Nicolas Bédère
email: nicolas.bedere@inrae.fr

Joëlle Dupont

Yannick Baumard

Christophe Staub

David Gourichon

Elleboudt Frédéric

Pascale Le Roy

Tatiana Zerjal

Frédéric Elleboudt

Genetic background of body reserves in laying hens through backfat thickness phenotyping

Keywords: body composition, body reserves, backfat thickness, ultrasonography, CT-scan, adipokines, genetic correlations, heritability, laying hens

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Introduction

One of the major challenges of the egg production sector is to extend the egg production period of laying hens, for ethical, environmental, and economical reasons (reviews: [START_REF] Bain | Increasing persistency in lay and stabilising egg quality in longer laying cycles. What are the challenges?[END_REF][START_REF] Preisinger | Innovative layer genetics to handle global challenges in egg production[END_REF]).

Laying hens have been selected for laying criteria for more than 60 years, resulting in animals able to maintain profitable egg production from approximately 20 to 80 weeks of age. The priority of, at least European stakeholders, is now to extend the laying period to 100 weeks of age, with the aim of producing 500 eggs per hen. This would further dilute the economic and environmental costs related to nonproductive life periods (such as growth and laying pauses), and reduce the number of hens by decreasing the breeder stock.

The late-laying period, which goes beyond 80 weeks of age, remains relatively unexplored for what is our understanding of its physiology, nutrition, and genetics. The existing literature on this laying period is notably scarce, providing limited insights into these aspects, therefore, further research and investigation are warranted to enhance our knowledge in these areas. Egg production is a major nutrient expenditure for layers (energy, protein, calcium...) and about 25% of the gross energy intake goes to egg production [START_REF] Larbier | L'oeuf et l'alimentation des poules pondeuses[END_REF][START_REF] Luiting | Genetic variation of energy partitioning in laying hens: causes of variation in residual feed consumption[END_REF]. Excessive investment in egg production may lead to different metabolic diseases, and the longer the production cycle, the higher the risk. Risk factors mainly involve genetics, physiology, nutrition and management [START_REF] Bain | Increasing persistency in lay and stabilising egg quality in longer laying cycles. What are the challenges?[END_REF]. For instance, extending the laying period makes hens more likely to develop hepatic steatosis, a disease responsible for egg production drop and obese conditions [START_REF] Bain | Increasing persistency in lay and stabilising egg quality in longer laying cycles. What are the challenges?[END_REF]. Therefore, we need to monitor both egg production and fattening in laying hens, to select balanced hens that can ensure cost-effective egg production while maintaining optimal fatness. The monitoring of egg production and the pedigree of laying hens has been facilitated by cage-rearing systems. In some regions of the world, cages are about to be banned and technical solutions are emerging for individual recording systems and relevant selection criteria for egg production in alternative systems [START_REF] Bécot | Sélection sur la ponte des poules en systèmes alternatifs à la cage: (Full text available in English)[END_REF]. Regarding fatness in chicken, like in other species, the gold standard and most common method to determine body composition are lethal and destructive because it is either a dissection with adipose tissue weighing or a chemical analysis of the shredded body. This phenotyping method is unsatisfactory because it requires the euthanasia of the animal, which raises ethical and practical problems. Indeed, the animals can no longer be used for genetic selection, except as collateral information when using allometric sequential slaughter designs to evaluate both states and dynamics of body reserves.

Alternative and non-invasive methods are now available to determine body composition in various species [START_REF] Lerch | Estimation of dairy goat body composition: A direct calibration and comparison of eight methods[END_REF][START_REF] Staub | Ultrasonographic measures of body fatness and their relationship with plasma levels and adipose tissue expression of four adipokines in Welsh pony mares[END_REF][START_REF] Xavier | The use of 3-dimensional imaging of Holstein cows to estimate body weight and monitor the composition of body weight change throughout lactation[END_REF]. In poultry, tomography has proven to be sufficiently accurate to be considered as a reference method for body composition, with phenotypic correlations above 0.80 in broilers [START_REF] Cobo | Mise en place d'une méthodologie en imagerie pour prédire des caractères de production chez les palmipèdes à foie gras[END_REF]Mellouk et al., 2018b). However, the routine use of tomography is difficult to implement on a large number of animals as it cannot easily be performed on the farm and because it requires sedation of the animal, which is time-consuming and costly and not without risk for the animals. The methods relying on ultrasonography have been used effectively to assess body fatness in chickens. A specific region was identified on top of the synsacrum where subcutaneous adipose tissue thickness was highly correlated to chemical analyses of the shredded body (r=0.92; Mellouk et al. 2018b), to the abdominal fat pad weight by dissection (r=0.86; Mellouk et al., 2018b) and the body fat volume estimated by tomography (r>0.84; Mellouk et al. 2018b;[START_REF] Grandhaye | Assessment of the body development kinetic of broiler breeders by non-invasive imaging tools[END_REF]. So far, body fatness traits recorded by ultrasonography were all tested on broilers while no data are available on laying hens. Despite belonging to the same species (Gallus gallus domesticus), broilers and layers have been subjected to separate and intense genetic selection for over 60 years. As a result, they differ greatly in terms of growth rate and energy metabolisms. In addition, selection and phenotype recording target different physiological stages, focusing on young animals in broilers and adults in layers.

As they age, layers tend to become fatter, and breeders aim to achieve a balanced target fat level: neither too thin nor too fat, to maintain sufficient body reserves in case of nutrient scarcity while avoiding unnecessary energy storage.

Consequently, the present study aimed to achieve several objectives. Firstly, it sought to test and validate the phenotyping by ultrasounds of the subcutaneous adipose tissue thickness on top of the synsacrum as an accurate indicator of the overall fatness of the layer hen. Secondly, it aimed to estimate the heritability of this new trait in laying hens. Finally, it aimed to study the phenotypic and genetic correlations between this trait and other traits from the breeding goal of most of the lines of laying hens, in order to evaluate its potential as a selection criterion.

Methods

Laying hen population and rearing condition

The laying hens used in this study belong to two experimental lines originating from the same Rhode Island Red population, divergently selected since 1976 on the residual feed intake, a trait for feed efficiency [START_REF] Bordas | Direct and correlated responses to divergent selection for residual food intake in Rhode island red laying hens[END_REF]. These lines were chosen for this study because the selection process on RFI has also led to marked differences in carcass adiposity with the efficient line (R-) being fatter than the inefficient (R+) one, despite a reduced feed intake [START_REF] El-Kazzi | Divergent selection for residual food intake in Rhode Island Red egg-laying lines: Gross carcase composition, carcase adiposity and lipid contents of tissues[END_REF]. The RFI was estimated as defined in [START_REF] Byerly | Feed Requirements for Egg Production[END_REF] and represents the difference between the observed feed intake and the expected one estimated based on known maintenance and production requirements.

In total, we used 394 animals, 215 from the R+ line and 179 from the R-line. There were 92 and 123 R+ phenotyped in 2019 and 2021 (from 9 sires and 38 dams in 2019, and 10 sires and 42 dams in 2021), and 75 and 104 R-in 2019 and 2021 (from 9 sires and 43 dams in 2019, and 9 sires and 41 dams in 2021). All animals were hatched in two batches at the INRAE Pôle d'Expérimentation Avicole de Tours (UE PEAT, Nouzilly, France ; https://doi.org/10.15454/1.5572326250887292E12). They were reared under standard farming conditions in floor pens until 17 weeks of age when 46 birds were euthanized for body composition recording (23 pullets per line), by neck cut and bleeding, immediately after head electrical stunning. The remaining animals were transferred to individual cages with a lighting regime set at 14 h of light per day, temperature was maintained between 19 and 21°C, and the hens were fed ad libitum a commercial diet (15.5% CP and 2,650 kcal of ME/kg) automatically distributed at 8:00 a.m. (Appendix 1). Egg production was recorded daily up to 53 weeks of age, when the hens were euthanized as described above. Because of the adaptation of the experimental facility to both the sanitary situation and lockdown policy caused by the COVID-19 pandemic, only the body weight (named BodyWeight) and last backfat thickness were recorded in birds from the batch 2019. Corp., Germany). During the scan, each hen was placed dorsally on the CT table. The X-ray source was set at 120 kV and 500 mA/s. In humans, fat tissue typically displays Hounsfield Units (HU) ranging from -150 to -50, although the exact limits do vary by individual and tissue type [START_REF] Kim | Body fat measurement in computed tomography image[END_REF]. To ensure these limits align with laying hens, thresholds were estimated. Specifically, for all images, two discs of 10 mm² were placed: one in the backfat tissue where the ulstrasonography was done, and another in the abdominal fat pad tissue. The minimum and maximum HU values were obtained for each disc. It is possible that non-fatty components in the region, such as blood protein, were also captured. Therefore, the thresholds were set at a 0.90 quantile for minimum values and a 0.10 quantile for maximum values. It resulted in a lower limit of -130 HU and an upper limit of -90 HU, aligning with the updated range of -123 to -89 HU reported in a recent study in humans [START_REF] Pop | Fat Hounsfield Unit Reference Interval Derived through an Indirect Method[END_REF]. The total volume of pixels within these bounds was used as the total volume of fat in the animal (example in Figure 1; trait named CT-TotFat). on high correlations with CT-TotFat (Mellouk et al. 2018b;[START_REF] Grandhaye | Assessment of the body development kinetic of broiler breeders by non-invasive imaging tools[END_REF]). The BackFat was recorded according to the same protocol: the plumage was soaked with soapy water and then spread, ultrasound gel was applied in contact with the epithelium and the probe was put in contact with the gel.

Phenotypes

Tomography as the Gold Standard for body composition

In-vivo indicators for body energy reserves

The entire recording process took about 1 min per hen and no feathers were plucked. BackFat was recorded 5 times at 129,192,218,289, and 371 days of age. The BodyWeight was recorded together with BackFat.

Blood Adipokines levels

A first blood sample was collected from the wing vein at 17 weeks of age and a second blood sample was collected during the neck bleeding at the slaughter process, at 53 weeks of age. The difference in blood sampling is not expected to bias the results, but it is a limitation of the experimental design. Plasma was isolated from blood after centrifugation (5000 g for 10 min at 4°C) and then stored at -20°C. Consequently, all hens had two blood samples available to determine adipokines concentrations. The concentrations of four adipokines (visfatin, adiponectin, chemerin, and ghrelin) were determined in the plasma using chicken-specific ELISA kits as previously described [START_REF] Barbe | A grape seed extract maternal dietary supplementation in reproductive hens reduces oxidative stress associated to modulation of plasma and tissue adipokines expression and improves viability of offsprings[END_REF]Mellouk et al., 2018b). Briefly, MBS269004 (sensitivity 5 pg/mL), MBS016609 (sensitivity 0.1 μg/mL), MBS738819 (sensitivity 0.1 ng/mL), and MBS2700427 (sensitivity 0.05 ng/mL) were used for visfatin, adiponectin, chemerin, and ghrelin, respectively (My BioSource, San Diego, USA). The experiment was performed following the manufacturer's protocol with an intra-assay coefficient of variation ≤ 8%, < 10%, < 5.6%, and < 12%, respectively. The absorbance was measured at 450 nm and then compared with reference values. The traits are named after the appropriated adipokines (visfatin, adiponectin, chemerin, and ghrelin).

Egg production

Egg production was recorded daily from the first egg laid until the end of the experiment (i.e. culling of the flock; trait named TotEggNum).

Statistical analyses

Models

To calculate genetic parameters (correlations and heritabilities), variance components were estimated using bivariate animal model analyses [START_REF] Henderson | Best linear unbiased estimation and prediction under a selection model[END_REF]. Commonly in bivariate analyses, both traits have the same two variance strata, genetic and residual, or three strata, genetic, animal, and sampling. This common model with two strata can be described as:

[ 𝐲 1 𝐲 2 ] = [ 𝐗 1 0 0 𝐗 2 ] [ 𝐛 1 𝐛 2 ] + [ 𝐙 1 0 0 𝐙 2 ] [ 𝐮 1 𝐮 2 ] + [ 𝐞 1 𝐞 2 ]
where 𝐲 1 and 𝐲 2 are vectors of the observed values for the first and second trait respectively, 𝐗 1 and 𝐗 2 are design matrices for fixed effects and 𝐛 1 and 𝐛 2 are vectors of values for fixed effects (details at the end of the section), 𝐙 1 and 𝐙 2 are design matrices for the additive genetic random effects and 𝐮 1 and 𝐮 2 are vectors of breeding values, and 𝐞 1 and 𝐞 2 are vectors of residual values. The variance components are fitted as 2x2 matrices of variances-covariances for each stratum:

𝑉𝑎𝑟 [ 𝐮 1 𝐮 2 ] = 𝐆 × 𝐀 where 𝐆 = [ 𝜎 𝑢1 2 𝜎 𝑢1𝑢2 𝜎 𝑢1𝑢2 𝜎 𝑢2
2 ] and 𝐀 is the additive genetic relationship matrix

𝑉𝑎𝑟 [ 𝐞 1 𝐞 2 ] = 𝐑 × 𝐈 where 𝐑 = [ 𝜎 𝑒1 2 𝜎 𝑒1𝑒2 𝜎 𝑒1𝑒2 𝜎 𝑒2
2 ] and 𝐈 is the identity matrix For a bivariate analysis where both traits have three strata, the model can be described as:

[ 𝐲 1 𝐲 2 ] = [ 𝐗 1 0 0 𝐗 2 ] [ 𝐛 1 𝐛 2 ] + [ 𝐙 1 0 0 𝐙 2 ] [ 𝐮 1 𝐮 2 ] + [ 𝐙 1 0 0 𝐙 2 ] [ 𝐩𝐞 1 𝐩𝐞 2 ] + [ 𝐙 1 0 0 𝐙 2 ] [ 𝐬𝐚𝐦𝐩 1 𝐬𝐚𝐦𝐩 2 ]
where fixed effects are the same as for the former model, variance components are fitted as 2x2 matrices for the genetic strata and the remaining variance is decomposed into an animal (non-genetic) stratum and a sampling stratum defined as:

𝑉𝑎𝑟 [ 𝐮 1 𝐮 2 ] = 𝐆 × 𝐀 where 𝐆 = [ 𝜎 𝑢1 2 𝜎 𝑢1𝑢2 𝜎 𝑢1𝑢2 𝜎 𝑢2
2 ] and 𝐀 is the additive genetic relationship matrix

𝑉𝑎𝑟 [ 𝐩𝐞 1 𝐩𝐞 2 ] = 𝐏 × 𝐈 where 𝐏 = [ 𝜎 𝑝𝑒1 2 𝜎 𝑝𝑒1𝑝𝑒2 𝜎 𝑝𝑒1𝑝𝑒2 𝜎 𝑝𝑒2 2
] and 𝐈 is the identity matrix

𝑉𝑎𝑟 [ 𝐬𝐚𝐦𝐩 1 𝐬𝐚𝐦𝐩 2 ] = 𝐒 × 𝐈 where 𝐒 = [ 𝜎 𝑠𝑎𝑚𝑝1 2 𝜎 𝑠𝑎𝑚𝑝1𝑠𝑎𝑚𝑝2 𝜎 𝑠𝑎𝑚𝑝1𝑠𝑎𝑚𝑝2 𝜎 𝑠𝑎𝑚𝑝2 2
] and 𝐈 is the identity matrix However, when one trait has two strata (𝐲 1 say) and the other has three strata (𝐲 2 say), the direct product variance structure breaks down; 𝜎 𝑒1 2 (𝜎 𝑒1𝑒2 ) cannot be partitioned into 𝜎 𝑝𝑒1 2 + 𝜎 𝑠𝑎𝑚𝑝1 2 (𝜎 𝑝𝑒1𝑝𝑒2 + 𝜎 𝑠𝑎𝑚𝑝1𝑠𝑎𝑚𝑝2 ). We can estimate four (three different) parameters:

𝜎 𝑒1𝑒2 * = 𝜎 𝑝𝑒1𝑝𝑒2 + 𝜎 𝑠𝑎𝑚𝑝1𝑠𝑎𝑚𝑝2 𝜎 𝑒1 2 * = 𝜎 𝑒1 2 -𝜎 𝑒1𝑒2 * = 𝜎 𝑝𝑒1 2 + 𝜎 𝑠𝑎𝑚𝑝1 2 -𝜎 𝑠𝑎𝑚𝑝1𝑠𝑎𝑚𝑝2 𝜎 𝑝𝑒2 2 * = 𝜎 𝑝𝑒2 2 -𝜎 𝑝𝑒1𝑝𝑒2 𝜎 𝑠𝑎𝑚𝑝2 2 * = 𝜎 𝑠𝑎𝑚𝑝2 2 -𝜎 𝑠𝑎𝑚𝑝1𝑠𝑎𝑚𝑝2
The phenotypic variance components are then given by:

𝜎 𝑡𝑜𝑡𝑎𝑙1 2 = 𝜎 𝑢1 2 + 𝜎 𝑒1 2 * + 𝜎 𝑒1𝑒2 * 𝜎 𝑡𝑜𝑡𝑎𝑙1𝑡𝑜𝑡𝑎𝑙2 = 𝜎 𝑢1𝑢2 + 𝜎 𝑒1𝑒2 * 𝜎 𝑡𝑜𝑡𝑎𝑙2 2 = 𝜎 𝑢2 2 + 𝜎 𝑝𝑒2 2 * + 𝜎 𝑠𝑎𝑚𝑝2 2 * + 𝜎 𝑒1𝑒2 *
The variance components were estimated using the average-information restricted maximum likelihood method (AI-REML algorithm; [START_REF] Gilmour | Average Information REML: An Efficient Algorithm for Variance Parameter Estimation in Linear Mixed Models[END_REF]. Reported heritability and standard error estimates are means calculated with all bivariate analyses. Genetic parameters were considered low between 0.00 and 0.25, moderate between 0.25 and 0.50, and high above 0.50. The fixed effects in the model include the genetic line to account for their mean differences (levels: R+ or R-), the effect of the batch (levels: 2019 or 2021) and the regression coefficient for the time of recording for the repeated trait. The genetic line was not used to stratify the random effects because preliminary analyses indicated that the variance components were similar in both lines. See the provided scripts "BEDERE_2023_ASREMLScript_bivariate_ 2x2strata.as", "[...]3x3strata.as", and "[...]3x2strata.as" for details.

Bartlett's test

Descriptive statistics of the data suggested a bimodal distribution of BackFat in both lines. This type of distribution may highlight the presence of a major gene controlling the trait. A simple test to detect a major gene effect on a trait is to test the homogeneity of the variances between families (Le [START_REF] Roy | Simple test statistics for major gene detection: a numerical comparison[END_REF].

A Bartlett test was performed to test this hypothesis, using the sire as the family identifier [START_REF] Bartlett | Some Examples of Statistical Methods of Research in Agriculture and Applied Biology[END_REF].

See the provided script "BEDERE_2023_RScript_BartlettTest.R" for details.

Programs used

Data handling, graphs, and the Bartlett test were performed in base R (R Core Team, 2023). Variance components and genetic parameters estimations were performed with ASReml 4.2 [START_REF] Gilmour | ASReml User Guide Release 4.2 Functional Specification[END_REF].

License: CC BY 2.0 The traits are named after their phenotypes: BackFat for dorsal subcutaneous adipose tissue thickness, BodyWeight for body weight, CT-TotFat for the total volume of pixels of fat components, then the blood Adipokines levels named after the appropriate adipokine, and TotEggNum for the total number of eggs laid.

Results & Discussion

Phenotypic description of the divergent lines

The two lines used, diverging for RFI, were very different regarding BackFat and chemerin blood concentration (Table 1, Figure 3): the R-line had a larger BackFat (+34%) and lower chemerin levels (-31%) than the R+ line. However, the lines were not significantly different regarding CT-TotFat, BodyWeight, Adiponectin, Ghrelin, Visfatin, and TotEggNum. For the P-value for CT-TotFat which is 0.23, given the difference in mean and the variance, there may be some lack of power in the analysis due to the fact that the tomography could be performed on one batch only. Previous studies about these lines reported that the R+ line (inefficient ones) has a higher feed intake, higher diet-induced thermogenesis and different endocrine responses, resulting in different lipid metabolism between the lines [START_REF] Gabarrou | Glucose-insulin relationships and thyroid status of cockerels selected for high or low residual food consumption[END_REF][START_REF] Gabarrou | Energy balance of laying hens selected on residual food consumption[END_REF][START_REF] Gabarrou | Diet-Induced Thermogenesis in Cockerels Is Modulated by Genetic Selection for High or Low Residual Feed Intake[END_REF][START_REF] Swennen | Further Investigations on the Role of Diet-Induced Thermogenesis in the Regulation of Feed Intake in Chickens: Comparison of Adult Cockerels of Lines Selected for High or Low Residual Feed Intake[END_REF] which could explain the observed differences in adiposity between lines.

Interestingly, in pigs, the selection on residual feed intake was also associated with a difference in BackFat with the efficient line being fatter, although the magnitude of difference was smaller [START_REF] Gilbert | Review: divergent selection for residual feed intake in the growing pig[END_REF].

The results on fat content observed in our study corroborate previous results about the R+ and R-lines, with the R+ being leaner than the R-. However, contrary to our expectation, no differences were observed in the blood level of adiponectin and ghrelin, which are hormones associated with feed intake acting as appetite-regulating signals (Mellouk et al., 2018a).

BackFat thickness is an indicator of body reserves

BackFat and CT-TotFat were genetically highly positively correlated, and phenotypically moderately positively correlated (Table 2). Previous studies reported a high phenotypic correlation between BackFat and CT-TotFat in chicken (r > 0.84; Mellouk et al. 2018b;[START_REF] Grandhaye | Assessment of the body development kinetic of broiler breeders by non-invasive imaging tools[END_REF]) but neither heritability, License: CC BY 2.0 nor phenotypic and genetic correlations with other traits of interest were calculated. The lower phenotypic correlation reported in Table 2 takes into account the effects of the model, which can influence the correlation estimate (genetic line, the batch, the repetition of recording, and the genetic and permanent environment variances). When we repeated the estimation using the same approach (i.e. Pearson correlation using raw data; Mellouk et al., 2018b;[START_REF] Grandhaye | Assessment of the body development kinetic of broiler breeders by non-invasive imaging tools[END_REF], we obtain a correlation value of 0.71 (0.77 in R+ and 0.60 in R-), which is consistent with the findings previoulsy published.

The BodyWeight and CT-TotFat were also highly positively correlated. However, BackFat and BodyWeight were moderately positively correlated. The overall results show that BackFat is a good indicator of fatness in adult layers, consistent with previous findings in young broilers, where it exibited a high phenotypic correlated with the fat fraction from chemical analyses of the shredded body (r=0.92) and the abdominal fat pad weight obtained by dissection (r=0.86; Mellouk et al., 2018b). Given the genetic correlations between BackFat, CT-TotFat, and BodyWeight, we can conclude that BackFat and BodyWeight hold different information related to fatness in chickens. Compared to CT-TotFat, BackFat offers notable advantages as an easy-to-record trait: it is fast to record, does not require the animal to be asleep, and can be done with a portable machine. Our results combined with these technical aspects, make BackFat a very good indicator trait of fatness in chicken.

Table 2: Mean heritability estimates (in diagonal together with their associated mean standard errors), genetic correlation estimates (below the diagonal together with their associated standard errors), and phenotypic correlations (above the diagonal with their associated standard errors) for traits related to body composition (CT-TotFat, BackFat, and BodyWeight), blood adipokine levels (Adiponectine, Chemerin, Ghrelin, and Visfatin), and egg production (TotEggNum) in two lines divergently selected for residual feed intake (efficient: R-, inefficient: R+).

Genetic background of BackFat thickness

BackFat displayed a moderate heritability (Table 2). The distribution of the values for BackFat in both lines displayed a large variance, with apparently two modes, which seems to become exacerbated with time (Figure 3). The sire-family variances were heterogeneous according to the Bartlett test License: CC BY 2.0 (P-value=0.008). Both the multimodal distribution and the heterogeneity of sire-family variance are evidence of a major gene effect (Le [START_REF] Roy | Simple test statistics for major gene detection: a numerical comparison[END_REF].

In quails, a study reported a low heritability of 0.17 for fat skin percentage (recorded as the fat content of the shredded skin) as an indicator similar to BackFat [START_REF] Lotfi | Genetic evaluation of carcass composition and fat deposition in Japanese quail[END_REF]. In pigs, BackFat displayed a high heritability (from 0.63 to 0.72; [START_REF] Cai | Selection response and genetic parameters for residual feed intake in Yorkshire swine1[END_REF][START_REF] Gilbert | Genetic parameters for residual feed intake in growing pigs, with emphasis on genetic relationships with carcass and meat quality traits[END_REF][START_REF] Suzuki | Genetic parameter estimates of meat quality traits in Duroc pigs selected for average daily gain, longissimus License: CC BY 2.0 muscle area, backfat thickness, and intramuscular fat content[END_REF] while in cattle, BackFat presented moderate ones (from 0.36 to 0.59; [START_REF] Arnold | Estimates of genetic parameters for live animal ultrasound, actual carcass data, and growth traits in beef cattle[END_REF][START_REF] Nkrumah | Genetic and phenotypic relationships of feed intake and measures of efficiency with growth and carcass merit of beef cattle1[END_REF][START_REF] Schenkel | Genetic parameters and breed differences for feed efficiency, growth, and body composition traits of young beef bulls[END_REF]. Many quantitative trait loci (QTL) associated with fatness in chickens are reported: there are 129 QTL listed in chickenQTLdb (https://www.animalgenome.org/QTLdb/chicken/) from 69 scientific articles. Some genes are known to be involved in lipogenesis and differently expressed in lean and fat broilers [START_REF] Bourneuf | Microarray analysis of differential gene expression in the liver of lean and fat chickens[END_REF][START_REF] Resnyk | Transcriptional analysis of abdominal fat in chickens divergently selected on bodyweight at two ages reveals novel mechanisms controlling adiposity: validating visceral adipose tissue as a dynamic endocrine and metabolic organ[END_REF]. Yet, major genes for BackFat were not explicitly identified, further analyses including segregation analyses and genome-wide association studies accounting for dominant effects would help to identify them.

Genetic background of other traits related to fatness

Moderate heritabilities were observed for CT-TotFat and BodyWeight (Table 2), with the latter aligning with previous studies reporting estimates ranging from 0.32 to 0.53 [START_REF] Rowland | Genetic analysis of production, physiological, and egg quality traits in heat-challenged commercial white egg-laying hens using 600k SNP array data[END_REF]Wolc et al., 2011[START_REF] Wolc | Genetic variability in residual variation of body weight and conformation scores in broiler chickens[END_REF]. Heritability in the R+ and R-lines may have changed a little because estimates for BodyWeight were reported to be 0.56 and 0.61 in females and males respectively in the 15 first generations [START_REF] Tixier-Boichard | Restricted Maximum Likelihood Estimates of Genetic Parameters of Adult Male and Female Rhode Island Red Chickens Divergently Selected for Residual Feed Consumption[END_REF]. Carcass percentage of fat displayed a moderate heritability in other studies using other chicken lines (0.43 to 0.55; [START_REF] Moreira | Integration of genome wide association studies and whole genome sequencing provides novel insights into fat deposition in chicken[END_REF][START_REF] Nunes | Genetic parameters for body weight, carcass chemical composition and yield in a broilerlayer cross developed for QTL mapping[END_REF].

The chosen adipokines in this study are known to be indicators of body reserve status and dynamics (review: Mellouk et al., 2018a). Adiponectin is used as an indicator of energy deficit: the leaner the bird the higher the level of adiponectin. Chemerin is used as an indicator of body lipid mobilization: the lower the abdominal fat pad, the higher the level of chemerin. Ghrelin is used as an indicator of general body reserves accretion: it is known to stimulate intake and growth hormone release. Visfatin is acting like a myokine in birds [START_REF] Krzysik-Walker | Is visfatin an adipokine or myokine? Evidence for greater visfatin expression in skeletal muscle than visceral fat in chickens[END_REF] and it is used as an indicator of lean body reserve status compared to body lipid reserves. The genetic background, particularly the genes coding for these proteins are well described. All adipokines except chemerin displayed very high heritability (Table 2). This indicates that genetics is the primary source of phenotypic variation, and that environmental fluctuations have minimal influence in our setup, where hens are housed in individual cages and fed ad libitum. We observed a significant increase in blood levels of adiponectin (P < 0.001) and visfatin (P = 0.007), a significant decrease in chemerin (P < 0.001), and no significant change in ghrelin (P = 0.14) between 17 and 53 weeks of age. It has been reported in turkeys that plasma levels of adiponectin, chemerin, and visfatin decrease during the laying period [START_REF] Diot | Expression of adiponectin, chemerin and visfatin in plasma and different tissues during a laying season in turkeys[END_REF]. A kinetic experimental design would be required to further investigate the effect of physiological stage on blood levels of adipokines. Blood adipokine levels are also known to vary with dietary intake and composition in broilers (Mellouk et al., 2018a(Mellouk et al., , 2018b)), but these were similar between hens in our experimental setup. Genetic parameters for chemerin could not be estimated because the estimated additive genetic variance was too close to the zero boundary. This means that almost none of the observed variance is due to genetics, despite a phenotypic coefficient of variation close to 40%. We hypothesize that there may be a single haplotype per line in the population, explaining why there is no genetic variance observed despite a significant difference in mean between the lines. Consequently, no genetic correlation with other traits could be estimated (explaining why chemerin is not in Table 2).

Adiponectin displayed a moderate and positive genetic correlation with Ghrelin, no correlation with Visfatin and TotEggNum, and moderate and negative genetic correlations with CT-TotFat, BackFat, and BodyWeight. This is consistent with its role in chicken: increased blood level of adiponectine is associated with decreased lipid deposition, decreased body weight and increased feed intake (Mellouk et al., 2018a).

Ghrelin displayed low and positive genetic correlations with Visfatin and TotEggNum, moderate and negative genetic correlations with BackFat and BodyWeight, and a high and negative genetic correlation with CT-TotFat. This is consistent with its role in chicken: increased blood level of ghrelin is associated with decreased feed intake and increased lipolysis [START_REF] Murugesan | Role of leptin and ghrelin in regulation of physiological functions of chicken[END_REF]. These correlations further support BackFat as a good indicator trait for fatness and energy reserves in chickens. Chemerin levels were significantly higher in the R+ line, which is consistent with the fact that it is associated with lower body fatness (Mellouk et al., 2018a). Visfatin displayed a low and positive genetic correlation with TotEggNum, and low-to-moderate and negative genetic correlations with CT-TotFat, BackFat, and BodyWeight. We were expecting a lower genetic correlation between visfatin and fat-related traits given its biological function: visfatin is acting like a myokine in chicken [START_REF] Krzysik-Walker | Is visfatin an adipokine or myokine? Evidence for greater visfatin expression in skeletal muscle than visceral fat in chickens[END_REF]. Increased blood levels of visfatin are associated with increased feed intake and body weight (lean part; Mellouk et al., 2018a). It is important to note the high standard errors reported for genetic correlations between adipokines and other traits, pinpointing they could gain from additional data.

Tradeoff between body reserves and egg production

The TotEggNum displayed a moderate heritability (Table 2). This phenotype is capturing two distinct biological processes: puberty (age at first laying) and laying rate. Total egg number displayed a low heritability in other studies (from 0.01 to 0.20; [START_REF] Bedere | Using egg production longitudinal recording to study the genetic background of resilience in purebred and crossbred laying hens[END_REF][START_REF] Liu | Genome-wide association analysis of egg production performance in chickens across the whole laying period[END_REF]Wolc et al., 2011a), but in most papers the early period (before 25 weeks of age) is skipped to start recording after the laying peak.

Again, the same trait in the first 15 generations was reported to be more heritable (h 2 = 0.48; Tixierboichard et al., 1995).

The genetic correlation of TotEggNum was moderate and negative with BackFat, and high and negative with BodyWeight (Table 2). These correlations suggest a tradeoff between body reserves and egg production in some populations. The genetic correlation between TotEggNum and BodyWeight was higher (-0.82) than that with CT-TotFat (-0.49) or BackFat (-0.34). This means that the genetic share between TotEggNum and BodyWeight is stronger than with CT-TotFat or BackFat. We hypothesize that this could be explained by a larger tradeoff, possibly including energy, minerals and protein, whereas the tradeoff between egg production and fatness would be limited to energy resources. The BodyWeight is partly composed of fat, consistent with the share of their genetic architecture, as indicated by the moderate-tohigh and positive genetic correlations between BackFat or CT-TotFat and BodyWeight. The few studies mentioning genetic correlations between egg production and body weight reported moderate and negative correlations (-0.29 to -0.42;[START_REF] Yoo | Genetic parameters for Oviposition time and interval in a white leghorn population of recent commercial origin[END_REF] or no correlation (Wolc et al., 2011b). The very high value estimated in our study may be a specificity of the R+ and R-lines, which is an unsusual population for the egg industry. Both the size and fatness are optimum-based breeding goals: a targeted neither too big nor too small size and fatness are desired, whereas egg production is mostly maximized. This means that the selection index must consider these genetic correlations to combine selection criteria such as TotEggNum, BackFat, and BodyWeight to breed multi-performing laying hens. In fact, if similar genetic correlations were found in commercial lines, including BackFat in the selection index would allow avoiding the indirect response of fatness to selection on egg production. Breeding companies may be interested in stabilizing fatness in chickens to avoid health, welfare and performance problems due to metabolic disorders associated with extreme conditions: leanness and obesity [START_REF] Baéza | Chicken lines divergent for low or high abdominal fat deposition: a relevant model to study the regulation of energy metabolism[END_REF][START_REF] Bain | Increasing persistency in lay and stabilising egg quality in longer laying cycles. What are the challenges?[END_REF].

Conclusion

To conclude, this study showed, on two Rhode Island lines diverging for feed efficiency differing also in fat content, that backfat thickness is a potentially accurate indicator of the overall fatness of laying hens.

Backfat thickness can be recorded repeatedly during the production cycle, creating opportunities to better understand body reserve dynamics in chickens. In addition, backfat thickness displayed a moderate heritability, implying that there is room for genetic improvement, probably canalization around an optimum to be defined. Both the bimodal distribution of the trait and the heterogeneity of the variances between families are signs of the presence of a major gene segregating backfat thickness in the population.

The genetic correlation with body weight was moderate, implying that backfat holds complementary genetic information about fatness that is currently not considered in breeding programs including body weight in their breeding goal. Finally, the genetic correlation with egg production was moderate and unfavorable. This correlation should be taken into account to avoid undesired responses to selection. It is important to keep in mind that all the reported results are based on particular genetic lines, divergently selected since 1976 on the residual feed intake. They need to be confirmed on regular commercial genetic lines to consider backfat thickness in the breeding goal.

License: CC BY 2.0

CRediT (Contributor Roles Taxonomy, https://credit.niso.org/)

Figure 1 :

 1 Figure 1: Example of CT-scan image visualizing the 3D axes (hen ID: PEA2021045179). The pixels set between -130 and -90 HU and colored in pink to display the fatty components

Figure 2 :

 2 Figure2: Ultrasound scan panoramic image of the dorsal subcutaneous adipose tissue thickness above the synsacrum, an example of the same hen as in Figure1(hen ID: PEA2021045179)

Figure 3 :

 3 Figure 3: Distribution of the raw values for BackFat according to age in both lines. R+ are in purple on the left side of the beanplot, R-are in orange on the right side of the beanplot. The dash is the mean for each level.

Table 1 :

 1 Summary statistics for traits related to body composition (CT-TotFat, BackFat, and BodyWeight), blood adipokines levels (Adiponectine, Chemerin, Ghrelin, and Visfatin), and egg production (TotEggNum) in two lines divergently selected for residual feed intake (efficient: R-, inefficient: R+).

	Trait 1		CT-TotFat (mm 3 )	BackFat (mm)	BodyWeight (g)	Adiponectin (µg/ml)	Chemerin (ng/ml)	Ghrelin (ng/ml)	Visfatin (ng/ml)	TotEggNum (count)
				Summary statistics of observed values
		number of records	111	580	771	233	233	233	233	192
		mean	540,367	6.1	2,339	2.4	39.5	57.0	58.0	158
	R+	standard deviation 167,107	1.8	323	0.4	15.9	12.1	6.6	43
		minimum	114,366	0.6	1,524	1.1	26.0	32.0	43.0	1
		maximum	955,873	11.5	3,234	3.2	78.0	88.0	75.0	218
		number of records	86	460	611	191	190	190	189	155
		mean	633,050	8.9	2,304	2.0	27.5	60.3	59.2	150
	R-	standard deviation 141,608	1.8	351	0.4	11.9	14.3	6.2	56
		minimum	380,231	1.9	1,375	1.0	3.0	34.0	43.0	0
		maximum	1,087,374	12.7	3,465	3.2	68.0	112.0	74.0	194
			Fixed

effect of the genetic line estimated by the mixed models (R+ compared to R-)

	Estimated effect	-80,028	-3.1	23	0.3	12.1	-2.6	-1.8	-4
	P-value	0.23	<.001	0.82	0.40	<.001	0.84	0.74	0.82
	1

CT-TotFat BackFat BodyWeight Adiponectin Ghrelin Visfatin TotEggNum CT-TotFat 0.27 (0.04)

			0.39	0.54	-0.24	-0.32	-0.06	-0.01
			(0.04)	(0.04)	(0.06)	(0.06)	(0.07)	(0.09)
	BackFat	0.91 (0.13)	0.38 (0.06)	0.31 (0.04)	-0.18 (0.08)	-0.23 (0.07)	-0.07 (0.06)	-0.01 (0.06)
	BodyWeight	0.67 (0.16)	0.39 (0.12)	0.42 (0.08)	-0.24 (0.07)	-0.28 (0.07)	-0.08 (0.07)	-0.17 (0.05)
	Adiponectin	-0.37 (0.15)	-0.28 (0.22)	-0.42 (0.18)	0.92 (0.02)	0.39 (0.05)	-0.03 (0.06)	0.01 (0.08)
	Ghrelin	-0.80 (0.16)	-0.44 (0.20)	-0.39 (0.18)	0.41 (0.06)	0.91 (0.02)	0.17 (0.06)	0.11 (0.08)
	Visfatin	-0.32 (0.19)	-0.19 (0.13)	-0.26 (0.21)	-0.05 (0.07)	0.18 (0.07)	0.80 (0.03)	0.27 (0.06)
	TotEggNum	-0.49 (0.34)	-0.34 (0.32)	-0.82 (0.19)	0.02 (0.16)	0.20 (0.20)	0.36 (0.15)	0.24 (0.05)

Acknowledgements

The authors are grateful to:

-the staff of the different research units that helped and lot: Baudouin Rivet, François Lecompte, Christelle Ramé, Marine Cirot, Mathilde Daudon, Marine Chahnamian, and Lola Guerche -the Animal Genetic Division of INRAE for supporting this study -Arthur Gilmour for his guidance in the bivariate analyses parametrization

Funding

This research was funded by the Animal Genetic Division of INRAE.

Ethics statement regarding animals

All data coming from living animals were recorded as part of the breeding program of INRAE Poultry experimental facility (UE PEAT, Nouzilly, France ; https://doi.org/10.15454/1.5572326250887292E12) conducted in compliance the French Ministry of higher education, research and innovation authorization (number agreement 02414.01). The traits involved are egg number, body weight, and backfat thickness.

The other traits were recorded post-mortem, after the animals were euthanized in compliance with national regulations pertaining to livestock production and according to procedures approved by the French Veterinary Services. The traits involved are body composition by tomography, blood adipokines concentrations, and carcass traits (e.g. abdominal fat pad weight).

Data, scripts, code, and supplementary information availability

Data are available online: https://doi.org/10.57745/HUQOXW Scripts and code are available online: https://doi.org/10.57745/HUQOXW

Conflict of interest disclosure

The authors declare that they comply with the PCI rule of having no financial conflicts of interest concerning the content of the article.

Appendix Appendix 1: Diet composition (AVRIL NUTRITION ANIMALE, Bruz, France)

Ingredients: wheat, soybean meal, corn, sodium carbonate, dried and soluble corn distillers grains, barley, monocalcium phosphate, sodium chloride, soybean oil, soybeans, wheat bran, rapeseed meal.

Additional feedstuff: vitamins (A: 10 000 UI/kg, D3: 3 000 UI/kg, E: 21 UI/kg), oligoelements (iron sulfate: 50.3 mg/kg, anhydrous calcium iodate: 1.5 mg/kg, copper sulfate: 10 mg/kg, manganese oxide II: 50 mg/kg, hydrated glycine manganese chelate 30 mg/kg, zinc oxide: 50 mg/kg, hydrated glycine zinc chelate 30 mg/kg, sodium selenite: 0.3 mg/kg), amino-acids (L-lysine sulfate: 545 mg/kg), digestibility enhancer (endo-1.4-beta-xylanase: 560 TXU/kg, endo-1.4-beta-glucanase: 250 TGU/kg, 3-phytase: 5000 FTU/kg) other (lutein extract: 6.0 mg/kg, carothenoids: 4.6 mg/kg, canthaxanthine: 2.0 mg/kg), grappeseed dried extract, organic acids).

Proximate analyses: 17.3% protein, 3.2% cellulose, 2.3% fat, 13.0% ashes, 0.9% Lysine, 0.4% Methionine, 3.9% calcium, 0.1% sodium, 0.4% phosphorus.


Rémi Beau
email: remi.beau@cnrs.fr

The Ecological Community: The Blind Spot of Environmental Virtue Ethics

Keywords: environmental virtue ethics, ecological community, collective virtues, relational self, egocentrism, ecocentrism, self-realization

Since their emergence in the 1980s, environmental virtue ethics (EVE) has aimed to provide an alternative to deontological and consequentialist approaches for guiding ecological actions in the context of the global environmental crisis. The deterioration of the ecological situation and the challenges in addressing collective action problems caused by global changes have heightened interest in these ethics. They offer a framework for meaningful individual actions independently of the commitment of other actors. However, by shifting the focus onto individuals, EVE appear to grapple with the tension between anthropocentrism and respect for nature, as well as between self-flourishing and concern for other living beings. This article argues that this difficulty is rooted in the neglect within EVE of the communitarian aspect of ancient virtue ethics. Drawing from Baird Callicott's ecocentric approach and Val Plumwood's works, it explores the possibility of conceiving ecological communities as collective frameworks in which both public and private virtues are defined and practiced.

Introduction

Since the 1980's, environmental ethics has significantly echoed the revival of virtue ethics in moral philosophy [START_REF] Callicott | Thinking Like a Planet: The Land Ethic and the Earth Ethic[END_REF]. While deontological and consequentialist approaches struggled to show concrete effects on the deterioration of the ecological situation, environmental virtue ethics (EVE) emphasized the role of character and flourishing in the environmental movement. Remaining faithful to the Aristotelian idea that "by doing the acts that we do in our transactions with other men we become just or unjust" [START_REF]Aristotle Complete Works of Aristotle[END_REF] (1103b15), EVE aim to extend this moral approach to our transactions with earth others. These reflections took the form of a series of studies aimed at identifying dispositions for action that could be described as 'environmental virtues' (e.g., humility, frugality, attentiveness) [START_REF] Hill | Ideals of Human Excellence and Preserving Natural Environments[END_REF][START_REF] Wensveen | Dirty Virtues: The Emergence of Ecological Virtue Ethics[END_REF][5]. Several approaches emerged in this regard. Some followed the path of eudaimonist virtue ethics, aiming to demonstrate how respect for the environment was a necessary condition for human flourishing [START_REF] Sandler | Character and Environment. a Virtue-Oriented Approach to Environmental Ethics[END_REF]. Others pursued agent-centred approaches, which sought to identify the motivations found in the actions of exemplary individuals that enable virtuous acts to be defined. The goal was to illustrate how some of the great figures in environmentalism (such as Henry David Thoreau, Aldo Leopold, Rachel Carson) embodied a particular environmental virtue [START_REF] Cafaro | Toward an Environmental Virtue Ethics[END_REF]. Now firmly established in the field of environmental ethics, virtue ethics are facing the most pressing questions that permeate the discipline, particularly those related to global changes. How to define moral obligations aimed at protecting a nature that is constantly changing? How to care for ecological communities in the Anthropocene, that is, in a world where climate change and the erosion of biodiversity continue to accelerate? This article will begin by showing in section 2 how environmental virtue ethics address these questions by proposing a response to the problem of collective action that lies at the heart of the "moral storm" [START_REF] Gardiner | A Perfect Moral Storm: The Ethical Tragedy of Climate Change[END_REF] triggered by global change. Section 3 will analyze two strong objections to this response, claiming that it amounts to a return to an anthropocentric and individualistic ethical framework. The following two sections will examine how some EVE have attempted to escape this criticism by demonstrating how the relational nature of the self implies consideration of earth others in self-realization. However, after pointing out the limitations of these relational approaches in light of the works of Val Plumwood and Baird Callicott, section 5 will advocate for shifting the focus away from the self to explore collectives in which environmental virtues can be exercised. Finally, section 7 will demonstrate how the deindividualization of EVE can involve characterizing collective virtues conceived on the scale of ecological communities.

A response to the problem of collective action

Virtue ethics have grown in significance within environmental discourse as the global dimension of the ecological crisis has become one of the primary challenges to environmental action. Indeed, on a global scale, environmental issues present a particularly challenging "collective action problem." This type of problem, extensively discussed in moral and political philosophy, characterizes situations in which cooperation that would benefit all parties involved is hindered by the risk that some agents may opt not to act, prioritizing their individual self-interests over the common good. An early formulation of this is to be found in David Hume's Treatise on Human Nature published in 1739:

"Two neighbours may agree to drain a meadow, which they possess in common; because it is easy for them to know each others mind; and each must perceive, that the immediate consequence of his failing in his part, is, the abandoning the whole project. But it is very difficult, and indeed impossible, that a thousand persons should agree in any such action; it being difficult for them to concert so complicated a design, and still more difficult for them to execute it; while each seeks a pretext to free himself of the trouble and expence, and would lay the whole burden on others." [START_REF] Hume | A Treatise of Human Nature[END_REF] (p. 538)

In this light, climate change emerges as an immense challenge for cooperation, as it affects all inhabitants of Earth and spans multiple generations. Furthermore, as philosopher Stephen Gardiner has pointed out, the characteristics of this global problem, particularly the spatial and temporal dispersion of its causes and effects, foster forms of 'moral corruption' and free-riding strategies among individual or collective actors [START_REF] Gardiner | A Perfect Moral Storm: The Ethical Tragedy of Climate Change[END_REF]. In the absence of binding regulations enforced by an international institution capable of sanctioning non-compliance, the prospect that the commitment of some may not be reciprocated by others fosters a sense of inaction. Without cooperation, the calculation of costs and benefits doesn't necessarily support committing to action, as it may be more disadvantageous to combat climate change than to do nothing in a world where cooperative partners are not reliable.

At various levels, this issue prompts the question of the significance of ecological commitment.

Particularly at the individual level, recognizing the imperative need to profoundly transform the production and consumption patterns of industrial societies, alongside the observation of collective inertia, threatens to undermine the definition of an ethical life project. Why should I change my way of living, eating, moving, traveling, or housing myself if I know that these changes will not have any impact on global changes? In this context, virtue ethics provide a solution by no longer tying motivation for ecological action to the success of cooperation. Instead, they advocate for the development of dispositions for action, seen as intrinsic qualities of virtuous individuals, and therefore pursued regardless of the actions of others. As noted by American philosopher Dale Jamieson, in the context of climate change, virtues "give us the resiliency to live meaningful lives even when our actions are not reciprocated [START_REF] Jamieson | Reason in a Dark Time: Why the Struggle Against Climate Change Failed -and What It Means for Our Future[END_REF]". Therefore, virtue ethics prove particularly relevant due to their ability to guide individual behavior in a context where environmental disruptions pose new moral challenges that cannot be effectively addressed on an individual basis.

Back to anthropocentrism and individual-centred approach?

However, such an ethical response, in turn, raises an important question. Does it ultimately lead to a departure from the realm of collective action, embodying an individual retreat into ethics, partly motivated by the failure of climate and environmental policies? Thus, EVE may not truly offer a solution to the problem of collective action in environmental matters. Instead, they might lead to giving up on attempting to address it and pose the question of the possibility for individuals to lead a good life, knowing that we have failed to address global environmental issues. In doing so, it would result in a refocusing of environmental ethics on human individuals.

In Radical Ecology: The Search for a Livable World [START_REF] Merchant | Radical Ecology: The Search for a Livable World[END_REF], Carolyn Merchant schematized the shift in ethical thinking brought about by the environmental ethics movement, describing a progression that began with an egocentric stance and moved towards ethics centred on relationships among members of the same eco-community. In light of what has just been said, would the emergence of EVE lead us to travel in the opposite direction?

In fact, from the outset, the resurgence of virtue ethics starting in the 1960s generally faced criticism that viewed this movement as a focus on self-concern at the expense of concern for others. To put it simply, some virtue ethics were accused of theorizing a form of moral selfishness. Canadian philosopher Thomas Hurka argued in this regard that Aristotelian-inspired virtue ethics were, at their core, egoistic because they ultimately made individual pursuit of eudaimonia the central core of ethics [START_REF] Hurka | Monism, Pluralism, and Rational Regret[END_REF]. In such a framework, he believed that the ultimate reasons for moral action always relate to one's own flourishing and not to the flourishing of others. According to him, they would fail to escape a form of moral solipsism: the idea that by placing the self at the center of ethics, they render concerns for others secondary or even inconsequential.

Within the field of environmental ethics, these approaches centred on moral agents presented a second issue. The pursuit of individual flourishing as theorized by eudaimonistic ethics also seemed to narrow the sphere of moral consideration, limiting concerns to humans alone. From this perspective, virtue ethics represented a return to moral anthropocentrism. For many proponents of the intrinsic value of nature, this focus disqualified them from their claim to be an alternative to non-anthropocentric environmental ethics. In this regard, Holmes Rolston highlighted the limitations of these approaches in the field of environmental ethics:

Environmental virtues, as achieved by humans, will initially involve concern for human quality of life. But our deeper ethical achievement needs to focus on values as intrinsic achievements in wild nature. These virtues within us need to attend to values without us. Perhaps one starts with a love of nature that is tributary to self-love. Later one discovers that this self-love is quite inclusive, for the health of myriad nonhumans is implicated, entwined with ours. One is called to an active concern and positive engagement with the object of encounter. The other cannot be seen simply as a source of personal transformation. We must make the model at least an ellipse with two foci: human virtue and natural value [START_REF] Rolston | Environmental Virtue Ethics: Half the Truth but Dangerous as a Whole[END_REF] (p. 69).

For Rolston, the reflection on human virtues can indeed demonstrate how self-concern can benefit others, especially non-human beings. The flourishing of humans indirectly involves, without being aimed at as a moral end, the flourishing of other forms of life. However, according to him, this position, which resembles a weak anthropocentrism, cannot replace the moral imperatives that stem from the recognition of the intrinsic value of nature, insofar as it requires respecting other earth beings, even if they don't contribute to any human's self-flourishing.

In his discussion of EVE, Callicott, in a similar line, critiques the strictly individualistic approach adopted by their theoreticians. He writes as follows:

The Aristotelian cast of the contemporary revival of virtue ethics, consistent with the hyperindividualism of Modern moral philosophy, renders contemporary environmental virtue ethics supportive of the first prong of the virtue ethics at which Leopold hints, the self-respect of the individual [START_REF] Callicott | Thinking Like a Planet: The Land Ethic and the Earth Ethic[END_REF] (p. 255-256).

In summary, EVE would represent a significant regression toward anthropocentrism and individualism compared to the ambitious thinking carried out in environmental ethics since the 1970s.

Relational approaches

Faced with these criticisms, EVE could rely on the arguments put forth by virtue ethics theorists to counter the objection of egoism. One of the most robust responses is based on the assertion that the flourishing sought by the virtuous individual is fundamentally relational. This involves affirming the primacy of relationships in the pursuit of flourishing. This is, for example, clearly articulated by the philosopher Christopher Toner in his response to John Hare's formulation of the self-centred objection: This, then, is the template for a non-self-centred eudaimonistic virtue ethics: the agent seeks to live a life of virtue, where the virtues are simply those traits the possession and exercise of which constitute flourishing for a rational agent of that sort, where to flourish is to stand in the right relation to 'objects' according to their degrees and kinds of goodness, and where the right relation is that which acknowledges the nature or status of each relatum, in such a way that it is held in regard at least in part for its own sake. It is not self-centred to seek one's own flourishing because such flourishing is essentially relational. Attachment to others is not secondary as Hare alleges, but is of the essence of flourish [START_REF] Toner | The Self-Centredness Objection to Virtue Ethics[END_REF] (p. 613).

Toner responds point by point to the previous objections. Because virtues are relational dispositions, they necessarily entail extending consideration to others than oneself. Furthermore, because a relationship cannot be considered good if it only contributes to the flourishing of one of the two related beings, virtue ethics must take into account the specificity of the good for each of these beings. The flourishing of others, which presupposes the recognition of their existence as moral agents who matter for themselves, no longer appears as secondary but as a necessary correlate of one's own flourishing.

The scope of this theoretical reflection on virtue ethics in general quite explicitly extends to the specific domain of environmental ethics. Indeed, taking into account the flourishing of others for their own sake appears to open up virtue ethics to considering the intrinsic value of non-human beings. In his proposal for EVE, Ronald Sandler responds precisely in this manner to the objection that "a Virtue-Oriented Approach Cannot Value Environmental Entities for Their Own Sake [START_REF] Sandler | Character and Environment. a Virtue-Oriented Approach to Environmental Ethics[END_REF] (p. 114)." The relational approach allows him to refute this criticism because, as he writes regarding certain environmental virtues: They are not excellent in themselves, abstracted from the rest of the world. They are excellences in relating to the world. The facts about us (the sort of beings we are) and our world (the sort of demands there are for beings like us) are what make particular character traits virtues or vices. The bases of the virtues, therefore, in elude entities with inherent worth and values "without us." Moreover, they justify responsiveness to all sorts of environmental entities, including landscapes, ecosystems, living things, and sentient beings. Thus, there is no danger of 'environmental virtue losing touch with values or worth in the natural world [START_REF] Sandler | Character and Environment. a Virtue-Oriented Approach to Environmental Ethics[END_REF] (p. 112-113). This conception of ethical inquiry strongly resonates with Arne Naess's moral perfectionism. The theory of the ecological self that he unfolds in his works appears indeed as a relational approach to the self. It asserts that the self is not a social atom separate from the relationships it forms with the world, but rather that it is fundamentally constituted by its relationships with others and with nature. In this framework, making self-realization the goal of ethics would not signify a retreat into a form of egoism and anthropocentrism. Instead, it would involve defining ethical work as a practice aimed at continually improving one's connection to the world. Naess writes:

The greater our comprehension of our togetherness with other beings, the greater the identification, and the greater care we will take. The road is also opened thereby for delight in the well-being of others and sorrow when harm befall them. We seek what is best for ourselves, but through the extension of the self, our 'own' best is also that of others. The own/not-own distinction survives only in grammar, not in feeling. [START_REF] Naess | Community and Lifestyle: Outline of an Ecosophy[END_REF] (p. 175) In summary, proponents of relational approaches counter the criticisms of EVE by arguing that these objections hold true only if one assumes a flawed conception of what constitutes a moral agent. In an atomistic ontology, a moral theory does not seem capable of simultaneously being agent-centred and other-centred. However, they contend that this apparent contradiction dissipates when one adopts a relational conception of the self.

Redefining or decentering the self

However, while they may offer an elegant response to the objections of egoism and anthropocentrism, relational approaches to virtues maintain a certain ambiguity concerning how they seem to describe the merging of personal interests and the interests of others. According to these propositions, the risk of theorizing a form of moral egoism dissipates when one understands that self-realization entails the realization of the others who constitute this self. In Naess's terms, self-realization cannot be selfish if it pertains to an ecological self. But how can we ensure that this is indeed an overcoming of egoism and not merely a resolution of the tension that may exist between self-flourishing and the flourishing of others through the absorption and dissolution of others' interests?

Analyzing the discussion between Naess and the existentialist philosopher Peter Reed on this matter, Val Plumwood highlights the indecisive nature of self-realization: On first glance, Naess's account does not appear to appeal to either fusion or to egoism -since we are supposed to defend not the self but the big Self as 'the totality of our identifications'. But, says Reed, there seem to be inconsistent requirements hidden here: we are supposed to retain a sense of our individuality as we work to save the big Self from destruction -but at the same time we are supposed to lose interest in our individuality as we cultivate our identification with the big Self.1 We are required to be egoists and also not egoists, to retain the intensity and defence drive of egoism, but also to abandon certain key differentiations between ourselves and others, in order to establish that equivalence between self and other which enables a transfer of our selfregarding motives. Naess's position, on closer inspection, ultimately is based on a kind of self-interest and upon a form of fusion or expulsion of difference -taking the form, as Naess explains in his reply, of identity of interests [START_REF] Plumwood | Environmental Culture: The Ecological Crisis of Reason[END_REF] (p. 199).

According to her, the model of identification that guides the realization of the ecological self ultimately leads not to a critique of egoism but to its extension through this expansion of the self.

In an article on the concept of self-realization in Naess's philosophy, Callicott presents a similar critique by arguing that Naess's thought has evolved from a genuinely relational conception of the self that was emerging in the foundational article on deep ecology [START_REF] Naess | The Shallow and the Deep Long-Range Ecology Movement. A Summary[END_REF] to a monist conception inspired by Hinduism. Regarding the former, he writes:

What is an ecological approach to being in the world? In the context of 'The Shallow and the Deep' article Self-realization is a relational conception of the self and, more importantly, an experience of oneself in relational terms. One is and one experiences oneself to be a 'knot in the biospherical net of intrinsic relations.' Or better: as a node or nexus in a skein of internal socio-environmental relations. One experiences 'the beauty and complexity of nature [as] continuous with [one]sel[f].' [START_REF] Callicott | Notes on "Self-Realization: An Ecological Approach to Being in the World[END_REF] (p. 239) However, according to Callicott, this conception fades later on, particularly in Ecology, Community and Lifestyle [START_REF] Naess | Community and Lifestyle: Outline of an Ecosophy[END_REF]. In this work, he observes an unification and merging of selves that he finds problematic and describes as follows:

In the metaphysics of Advaita Vedanta, the atman or Self at the core of oneself is equated with Brahman, the one universal Being at the core of all other selves (jivas) and indeed at the core of all phenomena whatsoever. In short, there is a unity in multiplicity and that unity is Atman-Brahman. There is, thus, an actual, literal common identity that oneself shares with other selves. My Self is also your Self; my Self and your Self are not two (advaita); they are one and the same. All Selves are one and the same Self. [START_REF] Callicott | Notes on "Self-Realization: An Ecological Approach to Being in the World[END_REF] (p. 244) From this perspective, it becomes apparent that relational approaches can lead ethical reflection into what Plumwood refers to as the "Ocean of continuity [START_REF] Plumwood | Feminism and the Mastery of Nature[END_REF] (p. 155)," which no longer allows for the recognition of the distinct differences of each moral agent. If everyone is directed on an ethical path that leads from the ego to the Self, this process takes the form of a "one-place relationship" [START_REF] Plumwood | Environmental Culture: The Ecological Crisis of Reason[END_REF] (p. 197) that does not acknowledge other beings as centers of resistance with their own interests and goods. Consequently, ethical reflection may fail to break free from the monological relationship between the self and nature that characterizes centric constructions of ethics. In the end, an EVE that follows this path would fall under the previous charge of defending egocentrism.

For Plumwood, this is the reason why virtue ethics should redirect the focus from self-flourishing towards interpersonal relations. Virtues, especially environmental virtues, are, in her view, dispositions to act against "the oppressive ideologies of domination and self-imposition that have formed our conceptions of both the other and ourselves" [START_REF] Plumwood | Environmental Culture: The Ecological Crisis of Reason[END_REF] (p. 194). She calls them counter-hegemonic virtues. Through the exercise of these virtues, moral agents cultivate a form of decentering that involves attention and openness to the alterity of the diversity of earth others. The horizon of this virtue ethics is a world without a single center, in which the diversity of beings, acting as nodes in the biospheric network, is recognized in their specificity as moral agents.

From counter-hegemonic to collective virtues

Against the holistic view of the merging of the moral self into a vast undifferentiated whole, Plumwood and Callicott emphasize the plurality of selves and non-human agents. From this perspective, they assert that adopting a relational ontology does not imply the absorption of divergent interests or their alignment towards a single process of flourishing. Furthermore, they argue that EVE's central concern should not be the individual flourishing of moral agents, but rather the characterization of a state of the community to which they belong conducive to establishing a coexistence that respects the diversity of interconnected beings.

In that sense, Callicott assesses that EVE's main shortcoming is precisely that it failed to draw on what ancient philosophy offered in terms of thinking about virtues not merely as individual or even relational traits but as attributes of collectives. Collective virtues, in his view, represent the unexplored dimension of the contemporary revival of virtue ethics in the environmental domain.

In his own analysis of the potential of virtue ethics to address environmental issues, Callicott outlines three levels: personal, professional, and societal [START_REF] Callicott | Thinking Like a Planet: The Land Ethic and the Earth Ethic[END_REF] (p. 158). The latter two differ from the first in that they refer to institutions or collective actors. Professional virtues are associated with ways of excelling in the practice of a profession. Societal virtues, on the other hand, characterize an entire community capable of cultivating traits that enable self-respect. According to Callicott, these virtues are particularly important because they allow us to address environmental problems at the collective level. However, EVE, including their relational versions, have not been able to provide satisfactory answers because they have primarily focused on personal virtues.

What exactly does Callicott mean by collective virtues? A rich philosophical debate on the definition or even the existence of this type of virtue and the nature of the groups capable of exercising them has emerged [START_REF] Fricker | Can There Be Institutional Virtues[END_REF][START_REF] Byerly | Collective Virtue[END_REF]. This debate intersects with the theoretical discussions initiated by Margaret Gilbert's work on collective actions and beliefs [START_REF] Gilbert | On Social Facts[END_REF]. Without going into the details of this discussion, it is sufficient to mention here that the concept of collective virtue does not imply granting personhood status to groups. Following Ryan and Meghan Byerly's proposal, a collective virtue can be defined as follows:

A collective C has a virtue V to the extent that the members of C are disposed, qua members of C, to behave in ways characteristic of V under appropriate circumstances [START_REF] Byerly | Collective Virtue[END_REF] (p. 46)

The collective dimension of these virtues is expressed in the fact that individuals or agents exercise them qua members of a community to which these virtues are attributed.

But what communities are we referring to here? In his discussion, Callicott mainly mentions the ancient model of the polis. His references are to Aristotle and Plato [START_REF] Callicott | Thinking Like a Planet: The Land Ethic and the Earth Ethic[END_REF] (p. 259-261). However, this anchoring raises serious difficulties, first of all, because these social forms no longer exist, and moreover, their highly unequal structure makes them difficult to mobilize as a normative horizon within contemporary societies. These difficulties are at the heart of Alasdair McIntyre's work. How can we revive a virtue ethics that depends on social conditions that have disappeared? The analysis of this question takes an aporetic turn in After Virtue [START_REF] Macintyre | After Virtue: A Study in Moral Theory[END_REF]. McIntyre does not believe that modern nation-states can embody social forms conducive to the development of a communal life oriented toward a common ideal of the good life that would allow for the definition of private and public virtues. He then retreats to small face-toface communities that he believes are the only remaining spaces for the development of a virtue ethics. However, does the idea of public virtues necessarily lead to this pessimistic and politically conservative conclusion? A first response can emphasize that virtue ethics may not be destined to embody the type of moral authority capable of ordering society, as McIntyre aspires to. In a version more compatible with democracy, it can be conceived more modestly as an ethical reflection that engages in public discourse about the organization of common life. A second response can draw inspiration from the critique addressed by Plumwood to McIntyre, in which she asserts that virtue ethics is not necessarily linked to the attempt to resurrect social structures from the past but can accompany the invention or recognition of other more emancipatory social forms [START_REF] Plumwood | Feminism and the Mastery of Nature[END_REF] (p. 186). Such a proposition allows us to redirect the discussion on environmental virtues toward the identification of collective virtues that can be exercised within ecological communities.

Collective virtues within ecological communities

Critiquing the individualistic approach of EVE, Callicott calls for a shift in thinking by suggesting that environmental virtues could pertain to collectives or communities. This notion prompts to delve further into two questions: At what collective scale is it relevant to locate environmental virtues? Which environmental virtues can be defined as collective virtues?

The first question has the merit of drawing attention to the contextualization of the reflection on environmental virtues. While the theorization of individual virtues has often leaned towards the abstract characterization of virtuous environmental individuals, the approach through collective virtues appears as a way to reposition ethical reflection within a specific place and among a community of members who feel at least partially responsible for the decisions and actions of this community. In this regard, Plumwood repeatedly emphasizes the contextual dimension of the virtue ethics she describes.

Because spatial proximity promotes attention to the ecological consequences of our actions, smallscale communities seem to provide an especially conducive framework for practicing an ethics that values the care given to the relationships that bind the diversity of living beings. Plumwood appears to align with McIntyre in this regard by focusing on small face-to-face communities. In her discussion of bioregionalism, she acknowledges how the movement has effectively highlighted the invisibilization of ecological relationships resulting from the remoteness of chains of production and distribution and the benefits of relocalizing production activities. She also subscribes to the idea that decentralizing decisionmaking, on the contrary, encourages the consideration of the effects of these decisions on the environment. Finally, she argues that true participatory democracy can only occur at this local level. She writes on this subject: Democracy can only be truly participatory at the level of the small, face-to-face community, people will be in a position to have the knowledge and motivation as well as the democratic and communicative means to make good ecological decisions, decisions that reflect their own extended long-term and familiar interests as well as those of their local ecologically-defined communities. [START_REF] Plumwood | Environmental Culture: The Ecological Crisis of Reason[END_REF] (p.

74)

In summary, Plumwood's analysis leads us to consider that local socio-ecological communities represent a good scale for the collective practice of environmental virtues. However, she also cautions against the risks of becoming confined to localism that could generate other forms of invisibility. She observes that the proximity to nature facilitated by small-scale communities does not necessarily guarantee "the first condition of the bioregionalist, the transparency to inhabitants of ecological relationships and dependencies [START_REF] Plumwood | Environmental Culture: The Ecological Crisis of Reason[END_REF] (p. 76)." From this perspective, while McIntyre tended to focus on goods internal to communities, Plumwood's concerns also revolve around embedding these communities within the broader network of socio-ecological relationships. According to her, environmental virtues must precisely help cultivate a "critical sense of place that can situate local relationships and communities in relation to wider communities" [START_REF] Plumwood | Environmental Culture: The Ecological Crisis of Reason[END_REF] (p. 77). This leads us to the second question raised concerning the identification of collective and environmental virtues. What kind of trait or disposition to action could members of an ecological community cultivate as members of that community and be described as an environmental virtue? Among the list of virtues identified by Plumwood, solidarity emerges as an interesting candidate in this context. Thus, while this reflection will need to be broadened by considering other collective virtues, such as conviviality, the end of this section will focus on the concept of solidarity, which has already found significant resonance in ecological thought [START_REF] Mathevet | Environmental Stewardship and Ecological Solidarity: Rethinking Social-Ecological Interdependency and Responsibility[END_REF]. While she does not explicitly define it as a collective virtue herself, solidarity does indeed meet the criteria of a virtue that characterizes a collective rather than an individual. One would describe a community as "solidary" if the members of that community act and make decisions that demonstrate empathy towards others and an acknowledgment of the interdependent situation in which all members of that community find themselves. As noted by Byerly, there is no individual-level analogue to solidarity, which is why it constitutes a specifically collective virtue [START_REF] Byerly | Collective Virtue[END_REF].

Plumwood assigns a pivotal role to solidarity in her virtue ethics. She carefully distinguishes it from unity in her concern for respecting differences. Analyzing the central role of this concept in Plumwood's work, Chaone Mallory writes:

Political solidarity for Plumwood is a relation in which one (or, as is more suitable for our purposes, the collective) does not claim an identification with the other-political solidarity describes a relation in which beings are motivated to act on behalf of others with whom one admits one does not (necessarily) share experiences, interests, worldviews, or subjectivity. However, those standing in solidarity become joined both with the object of solidarity and others involved in struggling for change through the shared recognition of injustice and oppression and through acting to change it. [START_REF] Mallory | Val Plumwood and Ecofeminist Political Solidarity: Standing with the Natural Other[END_REF] (p. 8) As Mallory suggests, ecological solidarity is a form of political solidarity in Plumwood's philosophy. It is indeed a collective and public virtue, in the sense of being a "character trait[s] that bring[s] us into virtuous relationships with our communities and environments" [START_REF] Treanor | Environmentalism and Public Virtue[END_REF] (p. 18). This approach has the merit of undoing most binary distinctions that seemed to render EVE ineffective, particularly the dichotomies between the individual and the collective, the private and the public, or ethics and politics. In addition, rather than in opposition, to the realization of an ecological self, the work on collective virtues can be interpreted as a process through which members of an ecological community care for the flourishing of the diversity of living beings within it.

Conclusion

During their initial phase of development, EVE were associated, both by their theorists and their critics, with the solitary practice of virtues that exemplified what a life respectful of the diversity of living beings on Earth could be. This analytical framework aligned with a common way of narrating the history of environmental conservation, which successively invokes the prominent figures of the environmental cause, such as Thoreau, Muir, Leopold, and Carson. Despite their claims to the contrary, these individualistic approaches to environmental virtue ethics are seen by many of their critics as a form of depoliticization of environmental action.

However, the contributions of Plumwood and Callicott to this debate introduce a different perspective within EVE. By reintroducing social forms and ecological communities in which environmental virtues can be practiced, they demonstrate how a virtue ethics approach can, on the contrary, resonate with new forms of politicization of ecological issues.