus at 2 DPI H1N1, although the isolation and titration of viral particles was successful in only 4/8 and 3/8 animals, respectively (Table 2). At 3 DPI H1N1, all infected pigs were shedding the virus, with similar titers in both groups. At the end of the experiment (7 DPI H1N1), viral genome was detected in 7/8 animals in each infected group, and spread in the upper and lower lobes since the diaphragmatic lobes of 6/8 animals in both coinfected groups were detected positive.

Plasma IgG concentrations

Feed restriction had no effect on variation of the plasma IgG concentrations (P FR and P I6FR .0.1) (Table 3). The concentrations of IgG varied with time in MH1N1 pigs, but not in C pigs (P I6FR ,0.01). Whatever the feeding level, the IgG concentrations in MH1N1 pigs were similar to those in C pigs before and 17 days after Mhp inoculation (P.0.1) but higher at 7 DPI H1N1 (P, 0.001).

Postprandial profiles of plasma glucose, urea and amino acids

At 3 DPI H1N1 and after overnight fasting, 200 g of feed were given to all animals and plasma was regularly sampled during 4 hours. Two and 3 pigs from MH1N1-AL and MH1N1-FR, respectively, were not included because of a non-fonctionnal catheter. Co-infected pigs had lower glycemia at fasted basal state, 15 and 30 min after the meal, then from 105 to 210 min (P I ,0.05) (Figure 3a). The peak of glucose was the highest in C-AL pigs around 30 min (P I6FR = 0.03) whereas at 60 min glycemia was greater in MH1N1-AL pigs than in the three other groups (P I6FR = 0.05). As a consequence, glucose AUC was also lower in MH1N1 pigs (P I = 0.015). Feed restriction did not affect glucose AUC but modified the profile of glycemia that was lower in FR pigs than in AL pigs from 15 to 60 min after the meal (P FR ,0.05).

Plasma urea increased linearly before reaching a plateau value at 150 min (Figure 3b). The effect of feed restriction and the interaction I6FR were not significant at any time. Urea AUC tended to be lower in MH1N1 pigs than in C pigs (P I = 0.06). From 60 to 240 min, plasma urea concentrations were lower (P I , 0.05) or tended (0.05,P I ,0.1) to be lower in MH1N1 than in C pigs.

All AA concentrations increased after the meal, to reach a peak between 30 min and 60 min, and decreased thereafter, as shown for threonine and arginine in Figure 4 (see Tables S2,S3 and S4 for other AA). Both threonine and arginine exhibited a singular response to co-infection. Indeed, their postprandial plasma concentrations were lower in MH1N1 than in control pigs at any time (P I ,0.05) and affected to a greater extent than the other AA.

Average plasma AUC calculated during the 4 hours following the meal test, including basal concentrations, are presented in Table 4. The average AUC of threonine, histidine and asparagine were lower in MH1N1 than in C pigs (P I ,0.05). For AA whose AUC were affected by feeding level only, tryptophan was lower in FR than in AL pigs (P FR = 0.04), whereas leucine, glutamate and ornithine were greater in FR than in AL pigs (P FR ,0.05). Feeding level and infection had cumulative effects on AUC of arginine, citrulline, and tyrosine: their AUC were lower in MH1N1 than in C groups (P I ,0.01). Arginine and citrulline AUC were also higher (P FR ,0.05) in FR than in AL pigs, by contrast to tyrosine AUC that was lower (P FR = 0.03). A significant I6FR interaction (P I6FR ,0.05) was found for a last group of AA corresponding to lysine, alanine, aspartate, glutamate, ornithine and proline. Aspartate, glutamate and ornithine AUC were greater in FR than in AL pigs (P FR ,0.003), but the difference was significant only for Control pigs. Alanine, proline and lysine AUC were not affected by co-infection and feed restriction, but feed restriction increased their AUC in Control pigs but not in MH1N1 pigs.

Discussion

In this study, we evaluated the impact of a feed restriction on the ability of pigs to resist and to tolerate a Mhp/H1N1 coinfection. More specifically, we investigated the changes in glucose, urea and AA metabolism caused by the feed restriction and/or the co-infection.

As expected, following H1N1 inoculation, Mhp pre-infected pigs that were fed ad libitum developed coughing, hyperthermia, increased respiratory rates and a weight loss due to reduced feed consumption. This was in agreement with the symptoms we observed in similar previous studies [START_REF] Deblanc | Pre-infection of pigs with Mycoplasma hyopneumoniae modifies outcomes of infection with European swine influenza virus of H1N1, but not H1N2, subtype[END_REF][START_REF] Deblanc | Preinfection of pigs with Mycoplasma hyopneumoniae induces oxidative stress that influences outcomes of a subsequent infection with a swine influenza virus of H1N1 subtype[END_REF]. In the present experiment, we showed that feed restriction induced a decrease in rectal temperature that could be caused by a decrease in heat production due to feed digestion [START_REF] Koong | Effects of plane of nutrition on organ size and fasting heat production in pigs[END_REF]. Following H1N1 infection, feed restricted pigs, as compared to pigs fed ad libitum, presented a shorter hyperthermia period, did not decrease their feed consumption and did not lose weight. Even if the feed restriction had no impact on pneumonia lesions, on Mhp and H1N1 spreading in lungs and on virus shedding in nasal swabs, these data suggest that a feed restriction would limit the clinical outcomes of flu in Mhp/SIV co-infected pigs and would have a beneficial impact on the zootechnical performance of these animals.

Interestingly, it can be noticed that both the FR and AL groups ate the same quantity of feed (on average 30 g of food per kg of body weight) during the 3 first days following the H1N1 inoculation but the MWG was lower in MH1N1-AL. If a difference in gastrointestinal tract emptying between AL and FR pigs is probably not an explanation, a difference in the mass of visceral tissues could not be ruled out [START_REF] Koong | Effects of plane of nutrition on organ size and fasting heat production in pigs[END_REF]. Otherwise, MH1N1-FR pigs might have developed a lower inflammatory response, as suggested by their shorter hyperthermia. Thus, metabolic disturbances caused by the inflammatory response may have been less pronounced for MH1N1-FR pigs than for MH1N1-AL pigs, which resulted in a higher MWG despite a similar feed intake. Indeed, the analysis of post-prandial nutrient variations suggests a difference in the utilization of glucose and AA between groups. Postprandial nutrient utilization was investigated after a meal standardized for size, duration and composition. Nutrients whose metabolism was altered by co-infection or feed restriction were those whose profile of appearance and disappearance in the plasma after the meal were altered in comparison to control groups. It appeared that postprandial glycemia profiles were greatly influenced by both feed restriction and co-infection. Generally speaking, gluconeogenesis is not a major metabolic pathway in fed growing pigs [START_REF] Nafikov | Carbohydrate and lipid metabolism in farm animals[END_REF] since the feed supplies a large amount of carbohydrate (more than 40%) and few lipids (less than 4%). Consequently, the postprandial glucose profile mainly reflects the fate of dietary glucose. Co-infection elicited a lower postprandial glucose appearance. This clearly reveals that infection leads to changes in glucose metabolism and partitioning between organs. Glucose utilization is enhanced during the inflammatory state to meet the high energy requirements associated with immune activation and inflammatory process. For example, the immune cells require energy for proliferation but the use of glucose as an energy source remains debatable as different types of immune cells could preferentially use glutamine [START_REF] Calder | Fuel utilization by cells of the immune system[END_REF][START_REF] Calder | Glucose metabolism in lymphoid and inflammatory cells and tissues[END_REF][START_REF] Newsholme | Why Is l-Glutamine Metabolism Important to Cells of the Immune System in Health, Postinjury, Surgery or Infection?[END_REF] or glucose [START_REF] Pithon-Curi | Glucose and glutamine utilization by rat lymphocytes, monocytes and neutrophils in culture: a comparative study[END_REF]. Furthermore, in vivo quantitative measurements of glucose fluxes in growing pigs showed that muscle glucose utilization is increased during inflammation, thereby contributing to reduce glycemia [START_REF] Bruins | Aspects of organ protein, amino acid and glucose metabolism in a porcine model of hypermetabolic sepsis[END_REF]. In FR pigs, the glycemia profile indicated that little glucose appeared in the blood and/or that glucose was rapidly removed from the blood. The lower postprandial glucose concentrations during the first hour following the meal may be related to an increased use of glucose for reconstitution of the glycogen pool in both the liver and the muscle [START_REF] Noah | Bioavailability of starch and postprandial changes in splanchnic glucose metabolism in pigs[END_REF].

Plasma urea is produced by the liver from the catabolism of AA derived from the absorption of dietary protein as well as the catabolism of body proteins. In our study, plasma urea concentrations increased during the first 4 postprandial hours as previously reported [START_REF] Zervas | Effects of dietary protein and oathull fiber on nitrogen excretion patterns and postprandial plasma urea profiles in grower pigs[END_REF], due to the catabolism of dietary amino acids. The lower plasma urea concentration reported in coinfected pigs was unexpected because inflammation is known to enhance muscle protein catabolism and urea excretion [START_REF] Jahoor | Chronic protein undernutrition and an acute inflammatory stimulus elicit different protein kinetic responses in plasma but not in muscle of piglets[END_REF]. The spontaneous reduced feed intake observed during the first 3 days after the inoculation of H1N1 virus in AL pigs could have prevented the increase in urea synthesis that is mainly influenced by protein intake [START_REF] Zervas | Effects of dietary protein and oathull fiber on nitrogen excretion patterns and postprandial plasma urea profiles in grower pigs[END_REF]. Alternatively, AA may have been metabolized by the liver for the synthesis of acute phase proteins [START_REF] Reeds | Do the differences between the amino acid compositions of acute-phase and muscle proteins have a bearing on nitrogen loss in traumatic states?[END_REF], thus preserving amino acids from catabolism. In the present study, inflammation was confirmed in MH1N1-FR and MH1N1-AL groups by measuring an increase in plasma haptoglobin concentrations after H1N1 inoculation, with same levels in both infected groups (data not shown), as similarly observed in a previous study [START_REF] Deblanc | Preinfection of pigs with Mycoplasma hyopneumoniae induces oxidative stress that influences outcomes of a subsequent infection with a swine influenza virus of H1N1 subtype[END_REF].

Not all AA profiles were affected by co-infection suggesting a specific impact of infection on some AA rather than a global effect on protein metabolism. As in co-infected pigs in our study, lower tyrosine plasma concentrations were reported in pigs suffering from clinical dysentery and could be related to the incorporation of this AA into acute phase proteins [START_REF] Jonasson | Blood concentrations of amino acids, glucose and lactate during experimental swine dysentery[END_REF]. Among essential AA, plasma concentrations of histidine, arginine and threonine were also reduced in co-infected pigs. Lower histidine concentrations were reported in weaned pigs infected with Escherichia coli but this effect was nonspecific since many other AA showed a similar response [START_REF] Yoo | Glutamine supplementation maintains intramuscular glutamine concentrations and normalizes lymphocyte function in infected early weaned pigs[END_REF]. The involvement of arginine in the immune and inflammatory processes is fully documented [START_REF] Floc | Modifications of protein and amino acid metabolism during inflammation and immune system activation[END_REF]. Arginine is Table 3. Plasma total IgG concentrations (g/L) during the time course of the experiment in control (C) and co-infected (MH1N1) pigs. converted into nitric oxide (NO) by the action of inducible NO synthase (iNOS), of which the activity is enhanced by influenza virus infection [START_REF] Perrone | Inducible nitric oxide contributes to viral pathogenesis following highly pathogenic influenza virus infection in mice[END_REF]. Some strains of Mycoplasma are also known to activate iNOS [START_REF] Kagemann | Impact of Mycoplasma hyorhinis infection on L-arginine metabolism: differential regulation of the human and murine iNOS gene[END_REF]. Moreover, cells infected with influenza virus use arginine to synthesize an arginine rich component [START_REF] Becht | Cytoplasmic synthesis of an arginine-rich nuclear component during infection with an influenza virus[END_REF]. Arginine, with glutamate, is also the precursor of citrulline synthesis in the gut [START_REF] Kaore | Citrulline: pharmacological perspectives and its role as an emerging biomarker in future[END_REF]. Lower arginine concentrations may also limit the synthesis of citrulline, for which the concentrations were reduced in MH1N1 compared to C pigs, contributing to glutamate accumulation in the plasma. The plasma arginine profile, like that of ornithine and citrulline, was affected by feed restriction. The metabolism of these three AA is strongly interconnected in the urea cycle that takes place in the liver, since both ornithine and citrulline are synthesized from arginine. Their greater postprandial concentrations in FR pigs could be related to urea synthesis, although the lack of effect of feed restriction on the urea profile did not corroborate this hypothesis. Threonine is abundant in glycoproteins such as intestinal and tracheal mucins [START_REF] Lien | Mucin output in ileal digesta of pigs fed a protein-free diet[END_REF][START_REF] Lloyd | Regulation of the synthesis of mucin glycoproteins in swine trachea explants[END_REF] and immunoglobulins [START_REF] Cuaron | Effect of lysine and threonine supplementation of sorghum gestation diets on nitrogen balance and plasma constituents in first-litter gilts[END_REF]. In healthy pigs, the intestinal tissues extract dietary threonine in great amounts [START_REF] Floc | Catabolism through the threonine dehydrogenase pathway does not account for the high first-pass extraction rate of dietary threonine by the portal drained viscera in pigs[END_REF] and gut inflammation increases intestinal threonine uptake and mucin synthesis [START_REF] Remond | Intestinal inflammation increases gastrointestinal threonine uptake and mucin synthesis in enterally fed minipigs[END_REF] thereby reducing threonine availability for the other tissues. In our experiment, it seems unlikely that the reduced appearance of threonine observed in the plasma during the postprandial period was due to an increased use by the gastrointestinal tract. Indeed, Mycoplasma and H1N1 target the respiratory rather than the gastrointestinal tract [START_REF] Opriessnig | Polymicrobial respiratory disease in pigs[END_REF][START_REF] Kuntz-Simon | Genetic and antigenic evolution of swine influenza viruses in Europe and evaluation of their zoonotic potential[END_REF]. Both pathogens enhance mucin secretion in the respiratory tract [START_REF] Mata | N-acetyl-L-cysteine (NAC) inhibit mucin synthesis and pro-inflammatory mediators in alveolar type II epithelial cells infected with influenza virus A and B and with respiratory syncytial virus (RSV)[END_REF][START_REF] Kim | Expression of secreted and membrane-bound mucins in the airways of piglets experimentally infected with Mycoplasma hyopneumoniae[END_REF], but no evidence that threonine is also abundant in mucins of the porcine respiratory tract has been reported yet. Finally, threonine could be used for the increased production of total immunoglobulins that was demonstrated in co-infected groups. In growing pigs, the adequate threonine dietary supply required for production of immunoglobulins may be higher than that required for growth [START_REF] Defa | Effects of dietary threonine on performance, plasma parameters and immune function of growing pigs[END_REF].

Interestingly, co-infection differently affected lysine, alanine, aspartate, glutamate, proline and ornithine if the pigs were fed ad libitum or feed restricted. In our experiment, both feed restriction and co-infection impaired growth rate and would help to explain the accumulation in the plasma of lysine, an essential AA mainly used for protein synthesis. In co-infected pigs, the absence of a cumulative effect of feed restriction on the plasma concentrations of those AA may be explained by the greater mean daily gain after H1N1 inoculation, and thus by the greater protein deposition, of MH1N1-FR pigs compared with MH1N1-AL pigs. Alternatively, these AA may have been used in metabolic pathways related to immune response and contributing to energy supply for alanine [START_REF] Imamura | Liver metabolism and glucogenesis in trauma and sepsis[END_REF], to nitrogen recycling and removal for glutamate, aspartate, proline and ornithine and to the synthesis of immune proteins for lysine [START_REF] Klasing | The care and feeding of an immune system: an analysis of lysine needs[END_REF]. Whether or not this could have contributed to the positive effect of feed restriction on the growth of infected pigs remains speculative.

In conclusion, our study showed a beneficial, even if limited, effect of feed restriction on the outcomes of H1N1 infection in pigs pre-infected with Mhp. We showed, for the first time, that both coinfection and feed restriction modified the postprandial kinetics of glucose and AA concentrations, revealing major changes in nutrient metabolism. Further studies will be necessary to investigate the role of AA such as threonine and arginine during co-infection and to find out if a supplemented feed would improve the resistance and the tolerance of the pig to the disease. For instance, the correlation between the magnitude of clinical responses and metabolic responses deserve to be studied on larger experimental groups. Nevertheless, these first results already indicate that feeding practices could provide a means to prepare animals to overcome an influenza infection, especially in the context of the Porcine Respiratory Disease Complex.

Figure 1 .

 1 Figure 1. Mean rectal temperatures (6C) of pigs. Mean rectal temperatures in co-infected (MH1N1) and control (C) pigs fed ad libitum (AL) or feed restricted (FR) : C-AL (black dashed line), C-FR (gray dashed line), MH1N1-AL (black solid line) and MH1N1-FR groups (gray solid line), from the day of Mhp inoculation (day 0) until the end of the experiment. Arrows indicate the day of the beginning of the feed restriction (day 15) and the day of H1N1 inoculation (day 21). doi:10.1371/journal.pone.0104605.g001

Figure 2 .a

 2 Figure 2. Mean daily weight gain (kg) of pigs after Mhp and H1N1 inoculations. Mean daily weight gains between Day (D) 14 and D21 (the week before H1N1 infection), between D21 and D24 (between 0 and 3 DPI H1N1), between D21 and D28 (between 0 and 7 DPI H1N1) and between D14 and D28, for control (C) or co-infected (MH1N1) groups fed ad libitum (AL) or feed restricted (FR): C-AL (hatched black), C-FR (hatched gray), MH1N1-AL (black) and MH1N1-FR (gray) groups. Mean daily weight gains with different letters are significantly different with P,0.05. doi:10.1371/journal.pone.0104605.g002

  Standard errors of the mean (SEM) of control (C) and co-infected (MH1N1) conditions are presented with ad libitum (AL) and feed restricted (FR) pigs pooled together (C-AL+C-FR vs. MH1N1-AL+MH1N1-FR) because the effect of the feeding level (-AL or -FR) and its interactions with the effects of infection (I) and time (T) were not significant. a,b : concentrations with different letters are significantly different with P,0.001. doi:10.1371/journal.pone.0104605.t003

Figure 3 .

 3 Figure 3. Postprandial kinetics of plasma glucose (3a) and urea (3b) concentrations (mg/L) measured in pigs. Average plasma concentrations were measured after an overnight fasting and then during the 4 hours following the ingestion of 200 g of feed, 3 days after H1N1 or allantoı ¨c fluid administration in co-infected (MH1N1) and control (C) pigs fed ad libitum (AL) or feed restricted (FR): C-AL (black dashed line), C-FR (gray dashed line), MH1N1-AL (black solid line) and MH1N1-FR groups (gray solid line). Values are means +/2 SD. P values for glucose: P I ,0.05 at fasted basal state, 15 and 30 min after the meal, then from 105 to 210 min; P FR ,0.05 from 15 to 60 min after the meal. P values for urea: P I ,0.01 from 60 to 240 min. doi:10.1371/journal.pone.0104605.g003

Figure 4 .

 4 Figure 4. Postprandial kinetics of plasma threonine (3a) and arginine (3b) concentrations (mM) measured in pigs. Average plasma concentrations were measured after an overnight fasting and then during the 4 hours following the ingestion of 200 g of feed, 3 days after H1N1 or allantoı ¨c fluid administration in co-infected (MH1N1) and control (C) pigs fed ad libitum (AL) or feed restricted (FR): C-AL (black dashed line), C-FR (gray dashed line), MH1N1-AL (black solid line) and MH1N1-FR groups (gray solid line). Values are means +/2 SD. P values for threonine: P I ,0.05 from basal to 240 min; P FR ,0.05 from 180 to 240 min; P I6FR ,0.05 from 180 to 240 min. P values for arginine: P I ,0.01 from basal to 240 min; P FR ,0.05 from 150 to 240 min; P I6FR ,0.05 from 180 to 240 min. doi:10.1371/journal.pone.0104605.g004

Table 1 .

 1 Experimental design.

	Groups	Nb. of pigs	Intra-tracheal inoculations		Feed from Day 15 to Day 28
			Day 0+Day 1	Day 21 (0 DPI H1N1)
	MH1N1-AL	8	Mycoplasma hyopneumoniae	H1N1	ad libitum
	C-AL	4	Friis broth medium	allantoı ¨c fluid	ad libitum
	MH1N1-FR	8	Mycoplasma hyopneumoniae	H1N1	40% feed restriction
	C-FR	4	Friis broth medium	allantoı ¨c fluid	40% feed restriction
	doi:10.1371/journal.pone.0104605.t001

PLOS ONE | www.plosone.org

August 2014 | Volume 9 | Issue 8 | e104605

Acknowledgments

The authors thank colleagues from Anses Ploufragan/Plouzane ´laboratory for helpful contributions: Andre ´Ke ´ranflec'h, Jean-Marie Guionnet and Yann Bailly for animal care and sampling; Ste ´phane Que ´guiner, Ste ´phane Gorin and Nicolas Barbier for technical help in the virology laboratory and tissue sampling at necropsy; Se ´verine Ferre ´for preparing the Mhp inoculum and performing Mhp quantification; Ste ´phanie Bougeard for her advice in statistical analysis. They also thank colleagues from INRA: Francis Le Gouevec and Alain Chauvin for the surgical procedure, Marion Girard, Nadine Me ´zie `re, Yolande Jaguelin, Franc ¸oise Thomas, Michel Lefe `bvre and Jean-Noe ¨l Thibault for help during blood sampling and for laboratory analyses. They are indebted to Jean-Noe ¨l Sialelli and Fabien Vautrin, FARM'APRO, France, for helpful discussions.

This work received financial support from FARM'APRO. FARM'APRO participated in the study design but had no role in the data collection and analysis, decision to publish, or preparation of the manuscript. Remuneration of the Anses' personnel from the unit ''Production of decontaminated pigs and testing'' was partially supported by the Conseil Ge ´ne ´ral des Co ˆtes d'Armor. This funder had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.

Competing Interests: The authors received funding from a commercial source (FARM'APRO) but this does not alter their adherence to PLOS ONE policies on sharing data and materials.

Supporting Information


Marion Tetegan

Anne C Richer-De-Forges

Bernard Verbeque

Bernard B Nicoullaud

Caroline Desbourdes

Alain Bouthier

Dominique D Arrouays

Isabelle I Cousin
email: isabelle.cousin@orleans.inra.fr

A C Richer De Forges

The effect of soil stoniness on the estimation of water retention properties of soils: A case study from central France

Keywords: available water content, water deficit, pedotransfer functions, soil thickness, soil stoniness

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

Introduction

To estimate the ability of soils to provide water to plants for their growth, soil scientists and agronomists determine the available water content (AWC) of the soil, which is likely to be exploited by the roots. The AWC is thus an important agronomic parameter for calculating soil water balance and for managing the irrigation of cultivated soils. Therefore, there is a need to explain the soil hydric characteristics to optimize crop growth and to reduce water transfer and losses deeper than the root system. As a consequence, mapping the available water content of soils at a regional scale is a key factor for evaluating land suitability and for developing models to forecast the requirements for irrigation needs and the risks to groundwater quality [START_REF] Paydar | A methodology for up-scaling irrigation losses[END_REF]; this mapping is especially important in the context of the climatic change that may alter water reservoir inputs and management in ways that reduce the water supply to irrigated areas [START_REF] Garcia-Ruiz | Mediterranean water resources in a global change scenario[END_REF]. As stated by [START_REF] Jensen | Deficit irrigation based on drought tolerance and root signalling in potatoes and tomatoes[END_REF], new strategies for conserving irrigation water are needed.

Nevertheless, the estimation of the soil available water content remains difficult for heterogeneous soils [START_REF] Frison | Soil hydraulic properties in relation to local rapid soil changes induced by field drainage: a case study[END_REF], especially stony soils, because it requires a precise knowledge of the water retention capacities of each of the main soil phases: the fine phase composed of soil particles smaller than 2 mm in diameter and the stony phase composed of rock fragments larger than 2 mm in diameter [START_REF] Corti | Classing the soil skeleton (greater than two millimeters): Proposed approach and procedure[END_REF][START_REF] Tetegan | The contribution of rock fragments to the available water content of stony soils: proposition of new pedotransfer functions[END_REF][START_REF] Wang | Modeling the Soil Water Retention Curves of Soil-Gravel Mixtures with Regression Method on the Loess Plateau of China[END_REF]. The calculation of the available water content of the fine phase can be performed through various pedotransfer classes (tables) and pedotransfer functions based on easily accessible soil characteristics such as the bulk density, carbon content and texture of fine earth (Al Majou et al., 2007, 2008a, 2008b;Bastet et al., 1999;[START_REF] Wösten | Pedotransfer functions: bridging the gap between available basic soil data and missing soil hydraulic characteristics[END_REF]. Pedotransfer classes are tables allowing to obtain the water content of the fine earth thanks to its texture, or by combining its texture and its structure (Al Majou et al., 2005;[START_REF] Bruand | Estimation des propriétés de rétention en eau des sols: utilisation de classes de pédotransfert après stratifications texturale et texturostructurale[END_REF]. The water retention properties of the stony phase have often been ignored when calculating the available water content of stony soils, although several studies have shown that rock fragments could be a significant water reserve for soil [START_REF] Gras | Contribution de certains éléments grossiers à l'alimentation en eau des végétaux[END_REF][START_REF] Poesen | Rock fragments in top soils: significance and processes[END_REF]. [START_REF] Tetegan | The contribution of rock fragments to the available water content of stony soils: proposition of new pedotransfer functions[END_REF] have also recently proposed new pedotransfer functions and pedotransfer classes to estimate the available water content of rock fragments in soils originating from sedimentary rocks. However, to our present knowledge, no attempt to map and globally assess the available water content of soils at a regional scale has been conducted using these new pedotransfer functions.

The objective of this paper was to use these new pedotransfer functions for estimating the contribution of rock fragments to the available water content of stony soils to i) produce a more accurate map of the available soil water content at the regional scale and, ii) assess if regional estimates of the total available soil water content using the knowledge of this contribution of rock fragments lead to substantial differences from previous approaches. Conducted on several soils of variable stoniness, for each soil type, the available water content was calculated both when considering and when disregarding the volume and the water retention properties of the rock fragments.

Materials and methods

Study area

The study was conducted on approximately 36,200 ha located 110 km southwest of Paris in the Patay area within "Petite Beauce". Intensive agriculture in the Petite Beauce often utilizes irrigation and 76 % of the surface area is occupied by cereals crops, mainly maize and wheat [START_REF] Nicoullaud | Modélisation spatiale à l'échelle parcellaire des effets de la variabilité des sols et des pratiques culturales sur la pollution nitrique agricole[END_REF]. The climate is temperate continental with an oceanic influence and is characterized by an evapotranspiration of approximately 780 mm calculated using the Penman-Monteith formula. The average annual temperature was 11°C from 1971 to 2000, and the average annual rainfall was 635 mm and ranged from 415 to 850 mm over the [START_REF] Macaire | Etude sédimentologique des formations superficielles sur le tracé de l'autoroute A10 entre Artenay et Meung sur Loire[END_REF]-2000period (Richer de Forges and Verbèque, 2003).

The Patay soil map and the soil database

A soil map (scale: 1/50,000) was designed by Richer de Forges and Verbèque ( 2003) with a mean density of observations equal to 1 auger-hole per 17 hectares. Quantitative data used to create the soil map (granulometry, carbon content, pH, etc…) were measured on approximately 100 soil profiles, using standard analyses, and stored in an associated database. Upper horizons were affected by peri-glacial winds that largely redistributed the fine material [START_REF] Macaire | Etude sédimentologique des formations superficielles sur le tracé de l'autoroute A10 entre Artenay et Meung sur Loire[END_REF][START_REF] Bourennane | Improving the kriging of a soil variable using slope gradient as external drift[END_REF], resulting in a rather homogeneous particle-size distribution of the fine earth (i.e., values of silt, clay, sand, etc...). Most of the soils consisted then of a silty-clay layer (approximately 60% silt and 30% clay) developed on a lacustrine limestone substrate, which was locally cryoturbated. [START_REF] King | Relationship of the presence of a non-calcareous clay-loam horizon to DEM attributes in a gently sloping area[END_REF] and [START_REF] Bourennane | Three dimensional analysis of soils and surface materials by electrical resistivity survey[END_REF] indicated that the soil units that formed on cryoturbated limestone deposits or on soft limestone deposits had the deepest silty-clay layer (up to 0.8 m deep), whereas the shallowest soils (approximately 0.3 m deep) were developed directly on hard calcareous bedrock. This latter bedrock is cracked [START_REF] Lorain | La géologie du calcaire de Beauce[END_REF], which could allow a root proliferation of cultures. The study area was classified into 23 main map units (MUs) according to i) the spatial variability of the soil characteristics, ii) the depth and type of limestone where the soil horizons have been developed, and iii) the thickness of the silty-clay layers. Non-stony soils were mainly arenosols, cambisols, fluvisols, luvisols, haplic planosols and vertisols (IUSS Working Group WRB, 2006). Stony soils were mainly rendzic leptosols, haplic calcisols and calcaric cambisols, with various quantities of rock fragments of different sizes, from gravels to blocks (IUSS Working Group WRB, 2006).

The MUs of the soil map consisted of one or several soil units corresponding to a synthetic soil profile defined in terms of soil and agronomic characteristics observed in the field [START_REF] Legros | Mapping of the soil[END_REF]. The information on the soil units was stored in the "DoneSol" soil database [START_REF] Gaultier | L'organisation et la gestion des données pédologiques spatialisées : le projet DONESOL[END_REF][START_REF] Grolleau | Le système d'information national sur les sols : DONESOL et les outils associés[END_REF]. From this database, we used the following soil characteristics to estimate the available water content of both the stony and non-stony soils: -the volume proportion and the texture of the fine phase, -the volume proportion, the lithological class (limestone, quartz, or flint, on the studied area) and the bulk density of the whole stony phase (gravels, pebbles, stones and blocks), -the thickness of the horizons -the nature of the underlying parent material -the surface proportion of each soil unit in each of the 23 MUs of the map of Patay.

To be comparable with the pedotransfer functions developed by [START_REF] Tetegan | The contribution of rock fragments to the available water content of stony soils: proposition of new pedotransfer functions[END_REF] for the estimation of the contribution of rock fragments to the available water content, the bulk density of the rock fragments located within the soil profile was used, rather than the bulk density of the bedrock, the latter that could be slightly different. We define the available water content as the quantity of water that can be used by plants for their growth, and we assume that it is equal to the difference between the water content at field capacity, and the water content at permanent wilting point, with the units cm 3 .cm -3 , as calculated in equation (1). Nevertheless, for practical reasons, it is also expressed as a depth (expressed in mm): it represents the available water within a soil horizon (see equation ( 2)) or within a soil profile (see equation ( 3)) and can be directly used in a water balance model.

Method used to calculate the available water content

The available water content for both the fine phase and the stony phase (AWCi) was calculated using equation ( 1): AWCi = θfc,iθwp,i

(1) where AWCi represents the available water content of the phase i in cm.cm -3 , θfc,i represents the volumetric soil water content at field capacity for phase i in cm.cm -3 , and θwp,i is the volumetric soil water content at the permanent wilting point for phase i in cm.cm -3 . For both the fine phase and the stony phase, the volumetric soil water content at field capacity was set equal to the soil water content at a matric potential of -100 hPa, as suggested by [START_REF] Bruand | Estimation des propriétés de rétention en eau des sols à partir de la base de données SOLHYDRO: Une première proposition combinant le type d'horizon, sa texture et sa densité apparente[END_REF]) and Al Majou et al. (2008a, 2008b) for the fine earth, and by [START_REF] Tetegan | The contribution of rock fragments to the available water content of stony soils: proposition of new pedotransfer functions[END_REF] for the stony phase. For both the fine and the stony phase, the volumetric soil water content at permanent wilting point was set equal to the soil water content at a matric potential of -15850 hPa, as typically required. For the fine phase, the volume soil water contents at -100 hPa and -15850 hPa were calculated from the pedotransfer classes of [START_REF] Bruand | Estimation des propriétés de rétention en eau des sols à partir de la base de données SOLHYDRO: Une première proposition combinant le type d'horizon, sa texture et sa densité apparente[END_REF]. These authors used the soil texture (Baize et al., 2009) from the French texture class triangle of [START_REF] Jamagne | The effect of soil stoniness on the estimation of water retention properties of soils: A case study from central France[END_REF] and separated topsoil and subsoil horizons in the soil profile. For our study, the dominant texture of the fine phase for each horizon of each soil unit was collected from the soil database and the position of each horizon in the soil profile was simplified by distinguishing only between topsoil and subsoil horizons. Table 1 presents the AWCi for the fine phase. For the stony phase, the volumetric soil water contents at -100 hPa and -15850 hPa were calculated using the pedotransfer classes proposed by [START_REF] Tetegan | The contribution of rock fragments to the available water content of stony soils: proposition of new pedotransfer functions[END_REF], who used informations only on the lithological class of the rock fragments and the bulk density of each rock fragment. In our study, the dominant lithological class of the rock fragments was collected from the soil database for each soil horizon. The bulk density of the rock fragments of each lithological class was taken from [START_REF] Tetegan | The contribution of rock fragments to the available water content of stony soils: proposition of new pedotransfer functions[END_REF]. The AWCi for the stony phase is presented in Table 2.

From the AWCi of both the fine and the stony phases given by Eq. ( 1), the available water capacity of a horizon n ( n h AWC ) was calculated in millimetres by using the equation Eq.

(2) proposed by [START_REF] Cousin | Influence of rock fragments on the water retention and water percolation in a calcareous soil[END_REF]:

n h n i i i i n h T P AWC AWC ×         × ∑ = = = 2 1 (2)
where n i P -collected from the soil database -represents the volume proportion of phase i (fine phase or stony phase) in horizon n and n h T represents the thickness in millimetres of the horizon n. 4). In the equation Eq. ( 3) N represents the number of horizons of the soil unit m. In the equation Eq. ( 4) N is the number of soil units composing the map unit t and m SU S is the surface proportion of the soil unit m in the map unit t.

∑ = = N N n h m SU AWC AWC 1 (3) ) ( 1 m SU N N m SU t MU S AWC AWC × ∑ = = (4)

Choice of a value of soil depth to calculate the AWC

A fundamental parameter in the estimation of the available water content is the soil depth. In our study, we calculated two values of the AWC to accommodate two definitions of the soil depth:

-For a soil scientist, the soil depth is the thickness of all the soil layers from the surface to the bedrock. The depth of the bedrock, designated here as the "real soil depth" was known from the soil database. The available water content map calculated using the real soil depth ( soil AWC ) can be used to generate a generic map that is useful for many environmental applications.

-For an agronomist, the soil depth corresponds to the rooting depth, which varies considerably with the plant type. In our study, we will focus our calculations on the maize crop because i) it is known for its high water requirement (to reach a maximum production, maize needs from 500 to 800 mm of water, depending on the climate) [START_REF] Doorenbos | Yield response to water[END_REF] and ii) it is cultivated over much of the Patay area and typically requires irrigation. The average rooting depth of maize is equal to 120 cm (B.S.I. [START_REF] Standards | Draft code of practice for the planning design and installation of irrigation schemes in the UK. Part 2. Irrigation water requirements[END_REF]. Therefore, knowing that: i) at the study area, the shallow stony soils are draining soils lying on a cracked calcareous bedrock [START_REF] Lorain | La géologie du calcaire de Beauce[END_REF]) enabling a storage of the drained water and giving enough spaces between cracks for root proliferation, ii) calcareous bedrock can provide nutrients to plants (Abou-el-Seoud and Abdel-Megeed, 2012; Korboulewsky et al., 2010a;[START_REF] Korboulewsky | Contribution of rock fragments to soil fertility[END_REF], iii) maize roots have a remarkable plasticity by increasing their root length, allowing to capture nutrient resources [START_REF] Li | Contribution of root proliferation in nutrient-rich soil patches to nutrient uptake and growth of maize[END_REF], a soil depth of 120 cm was used to calculate the AWC in this second case ( maize AWC ), regardless of the soil unit and the "real soil depth".

Depending on their locations, soil units exhibited different real soil depths, from a few centimeters to more than one meter (Fig. 1). For the soil AWC and maize AWC calculations, soils thicker than 120 cm were truncated to 120 cm. This truncation ranged from 2 to 31 cm.

For the maize AWC calculations, soils less than 120 cm thick were extended to 120 cm. This extension for soils shallower than 120 cm was mainly necessary for soils that were developed on limestone (cracked calcareous bedrock). This extension ranged from 1 to 109 cm with a median value of 38 cm. From the depth observed in the field to the depth of 120 cm, we accounted for the rocky parent material and hypothesized that its hydric properties were close to those of the rock fragments in the soils of Patay. This hypothesis was supported by experimental measurements of the water content in soil rock fragments at different depths in the soil profile and at different degrees of alteration [START_REF] Tetegan | The contribution of rock fragments to the available water content of stony soils: proposition of new pedotransfer functions[END_REF].

Evaluation of the role of rock fragments in the calculation of the available water content

The available water contents estimated for two soil depths ( soil AWC and maize AWC ) were calculated according to three scenarios.

• Scenario 1-Under this scenario, the stony soils were considered to contain active rock fragments; we accounted for both the volume proportion of the rock fragments and their hydric properties, which were estimated with the pedotransfer classes proposed by [START_REF] Tetegan | The contribution of rock fragments to the available water content of stony soils: proposition of new pedotransfer functions[END_REF]. This scenario is considered the reference scenario, where soil contains porous rock fragments that may store water. • Scenario 2-All soils were considered to be 100 % fine earth; the volume of the rock fragments in each soil horizon was replaced by an equivalent volume of fine earth. This scenario is similar to actual scenarios when the available water content is calculated over large scales, for example to create regional maps. In this situation, both the quantity and properties of rock fragments are neglected, and the proportion of rock fragments is replaced by an equivalent quantity of fine earth. • Scenario 3-The stony soils contained inert rock fragments; we accounted for the volume of the rock fragments but assumed that they could not to store water. This scenario is an intermediate scenario between scenarios 1 and 2; the proportion of rock fragments is high enough that it is usually not neglected in available water content calculations. Nevertheless, their hydric properties, especially their contribution to the available water content, are unknown. In this situation, the proportion of rock fragments is not neglected, but the rock fragments are replaced by nothing (not by the fine earth).

Scenarios 2 and 3 were each compared with the reference scenario: for each map unit, we have computed the difference between the reference scenario and another scenario with the following equation:

jref j AWC AWC AWC - = ∆
where AWCj represents the available water content calculated using scenario 2 or scenario 3 and AWCjref represents the available water content calculated using scenario 1. Negative To evaluate the global differences between these scenarios at the regional scale, we have also computed the Mean Error (ME), the Mean Absolute Error (MAE) and the Root Mean Square Error (RMSE), using the following equations:

∑ - = = N j jref j AWC AWC AWC N ME 1 ) ( 1 (6) ( ) ∑ - = = N j jref j AWC AWC AWC N MAE 1 1 (7)         ∑ - = = N j jref j AWC AWC AWC N RMSE 1 2 ) ( 1 , ( 8
)
where AWCj represents the available water content calculated using scenario 2 or scenario 3 and AWCjref represents the available water content calculated using scenario 1.

Exclusively related to the difference between scenarii on the AWC-calculation and not including the errors linked to parameters provided by "DoneSol" soil database, these ME, MAE and RMSE give a theoretical estimate of the quantitative relevance of the contribution of the hydric properties of the stony phase in those of stony soils.

The maps showing the available water content maps were designed with the ArcGis9 ® software.

Results and discussion

Soil stoniness and soil thickness

The real soil thickness in the study area varied from 11 cm to 151 cm (Fig. 1). The shallowest soils (thickness less than 50 cm, typically Rendzic leptosol) occupied 17% of the surface of the map. The largest area of the soil map (81%) was represented by soils with a thickness between 50 and 150 cm. The deepest soils (thickness greater than 150 cm, typically Haplic luvisol) represented only 2% of the surface of the map and were mainly situated in a band oriented from southwest to northeast (Fig. 1).

The soil stoniness was highly variable in space (Fig. 2a and2b). The rock fragments were mainly limestone (78 %), quartz (13%) and flint (9%) (Fig. 2a). The southeast part of the map was occupied by soils containing the smallest percentage of rock fragments (less than 10%), and was surrounded by rockier soils. Sixty-seven percent of the surface was covered by soils with a mean stoniness less than or equal to 10%, and 31% was covered by soils with mean stoniness values between 10% and 30% (Fig. 2b). Only 2% of the soil surface of the Patay area demonstrated stoniness above 30%.

The soil stoniness and the thickness were weakly correlated (R = 0.34) (Fig. 3). The thickest soils (thicker than 100 cm) were not systematically located in areas with the lowest proportion of rock fragments. However, as expected, the shallowest soils (thickness less than or equal to 30 cm) were located in areas with high rock fragments proportions, i.e., between 10% and 20% of the soil volume (Figs. 1 and2). The southwest to northeast band of the map was an area where the soil stoniness and thickness were approximately 20 -30 % and 100 -140 cm respectively.

After substituting the maize rooting depth for the real soil depth, we recalculated the stoniness values for all the MUs (Fig. 2c). In this case, 98 % of the surface area of the Patay map was represented by soils containing rock fragments. Over 50 % of the surface contained ) and the three scenarios defining the degree to which soil stoniness was considered in the calculations. For the real soil depth neither the range of the AWCsoil values nor the mean or median AWCsoil values varied significantly with the scenario (Table 3). As expected, however, the lowest values were recorded for scenario 3, which ignored the water retention properties of the stony phase. The lowest median value (scenario 3) was equal to 105 mm of water, which corresponded to 1.12 mm of water by cm of soil (Table 3). This value is close to 1 mm/cm, the AWC value usually used for loamy soils developed on loess [START_REF] Jamagne | The effect of soil stoniness on the estimation of water retention properties of soils: A case study from central France[END_REF]. The highest median soil AWC value was recorded for scenario 2: it was equal to 109 mm of water which corresponded to 1.20 mm of water by cm of soil.

Estimation of the soil available water content for the 3 scenarios

When the maize rooting depth was used as the soil depth to calculate the available water content ( maize AWC

), the range of maize AWC values and the mean and median maize AWC differed with the scenario (Table 3). The highest mean value of the

Spatial variability of the available water content calculated for the two depths and the 3 scenarios

The spatial variability of the available water content on the Patay map is presented in Figure 4. For the reference scenario (scenario 1), whatever the depth of calculation, the available water content ranged mainly from 50 to 150 mm for more than 70 % of the map surface (Figs. 4a and4a'). The main spatial feature of the maize AWC map was its relative homogeneity of the map, whereas the soil AWC map was more heterogeneous. For the soil AWC map, the minimum values -lower than 50 mm -were located in the south-central portion of the area, whereas the maximum values -larger than 150 mm -were rare (Fig. 4a). For scenarios 2 and 3, the minimum values of soil AWC and maize AWC -lower than 50 mm -were also in the south-central area. Regardless of the location, the available water content calculated when the rock fragments were considered to be inert (scenario 3) was systematically less than or equal to the available water content calculated when the soil was considered to contain fine earth only (scenario 2) (Figs. 4b, 4c, 4b'and4c').

Mapping differences in available water content estimates

The differences in available water content between scenarios 2 or 3 and the reference scenario are presented in figure 5. The soil AWC was systematically overestimated using scenario 2 compared with scenario 1 (Fig. 5a). By contrast, scenario 2 either underestimated or overestimated maize AWC compared with scenario 1 (Fig. 5a'). The overestimation, which covered 33 % of the surface area, was usually located in areas with a low stoniness, for example in non-stony soils of the southeast part of the map or in deep soils with a low stoniness, as in the southwest-northeast band. Nevertheless, the maize AWC was overestimated in some highly stony areas, presumably when rock fragments could store only a small quantity of water, less than that stored in the fine earth (see in the central part of the map on figure 5a'). Figure 5a' also shows that the maize AWC was underestimated on 67 % of the surface of the map. Depending on the texture of the fine earth and the lithological class of the rock fragments, the stony phase can store more water than the fine earth can. This situation occurred when the fine earth had a low available water content -sandy fine earth for example -, and when the rock fragments had a high available water content -porous limestone for example. In some stony areas, the maize AWC was underestimated by more than 50 % (Fig. 5a'). These areas had thin soils, and the maize AWC was calculated by assuming that much of the subsoil explored by the maize roots consisted of parent material. As explained in the materials and method sections, we have hypothesized that the available water content of the subsoil was the same as that of the rock fragments in the soil.

For the scenario 3, in which the stony phase was considered to be inert, the available water content (both soil AWC and maize

AWC

) was systematically underestimated. In areas with low rock fragments content (the southeast part of the soil map on Figs. 5b and 5b', for example), the differences between scenario 1 and scenario 3 were minor (less than 10 %), as expected. In some very stony areas (see the black arrow in Fig. 5b), the difference between scenarios 3

AWC

values that were lower in scenario 3 than in 1: in these cases, rock fragments can retain significant quantities of water and must not be neglected when evaluating the available water content of soil.

3.2.4. Effect of soil thickness and soil stoniness on the estimation of the available water content

The relationships between the thickness and soil stoniness of soils and the errors in the soil AWC values estimated using scenarios 2 and 3 compared with 1 are presented in Figures 6a and6b. As might be expected, the errors were the lowest not only for soils with a low stoniness, but also for the shallowest soils. No obvious relationship between the errors on the soil AWC estimation and the thickness or stoniness was observed.

Assumptions used to estimate the available water content at a regional scale

We have defined the available water content as the quantity of water that can be used by plant for their growth. It is typically defined as the difference between the water content at field capacity, and the water content at permanent wilting point. In our calculations, we have determined that the water content at field capacity could be considered equal to the water content at -100 hPa, as demonstrated by [START_REF] Bruand | Estimation des propriétés de rétention en eau des sols à partir de la base de données SOLHYDRO: Une première proposition combinant le type d'horizon, sa texture et sa densité apparente[END_REF] for fine earth, and by [START_REF] Tetegan | The contribution of rock fragments to the available water content of stony soils: proposition of new pedotransfer functions[END_REF] for rock fragments. Nevertheless, this value of water potential at field capacity, that has been shown to be true in our study could be changed and should be adapted in other contexts: for example, it could be adjusted to -330 hPa [START_REF] Bruand | Estimation des propriétés de rétention en eau des sols à partir de la base de données SOLHYDRO: Une première proposition combinant le type d'horizon, sa texture et sa densité apparente[END_REF] for more clayey soils, or, even -500hPa, as suggested by [START_REF] Whalley | Structural differences between bulk and rhizosphere soil[END_REF] for some crops, when available water contents are used in crop models.

For the permanent wilting point, we have used the classic value of water potential equal to -15850 hPa that can be calculated by pedotransfer functions and is commonly used by soil scientists. This value can be used with mature plants; however, it is known that the real value of permanent the wilting point depends on both the soil (especially the soil texture) and the physiological characteristics of the plant, as demonstrated by [START_REF] Czyz | Plant wilting can be caused either by the plant or by the soil[END_REF]. Plants from humid zones would not survive such low pF values and their permanent wilting point would be lower. In contrast, desert plants, if we choose an extreme, will survive extremely low pF values (about -163,000 hPa for Artemisia Herba Alba [START_REF] Gobat | Le sol vivant: Bases de pédologie, biologie des sols[END_REF] The results obtained here at a regional scale, where all the crops are considered to be maize, could then be different for other crops where the water potential at field capacity may be different for physical reasons, and both the water potential at the permanent wilting point and the soil depth could be different for agronomic and/or ecophysiological reasons.

Conclusions

The available water content of the stony soils of Patay was calculated using pedotransfer classes proposed by [START_REF] Bruand | Estimation des propriétés de rétention en eau des sols à partir de la base de données SOLHYDRO: Une première proposition combinant le type d'horizon, sa texture et sa densité apparente[END_REF] for the fine earth and [START_REF] Tetegan | The contribution of rock fragments to the available water content of stony soils: proposition of new pedotransfer functions[END_REF] for the rock fragments. We demonstrated that the AWC calculated by assuming that the stony soil was completely composed of fine earth led either to overestimations or underestimations of the real AWC calculated when both the proportion and the water -holding capacities of the stony phase are taken into account. We also demonstrated that the AWC calculated by assuming that the stony soils considered to consist of fine earth and nonporous rock fragments led to systematic underestimation (up to 20%) of the real AWC. Estimation of the available water content, and thus of the soil water deficit, was strongly dependent on value of the soil thickness which was considered in the calculations. From these maps and the associated databases, the water demand of crops in the study area can be calculated. In the context of climate change, which is expected to intensify droughts and increase the need for irrigation, this will enable a more accurate calculation of the water deficit and will be beneficial for farmers and regional planning of irrigation needs. If these maps are to be used as spatial data input for regional water balance modelling, our results demonstrate that failing to account for the rock fragments may lead to erroneous conclusions and unappropriated recommendations.

Abou-el-Seoud, I.I., and Abdel-Megeed, A., 2012. Impact of rock materials and biofertilizations on P and K availability for maize (Zea Maize) under calcareous soil conditions. Saudi Journal of Biological Sciences 19 (1), 55-63. Al Majou, H., Bruand, A., Nicoullaud, B., Duval, O., Dupont, J., 2005 Bruand, A., Duval, O., 2008b. Use of in situ volumetric water content to improve prediction of soil water retention properties. Can. J. Soil Sci. 88, 522-541. Baize, D., Girard, M.C., Jabiol, B., Rossignol, J.P., Eimberck, M., Beaudou, A., 2009. Référentiel pédologique 2008. Association Française pour l'Etude des sols. Coll. Savoir Faire. Quae Editions. France, p. 435. Bastet, G., Bruand, A., Voltz, M., Bornand, M., Quétin, P., 1999

List of tables:

Table 1: The pedotransfer classes used in the equation Eq. ( 1) for the calculation of the available water content (AWCi) of the fine phase as a function of both the position of the horizon in the soil profile and the texture of the fine earth texture (From Bruand et al., 2004). Table 2: Available water content (AWCi) of the stony phase calculated from the pedotransfer classes of [START_REF] Tetegan | The contribution of rock fragments to the available water content of stony soils: proposition of new pedotransfer functions[END_REF]. Table 3: Ranges of the soil available water content according to the different calculation scenarios. The scenario 1 is the reference scenario.

  Comment citer ce document : Tetegan, M., Richer-de-Forges, A., Verbeque, B., Nicoullaud, B., Desbourdes, C., Bouthier, A., Arrouays, D., Cousin, I. (Auteur de correspondance) (2015). The effect of soil stoniness on the estimation of water retention properties of soils: A case study from central France. Catena, 129, 95-102. , DOI : 10.1016/j.catena.2015.03.008 Version définitive du manuscrit publié dans / Final version of the manuscript published in : Catena, 2015, 129, 95-102 http://dx.doi.org/10.1016/j.catena.2015.03.008 Manuscrit d'auteur / Author Manuscript Manuscrit d'auteur / Author Manuscript Manuscrit d'auteur / Author Manuscript -5 -The available water content of a soil unit m ( m SU AWC ) was calculated for its soil profile as the sum of the n h AWC of each horizon (Eq. (3)). From the available water content of each soil unit ( m SU AWC ), the available water content of each map unit ( t MU AWC ) was calculated according to the equation Eq. (

  Comment citer ce document : Tetegan, M., Richer-de-Forges, A., Verbeque, B., Nicoullaud, B., Desbourdes, C., Bouthier, A., Arrouays, D., Cousin, I. (Auteur de correspondance) (2015). The effect of soil stoniness on the estimation of water retention properties of soils: A case study from central France. Catena, 129, 95-102. , DOI : 10.1016/j.catena.2015.03.008 Version définitive du manuscrit publié dans / Final version of the manuscript published in : Catena, 2015, 129, 95-102 http://dx.doi.org/10.1016/j.catena.2015.03.008 Manuscrit d'auteur / Author Manuscript Manuscrit d'auteur / Author Manuscript Manuscrit d'auteur / Author Manuscript -9 -soils with mean values of stoniness greater than or equal to 35%. The southeast part of the map remained the area containing soils with the fewest rock fragments (composing less than 20 % of the soil volume).

  3.2.1. Overview of the available water content calculated for the two depths and the 3 scenariosThe available water content of the soils in the area of Patay was calculated for each map unit using the two soil depths (

  2 and was equal to 124 mm. The range of maize AWC values was larger for the scenario 2 than for the reference scenario. Comment citer ce document : Tetegan, M., Richer-de-Forges, A., Verbeque, B., Nicoullaud, B., Desbourdes, C., Bouthier, A., Arrouays, D., Cousin, I. (Auteur de correspondance) (2015). The effect of soil stoniness on the estimation of water retention properties of soils: A case study from central France. Catena, 129, 95-102. , DOI : 10.1016/j.catena.2015.03.008Version définitive du manuscrit publié dans / Final version of the manuscript published in : Catena, 2015, 129, 95-102 http://dx.doi.org/10.1016/j.catena.2015.03.008 Manuscrit d'auteur Author Manuscript Manuscrit d'auteur / Author Manuscript Manuscrit d'auteur / Author Manuscript -10 -

  Comment citer ce document : Tetegan, M., Richer-de-Forges, A., Verbeque, B.,Nicoullaud, B., Desbourdes, C., Bouthier, A., Arrouays, D., Cousin, I. (Auteur de correspondance) (2015). The effect of soil stoniness on the estimation of water retention properties of soils: A case study from central France. Catena, 129, 95-102. , DOI : 10.1016/j.catena.2015.03.008 Version définitive du manuscrit publié dans / Final version of the manuscript published in : Catena, 2015, 129, 95-102 http://dx.doi.org/10.1016/j.catena.2015.03.008 Manuscrit d'auteur / Author Manuscript Manuscrit d'auteur / Author Manuscript Manuscrit d'auteur / Author Manuscript -11 -

Figure captions: Figure 1 :

 1 Figure captions: Figure 1: Map of the soil thickness in the area of Patay. Four synthetic characteristic soil profiles are represented (from Richer de Forges and Verbèque, 2003). Figure 2: Rock fragments content and types, in the area of Patay. a) Primary of rock fragments type; b) Rock fragments content observed in the field (information stored in the soil database ; c) Rock fragments content recalculated for a soil thickness of 120 cm. Figure 3: Relationship between the mean values of rock fragment content and the mean soil thickness for each map unit. Figure 4: Maps of the soil available water content (mm) calculated for the two soil depths and the three scenarios. a)

Figure 2 :

 2 Figure captions: Figure 1: Map of the soil thickness in the area of Patay. Four synthetic characteristic soil profiles are represented (from Richer de Forges and Verbèque, 2003). Figure 2: Rock fragments content and types, in the area of Patay. a) Primary of rock fragments type; b) Rock fragments content observed in the field (information stored in the soil database ; c) Rock fragments content recalculated for a soil thickness of 120 cm. Figure 3: Relationship between the mean values of rock fragment content and the mean soil thickness for each map unit. Figure 4: Maps of the soil available water content (mm) calculated for the two soil depths and the three scenarios. a) soil AWC

Figure 5 :

 5 Maps of the errors in the estimation of the soil available water content calculated for the two soil depths. a) Difference in soil AWC calculated for scenario 2 compared with scenario 1 ; b) Difference in soil AWC calculated for scenario 3 compared with scenario 1; a') Difference in maize AWC calculated for scenario 2 compared with scenario 1 ; b') Difference in maize AWC calculated for scenario 3 compared with scenario 1.

Figure 6 :

 6 Relationship between the errors in estimation of the available water content ( soil AWC ) and a) the soil thickness and b) the soil stoniness.

2.3 Calculation of the available water content at different scales 2.3.1. Choice of a definition and units for the available water content

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  Version définitive du manuscrit publié dans / Final version of the manuscript published in : Catena, 2015, 129, 95-102 http://dx.doi.org/10.1016/j.catena.2015.03.008 values represent under-estimations of the real available water content, whereas positive values represent over-estimations.

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  Comment citer ce document : Tetegan, M., Richer-de-Forges, A., Verbeque, B., Nicoullaud, B., Desbourdes, C., Bouthier, A., Arrouays, D., Cousin, I. (Auteur de correspondance) (2015). The effect of soil stoniness on the estimation of water retention properties of soils: A case study from central France. Catena, 129, 95-102. , DOI : 10.1016/j.catena.2015.03.008Version définitive du manuscrit publié dans / Final version of the manuscript published in :AWC was minor as well (less than 10 %): in these areas, the rock fragments were quartz or flint, which are rock fragments with a quasi-null water retention capacity. By contrast, large surface areas characterized by highly rocky soils exhibited

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soil

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estimation of water retention properties of soils: A case study from central France. Catena, 129, 95-102. , DOI : 10.1016/j.catena.2015.03.008

  . Performance of available pedotransfer functions for predicting the water retention properties of French soils, in: Comment citer ce document : Tetegan, M., Richer-de-Forges, A., Verbeque, B., Nicoullaud, B., Desbourdes, C., Bouthier, A., Arrouays, D., Cousin, I. (Auteur de correspondance) (2015). The effect of soil stoniness on the estimation of water retention properties of soils: A case study from central France. Catena, 129, 95-102. , DOI : 10.1016/j.catena.2015.03.008 Version définitive du manuscrit publié dans / Final version of the manuscript published in : Catena, 2015, 129, 95-102 http://dx.doi.org/10.1016/j.catena.2015.03.008

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