Laura Altenschmidt
email: laura.altenschmidt@u-psud.fr

Giulia Fornasieri

Eric Rivière

François Brisset

Romuald Saint-Martin

Anne Bleuzen
email: anne.bleuzen@u-psud.fr

Effect of alkali cations on the photomagnetic behavior of CoFe Prussian blue analogue nanoparticles embedded in ordered mesoporous silica

Keywords: Prussian blue analogues, Nanoparticles, Ordered Mesoporous Silica, Photomagnetism

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

Introduction

Prussian blue analogues (PBA) build a large family of compounds showing various properties (electrochemical [1,2], adsorption [3 ]…), among them magnetic switching. Thus, by applying an external stimulus, such as light [4][5][6], temperature [7,8] or pressure [9] on these compounds, it is possible to induce a charge transfer process in between the bimetallic pair which is accompanied by a change in the magnetic properties. Especially the CoFe PBA have been of particular interest in the past years [10][11][12][13]. During irradiation at low temperatures, the diamagnetic Co III (LS)-Fe II pairs are excited into a paramagnetic Co II (HS)-Fe III state and return to the initial state when the temperature is increased. This bistable behavior is of interest for the development of future applications, such as information storage devices [14,15].

Several studies aiming at understanding the origin of the switching properties in photomagnetic CoFe PBA powders and showing the general dependencies of these properties have shown the crucial role of interstitial alkali cations [12,[16][17][18][19]. The introduction of alkali cations such as Rb + or Cs + into the structure is accompanied by an increase of nitrogen atoms around the cobalt causing a decrease of the Co redox potential, which triggers a charge transfer and the generation of the photoswitchable diamagnetic Co III (LS)-Fe II pairs [20]. Moreover, not only the amount of diamagnetic pairs is crucial for the photomagnetic effect but also the amount of [Fe(CN)6] vacancies since the network needs to retain a certain degree of flexibility to adapt to the structural changes (Co II (HS) to ligand bond distance is longer) during the irradiation induced charge transfer. The more alkali cations are introduced in the interstitial sites, the more rigid the network becomes. It could also be shown that the photomagnetism not only depends on the amount but also on the nature of alkali cation used [16,20].

Further, for the elaboration of high-density storage devices, the reduction of the particle size and the control of the resulting properties is a necessary step. Our approach is the use of ordered mesoporous silica monolith with 2D hexagonal organization of the pores for the control of the different parameters (size, surrounding, chemical composition…) of the nanoparticles within the calibrated pores. The diameter of the pores, determined via transmission electron microscopy (TEM) as well as by nitrogen physisorption [21], can slightly vary from one synthesis to the other over the 5-6 nm range. A representative image of the hexagonal organization of the porous system and the filling of the pores with the monocrystalline PBA nanoparticles is shown in figures S1 and S2.

Pajerowski et al. [22] reported a dependence of the photomagnetic response of RbCoFe PBA nanoparticles depending on the particle size, showing that the efficiency of the photomagnetic effect decreases with particle size reduction. Our group has also previously studied the behavior of Rb2Co4[Fe(CN)6]3.3 nanoparticles embedded in mesoporous silica to understand the effect of the size reduction and the environment on the switching properties [23,24]. In comparison with the powder of the same chemical composition the mechanism behind the photomagnetic effect is the same at the scale of the bimetallic CoFe pair. However, the surface plays a peculiar role in the magnetic behavior due to its structure and composition.

It could be shown that the nanoparticles inside the silica matrix form a (Co III -Fe II )-core-(Co II -Fe III )-shell structure being responsible for the observed photomagnetic behavior.

In this work, we show that, it is possible to selectively vary the chemical nature of the alkali cations inserted in interstitial sites of CoFe nanocrystals while keeping the other parameters (size, surrounding) constant. We use the preparation method of nanocomposites which has been developed in our group in which ordered mesoporous silica monoliths with a 2D hexagonal organization of calibrated cylindrical pores contain PBA nanoparticles. This enables us to compare the RbCoFe and CsCoFe PBA which have been synthesized in the same conditions on the macro-and nanoscale and discuss the effect of the synthesis conditions and the size reduction on the different systems and their photomagnetic properties.

Material and methods

Nanoreactor synthesis:

The synthesis of the monoliths has already been described elsewhere [25]. In a typical procedure, a solution was prepared by dissolving the copolymer P123 (2.4 g) in the silica precursor TMOS solution (4 g) in a 30 mL polypropylene vial while stirring at 50 °C. An aqueous cobalt(II) nitrate acidic solution (80 mg, pH 1.4, 2 mL) was quickly added to the cooled solution. The mixture was stirred for 2 min 30 sec and then divided into four vials, which were sealed and aged for 1h in a 23 °C water bath. Finally, the vials were opened and left aging at 23 °C for 1 week to give homogeneous pink silica monoliths containing P123 and Co 2+ ions. Finally, the P123 copolymer was removed by calcination at 500 °C in air being accompanied by a color change to blue due to a change of the coordination sphere around the Co 2+ ion.

Photomagnetic Nanocomposites:

For the preparation of the photomagnetic nanocomposites either Rb + or Cs + were used as insertion cations. For all samples, a molar ratio of 2:1:2 for Rb/Cs:Co:Fe in the reaction solution was used. In the following these samples are called nanoRbCoFe and nanoCsCoFe.

During a typical synthesis, the blue calcined monolith was immersed in heptane. Then, a volume corresponding to 80% of the porous volume of a previously prepared acidified (80µl HNO3 per 1 mL) solution containing Rb + or Cs + ions and [Fe(CN)6] 3-complexes was added. The color change of the monolith from blue to dark purple is immediate, indicating the formation of the Prussian blue analogue. After 15 minutes the heptane is removed and the monolith is washed on average 5 times with distilled water until no coloration of the washing solution could be observed. Finally, the obtained nanocomposites are dried in air.

Photomagnetic References:

As references, the corresponding bulk photomagnetic materials with a molar ratio of Rb/Cs:Co:Fe of 2:1:2 in the reaction solution (as in the case of the nanocomposites) have been synthesized following the protocol in reference [16] with minor changes. In a typical synthesis, 400 mL of a 2.5 mmol/L cobalt(II)nitrate solution was added to 100 mL of a 20 mmol/L solution of hexacyanoferrate(III) and 20 mmol/L of either RbNO3 for RbCoFe or CsNO3 for CsCoFe. Both solutions were acidified with 3 drops of HNO3 (68%) each and the total addition time was 1 hour. The obtained purple precipitate was centrifuged 3 times, washed with distilled water and finally dried in air.

Instrumentation

Elemental analysis for the reference powders concerning the heavier elements (Rb, Cs, Fe and Co) was done by energy dispersive spectroscopy (EDS) using a Zeiss Sigma HD scanning electron microscope fitted with a SAMx Idfix EDS microanalysis system. The composition of the nanocomposites and the quantity of H, C and N was determined via standard microanalytical methods.

Infrared spectroscopy was performed in the range of 4000 cm -1 to 900 cm -1 on a Perkin Elmer Spectrum 100 in transmission mode with a resolution of 4 cm -1 . The compounds were mixed with nujol to obtain a suspension which was placed in between CaF2 windows.

Powder X-ray diffraction (Cu-Kα) was done on a Philips X'Pert Diffractometer in the 10-80 2θ range with a step size of 0.01 in the case of the powders and 0.033 for the nanocomposites.

Photomagnetic measurements were performed using a SQUID Quantum Design XL7 magnetometer. The ground samples (ca. 10 mg) were mixed with nujol, spread on cardboard and fixed in between two straws. For the references, the powder was diluted in silica to obtain the same Si/Co ratio as in the nanocomposites. These prepared samples were irradiated at 10 K inside the magnetometer with the help of a laser diode (635 nm, 50 mW/cm 2 ) until reaching the photostationary state. Afterwards, the magnetization value was measured while heating to 200 K with a sweeping rate of 2 K/min, followed by a measurement during the cooling process back to 10 K. The temperature dependence was determined in the 10-200 K range with a sweeping rate of 2 K/min before irradiation. For all the measurements, a field of 5000 Oe was applied.

Results and Discussion

Elemental analysis.

The experimental results of the elemental analyses and therefrom proposed chemical formulas taking into account the presence of well-defined entities and the electroneutrality of the solid are gathered in table 1 for the powdered reference compounds and in table 2 for the nanocomposites. Even though the synthesis conditions are the same for both reference samples, it can be seen that the Cs + ion enters in a higher amount into the PBA lattice. This behavior could already be observed in previous studies of powdered compounds [16] and can also be seen in the nanocomposites. However, since the PBA nanoparticles are highly diluted in a silica matrix the determination of the exact chemical composition is hindered and a large error range is assumed. From the elemental analysis we propose the formula RbxCo4[Fe(CN)6]8+x/3 with x = 1.9±0.5 for nanoRbCoFe and CsxCo4[Fe(CN)6]8+x/3 with x = 4±0.5 for nanoCsCoFe. In comparison to the corresponding reference powders, the amount of inserted alkali cation is slightly increased in the nanocomposites, which can be explained by the confinement effects of the silica matrix and/or higher concentration of the PBA precursors in the reaction medium.

Infrared spectroscopy.

The IR spectra of nanoRbCoFe and nanoCsCoFe are compared to those of the corresponding reference powder compounds over the 2200 to 2050 cm -1 range (CN stretching vibration) in Fig. 1a for the Rb system and in Fig. 1b for the Cs one. In the references only one main band at 2125 cm -1 corresponding to the Co III -NC-Fe II linkages can be seen regardless of the inserted alkali cation in agreement with previous studies. In excess of Rb + or Cs + ions, the amount of alkali cation inserted in the structure is enough to induce a total Co II Fe III to Co III Fe II electron transfer during the synthesis [16].

The nanocomposites also exhibit one main band but it differs from the one in the powdered compounds and also from one alkali cation to the other. In the Rb + ions containing sample (Fig. 1a), the main band at 2137 cm -1 has been assigned to cyanide ions in Co III -NC-Fe II linkages [24]. The slight shift of the band toward higher energies in the nanocomposites with regard to the powdered compound has already been observed in alkali cation free CoFe and NiFe PBA nanoparticles contained in a comparable silica matrix and has been explained by confinement and/or size effects [25]. In the Cs + ions containing nanocomposite (Fig. 1b), the main band is situated at 2125 cm -1 but it is significantly broader than the one observed on the spectrum of the powdered compound. The position of the band indicates the presence of CN ions in Co III -NC-Fe II linkages as in the powdered compounds and in the Rb nanocomposite. This shows that the nanoparticles contain Cs + ions and the Co II Fe III to Co III Fe II electron transfer occurred during their formation. Nevertheless, the broadness of the band indicates that the nanoparticles are made up of CN oscillators in a wider range of environments than in the powders and in the Rb + containing nanoparticles. From these observations we can conclude that, in contrast to the powdered compounds prepared in comparable conditions, the Co-NC-Fe linkages in the nanoparticles significantly dependent on the inserted alkali cation. This can be explained by different interactions between the bimetallic cyanide network and the alkali cation possibly in relation with the size of the latter combined with weaker structural cooperative effects within the bimetallic cyanide network in the nanoparticles, making the structure more sensitive to these interactions.

X-ray diffraction.

The diffraction patterns of the nanocomposites and corresponding reference powders are shown in Fig. 2. Around 24 ° a broad peak can be seen on the diffraction pattern of the nanocomposites, which is caused by the amorphous silica matrix in which the nanoparticles are embedded. Further, the diffraction pattern of the nanocomposites exhibit diffraction lines which are less intense and broader due to the dilution inside the silica and the reduced particle size. Nevertheless, it can be seen that those peaks all well agree with the obtained diffraction lines of the reference powders. Therefore, it can be concluded that in all cases the face-centered cubic PBA structure was formed.

With the Scherrer formula an average particle size of 6.2±0.5 nm for nanoRbCoFe and of 5.1±0.4 nm for nanoCsCoFe was approximated. Due to the strong broadening and the weak intensity of the diffraction peaks resulting from the dilution and small size of the particles, the error on the particle size is non negligible. It should be noted that the calculated size typically refers only to the crystallite size of the particles which in this case is similar to the particle size due to the monocristallinity of the PBA nanoparticles. In comparison, these values are very close to those of the diameter of the cylindrical pores of the silica monoliths and therefore are in agreement with the formation of spherical nanocrystals having a size which is determined by the shortest dimension of the nanoreactors (i.e. the diameter of the pores) [26]. The slight difference in the particle size can be explained by a possible slight variation of the pore diameter from one monolith to another.

From the (200), ( 220) and (400) diffraction lines, the lattice parameters a were calculated. For the Rb-containing samples a value of a = 9.96±0.01 Å for RbCoFe and a = 9.96±0.1 Å nanoRbCoFe was found. The values of the lattice parameter of the Rb nanoparticles inside the silica and the precipitated Rb powder are very close. It can be concluded, that the lattice parameter of the PBA structure is governed by the length of the Co III -NC-Fe II linkages with a minor effect of the particle size on it. It is known that if the crystal is composed of Co III -(LS) ions, the lattice parameter is typically around 9.96 Å and in the case of Co II -(HS) ions the bonds are longer and the lattice parameter is about 10.30 Å [20].

It has to be noticed here that in a previous study [24] we showed that Rb2CoFe PBA nanoparticles confined in the pores of similar ordered mesoporous silica monoliths consist of a Co III -Fe II core with a structure comparable to that of the bigger powder particles and a Co II -Fe III surface. These Co II Fe III surface species are not detected by X-ray diffraction. Due to much stronger intensity of CN stretching vibration when linked to Fe II rather than to Fe III ions, the identification of a contribution of Co II Fe III surface species on the IR spectra of nanoparticles mainly made of Co III Fe II species is not evident either.

In contrast, for the Cs samples this good agreement in between the lattice parameter value of the powdered reference compound and the nanoparticles is not observed. In Fig. 2b, it can already be seen that the peaks in the nanocomposite are shifted towards lower 2θ values. For CsCoFe a = 9.97±0.01 Å was calculated which is similar to the powdered rubidium reference. In the powdered samples containing a sufficient amount of Rb + or Cs + ions, the lattice parameter of the PBA structure is governed by the length of the predominant Co III -NC-Fe II linkages. The structure of the PBA seems to result from long-range order of the Co III -NC-Fe II linkages with a minor effect of the interactions of the inserted alkali cation with the surrounding lattice.

For the determination of the lattice parameter in nanoCsCoFe only the (220) and (400) diffraction lines could be used since the (200) cannot be seen due to the low intensity. This is in agreement with a study by Escax et al. [18] who showed that the ratio between the (200) and ( 220) diffraction line decreases with increasing amount of Cs + in the structure. In the diffraction pattern of nanoCsCoFe, the (200) line is not visible and the (220) line shows a high intensity, which is in good agreement with the results of the elemental analysis (section 3.1) that a high quantity of cesium entered the lattice structure.

Concerning the lattice parameter of the Cs-nanoparticles, a value of a = 10.12±0.1 Å was found, which is different from the one of the Cs powdered compound and also from the one of the PBA nanoparticles in the Rb nanocomposite. This value is significantly higher than that expected for a structure governed by the length of predominant Co III -NC-Fe II linkages. Since both Rb and Cs containing nanoparticles have on average the same size due to the confinement from the surrounding silica matrix and several monoliths containing RbCoFe PBA nanoparticles always exhibit a shorter lattice parameter [24], it can be concluded that the differences are not caused by the particle size only. The nature of the alkali cation also plays a role in its lengthening. We can propose two possible explanations for this lengthening: i) the diffracting domain of the Cs-containing nanoparticles is formed of a mixture of Co III -NC-Fe II and Co II -NC-Fe III linkages, ii) interactions with the bigger Cs + alkali cations slightly distort the structure made of Co III Fe II linkages, which is not the case in bigger particles due to collective structural effects. These two propositions are in line with the IR study (section 3.2). The large band centered around 2124 cm -1 reveals the existence of a large range of CN vibrators, which can be explained by competitive interactions between the CN bridges and the transition metal ions on the one hand and the CN bridges and alkali cations on the other hand and/or the presence of a mixture of Co III -NC-Fe II and Co II -NC-Fe III linkages in the bimetallic cyanide network. Work is in progress in order to clarify the electronic structure of the transition metal ions.

Even though the samples were all synthesized with an equal molar ratio in the initial solutions, only the Rb-samples seem to show a similar crystal structure on the macro-and nanoscale. The Cs-samples showed significant differences. Therefore, we can conclude that the lattice on the nanoscale depends not only on the amount of the inserted alkali cation and the accompanied charge transfer leading to the formation of Co III -(LS) ions but also on the nature of the cation.

Magnetic measurements.

The temperature dependence of the magnetization has been recorded over the 10-200 K temperature range for all samples before and after irradiation. In figure S3, the magnetization curves for the pure RbCoFe powder for two different irradiation times (λ = 635 nm, P = 50 W•cm -2 ) are shown.

When the sample is irradiated until the magnetization curve reaches a plateau (photostationary state), the expected magnetic behavior for the excited state of the CoFe PBA containing Rb + ions in sufficient amount can be observed with a Curie temperature at 21 K and a thermally activated relaxation of the excited state over the 100-150 K temperature range. The temperature range corresponding to the thermal relaxation of the photo-excited state can be decomposed into two areas: the 100-115 K temperature range corresponding to a rather sharp drop of the magnetization followed by a very progressive return of some residual excited species to the ground state over the 115-150 K temperature range. This behavior is due to a complete photo-induced Co III (LS)Fe II → Co II (HS)Fe III electron transfer in the irradiated part of the sample. It can be noticed that a sigmoidal shape of the time-dependent relaxation magnetization curves due to cooperative effects was already observed by Goujon et al. [27] in a comparable compound. But when the irradiation is stopped prior to reaching the photostationary state (Fig. S3b), a partially excited system is obtained. Regardless of the degree of conversion, the systems fully return to the ground state over the same temperature range with the same two steps. The slowness of the photo-excitation can be explained by the dark color of the powder, which strongly absorbs in the visible range, thereby leading to a partial excitation of the sample.

Wanting to avoid the strong self-absorbance of the pure powder, the RbCoFe powder was diluted in pure silica in the same PBA/silica ratio as in the nanocomposites. The obtained magnetization curves are shown in Fig. 3a. When the powdered sample is diluted in silica, several observations can be made: i) the phototransformation occurs faster than in the pure powder and ii) when the laser is cut prior to the completed excitation no difference in the relaxation can be found except a lower total reached magnetization due to the incomplete transformation. This behavior can be explained by the dilution effect of the silica. The radiation is rather scattered and readily diffused towards all the PBA particles within the matrix than getting absorbed by a compact stacking of PBA particles as it was the case in the pure sample. Regarding the trend of the relaxation (Fig. S4), as was the case for the fullytransformed non-diluted powder, three areas can be identified: i) the change in magnetic ordering at 21 K from ferrimagnetic ordered to disordered, ii) the sharp drop of the magnetization over the 90-115±2 K temperature range [27] and iii) a very progressive relaxation of residual pairs from 115 to 150±2 K.

Figure S5 shows the increase of the magnetization over time during irradiation of the diluted powder references. Consistent with the previous discussion, the magnetization in the diluted RbCoFe powder increases drastically after the beginning of irradiation revealing a fast charge transfer from iron to cobalt. In contrast, the diluted CsCoFe sample only shows a slow and overall small increase of the magnetization. The photomagnetic effect in CsCoFe is significantly decreased. The relaxation of the excited CsCoFe system is very progressive (Fig. S6) and also occurs at a lower temperature than in the Rb powder at 135±2 K. This and the reduced photomagnetic effect can be explained by the presence of three cesium ions at the interstitial sites of the cubic lattice causing the lattice to become more rigid due to the reduced amount of Fe(CN)6 vacancies and/or more strained due to stronger interactions between the bimetallic cyanide network and alkali cations. Thereby the charge transfer accompanied by an increase of the lattice parameter is hindered and more total energy is needed. Such a reduction of the efficiency of the photomagnetic effect in powdered compounds was already evidenced upon replacing Rb + by Cs + in a two alkali cations per unit cell-containing powder [20] and up to its disappearance in the derivative containing four Cs + cations per cell [16]. The magnetization curves of the nanocomposites before and after irradiation are shown in Fig. 4. The absolute values of the magnetization will not be discussed because the exact amount of nanoparticles in the fraction of sample used for the measurements is not exactly known. Both nanocomposite samples show an increase in the magnetization value during irradiation and therefore the photomagnetic effect is preserved at the nanoscale. The increase of the magnetization over time during irradiation is shown in Fig. S7. No significant difference between the two nanocomposites can be observed. After completed irradiation, the excited Co II (HS) Fe III pairs relax back into the Co III (LS)Fe II ground state when the temperature is increased. The relaxation temperature is defined as the temperature at which the two curves, before and after irradiation, intersect. For nanoRbCoFe the difference of the magnetization after and before irradiation returns to zero at 130±2 K and for nanoCsCoFe at 127±2 K. The relaxation of the photo-excited pairs is rather progressive and no significant difference can be seen in between the two nanocomposites (Fig. S8). This can be explained by the reduced size of the PBA nanoparticles, which are not large enough for the development of cooperative effects and for which surface effects predominate. Work is in progress to prepare silica matrices with various sizes of the pores, in which PBA nanoparticles with various sizes could be formed. Since the observed photomagnetic behavior is rather similar in both nanocomposites even though the XRD and IR data showed differences, the temperature dependence of XT in the range of 10-300 K for both nanocomposites has been measured (Fig. S9). A similar trend for the cesium nanocomposite and the rubidium one can be seen and therefore a possible thermal-activated charge transfer can be excluded. This observation has also been evidenced by Cafun et al. [20] in PBA powders. The differences in the electronic structure observed in the ground states of the nanocomposites seem not to have a significant impact on the photomagnetic effect. Surprisingly, the shape of the magnetization curves and therefore the photomagnetic effect is very similar regardless of the inserted alkali cation even though the structural characterizations (XRD, IR) showed significant differences in the ground state of the nanoparticles. This is all the more surprising that the photomagnetic effect of the two corresponding powders significantly depends on the nature of the inserted alkali cations. On the nanoscale, new effects and interactions between the alkali cation and the bimetallic cyanide network as well as the contribution of different species prevail, resulting in a different behavior than at the macroscale. A better knowledge of the effects of size reduction on cooperative effects would certainly help understand the behavior of these photomagnetic nanoparticles. Further studies are in progress to better understand these effects.

Conclusion

We were able to synthesize CoFe PBA nanoparticles containing alkali cations of different natures in the pores of mesoporous silica monoliths exhibiting a 2D hexagonal organization of the pores. As all other parameters were kept the same (size, crystallinity, surrounding), this enabled us to study the effect of a change of alkali cation in interstitial sites on the photomagnetic properties of the nanoparticles. Their properties were also compared to those of powders precipitated in aqueous solutions in the presence of same ratio of reactants.

The study shows that for both rubidium and cesium cations the nanoparticles exhibit a photomagnetic effect. Even though a sizable effect of the nature of the inserted alkali cation in powders on the photomagnetic effect has been reported in literature [20] and could also be observed in the here prepared rubidium and cesium references, we show that on the nanoscale the role of the inserted alkali cation is different. Whereas the big cesium cation hinders the photomagnetic effect when contained above a certain threshold in powders, such an effect does not persist at the nanoscale where cooperative and collective effects are reduced and surface contribution increases. Further studies of this behavior are needed to fully understand the interactions and contributions of the alkali cation on the nanoscale.

Fig. 1 .

 1 Fig. 1. IR-spectra of a) Rb-containing samples and b) Cs-containing samples.

Fig 2 .

 2 Fig 2. XRD pattern of a) Rb-containing samples and b) Cs containing samples. The peaks corresponding to the aluminum support in the nanocomposites (grey lines) are marked by an asterisk (*).

Fig. 3 .

 3 Fig. 3. Magnetization curves of a) RbCoFe and b) CsCoFe diluted in silica before (grey) and after (black) irradiation.

Fig. 4 .

 4 Fig. 4. Magnetization curves for a) nanoRbCoFe and b) nanoCsCoFe before (grey line) and after (black line) irradiation.

Table 1 :

 1 Chemical composition of the reference powders.

			Rb	Cs	Co	Fe	C	H	N
	RbCoFe Exp.	10.33 -	19.46 14.26 17.73 1.96 20.57
		Calc.	9.12 -	19.32 14.19 18.31 1.99 21.36
			Rb1.3Co4[Fe(CN)6]3.1 • 12 H2O
	CsCoFe	Exp.	-	27.40 14.57 12.13 16.49 1.13 19.23
		Calc.	-	25.84 14.79 12.96 16.72 1.14 19.5
			Cs3.1Co4[Fe(CN)6]3.7 • 9 H2O

Table 2 :

 2 Chemical composition of the nanocomposites.

		Rb	Cs	Co	Fe	C	H	N	Si
	nanoRbCoFe Exp.	0.52 -	0.76 0.56 0.88 1.56 0.88 37.74
	Calc.	0.53 -	0.77 0.60 0.78 1.59 0.91 38.43
		Rb1.9Co4[Fe(CN)6]3.3 @(SiO2)417(H2O)240
	nanoCsCoFe Exp.	-	1.89 0.75 0.57 0.89 1.54 0.90 37.59
	Calc.	-	1.70 0.75 0.72 0.92 1.55 1.08 37.86
		Cs4Co4[Fe(CN)6]4 @(SiO2)421(H2O)240

Acknowledgments

This research was carried out with the support of the University Paris-Sud and the CNRS.

Appendix A: Supplementary Data

Supplementary information (Figures S1-S9)

Conflict of Interest

The authors declare no conflict of interest.


Charlotte Paes

Laurence Fortun-Lamothe

K Bébin

J Duperray

C Gohier

E Guené-Grand

G Rebours

Patrick Aymard

Carole Bannelier

C Paës

K Bebin

A.-M Debrusse

T Gidenne

S Combes

Onset of feed intake of the suckling rabbit and evidence of dietary preferences according to pellet physical properties

Keywords: Rabbit, Weaning, Solid consumption, Pellet die, Feed preference ADFom, acid detergent fiber expressed exclusive of residual ash, aNDFom, neutral

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

Introduction

Despite consequent improvements in rabbit feed formulation and husbandry practices since the 1980s, a high incidence of digestive disorders around the time of weaning can still be observed. In this context, antibiotic use remains high on meat rabbit farms [START_REF] Méheust | Suivi des ventes de médicaments vétérinaires contenant des antibiotiques en France en[END_REF]. The cost of antimicrobials and the spread of antibiotic-resistant bacteria have increased the need for alternatives to antibiotic use.

Establishing a stable digestive microbiota at an early stage could enhance rabbit resilience to the stress of weaning [START_REF] Fortun-Lamothe | A review on the interactions between gut microflora and digestive mucosal immunity. Possible ways to improve the health of rabbits[END_REF][START_REF] Combes | Engineering the rabbit digestive ecosystem to improve digestive health and efficacy[END_REF]. Indeed, relationships between enteric infection and disruption of the normal microbiota (known as dysbiosis) have been highlighted in different species such as rabbit [START_REF] Sekirov | The role of the intestinal microbiota in enteric infection[END_REF][START_REF] Bäuerl | Changes in Cecal Microbiota and Mucosal Gene Expression Revealed New Aspects of Epizootic Rabbit Enteropathy[END_REF]. As in the case of human children and piglets [START_REF] Konstantinov | Microbial diversity studies of the porcine gastrointestinal ecosystem during weaning transition[END_REF][START_REF] Koenig | Succession of microbial consortia in the developing infant gut microbiome[END_REF], the rabbit kit digestive microbiota composition is strongly shaped by solid feed intake [START_REF] Padilha | Relationships between microflora and caecal fermentation in rabbits before and after weaning[END_REF][START_REF] Padilha | Caecal microflora and fermentation pattern in exclusively milk-fed young rabbits[END_REF]. Previous studies demonstrated that ingestion by kits of doe feces left in the nest can speed up colonization of microbial cecum [START_REF] Kovács | Effect of nursing method and faeces consumption on the development of bacetroides, lactobacillus and coliform flora in the caecum of the newborn rabbits[END_REF][START_REF] Combes | Coprophagous behavior of rabbit pups affects implantation of cecal microbiota and health status[END_REF]. Solid intake via ingestion of fecal pellets or feed offered in the nest can start 2-3 days after birth [START_REF] Gidenne | Capacité d'ingestion d'aliment sec par le lapereau au nid: Interaction avec l'ingestion de fèces dures maternelles[END_REF][START_REF] Combes | Coprophagous behavior of rabbit pups affects implantation of cecal microbiota and health status[END_REF][START_REF] Kacsala | Early solid additional feeding of suckling rabbits from 3 to 15 days of age[END_REF]. As opposed to fecal pellet ingestion, neonatal solid feed intake behavior has never been quantified.

Since newborn rabbits are able to ingest solid feed, and since this behavior can contribute to triggering the installation of microbiota, we hypothesize that providing palatable feed to the kits is an opportunity to strengthen rabbit gut health. However, in rabbit farming, kits only have access to pelleted feed when they start leaving the nest box at around 16-18 days [START_REF] Gidenne | Feeding strategy for young rabbit around weaning: a review of digestive capacity and nutritional needs[END_REF]. Moreover, no data exists to validate the ability of the physical properties of the common industrial pellet to encourage early feed intake.

Preliminary studies investigated the effect of pellet hardness on rabbit intake in rabbits from 2-to 5-weeks old, but this effect could have been combined with feed composition or pellet size effect [START_REF] Gidenne | Adaptation du lapereau à un aliment fibreux distribué avant sevrage: comportement d'ingestion, croissance et santé digestive[END_REF][START_REF] Travel | Préférences alimentaires du lapereau selon le diamètre du granulé, dans le cadre d'une alimentation séparée mère-jeunes[END_REF]. [START_REF] Maertens | Influence du diamètre du granulé sur les performances des lapereaux avant sevrage[END_REF] previously showed that pellet size had an effect on rabbit intake after weaning, with higher consumption when a large pellet diameter (4.8 mm) was offered, suggesting that the physical properties of feed are key factors to optimize solid feed intake.

The aims of this study were (i) to quantify kit feed intake from 8 to 35 d, and (ii) to determine the optimal pellet diameter and hardness necessary to stimulate the onset of feed intake. To reach these goals, an original feeding system in the nest was designed and nine pellets with different press die specifications (i.e., length and diameter) were offered in double-choice tests before weaning to evaluate kit feed preferences and intake.

Material and methods

The study was conducted by the Groupe d'Experimentation Cunicole (GEC) network 1 and the experiments were carried out at the PECTOUL Experimental Unit (INRA, Castanet-1 GEC is a French network of companies with expertise on rabbit production and nutrition. It includes CCPA, EVIALIS, INZO, MiXscience and TECHNA.

Tolosan, France). Animals were handled according to the recommendations for animal care in experimentation in agreement with the EU Directive 2010/63/EU [START_REF] Anonymous | Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used[END_REF] and the Journal Officiel de la République Française [START_REF] Anonymous | Décret n° 2013-118 du 1er février 2013 relatif à la protection des animaux utilisés à des fins scientifiques[END_REF].

Animal management and housing

Female rabbits (hybrid breed PS19; Hypharm, France) were inseminated with semen from male commercial breeds for two successive reproductive cycles (first two lactations).

The does and their litter (n = 60 and 63 for the first and second trial, respectively) were raised together in a ventilated breeding unit in individual wire cages (width: 61 x length: 69 x height: 61 cm). Cages were equipped to allow the mother and its litter to be fed separately. In that respect, a wire mesh partition separated the doe from the area containing the kits' feeders. The doe's feeder in the cage was elevated so that the kits could not access to the doe's feed (Fig. 1). Two days after parturition (day 2), nest quality was assessed using a 3-point scale: 1 represented a nest of weak quality in which only wood shavings were found; 2 represented a nest of medium quality in which doe fur was present but insufficient to fully cover the litter; and 3 corresponded to a nest of good quality in which doe fur entirely covered the litter. After nest quality assessment, the litter size was standardized to ten kits per doe by cross-fostering or culling. The doe could access to the nest once a day for suckling (controlled suckling between 8:00 and 9:00 am for 10 min) until day 21. After day 21, the doe could freely access to the nest. Two water nipples were available: one in the doe area and one in the litter space.

Feed was offered ad libitum to the youngs in the nest (width: 25 x length: 38 x height: 20 cm) from 3 to 17 days of age using two plastic cups (volume: 30 mL; Ø = 40 mm; height: 32 mm; GOSSELIN ® , Le Mans, France), placed opposite the doe's entrance (Fig. 2). The feed present in the nest was removed before suckling to prevent the doe from eating it, then weighed and finally reintroduced after suckling. Cups were carefully maintained in holders (6-cm high) and contained feed pellets that differed by their physical characteristics alone (same formulation).

From 15 to 35 d, the same kind of pellets was offered ad libitum outside the nest in the litter area using two feeders, one for each type of pellet tested. The kits' feeders were designed as presented by [START_REF] Fortun-Lamothe | Technical note : An original system to separately control litter and female feed intake without modification of the mother -young relations[END_REF], with triangular-shaped openings to limit waste. To avoid confusion between feed placement and pellet preference, the location of cups or feeders containing each pellet type was reversed when feeding measurements were performed. The feeding scheme is summarized in Fig. 3. Milk production was measured as the difference of female weight before and after suckling at 3 or 4, 7, 10, 14, 17 and 21 days. Litter weight after suckling was recorded at 3 or 4, 10, 14, 21, 28 days.

At weaning (35 days of age), kits were assigned to cages of five rabbits/cage (width: 78 x length: 46 x height: 30 cm) according to the double-choice preference test conducted and litter of origin. Individual rabbit weight was measured at weaning, at 50 and 64 days of age.

Following common feeding strategy in France, the animals were given restricted feed until 64 days in order to achieve an average daily gain of 40 g/day. Animals were then fed freely until slaughter (day 72). Rabbit mortality was recorded daily from 3 to 72 days of age.

Diet processing

Does were fed a commercial feed (without any antibiotics) adapted to the lactation stage (Table 1). The diet for suckling rabbit was formulated to meet the nutrient needs of both does and kits. The feed mash was manufactured by Euronutrition SAS (Saint-Symphorien, France). Nine kinds of pellets from different dies were then produced (Tecaliman, Nantes, France) from the same mash to ensure that pellets had identical chemical composition. The ingredients and chemical composition of the pelleted feeds are presented in Table 1. In the pelletizer, the meal was delivered to a conditioning chamber where the quantity and pressure of added steam were controlled to ensure that the mash was at 65°C before flowing into the mill die chamber. To process the pellets, the mash was forced through one of the nine dies with a specific channel length and diameter (Table 2). Die length-to-diameter ratio (also referred to as compression rate) is known to influence hardness [START_REF] Thomas | Physical quality of pelleted animal feed 1. Criteria for pellet quality[END_REF]. Therefore, die size was the parameter chosen to obtain the desired pellets: pellets with four different diameters and similar hardness in Trial 1 (A, B, C, D), and pellets with three different hardnesses for two given diameters in Trial 2 (E, F, G and C, H, I).

Experimental design

From 3 to 35 days, to assess solid feed preferences, each litter was offered two pellet types while they were still suckling the doe. A complete double-choice feeding protocol was performed in both trials. In the first trial, four pellets were tested in pairs, which resulted in six combinations (AB, AC, AD, BC, BD, and CD, where comparison treatment AB represented pellet A tested against pellet B, etc.). In this trial for diameter preference, we had run out of pellet A stock at 32 days, thus ending the preference trial. Hardness preference was assayed in Trial 2, comparing 2.5-mm-diameter pellets against each other and 4.0-mmdiameter pellets against each other. These comparisons of pellets with the same diameter but with different compression rates resulted in six comparison treatments (EF, EG, FG and CH, CI, HI). Pellet C was used in both trials. The litters were assigned to one double-choice treatment according to doe parity (only primiparous in Trial 1 and a parity rank of 1.9 ± 0.01 in Trial 2) and to kit weight (average body weight at 2 days was 66 ± 1 g per kit in Trial 1 and 72 ± 1 g per kit in Trial 2). Allocation inside the farm was also equally divided between the treatments. Each treatment had ten or eleven litters of ten rabbits at the beginning of the experiment.

Evaluation of the feed intake

Early feed intake was calculated daily based on the weight of feed offered and refused from 3 to 17 days of age in the first trial using a 0.001-g accuracy scale (OHAUS, Parsippany, NJ, USA). Water loss, measured as the difference of pellet weight between two days in the absence of animals, was measured the first days of the experiment for the four pellets A, B, C and D. After correction for these intrinsic changes, it was found that feed intake was negligible the first week. For this reason, feed intake was measured daily from 8 to 17 days in the second trial. When waste or cup spillage occurred, we considered feed consumption in the two cups as missing data. In these cases, all of the nest material was renewed to ensure that no pellets were left inside the nest. Remaining feed in the feeders was weighed at 18, 21, 25, 28, 32 and 35 days of age. If one kit was found dead between these dates, the remaining feed in the cage was weighed to recalculate feed intake.

Physical characteristics of pellets

Pellet length and diameter were measured with a caliper with a resolution of 0.5 mm (ROCH, France). Diameters obtained were in accordance with the die used during the pellet processing (Table 3), with the biggest pellet (pellet D) 3-fold larger than the smallest one (pellet A). Pellet hardness was determined with the Kahl Pellet Hardness Tester (AMANDUS KAHL GmbH & Co. KG, Reinbek, Germany) on a minimum of ten pellets. Each pellet was inserted between two bars with an increasing static pressure to determine the force needed to crack it [START_REF] Thomas | Physical quality of pelleted animal feed 1. Criteria for pellet quality[END_REF]. Force values obtained in Newtons were then expressed as a pressure variable to take account of the length and diameter of each pellet tested as follows:

H = (π * * ⁄ )
where H is the calculated hardness (MPa), is the measured hardness (N), is the length of the pellet (mm), and is the pellet radius (mm) Pellet durability is "the amount of fines returning from pellets after they were subjected to an abrasing action" [START_REF] Thomas | Physical quality of pelleted animal feed 1. Criteria for pellet quality[END_REF]. For each measurement, 200 g of feed pellets were tumbled in a SABE drum (SABE Distribution, Chauche, France) at 500 rpm for 20 seconds. After tumbling, the fines (particles of less than 2 mm) were removed.

Durability was expressed as the percentage of remaining pellets (ISO 17831-1:2015). This procedure was repeated three times per pellet type.

The particle size distribution was determined after water and ultrasonic treatments according to the Lebas and Lamboley procedure (1999). Water-holding capacity (WHC) and feed-swelling capacity (SC) were measured for each type of pellet to check for confusing effects since these properties affect satiation [START_REF] Tan | Effects of dietary fibers with high waterbinding capacity and swelling capacity on gastrointestinal functions, food intake and body weight in male rats[END_REF]. WHC was determined twice according to the Giger Reverdin (2000) method: 2 g of sample were poured into 10 mL distilled water and left to soak for 24 hours, so that water was in excess. After centrifugation, wet supernatant was removed and weighed to measure water absorption. WHC was calculated as the ratio of the supernatant weight to the sample dry matter weight. SC was obtained by dissolving 2 g of sample in 25 mL distilled water. After 1 hour at room temperature, the increased volume of the feedstuff due to hydration was measured to determine SC, as previously described by [START_REF] Arroyo | Effects of presentation and type of cereals (corn or sorghum) on performance of geese[END_REF]. WHC and SC were measured in triplicate.

Chemical analysis of the feeds

Chemical analysis was performed on the nine experimental pre-weaning pellets and on the commercial pellets provided to the does using the ISO methods and the procedures described by the European Group on Rabbit Nutrition [START_REF] Egran | Technical note: Attempts to harmonise chemical analyses of feeds and faeces, for rabbit feed evaluation[END_REF]. Dry matter (DM) was obtained after 24 h at 103°C (ISO 6496:1999) and ashes after 5 h at 550°C. Total nitrogen was determined with the DUMAS combustion method using an automatic analyzer (Vario El cube model; Elementar, Langenselbold, Germany) to approximate the crude protein content (N x 6.25) (ISO 16634-1:2008). Neutral detergent fiber (aNDFom) and acid detergent fiber (ADFom) were measured using the sequential method of [START_REF] Van Soest | Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition[END_REF] with a heatstable amylase pre-treatment (AFNOR, 1997). Results were expressed exclusive of residual ash. Lignin analyses were performed on ADFom residues by solubilization of cellulose with sulfuric acid (sa). Starch was determined with the enzymatic procedure described by [START_REF] Kozlowski | L'amidon, quel dosage pour quel échantillon[END_REF]: starch was solubilized in potassium hydroxide and then hydrolyzed with amyloglucosidase. Released glucose molecules were finally quantified with the hexokinase glucose 6-phosphate deshydrogenase system (D-Glucose ® ; Boëhringer, Mannheim, Germany). Intense ether washing was performed using the Tecator Soxtec system HT (FOSS, Hilleroed, Denmark -acid hydrolysis pre-treatment) to determine crude fat content (ISO 6492:1999). Gross energy was established with adiabatic combustion (Model C5000 adiabatic calorimeter; IKA, Staufen, Germany) (ISO 9831:1998).

Calculation and statistical analysis

Before weaning, statistical models include the litter as the experimental unit, whereas after weaning, the individual animal is the experimental unit. Feed intake values in the nest (8-17 days) that were three standard deviations away from the mean were removed. These outliers represented less than 1.5% of the dataset. For feed consumption after 17 days of age, the number of kits was adjusted assuming that dead animals did not consume feed two days before their death.

Voluntary feed intake measurements were used to determine feed preferences: relative consumption (RC) was calculated for each pellet within a litter, which is the percentage contribution of a pellet to the total feed intake. For example, in the treatment AB of a given litter, the RC of pellet A was calculated as follows:

RC A = [Amount of pellet A consumed/(Amount of pellet A consumed + Amount of pellet B consumed)] *100
This index was chosen to describe preferences because it is informative, intuitive and has practical appeal [START_REF] Lockwood | On the statistical analysis of multiple-choice feeding preference experiments[END_REF]. RC values could represent total aversion (0%), indifference (50%) or strong preference (100%; [START_REF] Seabolt | Feed preferences and performance of nursery pigs fed diets containing various inclusion amounts and qualities of distillers coproducts and flavor[END_REF]. If RC was significantly higher than the neutral value of 50%, then a preference for the given pellet was observed inside the treatment. RC values of the first trial were calculated as of 13 days of age because we encountered technical difficulties with the feeding system before this date (cups spilled by the rabbits). In the second trial, RC values could be calculated from 8 to 35 days.

Statistics were performed with R software (R Core Team, 2018). One sample t-test was performed to test the RC means compared to the neutral value of 50%. If the RC value did not fit a normal distribution, Arcsine transformation was used. Comparison treatment effect on feed intake, milk intake and growth performance was controlled to ensure that preference responses could be compared between treatments by using one-way analysis of variance. In the second trial, the intake of 2.5-mm pellets was also compared with the intake of 4-mm pellets with ANOVA procedure. A linear mixed model was fitted to investigate inter-litter variability of the feed consumption measurements in the second trial from 8 to 21 days since no milk data were available beyond this date. Daily milk data between 8 and 21 days were obtained with extrapolation of the farm measurements by modeling the lactation curve with a quadratic model [START_REF] Casado | Modelling the lactation curve of rabbit does: Towards a model including fit suitability and biological interpretation[END_REF]. Time, comparison treatment and nest quality scores were set as fixed categorical factors, and litter weight at equalization and milk consumption were set as quantitative cofactors; the litter was considered to be a random effect. A linear mixed procedure was also applied on post-weaning data with comparison treatment before weaning and rabbit sex as fixed effects, litter and fattening cage as random effects. Mortality data was analyzed using adjusted chi-square according to the Donner and Banting procedure (1989), so that litter cluster was accounted for [START_REF] Reed | Adjusted Chi-Square Statistics: Application to Clustered Binary Data in Primary Care[END_REF][START_REF] Princée | Exploring Studbooks for Wildlife Management and Conservation[END_REF].

Differences were considered to be significant when P ≤ 0.05. If P-value was less than 0.10 but greater than 0.05, then differences among means were considered to suggest a tendency.

Results

Pellet physical characteristics

Measurements of pellet physical characteristics are given in Table 3. In Trial 1, by ranking in increasing order of hardness, we obtained pellets D, A, B and C (pellet C was 36% harder than pellet D). In Trial 2, for the 2.5-mm-diameter pellet group, pellet G had a greater hardness than pellets E and F (+ 0.3 MPa), whereas for the 4-mm diameter, pellet C was softer than pellets H and I (-0.2 MPa). These differences in hardness were related to the die length-to-diameter ratio used: 5.6 for G (highest compression rate of its group) and 4.5 for C

(lowest compression rate of its group). Pellet D showed a notably low durability (85.7 ± 1.1%) compared to the other pellets (94.3 ± 1.9% on average).

Feed and milk intake

Considering the whole pellet intake, regardless of the comparison treatments in Trials 1 and 2, we observed that solid feed ingestion in the nest started at 8 days of age, with a mean of 0.03 ± 0.03 g of DM consumed per kit (Fig. 4). One fourth of the litters did not start solid feed intake at 8 days, while one day later, only 9% had not started. At 12 days of age, all the litters were consuming the pellets. Pellet intake progressively increased with age and reached 0.54 ± 0.19 g of DM per rabbit at 17 days. Between 15 and 17 days, the feed was accessible in both the nest and the feeders. The young rabbits preferred to consume feed in the nest rather than in the feeders during this period (+0.20 g of DM/rabbit from 15 to 17 days in the nest compared to the feeder origin; P = 0.003). Over the first 17 days of life, a rabbit consumed a total of 1.63 ± 0.76 g of DM in the nest and a total of 2.50 ± 0.44 g of DM from the pellets in both the nest and the feeders. In relation to fresh matter, the pellet intake from 8

to 17 days corresponded to 1.3% of the total milk intake over this period. The feed-to-milk intake ratio increased from 0.1% of the milk intake at the beginning of the experiment (day 8), to 2% of the milk intake at 17 days of age. Interestingly, post-natal feed intake was highly variable between litters (CV = 89% for the total feed intake at 12 days, for example). A statistical model was therefore built to determine which factors had an effect on the daily feed intake using data obtained in the second trial until 21 days of age. Daily milk intake and nest quality score did not affect the solid feed intake (NS). Instead, the body weight at 3 days of age (average weight at 3 days: 109 ± 9 g/rabbit) correlated positively with the litter's postnatal pellet consumption from 12 to 17 days (P=0.003), and the correlation coefficients ranged from 0.3 to 0.5 (Fig. 5). After 21 days of age, the amount of feed consumed quickly increased (Fig. 6) to reach 65 ± 14 g of DM per kit on average at weaning. Concomitantly, the milk yield curve reached a plateau at around 21 days (292 ± 75 g of milk/doe).

The total feed intake in the feeders was similar between comparison treatments (Table 4) except at the beginning of the first trial (15 -17 days). However, the precision of the mean estimates for this period was low as indicated by the standard error of the mean (SEM). We compared intake of 2.5-mm pellets vs. 4-mm pellets in Trial 2, a situation where no choice between diameters was given (EF, EG and FH treatments compared to CH, CI and HI treatments). Between 21 and 27 days, the 2.5-mm pellet intake tended to be higher than the 4mm pellet intake (P = 0.068), but no effect was observed from 15 to 35 days (P = 0.645).

Pellet preferences

RC value of the 6-mm-diameter pellet (D) was greater than 50% when offered with the 2-mm-diameter pellet (A) in the nest (P=0.002). No other preference was expressed in the nest within treatments of the first trial (Fig. 7). In contrast with what was observed in the nest, clear-cut aversion was found for pellet D compared to A, B (3.2 mm) and C (4 mm) in the feeders, with RC values below 50% of total feed intake (RC of 14% in the double-choice group AD, 28% in BD and 27% in CD; P<0.001; Fig. 7). Relative consumption of A was greater than 50% when it was offered against B (61%; P=0.021) and D (86%; P<0.001), and tended to be greater than 50% compared to C (67%; P=0.081).

In the second trial, RC values within the double-choice groups EF, EG and FG did not significantly differ from 50%, meaning that no preference was expressed throughout the whole trial (Fig. 8). In the nest, slight avoidance was noticed for I compared to C (RC of 40%; P=0.014, when compared to the 50% neutral value) but this response was no longer significant in the feeders (P=0.117). When H was present with I in the feeders, the latter was avoided since its relative consumption was less than 50% (39%; P=0.020).

Health and growth performances according to treatments

Throughout both trials, all animals remained healthy: the mortality was low (4.5% from 3 to 35 days and 1.8% from 36 to 72 days) and was not affected by the pellets offered before weaning (NS). Milk intake was similar between groups as well as feed intake from 18 days of age to weaning.

Growth performances are reported in Tables 5 and6. Rabbit weights before weaning were not affected by the comparison treatments (NS). Double-choice treatment had an effect on the daily weight gain between 21 and 35 days of age in the first trial (P = 0.039), but the Tukey follow-up test performed on the six group means was not significant for any pair of means. The gender did not affect growth after weaning, thus the sex effect was removed from the final model. Rabbit growth performances during the fattening period did not differ between comparison treatments.

Discussion

We hypothesized that stimulating early solid feed intake would contribute to preserving the health of young rabbits through a positive action on cecal microbiota implantation. We thus aimed to, first, quantitatively describe early feed intake and, second, to determine young rabbit pellet preferences according to their physical properties.

Characterization of feed intake patterns

To our knowledge, this study is the first to provide comprehensive quantitative data on the solid intake of suckling rabbits. A nest feeding system was especially developed to measure post-natal rabbit feed intake. Previous studies dealing with the feeding capacity of young rabbits provided gradual amounts of pellets in the nest. They visually observed solid intake [START_REF] Kacsala | Early solid additional feeding of suckling rabbits from 3 to 15 days of age[END_REF] or measured the consumption by counting the number of feed pellets left [START_REF] Gidenne | Capacité d'ingestion d'aliment sec par le lapereau au nid: Interaction avec l'ingestion de fèces dures maternelles[END_REF]. By providing quantitative feed intake records, this study provided new insights into the occurrence, magnitude and variability of this behavior.

Suckling rabbits were able to consume some feed pellets as of 8 days of age. Before this age, the diet of young rabbits depends on milk supply alone. The absence of solid intake during the first week might be explained by a lack of capability since mastication is functional as of 13 days and kits are still learning to masticate until 4 weeks of age [START_REF] Langenbach | A longitudinal electromyographic study of the postnatal maturation of mastication in the rabbit[END_REF]. Furthermore, while the olfactory system is fully developed at birth, the onset of visual functions is estimated to be at around 7-8 days of age [START_REF] Gottlieb | Ontogenesis of sensory function in birds and mammals[END_REF]. These ontogenetic parameters should be considered when developing feed adapted to the young rabbit, for example, by adding sensory functional ingredients in the feed or by offering feed with an optimal texture for the kit's masticatory system. In this study, pellet size and hardness were investigated since we assumed that rabbits would prefer soft and small pellets because they are easier to chew.

In the present study, we were able to determine the evolution of solid feed intake in kits. As previously described [START_REF] Gidenne | Feeding behaviour of rabbits[END_REF], the amount of solid feed ingested outside the nest remained low until 21 days, before a sharp increase. The early onset of solid feed intake allowed by a nest feeding system did not modify this general pattern. In the nest, the between-litter variability of the solid feed intake was high, especially in the early period. Our results showed that the maternal ability of the doe, assessed together with milk intake and nest quality, did not affect the early feeding in the nest or in the feeders. [START_REF] Bonachera | Milk intake in kits: not only the total amount matters[END_REF] found, on the contrary, that the higher the milk intake was during early lactation (0-17 days), the higher the feed intake (17-28 days) was, which suggests that high milk intake could promote solid intake. However, these authors did not study the correlation between the feed intake and the corresponding milk intake at an early stage (before 17 days of age). At this age, we hypothesized that other factors could explain dietary intake, including litter weight at standardization, which was positively correlated with the early feed intake of the litter. At the within-litter level, a similar conclusion was reached by [START_REF] Pajor | Consumption of solid food by suckling pigs: individual variation and relation to weight gain[END_REF] who studied the variation of solid feed intake of suckling piglets. They demonstrated that piglets of high birth weight ate more pre-weaning feed than their littermates of lower birth weight. This could be explained by greater needs and a more developed digestive system in heavier animals [START_REF] De Passillé | Relationships of weight gain and behavior to digestive organ weight and enzyme activities in piglets[END_REF]. However, these findings were not consistent with the research of [START_REF] Bruininx | Individually assessed creep food consumption by suckled piglets: influence on postweaning food intake characteristics and indicators of gut structure and hind-gut fermentation[END_REF] who did not report a relationship between the piglets' birth weight and individual early feed consumption. Better knowledge of the animal related factors that affect solid feed intake variability is of importance in order to increase both the number of rabbits that exhibit early feed intake and the levels of ingestion.

Pellet processing and effects on suckling rabbits' preferences

This study attempted to analyze separately diameter and hardness effects by using pellets with different diameters and constant compression rate, and vice versa. However, hardness differences were found between pellets of different diameters in Trial 1, whereas the pellet compression rates were similar. Inversely, for pellets in Trial 2 with the same diameter, different compression rates led to similar hardness. These unexpected results might be explained by the heterogeneity of the internal pellet structure inherently caused by the diverse raw materials that make it up. [START_REF] Thomas | Physical quality of pelleted animal feed 1. Criteria for pellet quality[END_REF] confirmed the large variation of fragmentation-type measurements for a given hardness device. For this reason, hardness measurements should be carefully interpreted. As expected, the pellet processing of this experiment did not alter pellet chemical composition and hydration properties, which suggests that post-ingestive effects were similar between feeds. Thus, dietary preferences were considered to be a good indicator of feed palatability, which is defined as the "physical and chemical properties of the diet that are associated with promoting or suppressing feeding behavior during the pre-absorptive or immediate post-absorptive period" [START_REF] Mcarthur | The Role of Palatability in the Food Intake Response of Rats Fed High-Protein Diets[END_REF]. In the first trial, results suggested a shift of preferences between the nest and the feeder, with a slight preference for the pellet with the greatest diameter over the lowest (6.0 mm over 2.0 mm) in the first days of the experiment, whereas the rabbits rejected 6.0-mmdiameter pellets a few days before weaning. The initial preference expressed for the 6.0-mm pellets may be explained by an overestimation of their intake due to their low durability index. Indeed, pellets of low durability produce a greater amount of fines, which are not consumed by the rabbits [START_REF] Maertens | Feeding systems for intensive production[END_REF] but are counted as consumed with food weighing method. Pellet durability could not be controlled with our experimental design during the pelletizing since die size has an intrinsic effect on durability [START_REF] Oduntan | Effect of speed, die sizes and moisture contents on durability of cassava pellet in pelletizer[END_REF]. In the feeders, we were able to rank the pellets by order of preference: first: 2.0-mm-diameter pellets; second: 3.0-and 4.0-mm-diameter pellets; and third: 6.0-mm-diameter pellets.

Regarding pellet physical properties, pellets with diameters ranging between 2.0 and 4.0 mm had similar hardness, durability and hydration properties, so we could therefore conclude that the higher palatability of 2.0-mm-diameter pellets was due to their small diameter. Pellet presentation can modify feed sensory characteristics such as appearance and feed texture sensation. Ease of prehension of small pellets may partly explain their attractiveness for the litters since rabbits are known to eat pellets one by one [START_REF] Maertens | Influence du diamètre du granulé sur les performances des lapereaux avant sevrage[END_REF]. Regarding the largest pellets, they were found to have a low durability index, which could negatively affect their palatability since rabbits do not like fines as previously discussed. Therefore, it remains unclear to what degree the large diameter of these pellets is responsible for their low palatability. In the second trial, rabbits slightly avoided eating the 4.0-mm-diameter pellet processed with the highest compression rate when facing another kind of 4.0-mm-diameter pellet. However, this avoidance was only visible in one of the two comparison combinations in the nest and in the feeders. Limited statistical power due to the sample size used in the present study (n = 10 litters) may have reduced the chance to detect moderate magnitude preferences for pellets with a lower compression rate both in the nest and the feeders. Physical measurements performed on the pellet with the highest compression rate did not highlight any specific characteristics when compared to other 4.0-mm-diameter pellets. Consequently, the effects of compression rate on pellet palatability still need to be investigated.

Overall, in the present study, no clear preference pattern in the nest was determined.

The small quantity of feed consumed in the nest, which requires high measurement accuracy, may explain why we did not detect preferences. This phenomenon can also be explained by the low activity of the kits during the two first weeks of life: their exploration of the nest is limited at that time and feed consumption probably occurred randomly. In contrast, in the feeders, suckling rabbits were able to differentiate pellets according to their diameter.

Stimulation of the early feed intake and implications

Currently, rabbit pellets are manufactured with a diameter of 3.0 or 4.0 mm (Acedo- et al., 2010). High palatability of 2.0-mm-diameter pellets in the present study suggests that a preferential use of small-diameter pellets could be advocated to stimulate early consumption. However, some studies have pointed out that when domestic caged rabbits expressed a preference for a specific feed rather than another, the daily feed intake could be similar between the two types of feed once they were offered alone [START_REF] Gidenne | Feeding behaviour of rabbits[END_REF].

Rico

Nevertheless, these studies were performed on fattening rabbits, and suckling rabbits may present specific feed intake regulations. In the first double-choice trial, total feed intakes of treatments providing the smallest pellet did not differ from the feed intake of other treatments, except between 15 and 17 days (however those results might be meaningless due to low precision). It could suggest that the higher palatability of the small pellets is not sufficient to increase feed intake. On the contrary, in the second trial in this study, there was a trend towards a higher feed intake in the feeders for the treatments with 2.5-mm pellets when compared to treatments with 4.0-mm pellets. Experiments where one kind of pellet is tested per litter (no-choice tests) are now required to ensure that the feed intake increases with small pellets and to evaluate further consequences on rabbits growth.

In the current study, the onset of solid feed was advanced by eight days compared to conventional rabbitries where the solid feed intake behavior starts at around 16 days of age [START_REF] Gidenne | Feeding strategy for young rabbit around weaning: a review of digestive capacity and nutritional needs[END_REF]. The substrates precociously reaching the gut from the solid feed are likely to stimulate the microbiota implantation process. Indeed, it was shown that intake of fecal pellets left behind by the doe in the first 10 days after delivery plays a role in cecal microbiota colonization [START_REF] Kovács | Effect of nursing method and faeces consumption on the development of bacetroides, lactobacillus and coliform flora in the caecum of the newborn rabbits[END_REF][START_REF] Combes | Coprophagous behavior of rabbit pups affects implantation of cecal microbiota and health status[END_REF], even if the ingestion is small (a mean of ten fecal pellets per litter with free milking conditions). The feed is also a key factor for microbiota activity, as highlighted by [START_REF] Kovács | Effect of nursing method and faeces consumption on the development of bacetroides, lactobacillus and coliform flora in the caecum of the newborn rabbits[END_REF] through an early weaning experiment: when the rabbit solid feed intake was stimulated as of 21 days of age by preventing milk supply, the cecal microbiota fermentation activity significantly increased. Seeding the microbiota of neonatal gut with healthy commensal bacteria is essential to optimize rabbit health since it its known that microbiota strongly interacts with mammal health [START_REF] Mage | B cell and antibody repertoire development in rabbits: The requirement of gut-associated lymphoid tissues[END_REF][START_REF] Combes | Pour des lapereaux plus robustes au sevrage : des bases biologiques aux leviers d'action en élevage[END_REF]). The effect of early feeding on the rabbit health needs to be further investigated since it could not be studied with our experimental design (all the litters had access to pellets as of 3 days of age).

Conclusion

Intake of supplemental solid feed provided in the nest was observed as of 8 days of age. The high variability of feed consumed during the early suckling period was partially explained by the litter weight at the beginning of the experiment: heavier litters were associated with higher solid feed intake at two weeks of age. Outside the nest, suckling rabbits were able to discriminate pellets according to their diameter with the following preferences: 2 mm > (3.2 and 4 mm) > 6 mm diameter pellets. The effect of compression rate on the kits' feeding behavior needs to be further investigated. In terms of implications, this research shows that it is possible to promote post-natal feed intake through a nest feeding system and that pellet palatability can be improved before weaning with an optimal presentation. The acceleration and the increase in the solid intake before weaning could contribute to the acquisition and maturation of the kit microbiota and, hence, contribute to improved health.

Declarations of interest: none

Fig. 1. Housing used to control separately the doe's feed intake and that of its litter, with a limited access to the nest box for the doe. On the top is mapped the view of the cage seen from the top. Below is represented the view of the same cage seen from left side. Fig. 2. View of the nest box when rabbits were 4-days old. The two cups on the right contained two types of feed pellets differing in either diameter (Trial 1) or compression rate (Trial 2). Fig. 3. Experimental design used to characterize suckling rabbit intake and pellet preferences. Fig. 4. Dynamic of feed consumption in 8 to 17-day-old rabbits receiving pellets in the nest as of 3 days of age (Trials 1 and 2). From 15 to 17 days, feed was also available in feeders outside the nest.

The intake is the sum of the consumptions of the two types of pellets tested. Results are presented as the mean and standard deviation. The different letters stand for significant differences (P<0.05). Fig. 5. Relationship between starter feed intake and litter weight (at 3 days of age) in Trial 2. The total feed intake from 12 to 14 days is represented on the left, and the total feed intake from 15 to 17 days is represented on the right. Dot size is proportional to milk consumption. r stands for the Pearson Correlation, P for the significance level of the correlation, and N for the sample size. Fig. 6. Feed and milk consumption of suckling rabbits receiving solid pelleted feed as of 3 days of age (Trials 1 and 2). For each litter, the intake is the sum of the consumptions of the two pellets tested.

Results are presented as the mean and standard deviation.

Fig. 7 .Fig. 8 .

 78 Fig.1. Housing used to control separately the doe's feed intake and that of its litter, with a limited access to the nest box for the doe. On the top is mapped the view of the cage seen from the top. Below is represented the view of the same cage seen from left side. Fig.2. View of the nest box when rabbits were 4-days old. The two cups on the right contained two types of feed pellets differing in either diameter (Trial 1) or compression rate (Trial 2). Fig.3. Experimental design used to characterize suckling rabbit intake and pellet preferences. Fig.4. Dynamic of feed consumption in 8 to 17-day-old rabbits receiving pellets in the nest as of 3 days of age (Trials 1 and 2). From 15 to 17 days, feed was also available in feeders outside the nest. The intake is the sum of the consumptions of the two types of pellets tested. Results are presented as the mean and standard deviation. The different letters stand for significant differences (P<0.05). Fig.5. Relationship between starter feed intake and litter weight (at 3 days of age) in Trial 2. The total feed intake from 12 to 14 days is represented on the left, and the total feed intake from 15 to 17 days is represented on the right. Dot size is proportional to milk consumption. r stands for the Pearson Correlation, P for the significance level of the correlation, and N for the sample size. Fig.6. Feed and milk consumption of suckling rabbits receiving solid pelleted feed as of 3 days of age (Trials 1 and 2). For each litter, the intake is the sum of the consumptions of the two pellets tested. Results are presented as the mean and standard deviation. Fig. 7. Relative pellet consumption in the nest (A) and in the feeders (B) for the first trial. The animals had the choice between two pellets that differed in diameter (A: 2 mm; B: 3.2 mm; C: 4 mm; and D: 6 mm). Results are presented as the mean and standard deviation. The symbols represent significantly different preference values ( †: P < 0.1; *: P < 0.05; **: P < 0.01; ***: P < 0.001). Fig. 8. Relative pellet consumption in the nest (A) and in the feeders (B) for the second trial. The young rabbits had the choice between two pellets of the same diameter (E, F, G: 2.5 mm, and C, H, I: 4 mm) that differed by compression rate. Results are presented as the mean and standard deviation. The symbols represent preference values significantly different by 50% (*: P<0.05).

Table 1

 1 Ingredients and chemical composition of the experimental meals. Premix provided per kg of complete feed: vitamin A, 10,000 IU; vitamin D3, 1000 IU; vitamin E, 50. Neutral detergent fiber expressed exclusive of residual ash. Acid detergent fiber expressed exclusive of residual ash.

	Kits a	S.E. a	Does

b c Containing 0.5% diclazuril. d e f Lignin obtained with sulfuric acid method.

Table 2

 2 Pellet press dies specifications used to process young rabbit feeds.

				Die specification
	Pellet	Trial	Channel diameter (mm)	Channel length (mm)	Length-to-diameter ratio
	A	1	2	10	5
	B	1	3.2	16	5
	C	1 and 2	4	18	4.5
	D	1	6	24	4
	E	2	2.5	10	4
	F	2	2.5	12	4.8
	G	2	2.5	14	5.6
	C	1 and 2	4	18	4.5
	H	2	4	20	5
	I	2	4	24	6

Table 3

 3 Physical characteristics of the pellets processed with nine different dies.

	668
	669
	670
	671
	672
	673
	674
	675
	676
	677
	678
	679
	680

b SC: Swelling capacity.

Table 4

 4 Effects of the physical properties of the pellets on the feed intake in the feeders from 15 to 35 days (weaning). Values are means of the amount of solid feed ingested per rabbit per day. Means denoted by a different letter indicate significant differences between treatments.

	Trial 1.
		Double comparison groups AB AC AD BC BD	CD	SEM P-value
	Feed intake (g of DM/rabbit/day)
	15-17 days 0.3 ab 0.4 ab 0.5 b 0.2 a 0.3 ab 0.2 ab 0.26	0.025
	18-24 days	5	5	6	4	4	5	1.29	0.257
	25-31 days 32	31	23	27	28	27	6.30	0.199
	32-35 days NC NC NC 56	59	57	NC	NC
	Trial 2.
		EF	Double comparison groups EG FG CH CI	HI	SEM P-value
	Feed intake (g of DM/rabbit/day)
	15-17 days 0.9	0.7 0.8 0.9 1.0	0.7 0.30	0.349
	18-24 days	6	6	5	5	4	5	1.63	0.387
	25-31 days 41	39	37	40	36	36	5.32	0.523
	32-35 days 62	59	61	61	63	52	10.7	0.563

NC: not calculable because we ran out of pellet A as of 32 days.

Table 5

 5 Effects of physical properties of the pellets on growth performance before weaning.

	Trial 1.
		AB	Double comparison groups AC AD BC BD	CD	SEM P-value
	Number of litters	10	8	10	10	10	10
	Live weight (g/rabbit)
	3 days	86	86	84	86	85	84	7	0.976
	10 days	171	176	168	173	177	174	16	0.941
	14 days	222	229	216	222	234	224	20	0.689
	21 days	302	317	303	311	326	309	28	0.687
	28 days	532	558	525	509	530	520	42	0.511
	Average daily gain (g/d)
	3 -21 days	12.0 12.8 12.2 12.5 13.4 12.5	1.4	0.627
	21 -35 days	38.6 38.4 33.8 33.6 35.6 35.9	3.3	0.039
	Feed conversion ratio
	21 -28 days	0.46 0.50 0.54 0.47 0.49 0.52 0.05	0.081
	28 -35 days	NC	NC	NC	1.04 1.04 0.97	NC	NC
	Trial 2.
		EF	Double comparison groups EG FG CH CI	HI	SEM P-value
	Number of litters	10	11	11	10	11	10
	Live weight (g/rabbit)
	4 days	110	112	108	109	105	107	7	0.713
	14 days	280	279	269	282	273	272	22	0.880
	21 days	381	385	378	387	378	372	31	0.960
	28 days	646	643	628	639	606	601	44	0.353
	Average daily gain (g/d)
	4 -21 days	15.9 16.1 15.9 16.4 16.0 15.5	1.5	0.967
	21 -35 days	43.2 40.1 40.6 40.1 40.8 37.0	3.5	0.140
	Feed conversion ratio
	21 -28 days	0.46 0.47 0.46 0.45 0.42 0.48 0.06	0.660
	28 -35 days	1.14 1.20 1.13 1.22 1.09 1.15 0.10	0.226

NC: not calculable because we ran out of pellet A as of 32 days.

Table 6

 6 Effects of physical properties of the pellets on rabbit growth performance after weaning.

	Trial 1.
		AB	Double comparison groups AC AD BC BD	CD	SEM P-value
	Number of rabbits at weaning	98	75	92	93	85	89
	Live weight (g/rabbit)
	35 days	842	859	777	782	824	812	58	0.118
	50 days	1225 1259 1195 1222 1227 1196	46	0.487
	64 days	1808 1907 1822 1824 1855 1798	67	0.517
	Average daily gain (g/d)
	35-50 days	25	27	28	29	27	26	3	0.259
	50-64 days	41	46	44	43	45	43	3	0.370
	Trial 2.
		EG	Double comparison groups FG EF CH CI	HI	SEM P-value
	Number of rabbits at weaning 102	102	74	91	89	81
	Live weight (g/rabbit)
	35 days	986	947	934	889	948	959	62	0.228
	50 days	1506 1510 1552 1511 1512 1477	43	0.591
	64 days	2132 2138 2218 2152 2130 2133	61	0.518
	Average daily gain (g/d)
	35-50 days	37	37	38	35	38	39.	2	0.784
	50-64 days	44	45	47	45	44	47	3	0.344

Acknowledgments

This work was supported by the Institut Carnot Santé Animale [eFeedIT-ICSA-2015].

The authors gratefully acknowledge the assistance of M. Moulis and J-M Bonnemere at the rabbit experimental unit, as well as the technical help of M. Segura for starch analysis (INRA, GenPhySE unit). They also would like to thank F. Enjalbert for constructive criticism of the manuscript (ENVT, GenPhySE unit).


MD Laura Olivier-Gougenheim

MD Caroline Freychet

MD, PhD Sophie Collardeau-Frachon

MD Chantal Roure-Sobas

MD, PhD Sylvie Di Filippo

MD Roberto Riva

MD, PhD Jean-Christophe Lega

MD, PhD Alexandre Belot
email: alexandre.belot@chu-lyon.fr

Alexandre Pr

Belot

Queries for progressive cerebral and systemic vasculitis

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.

A 9-year-old girl was admitted for a status epilepticus; neuroimaging revealed an intra-parenchymal hematoma and intraventricular hemorrhage. She was born with a complex congenital cardiopathy requiring ventriculopulmonary tube (RV-PA) within the first week of life. CNS arteriography showed a right sylvian artery aneurysm (Figure 1A). Despite being afebrile, endocarditis with mycotic CNS aneurysm was considered due to the presence of intracardiac prosthetic material and elevated inflammatory markers. There was no phenotypic trait for genetic collagen disorders. Echocardiography, and thoraco-abdomino-pelvic scans were normal. Successive blood cultures, universal PCR as well as Human Herpesvirus 1-5 PCR were all negative. The aneurysm was embolized; The hematoma was drained. Empiric antibiotic therapy was administered for six weeks. She recovered with a mild left residual hemiparesis.

One month later, she presented with diabetes, followed by an acute hepatitis. A systemic inflammatory disease was considered but specific autoantibodies (anti-GAD, anti-IA2, anti-ZnT8, ANA, anti-LKM, ANCA, AMA) were negative. Liver biopsy revealed features of non-specific subacute hepatitis without fibrosis nor granuloma.

Of note, peripheral naïve T cells were low as a possible consequence of early thymectomy and an inflammatory complication of immunodeficiency. 22q11.2 deletion syndrome was ruled out.

On three months follow-up, a new cerebral aneurysm was detected; this time involving the contralateral left sylvian artery (Figure 1B). This aneurysm was again embolized and the RV-PA conduit was changed.

Cultures of the explanted material remained sterile. Infectious evaluation was repeated and this time, included Q fever serology, which was positive as well as the PCR in the liver biopsy. The diagnosis of chronic Q fever was made. A one-year treatment with doxycycline and hydroxychloroquine was started. The girl made a complete recovery.

There are numerous causes of non-traumatic cerebral aneurysms in children comprising Marfan, vascular Ehlers-Danlos syndrome or fibromuscular dysplasia, primary cerebral / systemic vaculitis or infections. Negative blood cultures imply also consideration for fungi, staphyloccus and streptococcus variants, the gram-negative bacilli of the HACEK group, Bartonella species, Tropheryma whipplei and Coxiella burnetti.

Coxiella Burnetii is the intracellular pathogen causing the "query" fever, a zoonotic disease transmitted by dust aerosol of some contaminated animals (goat, sheep and cattle). Q Fever should be considered when querying multisystem disease with cerebral vasculopathy, even in the absence of fever.

Question:

A 9-year-old girl with a past medical history of congenital heart disease and early cardiac surgery presents with intra-parenchymal hemorrhage associated with an isolated cerebral aneurysm. She is afebrile, successive blood cultures and universal PCR remain negative but inflammation markers were increased (CRP=45mg/l and ESR=50mm/h). Three months later, the child represents with acute hepatitis and a contralateral cerebral aneurysm.

Query: What is the diagnosis?

Staphylococcal infection Q Fever Systemic Poly/Panateritis nodosa (PAN)

Kawasaki disease

Contributors

We each substantially contributed to the clinical management of the patient and to the writing up of the manuscript.

Declaration of interests

We declare no competing interests.

Figure 1 :

 1 Figure 1: Cerebral Angiographies