Adrien François

Iman Laziz

Stephanie Rimbaud

Denise Grebert

Didier Durieux

Edith Pajot

Nicolas Meunier

Early survival factor deprivation in the olfactory epithelium enhances activity-dependent survival

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Early survival factor deprivation in the olfactory epithelium enhances activity-dependent survival

Adrien François1,2,3 , Iman Laziz 1,2,3 , Stéphanie Rimbaud 1,2,3 , Denise Grebert 1,2 , Didier Durieux 1,2 , Edith Pajot-Augy 1,2 , Nicolas Meunier 1,2,3 www.jouy.inra.fr rI7 mRNA level is decreased after treatment. Odorization with octanal significantly reversed this decrease in a dose-dependent manner. The mRNA expression levels of 3 other ORs were statistically decreased by BQ treatment, one (U131) was significantly increased (odorant present in rat milk). The effect of BQ treatment was affected by octanal odorization only for rI7. Finally, coodorizaton with citral, a known antagonist of rI7, limited the effect of octanal odorization. While the number of OSNs on turbinates IIa, IIb and II was stable between control and treated animal, it was significantly affected in turbinates IV. When the dam is odorized with octanal the increased rI7-expressing neurons apoptosis in the OE of BQ treated animal is reversed.

  the olfactory mucosa of rats treated by intranasal antagonists of endothelin receptors : TUNEL Responses of olfactory mucosa to odorants from rats treated by intranasal antagonist of endothelin receptors : EOG Olfactory driven orientation behaviour of rats treated with ET receptor antagonists : Behaviour Test TUNEL signal was enhanced in all layers of the olfactory epithelium after the ET receptor antagonist treatment (twice a day during 7 days). OSNs population Real-time quantitative PCR 10 days old rats treated or not with ET receptor antagonist were monitored for their orientation toward mother's bedding. Treated animals scored better despite their lower EOG amplitude response to odorant and higher level of apoptosis in the olfactory neuron layer of their olfactory epithelium.



  Endothelin system inhibition in young rats leads to  Increased apoptosis in all cell layer  Decreased amplitude of odorant response recorded by EOG  Improved odour based orientation behaviour toward maternal smell  Modulation of OR mRNA level dependent of odorant presence  Endothelin acts as an anti-apoptotic factor in the olfactory mucosa. Its inhibition could lead to improved detection of odorants already present in the animal's environment by increasing their relative OSN sensitive population.

INRA, UR1197 Neurobiologie de l'Olfaction et Modélisation en Imagerie, Jouy-en-Josas, France;

IFR144, NeuroSud Paris, Gif-Sur-Yvette, France;

Université de Versailles Saint-Quentin en Yvelines, Versailles, France. INSTITUT NATIONAL DE LA RECHERCHE AGRONOMIQUE CENTRE de Jouy-en-Josas Unité NOeMI -Neurobiologie de l'olfaction et Modélisation en Imagerie Domaine de Vilvert • 78352 Jouy-en-Josas Cedex • FRANCE • email : adrien.francois@jouy.inra.fr


Stéphane Berciaud

Laurent Cognet

Philippe Tamarat

Brahim Lounis
email: b.lounis@cpmoh.u-bordeaux1.fr

Observation of intrinsic size effects in the optical response of individual gold nanoparticles

The Photothermal Heterodyne Imaging method is used to study for the first time the absorption spectra of individual gold nanoparticles with diameters down to 5 nm. Intrinsic size effects which result in a broadening of the Surface Plasmon Resonance are unambiguously observed. Dispersions in the peak energies and homogeneous widths of the single particle resonances are revealed. The experimental results are analyzed within the frame of Mie theory.

Noble metal nanoparticles are strong absorbers and scatterers of visible light due to Surface Plasmon Resonance (SPR). The corresponding resonant peak energies and linewidths are sensitive to the nanoparticle size, shape and nano-environment [START_REF] Kreibig | Optical properties of metal clusters[END_REF] . Moreover, plasmon resonances allow for the enhancement and manipulation of local electromagnetic fields at nanoparticle surfaces [START_REF] Moskovits | [END_REF][3][4] . Because of these singular optical features, noble metal nanoparticles stimulate great interests for implementations in photonics 5 or biotechnology 6,7 .

The spectral properties of the SPR have been extensively studied on ensembles of noble nanoparticles and compared to Mie theory 8,9 . For rather large nanoparticles (diameter D ≥ 20nm), the resonant peak energy E R experiences a red-shift with increasing sizes, due to retardation effects as well as to increasing contributions from multipolar terms. In addition, for nanoparticles larger than 50 nm, radiative damping of the collective electronic excitation significantly broadens the linewidth of the SPR.

For smaller nanoparticles (D < 20 nm), these extrinsic size-effects become negligible and intrinsic sizeeffects prevail. They account for size-dependent modifications of the dielectric constant with respect to the bulk values, due to additional surface damping [START_REF] Kreibig | Optical properties of metal clusters[END_REF]10 and translate into a broadening of SPR linewidths with decreasing sizes, and to a lower extent, into slight shifts of E R . Comparisons between theory and ensemble measurements are still a matter of debate because the inhomogeneities in nanoparticle size, shape and local environment experimentally hide the homogenous width of the SPR.

For large particles, scattering-based methods allowed to perform SPR measurements on individual particles and to study the extrinsic size-effects 11,12 . No intrinsic-size effects from additional surface damping were observed since a good agreement was found with Mie theory using the bulk values for the metal dielectric function 13 . Because the scattering cross-section of nanoparticles decreases as D 6 , these methods are limited to nanoparticles with D ≥ 20 nm. For smaller particles, different methods have been proposed to access the homogeneous SPR linewidth. Persistent spectral hole-burning techniques have recently been applied 14 : by the extrapolation of measurements performed at high intensities, homogeneous width values of a subpopulation of metal nanoparticles with a given resonant energy could be extracted from the inhomogeneous band. Concurrently, it was recently shown that plasmon resonance of single nanoparticles smaller than 10 nm can be measured by detecting the scattered field by one particle, through an interferometric method 15 . In that case, the measured signal scales as D 3 similarly to the absorption cross-section 16 .

In this letter, we used a recently developed photothermal far-field absorption-based method 17 , which allows to perform for the first time a systematic study of absorption spectra of individual gold nanoparticles with diameters down to 5 nm with a high signal-to-noise ratio. We found that a very small ellipticity in the nanoparticle shape results in a significant dispersion on the peak resonant energy E R .

We directly measured the homogeneous SPR linewidths and show that for nanoparticles with nm D 10 ≤ they are significantly affected by surface damping mechanisms leading to reduced SPR decoherence times.

We applied the Photothermal Heterodyne Imaging (PHI) method which allows for the detection of very small gold nanoparticles down to 1.4 nm in diameter 17 . It uses a combination of two laser beams: a intensity-modulated heating beam, close-to-resonance, and a cw off-resonance probe beam. Absorption of the heating beam by a nanoparticle induces a time modulated increase of the temperature in the vicinity of the nanoparticle. Propagation of the probe beam through the resulting time-modulated index of refraction profile, produces a frequency shifted scattered field which is detected by its beatnote with the probe field on a fast photodiode. The signal is thus directly proportional to the absorption cross section σ abs of the nanoparticle. The experimental setup is presented in Figure 1a. The heating beam was delivered by a tunable cw dye laser (Coherent 599, tuning range 515-580 nm i.e. photon energy range 2.41-2.14 eV). The intensity modulation of the beam (300 kHz) was performed by an acousto-optic modulator (AOM). Wavelength-dependent angular deviations were compensated by double pass through the AOM. The beam was linearly polarized and overlaid with a probe beam (HeNe laser, 633 nm) and focused on the sample.

In order to avoid any dependence of the relaxation dynamics of electrons on the absorbed power, we performed the experiments in the weak excitation regime where the average time between two successive absorption events is significantly longer than the relaxation times in metal nanoparticles (of the order of few ps) 18 . This corresponds to heating powers ranging from ≈ 1 µW to ≈ 500 µW (for D = 33 nm to D = 5 nm respectively), allowing to image the individual nanoparticles with signal-to-noise ratios greater than 10 at the peak resonant energies for 10ms integration times (see Figure 1b andc).

We prepared the samples by spin-coating solutions of gold nanoparticles (average diameter: 5 nm, 10 nm, 20 nm, 33 nm, diluted in 2% mass polyvinyl-alcohol (PVOH) aqueous solution) onto clean microscope coverslips. The dilution and spinning rates were chosen such that the final density of nanoparticles in the samples was less than 1 µm -2 . The size dispersion of the nanoparticles (~10%) was checked by transmission electron microscopy (TEM). A drop of viscous silicon oil was added on top of the samples to ensure homogeneity of heat diffusion. In the calculations, we assume that the refractive index around the nanoparticles was n m = 1.5.

Prior to spectra acquisitions, single nanoparticles were first located by imaging a 10×10 µm 2 area of the sample at fixed heating wavelength. For each single nanoparticle, spectra were then acquired by automatically sweeping the laser photon energy by steps of 10 meV across the resonance with a 2 s integration time per point. The photothermal signals were corrected for the wavelength dependence of the diffraction-limited laser spot size. The heating power was measured during the acquisitions and used to normalize the spectra.

Figure 2 shows examples of absorption spectra recorded for 4 individual nanoparticles of different sizes. One can already notice that both a blue-shift and a broadening of the SPR with decreasing particle sizes are clearly visible. The SPR spectra are asymmetric due to interband transitions. Indeed, in gold, the energy threshold for interband transitions lies at ~2.4 eV and is preceded by an absorption tail starting at about 1.8 eV 1 . Defining a full-width-at-half maximum of absorption spectra for gold nanoparticles would thus be delicate, and even impossible for very small particles. As a consequence, we extracted from each spectrum the peak resonant energy E R , and the homogenous red half-width at half maximum

2 1 2 1 E E R - = Γ
where

2 1
E is the photon energy for which the absorption is half of the peak value (see Figure 2a) 19,20 .

For each particle size, we built the histograms of E R and 2 1 Γ (Figure 3a to h) from more than 30 single particle SPR spectra. The distributions are unimodal, but fairly wide and will be used in the following to precise the overall features observed in Figure 2. We first compare in Figure 4a the average values of E R deduced from the histograms plotted in Figure 3a to d with the size dependence predicted by Mie theory. The red-shift experienced by E R for increasing sizes is well reproduced by the theory (Figure 4a, dotted line) using the bulk dielectric function of gold 21 . Concerning the dispersion of the measured values E R (standard deviations of 17, 20, 14, 18 meV for particles diameters of 33, 20, 10, 5 nm respectively), it cannot be explained by the dispersion in the nanoparticles diameters. On the contrary, a slight ellipticity of the particles which are randomly oriented with respect to the laser polarization can explain this dispersion. Indeed, Figure 3i to l reports the distributions of the particles aspect ratios deduced from TEM images. All distributions are narrow with a mean aspect ratio of 1.1 ± 0.1, and by extending the Mie theory to the case of these randomly oriented non-spherical particles 8,22 , we predict a dispersion in E R of ~20 meV. As a consequence, although variations of E R due to inhomogeneities of the dielectric environment cannot be completely ruled out, we find that the measured width of E R can fully be explained by the ellipticity of the nanoparticles.

Mie theory with the bulk dielectric functions only predicts a minor dependence of the SPR linewidth with particle size (dashed line in Figure 4b). In order to explain the experimental variations of 2 1 Γ with D (scattered dots in Figure 4b), surface effects have to be considered through a size-dependent dielectric

function 1 : ( ) ( ) ( ) D i D p IB ωγ ω ω ε ω ε + Ω - + = 2 2 1 ,
where Ω p is the bulk plasma frequency. ( ) Γ which are more pronounced for the smallest particles (standard deviations of 13, 15, 20, 25 meV for particles diameters of 33, 20, 10, 5 nm respectively). Nanoparticle ellipticity can lead to measurements with significantly broadened SPR lines 22 . However, since the aspect ratio of our particles is 1.1 (Figure 3i to l), we calculate that the ellipticity contributes for ~10 meV in the dispersion of the 2 1 Γ histograms. Therefore it accounts only partially for the measured dispersions. Heterogeneities in the interface decay channels 1 , for which the smallest nanoparticles are most sensitive can explain the additional dispersion.

D ,
For gold nanoparticles, because of the non-Lorentzian profile of the extinction cross section, the bandwidth usually used to define the plasmon dephasing time is the red half width of the absorption band 19,20 :

2 1 2 Γ = T
. We found values of T 2 ranging from 5.9 fs for 33 nm nanoparticles down to 4.1 fs for 5 nm nanoparticles. These values are larger than those deduced from ensemble measurements 19 . This difference can be attributed to inhomogeneous broadenings in ensemble measurements.

In conclusion, we have measured for the first time the absorption spectra of individual gold nanoparticules as small as 5 nm. We have clearly evidenced intrinsic size effect in the optical response of the smallest particles and accessed the homogeneous width of their SPR. Future developments include the study of silver nanoparticles for which interband damping plays minor role in the SPR bandwidth and the spectroscopy of complex structures made of small nanoparticles. The heating power was less than 500 µW and the integration time per pixel was 10 ms. Γ ((e)-(h)) extracted from single particle SPR spectra. The average size of the studied nanoparticles is 33 nm ((a), (e)), 20 nm ((b), (f)), 10 nm ((c), (g)) and 5 nm ((d), (h)). For each particle size, the histograms are built from more than 30 single nanoparticle SPR spectra. The aspect ratios histograms of the nanoparticles (33 nm (i), 20 nm (j), 10 nm (k) and 5 nm (l)) are extracted from TEM images (insets). The disparity in the total number of particles contributing to these histograms is due to variations in TEM samples preparation.

γmeV and v f = 1 . 4 1 . 1 Γ . One should mention that 2 1 Γ 1 Γof 2 1 Γ

 1411111 is due to interband transitions and assumed to be size independent, whereas the second is the Drude-Sommerfeld free electron term. It contains the size-dependent phenomenological damping constant: is the bulk damping rate and v f is the Fermi velocity of the electrons. For gold, nm fs -1 . The dimensionless size parameter A accounts for additional surface damping terms. More precisely, surface scattering is usually described in terms of inelastic collisions which shorten the electron mean free path when the nanoparticle sizes decrease. Additionally, for embedded nanoparticles, chemical interface damping, i.e. fast energy transfer between the nanoparticle and its close surrounding, may also damp the phase coherence of the collective oscillation 1, 14 when the particle plasmon energy is close to adsorbate energy levels. As the number of conduction electrons participating in the collective oscillation is proportional to the volume of the nanoparticle, the size dependent correction in ( ) D γ scales as the surface-to-volume ratio i.e. DUsing the Mie theory with the size-dependent dielectric function, we simulated the absorption spectra for the different particles sizes (solid lines in Figure2) and found 25 . 0 = A as the best value for the size parameter. From those simulations, we could then extract a theoretical red half-width at half maximum 2 contains the two damping contributions mentioned above.We now compare in Figure4, the size-dependence predicted by the Mie theory using 25 the influence of the uncertainties in the tabulated values of the bulk dielectric function21 ) with the measured average values of E R and 2 . First, Figure4(a) shows that introduction of the non-zero value of A does not significantly affect the expected values of E R which maintains the agreement between the measurements and Mie simulations. Second, the theoretical and experimental variations with D are shown in Figure4(b). We find for the same value of A , a good agreement between the experimental widths and Mie simulations, a clear signature of the presence of intrinsic size effects in the absorption spectra of small nanoparticles.Interestingly, we observe notable dispersions in the measured values of 2 1

Figure 1 :

 1 Figure 1: (a) Schematic of the experimental setup. The heating beam intensity is stabilized by an

Figure 2 : 1 Γ

 21 Figure 2: Normalized absorption spectra of single nanoparticles of various diameters. (a) 33 nm, (b) 20

Figure 3 :

 3 Figure 3: Histograms of E R ((a)-(d)) and 2 1

Figure 4 : 1 Γ

 41 Figure 4: Size dependence of E R (a) and 2 1 Γ (b). Experimental data (circles with standard deviations)

Figure 1 : 11

 111 Figure 1:

Figure 2 :

 2 Figure 2:

Figure 4 :

 4 Figure 4:

ACKNOWLEDGMENT: We thank O. Lambert and A. Brisson for the TEM measurements, V. Sandoghdar for valuable discussions, O. Labeau and G. A. Blab for their assistance and P. Morin (Coherent France) for the loan of the dye laser. This research was funded by the CNRS and the MENRT (ACI Nanoscience and DRAB) and by Région Aquitaine.


Audrey Didier

Marion Deloche

Florence Exbrayat-Vinson

Cindy Haller

Lionel Bardy

François Balfourier

Certification process of small grain cereals BRC in France

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

Mains improvements Development of a process approach Activities are managed annually by an action plan

Definitions of objectives and priorities in quality policy System's presentation in quality manual Redaction of BRC's Terms and conditions

Certification process of small grain cereals BRC in France

How we did it?

Project was funding through French biological, health and agronomy infrastructure (IBiSA) for 2 years with recruitment of a quality engineer for a non-permanent period of 15 months.

Small grain cereals BRC's members involved in certified activities were training to quality and responsibility of process was shared out each individual.

Collaborations / tools

Strong interactions with French plant BRC network, Exchanges on problematic and documentation with others certified French BRC, Implementation of AQ-Tools, developed by R. Cottin (CIRAD) to manage our quality system.

Regeneration and conservation

Reviewing and regular control of storage's conditions Active collection: Cold room 100m 3 ; at +4°C and 30% relative humidity Base collection: Freezer ; at -20°C as secure storage

Distribution of sample

New order request form was produced with required informations ie. intended use of genetic resources. The objectives are to get feed back on project using our genetic resources and on data produced. With certification, we have reinforced traceability of action from request to distribution.

To better understand the expectation of genetic resource's users, we carried out a satisfaction survey to genetics resources requester between September 2013 to August 2014. 43% of questionnaire was returned with a global satisfaction of 92%. Improvement suggestions from users were taking into account and introduce in our action plan or informations were provided in our documentary (quality manual, BRC's Terms and conditions).

To improve access to a list of resources and associated data available in our collection, an online database is updated regularly -SIReGal www.urgi.versailles.inra.fr/siregal Didier A., Deloche M., Exbrayat F., Haller C., Bardy L., Balfourier F.

The small grain cereals Biological Resources Centre (BRC) is connected to INRA Auvergne-Rhône-Alpes and is a part of the Joint research unit UMR 1095 Genetics, Diversity and Ecophysiology of Cereals. 2 axis is developed by BRC: -Management, at national level, of collections of wheat, barley, oat, rye, triticale and wild related species, -Enhancement of collections in research programs at national and international level.

Why going thought a certification process?

With the increase of the collection (currently 27 000 accessions) and associated data, the evolution of genetic resources regulation and new notion of collection of references, traceability requirement need to be reinforced. The development of quality system management was the occasion to redefine BRC's organization, taking into account of these news notions. Small grain cereals BRC obtained in June 2015 certification under NF S 96-900 standard, French standard specific to BRC management, for these activities of receipt, preparation, propagation, preservation and provision of biological resources.

Acquisition

New form to integrate accession with required informations ie. regulation status Humidity tests after harvest were also setting up to insure that seeds introduced in cold room are between 11% and 12% of relative humidity. Sample's viability is evaluated after multiplication by random sampling of 200 accessions and when distributed up to 400 accessions.

France Abroad

Fig. 3 :Fig. 4 :

 34 Fig. 1: Process mapping


Francesco Grisolia
email: grisolia@unicz.it.

Emanuele Ferragina
email: emanuele.ferragine@sciencespo.fr

Social Innovation on the Rise: yet another buzzword in time of austerity 1 ?

We challenge the idea that social innovation can constitute an effective strategy to counterbalance the retrenchment of public social provisions. The changes within the Italian healthcare system, one of the most important sectors of the welfare state, in which social innovation ideas have been widely discussed, are a clear example of a broader trend toward decentralisation and marketization. In conjunction with a new balance between public and private funding, these changes are contributing to increased inequalities and territorial disparities. A social innovation perspective, which is not embedded within a structural reform of the Italian welfare state and the health care system, might simply become a convenient buzzword to forward neoliberal ideology in a time of austerity.

Introduction

Over the last two decades social innovation has gained significant popularity 4 as a strategy to tackle new social risks [START_REF] Bonoli | The Politics of the New Social Policies. Providing Coverage Against New Social Risks in Mature Welfare States[END_REF][START_REF] Taylor-Gooby | New risks and social change[END_REF] including population ageing and its health correlates (Franz, Hochgerner and Howaldt, 2012;Hubert, 2010;[START_REF] Mulgan | Social Innovation. What it is, why it matters and how it can be accelerated[END_REF][START_REF] Mulgan | Innovations in Health: Approaches from the Regional Innovation Funds[END_REF][START_REF] Murray | The Open Book of Social Innovation[END_REF]OECD, 2011;Phills et al., 2009). However, as other concepts recently developed within the academic debateamong them, social capital [START_REF] Ferragina | Social Capital in Europe: a Comparative Regional Analysis[END_REF](Ferragina, , 2013a;;[START_REF] Ferragina | The Parabola of Social Capital: Requiem for a Theory?[END_REF][START_REF] Muntaner | Social Capital, Disorganized Communities, and the Third Way: Understanding the Retreat from Structural Inequalities in Epidemiology and Public Health[END_REF] social innovation might soon turn out to be simply another way to juxtapose the qualifier "social" to the private sector jargon (Phills, Deigleimer and Miller, 2008, p. 36), in order to avoid heated discussions on structural inequalities 5 .

Social innovation is commonly defined as new ideas (products, services and models) that simultaneously meet social needs and create new social relationships or collaborations. These innovations are considered both good for society and capable of enacting greater societal involvement in the provision of social services (Murray, Caulier-Griece and Mulgan, 2010, p.3) 6 . The Young Foundation, in order to distinguish between social and business innovation, stressed that social innovation is developed and diffused via organisations, whose primary purposes are not centred on mere profit maximisation (Mulgan et al., 2007, p. 8). The Bureau of European Policy Advisers more precisely defined social innovation as socially oriented in both ends and means (Hubert, 2010). According to these influential definitions, social innovation is characterised by: the capacity to address social needs that traditional policy seems increasingly unable to tackle; the empowerment of groups and individuals; and the willingness to change social relations. Hence, social innovation is often presented as a way to increase the quality of social services and their cost-effectiveness, offering equivalent, if not superior, outcomes despite considerable budget constraints.

The aim of this paper is to question the idea that social innovation can substantially address the deficiencies and inadequacies of the welfare state. In particular, discussing the case of the Italian health care system, we show that social innovation cannot counter-balance increasing cuts in social spending. The paper has two sections. The first sketches the political climate and context surrounding the Italian national and regional healthcare systems, in which the concept of social innovation rose as a fundamental element to support welfare reforms. The second analyses the changes within the Italian healthcare system as part of a wider trend of reforms in Europe that are contributing to increasing inequalities. We conclude by suggesting that a social innovation perspective, which is not embedded within a structural reform of the Italian welfare state and the health care system, might simply become a convenient buzzword, useful for advancing neoliberal ideology in a time of austerity,7 rather than a tool for addressing the vital social challenges we are facing today.

The context

The effectiveness, flexibility and economic sustainability of welfare states have been under severe scrutiny since the 1970s. More recently, the emergence of the welfare mix8 debate has primarily focused on the necessity to decrease state funding for social services while meeting, at the same time, rapidly growing welfare demands9 . This debate clearly overlaps with the spread of social innovation idea within policy-making and academia. In particular, there are two areas of convergence. First, the emphasis on individual and group empowerment is compatible with the reduction of public social spending, and consequently with the shift from "public to private social responsibility" [START_REF] Gilbert | Transformation of the Welfare State. The Silent Surrender of Public Responsibility[END_REF]. Secondly, social innovation is often presented by its advocates as a strategy to tackle pressing social needs more effectively and efficiently by "mobilising people's creativity" and making "better use of scarce resources" (Hubert, 2010, p. 7) than both the state and the market (Murray, Caulier-Grice and Mulgan, 2010, p. 3).

The concept of social innovation emerged10 during the 1990s in a political and economic context characterised by the end of Fordism and the attempt of neoliberalism to actively promote the reduction of public involvement. The diversification of suppliersinvolving liberalisation and out-sourcing of productionand the devolution of authority from central to local governments have been among the major trends across countries, thereby reshaping social services (Martinelli, 2012, p. 170). In Italy, the process was characterised by: a generalised aversion to public intervention, the representation of the welfare state as an obstacle to economic growth and the reframing of welfare in terms of residual policies (Pennacchi, 2008, p. 3-5).

Many scholars have considered the restructuring of the Italian welfare statecharacterised by devolution to local authorities and diversification of suppliers (through vertical and horizontal subsidiarity)as innovative by default; hence, inherently better than previous arrangements. Much empirical evidence shows, however, that this is not the case and that, on the contrary, strong exclusionary processes have accompanied these reforms (Martinelli, 2012, p. 175), diminishing the universality of the Italian health care system. Maino and Ferrera (2013, p. 323) have clearly pointed out the danger of a missed integration between the traditional, so-called 'first' welfare state (mostly publicly provided) and the emerging 'second welfare' (mostly private or community based). This missed integration is contributing to further reductions in the capacity of the welfare state to recalibrate its structure and, as a consequence, inequalities and territorial disparities (already in place) are rapidly increasing.

Italy is characterised by a 'peculiar' combination of profound territorial and socioeconomic divides 11 , on-going decentralization policies, declining state investments, and budget cuts (especially at the regional level) due to debt restructuring plans (the so-called "piani di rientro regionali"). However, education, health assistance and care services are the basis of social citizenship [START_REF] Marshall | Citizenship and Social Class: and Other Essays[END_REF], and for this reason, a reduction of public spending in these areas can fundamentally undermine universal access [START_REF] Martinelli | Social Innovation or Social Exclusion? Innovating Social Services in the Context of a Retrenching Welfare State[END_REF]. In this context of protracted austerity, the decentralisation of the Italian National Healthcare System (NHS) has contributed to widening territorial inequalities and reducing access to services. It has been highlighted that in order to avoid a zero-sum game, in which the eventual gains of the more efficient regions are mirrored by the losses in performance of the weaker areas, a renewed active role of the state in terms of spending and monitoring is urgently required (Pavolini and Vicarelli, 2012, p. 484-485). Instead, budget constraints have become a dominant concern, overshadowing the promotion and safeguard of public access to health care (Dirindin and Vineis, 2004, p. 214-215).

Discussion

Recent reports on the state of the Italian NHS (CEIS, 2013;OASI, 2013;Osservasalute, 2013) show, on the one hand, signs of economic recovery and budget rebalancing and, on the other hand, widening territorial inequalities. In Italy the NHS is less costly than in Britain, France and Germany, with less per capita spending and a smaller spending increase over the last decade (Cantù and Longo, 2013, p. 1). 12 Although, the Italian NHS deficit has been reduced and its financial sustainability in the medium term is not at risk, the EU Fiscal Compact imperatives have been translated into a state therapy of indiscriminate cuts, the so called "tagli lineari" ("linear cuts") (Cantù and Longo, 2013, p. 2). Hence, the real threat to the sustainability of the NHS, especially from the point of view of curtailing universal access, does not seem to arise from the overall deficit, but rather from the lack of investment in technology and infrastructure. More specifically, regional budgets have been rebalanced mainly by reducing the inputs, while neglecting outputs and service outcomes. 13 There is a lack of investment both at the national and local level, which is especially pronounced in the most deprived regions. While the 2003-2010 average per capita investment spending was 59 Eurosthe peaks were registered in Emilia-Romagna (111) and in the autonomous province of Trento (105)the expenditure in the southern regions was systematically below the national average, reaching the lowest levels in Calabria (20) and Apulia (29) (Cantù and Longo, 2013, p. 3). The prevailing focus on inputs reduction has translated into hiring and wage freezes 14 (especially in the regions subject to debt restructuring plans), a strategy that is likely to consolidate shortages and deficiencies in already inefficient regional systems.

11 For a discussion of different models of welfare state and healthcare systems, see [START_REF] Bertin | Modelli di welfare e sistemi sanitari: quali omogeneità e specificità[END_REF]. For an overview on health and social inequalities see Costa, Cislaghi andCaranci (2009), andNiero andBertin (2011). 12 The Osservasalute 2013 report confirms such condition. The total amount of public spending declined between 2009 and 2010 (from 103 to 101 billion euros), with a deepening trend between 2011 and 2012. http://www.salute.gov.it/imgs/C_17_notizie_1551_listaFile_itemName_0_file.pdf. 13 While the term output indicates the result of a specific healthcare provision (as performing an electrocardiogram), outcome represents the changes in individual, group or population health deriving from one or more interventions planned at various levels (a single surgical intervention, a service of instrumental diagnostics or a vaccination plan). Health outcomes may also stem from non-sanitary interventions, as the opening of a factory and its polluting effects on a certain area or, conversely, the construction of sports facilities in the same site, constituting a potential incentive to undertake physical activity. 14 The number of employees within public hospitals has significantly declined since 2010 (Rapporto Osservasalute 2013: 2). http://www.salute.gov.it/imgs/C_17_notizie_1551_listaFile_itemName_0_file.pdf.

In addition, declining public investment is not proportionally compensated by an increase in the out-of-pocket spending, which seems to be more influenced by personal income. The presumed inverse proportionality between private expenditure and public healthcare performance is not confirmed by empirical evidence in Italy: southern regions, characterised by lower levels of efficiency, also show the lowest levels of private spending in the country15. This might suggest that tight expenditure constraints and increased prescription charges are instigating an alarming tendency toward under-treatment and widening inequalities between the worse and the better off regions (Cantù and Longo, 2013, p. 4). As shown by two subsequent Ministry of Health reports on the fulfilment of LEA ("Livelli Essenziali di Assistenza", the scores of health care adequacy)16, there is a correlation between tight budget constraints and deteriorating quality standards (Ministero della Salute, 2013, p. 20;2014, p. 22-23;). In 2011 and 2012, nearly all the regions subject to debt restructuring plans -Abruzzo, Molise, Apulia, Campania, Calabria and Sicilyhave not been compliant, or just partially so, with basic healthcare provisions. This situation appears even more problematic if we consider how the economic crisis has not only reduced private spending, but also diminished the mobility of NHS users among regions17. Finally, there is a strong correlation between the regional poverty index and the inability of the regional healthcare systems to comply with the provision of basic services (measured through the LEA, see Table 1 andGraph. 1). 15 Out-of-pocket spending for prescription charges and drugs (although not inversely proportional to the shrinking of public investment) has more than doubled between 2003 (11,3 bln euro, 5,3% of GDP) and 2012 (23,7 bln , 12,2 %) (Rapporto Osservasalute 2013, http://www.salute.gov.it/imgs/C_17_notizie_1551_listaFile_itemName_0_file.pdf). 16 The acronym LEA (Basic Healthcare Provision) denotes the essential health services free or with a prescription chargewhich any citizen is entitled to receive from the NHS. The uniform supply of LEA in every Italian region was initially regulated by the Ministerial Decree (DM) 12 December 2001, in accordance with the Legislative Decree (D.Lgs.) 56/2000, defining a set of indicators to assess the quality of healthcare. The Ministerial Decree was subsequently replaced by the Prime Minister Decree of 23rd of April 2008. Current LEA includes 5700 prevention, care and rehabilitation services and provisions. The regional healthcare compliance with the parameters defined by the Ministry of Health is under the supervision of the LEA Committee. http://www.salute.gov.it/portale/ministro/p4_5_2_4_1.jsp?lingua=italiano&menu=uffCentrali&label=uffCentrali&id=1 151. 17 Whose social cost is entirely sustained by the patients and their families. (2014, p. 22-23).

TABLES AND GRAPHS

In this context of 'neoliberal austerity', a strong call in favour of social innovation might hide the attempt to shift public attention from structural deficiencies and disparities to individual and group responsibility, following the vision: "doing more with less" 18 . In order to guarantee universal coverage and universal social rights, however, the health care system cannot be managed with the logic of mere cost-effectiveness alone. A healthy population is the precondition for any well-functioning economy, not the other way around. As such, the enhancement of "politically motivated policies under the pretence of budget cuts" can be particularly dangerous in its consequences for population health [START_REF] Kleinert | Health in Europe -Successes, Failures, and New Challenges[END_REF]Horton, 2013, p. 1074).

The current economic difficulties, epidemiological changes and the foreseeable impact of population ageing suggest the need for substantial investments and structural transformations of the health care systemfor instance, the regeneration and upgrading of the existing facilities. The idea of implementing innovative procedures and services without any intervention within healthcare infrastructures seems, at best, quite optimistic (Cantù and Longo, 2013, p. 3). As noted by Caritas, an important charitable organization 19 , economic unease and social exclusion in Italy have been exacerbated by the on-going austerity policies. Such measures have weakened key sectors of the welfare state during a period of economic distress that demanded stronger social protection. Therefore, the promotion of 'innovative forms of solidarity' at the community level is simply not enough. Instead, a strengthening of institutional responsibilities to society's weakest members stands essential in combating new forms of poverty and social exclusion (Nanni, 2014). To conclude, we do not consider social innovation per se able to substantially tackle pressing social needs. Rather, the all-innovating and self-empowering jargon currently in vogue might disguise a dangerous inattention to structural inequalities, adversely affecting health outcomes across the board, but especially of the poorest. Among the therapies prescribed by the neoliberal orthodoxyliberalisation, deregulation, devolution, individual or group empowermentsocial innovation might soon reveal itself as a convenient buzzword, an eclectic concept to dissimulate political choices, legitimated by the doctrine of budgetary constraints. In our view, the redistribution of resources "from past to present generations"keeping constant the overall public spending -and the shift from a "transfer-based" to a "service-based" welfare state (for a discussion, see Ferragina, 2013b) would represent a truly innovative approach to Italian social policy, offering a credible and responsible alternative to the magic wand of social innovation. Less" (http://www.socialinnovation.ash.harvard.edu/welcome-to-innovation-cities-2.html). Welcome to the Project on Social Innovation is an online hub launched in 2011 by the Ash Centre for Democratic Governance and Innovation at the Harvard Kennedy School of Government. 19 Caritas is the pastoral branch of the Italian Episcopal Conference (CEI). According to its statute, CEI promotes the value of charity to contribute to "the integral development of man, to social justice and peace, with a particular attention for the destitute" (http://www.caritasitaliana.it/home_page/chi_siamo/00003684_Statuto.html).

Table 1

 1 Poverty Index and Scores Health Care Adequacy

		Poverty Index	Scores Health Care Adequacy (LEA)
	Italian Average	12.7
	Piedmont	7.3	186
	Lombardy	6	184
	Veneto	5.8	193
	Liguria	8.1	176
	Emilia Romagna	5.1	210
	Northern Regions	6.2
	Tuscany Umbria	6.8 11	193 171
	Marche	8.6	165
	Lazio	6.3	167
	Central Regions	7.1
	Abruzzo	16.5	145
	Molise	20.5	146
	Campania	25.8	117
	Apulia Basilicata	28.2 24.5	140 169
	Calabria	27.4	133
	Sicily	29.6	157
	Southern Regions	26.2
	R2	0.639

Graph. 1 Correlation Poverty Index and Scores Health Care Adequacy (LEA)

Source: ISTAT, "Net family income distribution indicators" (http://dati.istat.it/index.aspx?queryid=4836), and Ministero della Salute, Direzione Generale Programmazione Sanitaria

For a description of neoliberal ideology and its multiple manifestations, see[START_REF] Gamble | The Spectre at the Feast[END_REF] Harv ey (2005).

On the welfare mix debate see[START_REF] Ascoli | The New Partnership: The Changing Relationship between State and the Third Sector in the Scenario of New Social Policies in Italy[END_REF].

In addition, the corruption scandals alimented the need for greater transparency in the economic relations between local authorities and private suppliers(Ascoli, Pavolini and Ranci, 2002, p. 142-143).

Hubert (2010, p. 31) suggested that social innovation is rooted in the work of the founding fathers of sociology (i.e. Weber and Durkheim) and in the influential definition of innovation proposed by Schumpeter. In this regard, see als o[START_REF] Moulaert | Social Innovation: Institutionally Embedded, Territorially (Re)produced[END_REF] and[START_REF] Moulaert | The International Handbook of Social Innovation[END_REF].

1 This paper is part of the research project, Social capital, health and health care system in Calabria, co-financed by the European Social Fund (European Commission) and the Calabria Regional Administration. We are thankful to the Calabria Regional Administration


Sebastien Elis Abcd#
email: sebastien.elis@tours.inra.fr

Sandrine Freret Abcd

Alice Desmarchais Abcd

Virginie Maillard Abcd

Juliette Cognié Abcd

Eric Briant

Jean-Luc Touzé

Mickael Dupont

Philippe Faverdin

Véronique Chajès

Svetlana Uzbekova Abcd

Philippe Monget

Joëlle Dupont

Effect of a long chain n-3 PUFA-enriched diet on production and reproduction variables in Holstein dairy cows

Keywords: Dairy cows, Fertility, n-3 PUFA, Nutrition, Production

come

Effect of a long chain n-3 PUFA-enriched diet on

Introduction

Reproductive performance in cows is influenced by nutritional and metabolic status, as previously reviewed [START_REF] Butler | Nutritional interactions with reproductive performance in dairy cattle[END_REF][START_REF] Roche | The effect of nutritional management of the dairy cow on reproductive efficiency[END_REF][START_REF] Leroy | Nutrient prioritization in dairy cows early postpartum: mismatch between metabolism and fertility?[END_REF]. Therefore, dietary fat has been used to enhance reproductive status. It is well accepted that dietary fat directly affects dairy cow fertility rather than by modifying energy status of cows (Staples et al., 1998;[START_REF] Mattos | Effects of dietary fatty acids on reproduction in ruminants[END_REF]. Fatty acids (FA) and polyunsaturated FA (PUFA) were found to be important for various reproductive processes, especially as steroid hormone and prostaglandin precursors via cholesterol and arachidonic acid, respectively (Urlep and Rozman, 2013;Tessaro et al., 2015).

Among the PUFA, previous research has focused on the effect of omega-6 (n-6) and especially omega-3 (n-3) FA.

The n-3 PUFA belong to a family of biologically active FA. The n-3 PUFA are essential FA as mammals cannot synthesize n-3 PUFA have to be introduced into the body via the diet. The shortest member of this family, alpha-linolenic acid (ALA, C18:3), has an 18 carbon chain and can be converted by a series of desaturation and elongation reactions into more biologically active compounds [START_REF] Simopoulos | Omega-3 fatty acids in inflammation and autoimmune diseases[END_REF][START_REF] Plourde | Extremely limited synthesis of long chain polyunsaturates in adults: implications for their dietary essentiality and use as supplements[END_REF] of the n-3 family: eicosapentaenoic acid (EPA, C20:5) and docosahexaenoic acid (DHA, C22:6) [START_REF] Calder | Mechanisms of action of (n-3) fatty acids[END_REF]. The ALA can be found in several vegetarian sources, such as grass silage, linseed or dehydrated alfalfa. The conversion rate of ALA into EPA and DHA is low in mammals [START_REF] Burdge | Conversion of alpha-linolenic acid to longer-chain polyunsaturated fatty acids in human adults[END_REF]. Therefore, to increase concentrations of EPA and DHA in plasma of dairy cows, these two n-3 PUFA have to be directly introduced into the body via the diet.

The EPA and DHA are found in fish oil, as well as in microalgae oil [START_REF] Abughazaleh | Short communication: The effect of substituting fish oil in dairy cow diets with docosahexaenoic acid-micro algae on milk composition and fatty acids profile[END_REF].

Comment citer ce document : Elis, S., Fréret, S., Desmarchais, A., Maillard, V., Cognié, J., Briant, E., Touze, J.-L., Dupont, M., Faverdin, P., Chajès, V., Uzbekova, S., Monget, P., Dupont, J. ( 2016 The n-3 PUFA have a wide range of physiological roles, via receptors, sensors or cell membrane fatty acid composition (membrane order, lipid rafts etc…; reviewed in [START_REF] Calder | Mechanisms of action of (n-3) fatty acids[END_REF]). Among the physiological roles of n-3 PUFA, there is an effect on reproductive variables. There is a greater pregnancy rate because of a decrease in embryo mortality with feeding of a flaxseed diet [START_REF] Petit | Conception rate and reproductive function of dairy cows fed different fat sources[END_REF].

The feeding of an n-3 diet (enriched in ALA) led to an increase in the size of the preovulatory follicles [START_REF] Ambrose | Lower pregnancy losses in lactating dairy cows fed a diet enriched in alpha-linolenic acid[END_REF] and in number of small follicles (Zachut et al., 2010). The feeding of diets supplemented with n-3 PUFA (enriched in ALA) also had a tendency towards increasing conception rates [START_REF] Ambrose | Lower pregnancy losses in lactating dairy cows fed a diet enriched in alpha-linolenic acid[END_REF][START_REF] Dirandeh | Effects of different polyunsaturated fatty acid supplementations during the postpartum periods of early lactating dairy cows on milk yield, metabolic responses, and reproductive performances[END_REF]. One mechanism that could enhance fertility is that the n-3 diet decreases the concentration of prostaglandin F2 alpha (PGF2) in the uterus, thus potentially facilitating embryo implantation and reducing embryo mortality [START_REF] Mashek | The effects of feeding fish oil on uterine secretion of PGF2alpha, milk composition, and metabolic status of periparturient Holstein cows[END_REF].

However, some inconsistencies exist, likely due to dose and/or PUFA form differences, both fish oil (EPA, DHA) and flaxseed (ALA) diets were reported to decrease corpora lutea (CL) size, and consequently plasma progesterone concentrations, leading to a more unfavorable uterine environment for sustaining pregnancies and reduced fertility [START_REF] Hutchinson | Effect of supplementation with different fat sources on the mechanisms involved in reproductive performance in lactating dairy cattle[END_REF].

The objective of the present study was, thus, to assess the effect of a moderate amount of rumen-protected n-3 PUFA on production [body weight (BW), milk yield (MY), dry matter intake (DMI), energy balance (EB), milk fat content (MFC), milk protein content (MPC), subcutaneous fat thickness and metabolic variables, nonesterified fatty acids (NEFA), glucose, urea, as well as on ovarian variables, growing follicle numbers, conception rate after first AI (AI1)] of 25 Holstein dairy cows in which assessments occurred over two lactations. Long chain n-3 PUFA (DHA and EPA) were chosen because these molecules are the most efficient n-3

Comment citer ce document : Elis, S., Fréret, S., Desmarchais, A., Maillard, V., Cognié, J., Briant, E., Touze, J.-L., Dupont, M., Faverdin, P., Chajès, V., Uzbekova, S., Monget, P., Dupont, J. (2016). Effect of a long chain n-3 PUFA-enriched diet on production and reproduction variables in Holstein dairy cows. Animal Reproduction Science, 164, 121-132. DOI : 10.1016/j.anireprosci.2015.11.020

Page 5 of 37

A c c e p t e d M a n u s c r i p t

-5 -PUFA candidates [START_REF] Simopoulos | Omega-3 fatty acids in inflammation and autoimmune diseases[END_REF][START_REF] Deckelbaum | The omega-3 fatty acid nutritional landscape: health benefits and sources[END_REF]. Rumen-75 protected fish oil was used to provide DHA and EPA. To ensure a specific effect of 76 long chain n-3 PUFA, an n-6 PUFA diet was chosen as the control. It was 77 hypothesized that the n-3 diet could enhance not only the uterine environment (Mattos 78 et al., 2004) but also oocyte quality, independent of a secondary effect on production 79 or metabolism modifications. Therefore, the diets were fed from calving to 2 months 80 postpartum (2MPP), thus potentially affecting ovarian folliculogenesis and thus the 81 qualitative properties of oocytes released at the ovulation associated with AI1. 82

Comment citer ce document : Elis, S., Fréret, S., Desmarchais, A., Maillard, V., Cognié, J., Briant, E., Touze, J.-L., Dupont, M., Faverdin, P., Chajès, V., Uzbekova, S., Monget, P., Dupont, J. ( 2016 1. At the end of the experiment, a total of 23 cows received the n-3 diet and a total of 23 cows received the n-6 diet. Cows originated from a population previously selected for one female fertility QTL located on bovine chromosome 3, and carried either a favorable (Fertil+) or unfavorable (Fertil-) haplotype [START_REF] Coyral-Castel | Ovarian parameters and fertility of dairy cows selected for one QTL located on BTA3[END_REF]Coyral-Castel et al., 2012a). As this QTL affected reproductive variables during the first lactation, cows with each haplotype were equally distributed in the two dietary groups (n-3 diet group: 14

Fertil+ and 9 Fertil-cows; n-6 diet group: 15 Fertil+ and 8 Fertil-cows) and the haplotype effect was taken into account.

Experimental diets and dry matter intake

The composition of the two experimental diets was balanced for energy and protein contents as indicated in Table 2; the two diets were total mixed rations (TMR), with feed values being calculated using the INRA French feeding system

Comment citer ce document : Elis, S., Fréret, S., Desmarchais, A., Maillard, V., Cognié, J., Briant, E., Touze, J.-L., Dupont, M., Faverdin, P., Chajès, V., Uzbekova, S., Monget, P., Dupont, J. ( 2016 From 2MPP to the non-lactating period, the diet was the same for both groups and contained (% DM): 68% corn silage, 8% dehydrated alfalfa, 11% Sandifort® (wheat), 12% Sandilait® (soybean and rapeseed meal), 0.4% vitamins and minerals 5/23 and 0.5% calcium bicarbonate.

Body weight, milk yield and composition, energy balance

Body weight (BW) and milk yield (MY) were monitored daily during the milking period, from calving to 5 months postpartum (5MPP

Progesterone and metabolic assays, fatty acid composition of plasma and milk

Plasma progesterone was determined using an ELISA assay [START_REF] Canepa | Validation d'une methode immunoenzymatique pour le dosage de la progesterone dans le plasma des ovins et des bovins[END_REF] from 10 μL of undiluted plasma in samples collected three times a week from 15 days postpartum to the first artificial insemination (AI1) and then daily from the day of AI1 to 35 days after AI1.

For metabolic assays, plasma samples were collected once a week from caudal venipuncture before diet distribution, from 1-month antepartum (1MAP) to 5 months postpartum (5MPP). Plasma samples were stored at -20 °C until the assays were conducted. Plasma NEFA, urea and glucose were determined using enzymatic colorimetry on a multi-parameter analyzer (KONE Instruments Corporation, Espoo, Finland).

Plasma and milk fatty acid compositions were determined using gas chromatography, as previously described [START_REF] Chajes | Analysis of in vivo oocyte maturation, in vitro embryo development and gene expression in cumulus cells of dairy cows and heifers selected for one fertility quantitative trait loci (QTL) located on BTA3[END_REF][START_REF] Bernard | Effect of plant oils in the diet on performance and milk fatty acid composition in goats fed diets based on grass hay or maize silage[END_REF].

Plasma fatty acid composition was determined during the 2 years of the experiment for 13 cows in the n-3 diet group and 16 cows in the n-6 diet group, in 1 month antepartum (1MAP), 1 week postpartum (1WPP), 2 month postpartum (2MPP) and 5 month postpartum (5MPP) samples. Milk fatty acid composition was determined in

Comment citer ce document : Elis, S., Fréret, S., Desmarchais, A., Maillard, V., Cognié, J., Briant, E., Touze, J.-L., Dupont, M., Faverdin, P., Chajès, V., Uzbekova, S., Monget, P., Dupont, J. ( 2016

Subcutaneous adipose tissue thickness

Adipose tissue mobilization was assessed through subcutaneous fat thickness measurements in the sacral region using ultrasonographic examination with a linear probe (LA 332 3.5/10.0-MHz transducer; Mylab30vet; Esaote), as previously described [START_REF] Schroder | Invited review: Methods to determine body fat reserves in the dairy cow with special regard to ultrasonographic measurement of backfat thickness[END_REF]. Back fat thickness was measured at 1MAP, 15WPP, 1MPP, 2MPP and 5MPP during both years of the experiment.

Ovarian variables and fertility

During the feeding period of n-3 or n-6 diets, the ovarian follicular dynamics of cows (n = 46) was monitored three times a week, from 15 ± 2 days postpartum to 82 ± 23 days postpartum, with a trans-rectal ultrasonographic examination using a linear probe (LV 513 6.0/8.0-MHz transducer; Mylab30vet; Esaote) LA332 3.5/10.0-MHz, allowing for the detection and measurement of antral follicles, as previously described [START_REF] Coyral-Castel | Ovarian parameters and fertility of dairy cows selected for one QTL located on BTA3[END_REF]. Follicles with a diameter of 3 to 5 mm were counted and for all follicles greater than 5 mm, the diameter was recorded. Based on these repeated ultrasonographic examinations, the growth of each follicle was assessed in the two ovaries as previously described [START_REF] Sirois | Ovarian follicular dynamics during the estrous cycle in heifers monitored by real-time ultrasonography[END_REF][START_REF] Coyral-Castel | Ovarian parameters and fertility of dairy cows selected for one QTL located on BTA3[END_REF]. Ovarian follicles were classified according to diameter [START_REF] Lucy | Factors that affect ovarian follicular dynamics in cattle[END_REF]: small follicles (3 ≤ SF < 5 mm), medium follicles (5 ≤ MF < 10 mm) and large follicles (LF ≥ 10 mm). To be more accurate in the description of LF, this follicle size category was divided into LFa (10 ≤ LFa < 15 mm) and LFb (15 ≤ LFb <

Comment citer ce document : Elis, S., Fréret, S., Desmarchais, A., Maillard, V., Cognié, J., Briant, E., Touze, J.-L., Dupont, M., Faverdin, P., Chajès, V., Uzbekova, S., Monget, P., Dupont, J. ( 2016 -10 -calculated per week postpartum for each cow. Follicular cysts, defined as follicular structures with a diameter larger than 25 mm and from which ovulation did not occur or that did not regress spontaneously, were excluded from the analysis. Numbers of CL were also recorded.

Ovulations were determined from to two consecutive ultrasonographic examinations (separated by 2 or 3 days): when a pre-ovulatory follicle was detected at one examination, and had disappeared at the subsequent examination. Ovulations were always confirmed by the visualization of CL during examinations that occurred subsequent to the time when ovulation was detected. The first postpartum ovulation was recorded for each cow. For some cows, CL were visualized on the first postpartum examination. Estrous cycle durations were calculated between two consecutive ovulations, and the number of waves of ovarian follicular development was recorded.

Cows were artificially inseminated after 60 days postpartum with semen of the same bull, 12 h after visual detection of estrus (observation of standing estrus and/or repetition of secondary signs of estrus). One cow was not inseminated during the first year of the study because she never expressed any signs of estrus.

Pregnancy status was determined 21 d after AI1 by plasma progesterone assay [START_REF] Canepa | Validation d'une methode immunoenzymatique pour le dosage de la progesterone dans le plasma des ovins et des bovins[END_REF], and subsequently 35 and 90 d after AI1 using ultrasonographic examination. The percentage of non-fertilization and/or early embryo mortality (NF-EEM) was ascertained by the number of non-pregnant cows at 21 d after AI1 (cows with plasma progesterone < 1 ng/mL) divided by the total number of inseminated cows. The percentage of cows with a prolonged luteal phase or late embryo mortality (PLP-LEM) was ascertained by the number of non-pregnant cows at 35 d divided by the number of cows with plasma progesterone ≥ 1 ng/mL at 21 d after AI. First

Comment citer ce document : Elis, S., Fréret, S., Desmarchais, A., Maillard, V., Cognié, J., Briant, E., Touze, J.-L., Dupont, M., Faverdin, P., Chajès, V., Uzbekova, S., Monget, P., Dupont, J. ( 2016 was the interval from calving to AI1.

Statistical analyses

All statistical analyses were performed with SAS® software (SAS Institute Inc., 2011). Mean milk fatty acid concentrations at 2MPP during the first year of the experiment were compared between the groups of cows fed the n-3 and n-6 diets using a Student's t-test (TTEST procedure). All other variables (except NF-EEM, PLP-LEM, LEM-FM and CRAI1) were analyzed using a linear mixed model (MIXED procedure) for repeated measurements.

For plasma fatty acid composition, the model included the effects of year of experiment, diet, stage ante/postpartum and all interactions, with stage as a repeated effect within cow (year) with a CS covariance structure (repeated statement of the MIXED procedure). Haplotype was not included in the model.

For subcutaneous adipose tissue thickness, the model included the effects of year of experiment, diet, stage ante/postpartum, haplotype and all interactions, with the subcutaneous adipose tissue thickness value at 1MAP as a covariable, and with stage as a repeated effect within cow (year) with a CS covariance structure.

Concerning the parameters BW, MY, DMI and EB, the model included the effects of year of experiment, diet, week postpartum (WPP), haplotype and all interactions, with week postpartum as a repeated effect within cow (year) with a

Comment citer ce document : Elis, S., Fréret, S., Desmarchais, A., Maillard, V., Cognié, J., Briant, E., Touze, J.-L., Dupont, M., Faverdin, P., Chajès, V., Uzbekova, S., Monget, P., Dupont, J. ( 2016 For milk composition (MFC and MPC) during the first year of the experiment, the model included the effects of diet, WPP, haplotype and all interactions, with WPP as a repeated effect within cow (year) with a CS covariance structure and with the value of BW at WPP1 as a covariable.

For plasma metabolic variables (NEFA, glucose, urea), the model included the effects of year of experiment, diet, week ante/postpartum (WPP), haplotype and all interactions, with WPP as a repeated effect within cow (year) with a AR(1) covariance structure and with the value of the WPP-4 (week 4 antepartum) as a covariable.

For the follicular growth variables and number of CL, the model included the effects of year of experiment, diet, WPP, haplotype and all interactions, with WPP as a repeated effect within cow (year) with a AR(1) covariance structure.

For the day of first postpartum ovulation, estrous cycle duration, number of follicular waves and IFI, the model included the effects of year of experiment, diet, haplotype and diet x haplotype interaction.

The NF-EEM, PLP-LEM, LEM-FM and CRAI1 were analyzed using a logistic regression (GLIMMIX procedure) and the model included the effects of year of experiment, diet, haplotype and diet x haplotype interaction.

Least square means (lsmeans) estimated by the models were subsequently compared between diets using a Bonferroni adjustment for multiple comparisons.

Results are presented in tables and figures as lsmeans ± SEM, unless otherwise stated.

A difference with P ≤ 0.05 is considered significant and with 0.05 < P ≤ 0.10 is considered a tendency. NS means non-significant for P-values > 0.10.

Comment citer ce document : Elis, S., Fréret, S., Desmarchais, A., Maillard, V., Cognié, J., Briant, E., Touze, J.-L., Dupont, M., Faverdin, P., Chajès, V., Uzbekova, S., Monget, P., Dupont, J. ( 2016

Results and discussion

It was hypothesized that the n-3 diet would enhance oocyte quality independently from a secondary effect on production or metabolism modifications.

Efficiency of the diet: Plasma and milk fatty acid composition enrichment in n-3 PUFA

To assess the efficiency of the n-3 diet, plasma fatty acid composition (Table 3) was measured at different stages: before the n-3 diet period (1MAP), during the n-3 diet period (1WPP and 2MPP) and after the n-3 diet period (5MPP). As expected, only a few differences were reported between both diets. The EPA (C20:5 n-3) and DHA (C22:6 n-3), both supplied by the n-3 diet, were increased in the n-3 group at both 1WPP (P = 0.0001 and P = 0.01, respectively) and 2MPP (P < 0.0001).

Consequently, the n-6 PUFA-n-3 PUFA ratio was less with the n-3 group, at both 1WPP and 2MPP, by 29% (P < 0.0001) and 41% (P < 0.0001), respectively, compared to the n-6 group. Milk fatty acid composition was also assessed at 2MPP

during the first year (Table 4). Feeding of both EPA and DHA increased milk composition of these FA more than three-fold (P < 0.05).

Long chain n-3 EPA and DHA are thought to not be biohydrogenated to a significant extent by rumen microorganisms, except for the C18 n-3 fatty acids [START_REF] Ashes | Incorporation of n-3 fatty acids of fish oil into tissue and serum lipids of ruminants[END_REF] but there are inconsistencies in thought in this regard [START_REF] Castaneda-Gutierrez | Effect of supplementation with calcium salts of fish oil on n-3 fatty acids in milk fat[END_REF]. Thus, in the present study protected fish oil was chosen so as to maximize the proportion of EPA and DHA in plasma. The rumen-protected form (microencapsulated oil) is sufficient to enable a significant increase in plasma after the first WPP diet, even with the moderate amounts in the diet. The FO diet has been described to increase EPA and DHA in both plasma [START_REF] Ashes | Incorporation of n-3 fatty acids of fish oil into tissue and serum lipids of ruminants[END_REF] and milk

Comment citer ce document : Elis, S., Fréret, S., Desmarchais, A., Maillard, V., Cognié, J., Briant, E., Touze, J.-L., Dupont, M., Faverdin, P., Chajès, V., Uzbekova, S., Monget, P., Dupont, J. ( 2016 -15 - [START_REF] Cant | Effect of fish oil and monensin on milk composition in dairy cows[END_REF]. In addition, FO was reported to decrease the n-6:n-3 ratio in all tissues and milk fat except for subcutaneous and internal adipose tissue (Bilby et al., 2006b), as well as ovarian compartments (Zachut et al., 2010). Therefore, the n-3 diet of the present study might decrease the n-6:n-3 ratio in the reproductive tissues, consequently exerting the physiological effects previously described.

Production variables: BW, MY, DMI, fat thickness, EB, MFC and MPC

The BW and MY were recorded daily during 22 weeks after calving. The average BW was, throughout the period studied, 593.5 ± 9.2 kg in the n-3 group compared to 586.9 ± 9.1 kg in the n-6 group, with no difference between groups (diet effect P = 0.61; Figure 1A). The average MY was, throughout the period studied, 33.0 ± 0.8 kg/d in the n-3 group, compared with 34.4 ± 0.8 kg/d in the n-6 group, with no difference between groups (diet effect P = 0.25; Figure 1B).

The MFC and MPC were also assessed once a week during the first year of the experiment for 22 weeks after calving. The average MFC was, throughout the period studied, 46.3 ± 1.8 in the n-3 group compared with 48.5 ± 1.8 g/L in the n-6 group, with no difference between groups (diet effect P = 0.41; Figure 1C). The MPC differed between groups (P = 0.05) during the first week of lactation (36.5 ± 0.6 compared with 34.3 ± 0.9 g/L milk in the n-3 and n-6 groups, respectively; Figure 1D). Except for the difference reported at WPP1, MPC was not different between the n-3 and n-6 groups (29.7 ± 0.3 and 29.4 ± 0.3 g/L, respectively, diet effect P = 0.53).

The DMI and energy balance (EB) were measured during the first 8 weeks postpartum (period of n-3 and n-6 diet distribution). The DMI was not different between groups (diet effect P = 0.46), 13.5 ± 0.5 kg DM/day in the n-3 group compared to 14.0 ± 0.5 kg DM / day in the n-6 group (Figure 1E). There was no

Comment citer ce document : Elis, S., Fréret, S., Desmarchais, A., Maillard, V., Cognié, J., Briant, E., Touze, J.-L., Dupont, M., Faverdin, P., Chajès, V., Uzbekova, S., Monget, P., Dupont, J. ( 2016 and n-6 group, respectively, diet effect P = 0.90), consistent with the absence of differences for production variables between n-3 and n-6 groups, and all cows were still in negative EB at 2MPP.

The production changes (DMI, MY and MFC) that are common with the PUFA diet were not observed in the present study. Unsaturated FA are reported to decrease DMI and increase MY and MFC (reviewed in [START_REF] Rabiee | Effect of fat additions to diets of dairy cattle on milk production and components: a meta-analysis and meta-regression[END_REF][START_REF] Leroy | Dietary fat supplementation and the consequences for oocyte and embryo quality: hype or significant benefit for dairy cow reproduction?[END_REF]). In a study where supplementation of dairy cow diets with fish oil occurred, it was also reported that there was a tendency to increase MY in the fish oiltreated group [START_REF] Pirondini | Effect of dietary starch concentration and fish oil supplementation on milk yield and composition, diet digestibility, and methane emissions in lactating dairy cows[END_REF]. It is common to find a reduction of DMI, MY and changes in milk composition with feeding of the n-3 diet [START_REF] Lacasse | Addition of protected and unprotected fish oil to diets for dairy cows. I. Effects on the yield, composition and taste of milk[END_REF].

But with a rumen-protected form of fat, these adverse effects are less frequently encountered [START_REF] Lacasse | Addition of protected and unprotected fish oil to diets for dairy cows. I. Effects on the yield, composition and taste of milk[END_REF]. Most meta-analyzed studies compared fat supplementation to the control diet, which was usually enriched with saturated fat to provide an iso-energetic diet [START_REF] Rabiee | Effect of fat additions to diets of dairy cattle on milk production and components: a meta-analysis and meta-regression[END_REF], making the conditions of these studies different from conditions in the present study. In the present study, the control diet contained n-6 PUFA, which is the most similar control for an n-3 PUFA diet.

Therefore, the absence of differences in production variables in the present study is not surprising.

Fat thickness and plasma metabolic variables

To assess mobilization of the body reserve, subcutaneous adipose tissue thickness and plasma NEFA concentrations were measured prepartum and throughout lactation in the present study. Subcutaneous adipose tissue thickness was measured from 1MAP to 5MPP and was 0.46 ± 0.03 cm in the n-3 treatment group compared

Comment citer ce document : Elis, S., Fréret, S., Desmarchais, A., Maillard, V., Cognié, J., Briant, E., Touze, J.-L., Dupont, M., Faverdin, P., Chajès, V., Uzbekova, S., Monget, P., Dupont, J. ( 2016 -17 -with 0.47 ± 0.02 cm in the n-6 treatment group (diet effect P = 0.71; Figure 2A).

Plasma NEFA, glucose and urea concentrations were recorded from 4 weeks antepartum (WAP-4) to 22WPP. At WPP5, plasma NEFA concentrations were greater in n-3 groups, 1.1 ± 0.1 mM, compared with the n-6 group, 0.8 ± 0.1 mM (P = 0.039).

There were no other NEFA differences between the groups (diet effect P = 0.23).

Plasma glucose concentrations were at WAP-4: 3.7 ± 0.1 and 3.9 ± 0.1 mM in the n-3

and n-6 groups, respectively (Figure 2B). Plasma glucose concentrations were different at WPP1 (P = 0.030), when there was a decrease to 2.8 ± 0.1 and 2.4 ± 0.1 mM in the n-3 and n-6 groups, respectively. No other plasma glucose concentration differences were detected between the groups (diet effect P = 0.42). Plasma urea concentrations increased continuously from WAP-4 to WPP22 (Figure 2C). There was a tendency (P = 0.098) for increased plasma urea in the n-3 group at WPP2 compared with the n-6 group (5.5 ± 0.3 and 4.7 ± 0.3, respectively). There were no other urea differences between the groups (diet effect P = 0.84).

The absence of differences in adipose tissue mobilization between both groups is consistent with the absence of a difference in energy balance in the present study.

Plasma urea does not exceed the threshold that [START_REF] Butler | Review: effect of protein nutrition on ovarian and uterine physiology in dairy cattle[END_REF] described at the time of insemination (19 mg/dL -6.8 mM -for urea nitrogen in plasma), which can be deleterious for fertility [START_REF] Butler | Review: effect of protein nutrition on ovarian and uterine physiology in dairy cattle[END_REF]. The absence of differences in the glucose concentration between the n-3 and n-6 PUFA groups in the present study is consistent with findings in a previous study comparing diets containing flaxseed and sunflower seed [START_REF] Ambrose | Lower pregnancy losses in lactating dairy cows fed a diet enriched in alpha-linolenic acid[END_REF]. However, an increased glucose concentration with feeding of a n-3 PUFA diet compared to a n-6 PUFA diet was also reported [START_REF] Dirandeh | Effects of different polyunsaturated fatty acid supplementations during the postpartum periods of early lactating dairy cows on milk yield, metabolic responses, and reproductive performances[END_REF].

Comment citer ce document : Elis, S., Fréret, S., Desmarchais, A., Maillard, V., Cognié, J., Briant, E., Touze, J.-L., Dupont, M., Faverdin, P., Chajès, V., Uzbekova, S., Monget, P., Dupont, J. ( 2016 The absence of differences in plasma NEFA concentrations when feeding the n-3 and n-6 PUFA diets is relevant [START_REF] Mashek | The effects of feeding fish oil on uterine secretion of PGF2alpha, milk composition, and metabolic status of periparturient Holstein cows[END_REF][START_REF] Mashek | The effects of feeding fish oil on uterine secretion of PGF2alpha, milk composition, and metabolic status of periparturient Holstein cows[END_REF]. There are some inconsistencies in results between experiments. As reviewed by [START_REF] Leroy | Dietary fat supplementation and the consequences for oocyte and embryo quality: hype or significant benefit for dairy cow reproduction?[END_REF], fat feeding and fat supplementation increased plasma NEFA as a result of both DMI reduction and MY and MFC increases, consequently leading to an ovarian follicular environment that was more unfavorable to cow fertility. The absence of effects of dietary PUFA on production (DMI, MY, MFC) or metabolism variables (glucose, NEFA) in the present study is probably related to differences in amount of PUFA fed, type of dietary fat, and period of feeding of the diet [START_REF] Leroy | Dietary fat supplementation and the consequences for oocyte and embryo quality: hype or significant benefit for dairy cow reproduction?[END_REF] compared with previous studies. The diet used in the present study provided a moderate amount of rumen-protected fat (1% DM). Therefore, the lesser amount of fat and/or the fact that it was rumen-protected could reduce metabolic or production issues encountered when greater amounts of fat are fed. The control diet in the present study was an n-6 PUFA diet. The feeding of a saturated or mono-unsaturated diet has often been used as the control diet in previous studies with results being compared to those obtained with the n-3 diet. These differences may explain why, in the present study, there were not any effects on production or metabolic variables.

Ovarian variables and fertility at first insemination

The first postpartum ovulation occurred before 34.8 ± 5.3 days postpartum in the n-3 group, and before 32.3 ± 5.3 days postpartum in the n-6 group, with no difference between them (diet effect P = 0.74). Mean estrous cycle duration (23.3 ± 2.7 days in the n-3 group compared with 28.6 ± 2.5 days in the n-6 group, diet effect P = 0.15) and mean number of waves of follicular development per estrous cycle (2.1

Comment citer ce document : Elis, S., Fréret, S., Desmarchais, A., Maillard, V., Cognié, J., Briant, E., Touze, J.-L., Dupont, M., Faverdin, P., Chajès, V., Uzbekova, S., Monget, P., Dupont, J. ( 2016 Follicular growth (monitored by ultrasonographic examination of the ovaries three times a week) was analyzed from WPP3 to WPP10 (Figure 3). The total numbers of follicles during this 7 week period were 10.7 ± 0.5 and 10.0 ± 0.5 in the n-3 and n-6 groups, respectively (Figure 3A; diet effect P = 0.26). There was a trend towards increased follicle numbers at WPP8 in the n-3 group compared with the n-6 group (P = 0.08). The numbers of small follicles (SF) were 7.2 ± 0.4 and 6.7 ± 0.4 in the n-3 and n-6 groups, respectively (Figure 3B; diet effect P = 0.45). The numbers of medium follicles (MF) were 2.4 ± 0.1 and 2.2 ± 0.1 in the n-3 and n-6 groups, respectively (Figure 3C; diet effect P = 0.35). There was a trend for an increased number of MF at WPP7 in the n-3 group compared with the n-6 group (P = 0.09). The numbers of large follicles (LF) tended to be greater (Figure 3D) in the n-3 group (1.2 ± 0.1) compared with the n-6 group (1.0 ± 0.1; diet effect P = 0.06) throughout the period studied and there was a similar trend at both WPP6 and WPP7 (P = 0.07). The LF group was subdivided in two subgroups, LFa (10 ≤ diameter < 15 mm) and LFb (15 ≤ diameter < 25 mm). The numbers of LFa were 0.8 ± 0.0 and 0.7 ± 0.0 in the n-3

and n-6 groups, respectively, and did not differ throughout the period studied (diet effect P = 0.14; Figure 3E). The number of LFa was greater at WPP7 in the n-3 group compared with the n-6 group (P = 0.02). The number of LFb did not differ throughout the period studied between the n-3 and n-6 groups (0.4 ± 0.0 and 0.3 ± 0.0, respectively, diet effect P = 0.31; Figure 3F). However, the number of LFb in the n-3 group at 4WPP tended to increase compared with the n-6 group (P = 0.09). The numbers of CL were 0.9 ± 0.1 and 0.9 ± 0.1 in the n-3 and n-6 groups, respectively (diet effect P = 0.74; Figure 3G).

Comment citer ce document : Elis, S., Fréret, S., Desmarchais, A., Maillard, V., Cognié, J., Briant, E., Touze, J.-L., Dupont, M., Faverdin, P., Chajès, V., Uzbekova, S., Monget, P., Dupont, J. ( 2016 and n-6 groups (n = 23). The NF-EEM tended to be less in the n-3 (13.5%) compared with the n-6 group (38.8%, diet effect P = 0.09, Table 5). The PLP-LEM was not different between groups: 3/16 cows (15.8%) in the n-3 group and 2/14 cows (14.3%) in the n-6 group (diet effect P = 0.98). No difference was observed between groups for CRAI1 (Table 5): 72.9% in the n-3 group and 48.5% in the n-6 group at 35 d (diet effect P = 0.13) and 56.1% in the n-3 group and 48.1% in the n-6 group at 90 d after AI1 (diet effect P = 0.63). The LEM-FM was not different between groups: 3/16 cows (18.7%) in the n-3 groups and 0/12 cows (0%) in the n-6 group (diet effect P = 0.98).

Moreover, there were no differences with the interval to first insemination (IFI) between groups, 80.0 ± 5.1 days in the n-3 group, compared with 84.2 ± 5.0 days in the n-6 group (diet effect P = 0.56).

There was a trend for a decrease in NF-EEM in the n-3 group with fish oil supplementation compared to the control group in the present study. This difference is not attributable to a difference in IFI, which is similar in both groups. Rate of NF-EEM of the n-6 group and rates of PLP-LEM of both n-3 and n-6 groups were consistent with results from previous studies, between 20% and 40% and between 8% and 17.5%, respectively [START_REF] Humblot | Use of pregnancy specific proteins and progesterone assays to monitor pregnancy and determine the timing, frequencies and sources of embryonic mortality in ruminants[END_REF][START_REF] Diskin | Embryo death in cattle: an update[END_REF]Walsh et al., 2011).

These tendencies are consistent with results from previous studies that suggested a potential effect of the n-3 PUFA diet on reproductive variables: an increase in the number of small (Bilby et al., 2006c;Zachut et al., 2010) and medium follicles [START_REF] Petit | Milk production and composition, ovarian function, and prostaglandin secretion of dairy cows fed omega-3 fats[END_REF] and a trend for decreasing NF-EEM [START_REF] Ambrose | Lower pregnancy losses in lactating dairy cows fed a diet enriched in alpha-linolenic acid[END_REF][START_REF] Petit | Importance of dietary fat on reproduction of dairy cows[END_REF][START_REF] Dirandeh | Effects of different polyunsaturated fatty acid supplementations during the postpartum periods of early lactating dairy cows on milk yield, metabolic responses, and reproductive performances[END_REF]. The differences in numbers of follicles were moderate in the present study, but this finding was expected, as the n-6

Comment citer ce document : Elis, S., Fréret, S., Desmarchais, A., Maillard, V., Cognié, J., Briant, E., Touze, J.-L., Dupont, M., Faverdin, P., Chajès, V., Uzbekova, S., Monget, P., Dupont, J. ( 2016 associated with oocyte quality [START_REF] Diskin | Embryonic and Early Foetal Losses in Cattle and Other Ruminants[END_REF]Walsh et al., 2011).

Thus, in the present study, benefits of fish oil on NF-EEM could be through oocyte quality enhancement, as hypothesized in the literature (reviewed in [START_REF] Leroy | Dietary fat supplementation and the consequences for oocyte and embryo quality: hype or significant benefit for dairy cow reproduction?[END_REF]. For example, feeding of an ALA-rich n-3 diet appears to be a satisfactory approach for improving oocyte quality in dairy cows [START_REF] Moallem | Dietary alpha-linolenic acid from flaxseed oil improved folliculogenesis and IVF performance in dairy cows, similar to eicosapentaenoic and docosahexaenoic acids from fish oil[END_REF]. These potential beneficial effects could be amplified as a result of previously described physiologic mechanisms (lesser amounts of PGF2, uterine progesterone receptor gene expression increase) that could provide a more favorable uterine environment [START_REF] Mashek | The effects of feeding fish oil on uterine secretion of PGF2alpha, milk composition, and metabolic status of periparturient Holstein cows[END_REF]Bilby et al., 2006a).

Fertility at first service can be affected by milk production and metabolic variables which did not occur in the present experiment, where all production and metabolic variables analyzed were similar between groups and not affected by the n-3 PUFA diet. However, these data suggest that DHA and EPA provided by the diet might exert direct and specific actions on ovarian function independent of the metabolic and/or production variables.

Comment citer ce document : Elis, S., Fréret, S., Desmarchais, A., Maillard, V., Cognié, J., Briant, E., Touze, J.-L., Dupont, M., Faverdin, P., Chajès, V., Uzbekova, S., Monget, P., Dupont, J. ( 2016 The difference in NF-EEM between groups was notable but not significant.

One explanation is that the treatments imposed on both groups were expected to have beneficial effects on reproduction. This was expected because the n-6 PUFA diet was reported to reduce embryo mortality [START_REF] Petit | Importance of dietary fat on reproduction of dairy cows[END_REF], to increase the number of small [START_REF] Ponter | Effect of the type of dietary fatty acid (-linolenic acid or linoleic acid) on the number and the quality of oocytes collected by Ovum Pick-Up and on the production of embryos by dairy heifers[END_REF] and large follicles and estradiol concentrations in the follicular fluid (Zachut et al., 2010) and increase the size of the dominant follicle [START_REF] Robinson | Effects of dietary polyunsaturated fatty acids on ovarian and uterine function in lactating dairy cows[END_REF]. Feeding a more unsaturated source of FA improved fertilization and embryo development in lactating dairy cows while the metabolic status of the experimental animals was not changed [START_REF] Cerri | Effect of fat source differing in fatty acid profile on metabolic parameters, fertilization, and embryo quality in highproducing dairy cows[END_REF]. The n-6

PUFA diet is, however, the most desirable control group for the n-3 PUFA diet treatment as both contain polyunsaturated fatty acids, thus a difference in response between groups would indicate a specific effect of n-3 fatty acids. A comparison with a saturated diet will not allow for the discrimination between a beneficial effect of an unsaturated source compared with a PUFA source or a specific effect of n-3 fatty acids. The inclusion of the n-6 PUFA dietary treatment in the present study, as a result of positive effects on reproductive variables, made it more difficult to ascertain the beneficial n-3 dietary effects. Instead of using fish oil to provide EPA and DHA to dairy cows, another strategy could be used by supplying DHA from microalgae, which appears to replace at least in part the DHA from fish oil without leading to any adverse effect on production [START_REF] Abughazaleh | Short communication: The effect of substituting fish oil in dairy cow diets with docosahexaenoic acid-micro algae on milk composition and fatty acids profile[END_REF].

Conclusion

In the present research, effects were studied of a n-3 PUFA diet on production, metabolic and ovarian variables of dairy cows. No differences in production and metabolic variables were detected with the feeding of the n-3 PUFA diet compared

Comment citer ce document : Elis, S., Fréret, S., Desmarchais, A., Maillard, V., Cognié, J., Briant, E., Touze, J.-L., Dupont, M., Faverdin, P., Chajès, V., Uzbekova, S., Monget, P., Dupont, J. ( 2016 These effects do not appear to be related to production and metabolic variables.

Effects could, however, be associated with n-3 PUFA-specific dietary effects on the reproductive environment, with the supplementation period used (first 2 months postpartum) suggesting a potential effect on oocyte quality. Further studies are necessary to determine whether DHA or EPA enhances fertility in lactating dairy cattle and to investigate the mechanisms involved.

Comment citer ce document : Elis, S., Fréret, S., Desmarchais, A., Maillard, V., Cognié, J., Briant, E., Touze, J.-L., Dupont, M., Faverdin, P., Chajès, V., Uzbekova, S., Monget, P., Dupont, J. ( 2016 Comment citer ce document : Elis, S., Fréret, S., Desmarchais, A., Maillard, V., Cognié, J., Briant, E., Touze, J.-L., Dupont, M., Faverdin, P., Chajès, V., Uzbekova, S., Monget, P., Dupont, J. ( 2016 0.22 ± 0.02 a 0.26 ± 0.02 a 0.17 ± 0.02 a 0.18 ± 0.02 a 0.14 ± 0.02 a 0.21 ± 0.02 b 0.20 ± 0.03 a 0.24 ± 0.02 a C20:4n-6 5.57 ± 0.17 a 5.22 ± 0.16 a 4.53 ± 0.17 a 4.39 ± 0.16 a 3.29 ± 0.19 a 3.69 ± 0.17

  ). Effect of a long chain n-3 PUFA-enriched diet on production and reproduction variables in Holstein dairy cows.Animal Reproduction Science, 164, 121-132. DOI : 10.1016/j.anireprosci.2015.11

  ). Effect of a long chain n-3 PUFA-enriched diet on production and reproduction variables in Holstein dairy cows. Animal Reproduction Science, 164, 121-132. DOI : 10.1016/j.anireprosci.2015.11Animals and experimental design All experimental protocols were approved by the local ethics committee "Comité d'Ethique en Expérimentation Animale Val de Loire (CEEA VdL)" (protocol registered under ref. 2012-01-1), in accordance with European Directive 2010/63/EU on the protection of animals used for scientific purposes. Holstein cows (n = 25) were managed in loose housing and monitored over 2 consecutive years (21/25 cows were monitored during the second year) in the same dairy herd in a switchback design. Each cow received either n-3 or n-6 diets the first year, and the other diet the following year. For the first year of the experiment, cows were either in their first (n = 7) or second (n = 18) lactation. The number of cows used in the experimental design is presented in Table

  ). Effect of a long chain n-3 PUFA-enriched diet on production and reproduction variables in Holstein dairy cows. Animal Reproduction Science, 164, 121-132. DOI : 10.1016/j.anireprosci.2015.112007). The long chain n-3 fatty acid supplement (n-3 diet) was a rumenprotected encapsulated fish oil OMG750® (Kemin, Nantes, France) and the n-6 fatty acid supplement (n-6 diet) was toasted soybeans Danex® (Agralys, Blois, France). The supplements were manually mixed into the diet (at 1% DM for n-3 diet and at 1.8% DM for n-6 diet) and the TMR were distributed twice daily in individual weighing troughs (Insentec B.V., Marknesse, The Netherlands) as described in Coyral-Castel et al., 2012b. Cows were fed ad libitum and DMI was determined from the composition of the diets, the feed intake and the dry matter content of each ingredient included in the diets. The DMI was analyzed daily during the distribution of the n-3 or n-6 diets, from calving to 2 months postpartum (2MPP).

  ). Effect of a long chain n-3 PUFA-enriched diet on production and reproduction variables in Holstein dairy cows.Animal Reproduction Science, 164, 121-132. DOI : 10.1016/j.anireprosci.2015.11during the first year of the experiment in samples from six cows in the n-3 diet group and six cows in the n-6 diet group.

  ). Effect of a long chain n-3 PUFA-enriched diet on production and reproduction variables in Holstein dairy cows.Animal Reproduction Science, 164, 121-132. DOI : 10.1016/j.anireprosci.2015.11

  ). Effect of a long chain n-3 PUFA-enriched diet on production and reproduction variables in Holstein dairy cows. Animal Reproduction Science, 164, 121-132. DOI : 10.1016/j.anireprosci.2015.11(CRAI1) at 35 and 90 d after AI1 was ascertained by the number of pregnant cows (ultrasonographic examination) divided by the total number of inseminated cows. The percentage of cows with late embryo mortality or fetal mortality (LEM-FM) was ascertained by the number of non-pregnant cows at 90 d divided by the number of pregnant cows at 35 d. Interval to first insemination (IFI)

  ). Effect of a long chain n-3 PUFA-enriched diet on production and reproduction variables in Holstein dairy cows.Animal Reproduction Science, 164, 121-132. DOI : 10.1016/j.anireprosci.2015.11covariance structure and with the value of BW at WPP1 as a covariable for MY and DMI.

  ). Effect of a long chain n-3 PUFA-enriched diet on production and reproduction variables in Holstein dairy cows. Animal Reproduction Science, 164, 121-132. DOI : 10.1016/j.anireprosci.2015.11.020 Page 13 of 37 A c c e p t e d M a n u s c r i p t -13 -Comment citer ce document : Elis, S., Fréret, S., Desmarchais, A., Maillard, V., Cognié, J., Briant, E., Touze, J.-L., Dupont, M., Faverdin, P., Chajès, V., Uzbekova, S., Monget, P., Dupont, J. (2016). Effect of a long chain n-3 PUFA-enriched diet on production and reproduction variables in Holstein dairy cows. Animal Reproduction Science, 164, 121-132. DOI : 10.1016/j.anireprosci.2015.11.020 Page 14 of 37 A c c e p t e d M a n u s c r i p t -14 -

  ). Effect of a long chain n-3 PUFA-enriched diet on production and reproduction variables in Holstein dairy cows. Animal Reproduction Science, 164, 121-132. DOI : 10.1016/j.anireprosci.2015.11.020 Page 15 of 37 A c c e p t e d M a n u s c r i p t

  ). Effect of a long chain n-3 PUFA-enriched diet on production and reproduction variables in Holstein dairy cows.Animal Reproduction Science, 164, 121-132. DOI : 10.1016/j.anireprosci.2015.11between groups (-11.5 ± 0.8 and -11.6 ± 0.8 kg DM/day in the n-3

  ). Effect of a long chain n-3 PUFA-enriched diet on production and reproduction variables in Holstein dairy cows.Animal Reproduction Science, 164, 121-132. DOI : 10.1016/j.anireprosci.2015.11

  ). Effect of a long chain n-3 PUFA-enriched diet on production and reproduction variables in Holstein dairy cows.Animal Reproduction Science, 164, 121-132. DOI : 10.1016/j.anireprosci.2015.11

  ). Effect of a long chain n-3 PUFA-enriched diet on production and reproduction variables in Holstein dairy cows.Animal Reproduction Science, 164, 121-132. DOI : 10.1016/j.anireprosci.2015.11the n-3 group and 2.4 ± 0.2 waves in the n-6 group, respectively; diet effect P = 0.26) were also similar between groups.

  ). Effect of a long chain n-3 PUFA-enriched diet on production and reproduction variables in Holstein dairy cows. Animal Reproduction Science, 164, 121-132. DOI : 10.1016/j.anireprosci.2015.11assessed 21, 35 and 90 days after AI1 in the n-3 (n = 22)

  ). Effect of a long chain n-3 PUFA-enriched diet on production and reproduction variables in Holstein dairy cows. Animal Reproduction Science, 164, 121-132. DOI : 10.1016/j.anireprosci.2015.11affected follicle number in previous research (reviewed in Leroy et al., 2014). Moreover, the experimental design in the present study was meant to focus on oocyte quality, consequently affecting fertilization and/or early embryo development. The diet was provided between calving and 2MPP, thus during folliculogenesis in follicles from which ovulation of oocytes could occur around the time of AI. Potential consequences on oocyte quality can be observed by assessing pregnancy status 21 d and 35 d after AI1 to estimate the rates of NF-EEM and PLP-LEM. Pregnancy failures occurring after 24 d post-AI1 (late embryo or early fetal mortality) are not

  ). Effect of a long chain n-3 PUFA-enriched diet on production and reproduction variables in Holstein dairy cows.Animal Reproduction Science, 164, 121-132. DOI : 10.1016/j.anireprosci.2015.11

  ). Effect of a long chain n-3 PUFA-enriched diet on production and reproduction variables in Holstein dairy cows.Animal Reproduction Science, 164, 121-132. DOI : 10.1016/j.anireprosci.2015.11n-6 PUFA diet. An interesting finding in the present study is that the feeding of the n-3 PUFA diet tended to increase the number of large follicles and decrease the non-fertilization and/or early embryo mortality rate. Nevertheless, the conception rate at 35 and 90 d after AI1 was not affected by diet in the present study.

  ). Effect of a long chain n-3 PUFA-enriched diet on production and reproduction variables in Holstein dairy cows.Animal Reproduction Science, 164, 121-132. DOI : 10.1016/j.anireprosci.2015.11as well as Melanie Collin and Anne-Sophie Gross at the Institut 503Gustave Roussy for the fatty acid composition analysis in plasma. 504505Comment citer ce document : Elis, S., Fréret, S., Desmarchais, A., Maillard, V., Cognié, J., Briant, E., Touze, J.-L., Dupont, M., Faverdin, P., Chajès, V., Uzbekova, S., Monget, P., Dupont, J. (2016). Effect of a long chain n-3 PUFA-enriched diet on production and reproduction variables in Holstein dairy cows. Animal Reproduction Science, 164, 121-132. DOI : 10.1016/j.anireprosci.2015.11Comment citer ce document : Elis, S., Fréret, S., Desmarchais, A., Maillard, V., Cognié, J., Briant, E., Touze, J.-L., Dupont, M., Faverdin, P., Chajès, V., Uzbekova, S., Monget, P., Dupont, J. (2016). Effect of a long chain n-3 PUFA-enriched diet on production and reproduction variables in Holstein dairy cows. Animal Reproduction Science, 164, 121-132. DOI : 10.1016/j.anireprosci.2015.11., Fréret, S., Desmarchais, A., Maillard, V., Cognié, J., Briant, E., Touze, J.-L., Dupont, M., Faverdin, P., Chajès, V., Uzbekova, S., Monget, P., Dupont, J. (2016). Effect of a long chain n-3 PUFA-enriched diet on production and reproduction variables in Holstein dairy cows. Animal Reproduction Science, 164, 121-132. DOI : 10.1016/j.anireprosci.2015.11., Fréret, S., Desmarchais, A., Maillard, V., Cognié, J., Briant, E., Touze, J.-L., Dupont, M., Faverdin, P., Chajès, V., Uzbekova, S., Monget, P., Dupont, J. (2016). Effect of a long chain n-3 PUFA-enriched diet on production and reproduction variables in Holstein dairy cows. Animal Reproduction Science, 164, 121-132. DOI : 10.1016/j.anireprosci.2015.11.R., Burke, J.M., Thatcher, W.W., 1998. Influence of supplemental fats on reproductive tissues and performance of lactating cows. J. Dairy Sci. 81, 856-871. Tessaro, F.H., Ayala, T.S., Martins, J.O., 2015. Lipid Mediators Are Critical in Resolving Inflammation: A Review of the Emerging Roles of Eicosanoids in Diabetes Mellitus. Biomed Res Int 2015, 568408. Urlep, Z., Rozman, D., 2013. The Interplay between Circadian System, Cholesterol Synthesis, and Steroidogenesis Affects Various Aspects of Female Reproduction. Front Endocrinol (Lausanne) 4, 111. Walsh, S.W., Williams, E.J., Evans, A.C., 2011. A review of the causes of poor fertility in high milk producing dairy cows. Anim. Reprod. Sci. 123, 127-138. Zachut, M., Dekel, I., Lehrer, H., Arieli, A., Arav, A., Livshitz, L., Yakoby, S., Moallem, U., 2010. Effects of dietary fats differing in n-6:n-3 ratio fed to high-yielding dairy cows on fatty acid composition of ovarian compartments, follicular status, and oocyte quality. J. Dairy Sci. 93, 529-545.

Fig. 1 .

 1 Fig. 1. Production variables -Body weight (A) and milk yield (B) were monitored

Fig. 3 .

 3 Fig. 3. Ovarian follicular dynamics -Total number of follicles (A), the number of

Figure_1_cleanFigure_2_clean

  Figure_1_clean

Figure_3_clean

  Figure_3_clean

Table 1 :

 1 Number of cows per diet group and per year DM, dry matter; NE L , net energy for maintenance and lactation; PDIE, intestinal digestible proteins, when Energy is the limiting factor of microbial synthesis; PDIN, intestinal digestible proteins, when degraded N is the limiting factor of microbial synthesis

	n-3, n-3 polyunsaturated fatty acids; n-6, n-6 polyunsaturated fatty acids Table 2: Ingredients composing the experimental diets, and nutritional composition of the diets (mean ± SEM, n = 3 samples) Diet from calving to 2MPP n-3 n-6 Ingredient (% DM): corn silage 65.7 65.1 dehydrated alfalfa 8.3 8.3 Sandifort® (wheat) 10.3 10.2 Sandilait® (soybean and rapeseed meal) 13.7 13.6 Vitamins and minerals 5/23 0.6 0.6 calcium bicarbonate 0.4 0.4 encapsulated fish oil 1 ─── toasted soybeans ─── 1.8 Composition DM, % 38.8 ± 1.6 38.4 ± 1.3 NE L , MJ/kg DM 6.9 ± 0.1 7.0 ± 0.1 PDIE, g/kg DM 91.3 ± 4.0 88.9 ± 4.4 PDIN, g/kg DM 89.0 ± 6.6 86.1 ± 7.5 Neutral detergent fiber, % 32.0 ± 1.0 32.0 ± 1.4 Acid detergent fiber, % 20.8 ± 0.7 20.5 ± 0.9 Crude protein, % 12.8 ± 0.9 12.3 ± 1.0 Fat, % 2.8 ± 0.2 2.7 ± 0.1 n-3, n-3 polyunsaturated fatty acids; n-6, n-6 polyunsaturated fatty acids; 2MPP, two months Year 1 Year 2 Total n-3 13 10 23 n-6 12 11 23 postpartum; Diet Total 25 21 46

Table Comment

 Comment

citer ce document : Elis, S., Fréret, S., Desmarchais, A., Maillard, V., Cognié, J., Briant, E., Touze, J.-L., Dupont, M., Faverdin, P., Chajès, V., Uzbekova, S., Monget, P., Dupont, J. (

2016

). Effect of a long chain n-3 PUFA-enriched diet on production and reproduction variables in Holstein dairy cows. Animal Reproduction Science, 164, 121-132. DOI : 10.1016/j.anireprosci.2015.11.020

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Table 3 :

 3 Effect of n-3 (n = 13) or n-6 diet (n = 16) on plasma fatty acid composition (% of total fatty acid), expressed as lsmeans ± SEM

	Stage and diet

74 ± 0.62 a 18.38 ± 0.58 a 22.97 ± 0.62 a 22.86 ± 0.58 a 19.05 ± 0.67 a 20.96 ± 0.6 b 15.43 ± 0.89 a 17.84 ± 0.71 b MUFA 22.00 ± 0.65 a 21.30 ± 0.60 a 26.04 ± 0.65 a 25.62 ± 0.60 a 21.46 ± 0.70 a 23.29 ± 0.63 a 17.21 ± 0.92 a 19.76 ± 0.73 b C18:2n-6 16.58 ± 0.63 a 17.35 ± 0.58 a 19.33 ± 0.63 a 21.55 ± 0.58 b 23.93 ± 0.68 a 23.68 ± 0.61 a 26.86 ± 0.91 a 25.00 ± 0.2.37 ± 0.18 a 2.45 ± 0.21 a 3.63 ± 0.18 b 5.28 ± 0.27 a 4.92 ± 0.22 a C20:3n-9

Acknowledgements

The INRA (Institut National de la Recherche Agronomique), ApisGene and Région Centre Val de Loire financially supported this work. We would like to thank the technical staff of the research (PRC) and experimental (UEPAO) units in INRA Nouzilly (Christelle Ramé, Mickael Delanoue, Christophe Mouaze, Ludovic Métivier and Damien Capo) and two students (Sonia Roger and Anaïs Leport) who helped during experiment work and data analysis. The authors also acknowledge Thibaud Le Mouel, Marie Le Tilly and Colette Mustiere at the INRA Rennes for the NEFA and

Declaration of Interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.