R.-A Becerra-Arciniegas

R Narducci

G Ercolani

E Sgreccia

L Pasquini

M L Di Vona
email: divona@uniroma2.it

P Knauth

Model Long Side-Chain PPO-Based Anion Exchange Ionomers: Properties and Alkaline Stability

The stability of anion exchange membranes is paramount for the use in alkaline fuel cells. Long chain ionomers are supposed to be more alkaline-resistant with respect to short chain isomers. In this paper the synthesis, properties and stability of ionomers with a long side chain are investigated. Poly(2,6-dimethyl-1,4-phenylene)oxide (PPO) is chosen as backbone, due to its reported stability in alkaline conditions. The functional group is pentyl-ammonium with trimethylamine (TMA) or 1,4-diazabicyclo[2.2.2]octane (DABCO) as model amines. The synthesis is carried out via metalation reaction and is optimized as a function of temperature and time. The water uptake is relatively low, in accordance with the large hydrophobicity of the PPO backbone. The through-plane ionic conductivity is consistent with literature data; it amounts to 15.3 mS/cm at 80 °C for the TMA derivative. The mechanical properties are typical of ionomers below the glass transition temperature (for the TMA derivative at ambient humidity: Young Modulus = 1310 ± 30 MPa). The stability in alkaline conditions, studied by thermogravimetric analysis and measurements of ionic conductivity and ion exchange capacity, is higher than that of short-side chain ionomers with the same basic group. The decrease of ionic conductivity (57 vs 22% residual conductivity after 72 h in 2 M NaOH at 80 °C) and IEC is monitored showing that the degradation is fast in the first hours and may by described by second order kinetics. These results help in selecting high performance anion exchange membranes for electrochemical energy technologies.

Introduction

Anion exchange ionomers are used as innovative separators in alkaline membrane fuel cells [START_REF] Merle | Anion exchange membranes for alkaline fuel cells: A review[END_REF][START_REF] Couture | Polymeric materials as anion-exchange membranes for alkaline fuel cells[END_REF][START_REF] Hickner | Anion Exchange Membranes: Current Status and Moving Forward[END_REF] .

The major advantage of these devices is that the oxygen reduction reaction (ORR) can be catalysed in alkaline conditions by non-noble metals, such as Ag or Ni [START_REF] Hickner | Anion Exchange Membranes: Current Status and Moving Forward[END_REF][START_REF] Pan | Advances and challenges in alkaline anion exchange membrane fuel cells[END_REF][START_REF] Arges | Anion Exchange Membranes' Evolution toward High Hydroxide Ion Conductivity and Alkaline Resiliency[END_REF] . The use of anion exchange membranes (AEM) avoids the severe carbonation issues observed in alkaline fuel cells with liquid electrolytes. However, the practical application of AEM in fuel cells is hampered by their instability in alkaline environment due to the attack of the ammonium groups by the hydroxide ions [START_REF] Arges | Best Practices for Investigating Anion Exchange Membrane Suitability for Alkaline Electrochemical Devices: Case Study Using Quaternary Ammonium Poly(2,6-dimethyl 1,4-phenylene)oxide Anion Exchange Membranes[END_REF][START_REF] Vona | Anion-conducting ionomers: Study of type of functionalizing amine and macromolecular cross-linking[END_REF][START_REF] Sun | The Alkaline Stability of Anion Exchange Membrane for Fuel Cell Applications: The Effects of Alkaline Media[END_REF][START_REF] Nunez | Quantitative H-1 NMR Analysis of Chemical Stabilities in Anion-Exchange Membranes[END_REF] . Furthermore, the polymer backbone can also be attacked and chain scission processes occur [START_REF] Miyanishi | Ether cleavage-triggered degradation of benzyl alkylammonium cations for polyethersulfone anion exchange membranes[END_REF][START_REF] Pandey | A Highly Hydroxide Conductive, Chemically Stable Anion Exchange Membrane, Poly(2,6 dimethyl 1,4 phenylene oxide)-b-Poly(vinyl benzyl trimethyl ammonium), for Electrochemical Applications[END_REF] . The instability of the widely studied poly(arylene ether sulfones) (PSU) is promoted by the sulfone groups present in the backbone through their inductive effect increasing the reactivity in specific positions [START_REF] Becerra-Arciniegas | Alkaline stability of model anion exchange membranes based on poly(phenylene oxide) (PPO) with grafted quaternary ammonium groups: Influence of the functionalization route[END_REF] . AEM based on poly(2,6-dimethyl-1,4-phenylene oxide) (PPO) [START_REF] Pandey | A Highly Hydroxide Conductive, Chemically Stable Anion Exchange Membrane, Poly(2,6 dimethyl 1,4 phenylene oxide)-b-Poly(vinyl benzyl trimethyl ammonium), for Electrochemical Applications[END_REF][START_REF] Cheng | A mini-review on anion exchange membranes for fuel cell applications: Stability issue and addressing strategies[END_REF] are expected to have a higher alkaline stability in comparison with PSU, due to the less activated backbone for a nucleophilic attack [START_REF] Cheng | A mini-review on anion exchange membranes for fuel cell applications: Stability issue and addressing strategies[END_REF] .

The alkaline attack may occur by several mechanisms [START_REF] Sun | The Alkaline Stability of Anion Exchange Membrane for Fuel Cell Applications: The Effects of Alkaline Media[END_REF][START_REF] Nunez | Quantitative H-1 NMR Analysis of Chemical Stabilities in Anion-Exchange Membranes[END_REF][START_REF] Cheng | A mini-review on anion exchange membranes for fuel cell applications: Stability issue and addressing strategies[END_REF] . The most difficult to avoid is the nucleophilic substitution reaction S N 2: the rate of this bimolecular reaction is proportional to the IEC of the ionomer and the OH -concentration in the medium [START_REF] Becerra-Arciniegas | Alkaline stability of model anion exchange membranes based on poly(phenylene oxide) (PPO) with grafted quaternary ammonium groups: Influence of the functionalization route[END_REF][START_REF] Kreuer | A practical method for measuring the ion exchange capacity decrease of hydroxide exchange membranes during intrinsic degradation[END_REF] . The bimolecular Hofmann elimination E2 leads to the formation of an alkene. In sterically very unfavourable conditions, the monomolecular elimination reaction E1 can also occur. Various approaches are discussed in the literature to mitigate the alkaline degradation [START_REF] Merle | Anion exchange membranes for alkaline fuel cells: A review[END_REF][START_REF] Couture | Polymeric materials as anion-exchange membranes for alkaline fuel cells[END_REF][START_REF] Jasti | Stable and hydroxide ion conductive membranes for fuel cell applications: Chloromethyaltion and amination of poly(ether ether ketone)[END_REF][START_REF] Li | Highly Stable, Anion Conductive, Comb-Shaped Copolymers for Alkaline Fuel Cells[END_REF][START_REF] Li | Cross-linked comb-shaped anion exchange membranes with high base stability[END_REF][START_REF] Dang | Exploring Different Cationic Alkyl Side Chain Designs for Enhanced Alkaline Stability and Hydroxide Ion Conductivity of Anion-Exchange Membranes[END_REF][START_REF] Varcoe | Prospects for alkaline anion-exchange membranes in low temperature fuel cells[END_REF][START_REF] Jagur-Grodzinski | Polymeric materials for fuel cells: concise review of recent studies[END_REF][START_REF] Marino | Alkaline Stability of Quaternary Ammonium Cations for Alkaline Fuel Cell Membranes and Ionic Liquids[END_REF][START_REF] Pan | Hydrophilic side chain assisting continuous ion-conducting channels for anion exchange membranes[END_REF][START_REF] Narducci | Intrinsic" Anion Exchange Polymers through the Dissociation of Strong Basic Groups: PPO with Grafted Bicyclic Guanidines[END_REF][START_REF] Bauer | Anion-exchange membranes with improved alkaline stability[END_REF][START_REF] Zadok | Effect of Ammonium Cations on the Diffusivity and Structure of Hydroxide Ions in Low Hydration Media[END_REF] , including a change of the polymer backbone [START_REF] Bauer | Anion-exchange membranes with improved alkaline stability[END_REF][START_REF] Zhang | Highly stable anion exchange membranes based on quaternized polypropylene[END_REF][START_REF] Mohanty | Systematic Alkaline Stability Study of Polymer Backbones for Anion Exchange Membrane Applications[END_REF][START_REF] Lin | Quaternized triblock polymer anion exchange membranes with enhanced alkaline stability[END_REF] , the introduction of side chains [START_REF] Dang | Exploring Different Cationic Alkyl Side Chain Designs for Enhanced Alkaline Stability and Hydroxide Ion Conductivity of Anion-Exchange Membranes[END_REF][START_REF] Pan | Hydrophilic side chain assisting continuous ion-conducting channels for anion exchange membranes[END_REF][START_REF] Pan | Cationic Side-Chain Attachment to Poly(Phenylene Oxide) Backbones for Chemically Stable and Conductive Anion Exchange Membranes[END_REF][START_REF] Long | Hydroxide Degradation Pathways for Substituted Trimethylammonium Cations: A DFT Study[END_REF][START_REF] Liu | Tuning the properties of poly(2,6-dimethyl-1,4-phenylene oxide) anion exchange membranes and their performance in H-2/O-2 fuel cells[END_REF][START_REF] Zhu | Exploring backbone-cation alkyl spacers for multi-cation side chain anion exchange membranes[END_REF] , the presence of a secondary phase [START_REF] Vona | Anionic conducting composite membranes based on aromatic polymer and layered double hydroxides[END_REF][START_REF] Pizzoferrato | Layered Double Hydroxides Containing an Ionic Liquid: Ionic Conductivity and Use in Composite Anion Exchange Membranes[END_REF][START_REF] Derbali | Composite anion exchange membranes with functionalized hydrophilic or hydrophobic titanium dioxide[END_REF] , a delocalization of the positive charge [START_REF] Vona | Anion-conducting ionomers: Study of type of functionalizing amine and macromolecular cross-linking[END_REF][START_REF] Marino | Alkaline Stability of Quaternary Ammonium Cations for Alkaline Fuel Cell Membranes and Ionic Liquids[END_REF] , or a protection by steric hindrance [START_REF] Vona | Anion-conducting ionomers: Study of type of functionalizing amine and macromolecular cross-linking[END_REF][START_REF] Sun | Anion-Exchange Membranes for Alkaline Fuel-Cell Applications: The Effects of Cations[END_REF][START_REF] Liu | A facile strategy for the synthesis of guanidinium-functionalized polymer as alkaline anion exchange membrane with improved alkaline stability[END_REF] .

In this study, we investigate the alkaline stability of model long side-chain anion-conducting ionomers based on PPO with N-pentyl-N,N,N-trimethylammonium side groups, given that the theoretical work of Pivovar and coworkers shows that the most stable side-chain length corresponds to 4-5 CH 2 groups [START_REF] Long | Hydroxide Degradation Pathways for Substituted Trimethylammonium Cations: A DFT Study[END_REF] . Two model amines, small and flexible trimethylamine (TMA) and bulky and rigid 1,4-diaza-bicyclo[2.2.2]octane (DABCO), are used for functionalization. The water uptake, thermal, mechanical and electrical properties are reported before and after a harsh alkaline treatment in 2 M NaOH at 80 °C during various times.

Experimental section

Materials

Poly(2,6-dimethyl-1,4-phenylene)oxide (PPO, MW = 50000 g/mol) trimethylamine (TMA, 4.2 M in ethanol), 1,4-diazabicyclo[2.2.2] octane (DABCO), n-butyllithium (BuLi, 2,5 M in hexane), tetrahydrofuran (THF, anhydrous), 1,4-dibromobutane, and N-methyl-2-pyrrolidone (NMP) were reagent-grade, purchased from Sigma-Aldrich.

Ionomer synthesis

Synthesis of PPO-(CH 2 ) 5 Br. PPO (5 g, 41.7 meq) was dissolved in dry THF (500 mL) under nitrogen at 55 °C. The solution was cooled to RT and 25 mL of n-BuLi (2.5 M, 62.6 meq) was added dropwise until the appearance of a persistent light yellow coloration. The reaction was carried out during 3 h at two temperatures: 25 or 55 °C in order to optimize the final products (see Results and Discussion).

The solution was then cooled to -70 °C and 1,4-dibromobutane (15 mL, 125 meq) was added. A light yellow solution was immediately formed and the reaction was kept under stirring overnight at RT. The product was precipitated in ethanol and the yellow polymer was filtered, washed repeatedly with fresh ethanol and dried at 55 °C during 2 days.

The products were characterized by 1 H NMR. The degree of benzyl bromination was 0.24 at 25 °C and 0.35 at 55 °C.

Synthesis of PPO-(CH 2 ) 5 TMA and PPO-(CH 2 ) 5 DABCO.

The brominated polymer obtained at 55 °C (degree of benzyl bromination 0.35) was dissolved in NMP and the amine was added in excess of 5.5 (TMA) or 1.5 (DABCO) with respect to the degree of functionalization, due to the well-known volatility of TMA. The solution was kept at 80 °C for 72 h and then the polymer was precipitated in deionized water, filtered, washed with water and dried under vacuum at 60 °C.

Membranes were obtained by casting at 95 °C from NMP solution (0.3 g polymer in 25 mL solvent).

NMR spectroscopy 1 H NMR spectroscopy was performed using a Bruker Avance 400 apparatus (400.13 MHz) using deuterated solvents (CDCl 3 , DMSO-d 6 ). Chemical shifts were referenced to tetramethylsilane.

Ion exchange capacity

The IEC was determined by NMR spectroscopy (see Results) and by Mohr titration. For the Mohr titration, the chloride form was obtained by treating the AEM in 1 M NaCl solution during 24 h. The membranes were then washed carefully with deionized water to remove any excess NaCl. The Cl -anions contained in the membranes were exchanged with SO 4 2-by immersion in 1 M Na 2 SO 4 solution for 24 h. This solution was titrated using 0.02 M AgNO 3 . The end-point indicator was prepared dissolving 1 g of K 2 CrO 4 in 20 mL of water.

By Mohr titration, PPO-(CH 2 ) 5 -TMA had an IEC = 1.8 meq/g and PPO-(CH 2 ) 5 -DABCO an IEC = 1.3 meq/g.

Water uptake

The membrane mass was measured after 24 h immersion in deionized water at 25 °C, removing excess surface water by rapid blotting with absorbent paper (m wet ). The dry mass was obtained after drying in a desiccator over P 2 O 5 during 72 h (m dry ). The water uptake WU was calculated according to the equation:

The hydration numbers λ, which are the number of water molecules per ammonium group, are calculated using the IEC of the ionomer according to the equation:

M(H 2 O
) is the molar mass of water.

Ionic conductivity

Prior to the measurements, the membrane samples were immersed during 24 h in 2 M NaCl (Cl form) or 2 M NaOH (OH form) solution and then thoroughly washed in deionized water. In the latter case, the solution was kept under nitrogen to avoid contamination by CO 2 . The ionic conductivity was measured between 25 and 80 °C by impedance spectroscopy using a Biologic VSP300 equipment. The frequency range was between 1 Hz and 6 MHz and the a.c. voltage amplitude was 20 mV. The measurements were made on fully humidified samples in two-point through-plane configuration using a swagelok cell with electrode area A = 0.264 cm 2 . The membrane resistance R was determined at the intersection of the electrode arc with the real axis.

The membrane thickness d was determined before and after the measurements using a Mitutuyo micrometer.

The ionic conductivity was calculated according to the equation:

Mechanical measurements

The tensile stress-strain measurements were performed on membranes in Cl form using an ADAMEL Lhomargy DY30 apparatus at room temperature and ambient humidity ((50 +/-10) % RH) or at full humidification.

Thermogravimetry

High-resolution thermogravimetric analysis of membranes (Cl form) was made using a TA Q500 apparatus. The samples were placed in Pt sample holders. The experiments were perfomed in air flux between 50 and 600 °C. The maximum heating rate was 3 K/min.

Results and Discussion

Synthesis

Figure 1a shows the molecular formulas of long side-chain ionomers functionalized by TMA and DABCO. Butyllithium may attack PPO protons on two different positions in the side chain or in the main chain leading to the formation of a benzyl (A) or an aryl (B) carbanion (Figure 1b). The following reaction with 1,4-dibromobutane gives the bromine derivatives in benzylic or arylic position. The distribution of the products depends on the temperature and time. In our case, the time was fixed at 3 h while the temperature was varied (25 or 55 °C). At low temperature the ratio A:B (Figure 1b) was 2.4:1, while at higher temperature the ratio was 8.8:1. These data correspond to a competitive reaction under thermodynamic or kinetic control [START_REF] Chalk | Direct metalation of poly(2,6-dimethyl-1,4-phenylene ether)[END_REF] . The substitution at benzylic position appears to be more favored than that at the aromatic ring position under both kinetic and thermodynamic control, although a larger selectivity is observed under thermodynamic control. The larger stability of the benzyl anion can be ascribed to resonance delocalization of the negative charge that is not allowed for the sp2 centered aryl anion.

. °C (Figure 2a). The signals of the methylene group in  to bromine (2H, h and h') appear at 3.34 ppm for the benzyl derivative and at 3.48 ppm for the aryl one [START_REF] Wang | Alkaline Stability of Poly(Phenylene Oxide) Based Anion Exchange Membranes Containing Imidazolium Cations[END_REF] . The latter signal is not observable in the spectrum of the compound obtained at higher temperature (Figure 2b) due to the lower quantity of the aryl substitution. The integration of the signal f (2H, 1.40 ppm), present only in the derivative obtained from the attack in benzylic position (about 90% of the peaks h and h', Figure 2b), confirms that a part of the product is due to the formation of the aryl (B) carbanion (Figure 1b). The total degree of bromination DBr is calculated from the integration of the protons h and h' with respect to the protons a of PPO; DBr amounts to 0.34 and 0.39 at 25 and 55 °C respectively.

The reaction with tertiary amines was conducted only with the derivative obtained at higher temperature.

Figure 3a shows the 1 H NMR spectrum of PPO-(CH 2 ) 5 -TMA. The peak a is assigned to the hydrogens (2H) of the aromatic ring containing the pentyl chain originating from the attack of BuLi in benzylic position. The attack on the aryl moiety generates the signal b (approximatively 4%). The peak b is not integrable in the case of PPO-(CH 2 ) 5 -DABCO (Figure 3b). The degree of amination (DAM) of PPO-(CH 2 ) 5-TMA is obtained from the integration of the aromatic hydrogens of PPO (a, 2H), the hydrogens of methyl groups on the ammonium ions (i, 9H), and the internal hydrogens of the pentyl chain (e, f, g 6H) resulting in 0. The DAM is lower for DABCO showing a lower reaction yield probably due to the more bulky molecule that reduces the S N 2 reaction rate by steric hindrance. The IEC obtained from NMR using eq. 4 is 1.8 meq/g for TMA and 1.3 meq/g for DABCO in good agreement with the values obtained by Mohr titration (see experimental).

Properties

The water uptake and ion conductivity values of PPO-(CH 2 ) 5 -TMA and PPO-(CH 2 ) 5 -DABCO are reported in Table 1. The water uptake values are relatively low in comparison with PSU-based ionomers [START_REF] Kreuer | A practical method for measuring the ion exchange capacity decrease of hydroxide exchange membranes during intrinsic degradation[END_REF][START_REF] Pasquini | Effects of anion substitution on hydration, ionic conductivity and mechanical properties of anion-exchange membranes[END_REF] , certainly due to the very hydrophobic PPO backbone, known to exhibit the lowest water sorption among engineering plastics 41 , and increase relatively little with temperature. The room temperature data are consistent with similar PPO-based ionomers [START_REF] Arges | Mechanically Stable Poly(arylene ether) Anion Exchange Membranes Prepared from Commercially Available Polymers for Alkaline Electrochemical Devices[END_REF][START_REF] Parrondo | Synthesis and Alkaline Stability of Solubilized Anion Exchange Membrane Binders Based on Poly(phenylene oxide) Functionalized with Quaternary Ammonium Groups via a Hexyl Spacer[END_REF] . The hydration numbers are comparable with literature data for cross-linked copolymers [START_REF] Pandey | A Highly Hydroxide Conductive, Chemically Stable Anion Exchange Membrane, Poly(2,6 dimethyl 1,4 phenylene oxide)-b-Poly(vinyl benzyl trimethyl ammonium), for Electrochemical Applications[END_REF] , but clearly above ionomers made by a bromination route and with very long side chains [START_REF] Li | Highly Stable, Anion Conductive, Comb-Shaped Copolymers for Alkaline Fuel Cells[END_REF] . The higher hydration numbers for the DABCO derivative might be attributed to the larger volume of the DABCO molecule increasing the inter-chain space.

The conductivity data are comparable with recent literature [START_REF] Parrondo | Synthesis and Alkaline Stability of Solubilized Anion Exchange Membrane Binders Based on Poly(phenylene oxide) Functionalized with Quaternary Ammonium Groups via a Hexyl Spacer[END_REF][START_REF] Perez-Prior | DABCOfunctionalized polysulfones as anion-exchange membranes for fuel cell applications: Effect of crosslinking[END_REF] . They are also consistent with the conductivity reported at room temperature sulfonated PPO with an IEC = 2.63 meq/g (12 mS/cm 41 ), considering the lower mobility of hydroxide (at infinite dilution in water: 20.5 10 -5 cm 2 s -1 V -1 ) [START_REF] Kortüm | Treatise on Electrochemistry[END_REF][START_REF] Hamann | Electrochemistry[END_REF] vs. hydronium ions (36.3 10 -5 cm 2 s -1 V -1 ) [START_REF] Kortüm | Treatise on Electrochemistry[END_REF][START_REF] Hamann | Electrochemistry[END_REF] . The ion conductivity of the Cl form is below the OH form due to the lower mobility of the chloride ion (at infinite dilution in water: 7.9 10 -5 cm 2 s -1 V -1 ) [START_REF] Kortüm | Treatise on Electrochemistry[END_REF][START_REF] Hamann | Electrochemistry[END_REF] . The higher values for the TMA derivative are consistent with its higher IEC.

The activation energy of conductivity calculated for the OH form of PPO-(CH 2 ) 5 -TMA and PPO-(CH 2 ) 5 -DABCO are 0.11 and 0.13 eV, respectively, consistent with a conduction mechanism through hydrated channels [START_REF] Pandey | A Highly Hydroxide Conductive, Chemically Stable Anion Exchange Membrane, Poly(2,6 dimethyl 1,4 phenylene oxide)-b-Poly(vinyl benzyl trimethyl ammonium), for Electrochemical Applications[END_REF] . The activation energy for the Cl forms is higher (0.2 eV) in agreement with the expectation and the literature for halide ion conductors [START_REF] Pandey | A Highly Hydroxide Conductive, Chemically Stable Anion Exchange Membrane, Poly(2,6 dimethyl 1,4 phenylene oxide)-b-Poly(vinyl benzyl trimethyl ammonium), for Electrochemical Applications[END_REF][START_REF] Braglia | Electrochemical synthesis of thin, dense, and conformal anion exchange membranes with quaternary ammonium groups[END_REF] . 2. They are comparable to the properties of other aromatic polymer electrolytes [START_REF] Vona | Anion-conducting ionomers: Study of type of functionalizing amine and macromolecular cross-linking[END_REF][START_REF] Derbali | Composite anion exchange membranes with functionalized hydrophilic or hydrophobic titanium dioxide[END_REF][START_REF] Pasquini | Anion-conducting sulfaminated aromatic polymers by acid functionalization[END_REF][START_REF] Narducci | Low-Permeability Poly(ether Ether Ketone)-Based Ampholytic Membranes[END_REF][START_REF] Narducci | Mechanical Properties of Anion Exchange Membranes by Combination of Tensile Stress-Strain Tests and Dynamic Mechanical Analysis[END_REF] and are typical of stiff ionomers below their glass transition temperature. The glass transition temperature of pristine PPO is known to be very high (212 °C) 41 . The Young modulus is above 1 GPa at ambient humidity, related to the high stiffness and strength of pristine PPO (2.55 GPa and 63.4 MPa, respectively 41 ) weakened by the introduction of the ionic groups that attract water molecules, as observed in all ionomers [START_REF] Sgreccia | Self-assembled nanocomposite organic-inorganic proton conducting sulfonated poly-ether-ether-ketone (SPEEK)-based membranes: Optimized mechanical, thermal and electrical properties[END_REF] .

The high dielectric constant of water reduces the Van der Waals forces between the macromolecular chains, mainly attributed to dipole-dipole interactions of the benzene rings 41 , which are probed in the elastic domain. The lower Young modulus and yield stress at 100% RH is consistent with the plasticizing effect of water. The low elongation at break of all samples, irrespective of the water content, is in the order of magnitude of similar ionomers [START_REF] Pasquini | Anion-conducting sulfaminated aromatic polymers by acid functionalization[END_REF] . The yield stress and tensile strength are much lower for the DABCO derivative, which is attributable to the large and rigid DABCO molecule, which increases the distance between macromolecular chains, enabling larger hydration numbers and weakening the Van der Waals forces between chains, as discussed above.

Alkaline stability

The thermal behavior of AEM is investigated by high-resolution thermogravimetric analysis before and after a degradation treatment during 168 h in 2 M NaOH at 80 °C (Figure 5a andb).

Figure 5a shows the mass loss and Figure 5b the derivative thermograms. The peaks correspond to the principal mass loss phenomena. In pristine PPO-(CH 2 ) 5 -TMA, the large peak around 200 °C can be attributed to the loss of TMA; the mass loss of about 12%, integrated between 160 and 230 °C, is in good agreement with the degree of functionalization of 0.28 (corresponding to 11%). This asymmetric peak is significantly reduced after the alkaline treatment. The mass loss, integrated over the same temperature range, is 8%, corresponding to a decrease of the ammonium groups by about 40 %, In PPO-(CH 2 ) 5 -DABCO, the loss of DABCO (red curves) is observed at a slightly higher temperature around 230 °C.

The second peak at 365 °C (starting around 300 °C) in the pristine ionomers is the trace of the decomposition of methyl and pentyl groups. Considering the degree of functionalization of the ionomers, the mass loss should be 27% for TMA and 25% for DABCO in good agreement with the experiment. The very similar broad mass loss centered around 415 °C for PPO-(CH 2 ) 5 -TMA and PPO-(CH 2 ) 5 -DABCO can be attributed to the decomposition of partially oxidized and crosslinked PPO, as shown previously [START_REF] Li | High-resolution thermogravimetry of poly(2,6-dimethyl-1,4-phenylene oxide)[END_REF][START_REF] Ehlers | Thermal degradation of polymers with phenylene units in the chain. I. Polyphenylenes and poly(phenylene oxides)[END_REF] . The main-chain of PPO rearranges on heating in the degradation step, accompanied by the formation of C-C bonds, followed by the thermal degradation of the methylene bridges 41 . After the alkaline treatment, the PPO degradation shows instead a sharp double peak around 410 °C. Similar observations were reported previously [START_REF] Becerra-Arciniegas | Alkaline stability of model anion exchange membranes based on poly(phenylene oxide) (PPO) with grafted quaternary ammonium groups: Influence of the functionalization route[END_REF] . For PPO-(CH 2 ) 5 -DABCO, the alkaline degradation is continuous, showing that the bulky and rigid side group does not improve the alkaline stability, different from the results in short side chain ionomers [START_REF] Becerra-Arciniegas | Alkaline stability of model anion exchange membranes based on poly(phenylene oxide) (PPO) with grafted quaternary ammonium groups: Influence of the functionalization route[END_REF] .

In the literature [START_REF] Dang | Exploring Different Cationic Alkyl Side Chain Designs for Enhanced Alkaline Stability and Hydroxide Ion Conductivity of Anion-Exchange Membranes[END_REF] , a low degradation was reported for a similar long side chain PPO-(CH 2 ) 5 -TMA in 1 M NaOH; although we do not confirm this result completely, the degradation is anyway much lower than for the corresponding short side chain PPO-TMA ionomer [START_REF] Becerra-Arciniegas | Alkaline stability of model anion exchange membranes based on poly(phenylene oxide) (PPO) with grafted quaternary ammonium groups: Influence of the functionalization route[END_REF] , as shown in the normalized conductivity plot represented in Figure 7. The higher degradation rate observed in this work is certainly also related to the higher NaOH concentration, which is a parameter of the rate of second order reactions. The improvement of stability for the long-chain sample is evident in Figure 7; the spacer concept [START_REF] Tomoi | Synthesis and thermal stability of novel anion exchange resins with spacer chains[END_REF] is verified. Several hypothesis explaining the better stability can be formulated: i) the S N 2 reaction is less effective, because the primary alkyl halide presents a more than two order of magnitude lower reactivity than the benzyl halide [START_REF] Streitwieser | Solvolytic Displacement Reactions[END_REF] ; ii) the ether linkage in the backbone is less destabilized due to the long alkyl chain that screens the positive charge of the ammonium group; iii) the antiperiplanar conformation needed for the E2 reaction is less stable when a long chain is attached to ammonium [START_REF] Long | Hydroxide Degradation Pathways for Substituted Trimethylammonium Cations: A DFT Study[END_REF] . A simple conformational analysis by Newman projections confirms this result.

Conclusions

Anion exchange membranes based on PPO with a pendant side chain functionalized with TMA or DABCO are synthetized using a metalation route. The optimization of the reaction conditions allows obtaining ionomers with the pendant groups in benzylic position, which is the thermodynamically favoured product.

The properties and alkaline stability are investigated by several techniques. Stress-strain tests are typical of stiff polymers below their glass transition temperature. The water uptake is lower than that of PSU-based ionomers, but comparable with short side chain PPO. The DABCO derivative presents a higher water uptake due to the bulky molecule. The ionic conductivity and its activation energy are consistent with the mobility of the conducting ions. The stability in alkaline condition, studied by thermogravimetry, ion conductivity and IEC measurements, is clearly improved vs. short side chain ionomers. The alkaline degradation is rapid during the first hours of the reaction and shows a slower kinetics at longer times. Both observations can be reconciled with the kinetics of second order reactions, such as S N 2 or E2.

Figure 1 .

 1 Figure 1. a) Molecular formulas of long side-chain anion exchange polymers (substitution in benzylic position); b) reaction pathways for the synthesis of brominated PPO.

Figure 2 . 1 H

 21 Figure 2. 1 H NMR spectra of PPO-(CH 2 ) 5 Br in CDCl 3 obtained at a) 25 °C and b) 55 °C.

  28. The other signals overlap with the solvents (DMSO, H 2 O). The spectrum of PPO-(CH 2 ) 5 -DABCO is more complex. The peak l due to hydrogens in  to the nitrogen are shifted to highfield with respect to hydrogens near to the quaternary nitrogen (i) and overlap with the signals h of the CH 2 belonging to the pentyl chain. The integration of NMR peaks e, f, g of the DABCO derivative shows the absence of crosslinks in spite of the two amine groups in the molecule[START_REF] Bauer | Anion-exchange membranes with improved alkaline stability[END_REF] .

Figure 3 . 1 H

 31 Figure 3. 1 H NMR spectra of (a) PPO-(CH 2 ) 5 -TMA with DAM = 0.28 and (b) PPO-(CH 2 ) 5 -DABCO with DAM = 0.20 in DMSO d 6 .

  168 g/mol are respectively the degree of amination, the molar mass of the polymer backbone and the molar mass of the functional unit, which contains 4 CH 2 groups given that one CH 2 belongs to the PPO structure.

Figure 4 .

 4 Figure 4. Tensile stress-strain curves of membranes (Cl form) at 25 °C at RH = (50 ± 10) % and fully humidified (RH = 100 %).

Figure 5 .

 5 Figure 5. High-resolution thermogravimetric curves of PPO-(CH 2 ) 5 -TMA (Cl form) before and after alkaline degradation in 2 M NaOH at 80°C during 168 h and PPO-(CH 2 ) 5 -DABCO (Cl form). a) Mass loss curves, b) derivative curves.

Figure 6 Figure 6 .

 66 Figure 6 presents the decrease of the ionic conductivity of PPO-(CH 2 ) 5 -TMA and PPO-(CH 2 ) 5 -DABCO with the time of the alkaline treatment. Furthermore, the decrease of the IEC of PPO-(CH 2 ) 5 -TMA is represented. Inside the experimental uncertainty, the IEC and conductivity data are in qualitative agreement, showing a rapid degradation during the first hours and a slower kinetics at longer times. Both observations can be reconciled with the kinetics of second order reactions, such as S N 2 or E2, which are both possible given the presence of a β-hydrogen in the long side-chain AEM.

Figure 7 .

 7 Figure 7. Normalized conductivity dependence on the time of alkaline treatment in 2 M NaOH at 80 °C for long and short side-chain AEM 13 .

Table 1 .

 1 Ion conductivity σ, water uptake WU and hydration number λ of PPO-(CH 2 ) 5 -TMA (IEC = 1.8 meq/g) and PPO-(CH 2 ) 5 -DABCO (IEC = 1.3 meq/g). PPO-(CH 2 ) 5 -TMA PPO-(CH 2 ) 5 -DABCO Typical mechanical tensile stress-strain test curves for membranes in Cl form are shown in Figure 4. One notices a very good reproducibility, especially for PPO-(CH 2 ) 5 -TMA. The average mechanical properties are reported for the two polymers in Table

Table 2 .

 2 Average mechanical properties of PPO-(CH 2 ) 5 -TMA and PPO-(CH 2 ) 5 -DABCO: Young modulus E, yield stress YS and elongation at break ε.

	Sample	RH / %	E / MPa	YS / MPa	ε / %
	PPO-(CH 2 ) 5 -TMA	50	1310 ± 30	28.4 ± 0.3	3.3 ± 0.1
		100	820 ± 60	16.2 ± 3.4	3.5 ± 0.4
	PPO-(CH 2 ) 5 -DABCO	100	730 ± 140	4.2 ± 0.8	2.5 ± 0.5

Acknowledgment

There is no financial support from funding institutions to be acknowledged.


H Benhajali

† X Boivin

J Sapa

P Pellegrini

P Boulesteix

P Lajudie

F Phocas

Assessment of different on-farm measures of beef cattle temperament for use in genetic evaluation 1

Keywords: beef cattle, behavior, docility, genetic evaluation, genetic parameter, on-farm temperament

ABSTRACT:

The aim of this study was to find a simple measure for calf temperament discrimination, which can be useful as a selection criterion for on-farm French beef cattle breeding schemes. Behavioral records were registered at an average age of 5 and 7 mo, respectively, for 1,282 and 1,440 Limousin calves born in 24 French farms between August 2007 and April 2008. Measures were repeated for 810 calves at the 2 ages. The test procedure consisted of individually restraining the calves in a chute, then exposing them to a stationary human situated in front of the chute for 10 s. For every calf and each period of the test, the number of rush movements and the total number of movements were scored by visual appraisal using a continuous scale ranging from 0 (no movements) to 60 (continuous movements). Initial scores were also transformed to categorical scores and analyzed. Genetic correlation across ages were very high for all the traits (above 0.84 ± 0.20) suggesting that these traits are governed by the same pool of genes at the 2 ages. The corresponding phenotypic correlations were about 0.3 for all the measures. Heritabilities were moderate for all measures (from 0.11 to 0.31) with the total number of movements during weighing measured at 7 mo being the greatest. All the measures were highly correlated (from 0.73 ± 0.26 to 0.99 ± 0.02). Genetic correlation across sexes was not statistically different from 1. However, traits measured during weighing showed different genetic variance estimates for females and males. Similar results were obtained for the transformed categorical scores. According to these results, the total number of movements during weighing seems to be the most promising trait for on-farm genetic evaluation of French beef cattle temperament.

INTRODUCTION

There is currently a growing interest in European beef cattle for genetic improvement on temperament, defined as "the animal's behavioral response to handling by humans" [START_REF] Burrow | Measurements of temperament and their relationships with performance traits of beef cattle[END_REF]. This is in part reflective of the fact that less labor on farms has been an industry trend over last several decades, but also the need to fit the international request to improve animal temperament. Animal temperament has been shown to be moderately heritable in beef cattle and therefore could respond to selection [START_REF] Burrow | Measurements of temperament and their relationships with performance traits of beef cattle[END_REF]. In some countries, such as Australia or the United States, animals are already selected on their temperament using a chute test [START_REF] Tier | Potential for improving the docility of Limousin cattle in Australia[END_REF]. In these countries, animals receive little human contact and may be much more reactive to handling than European animals. In European husbandry conditions, this chute test may not reveal enough genetic variability to discriminate animals based on their temperament. In France, only Limousin bulls in performance and progeny test stations have been evaluated on a docility test in an arena involving direct human exposure [START_REF] Le Neindre | Individual differences in docility in Limousin cattle[END_REF]. However, this test is not suitable for use in standard farm conditions. Therefore, there is actually a need to establish an on-farm test which will allow for early selection of all the breeding stock in French beef cattle. In French progeny test stations, [START_REF] Grignard | Do beef cattle react consistently to different handling situations?[END_REF] found significant phenotypic correlations between animal responses in the chute test and in the docility test, confirming the chute test as a potential on-farm selection criterion for temperament. However, they suggested that the human presence in the chute test could need to be more clearly defined (e.g., in front of the chute). This study aimed at finding a practical, heritable, and repeatable measure of temperament that can be carried out during existing situations of on-farm performance recording.

MATERIALS AND METHODS

Animal Care and Use Committee approval was not necessary because the data were obtained from routine practices in nucleus herds.

Animals

This study was conducted between October 2006 and September 2008 in 24 French pedigree farms recruited by the French Limousin Breed Association to contribute to this project.

To ensure good family structure and connection across farms, 12 experimental bulls were chosen among the French Limousin bulls selected for AI use. Their semen was disseminated over the 24 cow herds to produce at least 40 calves per bull. The bulls were chosen on the basis of EBV calculated from behavioral criteria collected in progeny test station during the docility test [START_REF] Le Neindre | Individual differences in docility in Limousin cattle[END_REF] using daughters of AI bulls. These criteria are related to the general activity of the heifers during the different stages of the test. The criteria are described in detail in [START_REF] Phocas | Genetic correlations between temperament and breeding traits in Limousin heifers[END_REF] and included the running time, the number of escapes of the animal, and a 4-point score that indicates if 1) the animal could be restrained and stroked, 2) the animal was restrained but not stroked, 3) the animal could not be restrained, and 4) the animal was aggressive. The 12 AI bulls were chosen to have 6 bulls likely to produce calm calves and 6 bulls likely to produce agitated calves. The first objective of this disruptive selection was to be almost certain to capture any potential genetic variation in the chute test because the first aim of the study was to establish if this test may be useful for genetic evaluation of temperament under French husbandry conditions. It was also thought that it may compensate for some bias in the estimation of the genetic variance because no semen is available on the market (and consequently for on-farm experimentation) for bulls that are found aggressive either on performance or progeny test.

Limousin calves were weighed 3 times for purposes of on-farm genetic evaluation on weaning weight. The aim of the current study was to test temperament during these weighing procedures. As recommended by a preliminary study, we focused on the second and third weighing of animals, which occur on average at 5 and 7 mo of age. During this period of life, whatever their BW, animals are not limited in the number of movements they can do within the chute. Animals were tested twice to assess the consistency of their behav-ior over time, repeatability of the measurements, and to determine for practical purposes the recommended age of measurement for a possible genetic evaluation. Therefore, 2 visits per farm were performed between February 2008 and September 2008 during on-farm performance test weighing. A total of 2,141 Limousin calves were tested.

Test Procedure and Measures

The test was performed during on-farm performance test conducted by state agricultural service technicians. Calves were individually restrained in the chute, but the head of the calf was not captured within the head stanchion. The test consisted of assessing their behavior during weighing, then when exposed to a stationary human situated in front of the scale for 10 s. Three persons were involved in the test: the farmer who led animals to the chute, the weigher who also identified animals by their ear tag, and the experimenter who scored animals.

There were 2 periods in the test. The first period was during weighing, the experimenter was on the side of the chute next to the weigher. The duration of the first assessment period was at least 10 s. When, in a few cases, the weighing machine could not stabilize the measure within the 10 s because the animal was too agitated, a maximal score was given to the calf. In the second period, the experimenter stood in front of the animal (at the exit door of the chute, so as to be visible and identifiable as a human by calves) and stood motionless for 10 s. All the tests were performed by the same experimenter who was always dressed the same way (blue coveralls) in the 24 farms at the 2 visits.

Therefore, for each calf and each period of the test, the total number of movements and the number of rush movements were scored by visual appraisal using the same continuous scale (Figure 1) ranging from 0 (no movements) to 60 (continuous movements). These continuous measures were later converted to 6 categorical scores, and both were analyzed to determine which was the most appropriate for genetic evaluation purposes.

For each calf, 4 measures were recorded: the total number of movements during weighing (TW), the number of rush movements during weighing (RW), the total number of movements during exposure to the human (TH), and the number of rush movements during exposure to the human (RH). Every movement of the calf involving feet and every movement of the head that exceeded 30° of deviation were counted as true movements. Rush movements were defined as fast and sudden movements that were of short duration. Some calves were small enough to be able to turn around in the chute, and therefore the experimenter also noted the position (Pos) of the calf [head facing the exit (Pos = 0) or head facing the entrance door of the chute (Pos = 1)] during each period of the test.

Data Description

The first objective of the study was to test animals twice at an average age of 5 and 7 mo. However, because testing was done on-farm during weighing, animals were evaluated from 14 to 400 d of age. A preliminary analysis about the variations of measures according to the age of the calf was performed before the genetic analysis of the data. This analysis showed 4 main phases. Calves tested before 80 d of age were handled for the first time, and therefore, corresponding data were eliminated from the analysis (7.7% of records). Between 80 and 179 d of age, all the measures of temperament increased almost linearly with advancing age. Between 180 and 280 d, there was almost no age effect on the measures of temperament. After 280 d, all the measures decreased with advancing age. This may be due to the increase of the BW of the calf and its increasing difficulty to move in the chute (i.e., not enough space to move). Data after 280 d of age (8.3% of records) were also eliminated from the final data set because of this potential problem with calf size.

First, data editing for the analysis consisted of selecting records corresponding to animals tested between 80 and 280 d of age. Second, data editing consisted of selecting animals from known sires with at least 5 progeny records and from contemporary groups with at least 3 records per farm-management group. Pedigrees were obtained from the French National Cattle Genetic Database. Data related to calves that were progeny of unknown sires were also eliminated. The final research data set included data of 1,678 calves, offspring of 73 sires with 706 animals tested twice. Data were split into 2 data sets according to the age of the calf (Table 1). In the following, we refer as measures at age 1: those measurements recorded between 80 and 179 d of age, and measures at age 2: those measurements recorded between 180 and 280 d of age. Sex ratio (number of males/total number of animals) was 0.48 at the 2 ages. Hereafter, the name of a measure (e.g., TW) will be followed by the suffix 1 if measured at the age 1 (e.g., TW1) and the suffix 2 if measured at the age 2 (e.g., TW2).

Data Distribution

Data clearly followed a Poisson distribution because measures involved counting of the number of movements of each calf during a certain period of time. It was therefore easy to transform initial continuous variables into categorical ones. The measures TW, RW, TH, and RH were so transformed into categorical scores (CTW, CRW, CTH, and CRH, respectively) with 6 classes: 0 to 2 movements, 3 or 4 movements, 5 or 6 movements, 7 or 8 movements, 9 or 10 movements, and more than 10 movements.

Figures 2 and3 show data distribution for CTW and CRW at age 1. Almost 50% of the animals had 4 or fewer movements in total and at most 2 rush movements. Data distributions were similar at age 2 and during exposure to humans.

Preliminary Analysis for Fixed Environmental Effects

Preliminary analyses were performed to investigate the nongenetic factors influencing the traits using the GLM procedure for continuous variables and GEN-MOD procedure for discrete variables (SAS Inst. Inc., Cary, NC). Preliminary analyses of traits were carried out using a linear model that included the fixed effects of the farm (24 levels), sex of the calf (2 levels), type of birth of the calf (2 levels), dam parity (2 levels), Pos of the calf at birth at the start of the second period of the test in 2 levels (head facing the exit or the entrance door of the cage), age of the calf was used as a covariate or as a categorical variable (catage, 5 levels), the management group (11 levels) within farm, and the farm × sex, farm × parity, Pos × sex, type of birth × catage, parity × catage, sex × catage interactions. The weigher effect could not be tested because it was confounded with the farm effect. There were almost as many technicians as farms. Only significant effects from this set of preliminary analyses were included in the mixed model analysis aimed at estimating genetic parameters.

The models used assumed a normal distribution of data. However, data distribution was skewed at the left side for all the traits. Initial variables were transformed by Box-Cox transformation to ensure a normal distribution [START_REF] Neter | Applied Linear Statistical Models[END_REF] and to determine that significance of effects did not change compared with the untransformed measurements. The same fixed effects were significant when using the transformed variables showing that linear modeling was robust enough to handle the initial measures. This was also true for categorical variables, which were analyzed with Poisson regression models.

Genetic Analysis

For all traits (either continuous or discrete variables), estimates of variance components were performed under linear animal models by REML using the average information algorithm [START_REF] Gilmour | Average information REML, an efficient algorithm for variance parameter estimation in linear mixed models[END_REF] and AS-REML-R1 software [START_REF] Gilmour | ASREML Reference Manual[END_REF].

The following animal model was considered:

y = Xβ + Zu + e,
where y is the vector of observations, β is the vector of fixed effects and X is the corresponding incidence matrix, u is the vector of random genetic effects and Z is the corresponding incidence matrix, and e is the vector of random residual effects. Under the single trait model, random effects e and u were assumed to be normally distributed with expectation zero and variance matrix equal to I and A σ σ e a 2 2 , respectively. The matrix I is the identity matrix. The relationship matrix (A) between animals included 3 generations of ancestors that accounted for 5,363 animals in total. Fixed factors included in the models for the estimation of genetic parameters for the analyzed traits were the contemporary group (farm-management group interactions), the position of the calf during the test (Pos) for all the traits, the type of birth, the interaction farm × sex and Pos × sex for RW1 and TW1, the interaction farm × parity of the dam and calf age as a covariate for all the measures recorded at age 1.

To check if the accuracy of genetic evaluation can be significantly improved by including repeated measures, a repeatability model was used for each trait (combining data across age 1 and 2 into a single trait analysis and adding a permanent environmental effect of the animal to the previous animal model).

Heritabilities were first estimated for each of the 8 traits with observed and transformed data points (TW1, RW1, TH1, RH1, TW2, RW2, TH2, RH2, CTW1, CRW1, CTH1, CRH1, CTW2, CRW2, CTH2, and CRH2) using single trait models. Correlations between the same measures considered across ages were estimated using linear bivariate animal models. For each given age, a multitrait linear model including the 4 traits was initially tested to estimate genetic correlations between the different traits. Due to extremely high genetic correlations (above 0.95) between RW and TW or RH and TH measures, convergence could not be reached. Consequently, for each age, 3 different multitrait analyses were performed: 1) TH, TW, and RW, 2) RH, TW, and RW, and 3) RH and TH. Similar analyses were performed with categorical variables.

Categorical traits were also analyzed under threshold mixed models using the software CATKIT developed by V. Ducrocq (INRA, Jouy-en-Josas, France, personal communication) for French dairy cattle calving ease evaluation. For discrete traits, EBV were compared be- tween predictions of linear and threshold models. Because similar ranking of EBV were predicted (rank correlations over 0.99), multitrait analysis were performed under linear models whatever the nature (continuous or discrete) of the trait. Because a previous study (Sapa et al., 2006) showed some heterogeneity of variance across sexes, records of males and females were treated as different traits in bivariate analysis to determine whether or not the same biological roots were behind male and female temperament measures.

Reliability of each EBV (û) was derived as R = 1 -Var(u -û)/Var(u). Rank and Pearson correlations between EBV estimated for initial data and categorical data were performed for each trait using the procedure CORR within SAS. Probability values <0.05 were regarded as statistically significant.

RESULTS AND DISCUSSION

Descriptive Statistics

Descriptive statistics for all the traits are shown in Table 2. Except for TH, all trait means increased between age 1 and 2, which meant that older animals were the most agitated. On the contrary, the raw SD remained stable over ages and very large CV were served (from 70 to 100%).

Animals at age 2 were weighed for the third time of their life, and one might expect them to be less agitated with experience as increased amounts of handling experience often results in animals having decreased fear responses to new environments and handlers. Indeed, [START_REF] Crookshank | Effect of transportation and handling of calves upon blood serum composition[END_REF] and [START_REF] Kadel | Genetics of flight time and other measures of temperament and their value as selection criteria for improving meat quality traits in tropically adapted breeds of beef cattle[END_REF] showed that agitation and cortisol concentrations in cattle were decreased over multiple handling experiences. However, present results agreed with those reported by [START_REF] Müller | Consistency of flight speed and its correlation to productivity and to personality in Bos taurus beef cattle[END_REF] and [START_REF] Petherick | Productivity, carcass and meat quality of lot-fed Bos indicus cross steers grouped according to temperament[END_REF] who found increases in flight speed over repeated tests and [START_REF] Grandin | Behavioral agitation of cattle during handling is persistent over time[END_REF] who mentioned that European Continental cattle that were worked through a squeeze chute repeatedly in a single day became increasingly agitated. Different results between studies are likely due to different levels of animal fear and aver-sion to the test. The increase of agitation during weighing observed at age 2 may also be due to physiological changes with age.

Heritabilities and Genetic and Phenotypic Correlations

Estimates of heritabilities and genetic and phenotypic correlation across ages for all the measures are presented in Table 3. Results obtained for categorical variables analyzed under linear models were very similar to those obtained for the initial scores (Table 3), confirming that categorizing the continuous data did not create a significant loss of information. When analyzing categorical variables under threshold models, assuming an underlying normal variables, the heritability of the corresponding liabilities were slightly (+0.01 to 0.05) greater than the heritability estimated using linear models, as expected for multinomial variables [START_REF] Vinson | Overall classification as a selection criterion for improving categorically scored components of type in Holsteins[END_REF].

Heritabilities ranged from 0.11 to 0.22 at age 1 and 0.16 to 0.31 at age 2, which meant that all the measures were moderately heritable as it was often shown in the literature for temperament traits [START_REF] Shrode | Chute behavior of yearling beef cattle[END_REF][START_REF] Le Neindre | Individual differences in docility in Limousin cattle[END_REF][START_REF] Burrow | Genetic and environmental factors affecting temperament of zebu and zebu-derived beef cattle grazed at pasture in the tropics[END_REF][START_REF] Burrow | Variances and covariances between productive and adaptive traits and temperament in a composite breed of tropical beef cattle[END_REF][START_REF] Tier | Potential for improving the docility of Limousin cattle in Australia[END_REF][START_REF] Kadel | Genetics of flight time and other measures of temperament and their value as selection criteria for improving meat quality traits in tropically adapted breeds of beef cattle[END_REF][START_REF] Phocas | Genetic correlations between temperament and breeding traits in Limousin heifers[END_REF]. The greatest genetic variations were observed during weighing at age 2, and the total number of movements seems to better discriminate animals than the number of rush movements.

For each trait, genetic correlation between measures recorded at age 1 and age 2 was not significantly different from 1 (P < 0.01). This suggests that traits are governed by the same pool of genes at the 2 ages and could consequently be measured at either age for inclusion in a genetic evaluation of a given temperament trait. Heritabilities of traits recorded during exposure to the human were stable with age, contrary to those of traits recorded during weighing, which were greater at age 2 than age 1 (Table 3). Although the same pool of genes influenced the temperament phenotypes at both ages, a greater expression of genetic potential is observed at age 2 during weighing. Such age differences have been On-farm measures of beef cattle temperament already reported for flight speed heritability estimated by [START_REF] Burrow | A new technique for measuring temperament in cattle[END_REF], which was 0.54 at 6 mo and only 0.26 at 18 mo of age. For a given trait, phenotypic correlation between measures at age 1 and 2 corresponded to the trait repeatability because the same biological character was measured at the 2 ages. Repeatability was about 0.3 for all traits, indicating that environmental influences on these temperament traits changed greatly over time. This is similar to the repeatability of chute score (0.36) reported for tropically adapted beef [START_REF] Kadel | Genetics of flight time and other measures of temperament and their value as selection criteria for improving meat quality traits in tropically adapted breeds of beef cattle[END_REF] measured on average at 246 and 564 d of age and less than repeatability for Angus cattle (0.44; [START_REF] Johnston | Evaluation of flight time and crush score as measures of temperament in Angus cattle[END_REF] measured 73 d apart. Behavioral traits often have reduced stability over time, and the few studies that looked at the consistency of behavioral reactivity in beef cattle often reported moderate repeatability. This retesting variability can be explained 2-fold. First, the effect of the experience/ age of the animal is expected because juveniles are undergoing dramatic developmental change and therefore do not show repeatable behavior [START_REF] Bell | The repeatability of behavior: A meta-analysis[END_REF]. Second, environmental changes in animal management or testing conditions might also cause repeatability to decrease particularly with the increase of the time interval between observations. When this time is long, it is unlikely that the animals are of similar state (e.g., size, condition, dominance, social group) during both observations and are experiencing similar environments [START_REF] Bell | The repeatability of behavior: A meta-analysis[END_REF]. Repeated measures in this study were undertaken over a time interval of 3 mo, and in those 3 mo, the environment of the animal may have undergone several changes that could influence its behavior in the chute.

Heterogeneous Variance Components Between Sexes

Results showed no effect of calf sex on any of the studied traits. However, previous studies reported frequently phenotypic differences between sexes with females often more difficult to handle [START_REF] Tulloh | Behavior of cattle in yards: II. A study of temperament[END_REF][START_REF] Shrode | Chute behavior of yearling beef cattle[END_REF][START_REF] Sato | Factors associated with temperament of beef cattle[END_REF][START_REF] Gauly | Estimating genetic variability in temperamental traits in German Angus and Simmental cattle[END_REF][START_REF] Sapa | Genetic parameters between sexes for temperament traits in Limousin cattle[END_REF]. Even if genetic correlations were estimated close to unity between sexes for all traits indicating that the same pool of genes was involved in female and male temperament, large significant differences in genetic variability were estimated between male and female measures recorded during weighing. As shown in Table 4, females exhibited less genetic variability at age 1 than males and, on the contrary, greater genetic variability at age 2. However, genetic variability of measures recorded during exposure to the human was homogeneous across sexes. Female genetic variance changed substantially between the 2 periods of the test, which meant that they might be more sensitive to the human presence than males.

Results related to age 2 are similar with those reported by [START_REF] Sapa | Genetic parameters between sexes for temperament traits in Limousin cattle[END_REF] who found that heritabilities of running time and number of escapes during the docility test [START_REF] Le Neindre | Individual differences in docility in Limousin cattle[END_REF] were significantly greater for heifer temperament than for bull temperament. On the contrary, [START_REF] Sapa | Genetic parameters between sexes for temperament traits in Limousin cattle[END_REF] found no differences between sexes for the docility score itself, similar to [START_REF] Burrow | Variances and covariances between productive and adaptive traits and temperament in a composite breed of tropical beef cattle[END_REF] who found similar heritability of flight speed test for both sexes. This heterogeneity can be explained by differences between sexes in responses to environmental stress. [START_REF] Denise | Genetic parameter estimates for preweaning traits of beef cattle in a stressful environment[END_REF]De Nise and[START_REF] Denise | Genetic parameter estimates for postweaning traits of beef cattle in a stressful environment[END_REF] show that genetic parameters change in response to level of environmental stress, and the sexes respond differently to these conditions.

Estimates of Genetic Correlations Between Traits

Genetic correlations between categorical traits at age 2 are presented in Table 5. Similar results were obtained at age 1 and, whatever the age, for the initial continuous variables. At a given age, the 4 traits were genetically highly correlated to each other (from 0.77 to 0.99). The genetic correlations between the total number of movements and the number of rush movements during weighing or during exposure to the human were not significantly different from unity, suggesting that these measures represent exactly the same trait at the genetic level. Therefore, the quality of the movement can be ignored when assessing calf behavior in the chute. Almost all the scores used in the literature to assess the behavior of the calf by visual appraisal take in account the quality of the movement (e.g., degree of violence, speed; [START_REF] Burrow | Measurements of temperament and their relationships with performance traits of beef cattle[END_REF], which increases the amount of subjectivity accounting for in these measures. The total number of movements, in addition of its greater heritability, reduces risks of subjectivity when assessing calf temperament. Genetic correlations between measures recorded during the 2 periods of the test (during weighing and exposure to the human) were also very high at both ages (from 0.79 to 0.86, Table 5). The second test period where the calf is exposed to human presence extends the duration of the test and requires additional work besides the weighing procedure. Results showed that the reaction of the calf to the human exposure can be predicted by its reactivity during weighing and suggested that the test can be shortened to only the weighing period.

Residual correlations between the total number of movements and the number of rush movements mea-sured in the same period of the test were greater than 0.89 (Table 5). This was expected as measures were recorded simultaneously and were influenced by the same environmental effects as proved by the statistical analysis. On the contrary, residual correlations between measures recorded at different periods of the test did not exceed 0.38 (Table 5). That meant that animals were not experiencing exactly the same environment during weighing or exposure to the human. This can be explained by the presence of the human in the front of the cage but also by the increase of time spent in restraint.

Correlations Between EBV Estimated for Initial Scores and Categorical Traits

Correlations between EBV estimated for initial scores and the corresponding categorical variables were above 0.98 (P < 0.01) for all the measures recorded at both ages and also when using a repeatability model on the overall data set. As expected from estimates of variance components, scoring the behavior of the animal using a categorical score gave similar information and is easier to implement for further on-farm data collection than the continuous scale used in the present study. In addition, categories adequately discriminate animals as well as the initial continuous score. Therefore, it is recommended for the implementation of a routine ge- 2 CTW = total number of movements during weighing in categories; CRW = number of rush movements during weighing in categories; CTH = total number of movements during exposure to the human in categories; CRH = number of rush movements during exposure to the human in categories.

On-farm measures of beef cattle temperament netic evaluation that on-farm evaluators use a categorical scale rather than the continuous scale to assess the behavior of the calf in the chute.

Sire EBV Reliability

Mean reliability values of sire EBV and associated number of progeny records per sire are shown in Table 6. Because a minimum number of 25 progeny records is required in French on-farm breeding scheme for a proven bull, bull index accuracies were calculated for bulls above and under this threshold. For sires with more than 25 progeny records, EBV reliability was above 0.61 for all the traits using only measures recorded at age 2. The EBV reliability increased significantly when considering the repeated measures for the number of movements and the rush movements during exposure to the human (Table 6). Considering these traits as selection criteria, it will be better to test animals at both ages to increase the accuracy of selection (i.e., above 0.50). However, considering that sires with more than 25 progeny records had sufficient EBV reliability, a single observation of temperament per progeny is sufficient for routine genetic evaluation using field data.

Because of greater heritabilities, including the repeated measures only slightly increased the evaluation accuracy for the total number of movements and the number of rush movements measured during weighing if compared with a single measure evaluation performed at age 2. Therefore, repeat measures are not worth doing when we consider these traits as selection criteria for genetic evaluation.

Implications for the Implementation of Genetic Evaluation of Calf Temperament

The purpose of the current study was to collect field data that was as close as possible to practical conditions. All the measures of temperament used in this study were heritable and repeatable and could therefore respond to selection. The number of movements during weighing recorded at age 2 seems to be the most useful selection criterion. It had the greatest heritability and was easier to measure than the number of rush movements, which implied a qualitative evaluation of the movements of a calf. To improve the accuracy of the genetic evaluation, the analysis model should take in account the heterogeneity of variance components between sexes.

These results are a start to the practical use of genetic evaluation on calf temperament. However, before defining the selection criterion to be used on-farm, further studies are required to investigate the relationship between these traits recorded on-farm and those collected in progeny test station for the 12 bulls used for connection in this study. This will allow a better choice of the selection criterion to be used on-farm for genetic evaluation in terms of the breeding goals of breeders related to aggressiveness or wildness of animals.

Figure 1 .

 1 Figure 1. Illustration of the ruler scales used to score calf movements. The distance (mm) from origin to marking by evaluator was used to generate the continuous data points for the various measures of temperament. TW = total number of movements during weighing; RW = number of rush movements during weighing; TH = total number of movements during exposure to the human; and RH = number of rush movements during exposure to the human.

Figure 2 .

 2 Figure 2. Frequency distribution of the total number of movements during weighing at age 2 expressed in 6 categories. Age 2: measurements recorded between 180 and 280 d of age.

Figure 3 .

 3 Figure 3. Frequency distribution of the number of rush movements during weighing at age 2 expressed in 6 categories. Age 2: measurements recorded between 180 and 280 d of age.

Table 1 .

 1 Final data sets according to calf age

	Item	Data set 1	Data set 2
	No. of observations	1,113	1,271
	Age interval, d	80,179	180,280
	Average age ± SD, d	142 ± 25	218 ± 60
	No. of sires	73	65

Table 2 .

 2 Descriptive statistics for all the traits at ages 1 and 2 1

			Age 1		Age 2
	Trait	n	Mean ± SD	n	Mean ± SD
	Total No. of movements during weighing	1,279	24.8 ± 18.7	1,439	28.2 ± 18.8
	No. of rush movements during weighing	1,278	16.8 ± 17.2	1,439	22.0 ± 18.8
	Total No. of movements during exposure to the human	1,282	23.4 ± 16.5	1,440	22.7 ± 16.9
	No. of rush movements during exposure to the human	1,282	15.8 ± 15.7	1,440	16.8 ± 16.9
	Total No. of movements during weighing expressed in 6 categories	1,279	2.6 ± 1.5	1,439	2.9 ± 1.6
	No. of rush movements during weighing expressed in 6 categories	1,278	2.0 ± 1.3	1,439	2.4 ± 1.4
	Total No. of movements during exposure to the human expressed in 6	1,282	2.4 ± 1.4	1,440	2.4 ± 1.4
	categories
	No. of rush movements during exposure to the human expressed in 6	1,282	1.9 ± 1.1	1,440	2.0 ± 1.3
	categories
	1 Age 1: measurements recorded between 80 and 179 d of age; age 2: measurements recorded between 180 and 280 d of age.

Table 3 .

 3 Phenotypic and genetic parameters for the continuous and categorical scores across ages 1

		Phenotypic	Phenotypic					Genetic
		variance	correlation	Genetic variance	Heritability	correlation
				Between					Between
	Trait 2	Age 1	Age 2	ages	Age 1	Age 2	Age 1	Age 2	ages
	TW	264 ± 13	278 ± 13	0.27 ± 0.04	38 ± 22	87 ± 30	0.14 ± 0.08	0.31 ± 0.10	0.84 ± 0.20
	RW	222 ± 11	288 ± 14	0.32 ± 0.04	24 ± 16	81 ± 29	0.11 ± 0.07	0.28 ± 0.09	0.99 ± 0.19
	TH	247 ± 12	243 ± 11	0.32 ± 0.04	55 ± 20	41 ± 18	0.22 ± 0.08	0.17 ± 0.07	0.84 ± 0.21
	RH	217 ± 10	250 ± 11	0.31 ± 0.04	44 ± 17	47 ± 18	0.20 ± 0.08	0.19 ± 0.07	0.95 ± 0.19
	CTW	1.8 ± 0.1	2.0 ± 0.1	0.29 ± 0.04	0.33 ± 0.2	0.55 ± 0.2	0.16 ± 0.08	0.29 ± 0.10	0.94 ± 0.17
	CRW	1.3 ± 0.1	1.6 ± 0.1	0.29 ± 0.04	0.14 ± 0.1	0.37 ± 0.1	0.11 ± 0.07	0.23 ± 0.09	0.98 ± 0.19
	CTH	1.7 ± 0.1	1.7 ± 0.1	0.33 ± 0.04	0.34 ± 0.1	0.28 ± 0.1	0.20 ± 0.08	0.16 ± 0.07	1.00 3
	CRH	1.2 ± 0.0	1.4 ± 0.1	0.34 ± 0.04	0.18 ± 0.1	0.22 ± 0.1	0.15 ± 0.07	0.16 ± 0.07	1.00 3

Table 4 .

 4 Phenotypic and genetic parameters of all categorical traits according to sex of calf CTW = total number of movements during weighing in categories; CRW = number of rush movements during weighing in categories; CTH = total number of movements during exposure to the human in categories; CRH = number of rush movements during exposure to the human in categories.2 Age 1: measurements recorded between 80 and 179 d of age; age 2: measurements recorded between 180 and 280 d of age.

		n			Mean	Phenotypic variance	Heritability
	Trait 1,2	Male	Female	Male	Female	Male	Female	Male	Female
	Measure at age 1
	CTW	622	657	2.6 ± 1.5	2.6 ± 1.5	1.9 ± 0.1	1.8 ± 0.1	0.27 ± 0.14	0.05 ± 0.07
	CRW	622	656	2.0 ± 1.3	2.0 ± 1.2	1.2 ± 0.1	1.2 ± 0.8	0.24 ± 0.14	0.04 ± 0.07
	CTH	626	656	2.5 ± 1.4	2.4 ± 1.4	1.7 ± 0.1	1.7 ± 0.1	0.21 ± 0.11	0.19 ± 0.10
	CRH	626	656	1.9 ± 1.1	1.9 ± 1.2	1.1 ± 0.1	1.2 ± 0.1	0.18 ± 0.11	0.15 ± 0.09
	Measure at age 2
	CTW	697	742	2.9 ± 1.6	2.8 ± 1.6	1.9 ± 0.2	2.1 ± 0.1	0.14 ± 0.09	0.52 ± 0.15
	CRW	697	742	2.4 ± 1.5	2.3 ± 1.4	1.6 ± 0.1	1.6 ± 0.1	0.10 ± 0.08	0.50 ± 0.16
	CTH	699	741	2.5 ± 1.5	2.4 ± 1.4	1.9 ± 0.1	1.6 ± 0.1	0.17 ± 0.09	0.16 ± 0.09
	CRH	699	741	2.0 ± 1.3	2.0 ± 1.3	1.5 ± 0.1	1.4 ± 0.1	0.14 ± 0.09	0.19 ± 0.10

1

Table 5 .

 5 Genetic and residual correlations (respectively above and below the diagonal) between the different categorical traits recorded at age 2 1 for the Limousin calves

	Trait 2	CTW	CRW	CTH	CRH
	CTW		0.99 ± 0.01	0.79 ± 0.20	0.82 ± 0.21
	CRW	0.90 ± 0.01		0.73 ± 0.26	0.77 ± 0.20
	CTH	0.33 ± 0.01	0.38 ± 0.06		0.99 ± 0.02
	CRH	0.37 ± 0.04	0.36 ± 0.01	0.89 ± 0.01

1

Age 2: measurements recorded between 180 and 280 d of age.

Table 6 .

 6 Sire EBV reliability (mean ± SD) according to the number of recordings per progeny

		1 record/progeny (at age 2) 2	2 records/progeny
	Trait 1	≥25 progeny	<25 progeny	≥25 progeny	<25 progeny
	CTW	0.73 ± 0.12	0.39 ± 0.12	0.74 ± 0.13	0.42 ± 0.14
	CRW	0.70 ± 0.13	0.34 ± 0.13	0.71 ± 0.13	0.38 ± 0.14
	CTH	0.61 ± 0.14	0.27 ± 0.11	0.76 ± 0.09	0.46 ± 0.11
	CRH	0.64 ± 0.13	0.29 ± 0.11	0.69 ± 0.14	0.36 ± 0.14

The authors thank the program Agriculture et Développement Durable of the National Agency for Research and the French Limousin Breed Association for funding this study.

2 Corresponding author: florence.phocas@jouy.inra.fr Received

May 3, 2010. Accepted July 12, 2010.

at INRA Institut National de la Recherche Agronomique on August 26, 2013 www.journalofanimalscience.org Downloaded from

Age 1: measurements recorded between 80 and 179 d of age; age

2: measurements recorded between 180 and 280 d of age.2 TW = total number of movements during weighing; RW = number of rush movements during weighing; TH = total number of movements during exposure to the human; RH = number of rush movements during exposure to the human; CTW = total number of movements during weighing in categories; CRW = number of rush movements during weighing in categories; CTH = total number of movements during exposure to the human in categories; CRH = number of rush movements during exposure to the human in categories. 3 Fixed at the boundary value of the parameter space.Benhajali et al.

CTW = total number of movements during weighing in categories, CRW = number of rush movements during weighing in categories; CTH = total number of movements during exposure to the human in categories; CRH = number of rush movements during exposure to the human in categories.

Age 2: measurements recorded between 180 and 280 d of age.Benhajali et al.

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