dans le gras
Contexte et objectifs : Des études précédentes ont montré que l’environnement sanitaire peut modifier l’état de santé et la croissance des porcs. Les objectifs de cette étude sont d’induire un état inflammatoire chronique modéré chez le mâle entier par l’intermédiaire de conditions sanitaires dégradées, et surtout d’étudier les répercussions de l’état inflammatoire sur le développement sexuel et, in fine, sur la concentration en androsténone dans le gras.
Matériel et méthodes : L’étude porte sur 115 porcs croisés Piétrain x (Large White x Landrace), élevés en deux répétitions. De 81,3 ±5,9 à 116,7 ±4,5 kg (pendant 4 à 6 semaines), les porcs sont placés dans deux salles aux environnements contrastés. La salle sale présente une absence de vide sanitaire, une ventilation réduite, pas de nettoyage, et un mélange des porcs expérimentaux à d’autres bandes. La salle propre a des précautions d’hygiène renforcées. Des mesures relatives à l’état inflammatoire, à l’activité testiculaire et aux performances de croissance sont réalisées à J0 (1er jour de traitement), J27 et/ou à l’abattoir (J28, J35 ou J42). Il s’agit de la formule sanguine (J0 et J27), des concentrations plasmatiques de CRP, d’haptoglobine, de testostérone et d’œstradiol (J0 et J27), du cortisol salivaire (J0, J27 et jour de l’abattage) et de la teneur du gras dorsal en androsténone (J27 et jour de l’abattage). La température rectale, le poids vif et l’épaisseur de lard dorsal sont mesurés à J0, J27 et le jour de l’abattage. De plus, à l’abattoir, les lésions pulmonaires, nasales et stomacales sont évaluées, les testicules et les glandes de cowper sont pesés. La salle n’ayant que peu d’effet sur l’état inflammatoire des porcs, ils sont regroupés en fonction de leur état inflammatoire en deux classes (Infl- et Infl+) établies à partir d’une analyse en composantes principales incluant les mesures relatives à l’état inflammatoire : nombres de granulocytes et de lymphocytes, concentrations plasmatiques en haptoglobine et CRP, concentration salivaire en cortisol, température rectale. Les effets de l’état inflammatoire sur les variables relatives à la santé (notes de lésions), à l’activité testiculaire (concentrations plasmatiques de testostérone et d’œstradiol, poids des testicules et des glandes de Cowper) et aux performances de croissance (poids vif, épaisseur de lard dorsal,
Chapitre III – Etat inflammatoire
58
gain moyen quotidien) sont analysés par anova en incluant la répétition, la classe inflammatoire et leur interaction comme effets fixes dans les modèles d’analyse.
Résultats: La salle n’a pas d’influence sur la répartition des porcs entre les deux classes d’inflammation (P > 0,05). Les porcs du groupe au statut inflammatoire le plus élevé ont davantage de granulocytes, des concentrations en haptoglobine, CRP et cortisol plus fortes, des vitesses de croissance réduites et des scores de pneumonie plus élevés, en comparaison aux porcs dont le statut inflammatoire est plus faible. Aucun effet de l’état inflammatoire n’a été mis en évidence sur les concentrations plasmatiques en testostérone et en œstradiol, sur la concentration en androsténone du gras dorsal et sur le poids des organes sexuels (P > 0,05).
Discussion et conclusion :
Le modèle salles « propre et sale » n’a pas permis d’induire de différence claire d’état inflammatoire mais la variabilité entre animaux a été suffisante pour distinguer deux classes de porcs avec des états inflammatoires plus (Infl+) ou moins (Infl-) marqués.
L’état inflammatoire plus marqué des porcs Infl+ est probablement dû à un problème de pneumonie puisqu’il associé à une note de lésion pulmonaire plus élevée. Par ailleurs nos résultats sont en accord avec le fait que l’inflammation induit généralement une réduction de la vitesse de croissance (Johnson, 1997 ; Le Floc'h et al., 2006 ; Le Floc’h et al., 2009). Contrairement aux effets délétères d’une inflammation aigüe sévère sur l’activité testiculaire (Wallgren et al., 1993), nous n’avons observé aucun effet d’un état inflammatoire chronique de type modéré sur le développement sexuel de porcs mâles entiers en fin d’engraissement. L’intensité et la durée de l’inflammation pourraient expliquer ces différences de résultats.
A retenir : Un état inflammatoire modéré n’a pas d’effet sur l’activité testiculaire des porcs mâles entiers et donc sur la concentration en androsténone du tissu adipeux.
Influence of the inflammatory status of entire male pigs on
their pubertal development and fat androstenone
S. P. Parois
†, A. Faoüen
a, N. Le Floc’h and A. Prunier
PEGASE, Agrocampus Ouest, INRA, 35590 Saint-Gilles, France
(Received 22 June 2016; Accepted 28 September 2016)
Androstenone production increases during pubertal development and plays a major role in boar taint. The objective of the present study was to evaluate the effect of a subclinical inflammation on the pubertal development of boars and hence on fat
androstenone. Contrasted hygiene conditions were applied during rearing to increase the variability of the inflammatory status. Boars from a commercial cross line were allocated at 139 ± 0.9 days of age (Day 0) and 81.3 ± 5.9 kg of live weight either to Good (n=61) or Poor (n=54) hygiene conditions until slaughter at 172.9 ± 4.8 days of age and 116.7 ± 4.5 kg live weight.
Inflammatory status, growth and pubertal development were evaluated on Day 0, Day 27 and at slaughter by analysing the blood formula, plasma inflammatory proteins; testosterone and oestradiol, salivary cortisol, rectal temperature, live weight, back fat thickness, weight of reproductive organs and clinical scores of organs (lungs, stomach, snout). Fat was collected on Day 27 by biopsy and at slaughter to measure androstenone concentration. A principal component analysis including inflammatory indicators followed by a clustering procedure was performed to identify pigs with a high (Infl+ ,n= 50) or a low (Infl− ,n=65)
inflammatory status. Infl+ pigs had more granulocytes/ml, higher concentrations of haptoglobin, C-reative protein and cortisol (P<0.05), lower growth rate and higher lung pneumonia score. However, regardless of stage, the inflammatory status had no significant effect on plasma testosterone or oestradiol, fat androstenone or sexual organ development. Present data suggest that a mild inflammatory status has no influence on pubertal development or fat concentration of androstenone in boars.
Keywords: pigs, boar taint, health, steroids, sanitary challenge
Implications
In the context of the European ban on surgical castration without anaesthesia or analgesia by 2018, raising entire male pigs is a viable solution. The main drawback of this approach is boar taint, especially androstenone, an unplea-sant smell emitted during cooking. Androstenone production increases during pubertal development. Health via the inflammatory status may affect the sexual maturity of boars. The present data suggest that a mild inflammatory status has no influence on pubertal development and hence on fat androstenone but is associated with moderate health pro-blems and lower growth rate.
Introduction
Boar taint of pork products is the major issue to be addressed before converting the production of castrate males in entire male pigs. It is mainly due to the presence of two odorant
molecules: androstenone (or 5α-androst-16-en-3-one) (Patterson, 1968) and skatole (3-methylindole) (Vold, 1970 cited in Walstra, 1974). Genetic and nutritional effects on boar taint have been well studied. Skatole is produced by bacteria in the large intestine and can be easily altered by the diet (Wesoly and Weiler, 2012). Androstenone is produced in the testes by the Leydig cells whose activity is under the control of the pituitary hormone LH itself under the control of the hypothalamic gonadotropin-releasing hormone (Zamar-atskaia and Squires, 2009). Concentration of fat androste-none is highly heritable and the percentage of tainted carcasses can vary from 5% to 50% depending on the breed (Zamaratskaia and Squires, 2009). However, other factors such as inflammation caused by diseases could also play a role. For example, cytokines produced during inflammation decrease the synthesis of steroids by acting on the hypothalamo-pituitary-testicular axis (Diemeret al., 2003). In addition, digestive disorders seem to favour the production of skatole in the intestine (Skrlepet al., 2012).
It has been shown in pigs that applying degraded environ-mental hygiene conditions at weaning (Le Floc’het al., 2006)
†E-mail: severine.parois@inra.fr
aPresent address: Biomin France Sarl, 22440 Ploufragan, France
or in early fattening around 2 months of age (Le Floc’het al., 2014b) induces a moderate inflammatory state. This health challenge is less invasive and drastic than an lipopoly-saccharide injection and is more representative of what could happen in commercial farms.
Plasma concentration of acute phase proteins (Heegaard
et al., 1998) and blood formula (Le Floc’het al., 2014b) are good indicators of the inflammatory status of pigs. Cortisol concentration can also be an indirect indicator in relation to its anti-inflammatory properties (Chrousos, 1995). Rectal temperature can also be used as it usually rises when the immune system is stimulated. Lung lesions, deviation of the nasal wall, atrophy of the nasal turbinates as well as stomach ulcers can be added as they are witnesses of previous health problems with inflammatory components.
The purpose of the present study was to test the hypothesis that a moderate inflammatory state inhibits testicular activity of growing boars and hence reduces boar taint. To achieve this aim, we applied contrasted hygiene measures to induce variability in the inflammatory status of pigs and simulta-neously measured parameters of the inflammatory and health status and of the pubertal development.
Material and methods
The experiment was conducted according to the French guidelines for animal care and use (http://ethique.ipbs.fr/ sdv/charteexpeanimale.pdf; accessed 15 January 2011). Furthermore, the complete experimental procedure was evaluated (#2015061711416632) and approved by the local ethic committee (C2EA–07).
Animals and management
A total of 115 boars were studied at the experimental farm of INRA (35590 Saint-Gilles, France). The experiment was con-ducted in two identical replicates from September to March for the first replicate (replicate 1: n=56) and from December to June for the second replicate (replicate 2:
n=59). Piglets were weaned at 29.0 ± 0.9 days (mean ± SD). During the weaning period, they were housed in a single room on a slatted floor. Each pen (2.5 × 1.8 m) contained eight to 10 pigs originating from one or two distinct litters. Pigs were moved at 69 days of age to a fattening building where they were housed in a single room on a solid concrete floor. Each pen (1 × 2 m) contained two pigs originating from the same weaning pen. At 104 days of age, the two boars from each pen were separated but all remained in the same room. At 139 days of age (referred to hereafter as Day 0) and 81.3 ± 0.6 kg of live weight, boars were allocated to two distinct rooms with contrasted hygiene conditions until slaughter. The numbers of days that will be mentioned all along the paper were counted from Day 0. Both rooms were identical to the previous one and boars were allocated individually. Allocation was balanced for live weight and litter origin. The room with poor hygiene conditions (n=24 for replicate 1 and 30 for replicate 2) was previously soiled by other pigs, and weakly ventilated. Faeces and urine were not
removed and the room was not cleaned during the experi-mental period. The room with good hygiene conditions (n=32 for replicate 1 and 29 for replicate 2) was cleaned and disinfected before entry of the experimental pigs, cleaned every day, and correctly ventilated. Staff was required to wear specifically allocated, clean clothing and footwear when entering the Good room. Moreover, experi-mental pigs in the Good hygiene conditions were only housed with pigs from the present experiment whereas non-experimental pigs from other batches were added in the Poor hygiene conditions so that the total number of pigs for the same space was doubled. These contrasting conditions of hygiene were similar to those described by Le Floc’h
et al. (2014b). They led to different levels of NH3(on Day 23: 8.1 ± 1.1 and 12.9 ± 1.4 ppm, in the Good and the Poor conditions, respectively) that exceeded the recommended threshold for the health of pigs in the Poor room (Donham
et al., 2002); and H2S (on Day 23: 0.0 ± 0.0 and 2.2 ± 0.8 ppm, in the Good and the Poor conditions, respectively).
Animals were fed with pellets of standard composition (15.3% of total nitrogenous matter, 3019 Kcal of metabo-lized energy per kilogram of pellets and 9.5 g of lysine per kilogram of pellets). They had permanent access to water. They did not receive any antibiotics throughout their life.
In order to limit variability in live weight at slaughter, boars were slaughtered in three different batches for each replicate. Slaughter occurred on days 29, 35 or 42, starting with the heaviest boars. On average, pigs were 172.9 ± 4.8 days of age and 116.7 ± 4.5 kg of live weight. Several measures (see below) were taken on Day 27 (167 days of age and 110.5 ± 6.5 kg of live weight), before the first departure to slaughterhouse.
Measures and sample collection on live animals
Pigs were weighed at birth, at weaning, on Day 0, on Day 27 and before departure to the slaughterhouse. Thereafter, average daily gain (ADG) was calculated. On Day −1 and on Day 28, back fat thickness was measured using an ultra-sound scanner (ImaGo 90-1945) on both sides of the pigs (6.5 cm from the vertebral column) at the level of the last rib. Thereafter, the mean was calculated and used for statistical analyses.
Blood was collected in tubes containing EDTA (10 ml) and heparin (10 ml) on Day 0 and Day 27 from the external jugular vein, between 0930 and 1100 h, in the home pen. The sampling procedure took <2 min to limit the impact of stress response on blood parameters. Total numbers of lymphocytes and granulocytes were counted in the EDTA blood samples with a haematology automatic cell counter calibrated for pigs (MS-9; Melet Schloesing Laboratories, Osny, France). Thereafter, the blood samples were cen-trifuged at 2500 × g for 10 min at +4°C, and the plasma was stored at –20°C until analysis. Haptoglobin concentrations were determined in the heparinized plasma samples using a colorimetric method with an assay kit based on binding of haptoglobin to haemoglobin (Tridelta Ltd, Maynooth Co., Kildare, Ireland) which was developed for an automated Parois, Faoüen, Le Floc’h and Prunier
2
http://dx.doi.org/10.1017/S1751731116002329
Pontoise, France). Plasma C-reative protein (CRP) concentrations were measured in the heparinized samples using an ELISA kit developed for pig plasma (Genway Biotech, San Diego, CA, USA). Testosterone and oestradiol concentra-tions were measured in the heparinized plasma samples using ELISA kits (ST AIA-Pack Testosterone, ST AIA-Pack hsE2; Tosoh Corporation, Tokyo, Japan) developed for an automatic analyser (AIA 1800; Tosoh Corporation). The detection limits were 6.3 ng/ml, 0.28 ng/ml, 7 pg/ml and 0.005 mg/ml, for CRP, testosterone, plasma 17β-oestradiol and haptoglobin, respec-tively. For testosterone, inter-assay CVs were 6.0% and 2.5% at 0.09 and 1.45 ng/ml, respectively, and intra-assay CVs were 5.3% and 1.8% at 0.05 and 1.50 ng/ml, respectively. For 17β-oestradiol, inter-assay CVs were 4.5% and 3.4%, and intra-assay CVs were 3.2% and 1.4% at 67 and 764 pg/ml, respectively. For CRP, inter-assay CV was 5.3% at 5.3 μg/ml, and intra-assay CV was 11.2% at 56 μg/ml. For haptoglobin, intra-assay CVs were 5.3% and 6.3% at 0.6 and 1.3 mg/ml, respectively; and inter-assay CVs were 5.7% and 4.1% at 0.6 and 6.3 mg/ml, respectively.
Before blood sampling, saliva was collected for cortisol determination with Salivette® on Day 0, Day 27 and at slaughter. Cortisol was measured using an ELISA human kit (IBL International, Hamburg, DE, USA). The detection limit was 0.03 ng/ml. Intra-assay CVs were 4.5% and 1.9% at 0.11 and 26 ng/ml; and inter-assay CVs were 4.3% and 3.6% at 0.26 and 27 ng/ml, respectively.
On Day 27, a biopsy of fat was collected from the neck to measure androstenone using the material and methodology described by Baeset al. (2013). A piece of back fat was also sampled from the carcass immediately after slaughter. For samples collected at slaughter, androstenone was measured by HPLC as previously described in Batorek et al. (2012). For biopsy samples, a micro-method based on Batoreket al. (2012) and Baeset al. (2013) was developed. Correlation between the concentrations of androstenone measured with the ‘standard’ and the micro-method was high (r=0.95). The detection limit of both methods was 0.24 µg/g. Concentration of androste-none was expressed per gram of lipid fat.
Rectal temperature was measured on Day 0, Day 27 and at slaughter. Pigs were previously habituated to this procedure.
Measures and samples at slaughter
Shortly after slaughter, the lean meat percentage (LMP) and the thickness of subcutaneous fat (G2) were measured on carcasses and testes, and Cowper glands were weighed after trimming the surrounding tissues. Lung lesions, rhinitis and stomach ulcers were scored as described by Merlotet al. (2013) with a scale ranging from 0 (no lesion) to 28 (total pulmonary consolidation) for the lungs; 0 (no lesion) to 18 (total atrophy and severe deviation) for the snout; and 0 (smooth and normal mucous membrane) to 7 (wounded ulcer) for the stomach.
Statistical analyses
Statistical analyses were performed with the R software (R Core Team, 2015). Detection limits of the assays were
mental unit was the individual animal. The rectal tempera-ture, the number of granulocytes, concentrations of CRP, haptoglobin, cortisol, testosterone and 17β-oestradiol as well as the weight of both Cowper glands and the ratio between the weight of both Cowper glands and live weight at slaughter were normalized by logarithmic transformation. The live weight at various ages, ADG, the back fat thickness, the number of lymphocytes, the weight of both testes as well as the ratio between the weight of both testes and the live weight at slaughter were normally distributed without any transformation. Androstenone concentration and scores of lesions could not be normalized.
A principal component analysis (PCA) including inflam-matory indicators (numbers of granulocytes and lympho-cytes, rectal temperature, CRP and haptoglobin in plasma, and salivary cortisol measured on Day 0 and Day 27, all previously transformed for normality) followed by a clustering procedure was performed to identify groups of pigs with different inflammatory status. First, three missing values in health parameters (two for CRP Day 27 and one for haptoglobin Day 27) were imputed with a PCA (10 first dimensions kept for the imputation) with the function imputePCA of the R package missMDA (Josse and Husson, 2016). Thereafter, the functions PCA and hierarchical clustering on principal components (HCPC) of the R package FactoMineR (Le et al., 2008) were successively used. For HCPC, five dimensions were kept (eigenvalue >1) and two clusters were selected after considering the overall shape of the tree and the bar plot of the gain in inertia (Le et al., 2008). Clusters were well linked to the first dimension of the PCA (0.65) and were mainly determined by the plasma concentration of haptoglobin and the numbers of
Figure 1 Results of the principal component analysis (n = 115) representing relationships between the inflammatory indicators (numbers of granulocytes and lymphocytes, rectal temperature, C-reative protein (CRP) and haptoglobin in plasma, and salivary cortisol measured on Day 0 and Day 27, previously log-transformed for normality if needed). The biplot of the first two principal components jointly represent 45.29% of the total variation. lCRP = log(concentration of CRP in plasma + 1); lHaptoglobin = log(concentration of haptoglobin in plasma + 1); lCortisol = log(concentration of free cortisol in saliva); lT = log(rectal temperature); lGR = log(number of granulocytes in fresh blood); LYM = number of lymphocytes in fresh blood; Day 0 = first day of the treatment of hygiene conditions; Day 27 = 27 days after the beginning of the treatment.
lymphocytes and granulocytes on Day 27 (Figures 1 and 2). Pigs from Cluster 1 will be (hereafter) considered as having a low inflammatory status (Infl−) and those from Cluster 2 as having a moderate inflammatory status (Infl+).
To determine the effect of the hygiene conditions and replicate on the health and inflammatory status of pigs, Fisher’s pairwise comparisons were performed on the num-bers of animal per category. Comparison between clusters, for normal/normalized variables measured only once, was performed with the function lm of the R software. A covariate analysis with live weight on Day 0 was also performed for live weight and age at slaughter, ADG, back fat thickness, LMP and G2. Comparison between clusters, for normal/ normalized variables with repeated measures, was per-formed with the function lmer from the R package lme4 (Bates et al., 2015), with the animal being included as a random effect. The time (Day 0, Day 27, Day at slaughter) and the interaction between cluster and time were tested and removed whenP>0.05. The lsmeans function from the R package lsmeans (Lenth, 2016) was used to perform