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International audience; Many experimental data in several species clearly demonstrate the important genetic contribution to variations in HPA axis activity. The influence of corticosteroid hormones on adaptive processes and on production traits such as growth rate, feed efficiency, carcass composition and meat quality is a strong impetus to the search for the molecular bases of these differences for efficient genetic selection. Three main sources of genetic variability have been documented so far in farm animal species, the adrenal cortex sensitivity to ACTH regulating corticosteroid hormone production, the bioavailability of corticosteroid hormones and especially corticosteroid-binding globulin capacity, and glucocorticoid receptor function. The effect of single mutations may be dependent on the genetic background and genetic variation of cortisol levels may have different functional consequences depending on the molecular mechanisms responsible for this change. The next step will be to explore HPA axis variability from a system genetics perspective including the multiple sources of variation and their interactions. This multifactorial approach is a prerequisite to the use of the HPA axis phenotypes in the genetic selection for more productive and robust animals, with a high level of production of quality products.

Pig husbandry is known as an intensive breeding system, piglets being submitted to multiple stressful events such as early weaning, successive mixing, crowding and shipping. These stressors are thought to impair immune defences and might contribute, at least partly, to the prophylactic use of antibiotics. Robustness was recently defined as the ability of an individual to express a high-production potential in a wide variety of environmental conditions. Increasing robustness thus appears as a valuable option to improve resilience to stressors and could be obtained by selecting piglets upon their adrenocortical activity. In this study, we aimed at depicting the consequences of an acute social stress on the immune capacity of piglets genetically selected upon divergent hypothalamic-pituitary-adrenocortical (HPA) axis activity. For this purpose, we monitored neuroendocrine and immune parameters, in high- (HPA(hi)) and low- (HPAI(lo)) responders to ACTH, just before and immediately after a one-hour mixing with unfamiliar conspecifics. As expected, stressed piglets displayed higher levels of circulating cortisol and norepinephrine. Blood cell count analysis combined to flow cytometry revealed a stress-induced leukocyte mobilization in the bloodstream with a specific recruitment of CD8 alpha(+) lymphocytes. Besides, one-hour mixing decreased LPS-induced IL-8 and TNF alpha secretions in whole-blood assays (WBA) and reduced mononuclear cell phagocytosis. Altogether, our data demonstrate that acute social stress alters immune competence of piglets from both groups, and bring new insights in favour of good farming practices. While for most parameters high- and low-responders to ACTH behaved similarly, HPA(hi) piglets displayed higher number of CD4(+) CD8 alpha(-) T cells, as well as increased cytokine production in WBA (LPS-induced TNFa and PIL-induced IL-8), which could confer them increased resistance to pathogens. Finally, a principal component analysis including all parameters highlighted that overall stress effects were less pronounced on piglets with a strong HPA axis. Thus, selection upon adrenocortical axis activity seems to reduce the magnitude of response to stress and appears as a good tool to increase piglet robustness.

National audience; An unfavourable relationship between responses to stress and feed efficiency has been proposed. Parallel trials were run, including divergent lines for residual feed intake and divergent lines for plasma cortisol response 1 h after ACTH injection. At 6 weeks of age, 48 pigs from each of the four lines were injected with ACTH. Plasma cortisol was measured before injection, and 1 h and 4 h later. Growth rate, feed intake, feed efficiency, body composition and meat quality were measured during the growing-finishing period on the same pigs fed either a conventional diet (9.7 MJ net energy (NE) and 160 g crude protein/kg) or a diet with an increased proportion of dietary fibres to dilute NE and digestible amino acids (AA) by 10%. Comparison of the lines selected for feed efficiency showed that the response to ACTH was slightly stronger, and lasted longer, in the more efficient line (P < 0.05). During the feed challenge, the growth rate of the most efficient line decreased more with the diet with lower NE and AA contents than that of the less efficient line (P < 0.06). However, in terms of feed efficiency, the more efficient line remained more efficient. On the other hand, comparison of the 'response to ACTH' lines showed no difference for growth and feed intake during the growth period, but a higher carcass yield and a leaner body composition of animals with higher plasma cortisol after ACTH injection. Thus, the initial hypothesis of a negative association between feed efficiency and the HPA axis activity is not supported.; Une relation défavorable entre réponse au stress et efficacité alimentaire est souvent mise en avant. Deux lignées divergentes pour l’efficacité alimentaire, et deux lignées divergentes pour la réponse en cortisol plasmatique à une injection d’ACTH, indicateur de l’activité de l’axe corticotrope, ont été testées en parallèle. A 6 semaines d’âge, 48 porcs par lignée ont reçu une injection d’ACTH. Le cortisol plasmatique a été mesuré avant injection, 1 h et 4 h après injection. La vitesse de croissance, l’ingestion, l’efficacité alimentaire, la composition de carcasse et la qualité de la viande ont été enregistrées. La moitié des animaux a reçu un aliment croissance conventionnel (9,7 MJ d’énergie nette (EN) et 160 g de protéines brutes/kg), et l’autre moitié a été soumise à un challenge alimentaire avec un aliment contenant des fibres alimentaires pour diluer l’EN et les acides aminés digestibles dans une proportion de 10%. La réponse à l’ACTH a été légèrement plus forte, et prolongée dans le temps, dans la lignée la plus efficace (P < 0,05). La vitesse de croissance et l’ingéré de la lignée plus efficace soumise à un régime fibreux ont été plus diminués que ceux des porcs moins efficaces (P < 0,06), mais l’avantage d’efficacité alimentaire a été maintenu. D’autre part, la comparaison des lignées « réponse à l’ACTH » ne montre pas de différence pour les caractères de croissance et d’ingestion pendant la période de croissance, quel que soit l’aliment, mais montre une composition corporelle plus maigre des porcs ayant une concentration de cortisol plasmatique plus élevée. Ainsi, contrairement à l’hypothèse initiale, la sélection pour une meilleure efficacité alimentaire n’affecte pas négativement la réponse à l’ACTH, et la diminution de l’activité de l’axe corticotrope n’est pas favorablement corrélée aux performances.

Selection for feed efficiency is questioned as reducing the ability of farm animals to face stress and overcome challenges. The hypothalamic-pituitary-adrenocortical (HPA) axis activity, measured by the cortisol level in plasma after ACTH injection, has been proposed as an indicator of robustness. The objectives of this study were to evaluate 1. if a modified cortisol level in pigs alters their feed efficiency and their production performance, 2. if alternative dietary resources would affect these responses. Parallel trials including divergent lines for plasma cortisol 1 hour after ACTH injection and divergent lines for residual feed intake (RFI) were run during growth with a conventional diet and a diet with high fibres, low energy and low protein content. Selection for higher or lower plasma cortisol levels after stimulation of the adrenal gland did not impact growth and feed intake traits, but it had a significant impact on body composition and carcass yield, with improved composition in the high cortisol animals. The two lines had similar responses to an alternative diet, with decreased growth rate and feed intake, and increased feed conversion ratio. On the other hand, lines selected for divergent RFI had different responses to the alternative diet, the more efficient line having a more reduced growth rate with the diet with lower energy and AA contents. However, in terms of FCR it remained more efficient. The initial hypothesis of decreased efficiency associated to increased cortisol was not validated.

International audience; The plasma cortisol levels measured one hour after injection of ACTH reflects the hypothalamic-pituitary-adrenocortical (HPA) axis activity. The test was performed on piglets at 6 weeks of age. It was investigated as a potential selection criterion to improve pig robustness. From a base population of Large White pigs, two divergent lines were derived for 3 generations of selection, one selected for a high cortisol level (H line), one for a low cortisol level (L line). All breeders, as well as unselected animals, were also genotyped on a 70kSNP chip. At the 3rd generation of selection, the divergence between the two lines was about 5 genetic standard deviations. The high heritability of the trait was confirmed (h² = 0.64 ± 0.03). The genetic correlations with individual body weights and growth rates measured during lactation, post weaning and finishing periods were very low except for the genetic correlation between cortisol level and post-weaning growth rate (rg=-0.22 ± 0.10). The genome-wide association study revealed a quantitative trait locus influencing post-ACTH cortisol level, located on chromosome 2. It was suggested that divergent selection modified the frequency of NR3C1 alleles, favouring the hypersensitive glucocorticoid receptor in the low line.

National audience; The robustness of a piglet at weaning can be seen as its ability to express optimal growth without any health problems and regardless of weaning conditions. The aim of this study was to identify some biological markers measured around weaning that could predict growth of the piglet after weaning. Blood variables (n=62) describing immunity, stress, oxidative status and metabolism were measured at 26 and 33 days of age on piglets (n=288) from 16 commercial farms with contrasting health status (deteriorated: SAN- or good: SAN+). The robustness was approached by ADG from 26-47 days of age divided by live weight at 26 days (GMQR 26-47). Piglets were then classified according to the median of their farm in classes of low (GMQR-) or high (GMQR+) GMQR 26-47. The health status of the farm was associated with 37 of 67 variables measured (P < 0.05). Thus, piglets reared on SAN- farms showed higher activation of the immune system, mobilization of body reserves and oxidative stress after weaning than SAN+ piglets. Independent of the effect of health status, GMQR+ piglets showed lower mobilization of body reserves and a higher concentration in vitamin A, an antioxidant vitamin, after weaning compared to those of GMQR- piglets. Linear regression of GMQR explained 29% of the variance with 7 variables. Finally, this study shows that it is partly possible to predict the live weight 19 days after weaning from a reduced number of biological variables measured just before or 5 days after weaning (r2=0.72).; La robustesse du porcelet au sevrage peut être vue comme sa capacité à exprimer une croissance optimale sans manifester de problèmes de santé et ce quelles que soient les conditions de sevrage. L’objectif de cette étude était d’identifier des variables biologiques mesurées autour du sevrage permettant de prédire la croissance du porcelet après sevrage. Des variables sanguines (n=62) décrivant l’immunité, le stress, le statut oxydant et le métabolisme ont été mesurées à 26 et 33 jours d’âge sur des porcelets (n=288) issus de 16 élevages commerciaux de statuts sanitaires contrastés (dégradé : SAN- ou élevé : SAN+). La robustesse a été approchée par le GMQ entre 26 et 47 jours d’âge rapporté au poids à 26 jours (GMQR 26-47). Les porcelets ont été classés par rapport à la médiane de leur élevage dans les classes de GMQR 26-47 bas (GMQR-) ou haut (GMQR+). Le statut sanitaire de l’élevage était associé avec 37 des 67 variables sanguines et zootechniques (P < 0,05). Ainsi les porcelets des élevages SAN- montraient une plus grande activation de leur système immunitaire, une plus grande mobilisation de leurs réserves corporelles et un stress oxydant plus important après le sevrage que les porcelets SAN+. Indépendamment de l’effet du statut sanitaire, les porcelets GMQR+ montraient une moins forte mobilisation des réserves corporelles et une plus forte concentration en vitamine A, une vitamine antioxydante, après sevrage que les porcelets GMQR-. La régression linéaire du GMQR expliquait 29% de la variance avec 7 variables. En conclusion, ce travail montre qu’il est possible de prédire partiellement le poids 19 jours après sevrage à partir d’un nombre réduit de variables biologiques mesurées juste avant et 5 jours après le sevrage (r2=0,72).

Background Stress is a generic term used to describe non-specific responses of the body to all kinds of challenges. A very large variability in the response can be observed across individuals, depending on numerous conditioning factors like genetics, early influences and life history. As a result, there is a wide range of individual vulnerability and resilience to stress, also called robustness. The importance of robustness-related traits in breeding strategies is increasing progressively towards the production of animals with a high level of production under a wide range of climatic conditions and management systems, together with a lower environmental impact and a high level of animal welfare. The present study aims at describing blood transcriptomic, hormonal, and metabolic responses of pigs to a systemic challenge using lipopolysaccharide (LPS). The objective is to analyze the individual variation of the biological responses in relation to the activity of the HPA axis measured by the levels of plasma cortisol after LPS and ACTH in 120 juvenile Large White (LW) pigs. The kinetics of the response was measured with biological variables and whole blood gene expression at 4 time points. A multilevel statistical analysis was used to take into account the longitudinal aspect of the data. Results Cortisol level reaches its peak 4 h after LPS injection. The characteristic changes of white blood cell count to LPS were observed, with a decrease of total count, maximal at t=+4 h, and the mirror changes in the respective proportions of lymphocytes and granulocytes. The lymphocytes / granulocytes ratio was maximal at t=+1 h. An integrative statistical approach was used and provided a set of candidate genes for kinetic studies and ongoing complementary studies focused on the LPS-stimulated inflammatory response. Conclusions The present study demonstrates the specific biomarkers indicative of an inflammation in swine. Furthermore, these stress responses persist for prolonged periods of time and at significant expression levels, making them good candidate markers for evaluating the efficacy of anti-inflammatory drugs. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-4363-5) contains supplementary material, which is available to authorized users.

Previous studies using the inbred rat strains Lewis (LEW) and spontaneously hypertensive rats (SHR) led to the mapping of two quantitative trait loci, named Ofil1 (on chromosome 4 of the rat) and Ofil2 (on chromosome 7), for open-field inner locomotion, a behavioral index of anxiety. Studies using other strains showed that the region next to Ofil1 influences measures of not only anxiety but also ethanol consumption. In view of the high prevalence of psychiatric disorders such as anxiety and alcoholism, as well as the comorbidity between them, the present study was designed to better characterize the contribution of these two loci to complex emotional and consummatory responses. Rats deriving from an F2 intercross between the LEW and the SHR strains were selected according to their genotype at markers flanking the loci Ofil1 and Ofil2 and bred to obtain lines of rats homozygous LEW/LEW or SHR/SHR for each of the two loci, thus generating four genotypic combinations. These selected animals as well as purebred LEW and SHR rats of both sexes were submitted to a battery of tests including measures of locomotor activity, anxiety, sweet and bitter taste reinforcement and ethanol intake. Lewis rats displayed more anxiety-like behavior and less ethanol intake than SHR rats. Ofil1 (on chromosome 4) affected both the activity in the center of the open field and ethanol drinking in females only. These results suggest that Ofil1 contains either linked genes with independent influences on anxiety-related responses and ethanol drinking or a pleiotropic gene with simultaneous effects on both traits.

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Background Successful achievement of early folliculogenesis is crucial for female reproductive function. The process is finely regulated by cell-cell interactions and by the coordinated expression of genes in both the oocyte and in granulosa cells. Despite many studies, little is known about the cell-specific gene expression driving early folliculogenesis. The very small size of these follicles and the mixture of types of follicles within the developing ovary make the experimental study of isolated follicular components very difficult. The recently developed laser capture microdissection (LCM) technique coupled with microarray experiments is a promising way to address the molecular profile of pure cell populations. However, one main challenge was to preserve the RNA quality during the isolation of single cells or groups of cells and also to obtain sufficient amounts of RNA. Using a new LCM method, we describe here the separate expression profiles of oocytes and follicular cells during the first stages of sheep folliculogenesis. Results We developed a new tissue fixation protocol ensuring efficient single cell capture and RNA integrity during the microdissection procedure. Enrichment in specific cell types was controlled by qRT-PCR analysis of known genes: six oocyte-specific genes (SOHLH2, MAEL, MATER, VASA, GDF9, BMP15) and three granulosa cell-specific genes (KL, GATA4, AMH). A global gene expression profile for each follicular compartment during early developmental stages was identified here for the first time, using a bovine Affymetrix chip. Most notably, the granulosa cell dataset is unique to date. The comparison of oocyte vs. follicular cell transcriptomes revealed 1050 transcripts specific to the granulosa cell and 759 specific to the oocyte. Functional analyses allowed the characterization of the three main cellular events involved in early folliculogenesis and confirmed the relevance and potential of LCM-derived RNA. Conclusions The ovary is a complex mixture of different cell types. Distinct cell populations need therefore to be analyzed for a better understanding of their potential interactions. LCM and microarray analysis allowed us to identify novel gene expression patterns in follicular cells at different stages and in oocyte populations.

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Chantier qualité GA; We studied the effect of activation of serotonin 5-HT1A receptors with selective agonist 8-OH-DPAT (0.1, 0.5, and 1.0 mg/kg) on intraspecies aggression and freezing reaction (catalepsy) in male mice of catalepsy-resistant AKR/J and two catalepsy-prone strains CBA/Lac and congenic AKR. CBA-D13Mit76. The latter strain differs from AKR strain only by terminal chromosome 13 fragment transferred from CBA strain and containing a locus determining predisposition to catalepsy and a gene encoding 5-HT1A receptor. 8-OH-DPAT in a low dose (0.1 mg/kg) affecting primarily presynaptic receptors suppressed aggressive behavior in CBA mice, but had no effect on the time of cataleptic freezing. At the same time, this dose of the drug produced no significant effect on aggression in AKR and AKR. CBA-D13Mit76 mice, but significantly attenuated freezing in AKR. CBA-D13Mit76 mice. High doses of 8-OH-DPAT (0.5 and 1 mg/kg) which affected mainly postsynaptic receptors inhibited catalepsy in CBA and AKR. CBA-D13Mit76 mice and in a dose of 1 mg/kg it suppressed aggression in all tested mouse strains. We concluded that the genome of the recipient strain (AKR) modulated the involvement of 5-HT1A receptors into the regulation of aggression and catalepsy in mice.

Background Attention deficit hyperactivity disorder (ADHD) is a complex neuropsychiatric disorder with a substantial genetic component. The Spontaneously Hypertensive Rats (SHR), considered as a good animal model of ADHD, also show less anxiety-like behaviors than Lewis (LEW) rats. The use of these inbred rat strains led us to the mapping of two quantitative trait loci (QTL), named Ofil1 (on chromosome 4) and Ofil2 (on chromosome 7), related to locomotion in the central and aversive area of an open field. Herein, we examined whether LEW and SHR rats differ in the acoustic startle reflex, a test used to study the neurobiology of anxiety, and in the prepulse inhibition of the startle response, which is known to be impaired in ADHD patients. The effect of the two aforementioned loci on these behavioral responses was also studied. Methods For this latter purpose, rats deriving from an F2 intercross between the LEW and SHR strains were selected according to their genotype at markers flanking the QTLs and bred to obtain lines of rats homozygous LEW/LEW or SHR/SHR for each of the two loci, thus generating 4 genotypic combinations. Results The SHR rats displayed decreased startle and prepulse inhibition levels when compared to LEW rats. Ofil2 affected prepulse inhibition in female rats only. Conclusion The results suggest that the LEW and SHR strains are appropriate for studying mechanisms of sensorimotor gating and indicate that the locus Ofil2 on rat chromosome 7 contain genes controlling prepulse inhibition, a neuro-behavioral trait of ADHD.

The High-Ethanol Preferring line of rats (HEP), recently selected by R.D. Myers, is characterised by a high voluntary consumption of alcohol (3-4 g/kg/day for males and 6-8 g/kg/day for females, when a 10% ethanol solution is available as a choice vs. water) and a high sensitivity to taste reinforcement (saccharin, quinine). Our previous data obtained with HEP rats showed no evidence of development of dependence after long-term sustained alcohol intake. In this study, we subjected these rats to several long-term administration protocols suggested to favour the development of alcohol dependence, including multiple alcohol concentrations or sweetened alcohol solutions (ethanol 10% or 20%+saccharin), and deprivation periods. The results showed no increase in alcohol consumption, no shift of preference for alcohol solutions when offered as a free choice vs. a preferred saccharin solution, and a very limited alcohol-deprivation effect when alcohol is made available after a period of deprivation, the three criteria used to demonstrate the development of dependence. Regardless of the method used, HEP rats failed to show dependence after long-term, heavy ethanol consumption. Resistance to ethanol dependence may in fact be genetically influenced and the HEP rat appears as a valuable model to search for factors involved in the transition from alcohol abuse to dependence.

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