Fumonisins alone or mixed with other fusariotoxins increase the C22-24:C16 sphingolipid ratios in chicken livers, while deoxynivalenol and zearalenone have no effect.

Poultry feed is often contaminated with fumonisins, deoxynivalenol, and zearalenone, which can result in oxidative damage, inflammation and change in lipid metabolism. Although sphingolipids play key roles in cells, only the effects of fumonisins on the sphingolipidome are well-documented. In chickens, fumonisins have been shown to increase the sphinganine to sphingosine ratio and the C22-24:C16 sphingolipid ratio, which has been proposed as a new biomarker of toxicity. In this study, we used UHPLC-MSMS targeted analysis to measure the effect of fusariotoxins on sphingolipids in the livers of chickens fed with diets containing fusariotoxins administered individually and in combination, at the maximum levels recommended by the European Commission. Chickens were exposed from hatching until they reached 35 days of age. This study revealed for the first time that fumonisins, deoxynivalenol, and zearalenone alone and in combination have numerous effects on the sphingolipidome in chicken livers. A 30-50 % decrease in ceramide, dihydroceramide, sphingomyelin, dihydrosphingomyelin, monohexosylceramide and lactosylceramide measured at the class level was observed when fusariotoxins were administered alone, whereas a 30-100 % increase in dihydroceramide, sphingomyelin, dihydrosphingomyelin, and monohexosylceramide was observed when the fusariotoxins were administered in combination. For these different variables, strong significant interactions were observed between fumonisins and zearalenone and between fumonisins and deoxynivalenol, whereas interactions between deoxynivalenol and zearalenone were less frequent and less significant. Interestingly, an increase in the C22-24:C16 ratio of ceramides, sphingomyelins, and monohexosylceramides was observed in chickens fed the diets containing fumonisins only, and this increase was close when the toxin was administered alone or in combination with deoxynivalenol and zearalenone. This effect mainly corresponded to a decrease in sphingolipids with a fatty acid chain length of 16 carbons, whereas C22-24 sphingolipids were unaffected or increased. In conclusion the C22-24:C16 ratio emerged as a specific biomarker, with variations dependent only on the presence of fumonisins.

Research Note: Intestinal avian defensin 2 and robustness of chicks.

Poultry production is an important agricultural sector for human food worldwide. Chicks after hatch often face health problems leading to economic losses that are deleterious for breeders. Avian defensin 2 (AvBD2) is a prominent host defense peptide of the intestinal mucosa of cecum and is involved in the resistance of poultry to bacterial pathogens. This peptide could thus represent an innate immunity marker of robustness of birds. To test this hypothesis by comparing fast-growing and slow-growing lines in different conditions of breeding, the chick's cecal AvBD2 content was analyzed according to animal quality and immunity indicators. Chick's cecal tissue sections labeled by immunohistochemistry with newly developed specific antibodies revealed the localization of AvBD2 in the mucosa with high individual variability, without showing differences attributable to quality indicators, but interestingly showing inverse correlation with seric IgM levels in the fast-growing line. The availability of our anti-AvBD2 antibodies to the scientific community opens perspectives to identify the cellular sources of this defensin in the cecal mucosa and to investigate the organization and function of innate immune arsenal of birds.

Presence and Depletion of Sulfadiazine, Trimethoprim, and Oxytetracycline into Feathers of Treated Broiler Chickens and Impact on Antibiotic-Resistant Bacteria.

The valorization of poultry byproducts, like feathers (processed to feather meal), in animal feed could contribute to the presence of veterinary drugs, including antibiotics. An animal study was carried out to study the fate of sulfadiazine, trimethoprim, and oxytetracycline in feathers, plasma, and droppings of broiler chickens. Cage and floor housing, different from current farm practices, were studied. Samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A longer presence of antibiotics was observed in feathers compared to plasma, with sulfadiazine being present the most. The internal presence (via blood) and the external presence (via droppings) of antibiotics in/on feathers were shown. Analysis of Escherichia coli populations, from droppings and feathers, highlighted that resistant bacteria could be transferred from droppings to feathers in floor-housed animals. The overall results suggest that feathers are a potential reservoir of antimicrobial residues and could contribute to the selection of antibiotic-resistant bacteria in the environment, animals, and humans.

Targeted Analysis of Sphingolipids in Turkeys Fed Fusariotoxins: First Evidence of Key Changes That Could Help Explain Their Relative Resistance to Fumonisin Toxicity.

The effects of fumonisins on sphingolipids in turkeys are unknown, except for the increased sphinganine to sphingosine ratio (Sa:So) used as a biomarker. Fumonisins fed at 20.2 mg/kg for 14 days were responsible for a 4.4 fold increase in the Sa:So ratio and a decrease of 33% and 36% in C14-C16 ceramides and C14-C16 sphingomyelins, respectively, whereas C18-C26 ceramides and C18-C26 sphingomyelins remained unaffected or were increased. Glucosyl- and lactosyl-ceramides paralleled the concentrations of ceramides. Fumonisins also increased dihydroceramides but had no effect on deoxysphinganine. A partial least squfares discriminant analysis revealed that all changes in sphingolipids were important in explaining the effect of fumonisins. Because deoxynivalenol and zearalenone are often found in feed, their effects on sphingolipids alone and in combination with fumonisins were investigated. Feeding 5.12 mg deoxynivalenol/kg reduced dihydroceramides in the liver. Zearalenone fed at 0.47 mg/kg had no effect on sphingolipids. When fusariotoxins were fed simultaneously, the effects on sphingolipids were similar to those observed in turkeys fed fumonisins alone. The concentration of fumonisin B1 in the liver of turkeys fed fumonisins was 0.06 µmol/kg. Changes in sphingolipid concentrations differed but were consistent with the IC50 of fumonisin B1 measured in mammals; these changes could explain the relative resistance of turkeys to fumonisins.

Identification by volatolomics of hydrocarbon, oxygenated, sulfur and aromatic markers of livestock exposure to α-hexabromocyclododecane.

Volatile organic compounds (VOC)-based metabolomics, or volatolomics, was investigated for revealing livestock exposure to chemical contamination. Three farm animals, namely laying hens, broilers, and pigs, were experimentally exposed to 5 or 50 ng α-HBCDD g-1 feed. Liver and egg yolk for hens were analysed by headspace-SPME-GC-MS to reveal candidate markers of the livestock exposure to α-HBCDD. For hens, 2-butanol was found as marker in egg. In liver, twelve VOCs were highlighted as markers, with three aromatic VOCs - styrene, o-xylene, α-methylstyrene - highlighted for the two α-HBCDD doses. For broilers, six markers were revealed, with interestingly, styrene and phenol which were also found as markers in hen liver. For pigs, ten markers were revealed and the seven tentatively identified markers were oxygenated and sulfur VOCs. The candidate markers tentatively identified were discussed in light of previous volatolomics data, in particular from a γ-HBCDD exposure of laying hens.

Methodologies to Assess the Bioactivity of an Herbal Extract on Immunity, Health, Welfare and Production Performance in the Chicken: The Case of Melissa officinalis L. Extract.

The potential of herbal extracts containing bioactive compounds to strengthen immunity could contribute to reducing antimicrobial use in poultry. This study aimed at developing a reliable and robust methodological pipeline to assess the ability of herbal extracts to strengthen chicken innate defenses, especially concerning inflammation and oxidative stress. This methodology was applied to Melissa officinalis L. (MEL) extract, recognized for its biological activities including antioxidant and anti-inflammatory properties. Different methods were used to (1). guarantee the quality of MEL extract and its capacity to stimulate the innate immune system; (2). evaluate the relevance of an ex vivo model to mimic inflammatory and oxidative stress challenges to replace LPS injection in chickens; (3). analyse the effects of feed supplemented with MEL extract on inflammation and oxidative stress induced ex vivo; (4). assess the effects of MEL extract on the redox balance, health, welfare and performance in broilers exposed to suboptimal starting conditions through a large-scale approach. The quality of MEL extract preparations, through phytochemical quantification of rosmarinic acid (RA), revealed varying concentrations of RA in the different MEL extracts. RA concentrations remained stable for at least 9 months and in feed three months after incorporating MEL extract. When incubated with chicken cell lines MEL extract showed potential metabolic activation and ability to stimulate immune functions but induced cytotoxicity at high concentrations. The original ex vivo model of inflammation developed on chicken blood cells enabled inflammation and oxidative stress biomarkers to be expressed and revealed antioxidative and anti-inflammatory properties of blood cells from chickens fed MEL extract. The experimental model of chicken suboptimal starting conditions validated beneficial effects of MEL extract on the redox balance and also evidenced improved performance during the growth phase, a tendency for fewer muscle defects but a higher severity of pododermatitis lesions without affecting other welfare indicators. This study grouped methods and tools that could be combined according to the plant extract, the needs of professionals working in poultry production systems and staff responsible for animal health, welfare and feeding.

Detection of Frozen-Thawed Duck Fatty Liver by MALDI-TOF Mass Spectrometry: A Chemometrics Study.

The marketing of poultry livers is only authorized as fresh, frozen, or deep-frozen. The higher consumer demand for these products for a short period of time may lead to the marketing of frozen-thawed poultry livers: this constitutes fraud. The aim of this study was to design a method for distinguishing frozen-thawed livers from fresh livers. For this, the spectral fingerprint of liver proteins was acquired using Matrix-Assisted Laser Dissociation Ionization-Time-Of-Flight mass spectrometry. The spectra were analyzed using the chemometrics approach. First, principal component analysis studied the expected variability of commercial conditions before and after freezing-thawing. Then, the discriminant power of spectral fingerprint of liver proteins was assessed using supervised model generation. The combined approach of mass spectrometry and chemometrics successfully described the evolution of protein profile during storage time, before and after freezing-thawing, and successfully discriminated the fresh and frozen-thawed livers. These results are promising in terms of fraud detection, providing an opportunity for implementation of a reference method for agencies to fight fraud.

Toxic Effects of Fumonisins, Deoxynivalenol and Zearalenone Alone and in Combination in Ducks Fed the Maximum EUTolerated Level.

Toxic effects among fumonisins B (FB), deoxynivalenol (DON) and zearalenone (ZEN) administered alone and combined were investigated in 84-day-old ducks during force-feeding. 75 male ducks, divided into five groups of 15 animals, received daily during the meal a capsule containing the desired among of toxin. Treated animals received dietary levels of toxins equivalent to 20 mg FB1+FB2/kg (FB), 5 mg DON/kg (DON), 0.5 mg ZEN/kg (ZEN) and 20, 5 and 0.5 mg/kg of FB, DON and ZEN (FBDONZEN), respectively. Control birds received capsules with no toxin. After 12 days, a decrease in body weight gain accompanied by an increase in the feed conversion ratio was observed in ducks exposed to FBDONZEN, whereas there was no effect on performances in ducks exposed to FB, DON and ZEN separately. No difference among groups was observed in relative organ weight, biochemistry, histopathology and several variables used to measure oxidative damage and testicular function. A sphinganine to sphingosine ratio of 0.32, 1.19 and 1.04, was measured in liver in controls and in ducks exposed to FB and FBDONZEN, respectively. Concentrations of FB1 in liver were 13.34 and 15.4 ng/g in ducks exposed to FB and FBDONZEN, respectively. Together ZEN and its metabolites were measured after enzymatic hydrolysis of the conjugated forms. Mean concentrations of α-zearalenol in liver were 0.82 and 0.54 ng/g in ducks exposed to ZEN and FBDONZEN, respectively. ß-zearalenol was 2.3-fold less abundant than α-zearalenol, whereas ZEN was only found in trace amounts. In conclusion, this study suggests that decreased performance may occur in ducks exposed to a combination of FB, DON and ZEN, but does not reveal any other interaction between mycotoxins in any of the other variables measured.

Zearalenone and Metabolites in Livers of Turkey Poults and Broiler Chickens Fed with Diets Containing Fusariotoxins.

Zearalenone (ZEN) and metabolites were measured in livers of turkeys and broilers fed a control diet free of mycotoxins, a diet that contained 0.5 mg/kg ZEN (ZEN diet), and a diet that contained 0.5, 5, and 20 mg/kg of ZEN, fumonisins, and deoxynivalenol, respectively (ZENDONFB diet). The feed was individually distributed to male Grade Maker turkeys from the 55th to the 70th day of age and to male Ross chickens from the 1st to the 35th day of age, without any signs of toxicity. Together, the free and conjugated forms of ZEN, α- and ß-zearalenols (ZOLs), zearalanone (ZAN), and α- and ß-zearalanols (ZALs) were measured by UHPLC-MS/MS with [13C18]-ZEN as an internal standard and immunoaffinity clean-up of samples. ZAN and ZALs were not detected. ZEN and ZOLs were mainly found in their conjugated forms. α-ZOL was the most abundant and was found at a mean concentration of 2.23 and 1.56 ng/g in turkeys and chickens, respectively. Consuming the ZENDONFB diet significantly increased the level of total metabolites in the livers of chickens. Furthermore, this increase was more pronounced for the free forms of α-ZOL than for the conjugated forms. An investigation of the presence of ZEN and metabolites in muscle with the methods validated for the liver failed to reveal any traces of these contaminants in this tissue. These results suggest that concomitant dietary exposure to deoxynivalenol (DON) and fumonisins (FB) may alter the metabolism and persistence of ZEN and its metabolites in the liver.

A PBPK model to study the transfer of α-hexabromocyclododecane (α-HBCDD) to tissues of fast- and slow-growing broilers.

A physiologically based pharmacokinetic (PBPK) model was developed to investigate the production-specific factors involved in the transfer of α-hexabromocyclododecane (α-HBCDD) to broiler meat. The model describes growth and lipid deposition in tissues of fast- (FG) and slow- (SG) growing broilers from hatching to slaughter and simulates the exposure through the ingestion of contaminated feed or expanded polystyrene insulation material. Growth parameters were obtained from the literature while parameters relative to uptake, distribution, and elimination of α-HBCDD were adjusted using results of a previous experiment involving broilers exposed through feed throughout the rearing period or allowed to depurate before slaughter. The model was used to compare the two main edible tissues, breast and leg meat, as well as skin, and to investigate the variability within strain. Between strains and within strain, α-HBCDD assimilation efficiency (AE) is higher when the animals are slaughtered young or heavy. However, increasing slaughter age will lower α-HBCDD concentration in tissues, due to dilution. Based on fresh weight, the concentration of α-HBCDD in breast muscles and skin tends to be lower in SG than in FG broilers (-30 to +10%), while it is 10% to 80% higher in leg muscles. Compared to breast muscles, consuming leg muscles would elicit an exposure 9 and 16 times higher in FG and SG broilers, respectively. The consumption of skin together with muscles would multiply the exposure by up to 36 times compared to breast muscle alone. In case of acute exposure, the α-HBCDD concentration in tissues increased sharply, all the more since the animals are lighter in weight, and then decreased rapidly. In FG broilers, dilution through growth contributed for up to 37%, 28% and 97% to the decontamination of breast muscles, leg muscles and skin, respectively, depending on the duration of depuration before slaughter.

Toxicity of Fumonisins, Deoxynivalenol, and Zearalenone Alone and in Combination in Turkeys Fed with the Maximum European Union-Tolerated Level.

Surveys of mycotoxins worldwide have shown that deoxynivalenol (DON), fumonisins (FB), and zearalenone (ZON) are the most abundant Fusarium mycotoxins (FUS) in European poultry feed, in both the level and the frequency of contamination. Previous studies reported that a combination of FUS at concentrations that individually are not toxic may negatively affect animals. However, although toxic thresholds and regulatory guidelines exist for FUS, none account for the risk of multiple contamination, which is the most frequent. The aim of this study was to compare DON, FB, and ZON toxicity, alone and in combination, in male turkey poults. Ground cultured toxigenic Fusarium strains were incorporated in corn-soybean-based feed in five experimental diets: control diet, containing no mycotoxins, DON diet (5 mg DON/kg), FB diet (20 mg FB1 + FB2/ kg), ZON diet (0.5 mg ZON/kg), and DONFBZON diet (5, 20, and 0.5 mg/kg of DON, FB1 + FB2, and ZON, respectively). Seventy male Grade Maker turkeys were reared in individual cages on mycotoxin-free diets from 0 to 55 days of age. On the 55th day, the turkeys were weighed and divided into five groups each comprising 14 birds. Each group was fed one of the five experimental diets for a period of 14 days. On the 70th day of age, feed was withheld for 8 hr, at which time a blood sample was collected, and then all the turkeys were killed, autopsied, and different tissues sampled. The weight of the different organs, analyses of performance, biochemistry, histopathology, oxidative damage, and testis toxicity revealed no significant effects attributable to FUS. Measurement of sphingolipids in the liver revealed an increase in the sphinganine to sphingosine ratio in turkeys fed diets containing FB, but had no apparent consequences in terms of toxicity. Finally, only slight differences were found in some variables and the results of this study showed no interactions between DON, FB, and ZON. Taken together, results thus suggest that the maximum tolerated levels established for individual contamination by DON, FB, and ZON can also be considered safe in turkeys fed with combinations of these FUS for a period of 14 days.

Toxicidad de fumonisinas, deoxinivalenol y zearalenona solos y en combinación en pavos alimentados con el nivel máximo tolerado por la Unión Europea. Investigaciones sobre micotoxinas en todo el mundo han demostrado que el deoxinivalenol (DON), las fumonisinas (FB) y la zearalenona (ZON) son las micotoxinas de Fusarium (FUS) más abundantes en la alimentación avícola europea, tanto en el nivel como en la frecuencia de la contaminación. Estudios anteriores informaron que una combinación de micotoxinas de Fusarium a concentraciones que individualmente no son tóxicas puede afectar negativamente a los animales. Sin embargo, aunque existen umbrales tóxicos y pautas regulatorias para las micotoxinas de Fusarium, ninguno tiene en cuenta el riesgo de contaminación múltiple, que es lo más frecuente. El objetivo de este estudio fue comparar la toxicidad deoxinivalenol, fumonisinas, y zearalenona, solas o en combinación en pavos machos. Cepas toxigénicas de Fusarium cultivadas en suelos fueron incorporadas en alimentos a base de maíz y soya en cinco dietas experimentales: dieta de control, que no contiene micotoxinas, dieta DON (5 mg DON/kg), dieta FB (20 mg FB1+FB2/kg), dieta ZON (0.5 mg de ZON/kg) y dieta DONFBZON (5, 20 y 0.5 mg/kg de deoxinivalenol, fumonisinas 1 y 2 y zearalenona, respectivamente). Setenta pavos machos Grado Maker fueron criados en jaulas individuales con dietas libres de micotoxinas de 0 a 55 días de edad. En el día 55, los pavos se pesaron y se distribuyeron en cinco grupos, cada uno con 14 aves. Cada grupo fue alimentado con una de las cinco dietas experimentales durante un período de 14 días. En el día 70 de edad, el alimento se retuvo durante 8 horas, momento en el que se recolectó una muestra de sangre, y luego se sacrificaron todos los pavos, se les realizó la necropsia y se tomaron muestras de diferentes tejidos. El peso de los diferentes órganos, los análisis de rendimiento, la bioquímica, la histopatología, el daño oxidativo y la toxicidad en testículos no revelaron efectos significativos atribuibles a micotoxinas de Fusarium. La medición de esfingolípidos en el hígado reveló un aumento en la relación de esfinganina con relación a la esfingosina en pavos alimentados con dietas que contenían fumonisinas, pero no tuvo consecuencias aparentes en términos de toxicidad. Finalmente, solo se encontraron ligeras diferencias en algunas variables y los resultados de este estudio no mostraron interacciones entre deoxinivalenol, fumonisinas y zearalenona. Tomados en conjunto, los resultados sugieren que los niveles máximos tolerados establecidos para la contaminación individual por deoxinivalenol, fumonisinas y zearalenona también pueden considerarse seguros en pavos alimentados con combinaciones de estas micotoxinas de Fusarium durante un período de 14 días.

Fumonisin B1, B2 and B3 in Muscle and Liver of Broiler Chickens and Turkey Poults Fed with Diets Containing Fusariotoxins at the EU Maximum Tolerable Level.

Although provisional maximum tolerable daily intake and recommended guidelines have been established for fumonisins (FB) in food, few data are available concerning levels of FB in edible animal tissues. Such data are of particular interest in avian species that can tolerate relatively high levels of fumonisins in their feed. Also, even if multiple contamination of animal feed by toxins produced by Fusarium is very frequent, little is known about the consequences of multiple contamination for FB levels in tissues. The aim of this study was to analyze the concentrations of FB in the muscle and liver of chickens and turkeys fed with FB alone and with FB combined with deoxynivalenol (DON), and with zearalenone (ZEN). Experimental diets were formulated by incorporating ground cultured toxigenic Fusarium strains in corn-soybean based feeds. Control diets were free of mycotoxins, FB diets contained 20 mg FB1+FB2/kg, and FBDONZEN diets contained 20, 5, and 0.5 mg/kg of FB1+FB2, DON, and ZEN, respectively. Animals were reared in individual cages with free access to water and feed. The feed was distributed to male Ross chickens from the 1st to the 35th day of age and to male Grade Maker turkeys from the 55th to the 70th day of age. On the last day of the study, the birds were starved for eight hours, killed, and autopsied for tissues sampling. No sign of toxicity was observed. A UHPLC-MS/MS method with isotopic dilution and immunoaffinity clean-up of samples has been developed for analysis of FB in muscle (n = 8 per diet) and liver (n = 8 per diet). Only traces of FB that were below the LOQ of 0.25 µg/kg were found in most of the samples of animals fed with the control diets. Mean concentrations of FB1, FB2, and FB3 in muscle were 17.5, 3.39, and 1.26 µg/kg, respectively, in chickens, and 5.77, 1.52, and 0.54 µg/kg in turkeys, respectively. In the liver, the respective FB1, FB2, and FB3 concentrations were 44.7, 2.61, and 0.79 µg/kg in chickens, and 41.47, 4.23, and 1.41 µg/kg, in turkeys. Cumulated level of FB1+FB2+FB3 in the highly contaminated samples were above 60 and 100 µg/kg in muscle and liver, respectively. The concentrations of FB in the tissues of animals fed the FBDONZEN diet did not greatly differ from the concentrations measured in animals fed the diet containing only FB.

Lack of Toxic Interaction Between Fusariotoxins in Broiler Chickens Fed throughout Their Life at the Highest Level Tolerated in the European Union.

Fusarium mycotoxins (FUS) occur frequently in poultry diets, and regulatory limits are laid down in several countries. However, the limits were established for exposure to a single mycotoxin, whereas multiple contamination is more realistic, and different studies have demonstrated that it is not possible to predict interactions between mycotoxins. The purpose of this study was thus to compare the toxic effect of deoxynivalenol (DON), fumonisins (FB) and zearalenone (ZON), alone and in combination on broiler chickens, at the maximum tolerated level established by the EU for poultry feed. Experimental corn-soybean diets incorporated ground cultured toxigenic Fusarium strains. One feed was formulated for chickens 0 to 10 days old and another for chickens 11 to 35 days old. The control diets were mycotoxin free, the DON diets contained 5 mg DON/kg, the FB diet contained 20 mg FB1 + FB2/kg, and the ZON diet contained 0.5mg ZON/kg. The DONFBZON diet contained 5, 20, and 0.5 mg/kg of DON, FB1 + FB2, and ZON, respectively. Diets were distributed ad libitum to 70 broilers (male Ross PM3) separated into five groups of 14 chickens each reared in individual cages from one to 35 days of age. On day 35, after a starvation period of 8 h, a blood sample was collected, and all the animals were killed and autopsied. No difference between groups that could be attributed to FUS was observed in performances, the relative weight of organs, biochemistry, histopathology, intestinal morphometry, variables of oxidative damage, and markers of testicle toxicity. A significant increase in sphinganine and in the sphinganine to sphingosine ratio was observed in broilers fed FB. Taken together, these results suggest that the regulatory guidelines established for single contamination of broiler chickens fed with DON, FB, and ZON can also be used in the case of multiple contamination with these toxins.

Effect of different levels of feed restriction and fish oil fatty acid supplementation on fat deposition by using different techniques, plasma levels and mRNA expression of several adipokines in broiler breeder hens.

BACKGROUND: Reproductive hens are subjected to a restricted diet to limit the decline in fertility associated with change in body mass. However, endocrine and tissue responses to diet restriction need to be documented. OBJECTIVE: We evaluated the effect of different levels of feed restriction, with or without fish oil supplementation, on metabolic parameters and adipokine levels in plasma and metabolic tissues of reproductive hens. METHODS: We designed an in vivo protocol involving 4 groups of hens; RNS: restricted (Rt) unsupplemented, ANS: ad libitum (Ad, receiving an amount of feed 1.7 times greater than animals on the restricted diet) unsupplemented, RS: Rt supplemented, and AS: Ad supplemented. The fish oil supplement was used at 1% of the total diet composition. RESULTS: Hens fed with the Rt diet had a significantly (P < 0.0001) lower growth than Ad hens, while the fish oil supplementation had no effect on these parameters. Furthermore, the bioelectrical impedance analysis (BIA) and the fat ultrasonographic examinations produced similar results to the other methods that required animals to be killed (carcass analysis and weight of adipose tissue). In addition, the Rt diet significantly (P < 0.05) decreased plasma levels of triglycerides, phospholipids, glucose and ADIPOQ, and fish oil supplementation decreased plasma levels of RARRES2. We also showed a positive correlation between insulin values and ADIPOQ or NAMPT or RARRES2 values, and a negative correlation of fat percentage to RARRES2 values. Moreover, the effects of the Rt diet and fish oil supplementation on the mRNA expression depended on the factors tested and the hen age. CONCLUSIONS: Rt diet and fish oil supplementation are able to modulate metabolic parameters and the expression of adipokines and their receptors in metabolic tissue.

Food restriction but not fish oil increases fertility in hens: role of RARRES2?

Overfed hens selected for their rapid growth become fatter and develop reproductive disorders. Herein, we aimed to demonstrate that food restriction leading to a weight reduction and/or a supplementation with fish oil may be effective in preventing reproductive disorders through the regulation of adipokine expression in broiler hens. This study included four groups of food restricted (Rt) or ad libitum hens (Ad, feeding at a rate 1.7 times greater than Rt hens) supplemented or unsupplemented with fish oil (1%). The Rt diet significantly increased plasma chemerin (RARRES2) levels during the laying period, delayed sexual maturity by one week and improved egg quality and fertility. These effects were associated with higher progesterone production in response to IGF1 (or LH) in cultured granulosa cells and in vivo egg yolk, as compared with Ad hens. Fish oil supplementation had similar effects to the Rt diet on progesterone (P < 0.05), but without any effect on fertility. Using RT-PCR, we found that RARRES2 levels were lower in theca cells of Rt hens and NAMPT levels were increased by the fish oil supplementation. A significant positive correlation between RARRES2 expression in granulosa cells and the weight of F1 preovulatory follicle was observed, as well as a negative correlation of plasma RARRES2 levels with hatchability. Thus, food restriction but not fish oil supplementation improved fertility, and this was associated with variations in RARRES2 plasma and ovarian expression in hens.

Comparison of common components analysis with principal components analysis and independent components analysis: Application to SPME-GC-MS volatolomic signatures.

The aim of this work is to compare a novel exploratory chemometrics method, Common Components Analysis (CCA), with Principal Components Analysis (PCA) and Independent Components Analysis (ICA). CCA consists in adapting the multi-block statistical method known as Common Components and Specific Weights Analysis (CCSWA or ComDim) by applying it to a single data matrix, with one variable per block. As an application, the three methods were applied to SPME-GC-MS volatolomic signatures of livers in an attempt to reveal volatile organic compounds (VOCs) markers of chicken exposure to different types of micropollutants. An application of CCA to the initial SPME-GC-MS data revealed a drift in the sample Scores along CC2, as a function of injection order, probably resulting from time-related evolution in the instrument. This drift was eliminated by orthogonalization of the data set with respect to CC2, and the resulting data are used as the orthogonalized data input into each of the three methods. Since the first step in CCA is to norm-scale all the variables, preliminary data scaling has no effect on the results, so that CCA was applied only to orthogonalized SPME-GC-MS data, while, PCA and ICA were applied to the "orthogonalized", "orthogonalized and Pareto-scaled", and "orthogonalized and autoscaled" data. The comparison showed that PCA results were highly dependent on the scaling of variables, contrary to ICA where the data scaling did not have a strong influence. Nevertheless, for both PCA and ICA the clearest separations of exposed groups were obtained after autoscaling of variables. The main part of this work was to compare the CCA results using the orthogonalized data with those obtained with PCA and ICA applied to orthogonalized and autoscaled variables. The clearest separations of exposed chicken groups were obtained by CCA. CCA Loadings also clearly identified the variables contributing most to the Common Components giving separations. The PCA Loadings did not highlight the most influencing variables for each separation, whereas the ICA Loadings highlighted the same variables as did CCA. This study shows the potential of CCA for the extraction of pertinent information from a data matrix, using a procedure based on an original optimisation criterion, to produce results that are complementary, and in some cases may be superior, to those of PCA and ICA.

Liver volatolomics to reveal poultry exposure to γ-hexabromocyclododecane (HBCD).

Hexabromocyclododecane (HBCD) is a critical emerging brominated flame retardant to which consumers can be exposed at high doses through a single food intake. Based on an animal experiment involving 3 groups of laying hens fed during 70 days with a control diet or γ-HBCD-contaminated diets at 0.1 or 10 µg γ-HBCD g-1 feed, this study aims to use the volatolome of biological samples for revealing markers of livestock exposure to HBCD. Liquid chromatography-tandem mass spectrometry was used to monitor the time-course of HBCD levels in bodily samples. Each liver was analyzed by solid-phase microextraction-gas chromatography-mass spectrometry for volatolome profiling. After 70 days, γ-HBCD concentrations in egg yolk, fat, liver and serum reached 54 ± 4, 85 ± 6, 31 ± 6, and 32 ± 4 ng g-1 lw, respectively, for the low exposure level and 4.6+/5.7, 7.8+/6.5, 3.9+/3.0 and 3.9+/6.1 µg g-1 lw, respectively, for the high exposure level. Isomerization of γ-HBCD into α- and ß-HBCD was observed in all tissues, at least for the high exposure level. Volatolome data allowed a significant discrimination between control and exposed animals whatever the feed contamination load, demonstrating a liver metabolic response to γ-HBCD exposure. The relevance of the twenty nine volatile exposure markers tentatively identified was discussed in light of literature data.

Hens can ingest extruded polystyrene in rearing buildings and lay eggs contaminated with hexabromocyclododecane.

The overall concentration of hexabromocyclododecane (HBCDD) in eggs is low although abnormally high concentrations exceeding 3000 ng g-1 lw have been reported. In order to test whether these contaminations may originate from the ingestion of insulating materials in rearing buildings, a group of 55 hens raised in a collective cage was provided with a 64-g piece of extruded polystyrene (XPS, 2.59% HBCDD of which 75, 15 and 10% as α-, ß- and γ-HBCDD, respectively). Hens entirely consumed the piece within 3 days, leading to a mean daily exposure of 4.7 mg HBCDD per kg body weight. Whole egg HBCDD concentration reached a maximum of 1037 ng HBCDD g-1 fresh weight (fw), recorded 2 days after the piece had disappeared, and decreased down to 86 ng g-1 fw within the 19 following days. In all these samples, HBCDD was made of 98.7 ± 0.7 and 1.3 ± 0.6% α- and ß-HBCDD, respectively, and 0.1% γ-HBCDD when quantified; it was enriched in (-)α- and (+)ß-HBCDD with enantiomeric fractions of 0.438 ± 0.009 and 0.579 ± 0.030, respectively. HBCDD was quantified in all the individual eggs collected the last day of experiment at concentrations ranging between 0.47 and 1361 ng g-1 fw, according to a lognormal distribution. The ingestion of XPS in degraded rearing buildings is thus a plausible cause of on-farm egg contamination by HBCDD which should be strictly avoided.

Micropollutants and chemical residues in organic and conventional meat.

The chemical contamination levels of both conventional and organic meats were assessed. The objective was to provide occurrence data in a context of chronic exposure. Environmental contaminants (17 polychlorinated dibenzodioxins/dibenzofurans, 18 polychlorinated biphenyls (PCBs), 3 hexabromocyclododecane (HBCD) isomers, 6 mycotoxins, 6 inorganic compounds) together with chemical residues arising from production inputs (75 antimicrobials, 10 coccidiostats and 121 pesticides) have been selected as relevant compounds. A dedicated sampling strategy, representative of the French production allowed quantification of a large sample set (n=266) including both conventional (n=139) and organic (n=127) raw meat from three animal species (bovine, porcine, poultry). While contamination levels below regulatory limits were measured in all the samples, significant differences were observed between both species and types of farming. Several environmental contaminants (Dioxins, PCBs, HBCD, Zn, Cu, Cd, Pb, As) were measured at significantly higher levels in organic samples.

Enantiomer-specific accumulation and depuration of α-hexabromocyclododecane (α-HBCDD) in chicken (Gallus gallus) as a tool to identify contamination sources.

A LC-ESI(-)-HRMS method dedicated to the analysis of 6 HBCDD enantiomers at trace levels in animal matrices was developed, using a cellulose based stationary phase with a particle size of 2.5 µm. This method was applied to a sample set derived from a kinetic study of α-HBCDD previously conducted in fast- and slow-growing chickens (Gallus gallus domesticus, n = 49, plus controls), in order to study the enantiomer specific accumulation and depuration of α-HBCDD in various tissues. Regarding abdominal adipose tissue, muscle and liver, the average enantiomeric fractions of α-HBCDD (EFα) for continuously exposed groups ranged between 0.434 and 0.467, with standard deviations below 0.014, showing a significant enrichment in (-)α enantiomer even accentuated for slow growing individuals during depuration with EFα reduced by about 0.020. Similar trends were observed for pooled plasma. Then, EFα of circulating plasma α-HBCDD appeared to closely reflect EFα in storage tissues and liver, suggesting some equilibrium. The racemic elimination of α enantiomer in excreta during the contamination phase indicated that no preferential gastrointestinal absorption took place. By contrast, preferential excretion of (-)α-HBCDD from the circulating compartment to the intestinal lumen occurred during the depuration. Finally, the method was applied to samples collected in three chicken farms, selected for total HBCDD levels in muscle in the ng/g range, as a tool to determine whether the contamination occurred ante- or post-mortem, according to the chiral signature. Ante-mortem contamination was hypothesised for 2 farms, with feed being excluded as contamination source.