Christina C Nielsen-Leroux
email: christina.nielsen@jouy.inra.fr

Sophie S Gaudriault

Nalini N Rama Rao

Didier D Lereclus

Alain Givaudan

Nalini Ramarao

How the insect pathogen bacteria Bacillus

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

Introduction

Several entomopathogenic bacteria are known [1 ,2 ] but the most studied are Bacillus thuringiensis, Photorhabdus and Xenorhabdus. B. thuringiensis is a Gram-positive sporeforming bacterium that has been used for more than 50 years as bio-insecticide against insect pests in crop and forestry [START_REF] Bravo | Bacillus thuringiensis: a story of a successful bioinsecticide[END_REF][START_REF] Sanchis | From microbial sprays to insect-resistant transgenic plants: history of the biospesticide Bacillus thuringiensis. A review[END_REF]. B. thuringiensis is closely related to Bacillus anthracis, the etiological agent of anthrax, and to Bacillus cereus, an opportunistic human pathogen causing foodborne gastroenteritis [START_REF] Kolstø | What sets Bacillus anthracis apart from other Bacillus species?[END_REF]. Photorhabdus and Xenorhabdus species include Gram-negative symbiotic bacteria associated with entomopathogenic nematodes also used as alternatives to pesticides to control numerous insect pests [START_REF] Ehlers | Mass production of entomopathogenic nematodes for plant protection[END_REF].

Most studies have focused on the specific insecticidal toxins produced by these bacteria (Cry and Cyt toxins for B. thuringiensis [START_REF] Bravo | Bacillus thuringiensis: a story of a successful bioinsecticide[END_REF][START_REF] Bravo | Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control[END_REF] and Tc toxins for Photorhabdus and Xenorhabdus [START_REF] Ffrench-Constant | Insecticidal toxins from Photorhabdus bacteria and their potential use in agriculture[END_REF]). The role of other virulence and adaptation factors has been less investigated. Here, we highlight what is actually known about the factors necessary to cope with host tissue and host defence mechanisms. We aim to point out common infection strategies used by these bacteria. The issue is also to show that insect models, larger than Drosophila [START_REF] Vodovar | Drosophila: a polyvalent model to decipher host-pathogen interactions[END_REF], mainly lepidopteran larvae can also be useful to improve the understanding of bacterial infection processes [10 ,11 ,12 ].

Infection process

A bacterial pathogen must be able to colonize its host by coping with different mechanical and physical barriers as well as cellular and humoral defence mechanisms (see Box 1). In nature, B. thuringiensis spores or vegetative cells, are taken up by the larva by ingestion or more accidentally by wounding through the cuticle (see Figure 1 for a description of B. thuringiensis-insect interaction). By contrast, natural infection involving Photorhabdus/Xenorhabdus mainly starts from the body cavity, since they are released at that site from the nematode hosts (see Figure 1 for a description of Photorhabdus/ Xenorhabdus infection process). Then, the two groups of bacteria discussed here will deal with the various larval compartments/tissues at different moments post infection. Therefore, we describe, in the following sections, the expression and effects of various bacterial factors, at different stages of the infectious process.

Gut adaptation and intestinal barrier degradation

Photorhabdus and Xenorhabdus are vectored by the nematode through the gut barrier and get direct access to the hemocoel. However, the two bacteria have orally active insecticidal toxins (see Box 2). For B. thuringiensis, the oral infection is the norm and success to breach the gut barrier is mainly depending on the action of Cry toxins, which both permit B. thuringiensis spores to germinate and also to weaken the midgut cells. However the relative importance of Cry toxins and the bacteria itself depends on the insect species [START_REF] Heimpel | The site of action of crystalliferous bacteria in lepidotera larvae[END_REF] (see Figure 1 and Box 2 for details). In the gut, the newly germinated vegetative bacteria need to compete with the commensal gut microbiota and to resist to host factors like antimicrobial peptides in order to persist in the larval gut lumen. The factors involved in these resistance mechanisms are surface properties like the lysozyme, resistant peptidoglycan structure of B. thuringiensis and the production of bacteriocins [START_REF] Cherif | Characterization and partial purification of entomocin 110, a newly identified bacteriocin from Bacillus thuringiensis subsp. Entomocidus HD110[END_REF], which can also explain the reduction of the gut microbiota predominantly composed of Enterococcus sp. in lepidopteran larvae [START_REF] Jarosz | Gut flora of Galleria mellonella suppressing ingested bacteria[END_REF].

Beside the plasmid-encoded Cry toxins (see Box 2), the B. thuringiensis genome contains many genes, which might take part in gut pathogenesis. The role of the PlcR regulon in virulence of B. thuringiensis and B. cereus was demonstrated by using G. mellonella larvae [START_REF] Salamitou | The plcR regulon is involved in the opportunistic properties of Bacillus thuringiensis and Bacillus cereus in mice and insects[END_REF]. Indeed, deletion of the global transcriptional activator, plcR, drastically reduces bacterial virulence in orally infected insects, but not by injecting the bacteria into the larval hemocoel [START_REF] Salamitou | The plcR regulon is involved in the opportunistic properties of Bacillus thuringiensis and Bacillus cereus in mice and insects[END_REF][START_REF] Fedhila | The Bacillus thuringiensis PlcR-regulated gene inhA2 is necessary, but not sufficient, for virulence[END_REF], indicating that the PlcR regulon plays a key role in pathogenicity in the early stage of infection by oral route. PlcR controls the expression of 45 genes encoding secreted virulence factors including the pore forming enterotoxins complexes (Hbl and Nhe), hemolysins known for cytotoxic and hemolytic activities, antimicrobial peptides, and various degradative enzymes, which might allow the bacteria to develop in the intestinal environment and to damage the intestinal barrier [START_REF] Gohar | The PlcR virulence regulon of Bacillus cereus[END_REF]. Histological analyses indicate that B. thuringiensis colonize and damage the gut epithelium resulting in rupture of the gut integrity and the crossing of the gut barrier. Interestingly, a plcR mutant is blocked at the gut epithelial and transcriptional gfp promoter fusions showed that PlcR-regulated factors are expressed in the insect gut (Nielsen-LeRoux, unpublished data). However, deletion of enterotoxin genes (Nhe, Hbl complexes and CytK) did not significantly alter the insecticidal properties of B. thuringiensis towards three lepidopteran insects [START_REF] Klimowicz | A quadrupleenterotoxin-deficient mutant of Bacillus thuringiensis remains insecticidal[END_REF]. Then, further studies are The B. thuringiensis-based insecticidal products are a mixture of spores and insecticidal toxins forming a crystal inclusion. These toxins, Cry (also called delta endotoxins) and Cyt proteins, are often encoded by plasmid carried genes. They are active against larval stages of many insect orders and other invertebrates like nematodes [START_REF] Van Frankenhuyzen | Insecticidal activity of bacillus thuringiensis crystal proteins[END_REF]. The mode of action of Cry toxin has been extensively studied for many insect species. The general procedure is shown in Figure 1 insert and several families of specific midgut receptors have been identified [START_REF] Soberon | Signaling versus punching hole: how do Bacillus thuringiensis toxins kill insect midgut cells?[END_REF]. Meanwhile other indirect roles of Cry toxins: like the binding to the peritrophic matrix [START_REF] Rees | Peritrophic membrane contribute to bt Cry delta-endotoxion susceptibility in Lepidoptera and the effect of Calcofluor[END_REF] or the possible interaction with the commensal gut microbiota in some insects [START_REF] Mason | From commensal to pathogen: translocation of Enterococcus faecalis from the midgut to the hemocoel of Manduca sexta[END_REF] need more attention. Cyt toxins are present in a lower number of strains than Cry toxins and they are less specific as they interferes with several classes of cells including mammals. In many insects, the Cry toxin alone can kill the insect larvae (used in transgenic crops expression the Cry toxins) while for others the presence of the bacteria is needed [START_REF] Li | Importance of spores, crystals, and @-endotoxins in the pathogenicity of different varieties of Bacillus thuringiensis in Galleria mellonela and Pieris brassicae[END_REF]. Genes encoding Vip toxins (vegetative insecticidal proteins) have been found in various B. thuringiensis strains, but the expression and mode of action have been poorly studied (see (http:// www.lifesci.sussex.ac.uk/Home/Neil_Crickmore/Bt/) website for updated number of toxins). Although the main role of these toxins are related to gut cell interactions in the living insect, an eventual role during expression in the insect cadaver cannot be excluded.

Although Xenorhabdus and Photorhabdus directly enter within hemocoel following the release by nematode hosts, these bacteria, like B. thuringiensis, have oral insecticidal toxins (such as the 'toxin complexes' (Tc) and the 'Photorhabdus insect-related' (Pir) toxins) triggering massive damage to the gut epithelium of insect [START_REF] Ffrench-Constant | Insecticidal toxins from Photorhabdus bacteria and their potential use in agriculture[END_REF]. Despite intensive research on insecticidal toxin family, their reliable involvement in a precise step of the life cycle of the bacterial symbionts remained enigmatic. Indeed, P. luminescens Tc toxins inhibit phagocytosis of bacteria when added on insect hemocyte monolayers and recently, the biologically active components of the Tc toxins have been characterized as ADP-ribosyltransferases, which modify actin and Rho GTPases [START_REF] Lang | Photorhabdus luminescens toxins ADP-ribosylate actin and RhoA to force actin clustering[END_REF]. Because Tc toxins have also insecticidal activities after injection into insects, it is likely that the Tcdependent inhibition of phagocytosis takes place in the hemolymph during bacterial infection. More surprisingly is the discovery of oral insecticidal activity of the universally occurring chaperon molecule GroEL in X. nematophila. GroEL protein has no effect when injected into the hemocoel or when added to cultures of insect hemocytes [START_REF] Joshi | An insecticidal GroEL protein with chitin binding activity from Xenorhabdus nematophila[END_REF]. It is noteworthy that the XnGroEL protein and the insecticidal Tc-like complexes of the insect pathogen, Yersinia entomophaga, have chitin binding and chitinase activities, respectively. Busby et al. (2011) [START_REF] Busby | Structural analysis of Chi1 chitinase fromYen-Tc: the multisubunit insecticidal ABC toxin complex of Yersinia entomophaga[END_REF] propose that chitinase proteins form an integral part of the Tc complexes in Photorhabdus and Xenorhabdus as well. It is tempting to speculate that chitinase activity acting on the peritrophic membrane may help toxins or bacteria to reach gut epithelial cells. According to the biology of nematode symbionts, we can also propose that chitinase associated toxins may be also required in late infection in the cadaver compartment by contributing to the degradation of the insect cadaver exoskeleton. Moreover, Tc-like encoding genes have been recently found in some B. thuringiensis strains indicating a conserved and probably important function of these toxins, which would merit further attention in the B. thuringiensis life cycle [START_REF] Blackburn | The occurrence of Photorhabdus-like toxin complexes in Bacillus thuringiensis[END_REF].

Box 1 Larval anatomy and basic defense.

The lepidopteran larvae commonly used for infection analysis are Galleria mellonella, Spodoptera littoralis and Manduca sexta. The first insect barrier to overcome from the environment is the cuticle (skin), next the digestive tract, which is composed of a mouth, a proventricule, a forgut, a midgut and a hindgut. A sort of protective chitin rich web called peritrophic matrix, which separates the insect midgut from direct contact to food and pathogens [START_REF] Hegedus | New insights into peritrophic matrix synthesis, architecture, and function[END_REF], covers a large part of this 'tube' (see also Figure 1). The digestive tube is surrounded by the hemocoel (larval body cavity), which is mainly composed of hemolymph (blood). In the hemocoel are located fat body cells (liver function), circulating hemocytes and humoral immune factors. Hemocytes are key elements in cellular immune responses, which include phagocytosis, nodulation and encapsulation. Humoral factors include, among others, antimicrobial peptides, lysozyme, clothing mechanisms and prophenoloxidase, which are involved in the melanization process [START_REF] Jiang | Immunity in lepidopteran insects[END_REF]. Antimicrobial peptides and lysozyme can also be found in the gut. Thus, if an infection by B. thuringiensis results in generalized septicaemia, it indicates that the bacteria have been capable to survive and develop inside the digestive tube, notably by competing with natural gut flora, and had won access to the insect hemocoel by degrading of the peritrophic matrix and midgut cells. Survival and development in the hemocoel of B. thuringiensis, Photorhabdus and Xenorhabdus indicate that these bacteria produce factors, which can cope with the above mentioned cellular and humoral immune effectors.

needed to show the precise role of the PlcR-regulated factors in insect gut infection, like searched by a RT-PCR analysis [START_REF] Kyei-Poku | Detection of Bacillus cereus virulence factors in commercial products of Bacillus thuringiensis and expression of diarrheal enterotoxins in a target insect[END_REF]. In addition, to understand exactly how the bacteria cross the gut, including the peritrophic matrix (see Box 1), it is important to fully elucidate the relative impact of Cry toxins and the other B. thuringiensis bacterial factors, (like the metalloprotease Enhancin [START_REF] Fang | Bacillus thuringiensis Bel protein enhances the toxicity of Cry1Ac protein to Helicoverpa armigera larvae by degrading insect intestinal mucin[END_REF]), on these barriers. General natural larval infection and life cycles of B. thuringiensis, Photorhabdus and Xenorhabdus. B. thuringiensis is primary an oral pathogen and the spores along with the crystal toxin are acting together to kill the insect. When ingested by a susceptible insect, the crystal inclusions are solubilized in the gut and protoxins are activated by digestive enzymes (steps 1, 2, and insert). The activated toxins (small blue circles) are then able to cross the peritrophic barrier and to bind different classes of midgut receptors (Box 2, insert and [START_REF] Bravo | Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control[END_REF]). This results in the formation of pores in the epithelial midgut cells and impairment of intestinal function. This creates favorable conditions for spore-germination and development of vegetative growth (steps 2 and 3). Bacteria multiply and produce various virulence factors allowing access to the hemocoel [START_REF] Raymond | Bacillus thuringiensis: an impotent pathogen?[END_REF]. They adapt to this new compartment and produce new virulence factors to combat the host defenses (step 3). This may allow the bacteria to cause septicemia, resulting in insect death (step 4). Within the host cadaver the B. thuringiensis cells have to cope with novel growth conditions to complete their development, ending with the production of resistant spores and Cry toxin crystals able to disseminate in the environment to start a new cycle of infection in another host. Xenorhabdus and Photorhabdus spp. are pathogens of numerous insects and are mutualists of nematodes from the family Heterorhabditidae and Steinernematidae, respectively. The life cycle of Photorhabdus and Xenorhabdus begins and ends with the colonization of the intestinal tract of nonfeeding stage of the nematode known as the infective juvenile (IJ) [START_REF] Forst | Xenorhabdus and Photorhabdus spp.: bugs that kill bugs[END_REF]. Actually, the IJ stage nematodes generally use natural openings of insect larvae (mouth and anus) as main routes of entry (step 1), and end up in digestive track (gut; step 2) before entering in the hemocoel (step 3). However, the nematode vector of Photorhabdus, Heterorhabditis, can also enter into host's body by puncturing the larval cuticle (for review see [START_REF] Castillo | Insect immune responses to nematode parasites[END_REF]). Within the hemocoel, nematodes release their bacterial symbionts that undergo insect infection (step 3). In the insect cadaver, occur bacterial multiplication and nematode reproduction (step 4). During this life cycle, Photorhabdus and Xenorhabdus must successfully accomplish three distinct roles within the insect host: (i) overcome insect immune response, and kill insects, (ii) produce nutrients from the insect cadaver to facilitate development of the nematode, and (iii) colonize the IJ stage of the nematode [START_REF] Goodrich-Blair | Mutualism and pathogenesis in Xenorhabdus and Photorhabdus: two roads to the same destination[END_REF]. After rounds of nematode reproduction, progeny nematodes receive uncharacterized environmental cues that stimulate the development of a new generation of IJs colonized by the bacteria before emerging of several hundred thousand IJs from an insect cadaver.

Inside the hemocoel

Entomopathogenic bacteria entering in the hemolymph compartment have to face the insect immune system consisting of several defensive mechanisms that parallel many aspects of the vertebrate innate immune system (see Box 1).

Facing the humoral immunity

As part of the humoral response, insects produce a range of AMPs that target bacterial envelope components [START_REF] Nappi | Cytotoxicity and cytotoxic molecules in invertebrates[END_REF]. P. luminescens and B. thuringiensis counteract the humoral AMP response through constitutive modifications of their surface properties. In Salmonella, the addition of 4-aminoarabinose moieties on the negatively charged lipopolysaccharides LPS, requires the products of the two component system PhoPQ-regulated pmrHFIJKLM operon [START_REF] Gunn | Identification of PhoP-PhoQ activated genes within a duplicated region of the Salmonella typhimurium chromosome[END_REF][START_REF] Guo | Regulation of lipid A modifications by Salmonella typhimurium virulence genes phoP-phoQ[END_REF]. In P. luminescens, mutation of either phoP or the pmrK homologue, pbgE1, resulted in both an increased sensitivity to cationic AMPs and decreased virulence towards insects [START_REF] Derzelle | The PhoP-PhoQ two-component regulatory system of Photorhabdus luminescens is essential for virulence in insects[END_REF]26]. The dlt operon encodes proteins that alanylate the teichoic acids on the surface of Gram-positive bacteria, which neutralizes their negative charge and thus prevent attack by cationic AMPs. In B. cereus, and B. thuringiensis, a dlt null mutant is sensitive to molecules from the insect humoral immune system such as lysozyme, and the cationic AMP, cecropin, from the Fall Armyworm, Spodoptera frugiperda and shows reduced virulence towards lepidopteran insects [27 ]. The strategy used by X. nematophila to counteract cationic AMPs is to prevent their production: cecropin transcript levels are induced after injection of non-pathogenic Salmonella enterica, but not after injection of X. nematophila [28,29]. This suppression effect is under the control of the global regulators Lrp and CpxR [30 ], but the effectors responsible for the interference have yet to be identified. Many entomopathogenic bacteria secrete proteases that degrade antimicrobial peptides. The role of the B. thuringiensis metalloprotease InhA1 in inhibition of AMP activity and full virulence following injection into the hemocoel have been reported [START_REF] Dalhammar | Characterization of inhibitor A, a protease from Bacillus thuringiensis which degrades attacins and cecropins, two classes of antibacterial proteins in insects[END_REF]. Likewise, the Photorhabdus metalloprotease PrtA destroys commercial cecropins A and B and inhibits insect antibacterial activity when inoculated within lepidoptera [START_REF] Cabral | Purification and characterization of two distinct metalloproteases secreted by the entomopathogenic bacterium Photorhabdus sp. strain Az29[END_REF]. Therefore, both P. luminescens and B. thuringiensis/B. cereus are recognized by the insect immune system but overcome it by counteracting the action of antimicrobial peptides by multiple ways.

Facing the cellular immunity

Photorhabdus, Xenorhabdus and B. thuringiensis also interfere with insect cellular immunity. The cellular response is mediated by circulating hemocytes and includes both phagocytosis and nodulation (see Box 1). The latter response involves the aggregation of hemocytes around the invading microorganisms. Photorhabdus, Xenorhabdus and B. thuringiensis produce an array of cytolysins/hemolysins and toxins, some of which have been shown to induce necrosis or apoptosis when added to cultures of insect-immune cells (see Table 1). P. luminescens has a type III secretion system (TTSS) required for resistance to phagocytosis and encapsulation in nodules. This locus encodes an effector molecule (LopT), which is a GTPasemodifying cytotoxin. Interestingly, the expression of the lopT gene coincides with the T3SS-dependent inhibition of phagocytosis in the course of insect infection, indicating a possible role for LopT to prevent uptake of P. luminescens by hemocytes [START_REF] Brugirard-Ricaud | Site-specific antiphagocytic function of the Photorhabdus luminescens type III secretion system during insect colonization[END_REF]. By contrast, the genomes of Xenorhabdus nematophila and Xenorhabdus bovienii do not encode homologues of a dedicated TTSS [START_REF] Chaston | The entomopathogenic bacterial endosymbionts Xenorhabdus and Photorhabdus: convergent lifestyles from divergent genomes[END_REF]. Nevertheless, the X. nematophila flagellar secretion system (flagellar TTSS) is necessary for secretion of at least one non-flagellar protein, a lipase [START_REF] Park | Co-regulation of motility, exoenzyme and antibiotic production by the EnvZ-OmpR-FlhDC-FliA pathway in Xenorhabdus nematophila[END_REF][START_REF] Lanois | FliZ, a flagellar regulator, is at the crossroads between motility, haemolysin expression and virulence in the insect pathogenic bacterium Xenorhabdus[END_REF][START_REF] Richards | Xenorhabdus nematophila lrhA is necessary for motility, lipase activity, toxin expression, and virulence in Manduca sexta insects[END_REF]. In addition, the flagellar regulator, FliZ, controls the expression of the XaxAB and XhlAB hemolysins encoding-genes [START_REF] Lanois | FliZ, a flagellar regulator, is at the crossroads between motility, haemolysin expression and virulence in the insect pathogenic bacterium Xenorhabdus[END_REF].

XaxAB is the prototype of a new family of hemolysins with apoptotic and pore-forming activities in mammalian and invertebrate cells [START_REF] Vigneux | The xaxAB genes encoding a new apoptotic toxin from the insect pathogen Xenorhabdus nematophila are present in plant and human pathogens[END_REF]. The second FliZ-dependent hemolysin, XhlA, is a cell surface-associated hemolysin belonging to the two-partner secretion system family [START_REF] Cowles | Expression and activity of a Xenorhabdus nematophila haemolysin required for full virulence towards Manduca sexta insects[END_REF]. While XhlA and XaxAB recombinant proteins mediated lysis of insect immune cells [START_REF] Vigneux | The xaxAB genes encoding a new apoptotic toxin from the insect pathogen Xenorhabdus nematophila are present in plant and human pathogens[END_REF][START_REF] Cowles | Expression and activity of a Xenorhabdus nematophila haemolysin required for full virulence towards Manduca sexta insects[END_REF], only XhlA has been shown to be required for the full virulence of X. nematophila in lepidoptera. B. thuringiensis also produces an hemolysin, HlyII, which is a pore forming toxin able to induce lysis of mouse macrophages and insect hemocytes by apoptosis [START_REF] Tran | Haemolysin II is a Bacillus cereus virulence factor that induces apoptosis of macrophages[END_REF]. A protein (CwpFM) with homology to cell wall peptidases in several bacteria, notably B. subtilis, was shown to be important for full virulence in Galleria, and to induce macrophage vacuolization. However its precise role during insect infection still remains to be elucidated [START_REF] Tran | CwpFM (EntFM) is a Bacillus cereus potential cell wall peptidase implicated in adhesion, biofilm formation, and virulence[END_REF]. InhA1, is a metalloprotease localized in the Bacillus exosporium, and allows B. thuringiensis spores to escape from murine macrophages [START_REF] Ramarao | The InhA1 metalloprotease allows spores of the B. cereus group to escape macrophages[END_REF]. A similar role in the insect cells still needs investigations. Thus, these factors allow B. thuringiensis to counteract the bactericidal properties of host phagocytic cells. As for Photorhabdus and Xenorhabdus, a mutation in the flagellar apparatus of B. thuringiensis has a pleiotropic effect. Indeed, the deletion of FlhA flagellar body protein resulted in decrease in virulence following both oral and hemocoel infection, probably owing to the combined effect of reduced cell adherence and less secreted virulence factors [START_REF] Bouillaut | FlhA influences Bacillus thuringiensis PlcRregulated gene transcription, protein production, and virulence[END_REF].

Competing with the host for iron

Another part of innate host response to infection is the sequestering of iron sources to limit bacterial growth [START_REF] Cherayil | The role of iron in the immune response to bacterial infection[END_REF]. Several factors involved in iron acquisition contribute to the virulence of B. thuringiensis and Photorhabdus (see Table 1). The recently identified surface protein of B. thuringiensis, IlsA [START_REF] Fedhila | Identification of Bacillus cereus internalin and other candidate virulence genes specifically induced during oral infection in insects[END_REF], is involved in iron uptake from ferritin and heme. An IVET system and gfp transcriptional fusions showed that the ilsA gene is specifically transcribed when bacteria are within the hemocoel and the ilsA mutant is affected in its virulence towards Galleria [45 ]. The regulation of the hlyII hemolysin gene mentioned above might involve the ferric uptake repressor, Fur [START_REF] Harvie | Bacillus cereus Fur regulates iron metabolism and is required for full virulence[END_REF]. HlyII induces lysis of cells known to sequester iron during infection, and may thus participate in the release of iron. Furthermore, it was proposed that iron might constitute a signal to bacteria for the transition between the living insect infection and the cadaveric stage. Indeed, Jubelin et al. [START_REF] Jubelin | Studies of the dynamic expression of the Xenorhabdus FliAZ regulon reveal atypical iron-dependent regulation of the flagellin and haemolysin genes during insect infection[END_REF] found that expression of xhbF, a X. nematophila siderophore-related gene, was strongly upregulated in the early stages of infected larvae and downregulated in moribund larvae. The monitoring of virulence gene expression during insect infection process also revealed that xaxAB and xhlBA hemolysin genes are expressed concomitantly with the increase in iron availability detected at the time of insect death suggesting that iron constitutes a signal governing X. nematophila adaptation to shifting host environments.

In the cadaver Degradation of the host tissues

In addition to the PlcR-regulated proteins, B. thuringiensis strains produce several chitinases [START_REF] Liu | Chitinase B from Bacillus thuringiensis enhancing potential and its antagonism and insecticidal[END_REF], which might be involved in degradation of the chitin rich peritrophic matrix and parts of cuticle. The recently described quorum sensor NprR activates the transcription of a gene encoding an extracellular protease, NprA, which is the main extracellular component during sporulation (in vitro) [START_REF] Perchat | A cellcell communication system regulates protease production during sporulation in bacteria of the Bacillus cereus group[END_REF]. The massive production of this neutral protease might provide nutrients allowing the bacterial population to achieve a complete sporulation, which takes place during the late stage of the infection in the insect cadaver. Moreover, it was shown that NprR controls the expression of several genes encoding chitinases. These enzymes and a lipopeptide also regulated by NprR, contribute to the development of B. thuringiensis during the late stage of infection promoting the saprophytic lifestyle of B. thuringiensis [START_REF] Dubois | Necrotrophism is a quorum-sensing-regulated lifestyle in Bacillus thuringiensis[END_REF]. Likewise, the metalloprotease InhA1 is produced during stationary phase [START_REF] Grandvalet | Identification of genes involved in the activation of the Bacillus thuringiensis inhA metalloprotease gene at the onset of sporulation[END_REF] and may then contribute to the degradation of host tissue. Xenorhabdus and Photorhabdus also secrete several proteins such as proteases, lipases, hemolysins and chitinases that may participate in the transformation of the insect cadaver into nutrients [30 ,51]. Immuno-histochemistry revealed that on the basal side of the gut (facing the hemocoel), P. luminescens abundantly secretes two virulence factors, the gut-active toxin complex A (Tca) and the metalloprotease, PrtA, which may facilitate the rapid destruction of the gut tissue. Moreover, PrtA is detectable in the late step of septicemia when the insect is already dead [START_REF] Silva | Bacterial infection of a model insect: Photorhabdus luminescens and Manduca sexta[END_REF] and may consequently participate in the digestion of host components. By contrast, a recent study shows that an ortholog of Photorhabdus PrtA peptidase is produced by Xenorhabdus in the early stage of infection in Galleria mellonella [START_REF] Massaoud | Proteolytic enzyme production by strains of the insect pathogen xenorhabdus and characterization of an early-log-phasesecreted protease as a potential virulence factor[END_REF].

Elimination of competitors and emergence from the cadaver

The current view is that, in the insect cadaver, the numerous antimicrobial molecules (bacteriocins, secondary metabolites) produced by Xenorhabdus and Photorhabdus eliminate bacteria that might antagonize the growth of the nematode partner and compete for nutrient resources [START_REF] Bode | Entomopathogenic bacteria as a source of secondary metabolites[END_REF]. This was actually demonstrated for the phage-tail like bacteriocin of X. nematophila that is required for protecting the nematode partner, Steinernema carpocapsae, against bacterial competitors in G. mellonella [START_REF] Morales-Soto | The xnp1 P2-like tail synthesis gene cluster encodes Xenorhabdicin and is required for interspecies competition[END_REF]. Interestingly, among those antimicrobial molecules, the hydroxy-stilbene, produced by P. luminescens, and the benzylideneacetone, produced by X. nematophila, have a dual function both as inhibitors of the phenoloxidase activity and as antimicrobials against microbial competitors in the insect cadaver [START_REF] Eleftherianos | An antibiotic produced by an insect-pathogenic bacterium suppresses host defenses through phenoloxidase inhibition[END_REF][START_REF] Song | Bacterial metabolites of an entomopathogenic bacterium, Xenorhabdus nematophila, inhibit a catalytic activity of Phenoloxidase of the diamondback moth, Plutella xylostella[END_REF]. Chitinases of Xenorhabdus and Photorhabdus may also be required at the late stage of infection in the cadaver to inhibit fungi invaders [START_REF] Chen | Chitinase activity of Xenorhabdus and Photorhabdus species, bacterial associates of entomopathogenic nematodes[END_REF]. Likewise, a number of antimicrobial products including the PlcR-regulated peptides Spp may play a role both in the gut environment and in the cadaver to compete against invaders and commensal organisms. This also suggests that some PlcR-regulated factors might be functional at various steps during the B. thuringiensis lifecycle. It has been demonstrated that a NprR-dependent lipopeptide is essential to ensure the survival of B. thuringiensis in the insect cadaver [START_REF] Dubois | Necrotrophism is a quorum-sensing-regulated lifestyle in Bacillus thuringiensis[END_REF]. However, the precise role of this molecule remains to be elucidated. The NprR-regulated chitinases could also contribute to the degradation of the insect cadaver exoskeleton and to the dispersion of B. thuringiensis spores and crystals in the environment [START_REF] Raymond | Bacillus thuringiensis: an impotent pathogen?[END_REF].

Future challenges

Over the past, the field of insect-microbes interaction has made spectacular progress revealing the sophisticated dialogue between bacterial pathogens and the infected host cells. Insects provide powerful infection models to identify virulence and adaptation factors and toxins from pathogenic bacteria [12 ,59]. Here we show that three entomopathogenic bacteria share common effectors although parts of their infection strategies are different (Table 1 and Figure 1). Some factors contribute to pathogenesis at several infection sites and stages, suggesting both a local adaptation and different regulatory mechanisms of these effectors. Notably, the role of pore-forming hemolysins and metalloproteases are crucial elements, as also recently reported for another entomopathogenic bacteria, Pseudomonas entomophila [START_REF] Opota | Monalysin, a novel ss-pore-forming toxin from the Drosophila pathogen Pseudomonas entomophila, contributes to host intestinal damage and lethality[END_REF]. Today, the challenge is to study the properties of bacteria in the dynamic context of their interaction with host throughout the infection process. We believe that these insect models can be used to study various cutting edge aspects like coinfections, notably the interplay with commensal flora and other pathogens. Furthermore, insects provide an experimentally tractable organism and intra-vital imaging techniques enable the temporal and spatial visualization of events occurring within the full complexity of the living hosts. Meanwhile, to reach a real level of pathogen-insect dialogue, genetics tools and genome sequencing of the lepidopteran infection models (Spodoptera littoralis, Galleria mellonella, Manduca sexta) are needed. To increase the understanding of the infectious cycle at the tissue level, it is tempting to propose that these bacteria and the caterpillar host are further tools in 'tissue microbiology' [START_REF] Richter-Dahlfors | Tissue microbiology provides a coherent picture of infection[END_REF]. Indeed, by the association of several molecular, cellular, histo-pathological and imaging approaches, these small animals permit to visualize various tissue barriers on the same histological sample section. In addition, as several factors essential for tissue degradation and host cell death are similar to mammal pathogen effectors (pore-forming toxins and immune evasion compounds), the study of the bacteria-insect interactions is possibly a powerful approach to provide new valuable insights into factors and mechanisms involved in human pathogenesis. 28. Ji D, Kim Y: An entomopathogenic bacterium, Xenorhabdus nematophila, inhibits the expression of an antibacterial peptide, cecropin, of the beet armyworm, Spodoptera exigua.

J

Box 2

 2 Orally active insecticidal toxins and degradation of gut barriers, what else?

  Figure 1

  26. Bennett HPJ, Clarke DJ: The pbgPE operon in Photorhabdus luminescens is required for pathogenicity and symbiosis. J Bacteriol 2005, 187:77-84. 27. Abi Khattar Z, Rejasse A, Destoumieux-Garzo ´n D, Escoubas J, Sanchis V, Lereclus D, Givaudan A, Kallassy M, Nielsen-Leroux C, Gaudriault S: The dlt operon of Bacillus cereus is required for resistance to cationic antimicrobial peptides and virulence in insects. J Bacteriol 2009, 191:7063-7073. This study demonstrated that the dlt mutant of Bacillus cereus has a attenuated phenotype when injected into the hemocoel of Galleria mellonella and Spodoptera littoralis. It is also the first report of the presence of a dlt-like locus in Gram-negative bacteria including Photorhabdus luminescens.

Table 1 B

 1 . thuringiensis, Photorhabdus and Xenorhabdus effectors influencing the infection process in lepidopteran larvae

	Bacteria		Class of effectors	Mutant	Potential site(s) of effect and/or target(s) in the insect b	References
				virulence a
		Name	Short function/role		Gut	Hemocoel	Cadaver
	Regulators
	B. thuringiensis PlcR	Quorum-sensing pleiotropic regulator,	I: VIR	Activates the expression		[16,18]
			early phase of infection	O: NON-VIR	of 45 genes, intestinal
					cell cytotoxicity
		NprR	Quorum-sensing pleiotropic regulator I: VIR			Nutriment acquisition,	[48,49 ]
				O: VIR			saprophytic
							development
		Fur	Ferric uptake regulator	O: ATT		Regulates the expression of	[46]
						genes involved in iron
						uptake and virulence
	Photorhabdus PhoPQ	Two component system	I: NON-VIR		AMP resistance	[25]
		LuxS	Quorum-sensing auto-inducer	I: ATT		ROS resistance	[75]
			2 synthesis protein
		HcaR	LysR-type transcriptional regulator	I: ATT		ROS resistance	[76]
		HexA	LysR homolog A regulator	I: ATT			[77]
	Xenorhabdus	FlhD	Master regulon of the flagellar cascade I: ATT		Production of hemolysins,	Production of lipases [78]
						motility
		LrhA	LysR homolog A regulator, positive	I: ATT			Production of lipases,	[37]
			activator of flhD				Tc-like toxins
		Lrp	Leucine-responsive regulatory protein,	I: ATT		Production of hemolysins,	Production of lipases	[79]
			positive activator of lrhA and flhD			motility	and antimicrobial
							activities
		CpxRA	Signal transduction system, positive	I: ATT		Inhibition of hemolysin	Production of lipases,	[80]
			activator of lrhA			production; AMP resistance	inhibition of protease
						and nodules formation	and antimicrobial
							activity production
	Insecticidal toxins (effect of purified toxins and/or mutants)
	B. thuringiensis Cry	Cristal insecticidal toxins, pore	I: Vir	Degradation of insect		[7]
			forming toxins	O: ATT/	intestinal epithelial cells
				NON-VIR
		Vip	Vegetative Insecticidal Proteins, pore	O: ATT	Degradation of insect		[81]
			forming toxins		intestinal epithelial cells
	Photorhabdus/	Tc/Xpt (whole complex) Insecticidal protein complex (		Oral toxicity of the PP	Phagocytosis inhibition	[71,82-84]
	Xenorhabdus		proteins A, B, C)			effect of the PP on
						insect hemocytes
	Photorhabdus TcA	Tetrameric homoprotein forming a		After oral ingestion of the		Degradation of	[11 ,83]
			pore and probably involved in TcC		PP, degradation of insect		intestinal epithelial
			translocation		intestinal epithelial cells		cells; expressed
							in the gut

PirAB

Insecticidal toxin (Cry and juvenile hormone esterase domains)

Oral toxicity of the PP

[START_REF] Duchaud | The genome sequence of the entomopathogenic bacterium Photorhabdus luminescens[END_REF][START_REF] Waterfield | The Photorhabdus Pir toxins are similar to a developmentally regulated insect protein but show no juvenile hormone esterase activity[END_REF]

Pit Insecticidal toxin (Cry34 domain) Toxicity after hemocoel injection

[START_REF] Li | Expression and activity of a probable toxin from Photorhabdus luminescens[END_REF]

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Current Opinion in Microbiology 2012, 15:1-12

Table 1 (

 1 Continued ) Potential site(s) of effect and/or target(s) in the insect b References Please cite this article in press as: Nielsen-LeRoux C, et al.: How the insect pathogen bacteria Bacillus thuringiensis and Xenorhabdus/Photorhabdus occupy their hosts, Curr Opin Microbiol (2012), http://dx.doi.org/10.1016/j.mib.2012.04.006 I: by injection into the hemocoel; O: per os; VIR: full virulence; ATT: attenuated; NON-VIR: non virulent at tested doses.b AMP: anti-microbial peptides; ROS: reactive oxygen species; PP: purified protein.

	Current Opinion in Microbiology 2012, 15:1-12	Bacteria Xenorhabdus Hemolysins B. thuringiensis Hbl Name GroEL MrxA HlyII Photorhabdus/ Xenorhabdus PhlA/XhlA	Class of effectors Short function/role Molecular chaperone Soluble monomeric pilin Tripartite toxin composed of 3 proteins B, L1 and L2. Pore forming toxin. Pore forming toxin, virulence factor Hemolysin belonging to the two-partner secretion (TPS) family	Mutant virulence a O: VIR I: ATT I: VIR (Photo) I: ATT (Xeno)	Gut Oral toxicity of the PP Chitin binding activity Oral toxicity of the PP Hemolytic and cytotoxic to epithelial (mammals)	Hemocoel Pore forming-toxin activity of PP Induces apoptosis of insect hemocytes, Hemolytic activity in low-iron conditions (Photorhabdus) or in high-iron conditions	Cadaver	[72] [88] [19,89] [40,90] [10 ,39,91]
							(Xenorhabdus)
		Xenorhabdus	XaxAB	Binary cytotoxin	I: VIR		Necrotic and apoptotic	Late expression	[10 ,38]
							activities on insect	in cadaver
							hemocytes
		Effectors interfering with eukaryotic signals
		B. thuringiensis SPS	Involved with Glucose-6-phosphate	I: VIR	Specifically expressed			[104]
				uptake	O: VIR	in the gut
			IlsA	Involved in iron acquisition from	I: ATT		Specifically expressed in		[12 ,45 ]
				heme and ferritin	O: ATT		the hemocoel, iron regulated
			Yvrc	Iron dicitrate uptake	O: ATT				[92]
		Photorhabdus Cif	Cyclomodulin	I: VIR		PP triggers inhibition of		[93]
							proliferation and apoptosis
							in insect cell
			SctC	Secretin of a type 3 secretion	I: VIR		Nodule formation and		[33]
				system (TTSS)			phagocytosis by
							macrophage cells
			LopT	Effector of the type 3 secretion	I: VIR		PP triggers the release of		[33]
				system (TTSS)			RhoA and Rac from insect
							cell membrane
			Mcf	Protein with BH3 domain, which		PP triggers apoptosis in	PP triggers apoptosis in		[94,95]
				is sufficient to allow Escherichia coli		the midgut epithelium	hemocytes and inhibition
				both to persist within and kill an insect			of phagocytosis
				(makes the caterpillars floppy)
			ExbD	Component of the TonB complex	I: ATT		Virulence of the mutant		[96]
				involved in uptake of siderophores			rescued by FeCl 3 injection
	www.sciencedirect.com	YfeABCD Antimicrobial molecules Photorhabdus Hydroxy-stilbene	Divalent cation transporter Antimicrobial molecule whose production is phenylalanine ammonia-lyase (StlA) and polyketide synthetase-dependant	I: ATT I: ATT (sltA mutant)		Virulence of the mutant rescued by FeCl 3 injection Inhibition of phenoloxidase activation	Antibiotic activity against Gram negative and positive bacteria	[96] [55]

a

  Insect Physiol 2004, 50:489-496. 29. Park Y, Herbert EE, Cowles CE, Cowles KN, Menard ML, Orchard SS, Goodrich-Blair H: Clonal variation in Xenorhabdus nematophila virulence and suppression of Manduca sexta immunity. Cell Microbiol 2007, 9:645-656. 30. Richards GR, Goodrich-Blair H: Masters of conquest and pillage: Xenorhabdus nematophila global regulators control transitions from virulence to nutrient acquisition. Cell Microbiol 2009, 11:1025-1033. This review emphasizes regulatory cascades involved in coordinating transitions between various stages of the X. nematophila life cycle: infection, reproduction and transmission.

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Please cite this article in press as: Nielsen-LeRoux C, et al.: How the insect pathogen bacteria Bacillus thuringiensis and Xenorhabdus/Photorhabdus occupy their hosts, Curr Opin Microbiol (2012), http://dx.doi.org/10.1016/j.mib.2012.04.006

Please cite this article in press as: Nielsen-LeRoux C, et al.: How the insect pathogen bacteria Bacillus thuringiensis and Xenorhabdus/Photorhabdus occupy their hosts, Curr Opin Microbiol (2012), http://dx.doi.org/10.1016/j.mib.2012.04.006 Current Opinion in Microbiology 2012, 15:1-12 www.sciencedirect.com

Acknowledgements

Thanks to INRA departments MICA and SPE for financial support; and to former and actual group members as well as to the many colleagues whose work is contributing to the exciting area of science highlighted in this review.


Marianne Vidament
email: marianne.vidament@tours.inra.fr

Michèle Magistrini

Yoann Le Foll

Nicolas Levillain

Jean-Marie Yvon

Guy Duchamp

Elisabeth Blesbois

Temperatures from 4 to 15 °C are suitable for preserving the fertilizing capacity of stallion semen stored for 22 h more in INRA96 extender

Keywords: Horse, Spermatozoa, Storage, Fertility, Motility, Acrosome reaction

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

Introduction

Storage of chilled sperm is very important to optimize the management of reproduction in the equine species. However, better knowledge of this process is still needed in order to improve its efficiency in laboratory and also under field conditions. Temperature and duration of storage, extender, cooling curve, number of sperm per artificial insemination (AI) dose, 2 number of AI per cycle, interval between AI and ovulation affect the fertilizing capacity of stored equine semen.

Equine semen is usually stored for 12-48h at 4-5°C. Cooling to 4-5°C decreases the metabolic activity of spermatozoa, reduces microbial growth and consequently maintains the viability of sperm for extended periods of time [START_REF] Katila | Procedures for handling fresh stallion semen[END_REF]. Lower temperatures (0 and 2 °C) have been shown to reduce the motility of equine semen kept for 12 h in milk-based [START_REF] Moran | Determination of temperature and cooling rate which induce cold shock in stallion spermatozoa[END_REF] or 24 h in milk-free extender [3]. In contrast, motility of semen stored at constant temperatures ranging from 4°C to 25°C can be maintained for short periods in most extenders. High variability of in vitro results has been observed for longer periods of storage depending on stallions and experimental conditions [START_REF] Moran | Determination of temperature and cooling rate which induce cold shock in stallion spermatozoa[END_REF][3][START_REF] Varner | Effects of cooling rate and storage temperature on equine spermatozoal motility parameters[END_REF][START_REF] Varner | Fertilizing capacity of equine spermatozoa stored for 24 hours at 5 or 20 degrees C[END_REF][START_REF] Brinsko | Effects of transport container and ambient storage temperature on motion characteristics of equine spermatozoa[END_REF][START_REF] Aurich | Recent advances in cooled-semen technology[END_REF]. High variability in fertility was even more obvious [START_REF] Varner | Fertilizing capacity of equine spermatozoa stored for 24 hours at 5 or 20 degrees C[END_REF][START_REF] Palmer | Factors affecting stallion sperm survival and fertility[END_REF][START_REF] Batellier | Advances in cooled semen technology[END_REF].

The interactions between temperature, extender and air exposure could be important for the success of storage. Semen stored at 4°C in a milk-based extender (INRA82) exhibited higher motility under anaerobic or semi-anaerobic exposure compared to aerobic exposure and to semen stored in a modified Hanks' salts extender in any condition of air exposure [3]. In contrast, semen stored at 15°C exhibited higher motility under aerobic exposure compared to anaerobic exposure in this modified Hanks' salts extender. INRA96 extender (modified Hanks' salts solution supplemented with native phosphocaseinate) was very effective in maintaining the fertilizing capacity of equine sperm stored for 24 h at 4°C without air exposure or for 72 h at 15°C with air exposure in our standardized laboratory conditions [START_REF] Batellier | Advances in cooled semen technology[END_REF][START_REF] Batellier | Delayed insemination is successful with a new extender for storing fresh equine semen at 15 degrees C under aerobic conditions[END_REF]. In contrast, in another extender based on defined milk protein (Equipro), Price et al. [START_REF] Price | Effects of oxygen exposure and gentamicin on stallion semen stored at 5 and 15 degrees C[END_REF] observed an effect of temperature on motility and membrane integrity of equine sperm (15°C being less efficient than 5°C) and no effect of air exposure after 72 h of storage or more. Therefore new information was needed to explore under fixed laboratory conditions the combined effects of temperature and exposure to air for a long storage time.

Optimized semen storage conditions are also a challenge in the field practice of AI. However field conditions of semen storage may not be strictly compared to laboratory conditions. As an example, semen transportation between distant sites is a current practice that affects the semen storage temperature [START_REF] Heckenbichler | Quality and fertility of cooled-shipped stallion semen at the time of insemination[END_REF]. During shipment of semen, storage conditions, and especially temperature, are not remained constant which introduces a new variable. Douglas-Hamilton [START_REF] Douglas-Hamilton | A field study of the fertility of transported equine semen[END_REF] developed the reusable plastic container Equitainer II TM to be used with frozen cans, which is considered as the gold standard of storage containers. Different types of disposable container are now available for transportation of equine semen. They are relatively small and cheap with thermic performances that depend on ambient temperature and on the performance of the cooled or ice packs used [START_REF] Brinsko | Effects of transport container and ambient storage temperature on motion characteristics of equine spermatozoa[END_REF][START_REF] Heckenbichler | Quality and fertility of cooled-shipped stallion semen at the time of insemination[END_REF][START_REF] Katila | Comparison of three containers used for the transport of cooled stallion semen[END_REF][START_REF] Malmgren | Effectiveness of two systems for transporting equine semen[END_REF]. Because of heat exchange between the ice packs, the container (and its contents) and the ambient air, the initial inner temperature of storage in the container decreases, then reaches a minimum before increasing slowly when the ice packs begin to warm up. So the inner temperature in these small containers is variable. In vitro studies made with stallion semen diluted in milk-based extenders and stored for 24 h in different types of disposable containers at different ambient temperature suggest that motility is affected if the inner temperature in the container is below 2°C or above 20 °C [START_REF] Brinsko | Effects of transport container and ambient storage temperature on motion characteristics of equine spermatozoa[END_REF][START_REF] Malmgren | Effectiveness of two systems for transporting equine semen[END_REF][START_REF] Mckinnon | Effect of ambient temperature and container on temperature of extended equine semen[END_REF][START_REF] Avanzi | Efficiency of different cooling and storage systems for maintaining equine semen viability in a hot environment[END_REF][START_REF] Nunes | Efficiency of short-term storage of equine semen in a simple-design cooling system[END_REF][START_REF] Hueck | Untersuchungen zur Flussigkonservierung von Pferdesperma unter Verwendung verschiedener Kuhl-and Transportssyteme -Laborstudie. Investigation on the storage of fresh stallion semen, using different cooling and transport systems. A laboratory study[END_REF]. Consequently, Brinsko et al. [START_REF] Brinsko | Effects of transport container and ambient storage temperature on motion characteristics of equine spermatozoa[END_REF] suggested that stallion semen may be able to tolerate a wider range of cooling rates and storage temperatures than previously thought. Although these small containers are used more and more in the equine industry [START_REF] Aurich | Recent advances in cooled-semen technology[END_REF][START_REF] Heckenbichler | Quality and fertility of cooled-shipped stallion semen at the time of insemination[END_REF], there is no study presenting standardized fertility data with the use of these containers at different ambient temperatures. Thus, we hypothesized that equine semen stored in INRA96 could withstand variable temperatures between 4 and 15°C and variable conditions of air exposure, without modifying 3 3 its fertilizing capacity. We chose two conditions to test this hypothesis: 1) optimized semen treatment with strictly controlled temperature and artificial insemination procedure (laboratory conditions), 2) conditions that mimic field conditions, using disposable containers and less favorable AI conditions but that are fixed to ensure interpretable results (fixed field conditions). In the laboratory conditions experiment, we measured the effects of long semen storage duration (72 h) with two fixed conditions (5°C without air exposure versus 15°C with air exposure) on semen quality measured in vitro, and in vivo by fertility of semen inseminated shortly (~ 6 h) before ovulation (Experiment 1). In the fixed field conditions experiment, we explored the effect of variable temperatures without air exposure, and used disposable containers for a shorter storage of 22 h (after this time, inner temperature is no more controlled) and a larger interval between insemination and ovulation (~ 17 h) (Experiment 2).

Materials and Methods

General procedures

Reagents and chemicals

The extender INRA96, purchased from IMV (L'Aigle, France), is based on modified Hanks' salts, 67 mM glucose, 126 mM lactose, native phosphocaseinate (27 g/L, a milk purified fraction, composed of total micellar caseins) and is supplemented with penicillin (50 IU/mL) and gentamicin (50 µg/mL) [START_REF] Batellier | Delayed insemination is successful with a new extender for storing fresh equine semen at 15 degrees C under aerobic conditions[END_REF]. Sperm washing medium (HH) was composed of Hank's salts. For the hypoosmotic test (HOS test), a hypoosmotic medium (50 mOsm/L) was prepared with the same composition as HH but without NaCl. For induction of the acrosome reaction, ionophore A23187 (reference C7522, Sigma, Saint Quentin Fallavier, France) was diluted in dimethyl sulfoxide at 1 mg/mL. Acrosome staining was carried out using fluoresceinated Pisum Sativum agglutinin (PSA-FITC, reference L0770, Sigma) diluted in HH. All the above solutions were stored at -20°C.

Animals

Male Welsh ponies were housed individually at INRA Nouzilly. One hundred and twenty six female Welsh ponies (250-450 kg, 4-16 years old) were housed at INRA Nouzilly. Before ovulation, all mares were housed in pens of 4-5 and fed with the same concentrate and straw. After, they were kept in the same pasture and fed with grass and minerals until Day 14 postovulation. In this herd, overall per-cycle pregnancy rate per individual mare was higher than 50%. The authors of this study have read the policy of this journal relating to animal ethics and confirm that their study complies.

Semen collection, dilution and storage

Semen of all stallions was routinely collected with a closed-ended vagina before and during all the experiments. After gel filtration, sperm concentration was estimated photometrically on 2 aliquots. Semen was diluted rapidly in INRA96 maintained at 37°C, at 20 x 10 6 spermatozoa per mL. Doses for AI or for sperm evaluation consisted in 10 mL of diluted semen in 20-mL syringes. Syringes were always stored horizontally. Diluted semen was then cooled at different temperatures and under different air exposures, as fixed within each experiment. After storage, the doses were gently stirred, then either inseminated without rewarming or used for in vitro evaluation. Within each experiment, the Control group was the storage condition closer to the routinely conditions used through the world : 4-5°C without air exposure.

Insemination and evaluation of fertility

Follicular growth and ovulation of mares were monitored daily during estrus by transrectal ultrasonography. When mares presented growing follicles of 33 mm, induction of ovulation was performed with an intravenous injection of 15 mg of Crude Extract Equine Gonadotropin. This substance induces ovulation in 36 h in 80% of cycles [START_REF] Duchamp | Alternative solutions to hCG induction of ovulation in the mare[END_REF]. Within an experiment, mares were randomly assigned to stallion and to group according to the time of induction of ovulation. A maximum of 4 mares (= 4 cycles) were inseminated per ejaculate and per group.

Then, mean number of inseminated cycles per ejaculate per group within stallion was ≈ 2. Uterine body inseminations were made with 200 x 10 6 total sperm in 10 mL, once before ovulation. The time interval between induction of ovulation and AI was fixed within each experiment. All cycles during which mares ovulated too early, too late, more than once, or not at all were eliminated. Pregnancy was diagnosed ultrasonographically 14 to 16 days after ovulation. Fertility was measured according to the per-cycle pregnancy rate (PC), defined as the total number of pregnancies divided by the total number of estrus cycles. Most mares were inseminated on one cycle only. After pregnancy diagnosis, all mares received an injection of prostaglandin between the first and the second cycle. Some mares were inseminated on a second cycle to achieve the number of cycles, always with the sperm of another stallion, the group was assigned at random as for the first cycle.

Evaluation of sperm

After storage, semen of AI doses was warmed in a water-bath at 37°C for 10 minutes prior to any evaluation. During in vivo part of Experiments 1 and 2, motility parameters were routinely evaluated. For Experiment 1, these results are presented in Results section. For Experiment 2, due to management constraint, in vitro tests (motility, HOS test and acrosome evaluation) were performed just after the in vivo part, on split ejaculates of the same males.

Care was taken to check that motility parameters were equivalent during the in vivo and the in vitro parts of Experiment 2.

Motility (Experiments 1 and 2)

The sperm motility characteristics were measured by a computer-assisted motility analyzer (CASA) (HTM-IVOS, version 10.9, Hamilton Thorn Research, Beverly, MA, USA). Rapid motility was defined as the percentage of motile spermatozoa with average path velocity (VAP) higher than 40 m/sec. The parameter settings for this HTM-IVOS system were: 30 frames acquired at 60 frames per second; sorting on 16 frames/30 to define a track; minimum contrast 80; minimum cell size 4 pixels; lower VAP cut-off 20 m/s; VAP cut-off for progressive and rapid cells 40 m/s and straightness cut-off for progressive cells 80.

Hypoosmotic swelling test (HOS test) (Experiment 2)

For the HOS test, two aliquots of 1 mL semen was centrifuged at 600 g for 5 min and the pellet was resuspended in 1 mL of hypoosmotic medium (see 2.1. Reagents and chemicals) and incubated at 37°C for 15 min, then stored at 4°C. Two hundred spermatozoa per aliquot were observed under a phase-contrast microscope and were classified as non-swollen, slightly swollen or highly swollen [START_REF] Jeyendran | Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics[END_REF]. The results were expressed as percentage of swollen (including slightly and highly swollen) spermatozoa.

Acrosome evaluation (Experiment 2)

The acrosome reaction was performed according to an adapted method from Varner [START_REF] Varner | Induction of the acrosome reaction in stallion spermatozoa: effects of incubation temperature, incubation time, and ionophore concentration[END_REF]. Briefly, four aliquots of 1 mL semen were incubated at 37°C for 2 hours, with (two aliquots) or without 6 µM ionophore A23187 (two aliquots) [START_REF] Magistrini | Motility, triple stain and electron microscopic analysis of spermatozoa treated with ionophore A23187 for in vitro fertilization[END_REF]. Acrosome staining with PSA-FITC, adapted from Morales & Cross [START_REF] Morales | A new procedure for determining acrosomal status of very small numbers of human sperm[END_REF] and validated for equine sperm on filters or in suspension [START_REF] Casey | Validation of an acrosomal stain for equine sperm that differentiates between living and dead sperm[END_REF][START_REF] Meyers | Capacitation in vitro of stallion spermatozoa: comparison of progesterone-induced acrosome reactions in fertile and subfertile males[END_REF][START_REF] Guitton | Flow cytometric evaluation of acrosomal status in equine spermatozoa using FITC-Pisum sativum agglutinin[END_REF], was then performed. After washing with HH solution, the spermatozoa were fixed with 1% paraformaldehyde (final v:v) (10 min at room temperature). After further washing, spermatozoa were diluted in 40 % ethanol (final v:v) for 30 min at 4°C. After another washing, the spermatozoa were then incubated with PSA-FITC (25µg/mL). One hundred spermatozoa per tube were observed under a fluorescence microscope and were scored according to the classification derived from Cummins [START_REF] Cummins | A test of the human sperm acrosome reaction following ionophore challenge. Relationship to fertility and other seminal parameters[END_REF]: 1) complete staining of the acrosome, 2) partial or patchy staining of the apical segment of the acrosome, with complete staining of the equatorial segment, 3) staining of the equatorial segment only, 4) others (e.g. faint staining of the whole sperm, bright staining all over the head, bright staining of the postacrosome only). Pattern 1 was considered as intact acrosome. Patterns 2 and 3 were considered as acrosome reactions (AR). The difference between the percentages of AR for ionophore treatment and controls was called Diff. AR.

Preparation of semen

In July, semen of 5 pony stallions was prepared and diluted in INRA 96 as described in 2.1.3. When maintained at 5°C, AI doses were stored in 20-mL syringes without air exposure: syringes were loaded with 10 mL diluted sperm and no air. Then these syringes were inserted in a foam cylinder before being stored at 5°C horizontally (Group 5 (A-) = Control). When maintained at 15°C, AI doses were stored in 20-mL syringes under air exposure; syringes were loaded with 10 mL extended sperm and 10 mL air. These syringes without protection were put directly in the 15°C chamber and stored horizontally ( [START_REF] Batellier | Advances in cooled semen technology[END_REF] (Group 15 (A+)). In this last condition, the sample has a large contact with air, but the oxygen pressure between the surface and the bottom of diluted sample could be different. Semen was collected and stored in temperature chambers on Day 0, between 3.00 and 4.00 p.m. Ovulation was induced on Day 2 between 9.00 and 11.00 a.m. On Day 3, mares were checked for absence of ovulation between 9.00 and 11.00 a.m. and inseminated between 3 and 4.00 p.m. On Day 4, mares were checked for ovulation between 9.00 and 11.00 a.m. So the interval between AI and ovulation was assumed to be approximately 7 h. To achieve a total of 80 cycles, 66 mares were inseminated during 1 cycle, and 7 mares during 2 cycles.

In vitro measurements

During the fertility trial, AI doses of 3 ejaculates per stallion were prepared and cooled according to the two conditions, then examined for motility. The disposable containers were loaded similarly at each use: with the two filled syringes and the two ice packs (maintained at -20°C for almost 24 h before use). Then, they were closed by the cardboard cover and placed at different ambient storage temperatures. For this experiment, we chose a storage time of 22 h since at elevated ambient temperatures, the semen temperature was controlled only for approximately 24 h by the containers.

The cooling and warming curves

Four ambient temperature (7°C, 17°C, 27°C, 39°C) that are commonly observed during semen transportation in France (outside or inside cars and vans ), were chosen to obtain internal temperatures of 1) less than 0°C for a few hours for the lowest temperature, 2) ≈ 4°C (as a control), 3) ≈ 8 to 10°C and 4) ≈ 15°C (Figure I). At these 4 ambient temperatures, in a syringe with 10 mL diluted semen loaded in the disposable container, cooling down from 30 to 25°C was similar and was achieved in 10 min. At an ambient temperature of 7°C, a minimal temperature of ~ -0.5°C was obtained in 2 h 50 min in the semen; the temperature was below 0°C for ~ 2 h 30 min and was ~ 3°C after 22 h

Comment citer ce document : Vidament, M. (Auteur de correspondance), Magistrini, M., Le Foll, Y., Levillain, N., Yvon, J.-M., Duchamp At an ambient temperature of 7°C, when 2 syringes of semen were loaded in the container with 1 ice pack at -20°C and 1 ice pack at 4°C, minimal temperatures of 1.6°C and 4.9°C, and temperatures at 22 h of 3.5°C and 5.2°C were recorded in the syringe near the ice pack at -20°C and in the syringe near the ice pack at 4°C, respectively. At this same ambient temperature of 7°C, when 2 syringes of semen were loaded in the container with 2 ice packs at 4°C, minimal temperature and temperature at 22 h were similar: 6.6°C.

Fertility trial

From mid June to end of July, extended semen was loaded into containers on Day 0, at 12.00 a.m. Ovulation was induced on Day 0, at 3.00 p.m. On Day 1, mares were checked for absence of ovulation and inseminated at 10.00 a.m. On Day 2, mares were checked for ovulation before 9.00 a.m. So the interval between AI and ovulation was assumed to be approximately 17 h. To achieve a total of 96 cycles, 52 mares were inseminated during 1 cycle, and 22 mares during 2 cycles.

In vitro measurements

In August of the same year, two ejaculates per stallion were split, cooled and stored at the 4 temperatures. After 22 h of storage, motility, membrane integrity (HOS test), acrosome integrity and responsiveness to ionophore A23187 were evaluated.

Statistical analysis

Differences between fertility percentages (PC) were tested with a linear categorical data analysis, considering effects of treatment, stallion and treatment by stallion interaction (CATMOD, SAS Institute Inc., Cary, NC, USA). The procedure analyzed response functions (marginal probabilities) and the partitioning of variation into various sources, using weighedleast-square-estimation of parameters calculated on real values. If the residual and the treatment by stallion interaction were not significant, the model was considered as valid. Then the analysis determined whether there is a difference between all stallions and all treatments.

In the case of significant difference between treatments, an estimation of each treatment and its significance compared to the overall mean of the file (named intercept) were also calculated.

Comment citer ce document : Vidament, M. (Auteur de correspondance), Magistrini, M., Le Foll, Y., Levillain, N., Yvon, J.-M., Duchamp General differences between means of sperm characteristics were tested by a factorial analysis for balanced data (GLM procedure of SAS, SAS Institute Inc., Cary, NC, USA) considering the effects of sperm treatment, stallion and ejaculate within stallion. The interaction stallion by sperm treatment was used as an error term. Differences between means (stallions, sperm treatments) were further separated by a Student-Newman-Keuls procedure (in GLM) using the same error term. Statistical significance was assumed at P < 0.05.

Results

3.1. Fertility and in vitro quality of semen cooled and stored for 72 h at 5°C without air or at 15°C under air exposure, in laboratory conditions (Experiment 1)

Fertility (PC) obtained with semen stored for 72 h was high (60 % and more) and no difference was evidenced between the two storage conditions, 5°C without air or 15°C with air (Table 1) (the statistical model was valid, P= 0.7 on estimated values).

All the motility characteristics analyzed were similar between these two conditions (Table 2), except VCL and ALH which were significantly higher (P≤0.05) when semen was stored at 15°C with air. Place of tables 1 and 2 3.2. Fertility and in vitro quality of semen cooled and stored for 22 h without air in disposable containers at different ambient temperatures reproducing field conditions (Experiment 2)

Fertility obtained with semen stored for 22h without air exposure, at variable temperatures ranging from 4 to 10°C (ambient temperatures 17°C or 27°C; semen at 4 to 7°C or at 8 to 10°C) was similar while lower temperature (ambient temperature 7°C; semen at -0.5 to 3°C) significantly altered fertility (Table 3) (the statistical model was valid, P=0.002 on estimated values).

As for fertility results, motility, membrane integrity (HOS test) and acrosome integrity /responsiveness (Diff. AR) were similar for semen stored between 4 and 10°C (4 to 7°C and 8 to 10°C) (Table 4). In addition, the higher semen temperature of 12 to 15°C (ambient temperature 39°C) gave also similar results. The temperature -0.5 to 3°C confirmed its harmful effect by lower levels of acrosome integrity and responsiveness (Diff. AR). More precisely, at this temperature, spermatozoa showed more spontaneous acrosome reactions (P=0.02) and more "other" acrosomes (P=0.02) in control tubes than in the other groups. Surprisingly, curvilinear velocity (VCL) and head amplitude (ALH) were higher after storage between -0.5 to 3°C (P< 0.01) (Table 4). Place of tables 3 and 4

. Discussion

Despite the fact that some authors have demonstrated that stallion semen was better stored at 4°C than at higher temperatures after 24 h of storage or more [START_REF] Varner | Effects of cooling rate and storage temperature on equine spermatozoal motility parameters[END_REF][START_REF] Price | Effects of oxygen exposure and gentamicin on stallion semen stored at 5 and 15 degrees C[END_REF], previous results obtained in our laboratory [START_REF] Batellier | Advances in cooled semen technology[END_REF] suggested that equine semen stored in INRA96 could tolerate variable storage temperatures and degrees of air exposure. Recently, this extender has been shown to be also very efficient in maintaining fertility after freezing [START_REF] Pillet | Freezing stallion semen in INRA96®-based extender improves fertility rates in comparison with INRA82[END_REF]. However, a fertility comparison of semen stored without freezing at different temperatures in the extender INRA96 either for a long time (72 h) or in disposable containers was still lacking. We clearly show in the present study that, under laboratory conditions, semen stored for 72 h either at 5°C without air exposure or at 15°C with air exposure gave similar fertility results. These results confirmed our in vitro measurement (Table 2) with two exceptions: VCL and ALH were lower after 5°C without air exposure than at 15°C under air exposure. This difference of sperm motion could be possibly explained by differences in oxygen access for energetic metabolism. Although the respective effects of temperature and exposure to air could not be separated here, conservation of motility without air exposure is certainly dependent on glycolysis and possibly glycogen mobilization as suggested in other species [START_REF] Ballester | Evidence for a functional glycogen metabolism in mature mammalian spermatozoa[END_REF][START_REF] Krzyzosiak | Measurements of bovine sperm velocities under true anaerobic and aerobic conditions[END_REF][START_REF] Krzyzosiak | Effect of sperm number and oxygenation state of the storage media on in vitro fertility of bovine sperm stored at ambient temperature[END_REF] and in horses [3,[START_REF] Vasconcelos | Metabolic evaluation of cooled equine spermatozoa[END_REF]. All these results suggest that equine sperm can use both aerobic and anaerobic pathways to survive, depending on the conditions of storage.

One of the main results of the present study concerns the adaptation of stallion semen storage to fixed field conditions. Experiment carried out in field conditions of semen transport and storage (22 h in disposable containers), involving large variations in temperature without air exposure and less favorable conditions of AI showed similar fertility results when the storage temperature ranged from 4°C to 10°C, and similar in vitro semen quality in the range 4°C to 15°C. In a recent field study on cooled semen of 67 stallions shipped in disposable containers to one center, Heckenbichler [START_REF] Heckenbichler | Quality and fertility of cooled-shipped stallion semen at the time of insemination[END_REF] found no effect of semen temperature upon arrival (4°C to 22°C, mean 9.8°C) on subsequent fertility, but number of sperm per AI, extender, storage time and AI-Ovulation interval were not fixed. In most disposable containers, after initial cooling, semen is usually maintained more or less at the same temperature up to 24 h when the ambient temperature is near 20°C [START_REF] Brinsko | Effects of transport container and ambient storage temperature on motion characteristics of equine spermatozoa[END_REF][START_REF] Katila | Comparison of three containers used for the transport of cooled stallion semen[END_REF]. This is also the case in our study but for a wider range of temperatures (Figure I). So the results of experiments with passive containers used during short time can, at least partly, be compared to those obtained in the laboratory at constant temperature. Thus we showed that, even in experimental field conditions, the tolerance to variable temperatures of stallion sperm stored without air ranged clearly between 4°C and 10°C as tested by fertility, or possibly more widely, as suggested by the successful in vitro tests made also on semen stored at 12°C to 15°C.

However, we demonstrated that there are clear lower limits for the temperatures inside the containers. Temperatures near or below 0°C were not previously recommended [START_REF] Moran | Determination of temperature and cooling rate which induce cold shock in stallion spermatozoa[END_REF]3,[START_REF] Katila | Comparison of three containers used for the transport of cooled stallion semen[END_REF][START_REF] Hueck | Untersuchungen zur Flussigkonservierung von Pferdesperma unter Verwendung verschiedener Kuhl-and Transportssyteme -Laborstudie. Investigation on the storage of fresh stallion semen, using different cooling and transport systems. A laboratory study[END_REF][START_REF] Nishikawa | Temperature and viability of spermatozoa[END_REF]. But Katila [START_REF] Katila | Comparison of three containers used for the transport of cooled stallion semen[END_REF] and Brinsko [START_REF] Brinsko | Effects of transport container and ambient storage temperature on motion characteristics of equine spermatozoa[END_REF] demonstrated that storage below 0°C in transport containers for 4 to 8 h was not so detrimental for motility characteristics measured after 24 h of storage. However, we showed that such storage (2.5 h just below 0°C) is very deleterious because it affects fertility. Fertility, in this case, was strongly decreased for all stallions tested, even the most fertile. Another interesting point of our study was to combine fertility results with a wide range of in vitro tests. After storage at the lowest temperature, motility characteristics were not modified, except VCL and ALH that were higher. However, acrosome quality was clearly decreased in our study, thus confirming the sensitivity of stallion acrosomes to thermic stress. It is important to notice that this reduction of sperm quality may usually not be detected in field conditions where only motility evaluation by microscopy is performed upon arrival. Thus motility control after transport could give false information about the fertility potential of sperm cells. This also means that the measurement of storage temperature not only on arrival but also throughout transport would be very important for the successful use of the transport containers.

Finally, all these results can help to manage semen storage in such containers. In France, external temperatures during the breeding season can be very variable, especially in March and April (range: -5°C to 25°C). Moreover, at the same time of the year, temperature during semen transport in vans and cars may occasionally be much higher (more than 40 °C, up to 50°C). McKinnon & Walker [START_REF] Mckinnon | Effect of ambient temperature and container on temperature of extended equine semen[END_REF] showed that there is no disposable container able to maintain a correct temperature at ambient temperature of 50°C and so high temperatures should simply be avoided (vans and cars should not be parked in the sun). In our study, the manufacturer's recommendation for using the disposable container was to freeze 2 ice packs at -20°C before loading them and the sperm into the container. But when ambient temperature is below 7°C, we suggest to use 2 ice packs at 4°C to avoid too low storage temperature. Finally, as ambient temperatures during transportation are often not known, and in view of the above discussion, it is certainly better to manage the storage conditions so that the sperm is stored at temperatures higher than 2°C inside the container.

Motility and velocity of equine sperm are often impaired progressively along storage, this has been confirmed in extenders as INRA96 and equivalent ([3,11,35]. Although motility characteristics are often considered in equine to be predictive of the fertility capacity of the stallions at collection or after semen cooling [START_REF] Heckenbichler | Quality and fertility of cooled-shipped stallion semen at the time of insemination[END_REF][START_REF] Kenney | Stallion semen characteristics for predicting fertility[END_REF][START_REF] Jasko | Evaluation of stallion semen[END_REF], some authors estimated that conventional laboratory tests for semen evaluation are insufficient [START_REF] Varner | Developments in stallion semen evaluation[END_REF][START_REF] Moce | In vitro evaluation of sperm quality[END_REF][START_REF] Pillet | High fertility rates with stallion sperm cryopreserved in INRA96® based extender are not predicted by in vitro parameters[END_REF]. In the two experiments, we observed the same phenomenon for one group (Group 15°C(A+) in Experiment 1 and Group -0.5 to 3°C(A-) in Experiment 2) : VCL and ALH were increased whereas RAPID_PCT, PROG_PCT and STR were similar, suggesting strong lateral movement of the head and less pronounced forward movement. As the group with increased VCL and ALH showed similar fertility (Experiment 1) or lower fertility (Experiment 2) as the Control group, we could postulate that these motility characteristics could not be considered as the sign of a better quality after storage. Higher VCL was also observed by Brinsko [START_REF] Brinsko | Effects of transport container and ambient storage temperature on motion characteristics of equine spermatozoa[END_REF] and by Katila [START_REF] Katila | Comparison of three containers used for the transport of cooled stallion semen[END_REF] (tendency) after 24 h storage in some of their containers, when internal temperature was below or very close to 0°C. LeFrapper et al. [START_REF] Lefrapper | Comparison of various extenders for storage of cooled stallion spermatozoa for 72 hours[END_REF] also observed higher VCL values in addition to decreased PROG_PCT compared to 0 h values, during storage at temperature less than 7°C, in different extenders including INRA96. The major subpopulation of equine spermatozoa identified by high linearity and low VAP and ALH values at 0 h decreased during storage at 4°C [START_REF] Quintero-Moreno | Identification of sperm subpopulations with specific motility characteristics in stallion ejaculates[END_REF]. In conclusion, during storage, high velocities and ALH, that were not associated with high percentage of motile sperm cells (total and progressive motility) and high straightness, could be considered as doubtful criteria of quality of equine semen after storage.

All these observations could suggest the following sequence of modification of the motion characteristics of equine sperm during storage and aging : after a first phase of motion characteristics considered as an adaptation of the sperm cells to their environmental conditions, a subpopulation of spermatozoa (the less fertile?) could have their motility parameters modified from coordinated to non-coordinated movements (strong lateral and less forward movements), leading to increased ALH and VCL and unchanged RAPID_PCT and VAP. Then in a third phase, this subpopulation presents a very strong reduction of motility, leading to reduced motility characteristics. We may question whether these changes could be attributable either to "capacitation like" events during storage or to hyperactivation. Capacitation includes successive physiological events leading to membrane reorganization then to modification of its permeability to ions. When the spermatozoa reach the fertilization site, the zona pellucida of the oocyte primes the sperm to initiate the acrosome reaction [START_REF] Gadella | Capacitation and the acrosome reaction in equine sperm[END_REF] and motility hyperactivation may be observed. In Experiment 2, spontaneous acrosome reaction rate was higher at the lower temperature (-0.5 to 3°C) and was associated with the motility variations discussed above. However, movements observed after storage were quite different from those observed during hyperactivation of equine spermatozoa incubated in capacitation medium at 37°C (star-shaped movement pattern, VSL and STR decrease associated with ALH increase) [START_REF] Mcpartlin | Hyperactivation of stallion sperm is required for successful in vitro fertilization of equine oocytes[END_REF]. So whether the observed changes are related to "capacitation like" process, or to membrane or intracellular alterations, energetic or mitochondrial abnormalities has still to be explored.

11

Another interesting result is the alteration of acrosome integrity observed with the less fertile treatment in the second experiment. "Other" patterns of acrosomes were observed after in vitro storage. Whether these "other" patterns of acrosomes, observed by fluorescent microscopy, are specific to the storage conditions, or whether they represent normal degenerative forms, should now be explored. In addition, a higher level of spontaneous acrosome reactions was observed with this treatment, suggesting that the acrosome is especially sensitive to storage temperature injury. Taken together, these results suggest the need to be aware of two possible increasing defects visible after storage: 1) excessive individual sperm head movements and 2) acrosome injuries.

The fertility results obtained in our study were high in the laboratory conditions experiment (PC ~ 60% after 72 h storage) and also in the fixed field conditions experiment (PC ~ 50% after 22 h storage at 4 to 10°C). This is consistent with previous results with the same extender [START_REF] Batellier | Advances in cooled semen technology[END_REF][START_REF] Batellier | Delayed insemination is successful with a new extender for storing fresh equine semen at 15 degrees C under aerobic conditions[END_REF] and higher than recent field results observed after shipping semen in disposable containers (PC ~ 40%, storage time around 24 h, various extenders, 67 stallions) [START_REF] Heckenbichler | Quality and fertility of cooled-shipped stallion semen at the time of insemination[END_REF]. Despite the difference in storage time between our 2 experiments, we suggest that these high fertility levels could be due to 1) the AI-Ovulation interval, more favorable in the longer storage experiment (~ 7 h) and 2) the set of experimental mares. However, one has to have in mind that in "traditional" field conditions, the fertility rate could be lower because of more variable and less favourable conditions.

Conclusion

The present study shows that stallion semen may tolerate various air exposure and a wide temperature range (4°C to 15°C) during in vitro storage. Temperatures close to 0°C are detrimental, even for short periods of time (2 to 3 hours). The use of disposable containers for the transportation of stallion sperm may be encouraged for 22 h storage, provided that the temperature inside the container does not fall below 4°C.

Comment citer ce document : Vidament, M. (Auteur de correspondance), Magistrini, M., Le Foll, Y., Levillain, N., Yvon, J.-M., Duchamp

  Comment citer ce document : Vidament, M. (Auteur de correspondance), Magistrini, M., Le Foll, Y., Levillain, N., Yvon, J.-M., Duchamp, G., Blesbois, E. (2012). Temperatures from 4 to 15 °C are suitable for preserving the fertilizing capacity of stallion semen stored for 22 h ormore in INRA96 extender. Theriogenology, 78 (2), 297-307. DOI : 10.1016/j.theriogenology.2012.01.018 Version postprint Comment citer ce document : Vidament, M. (Auteur de correspondance), Magistrini, M., Le Foll, Y., Levillain, N., Yvon, J.-M., Duchamp, G., Blesbois, E. (2012). Temperatures from 4 to 15 °C are suitable for preserving the fertilizing capacity of stallion semen stored for 22 h more in INRA96 extender.

Theriogenology, 78 (2), 297-307. DOI : 10.1016/j.theriogenology.2012.01.018 10 10

Table 1 :

 1 , G., Blesbois, E. (2012). Temperatures from 4 to 15 °C are suitable for preserving the fertilizing capacity of stallion semen stored for 22 h ormore in INRA96 extender. Temperature curves in the sperm dose loaded in the disposable container placed at different ambient temperatures, after samples reached 25°C (Experiment 2) . Each bar on x axis represents 20 min. Lowest temperature was obtained in 2 h 50 min, 2 h 30 min, 2 h 20 min and 2 h 00 min, in container stored at 7, 17, 27 and 39°C, respectively. Per-cycle pregnancy rate (PC) after one insemination with semen stored for 72 h either at 5°C without air exposure (5(A-)) or at 15°C with air exposure (15(A+)) (interval IAovulation: 7 h) (Experiment 1) Temperatures (°C) of the semen in the disposable container : lowest temperature and temperature after 22 h storage. Cooling curves are presented in Figure I. b Weighted least square estimates $ : Within the row, significant difference from the estimate of the mean percentage of all treatments (intercept): 41%, P< 0.002

		40				12 to 15°C (container at ambient temperature of 39°C) 8 to 10°C (container at ambient temperature of 27°C)
		35				4 to 7°C (container at ambient temperature of 17°C)
		30				-0,5 to 3°C (container at ambient temperature of 7°C)
	Temperature (°C)	10 15 20 25
		5
		0
		-5
		00:00	01:00	02:00	03:00	04:00	05:00	06:00	07:00	08:00	09:00	10:00	11:00	12:00	13:00	14:00	15:00	16:00	17:00	18:00	19:00	20:00	21:00	22:00
											Time (hours)
												Group		Group
								Stallion			5(A-)		15(A+)
								A			3 / 8			1/ 8
								B			4 / 8			3 / 8
								C			5 / 8			7 / 8
								D			5 / 8			7 / 8
								E			7 / 8			7 / 8
								Total			24 / 40		25 / 40
							Observed PC (%)		60			63
						Estimated PC a (%)	60			63
					a Weighted least square estimates

Theriogenology, 78 (2), 297-307. DOI : 10.1016/j.theriogenology.2012.01.018 Version postprint Comment citer ce document : Vidament, M. (Auteur de correspondance), Magistrini, M., Le Foll, Y., Levillain, N., Yvon, J.-M., Duchamp, G., Blesbois, E. (2012). Temperatures from 4 to 15 °C are suitable for preserving the fertilizing capacity of stallion semen stored for 22 h more in INRA96 extender. Theriogenology, 78 (2), 297-307. DOI : 10.1016/j.theriogenology.2012.01.018 15 15 Figure I : a

Acknowledgments

The authors thank B. Bruneau, Y. Gaude, all the staff of the equine facilities in INRA Nouzilly (UEPAO), S. Francon, C. Marquet, P. Gatesoupe, X. Druart. N. Levillain and Y. Le Foll were students of the National Veterinary Schools of Nantes and Alfort, respectively. Grant support: This study was supported by Les Haras Nationaux/IFCE (French National Studs/ French Institute of Horse and Riding) and by INRA (Institut National de la Recherche Agronomique).

Comment citer ce document : Vidament, M. (Auteur de correspondance), Magistrini, M., Le Foll, Y., Levillain, N., Yvon, J.-M., Duchamp