Agnès Cornu

Effect of down-regulation of cinnamyl alcohol dehydrogenase on cell wall composition and on degradability of tobacco stems

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.

Effect of modification of the O-methyltransferase activity on cell wall composition, ultrastructure and degradability of transgenic tobacco

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 Maule 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.

Variation in content and composition of phenolic compounds in permanent pastures according to botanical variation.

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.

Odor compounds in cheese made from the milk of cows supplemented with extruded linseed and α-tocopherol.

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.

Immunocytochemical localisation of para-coumaric acid and feruloyl-arabinose in the cell walls of maize stem

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.

Rice straw degradation and biomass synthesis by rumen micro-organisms in continuous culture in response to ammonia treatment and legume extract supplementation

Abstract 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.

Traceability of grass feeding in beef: terpenes, 2,3-octanedione and skatole accumulation in adipose tissue of young bulls.

The development of analytical methods to verify the production system of meat products requires the identification of biomarkers that can trace the products 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.

Immunocytochemical localization of arabinoxylans in the cell wall of maize apical internode after microbial degradation in the rumen

Summary— 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.

Proton transfer reaction time-of-flight mass spectrometry: A high-throughput and innovative method to study the influence of dairy system and cow characteristics on the volatile compound fingerprint of cheeses

The aim of this work was to study the effect of dairy system and individual cow-related factors on the volatile fingerprint of a large number of individual model cheeses analyzed by proton transfer reaction time-of-flight mass spectrometry (PTR-ToF-MS). A total of 1,075 model cheeses were produced using milk samples collected from individual Brown Swiss cows reared in 72 herds located in mountainous areas of Trento province (Italy). The herds belonged to 5 main dairy systems ranging from traditional to modern and the cows presented different daily milk yields (24.6±7.9kg × d(-1)), stages of lactation (199±138 d in milk), and parities (2.7±1.8). The PTR-ToF-MS revealed 619 peaks, of which the 240 most intense were analyzed, and 61 of these were tentatively attributed to relevant volatile organic compounds on the basis of their fragmentation patterns and data from the literature. Principal component analysis was used to convert the multiple responses characterizing the PTR-ToF-MS spectra into 5 synthetic variables representing 62% of the total information. These principal components were related to groups of volatile compounds tentatively attributed to different peaks and used to investigate the relationship of the volatile compound profile obtained by PTR-ToF-MS to animal and farm characteristics. Lactation stage is related to 4 principal components which brought together 52.9% of the total variance and 57.9% of the area of analyzed peaks. In particular, 2 principal components were positively related to peaks tentatively attributed to aldehydes and ketones and negatively related to alcohols, esters, and acids, which displayed a linear increase during lactation. The second principal component was affected by dairy system; it was higher in the modern system in which cows received total mixed rations. The third principal component was positively related to daily milk production. In summary, we report the first application of this innovative, high-throughput technique to study the effects of dairy system and individual animal factors on volatile organic compounds of model cheeses. Individual cheesemaking procedures together with this spectrometric technique open new avenues for genetic selection of dairy species with respect to both milk and cheese quality.

Identification of quinoline, carboline and glycinamide compounds in cow milk using HRMS and NMR

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.