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Secondary compounds of grassland and forage plant species such as vitamins or phenolic compounds are involved in different health-promoting effects in animals. However, information on their concentration and composition in forage plant species remains scarce. The objective of this study was to characterize the composition of secondary compounds of seven grazed cover crop plant species harvested at two stages of growth. Carotenoids and tocopherols were characterized and quantified using ultraperformance liquid chromatography with a photodiode array, and soluble phenolic compounds were characterized using high-performance liquid chromatography with diode-array detection. All species were rich in carotenoids, especially at the vegetative stage, even if the concentrations varied between plant species. Variations in tocopherol concentrations and phenolic composition were more important between plant species than between stages within species. Among the plant species tested, sainfoin (Onobrychis viciifolia Scop) contained the most secondary metabolites.

International audience; Olive oil production yields a considerable amount of wastewater, a powerful pollutant that is currently discarded but could be considered as a potential source of valuable natural products due to its content in phenolic compounds and other natural antioxidants. The aim of this work was to explore the variability in olive mill wastewater composition from Algerian olive oil mills considering extraction processes (traditional discontinuous press vs 3-phases centrifugal system) and olive varieties (Azerraj, Sigoise, Chemlal). Whereas pH, dry or organic matter content didn’t vary, there was a significant difference in ash content according to extraction process and olive variety. Carotenoid content was 2.2-fold higher with 3-phases than with press systems whereas tocopherol content was not significantly different. Among the phenolic compounds quantified, tyrosol was usually the most abundant whereas oleuropein concentrations were highly variable. Differences in phenolic compound concentrations were more pronounced between olive varieties than between processes.

International audience; Introduction In the search for natural alternatives to synthetic chemicals able to mitigate methane emission by ruminants, bioactive plant secondary metabolites are valuable candidates. However, these phytochemicals come in myriad chemical structures, and any one plant may contain hundreds of them. Even in plant extracts containing tannins, saponins or essential oils, it is difficult to link the presence of a compound or combination to the plant's activity. Here we focus on low-molecular-weight (0.5) were discarded before data mining to avoid false-positives. With the minimum thresholds of 5 for S and 0.5 for C, there were 205 candidate peaks. In a first strategy, results were filtered via the constraints of a) co-occurrence of the peak in the 280 and 320 nm matrices and b) C > 0.65, which narrowed the candidate peaks down to 28. In a second strategy, the constraints were that the peaks had to be major (i.e. more than 10 times the area of the median peak) and present in the plants that showed high antimethanogenic effect (outliers), which narrowed the candidate peaks down to 24. Combining the two strategies resulted in 7 candidate peaks. One peak was easily identified as gallic acid. Based on absorbance spectrum between 200 and 400 nm, three others were cinnamic acid derivatives and two were flavonols. Conclusion Association rules mining was able to select a compact number of peaks making identification feasible. The effect of these pure compounds now has to be verified for proof of the concept. While the algorithm works with qualitative data, using strategy which selects among the major peaks of the profiles serves to integrate the quantitative aspect. Acknowledgements We thank ethnobotanist G. Lalière and the Conservatoire Botanique National du Massif Central for plant collection.

International audience; Propolis is a product that is rich in phenolic compounds and can be utilized in animal nutrition as a dietary additive. In this study, the effects of a Brazilian green propolis extract on rumen fermentation and gas production were determined. The fate of propolis phenolic compounds in the rumen medium was also investigated. Fermentation was done in 24-h batches over three periods. Inoculates were obtained from cows fed on grassland hay and concentrate. Propolis extract in a hydroalcoholic solution was applied at increasing doses to the substrate (1 to 56 g/kg). The fermentation substrate consisted on a mixture of alfalfa hay, soybean meal, and wheat grain mixture in dry matter. After 24 h of fermentation, seven new compounds were observed in the medium in amounts that correlated to the propolis dose. The dose of propolis extract linearly decreased the pH of the medium and linearly increased propionate production, which reduced the acetate-to-propionate ratio and influenced the total production of short-chain fatty acids. Propolis also linearly reduced methane production and increased the carbon dioxide-to-methane ratio. Ammonia nitrogen levels and in vitro digestibility of organic matter were similar among the treatments. The combination of increased propionate production and decreased methane production suggests better energy utilization from the feed.

Les consommateurs sont demandeurs d’informations et de garanties sur les conditions d’élevage des animaux et en particulier sur leur alimentation. Ces préoccupations conduisent les filières animales à prendre des engagements sur les conditions d’élevage et d’alimentation des animaux, engagements spécifiés dans des cahiers des charges. Il apparaît ainsi un besoin d’outils analytiques permettant de garantir le respect de ces engagements et/ou d’aider à les instruire. La nature de l’alimentation influence fortement la composition des tissus et produits animaux. Ceci est dû à des composés spécifiques qui sont soit directement transférés de l’aliment au produit, soit transformés ou produits par les microorganismes du rumen ou le métabolisme animal sous l’effet de l’alimentation. Certains de ces composés peuvent donc, en retour, être utilisés pour tracer l’alimentation. De plus, les différences de composition des tissus et produits provoquent des modifications de leurs propriétés optiques, qui peuvent également être mises à profit. Cet article présente les différentes approches analytiques développées actuellement, les principaux résultats obtenus lors de la comparaison d’alimentations contrastées, puis lors de l’alternance de régimes alimentaires avec les questions de latence et de persistance des biomarqueurs d’intérêt, et enfin quelques exemples d’application sur le terrain avec certaines filières animales intéressées par ces outils., There is currently an increased consumer demand for information on herbivore production factors, particularly animal diet. To meet these demands, farmers and commercial entities develop specifications via quality labels. There is therefore a need for analytical tools that may guarantee that the specification commitments have been fully met or to help constructing them. The nature of the diet strongly influences the composition of the animal tissues and products, which is due to specific compounds that are directly transferred from the feed to the end product or that are transformed or produced by ruminal microorganisms or the animal’s metabolism under the effect of specific diets. Some of these compounds can therefore be used as diet markers. Moreover, differences in the meat and milk composition induce diffe-rences in their optical properties, therefore in their spectral features, which can also be used for diet authentication. This paper reviews the different approaches that have been investigated, some leading results concerning the comparison of contrasting feeding situations, together with the persistence and latency of appearance of some diet markers in the event of animal diet changes, and lastly some examples of implementation in the field with farmers that are interested in such methods.

The extent of disappearance of 10 mono- and sesquiterpenes in presence of mixed rumen micro-organisms has been measured in 24-h batch cultures. The molecules that were tested are frequently found in the plants consumed by ruminants in highland pastures (limonene, β-myrcene, β-ocimene, α-pinene, sabinene, γ-terpinene and thymol) or are potential markers of the diet (camphene, β-caryophyllene and α-copaene). All terpenes were tested at 2 µL mL−1, except α-copaene and thymol, which were tested at 0.9 µL mL−1 and 2 mg mL−1, respectively. Camphene and thymol were not degraded to a significant extent. The disappearance of part of β-caryophyllene, (−)-limonene and γ-terpinene could not be attributed to the direct action of rumen micro-organisms but rather than to an effect of the extracellular medium. In contrast, a third group of one sesquiterpene (α-copaene) and four monoterpenes (myrcene, β-ocimene, α-pinene and sabinene) was extensively degraded by rumen micro-organisms, at rates of 1.6, 4.5, 3.5, 4.8 and 5.4 µmol mL−1 inoculum day−1, respectively. The preliminary exposure of rumen micro-organisms to a specific blend of essential oils compounds, containing mainly thymol, guajacol and limonene, increased the extent of disappearance of γ-terpinene only. Copyright © 2007 Society of Chemical Industry

The chemical composition, intake, digestibility, ultrastructure and microbial degradation of rice straw from Camargue were compared with barley straw. These variables were observed in two different herbivore digestive ecosystems: the sheep rumen and the donkey caecum. The two straws differed essentially in their ash content, which was three times higher in rice, owing to its silica content. Other chemical components were comparable, except a higher phenolic acids-to-lignin ratio in rice. Rice straw was better ingested than barley straw. Organic matter and neutral detergent fibre digestibilities were the same in both straws. Dry matter and cell wall disappearances could be adjusted to the exponential modelling equation with lag time, and differed between animals but not between straws. The sheep rumen had a higher extent of degradation, but the donkey caecum had a higher degradation rate. Statistical analysis revealed that cell-wall components degradation was similar in the two straws except for ferulic acid, which was more degraded in rice straw. Scanning electron microscopy showed important differences in parenchyma degradation, which was much more effective in rice. Copyright © 2003 Society of Chemical Industry

Immunogold labelling of para-coumaric acid was performed on the base and the top of the apical internode of the stem of Co125 and W401 maize, harvested at anthesis + 5 days, after incubation in the rumen in nylon bags for 4, 8 and 24 h. The description of labelling evolution during rumen degradation was permitted by the quantification of the intensity of labelling in four tissues: sclerenchyma, fibres (surrounding vascular bundles), xylem and parenchyma. The results are discussed in relation to maize digestibility.

Two phenolic compounds, p-coumaric acid and feruloyl-arabinose, were localised by immunocytochemistry in the cell walls of the apical internode of two lines of maize (Co125 and W401) of different digestibility. The compounds were detected at two stages of cell maturity in the lignified tissues (sclerenchyma, fibres and xylem) and in the medullary parenchyma, which, in the samples studied, was not lignified. p-Coumaric acid is a phenolic acid associated with lignins, which confer resistance on plant cell walls to microbial degradation in the rumen. Feruloyl-arabinose is a compound associated with xylans, the principal hemicelluloses in Gramineae, which are potentially degradable. Labelling of p-coumaric acid decreased in both maize lines with cell age and as the cell walls became lignified. The mass of lignin deposited in the cell walls masked p-coumaric acid, thereby making it less accessible to the antibodies. There was an inverse relationship in the labelling of p-coumaric acid and feruloyl-arabinose. Feruloyl-arabinose was more heavily labelled as the plant cell walls matured in all the lignified tissues of both maize lines and in the parenchyma of the less digestible line. All tissues except the parenchyma were more heavily labelled with both sera in Co125, the more digestible line. © 1998 Society of Chemical Industry.

The effect of down-regulation of tobacco cinnamyl alcohol dehydrogenase (CAD) on cell wall composition and degradability has been assessed. CAD activity was only 20, 16, 14 and 7%, relative to the controls, in four populations of plants (designated 40-1, 40-2, 48 and 50, respectively) transformed with CAD antisense mRNA. Cell wall residues of stem samples were analysed for polysaccharide composition, gravimetric and acetyl bromide lignins and lignin nitrobenzene oxidation products. In situ disappearance and cellulase solubility of both initial dry matter and CWR were determined. The populations of plants with depressed CAD activity showed no change in lignin content but some consistent changes in cell wall composition and digestibility were identified. The syringyl content of lignins decreased and the syringaldehyde to vanillin ratio (S/V) was consequently reduced. Dry matter degradability, as measured by both methods, was significantly improved in all CAD-depressed samples except for population 40-1, which was the least CAD-depressed. Increased in situ disappearance of cell wall (ISCWD) was found in all plants exhibiting more than 80% CAD down-regulation and was maximal (7 percentage units) in population 50 which had the greatest CAD depression. The rates of ISCWD increased slightly in some populations (40-2 and 50). The relationship between S/V and ISCWD was significant (R = -0·68) only in the samples from a selected population of mature, most depleted plants. Other modifications may therefore also contribute to the improvement in degradability. However the changes in lignin composition that were observed in CAD-depressed tobacco are largely similar to those seen in some maize and sorghum mutants with altered lignification and improved digestibility. These data therefore suggest that depressing CAD activity may be an effective method for improving digestibility in forage crops. © 1998 SCI.

International audience; The aim of this work was to characterise new UV-absorbing compounds (UAC) in cow milk in order to gain an overview of the molecular diversity of the minor bioactive constituents, that could be used to trace animal feed or that potentially affect milk quality. UAC were extracted from lyophilized milks, partitioned using SPE C-18 cartridges, purified by semi-preparative HPLC then analysed by HPLC/DAD/HRMS in both ESI− and ESI+ ionisation mode. Compounds that remained unidentified after comparison with UV and MS databases were analysed by 1D and 2D NMR techniques. The identification and structural elucidation of N-cinnamoylglycine, 2,4-, 2,6-, 2,8-quinolinediols, and 1-methyl-3-carboxy-beta-carboline are described.

The down regulation of cinnamyl alcohol dehydrogenase (CAD) activity in tobacco stems resulted in improved degradability without changes in cell wall composition. In this study, some changes were evidenced in cell wall solubilization by NaOH, cellulase, and DMSO treatments. Transgenic plants had an altered cell wall structure, the components of which were more accessible to alkali extraction. In plants at the same stage of maturity, cell wall degradability was linked to the yield of acid-precipitated fraction of the NaOH-soluble extract. Aldehydes that should have been incorporated in the cell wall were possibly detected in the different extracts. Guaiacyl and syringyl monomers were distributed differently in the controls and the antisense saponified residues and acid-precipitated fractions. It is suggested that polymeric lignin structure differs. These findings may help in the understanding of the improvement of cell wall digestibility. Keywords: Nicotiana tabacum; cinnamyl alcohol dehydrogenase; lignin ...

The combined effects of lucerne (Medicago sativa L.) extract supplementation and ammonia treatment of rice straw (Oryza sativa, variety Thaibonnet) on the ruminal digestion of cell wall components were investigated in six continuous culture systems using a randomised complete block design. Data were fitted to second-order polynomial models. Untreated rice straw had higher contents of ash-free cell wall residues (CWR; 763 v. 687 g/kg dry matter (DM)) and non-cellulosic sugars (191 v. 166 g/kg DM) than treated rice straw. Ammoniation preferentially removed xylose, which resulted in a lower xylose-to-arabinose ratio (5.1 v. 5.8). In absence of lucerne supplementation and ammoniation, degradability coefficients were 0.54, 0.46, 0.58, 0.54, 0.42 and 0.60 for cellulose-glucose, xylose, arabinose, galactose, mannose and uronic acids, respectively. Both factors had significant effects on the microbial degradation of structural polysaccharides. With lucerne extract at an optimal level, ammonia treatment increased ash-free cell wall degradation by more than 10%. The degradability coefficients were increased by ammoniation without any significant interaction with lucerne extract, except for glucose, whose degradability was mostly influenced by lucerne extract in a curvilinear way. The comparison of regression coefficients in cell wall and CWR models suggested that ammoniation improved the degradabilities of xylose, galactose and mannose by partly solubilising the corresponding hemicelluloses and by improving the susceptibility of the remaining fraction to microbial attack, whereas it increased the degradability of arabinose only by favouring microbial attack.

Phenolic constituents (lignins and phenolic acids) and carbohydrates are assembled in a tight architecture which differs according to the plant species. During cell wall digestion, the hydrolysis kinetics differ between carbohydrates and seem to depend chiefly on the content and organisation of tissue phenolics. Among the phenylpropanoids, ferulic acid is released more quickly than p-coumaric acid. Lignins remain largely in the cell walls. They also undergo transformations, chiefly solubilisation as lignin-carbohydrate complexes. The limiting effect of lignins on cell wall degradation increases with increasing content. However, their effect on degradation might also depend on qualitative factors such as lignin structure and polymer organisation in walls and tissues. When various grasses (normal and selected genotypes), or grasses and legumes are compared, correlations between certain factors such as lignin uncondensed fraction, syringyl units or phenolic acids contents and cell wall degradation emerge but not clear causal relationship has been shown. Nonetheless, other structural characteristics, related to the alkali reactivity of lignins, seem to have a stronger influence on cell wall degradation. Phenylpropanoids seem to act mainly as a physical and (bio)chemical barrier to the action of the microbial enzymes. In addition, their reactivity as phenolic compounds and their hydrophobicity seem to play a role. Digestion is not limited only by phenolics. The factors that limit glycanolysis—the accessibility, crystallinity and capillary structure of cellulose and the branching of hemicelluloses—seem to have little or no effect on cell wall degradation in vivo. In contrast, other antiquality substances (tannins, cutin and silica), plant antomy, environmental factors, factors modulating microbial growth and animal physiology influence cell wall utilisation. Future research in this field should focus on the effects of phenolic structure and of cell wall and tissue organisation on carbohydrate degradation.

International audience; The development of analytical methods to verify the production system of meat products requires the identification of biomarkers that can trace the product's origin, and secondly the factors that govern the deposition of these markers in animal tissue need to be defined. In this study, 2,3-octanedione, skatole and terpenes were selected as biomarkers, and their deposition was investigated in bull calves reared under three different strategies. All of the animals were reared indoors until approximately 150 days of age. They were suckled twice a day by their mothers, and both calves and cows had free access to cocksfoot hay. Then the first two groups of animals were kept indoors, suckled by their mothers twice a day and received either cocksfoot hay (HL) or freshly cut-green herbage (GL) and a limited quantity of concentrate. The third group of calves (PH) was kept on pasture with their mothers and offered concentrate ad libitum. The pasture supporting the PH animals was highly diversified, containing several terpene-rich plant species, whereas the herbage for the GL animals contained no species known to be aromatic. Perirenal and subcutaneous adipose tissues were analysed for volatile compounds. The perirenal fat was found to be more responsive to the treatment and a more reliable substrate than the subcutaneous adipose tissue. Higher levels of 2,3-octanedione (P < 0.05) were found in PH and GL than in HL fat (6.56, 6.51 and 5.77 area arbitrary units, respectively, in perirenal fat), confirming the ability of this molecule to trace green herbage feeding. Skatole was detected in the perirenal and subcutaneous fat of all the animals. Animals receiving high concentrate level (PH group) presented lower (P < 0.05) skatole values (5.83 area arbitrary units in perirenal fat) than animals receiving low concentrate level (HL and GL groups, 6.23 and 6.71 area arbitrary units, respectively, in perirenal fat). Terpenoids, and especially sesquiterpenes, were found at higher levels and diversities in the PH than in the GL and HL animals. Two monoterpenoids allowed group discrimination considering perirenal or subcutaneous fat without distinction, whereas 11 and 5 sesquiterpenoids from perirenal and subcutaneous fat, respectively, allowed it.

International audience; Phenolic compounds contribute to the micronutrient composition of pasture, which in turn may affect animal product composition. To assess the importance and variations in content of these compounds, the polyphenolic and botanical compositions of 24 permanent pastures located in one lowland and two upland regions were studied at equivalent stages of growth. Phenolic fractions were characterized and quantified using HPLC-PDA-ESI-QToF, and the total content was determined by colorimetry over each whole pasture. A rise in altitude was accompanied by a marked increase in total phenolic content, linked to changes in botanical composition, but did not have any influence on the distribution according to molecular class. For all of the pastures, significantly different patterns due to qualitative and quantitative differences among the 92 separate peaks were observed with 31 compounds identified. The involvement of certain plants in the variations of content and composition in phenolic compounds of pastures was statistically evaluated.

International audience; The last decade has seen important developments in the use of carotenoid pigments to authenticate pasture-feeding in ruminants. However, dehydrated alfalfa is sometimes incorporated in grain-based concentrates fed to stall-raised lambs, which may affect the reliability of the pasture-feeding authentication methods based on carotenoids in plasma and fat, due to significant residual carotenoid levels post-dehydration. The aim of this study was to examine whether other compounds can give additional information to authenticate diet and discriminate pasture-fed lambs from lambs fed high levels of alfalfa indoors. Two feeding treatments were compared: pasture-feeding (P) v. stall-feeding with dehydrated alfalfa (A). Each treatment group consisted of seven male Romanov3Berrichon lambs. Pasture-fed (P) lambs grazed a permanent graminaceae-rich pasture maintained at a leafy, green stage, offered ad libitum; they received no supplementation at pasture. A-group lambs were individually penned and fed dehydrated alfalfa and straw; their feed level was adjusted to achieve a similar growth pattern as for P-group lambs. Plasma carotenoid concentration was measured at slaughter by spectrophotometry. The reflectance spectrum of perirenal and subcutaneous caudal fat was measured at 24-h post mortem and used to calculate an index (absolute value of the mean integral (AVMI)) quantifying light absorption by carotenoid pigments present in the fat. The nitrogen (N) stable isotopes ratio (d15N) in both feed and longissimus dorsi muscle was measured by isotopes ratio mass spectrometry (IRMS). Volatile compounds were analyzed in perirenal fat for five randomly chosen lambs per treatment, using dynamic headspace–gas chromatography–mass spectrometry. Plasma carotenoid concentration and AVMI of the fat did not differ significantly between P- and A-group lambs, but there were significant between-treatment differences in meat d15N values and in the terpene profiles of perirenal fat. A discriminant analysis performed using three compounds in different animal tissues (d-cadinene in perirenal fat, d15N value of the meat and plasma carotenoid concentration) clearly separated pasture-fed lambs from lambs fed high levels of alfalfa indoors.

Cantal cheese was made with either raw or pasteurized milk from cows fed either pasture or a hay and concentrate diet, and was analyzed after 3- and 6-month aging. The cheese aroma was rather mild for pasteurized-milk cheese and rather strong for raw-milk cheese, and was little affected by the cows’ feeding patterns. Gas chromatography of the volatile compounds coupled with a new 8-way olfactometry device revealed 42 odorous peaks. The major odour-active compounds were butanoic acid, 2,3-butanedione, 3-methylthio-propanal, hexanal, acetic acid, isobutanoic acid, and 1-octen-3-ol. The intensity of butanoic acid was much stronger in raw-milk than pasteurized-milk cheese. The intensities of isobutanoic acid, pentanal, E-2-hexenal, cyclohexanone, tetramethylpyrazine and γ-heptalactone were lower in the 6-month-aged than the 3-month-aged cheeses, and only ethylbenzene intensity was higher. Isopentanoic acid odour was weaker when the milk had been obtained from cows fed a pasture diet.

International audience; Three diets for cows were used to evaluate the effect of extruded linseed (EL) or extruded linseed plus alpha-tocopherol (ELVE) supplementation of a maize silage diet (CO) on the odor-active compounds of Saint-Nectaire cheese. Cheese odor and flavor profiles were studied by sensory analysis. The volatile compounds were extracted by purge and trap and separated by gas chromatography. The odor compounds were detected and identified using an 8-way olfactometric device and a mass spectrometer. Twenty-nine volatile compounds were considered as contributing to the odor of Saint-Nectaire cheese. Half the compounds identified were known to be lipid degradation products but not all of them were affected by the diet. Among the markers of unsaturated fatty acid degradation, hexanal was not affected, whereas heptanal was increased more by the ELVE diet (6 times) than by the EL (3 times) diet. The ELVE diet led to cheeses with butanoic acid and heptanal odor peaks that were, respectively, 2 and 6 times higher than with the CO diet, which explained the lower milk odor and flavor scores obtained by sensory analysis on ELVE cheese. Although the cheese-making date had a greater effect than the diet on the aromatic profiles of the cheese, principal component analysis showed that the differences between cheeses obtained on the 3 diets were repeatable. The EL diet successfully enhanced cheese nutritional value without noticeably changing its flavor. alpha-Tocopherol supplementation was found to be unnecessary, as no oxidized odor was found.

The aim of this experiment was to study the effect of the addition, to milk, of an essential oil (EO) obtained from the hydrodistillation of plants collected from a mountain natural pasture on the milk and cheese sensory properties. The EO was mainly composed of terpenoid compounds (67 of the 95 compounds identified) as well as ketones, aldehydes, alcohols, esters, alkanes, and benzenic compounds. In milk, the addition of this EO at the concentration of 0.1 microL/L did not influence its sensory properties, whereas at 1.0 microL/L, sensory properties were modified. In cheeses, the effect of adding EO into milk was studied in an experimental dairy plant allowing the production of small Cantal-type cheeses (10 kg) in 3 vats processed in parallel. The control (C) vat contained 110 L of raw milk; in the other 2 vats, 0.1 microL/L (EO1) or 3.0 microL/L (EO30) of EO were added to 110 L of the same milk. Six replicates were performed. After 5 mo of ripening, chemical and sensory analyses were carried out on the cheeses, including determination of the volatile compounds by dynamic headspace combined with gas chromatography-mass spectrometry. The EO did not influence the sensory properties of the cheeses at the lower concentration (EO1). However, the EO30 cheeses had a more intense odor and aroma, both characterized as "mint/chlorophyll" and "thyme/oregano." These unusual odors and aromas originated directly from the EO added. In total, 152 compounds desorbing from cheese were found, of which 41 had been added with the EO; in contrast, 54 compounds of the EO were not recovered in the cheese. Few volatile compounds desorbing from cheeses, other than the added compounds, were affected by EO addition. Among them, 2-butanol, propanol, and 3-heptanone suggested a slight effect of the EO on lipid catabolism. The antimicrobial activity of terpenes is not or is only marginally involved in the explanation of the influence of the botanical composition of the meadows on the pressed cheeses sensory properties.

The first objective of the present paper was to assess the potential of both isotopic ( (18)O/ (16)O in milk water) and molecular biomarkers (terpenes, fatty acids, carotenoids, and vitamins) and milk color to discriminate the production zone (lowland or upland areas) from which 49 tanker bulk milks were collected over one year from a total of 204 farms. The milk water (18)O enrichment was higher in lowland (500 m altitude) than in upland (700 m altitude), but the delta (18)O values failed to discriminate systematically the production zone at the scale of the year because of its high variability related to the sampling period. In contrast with vitamins A and E, carotenoids, and milk color measurements, terpenes and fatty acids were confirmed to be relevant tracers of the production zone. The milk compounds with the strongest discriminative potential were fatty acids, which were determined by high-resolution gas chromatography. The calculation of fatty acid ratios, which permits the limitation of using fatty acid relative quantity expressed in percentage of total fatty acids to be overcome, was shown to be particularly relevant in discriminating upland from lowland milk ratios. The selection of two pairs of ratios, namely, iso-C17:0/C18:3 n-3 and iso-C15:0/iso-C14:0, enabled the authentication of 100% of the highland versus lowland milks whatever the season. The second objective was to evaluate the relevance of fatty acid composition to discriminate milks according to the proportion of corn silage in the diets of dairy cows. The selection of two fatty acids ratios, namely, trans11 cis15-C18:2/trans11-C18:1 and cis9-C16:1/iso-C16:0, enabled the correct classification of 100% of the milk samples according to the proportion of corn silage in the basic fodder rations (25% vs30%). The relationship between the milk production zone and the type of forage fed to the cows is discussed.

International audience; Besides supporting cattle feeding, grasslands are home to a diversity of plants and insects that interact with each other by emitting volatile compounds. The aim of this work was to develop a method to determine permanent grassland odorscape and relate it to flower-visiting insects. Two grasslands were chosen for their contrasting levels of botanical diversity, resulting from differing grazing managements. Measurements were made over two periods of three consecutive days at the beginning of grazing, and just after the cows had left the plots. Volatile compounds were trapped using solid-phase microextraction (SPME) fibers exposed eight hours a day in three exclosures per plot, and then analyzed by gas-chromatography-mass spectrometry (GC-MS). Insects were trapped using pan traps and a net, sorted and counted. The open air SPME method yielded volatile compound profiles that were richer than maize field profiles, comprising the common green leaf volatiles (GLV) and more specific ones. Differences between the odorscapes of the two grasslands were found, but they were not as marked as expected from their botanical composition. By contrast, there were sharp differences between the two periods, resulting from the combined effects of changes in weather conditions, plant phenological stage and grazing progress. Several correlations between insect counts and volatile compounds were found. Although their correlation coefficients were low, some of them were confirmed when tested by Spearman rank correlation, and could be logically explained. This method of grassland odorscape deserves to be developed because it can provide information on many aspects of grassland function and on the stresses that grassland plants undergo.

Terpene profiles in cheese can be considered a ‘terroir’ fingerprint as the information contained in it should enable the pastures on which the animals were fed to be recognised. Yet a certain elasticity of the signature must be taken into account when determining authentication strategies, since products acknowledged as containing a common signature may have undergone certain procedures, such as cheese making and milk pasteurisation, that could have potentially altered their terpene profiles. In this study, Cantal and Saint-Nectaire-type cheeses were made from both raw and pasteurised milk from the same herd of dairy cows that had been grazed on natural grassland. Cheeses from raw and pasteurised milk were made from the same milking on the same days. Cantal and Saint-Nectaire-type cheeses were made on 4 different days, alternatively over four weeks. The terpenes in the cheese fat were analysed by dynamic headspace/gas chromatography/mass spectrometry. A great diversity of monoterpenes, sesquiterpenes and oxygen-containing derivatives were identified. The major terpenes identified in most cheeses were β-caryophyllene, α- and β-pinene and limonene. Milk pasteurisation did not induce changes in the terpene profile of the cheese. Significant differences (p < 0.001) were observed between Cantal and Saint-Nectaire cheeses: α-pinene, β-myrcene and β-phellandrene were, respectively, three, five and five times more abundant in Cantal cheese, while tricyclene, α-phellandrene and geraniol were found exclusively in Cantal cheese. In contrast, unidentified sesquiterpenes with retention indices (KI) = 1342 and 1511, α-cubebene, longifolene and γ-elemene were more abundant or exclusively found in Saint-Nectaire cheese. A significant relationship with the date of milking (p < 0.01) was observed for α-pinene and tricyclene in Cantal, for β-myrcene, δ-3-carene, p-cymene and α-terpinene in Saint-Nectaire cheese. Copyright © 2005 Society of Chemical Industry

Supplying the consumers with guarantees concerning the feed given to animals needs reliable methods for characterizing meat and milk. Bodies operating product certification systems also require control tools, to be able to guarantee objectively that specification commitments have been fully met. This paper reviews the current state of knowledge concerning the traceability of animal feeding diets in the meat and milk of small ruminants. It presents the potential tracers and different methods that have been studied, together with recent results. Plant biomarkers such as carotenoids, terpenes and phenolic compounds, animal metabolites such as 2,3-octanedione, skatole, fatty acids and ratios of oxygen, carbon and nitrogen stable isotope, are potential tracers in meat and milk or animal tissues, of animal feeding diets. Terpenes, phenolic compounds and ratios of stable isotope are also potential tracers of the geographical origin of milk and meat. Global approaches, especially near infrared spectroscopy and functional genomics are just emerging and need further experimental evaluation. These techniques already allowed to discriminate among products obtained in contrasting feeding conditions. Intermediate situations, for example, in case of modification of animal's diet, may be less easily recognized and may require the combination of tracing methods. In particular, the persistence of tracers when animals are stall-fed a concentrate-based diet after pasture, and its implications for traceability are discussed. Further directions for research are finally highlightened.

International audience; Volatile compounds were analysed in subcutaneous fat from lambs raised and finished on grass (GG); raised and finished on concentrates (SS); raised on grass and finished on concentrates for a long (GS1) or a short (GS2) period. Whereas 3-methylindole (skatole), a compound that has been described as a good discriminator of grass feeding, was unaffected by the diet (P>0.05). 2,3-octanedione was lower (P

The effects of ammonia treatment and fresh legume extract supplementation on the metabolism of rumen micro-organisms maintained on rice straw ( Oryza sativa , variety Thaibonnet) in dual outflow fermenteurs were investigated. Alfalfa ( Medicago sativa L.) was chosen as a model legume, similar in biochemical composition to cowpea ( Vigna unguiculata ). Diets provided comparable amounts of readily available N. The interaction between ammonia treatment and legume extract supplementation was generally negligible. The relative productions of VFA (in moles/100 moles of hexoses fermented) were oriented to an increased proportion of C 4 to the detriment of C 2 by ammonia pre-treatment (+100% versus −28% at the intermediate extract input level), which also lowered the relative production of CH 4 by 36%. The true degradability of organic matter (OM) was 27% higher with treated rice straw (TRS) than with untreated rice straw (URS). The degradabilities of ash-free neutral detergent fibre (NDF) and acid detergent fibre (ADF) ranged from 30 to 50% and from 26 to 48%, respectively. At low levels of extract supplementation, the degradabilities of ash-free NDF and ADF tended to be lower with TRS than with URS (30% versus 46% in the case of ADF), while at high levels of the alfalfa extract they were not modified by straw pre-treatment. The outflow of non-ammonia nitrogen (NAN) was higher with TRS (+41% at the intermediate legume input rate). This increased NAN outflow was essentially of microbial origin. Alfalfa extract supplementation also increased net production of microbial protein. The efficiency of microbial protein synthesis was strongly increased by ammonia pre-treatment and by extract supplementation, in a linear manner. Both treatments were beneficial to rumen microbial metabolism in vitro, while acting independently on microbial functions. Ammonia treatment induced the most significant changes, especially in favouring extensive biomass synthesis.

The volatile components from nine plants growing on natural grasslands in Auvergne, central France, selected for the broad qualitative and quantitative diversity of their terpenoid fractions, were analyzed by high-resolution gas-phase chromatography and mass spectrometry (HRGC-MS) after static headspace solid-phase microextraction (SHS-SPME). SHS-SPME allowed all the plant material to be analyzed under the same conditions despite its wide-ranging composition. This is not always possible with other extraction methods. Using an apolar poly(dimethylsiloxane) (PDMS) phase, numerous terpenoid hydrocarbons, together with alcohols, cyclic ethers, and esters, were extracted. Its ease of use and the high resolution of the chromatographic profiles obtained make SHS-SPME well suited to the rapid characterization of the main components of the volatile fraction of plants. Of the nine plants studied, four (Meum athamanticum, Pimpinella saxifraga, Achillea millefolium, and Thymus pulegioides) exhaled more than 60 different volatile components. Certain terpenes present in large amounts in these plants might help link dairy products to grazing pasture, thus improving food traceability.

International audience; In Asia and Africa, rice straw enters into livestock feeds as the main constituent. It is sometimes associated with fresh legume material, from local garden farming or from legume-cereal rotations, but the possible benefits of this practice need to be estimated more precisely, with special attention to the influence of cytoplasmic contents other than nitrogen or macrominerals on rumen microbial metabolism. In the present study, fresh Medicago sativa extract (AE) was chosen as a model of cytoplasmic contents from tops of tropical legumes such as Vigna unguiculata. It was obtained from whole plants harvested at the beginning of flowering (primary growth). The effects of AE supplementation on a rumen microbial community maintained on a diet made of rice straw and inorganic nitrogen (CP: 73.3 g$\cdot$kg$^{-1}$ DM) were assessed in dual outflow continuous culture. Three input levels were applied in order to detect curvilinear effects: 0, 0.227 and 0.455 ml$\cdot$g$^{-1}$ straw DM. The pH and the redox potential varied from 6.72 to 6.84, and from -335 to -370 mv respectively. The addition of AE had minor effects on fermentation variates. The true degradability of dietary OM and the degradabilities of ash-free NDF and ADF were not modified by AE. Alfalfa extract lowered the outflow of fermented OM (FOM) by 14% while it enhanced the microbial OM outflow (+33%) and the efficiency of microbial protein synthesis (15 to 26 g N$\cdot$kg$^{-1}$ FOM). This effect was curvilinear, and appeared negligible above 45-50% of the maximal extract input rate. This legitimised the addition of fresh legume materials to the diet even in small amounts.; Les effets quantitatifs de l'apport d'extrait de luzerne sur la dégradation de la paille de riz, la fermentation et la synthèse de biomasse par les micro-organismes du rumen in vitro. En Asie et en Afrique, l'affouragement des ruminants en paille de riz est parfois associé à des déchets de maraîchage ou à des légumineuses (Niébé). Les bénéfices éventuels de cette pratique doivent être vérifiés, en portant une attention particulière à l'action des composés cytoplasmiques autres que les protéines et les minéraux majeurs sur le métabolisme des micro-organismes du rumen. Le contenu cytoplasmique du modèle choisi est un extrait par pressage d'une luzerne (Medicago sativa) récoltée en début de floraison (EL). Nous avons étudié l'effet de l'EL sur une communauté de micro-organismes du rumen maintenue en fermenteurs à double effluent sur une ration paille de riz + azote inorganique (MAT : 73.3 g$\cdot$kg$^{-1}$ MS). Trois niveaux d'apport ont été appliqués pour détecter d'éventuels effets curvilinéaires : 0 ; 0,227 ; 0,455 ml$\cdot$g$^{-1}$ MS paille. Le pH et le potentiel redox ont varié de 6,72 à 6,84, et de -335 à -370 mv respectivement. L'EL n'a pas significativement modifié les variables fermentaires, ni la dégradabilité réelle de la MO, ou les dégradabilités du NDF et ADF déminéralisés. L'extrait de luzerne a diminué le flux de MO fermentée (MOF) de 14 % tandis qu'il a accru le flux de MO microbienne (+ 33 %) et l'efficacité de la synthèse de protéines microbiennes (de 15 à 26 g N$\cdot$kg$^{-1}$ MOF). Cet effet a été curvilinéaire et paraît négligeable au delà de 45-50 % du niveau d'apport maximal. Dans notre essai, l'EL n'a pas significativement modifié l'apport d'énergie et d'azote aux micro-organismes. Son action catalytique sur le métabolisme microbien justifie l'apport de matière végétale fraîche dans la ration même en faible quantité.

Apical internodes of tall fescue (Festuca arundinacea Schreb. var. Clarine) harvested at flowering were sectioned into 5 or 10 equal parts to study in situ degradability and cell wall composition, respectively. The basal (youngest) section had the greatest primary wall content. Cell walls in the upper (older) sections had the highest xylose/arabinose ratio and lignin content and a lignin rich in syringyl units, all typical of extensive secondary wall development. Almost all of the p-coumaric (p-CA) and about half of the ferulic acid (FA) were released by 1 M NaOH and presumed to be ester-linked. The total FA content was approximately double that of p-CA in all sections other than the youngest with a distribution similar to that of total p-CA. However, the ratio of esterified to ether and ether plus ester linked (EtEt+Es) FA differed with age. Whereas the esterified form remained essentially constant ( approximately 4.5 g/kg of cell wall), EtEt+Es ferulate increased with increasing age of the tissue and was significantly related to lignin deposition (r = 0.79, P0.01). The extent of cell wall degradation after 48 h of incubation in the rumen was inversely related to maturity, falling from 835 g/kg of dry matter in the youngest section to 396 g/kg in the oldest. Both the rate and extent of cell wall degradation were significantly negatively related to the ratio of xylose to arabinose, lignin content, proportion of syringyl units present in lignin, and concentration of EtEt+Es FA present. A positive relationship between EtEt+Es FA was also found, with the rate (P0.01) being better correlated than the extent (P0.05) of cell wall degradation. Application of the newly extended internode model to fescue produced results consistent with the view that both the lignin content and the extent to which lignin was covalently bound to the other wall polymers crucially influenced the rate and extent of degradation.

International audience; Immunogold labelling of feruloyl-arabinose was performed on the base and the top of the apical internode of the stem of Co125 and W401 maize, harvested at anthesis +5 days, after incubation in the rumen in nylon bags for 4, 8 and 24h. The description of labelling evolution during rumen degradation was permitted by the quantification of the intensity of labelling in four tissues: sclerenchyma, fibres, xylem and parenchyma. The results are discussed in relation to maize digestibility.

Arabinoxylans were localised by immunocytochemistry using polyclonal antibodies in the cell walls of the apical internode of maize after degradation in the rumen. In order to understand the significance of arabinoxylan in digestibility property, two lines of maize differing in digestibility were used. Wide variations in the intensity of labelling were observed in the four tissues studied (sclerenchyma, fibres, xylem and parenchyma) from the first hours of incubation in the rumen. Incubation time in the rumen greatly influences the intensity of labelling.

International audience; The effect of O-methyltransferase (OMT) cDNA modulation on cell wall composition, ultrastructure and rumen degradability was measured on transgenic tobacco (Nicotiana tabacum). The expression of OMT cDNA in antisense orientation (AS) inhibited OMT activity by 92% whereas expression of sense constructs led to plants either co-suppressed (CS, 98% inhibition) or overexpressing OMT activity. The cell wall residues of stems were analysed for lignin content, products of nitrobenzene oxidation (NBO) and polysaccharide content. Degradability was determined by a cellulase method. Sections of stem were stained by acid phloroglucinol and Mäule reactant. Stem samples were incubated in the rumen for 8, 24 and 48 h and observed by scanning electron microscopy (SEM). Compared to controls, OMT-depleted stems showed decreased hemicellulose content but unchanged lignin content. In contrast, syringyl units decreased by 40 and 90% in AS and CS samples respectively and NBO content followed a similar trend. Dry matter cellulase degradability was significantly improved by 3·5 and 5·6 percentage units in AS and CS samples respectively. SEM showed a greater bacterial colonisation in these samples and indicated a higher rate of rumen degradability in CS tissues than in controls. Overexpressing plants had a composition and a degradability similar to that of controls. For all the plants studied, the improvements in dry matter degradability were closely linked to the syringyl to guaiacyl ratio or to the NBO content. The modifications observed in down-regulated tobacco were similar to those produced by bm3 maize mutation, but without lignin decrease. Genetic modifications should therefore be considered for improving forage digestibility.

Suppl. 1; International audience

Lignin-carbohydrate complexes (LCCs) are recognised as key structures in forage degradability. Apart from ester bonds involving phenolic acids, which seem to play a major role in grasses, little is known about the other types of linkages that must exist but have proved difficult to demonstrate. The chemical nature of possible LCC linkages is presented and the various mechanisms through which LCCs in the cell-wall architecture may interfere with carbohydrate utilisation by rumen microorganisms are discussed.

[U- 14 C]Phenylalanine (Phe * ) or [O- 14 CH 3 ]sinapic acid (Sin * ) was infused in wheat stems under the node of the apical fraction, with and without leaf, or at midinternode. ( 14 C-Lignin)Pronase-treated cell walls of the upper and lower halves of the internode were prepared and analyzed. With Phe * , the labeling had a greater variability than with Sin * and a different distribution in the organs; cell wall residues were labeled on lignin units with a high specific radioactivity, on phenolic acids, and on residual proteins

Suppl. 1; International audience