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See detailQuantification of four Isoflavones in Forages with UPLC®-MS/MS, using the Box-Behnken Experimental Design to Optimize Sample Preparation
Daems, Frédéric ULg; Romnee, Jean-Michel; Rasse, Catherine et al

in Chromatographia (in press)

A performant method for the simultaneous quantification of daidzein, genistein, formononetin and biochanin A in forages using an UPLC®-MS/MS was developed and fully validated. The ultrasound-assisted ... [more ▼]

A performant method for the simultaneous quantification of daidzein, genistein, formononetin and biochanin A in forages using an UPLC®-MS/MS was developed and fully validated. The ultrasound-assisted extraction and enzymatic hydrolysis used in the sample preparation step were optimized using the Box-Behnken experimental design. The optimal extraction conditions used for a representative mix of forage plants were 80°C, 10 min and 55% methanol, and for hydrolysis they were 20°C, 18 h and pH=6. The chromatographic separation was achieved using an Acquity® UPLC® HSS T3 column, with a water/methanol linear gradient containing 0.01% of formic acid at a 0.55 mL min-1 flow rate. The four isoflavones were detected by ESI mass spectrometry in positive ion MRM mode. The method allows high throughput analyses of samples and showed an adequate linear regression model for all isoflavones over a range from 5 to 125 ng mL-1. There was good intra- and inter-day precision (≤ 8.2% and ≤ 7.6%) and accuracy (≤ 11.4% and ≤ 7.1%). The recovery rates were in an acceptable range of 70-120%, except for biochanin A, where the rate was about 50%. Good method repeatability was also observed, and there was no matrix effect or carry-over problem. The sample extracts were stable for at least 6 days of storage at -21°C and 6°C. The method proved to be sensitive, precise and accurate for discriminating a wide variety of forages likely to be grazed by ruminants according to their isoflavone content and to observe the impact storage process on isoflavone content in forages. [less ▲]

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See detailAnalytical methods used to quantify isoflavones in cow's milk: a review
Daems, Frédéric ULg; Romnee, Jean-Michel; Heuskin, Stéphanie ULg et al

in Dairy Science and Technology (2016)

This paper provides an update and comprehensive review of the analytical methods used for quantifying isoflavones and their metabolites in cow’s milk. Isoflavones are secondary plant metabolites that are ... [more ▼]

This paper provides an update and comprehensive review of the analytical methods used for quantifying isoflavones and their metabolites in cow’s milk. Isoflavones are secondary plant metabolites that are similar to 17 β-estradiol in chemical structure. They form one of the most common categories of phytoestrogens. Numerous health benefits have been attributed to isoflavones, but many of these compounds are also considered to be endocrine disruptors, with adverse effects on health. These contradictory trends offer an attractive prospect for future research, and therefore, sensitive and reliable analytical methods are required to clarify various issues about isoflavones. For this review, a structured methodology was used to select 26 relevant articles published between 2005 and 2015 from the Scopus and CAB Abstract databases. The review discusses individual steps of the analytical procedures described in these articles, including sample preparation, instrumental analysis and validation. The most commonly used analytical procedure is sample preparation involving liquid-liquid extraction and an enzymatic hydrolysis step followed by liquid chromatography with mass spectrometry analysis. Currently, however, there is no standardized procedure for the sample preparation and analysis of isoflavones in milk. [less ▲]

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See detailChanges in the isoflavone concentration in red clover (Trifolium pratense L.) during ensiling and storage in laboratory-scale silos
Daems, Frédéric ULg; Decruyenaere, Virginie ULg; Agneessens, Richard et al

in Animal Feed Science & Technology (2016)

Isoflavones constitute one of the most common categories of nonsteroidal estrogen-like substances belonging to the broad group of phytoestrogens. The highest concentrations in the plant kingdom are found ... [more ▼]

Isoflavones constitute one of the most common categories of nonsteroidal estrogen-like substances belonging to the broad group of phytoestrogens. The highest concentrations in the plant kingdom are found in the Fabaceae family. They have become a focus of research because of their estrogenic or anti-estrogenic effect and potential impact on human health. In recent years, several studies have focused on the impact of biotic and abiotic factors and farming management on the isoflavone concentration in plants and their impact on the composition of cow’s milk. Nevertheless, knowledge about the effect of the ensiling process on isoflavone concentration remains limited. The objective of this work was to study the evolution of the concentrations of four compounds (daidzein, formononetin, genistein and biochanin A) in red clover (Trifolium pratense L.) ensiled at harvest. The impact of the field-drying process was also assessed. The plant material was ensiled in laboratory scale silos using vacuum-packed plastic bags and stored over a 6-month period. The quality of the silages was checked throughout the experiment by determining the pH, lactic acid concentration, volatile fatty acids, crude proteins, cellulose and other chemical characteristics. The isoflavone concentration in fresh plant material was 2050, 1766, 306 and 127 µg/g DM for formononetin, biochanin A, genistein and daidzein, respectively. After 4 days of drying in the field, no significant change in isoflavone concentration was found, except for daidzein, which increased twofold. The laboratory-scale silos experiment, however, showed a decrease in isoflavone concentration during the first 2 weeks, followed by stabilization over the 5 remaining months. The concentrations fell by 26, 39, 66 and 73% for daidzein, genistein, biochanin A and formononetin, respectively. Animals fed with silage would therefore absorb fewer isoflavones than those fed with fresh plant material or hay. [less ▲]

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See detailValidation d'une méthode d'analyse quantitative de l'équol par UPLC®-MS/MS dans le lait de vache - Application aux laits commercialisés en Wallonie (Belgique)
Daems, Frédéric ULg; Lognay, Georges ULg; Romne, Jean-Michel et al

Conference (2015, May 27)

Dans le cadre d’un projet intitulé «PhytoHealth », une méthode d’analyse quantitative de l’équol dans le lait a été développée et validée selon les recommandations de l’EMA/CVMP/VICH/463202/2009. Celle-ci ... [more ▼]

Dans le cadre d’un projet intitulé «PhytoHealth », une méthode d’analyse quantitative de l’équol dans le lait a été développée et validée selon les recommandations de l’EMA/CVMP/VICH/463202/2009. Celle-ci est constituée d’une étape d’hydrolyse enzymatique, suivie d’une double extraction liquide/liquide et d’une analyse par chromatographie liquide ultra performante couplé à un spectromètre de masse en tandem (UPLC®-MS/MS). Une fois les différents paramètres tels que la fiabilité, sensibilité et/ou robustesse de la méthode confirmées, celle-ci a permis, pour la première fois, d’estimer la teneur en équol d’ échantillons de laits commercialisés en Wallonie. Une Cinquantaine de laits ont été prélevés aléatoirement dans les commerces durant une période définie. Tous les échantillons possédaient une teneur en équol ≥ 10ng.mL-1. En comparant les valeurs moyennes, on a constaté qu’il n’y avait pas de différence entre les 4 catégories de laits analysés (entier, demi-écrémé, écrémé et cru). Par contre, une différence hautement significative a pu être observée entre les laits provenant de la filière biologique et ceux de la filière conventionnelle. Dans le premier cas, les teneurs varient entre 10 et 50 ng.mL-1 par contre, dans le second, elles varient entre 70 et 130 ng.mL-1. Cet impact de la filière biologique sur la teneur en équol dans les laits a pu être confronté à d’autres études européennes et confirmé. [less ▲]

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See detailApplication du plan d’expérience de Box-Behnken et de la méthodologie des surfaces de réponses pour optimiser une méthode de quantification de phyto-œstrogènes dans les fourrages
Daems, Frédéric ULg; Lognay, Georges ULg; Romnee, Jean-Michel et al

Conference (2015, May 27)

Dans le cadre d’un projet de valorisation des prairies, intitulé « PhytoHealth », une méthode d’analyse quantitative constituée d’une étape d’extraction à l’aide d’un bain à ultrasons, suivi d’une ... [more ▼]

Dans le cadre d’un projet de valorisation des prairies, intitulé « PhytoHealth », une méthode d’analyse quantitative constituée d’une étape d’extraction à l’aide d’un bain à ultrasons, suivi d’une hydrolyse enzymatique et d’une analyse par UPLC®-MS/MS a été développée pour assurer le dosage de phyto-œstrogènes dans les fourrages. Pour alléger l’optimisation de la préparation des échantillons, la méthodologie des surfaces de réponses a été utilisée. Un plan d’expérience de Box-Behnken a été appliqué aux étapes d’extraction et d’hydrolyse. Parmi les différents facteurs pris en compte, les impacts de la nature du solvant utilisé lors de l’extraction ainsi que la température et le pH lors de l’hydrolyse, ont clairement pu être mis en évidence. L’ensemble des données générées a également permis de définir des conditions optimales d’extraction et d’hydrolyse pour chacun des composés. Ces conditions n’étant pas identiques pour chacun d’entre eux, un compromis a été trouvé afin de pouvoir analyser l’ensemble de ces composés simultanément. L’ensemble de la méthode a ensuite été validée selon les recommandations de l’EMA/CVMP/VICH/463202/2009, pour confirmer sa fiabilité et robustesse. Cette dernière, combinée au model d’ensilage en sacs sous vide, a permis pour la première fois de suivre la cinétique d’évolution de 4 phyto-œstrogènes (daidzéine, génistéine, formononétine et biochanin A) durant une période d’ensilage. [less ▲]

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See detailUPLC®-MS/MS, an analytical tool for the accurate and rapid quantification of phytoestrogen metabolites in milk
Daems, Frédéric ULg; Jasselette, Christophe; Romnee, Jean-Michel et al

in Biotechnologie, Agronomie, Société et Environnement = Biotechnology, Agronomy, Society and Environment [=BASE] (2015, April 17), 19(2), 121-122

The term ‘phytoestrogen’ is used to define a wide variety of nonsteroidal compounds that occur naturally in many plants. When they are absorbed by cows, these polyphenolic compounds undergo ... [more ▼]

The term ‘phytoestrogen’ is used to define a wide variety of nonsteroidal compounds that occur naturally in many plants. When they are absorbed by cows, these polyphenolic compounds undergo biotransformation by specific bacteria present in the gastrointestinal tract. The resulting metabolites are absorbed in the organism and some are excreted in milk. The impact of these compounds on human health divides opinion. Some scientists believe that they might have adverse health effects. Others believe the opposite, especially with regard to their microbial metabolites, such as equol. Whatever one's view on the subject, scientists need accurate, sensitive and rapid analytical methods in order to continue the research on clarifying the phytoestrogen issue. Cow's milk is an animal product that is common in the human diet, and it is therefore important to evaluate its content of phytoestrogen metabolites. In order to study the human intake of equol via commercial milk, an analytical method was developed and validated following EMA/CVMP/VICH/463202/2009 guidelines. Enzymatic hydrolysis was used to release the equol. It was then extracted using double liquid/liquid extraction and analyzed using UPLC®-MS/MS, with an analysis runtime of only 5 min. This analytical method produced a linear calibration curve with a high correlation coefficient (R2≥0.996) between 5 and 1,000 ng mL−1. Good intra- and inter-day precision (RSDs≤5.3% and ≤5.2%, respectively) and accuracy (bias≤8.6%) were achieved. The recovery rate differed slightly among the different types of milk, ranging between 60.6±1.09% and 82.3±5.21%. Good method repeatability was observed (RSDs<15%). There was neither a matrix effect nor a carry-over effect, and the sample extracts were stable during storage for at least 7 days at −21°C and 5°C. In order to apply the proposed method and obtain an initial estimate of equol concentration in cow’s milk in Belgium, 44 samples of various brands found in several supermarkets and 5 raw milk samples from Walloon farms were analyzed. Equol was found in all the samples analyzed, with a concentration ranging from 10 to 50 ng mL−1 for conventional milk and from 70 to 130 ng mL−1 for organic milk. These results were compared with other European studies and the same trend was observed. The results obtained show the specificity, sensitivity and precision of this method for analyzing oestrogenic metabolite-equol in cow’s milk. This study should be extended to include a greater number of samples and be implemented over a longer period to see if there is a correlation between livestock practice and/or sample origins with the equol content in milk. The addition of other phytoestrogen compounds with this method, using UPLC®-MS/MS technology, could also lead to more reliable monitoring of these compounds in dairy production. [less ▲]

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See detailGenetic correlations between methane production and milk fatty acid contents of Walloon Holstein cattle throughout the lactation
Vanrobays, Marie-Laure ULg; Vandenplas, Jérémie ULg; Bastin, Catherine ULg et al

Poster (2015, April 16)

Methane (CH4) from ruminal fermentation is the major greenhouse gas produced by dairy cattle which contributes largely to climate change. Production of CH4 also represents losses of gross energy intake ... [more ▼]

Methane (CH4) from ruminal fermentation is the major greenhouse gas produced by dairy cattle which contributes largely to climate change. Production of CH4 also represents losses of gross energy intake. Therefore, there is a growing interest in mitigating these emissions. Acetate and butyrate have common bio-chemical pathways with CH4. Because some milk fatty acids (FA) arise from acetate and butyrate, milk FA are often considered as potential predictors of CH4. However, relationships between these traits remain unclear. Moreover, the evolution of the phenotypic and genetic correlations of CH4 and milk FA across days in milk (DIM) has not been evaluated. The main goal of this study was to estimate genetic correlations between CH4 and milk FA contents throughout the lactation. Calibration equations predicting daily CH4 production (g/d) and milk FA contents (g/100 dL of milk) from milk mid-infrared (MIR) spectra were applied on MIR spectra related to Walloon milk recording. Data included 243,260 test-day records (between 5 and 365 DIM) from 33,850 first-parity Holstein cows collected in 630 herds. Pedigree included 109,975 animals. Bivariate (i.e., CH4 production and one of the FA traits) random regression test-day models were used to estimate genetic parameters of CH4 production and 7 groups of FA contents in milk. Saturated (SFA), short-chain (SCFA), and medium-chain FA (MCFA) showed positive averaged daily genetic correlations with CH4 production (from 0.25 to 0.29). Throughout the lactation, genetic correlations between SCFA and CH4 were low in the beginning of the lactation (0.11 at 5 DIM) and higher at the end of the lactation (0.54 at 365 DIM). Regarding SFA and MCFA, genetic correlations between these groups of FA and CH4 were more stable during the lactation with a slight increase (from 0.23 to 0.31 for SFA and from 0.23 to 0.29 for MCFA, at 5 and 365 DIM respectively). Furthermore, averaged daily genetic correlations between CH4 production and monounsaturated (MUFA), polyunsaturated (PUFA), unsaturated (UFA), and long-chain FA (LCFA) were low (from 0.00 to 0.15). However, these genetic correlations varied across DIM. Genetic correlations between CH4 and MUFA, PUFA, UFA, and LCFA were negative in early lactation (from -0.24 to -0.34 at 5 DIM) and increased afterward to become positive from 15 weeks till the end of the lactation (from 0.14 to 0.25 at 365 DIM). Finally, these results indicate that genetic and, therefore, phenotypic correlations between CH4 production and milk FA vary following lactation stage of the cow, a fact still often ignored when trying to predict CH4 production from FA composition. [less ▲]

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See detailValidating the use of an ultra-performance liquid chromatography with tandem mass spectrometry method to quantify equol in cow's milk
Daems, Frédéric ULg; Jasselette, Christophe; Romnee, Jean-Michel et al

in Dairy Science & Technology (2015)

Cow’s milk can be used as a potential source of equol in the human diet. In order to study human intake, however, it is necessary to develop a reliable and sensitive analytical method. This paper reports ... [more ▼]

Cow’s milk can be used as a potential source of equol in the human diet. In order to study human intake, however, it is necessary to develop a reliable and sensitive analytical method. This paper reports on the validation of an analytical method using ultra-performance liquid chromatography coupled with a tandem mass spectrometry detector to quantify the equol in commercial milks (raw, whole, semi-skimmed, and skimmed milk). The equol was initially released using enzymatic hydrolysis, and it was then extracted using a double liquid/liquid extraction. The analytical method produced a linear calibration curve with a high correlation coefficient (R 2 ≥ 0.996) between 5 and 1,000 ng.mL−1. Good intra- and inter-day precision (≤5.3% and ≤ 5.2%, respectively) and accuracy (≤8.6%) were achieved. The recovery rate differed slightly among the different types of milk, ranging between 60.6 ± 1.09% and 82.3 ± 5.21%. Good method repeatability was observed (<15%). There was neither matrix effect nor carry-over effect, and the sample extracts were stable for at least 7 days of storage at −21 °C and 5 °C. The method proved to be specific, sensitive, precise, and accurate and was used for the first time to quantify the equol content in Belgian commercial cow’s milk. In all the samples analyzed, equol was present at a concentration ≥10 ng.mL−1 and had a significantly higher content in organic than in conventional milk. The study also found that the mean concentrations of equol were similar for each type of commercial conventional cow’s milk. [less ▲]

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See detailAssessing resilience of dairy cattle by studying impact of heat stress on predicted feed intake
Vanrobays, Marie-Laure ULg; Hammami, Hedi ULg; Laine, Aurélie ULg et al

in Proceedings of the Third DairyCare Conference 2015 (2015)

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See detailHot topic: Innovative lactation-stage-dependent prediction of methane emissions from milk mid-infrared spectra
Vanlierde, Amélie; Vanrobays, Marie-Laure ULg; Dehareng, Frédéric et al

in Journal of Dairy Science (2015), In press

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See detailShort communication: Novel method to predict body weight of primiparous dairy cows throughout the lactation
Vanrobays, Marie-Laure ULg; Vandenplas, Jérémie ULg; Hammami, Hedi ULg et al

in Journal of Dairy Science (2015), 98

Body weight (BW) of dairy cows can be estimated using linear conformation traits (calculated BW; CBW), which are generally recorded only once during a lactation. However, predicted BW (PBW) throughout the ... [more ▼]

Body weight (BW) of dairy cows can be estimated using linear conformation traits (calculated BW; CBW), which are generally recorded only once during a lactation. However, predicted BW (PBW) throughout the lactation would be useful, e.g., at milk-recording dates allowing feed-intake prediction for advisory purposes. Therefore, a 2-step approach was developed to obtain PBW for each milk-recording date. In the first step, a random-regression test-day model was used with CBW as observations to predict PBW. The second step consisted in changing means and (co)variances of prior distributions for the additive genetic random effects of the test-day model by using priors derived from results of the first step to predict again PBW. A total of 25,061 CBW from 24,919 primiparous Holstein cows were computed using equations from literature. Using CBW as observations, PBW was then predicted over the whole lactation for 232,436 dates corresponding to 207,375 milk-recording dates and 25,061 classification dates. Results showed that using both steps (the 2-step approach) provided more accurate predictions than using only the first step (the one-step approach). Based on the results of this preliminary study, BW of dairy cows could be predicted throughout the lactation using this procedure. These predictions could be useful in milk-recording systems to compute traits of interest (e.g., feed-intake prediction). The developed novel method is also flexible because actual direct measurements of BW can also be used together with CBW, the prediction model being able to accommodate different levels of accuracies of used BW phenotypes. [less ▲]

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See detailInnovative lactation stage specific prediction of CH4 from milk MIR spectra
Vanlierde, Amélie; Vanrobays, Marie-Laure ULg; Dehareng, Frédéric et al

Conference (2014, August 28)

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See detailUsing milk spectral data for large-scale phenotypes linked to mitigation and efficiency
Soyeurt, Hélène ULg; Vanlierde, Amélie; Vanrobays, Marie-Laure ULg et al

Conference (2014, August 26)

Even if producing milk efficiently has always been a major concern for producers, the direct environmental impact of their cows is becoming a novel one. Traits linked to this issue were identified as ... [more ▼]

Even if producing milk efficiently has always been a major concern for producers, the direct environmental impact of their cows is becoming a novel one. Traits linked to this issue were identified as methane emission (CH4), dry matter intake (DMI) and feed efficiency (FE); however they are available on a small scale. Researches showed that CH4 could be predicted from milk mid-infrared (MIR) spectra, allowing large-scale recording at low cost. The main objective of this study was to show, using a modelling approach, that DMI and FE could be derived from milk MIR spectra. For that, knowledge of body weight (BW) is required; however it was unknown in this study. Derived procedure was based on milk yield and composition, MIR CH4, and modelled standard animal requirements, allowing the prediction of expected BW. An external validation was conducted based on 91 actual records. 95% confidence limit for the difference ranged between -0.66 and 18.84 kg for BW, from -0.02 to 0.26 kg/day for DMI, and from -0.02 to 0.002 kg of fat corrected milk/kg DM for FE. Root mean square errors were 39.66 kg, 0.56 kg/d, and 0.03 kg/DM for the 3 studied traits. P-value for the t-test was not significant for BW and DMI. This suggests the possibility to obtain expected BW and therefore DMI from MIR spectra. Single trait animal test-day models used 1,291,850 records to assess the variability of studied traits. Significant variations were observed for the lactation stage, parity, genetics, and age. These findings were in agreement with the literature except for early lactation. This suggests in conclusion that the MIR information gave similar results for DMI and CH4 for the major part of lactation. The use of this novel method to predict expected BW offers new possibilities interesting for the development of genomic and genetic tools. [less ▲]

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See detailUsing milk spectral data for large-scale phenotypes linked to mitigation and efficiency
Soyeurt, Hélène ULg; Vanlierde, Amélie; Vanrobays, Marie-Laure ULg et al

in Book of abstracts of the 65th annual meeting of the European Federation of Animal Science (2014, August)

Even if producing milk efficiently has always been a major concern for producers, the direct environmental impact of their cows is becoming a novel one. Traits linked to this issue were identified as ... [more ▼]

Even if producing milk efficiently has always been a major concern for producers, the direct environmental impact of their cows is becoming a novel one. Traits linked to this issue were identified as methane emission (CH4), dry matter intake (DMI) and feed efficiency (FE); however they are available on a small scale. Researches showed that CH4 could be predicted from milk mid-infrared (MIR) spectra, allowing large-scale recording at low cost. The main objective of this study was to show, using a modelling approach, that DMI and FE could be derived from milk MIR spectra. For that, knowledge of body weight (BW) is required; however it was unknown in this study. Derived procedure was based on milk yield and composition, MIR CH4, and modelled standard animal requirements, allowing the prediction of expected BW. An external validation was conducted based on 91 actual records. 95% confidence limit for the difference ranged between -0.66 and 18.84 kg for BW, from -0.02 to 0.26 kg/day for DMI, and from -0.02 to 0.002 kg of fat corrected milk/kg DM for FE. Root mean square errors were 39.66 kg, 0.56 kg/d, and 0.03 kg/DM for the 3 studied traits. P-value for the t-test was not significant for BW and DMI. This suggests the possibility to obtain expected BW and therefore DMI from MIR spectra. Single trait animal test-day models used 1,291,850 records to assess the variability of studied traits. Significant variations were observed for the lactation stage, parity, genetics, and age. These findings were in agreement with the literature except for early lactation. This suggests in conclusion that the MIR information gave similar results for DMI and CH4 for the major part of lactation. The use of this novel method to predict expected BW offers new possibilities interesting for the development of genomic and genetic tools. [less ▲]

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See detailInnovative lactation stage specific prediction of CH4 from milk MIR spectra
Vanlierde, Amélie; Vanrobays, Marie-Laure ULg; Dehareng, Frédéric et al

in Book of abstracts of the 65th annual meeting of the European Federation of Animal Science (2014, August)

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See detailDétermination de la teneur en équol dans les laits commercialisés en Wallonie (Belgique)
Daems, Frédéric ULg; Jasselette, Christophe; Franckson, Delphine et al

Poster (2014, June 13)

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See detailEnrichir le lait de vache en équol par la consommation de fourrages prairiaux spécifiques
Franckson, Delphine; Daems, Frédéric ULg; Decruyenaere, Virginie et al

Poster (2014, March 16)

Detailed reference viewed: 46 (10 ULg)