Academic literature on the topic 'In vitro gas production'

The kinetics of gas production during ruminal fermentation may provide valuable information about foods that can be used to formulate diets and model animal responses. However, measurement of digestion kinetics is affected by methodology and techniques must be established that provide accurate and precise estimates of kinetic parameters. Because gas production measurements provide the opportunity to estimate the digestion kinetics of both soluble and insoluble matter in foods, it would be desirable to use this technique on a wide variety of forages, grains, supplements, and by-product foods. Applying an in vitro technique to such a wide variety of substrates raises questions about the type of inoculum that should be used. The objective of our study was to evaluate the effects of donor animal and its diet on the measurement of gas production kinetics using both forage and concentrate substrates.

Abstract Research objectives were to evaluate and to analyze in vitro gas production and cinetical fermentation of Kupang’s local feed. The feed materials were dried and grinded for nutrient and gas produced analysis. Collected data were chemical composition, in vitro gas production, cinetical fermentation, short chain fatty acids, energy metabolic, and organic matter digestation. The result showed that the materials have different gas production. Commonly, the higher the carbohydrate content the higher the gas production; the higher the protein and the fat content the lower the gas production. Total gas productions of each material after 96 hours incubation were 53.88 ml/0.5 g DM for the high fat content feed, 92.26 and 159.25 ml/0.5 DM for the high protein and high carbohydrate content. Regarding total production, the fermented fraction gas productions were 44.662; 82.54 and 153.462 ml/0.5 g DM. The short chain fatty acids produced were 0.31; 0.51 and 1.28 mM. The feed organic matter digestations produced were 42.2; 55.91 and 68.8 % for each feed with a high content of fat, protein, and carbohydrate. Conclusively comparing with the protein and carbohydrate feed sources, the higher the fat content the lower the gas production, short chain fatty acids, and organic matter digestion.

Dry matter (DM) degradation of wheat straw (WS), barley straw (BS), lucerne hay (LH) and maize silage (MS) was determined using two different techniques: (i) in vitro gas production and (ii) nylon bag degradability technique. In vitro gas production and in situ DM disappearance were measured after 3, 6, 12, 24, 48, 72 and 96 hours of incubation. In situ and in vitro DM degradation kinetics was described using the equation y = a + b (1 – e^ct). In all incubations there were significant (P < 0.001) correlations between gas production and in situ DM disappearance or estimated parameters ((a + b)_ga_s and (a + b)_is or (a + b)_gas and EDMD_is) whereas there were no significant (P > 0.05) correlations between c_gas and c_is or b_gas and b_is. Gas production from the insoluble fraction (b) alone explained 98.3% of the variation of EDMD. The inclusion of gas production from the quickly soluble fraction (a) and rate constant (c) of gas production in the regression equation improved the accuracy of EDMD prediction. The correlations between the results of both methodologies seem to be sufficiently strong to predict degradability parameters from gas production parameters. It was concluded that the in vitro gas production technique has good potentiality to predict in situ DM disappearance and some DM degradation parameters.    

The stoichiometry of fermentation was investigated in vitro with wheat and maize grains. Gas production proved to be an accurate index of VFA production and change in pH. Gas and total VFA production from wheat were strongly correlated with starch disappearance. On a stoichiometric basis, 66% of gas and 64% of VFAs produced from wheat were accounted for by starch fermentation. With maize only 18% of gas and 23% of VFAs produced were accounted for by starch disappearance. There were significant differences between grain species in rates of gas production (P < 0.001), being ranked in the order wheat > triticale, oats > barley > maize > rice, sorghum. Effects of varieties and growing sites on gas production were significant with wheat, oats, maize and sorghum. With barley, only varietal effects were significant (P < 0.001). With maize and sorghum, there were significant variety by site interactions.

Methodology can play a critical rôle in the measurement of digestion kinetics, especially when the objective is to define kinetic parameters for use in formulating rations or modelling animal responses. Measurement of gas production kinetics provides the opportunity to evaluate the rate of digestion of the soluble, more rapidly fermenting fractions of foods but has the potential for being more sensitive to the in vitro procedure used. Differences among procedures that have little impact on digestion of dry matter after 48 h of incubation, may have dramatic effects on fermentation of soluble matter during the first 20 h. Our objective was to develop a method for measuring the kinetics of gas production that would minimize any detrimental effects associated with the in vitro system and provide estimates of digestion kinetics that can be used to both describe foods for ration formulation systems and provide parameters for models of ruminal digestion.

AbstractThe relationship between in vitro gas production, concomitant in vitro apparent and true DM degradability has been examined in forty-two roughages. The partitioning of truly-degraded substrate between gas volume and microbial biomass yield and 15N incorporation into cells was also investigated. The relevance of this partitioning for the regulation of DM intake (DMI) was examined for fifty-four roughages. The results can be summarized as follows. In vitro gas production and in vitro apparent and true degradability are highly correlated (P<0.0001), r being 0.96 and 0.95 respectively. There is an inverse relationship between in vitro gas production and microbial biomass yield (r—0.67, (P<0.0001) and also 15N enrichment (P<0.001)when the variables were related to a given unit of substrate truly degraded. Selecting roughages by in vitro gas production may well be a selection against maximum microbial yield and a combination of in vitro gas volume measurements with a complementary determination of the substrate truly degraded is proposed, to calculate a partitioning factor (PF) reflecting the variation of short-chain fatty acid production per unit substrate degraded. PF is calculated as the ratio, substrate truly degraded: gas produced by it. PF was highly significant (P<0.0001) in DMI prediction when included in stepwise multiple correlations together with in vitro gas volume variables reflecting the extent and rate of gas production; 11 % of the variation in DMI was accounted for by the PF. The total model, including extent and rate of gas production and the PF, accounted for 84 % of the variation in DMI. Roughages producing proportionally less gas per unit substrate truly degraded had higher feed intakes.

The contents of phenolic compounds, protein precipitation capacity (PPC) and in vitro gas production of tropical browse species were evaluated. The stoichiometric relationship between in vitro gas measured on incubation of tannin-containing browses in buffered rumen fluid and calculated from short chain fatty acid (SCFA) production was investigated. Crude protein (CP) contents in the browses ranged from 54 to 300 g/kg dry matter (DM). Total phenol (TP), tannins (T) and condensed tannins (TP and T as tannic acid equivalent; CT, as leucocyanidin equivalent) ranged from 17–250, 7–214, and 0–260 g/kg DM respectively, and PPC from 0 to 1066 μg BSA precipitated/g DM. CP content of browses was negatively correlated with TP, T, CT and PPC. A significant correlation was observed between per cent change in gas production on addition of polyethylene glycol (PEG) and the contents of phenolics (r = 0.76 for both TP and T). Addition of PEG to tannin-containing browses increased in vitro gas production. PPC was significantly correlated with TP (r = 0.87; P<0.001), T (r = 0.83; P<0.001), and CT (r = 0.41; P<0.05). A good relationship (R2 = 0.94; P<0.001) was observed between measured in vitro gas production and that calculated from SCFA. The molar proportions of SCFA were not affected by the inclusion of PEG (P>0.05). The relationship between in vitro gas measured on incubation of browse leaves and that calculated from SCFA allows prediction of SCFA from gas production.

At the Forage Research Centre, Swedish University of Agricultural Sciences in Umeå a technique has been developed to describe the degradation of feeds in ruminant animals. The development of this technique has been made in collaboration with Dr J.W. Cone, Nutrition and Food, Animal Sciences Group of Wageningen UR, Lelystad, the Netherlands. Experiments have been performed in laboratories where feed samples have been incubated with rumen fluid and the amount of gas produced during the digestion has been measured continuously. These feed samples were analysed at three separate occasions. The purpose of this thesis was to identify and describe statistical procedures for detecting differences between feeds analysed within the same laboratory as well as differences between the same feeds analysed in two different laboratories (in Sweden and the Netherlands). To determine the rate of digestion and to describe to what extent feeds are digested in the rumen a gas production model was fitted, using non linear regression. In order to test whether there are significant differences between the feeds, three methods were applied. For each method, the variances were estimated differently. In the first method, Hotelling’s T2 tests and two sample t tests were performed. From these tests, differences between the feeds that were analysed within the same laboratory were detected whereas no differences between the same feeds analysed in two different laboratories could be found. In the other two methods, tests were performed using an assumption of normality. These two methods detected a larger number of differences between the feeds than the first method, primarily due to extremely underestimated variances. The first method is to be preferred since the estimated variances in this method are unbiased. This causes the result to be more reliable. For future experiments it is recommended that the feed samples are analysed at considerably more than three occasions. This would lead to better estimations in the first method and consequently the result would be enhanced.

The aim of this thesis was to investigate the suitability and application of an in vitro gas production technique as a routine analytical tool in animal nutrition studies. Firstly, the effect of several biological, chemical and physical factors on the measurement of gas production were investigated. These factors included incubation temperature, head-space pressure, shaking movement of the in vitro cultures, the nature of the feedstuff (chemical composition, particle size and drying process), the source of the microbial inoculum and the apparatus used to measure gas production. Secondly, two potential applications for the technique were investigated; (1) as a routine feed analysis tool for the prediction of the digestible energy (DE) content of equine feeds and (2) as a screening method for investigating the potential use of novel feed additives. The first application was investigated by incubating sixteen feedstuffs of known DE. Gas production parameters, DM loss and VFA production were then used to derive prediction equations. The best prediction equation was DE = -0.68 + 0.01087 DML + 6.82 Z - 2.297 log L_T (R² = 0.878; RSD = 0.99; where DML is dry matter loss in vitro, Z is a rate parameter and L_T is the lag time for gas production). The second application was investigated using four antibiotics; monensin, avoparcin, penicillin G and chloramphenicol in the automated gas production system. Differences in gas production profiles were detected between antibiotic supplemented and control fermentations suggesting that the technique can be used to screen feed additives. Results presented in this thesis inform the development of standardised methodologies and procedures for use during in vitro gas production studies, thus enabling this technique to be adopted as a robust, reliable and routine analytical tool in animal nutrition studies.

This study was aimed to assess the effect of fecal inocula from horses fed on concentrate (restricted amount daily) and oat straw (ad libitum) supplemented with fibrolytic enzymes on in vitro hindgut activity. Cellulase (CE), xylanase (XY) and CE+XY (1:1 v/v; CX) were tested at three levels (μL/g DM): 0, 1 and 3, in addition to control without enzyme addition (EP0). Fecal inocula were collected from 16 Quarter Horse mares supplemented with enzyme at 0 (FCOwithout enzyme), or fed 10 ml enzyme/mare/day of CE (FCE), XY (FXY) or CE+XY (1:1 v/v; FCX) for 15 days. The fecal content mixed with the culture media were used for incubation in bottles containing 1 g DM of substrate (a mixture of concentrate and oat straw (1:1 DM)). Gas (GP), methane (CH4) and carbon dioxide (CO2) productions were measured at 2, 4, 6, 8, 10, 12, 24 and 48 h post-incubation. Interactions occurred (P˂0.05) between fecal type × enzyme product for the asymptotic GP, the rate of GP, CH4 production, and fermentation kinetic parameters. Moreover, interactions were observed (P˂0.05) between fecal type × enzyme product × enzyme dose for the rate of GP, CH4 production and DM digestibility. Xylanase at 3 μL/g DM with FXY fecal increased (P˂0.05) the asymptotic GP and calculated fermentation parameters. At 24 h and 48 h and without enzyme, FCX and FXY, respectively, had the highest (P˂0.05) CH4 production. It can be concluded that xylanase enzyme at 3 μL/g DM was the most effective compared to other treatments.

The purpose of this thesis was to adapt the in vitro gas production technique (IVGPT) to estimate the rumen degradation profile of feed protein and to investigate the effect of sample preparation on the estimated kinetics of silage carbohydrate and protein degradation in the rumen.

The influence of microbial activity on the pattern of gas production and the possibility of developing a protocol for standardising the microbial activity of ruminal fluid were investigated (chapter 2). Firstly, ruminal fluid was diluted 1:2, 1:5, 1:9 and 1:20 (v/v) with buffer solution and the in vitro gas production from cellulose, glucose, hay and ryegrass studied. Gas production at 4, 24 and 120h were significantly decreased as the ratio of buffer solution to ruminal fluid increased. For each substrate, incubation with ruminal fluid diluted 1:2 (v/v) gave a higher (P<0.5) asymptotic value 'B', rate 'C' of gas production and lower lag time than when incubated in the 1:5, 1:9 and 1:20 dilutions. Secondly, the effect of frequency of ovine ruminal sampling on microbial activity and substrate fermentation was investigated. The bacterial DM, bacterial absorbance and blank's gas volume were significantly affected by daily sampling of ruminal fluid. Daily sampling of rumen fluid did not significantly affect the gas production degradability parameters (B,C and Lag) until the bacterial DM fell below 0.09 g DM/10 cm³ strained rumen fluid. Bacterial DM was strongly related to the absorbance of ruminal fluid (R² = 0.99, p<0.001), suggesting that standardisation of the level of microbial activity between days and laboratory can be achieved by estimating the bacterial DM from the absorbance of the strained ruminal fluid. The influence of donor diet on initial microbial concentration and gas production degradability was studied (chapter 3). Bacterial DM, bacterial absorbance and the blanks' gas volume increased significantly as the ratio of sheep pellet to hay increased. The gas production degradability parameters (B, C and Lag) were also affected by changing the donor diet ratio of sheep pellets and hay. NDF digestibility of cellulose and hay was not significantly affected by donor diet. Bacterial DM was strongly related to the absorbance of ruminal fluid and the blanks' gas volume (R² = 0.99, p<0.001). These results suggest that changing the ratio of concentrate to hay reduced the initial bacterial concentration and affected the gas production degradability parameters.

ABSTRACT: The study aimed to assess the nutritive value in vitro of 4 feeds (grains and forages) commonly used in horses nutrition in Mexico, in the absence or presence of Saccharomyces cerevisiae at 4 mg/g DM. Fecal inoculum was obtained from 4 adult English Thoroughbred horses fed on restricted amount of concentrate and oat hay ad libitum. The incubated substrates included were corn gluten meal, soybean meal, oat grain and alfalfa hay. Gas production was recorded at 2, 4, 6, 8, 10, 12, 14, 24, 48 and 70 h using the Pressure Transducer Technique. Some ingredient × yeast interactions were observed (P < .020) for the asymptotic gas production (GP) and GP at 48 and 70 h of incubation. Yeast addition increased (P < .001) the asymptotic GP of concentrates compared to forages. Concentrate feeds had higher (P < .05) GP and lower (P < .001) rate of GP compared to forages without yeast. From 24 to 70 h of incubation, forages with or without yeast had lower (P < .05) GP compared to concentrates with yeast addition. Forages had higher fermentation pH compared to concentrates, but lower (P < .05) metabolizable energy (ME), in vitro organic matter digestibility (IVOMD) and microbial protein production (MBP) compared to concentrates. Yeast addition increased (P < .05) the asymptotic GP of oat grain and soybean meal, without affecting the rate of GP or lag time of both. Yeast treatment improved fermentation of feeds with higher effects on concentrates compared to forage. It was concluded that concentrate feeds had higher nutritive value than forages commonly fed to horses

Purple threeawn (Aristida purpurea Nutt.) is a native bunch grass that is avoided by grazers. It is capable of dominating old cropland and overgrazed pastures, limiting livestock carrying capacity, and degrading wildlife habitat. Traditional management tools have had little impact on threeawn dominance in semiarid regions of the west. Our objectives were to: 1) assess fire and nitrogen treatment effects on threeawn forage quality at various phenological stages to test their potential as pretreatments in a grazing strategy and 2) examine a threeawn-dominated plant community?s response to prescribed fire, nitrogen addition, and clipping. Fire improved threeawn forage quality with greater improvements in early phenological stages. Nitrogen had little effect on forage quality. Fire and nitrogen reduced threeawn while increasing cool season grasses. Light and moderate clipping following fire did not improve the efficacy of fire. Fire appears to an effective preliminary treatment to improve the chance of herbivory.

Due to the warming of the climate system, public opinion has increased its interest in greenhouse gas, with particular predilection for methane (CH4 ) and their relationship with livestock sector. For this reason, several researches have proposed different nutritional strategies to reduce methane production in ruminants. The two main aims of this thesis were: i) to study methodological factors affecting in vitro CH4 production and to develop a new approach to measure in vitro CH4 production; ii) to evaluate effectiveness of some nutritional strategies on in vitro CH4 reduction. The objectives have been achieved through the research activities and reported in five experimental contributions. The first contribution aimed to analyse specific factors affecting in vitro gas production (GP), CH4 production and their ratio by a meta-analysis approach using 274 control treatments from 39 scientific papers published over 12 years. The common factors included in the analysis were: the pressure in the GP equipment (constant or increasing), the incubation time (24 or ≥48 h), the timing of rumen fluid collection (before or after feeding of donor animals), the presence of N in the buffer solution (presence or absence), and the ratio between amount of buffered rumen fluid and feed sample (<130 or 130-140 or >140 ml/g DM). The NDF content of feed sample incubated (392 ± 175.3 g/kg) was considered as a continuous variable. Results showed that GP measures were influenced mainly by the incubation time; values of CH4 production were affected mainly by the ratio between amount of buffered rumen fluid and feed sample; values of CH4 proportion (CH4 /GP) were mainly affected by timing of rumen fluid collection. Results suggest that factors considered in the meta-analysis explained a significant part (about 65%) of variability of GP and CH4 measures obtained in vitro. Thus, a higher standardization of specific methodological protocols would be desirable in order to compare results of different trials. The second contribution compared two in vitro techniques (closed or vented bottles) of sampling and measuring CH4 production from ruminant feeds. Five feeds (meadow hay, ryegrass hay, corn grain, dry sugar beet pulp, and expeller flaxseed) were tested. The experimental design was: 2 incubations×5 feeds×3 replications×2 gas sampling techniques, plus 4 blanks (bottles without feed sample), for a total of 64 bottles incubated. Half of the bottles were not vented during the incubation, whereas the remaining were vented at a fixed pressure and gas was collected into a tight bag connected to each bottle. At the end of each incubation, gas samples were sampled from the headspace of closed bottles or from headspace and bags of vented bottles and analyzed for CH4 concentration. GP values were adjusted or not for the amount of CO2 solubilized in the fermentation fluid. Values of CH4 concentration (ml CH4 /100 ml gas) and production (ml CH4 /g DM) were computed using corrected or uncorrected GP values. Closed bottles showed lower uncorrected GP (−18%) compared with vented bottles, whereas the correction for dissolved gas reduced but did not remove differences between techniques. Closed bottles showed unadjusted CH4 concentrations 23% greater than that of vented bottles, but the adjustment of measurements for solubilized CO2 reduced but did not remove this difference. Adjusted CH4 production was not influenced by technique. Closed bottles provide good measurements of CH4 production but not of GP. Venting of bottles at low pressure permits a reliable evaluation of total GP and CH4 production. The third contribution evaluated the influence that changes in main dietary constituents (starch:ADF ratio, protein and lipid contents) could have on in vitro GP, CH4 concentration and production. All changes have been made within ranges of diets used in intensive farms of the North Italy. A reference diet was used (273, 361, 158, and 33 g/kg DM of starch, aNDF, CP, and lipids, respectively). Other 6 diets had a smaller or a greater starch:ADF ratio (0.40 or 1.77, respectively), CP content (115 or 194 g/kg DM, respectively), or lipid content (26 or 61 g/kg DM, respectively), compared to the reference diet. The experimental design was: 4 incubations × 7 diets × 5 replications, plus 20 blanks (5 blanks/run), for a total of 160 bottles. Bottles were vented at fixed pressure and at the end of each incubation, gas samples were collected from the bottle headspace and analyzed for CH4 . The proportion of CH4 lost with venting was estimated. An increasing starch:ADF ratio reduced GP per g DM and per g TDMd, increased the amount of CH4 produced per g DM, whereas CH4 produced per g TDMd was unchanged. The increase of CP content decreased GP, whereas CH4 production was reduced in low protein diet only when expressed as proportion of GP. The increase of lipid content reduced GP per g DM, but no influences were observed on CH4 values. Results of this study showed that the magnitude of effects exerted by the dietary changes on in vitro fermentation was small. The fourth contribution aimed to explore effects of 4 pure plant extracts (allyl-sulfyde; cinnamaldehyde; eugenol; limonene) and 1 synthetic compound (monensin), all with antimicrobial properties, added to a commercial diet for dairy cows, on GP, CH4 concentration and production. Two dosages were tested: 3 or 30 mg/g of diet for pure plant extracts, 0.015 or 0.030 mg/g of diet for monensin. Incubation procedures were the same as the previous experiment. The experimental design was: 4 incubations×5 additives×2 dosages×3 replications, plus 12 bottles as control (without additive; 3/run) and 12 bottles as blanks (3/run), for a total of 144 bottles. Results showed that low dosages of all compounds did not exert any effect on in vitro GP and CH4 production. Compared to the control, high dosage of allyl-sulfyde, cinnamaldehyde, eugenol, limonene, and monensin significantly reduced in vitro GP (ml/g DM; -16%, -12%, -9%, -38%, -12%, respectively). In vitro CH 4 production was significantly reduced only by high dosage of allyl-sulfyde, cinnamaldehyde, limonene, and monensin (-32%, -12%, -43%, -18%, respectively, compared to the control). Only high dosage of allyl-sulfyde and limonene significantly reduce CH4 proportion (-18%, -12% respectively, compared to the control). The most promising results were observed for cinnamaldehyde, as the depression of CH4 production was not accompanied by a reduction of in vitro degradability. The fifth contribution investigated the possibility to reduce in vitro gas and CH4 production by combining a direct inhibitor of methanogenisis (chloroform) with two indirect inhibitors: i) nitrate, acting as a H2 sink, and ii) saponins, acting as antiprotozoal agent. The experiment was conducted using a semi-continuous flow system (RUSITEC ®). A diet, based on grass hay, was incubated alone (diet 1) or supplemented with 31.5 g/kg of nitrate (diet 2) or with 50 g/kg of saponins extracted from ivy extract (diet 3). Three diets were incubated without or with chloroform (2 µL/L), obtaining a total of 6 dietary treatments. Each of the 6 dietary treatments was incubated in four vessels, for a total of 24 vessels. Each vessel was inoculated with 800 ml of buffered rumen fluid. Whole incubation lasted 21 d. Total GP (on average 2.56 L/d) was not affected by diet or chloroform addition. When incubated alone, chloroform, nitrate and saponins reduced daily CH4 production by 96, 66 and 22%, respectively. However, when chloroform was combined with indirect inhibitors, neither nitrate or saponins had any additional effect in chloroform treated vessels on in vitro CH4 production. Results suggest that no synergistic effects emerged between the direct inhibitor and the two indirect inhibitors.
Il recente scenario del riscaldamento climatico globale, ha portato ad un maggiore interesse dell’opinione pubblica riguardo ai gas che causano l’effetto serra, ponendo particolare attenzione al gas metano (CH4 ) e alla sua relazione con il settore zootecnico. Per questa ragione, numerosi gruppi di ricerca hanno proposto delle strategie nutrizionali per ridurre le emissioni di CH4 da parte dei ruminanti. Nella presente tesi sono stati sviluppati due obiettivi principali. Il primo riguardava lo studio dei fattori metodologici che influenzano la produzione in vitro del CH4 e lo sviluppo di un nuovo approccio per la misurazione dello stesso gas prodotto durante delle fermentazioni in vitro. In secondo luogo, si è inteso valutare l’efficacia di alcune strategie nutrizionali sulla riduzione della produzione di CH4 . Tali obiettivi sono stati raggiunti attraverso cinque diverse attività di ricerca. Nel primo lavoro è stato valutato l’effetto che specifici fattori metodologici possono avere sulla produzione in vitro di gas e di CH4 (espresso sia in termini di produzione che di proporzione). Questa analisi è stata effettuata attraverso un approccio di meta-analisi utilizzando 274 osservazioni, che rappresentavano i trattamenti di controllo di 39 articoli scientifici pubblicati negli ultimi 12 anni. I fattori considerati erano: la pressione che si forma nello spazio di testa dello strumento utilizzato per le fermentazioni in vitro (costante o incrementale), il tempo di incubazione (24 o ≥48 ore), il momento di raccolta del liquido ruminale (prima o dopo la somministrazione del pasto agli animali donatori), la presenza di azoto nella composizione della saliva artificiale (presenza o assenza), e il rapporto tra la miscela di liquido ruminale e saliva artificiale sul campione alimentare incubato (130 or 130-140 or >140 ml/g DM). Questi cinque fattori sono stati considerati come variabili discrete, invece il contenuto di NDF del campione alimentare incubato (392 ± 175.3 g/kg) è stato considerato come variabile continua. I risultati hanno mostrato che la produzione totale di gas è principalmente influenzata dal tempo di incubazione, mentre la produzione di CH4 è influenzata soprattutto dal rapporto tra la miscela di liquido ruminale e saliva artificiale sul campione alimentare incubato. Quando invece il CH4 viene espresso in termini di proporzione sul gas totale prodotto, i valori sono principalmente influenzati dal momento di raccolta del liquido ruminale. Inoltre, questa meta-analisi dimostra che i fattori considerati spiegavano una parte considerevole (circa il 65 %) della variabilità dei dati di gas e CH4 prodotti durante le fermentazioni in vitro. Quindi, sarebbe desiderabile una maggiore standardizzazione dei protocolli metodologici internazionali, in modo da facilitare il confronto di dati ottenuti in diversi sperimentazioni. Il secondo contributo sperimentale ha inteso mettere a punto una procedura di raccolta e di misurazione del CH4 prodotto, utilizzando due diverse tipologie di fermentazione in vitro: i) un “sistema aperto”, con sfiato regolare del gas all’interno di un sacchetto connesso alle bottiglie di fermentazione, dal quale viene prelevato il campione di gas per l’analisi del CH4 ; ii) un “sistema chiuso”, con accumulo progressivo dei gas di fermentazione nello spazio di testa delle bottiglie, dal quale viene prelevato il campione per l’analisi del CH4 . Per le fermentazioni sono stati usati cinque alimenti singoli utilizzati nell’alimentazione dei ruminanti (fieno polifita, loietto, farina di mais, panello di lino e polpe di bietola). Il disegno sperimentale prevedeva: 2 incubazioni × 5 alimenti × 3 replicazioni per alimento × 2 tecniche di campionamento del gas, più 4 bianchi (bottiglie incubate senza campione alimentare), per un totale di 64 bottiglie incubate. Metà delle bottiglie non venivano sfiatate, mentre le rimanenti venivano sfiatate a pressione fissa e il gas era raccolto in un sacchetto a tenuta connesso ad ogni bottiglia. Alla fine di ogni incubazione, il gas veniva campionato dallo spazio di testa delle bottiglie utilizzate per il sistema chiuso o dallo spazio di testa e dal sacchetto delle bottiglie utilizzate per il sistema aperto. Tutti i campioni sono stati poi analizzati per quantificare la concentrazione di CH4 . I valori di gas prodotto venivano o meno corretti per la quantità di CO2 che si era disciolta nel liquido di fermentazione. I valori di produzione (ml CH4 /g DM) e di proporzione (ml CH4 /100 ml gas) di CH4 sono stati calcolati utilizzando i valori corretti o non corretti di produzione di gas totale. Il sistema chiuso ha mostrato una produzione di gas totale non corretto inferiore (-18%) rispetto al sistema aperto, mentre la correzione del gas ha ridotto ma non rimosso le differenze tra le due tecniche. Le bottiglie chiuse hanno mostrato una proporzione di CH4 non corretto superiore (+23%) rispetto al sistema aperto, mentre la correzione ha ridotto ma non rimosso le differenze tra le due tecniche. La produzione di CH4 corretto non è stata influenzata dalla tecnica utilizzata. Concludendo, il sistema chiuso non offre buone misurazioni della produzione del gas, mentre il sistema aperto consente una valutazione attendibile sia del gas che del CH4 prodotto. Nel terzo contributo sperimentale è stato valutato l’effetto che le variazioni quantitative dei principali componenti chimici (rapporto amido:ADF, contenuto proteico e contenuto lipidico) di diete per vacche da latte, posso avere sulla produzione di gas e CH4 . Le variazioni sono state fatte tenendo conto degli intervalli di fibra, amido, proteina e lipidi effettivamente utilizzate negli allevamenti intensivi del Nord Italia. La dieta di riferimento utilizzata aveva la seguente composizione chimica: 273, 361, 158, e 33 g/kg SS di amido, NDF, CP, e lipidi, rispettivamente. Le altre 6 diete avevano un minore o maggiore rapporto di amido:ADF (0.40 or 1.77, rispettivamente), o di contenuto proteico (115 or 194 g/kg DM, rispettivamente), o di contenuto lipidico (26 or 61 g/kg DM, rispettivamente), rispetto alla dieta di riferimento. Il disegno sperimentale prevedeva: 4 incubazioni × 7 diete × 5 replicazioni per dieta, più 20 bianchi (bottiglie incubate senza campione alimentare), per un totale di 160 bottiglie incubate. Il gas prodotto veniva regolarmente sfiatato a pressione fissa e alla fine di ogni incubazione un campione di gas veniva raccolto dallo spazio di testa delle bottiglie e veniva analizzato per misurare la concentrazione di CH4 . La quantità di CH4 perso durante lo sfiato del gas di fermentazione è stata stimata. I risultati mostrano che all’aumentare del rapporto amido:ADF si riduce la produzione di gas (per g di SS e per g di degradabilità "vera" della SS), aumenta la produzione di CH4 (per g di SS), mentre la produzione di CH4 espressa come g di degradabilità "vera" della SS non varia. All’aumentare del contenuto proteico si riduce la produzione di gas, mentre la proporzione di CH4 è stata ridotta solo nella dieta ipoproteica. L’aumento del contenuto lipidico ha ridotto la produzione di gas (per g di SS), ma non ha influenzato i valori di CH4 . Si può quindi concludere che l'entità degli effetti, esercitati dalle variazioni quantitative dei principali componenti chimici delle diete sulla fermentazione in vitro, è stata quasi inesistente. Il quarto lavoro ha inteso valutare l’effetto di quattro estratti puri di piante (allil-sulfide, cinnamaldeide, eugenolo e limonene) e di un composto sintetico (monensin), tutti con proprietà antimicrobiche, utilizzati come additivi di una dieta per vacche da latte, sulla produzione di gas e CH4 . Sono stati utilizzati due diversi dosaggi: 3 or 30 mg/g di per gli estratti puri di piante, 0.015 or 0.030 mg/g di dieta per il monensin. Le procedure di incubazioni utilizzate erano le stesse dell’esperimento precedente. Il disegno sperimentale prevedeva: 4 incubazioni × 5 additivi × 2 dosaggi × 3 replicazioni, più 12 bianchi (bottiglie incubate senza campione alimentare, 3 per incubazione), e 12 controlli (dieta incubata senza additivi, 3 per incubazione) per un totale di 144 bottiglie incubate. I risultati hanno mostrato che tutti i composti testati con il basso dosaggio non hanno mai influenzato la produzione in vitro di gas e CH4 . Rispetto al controllo, gli alti dosaggi di allil- sulfide, cinnamaldeide, eugenolo, limonene, e monensin hanno ridotto significativamente la produzione in vitro di gas (ml/g DM; -16%, -12%, -9%, -38%, -12%, rispettivamente). La produzione in vitro di CH4 è stata significativamente ridotta solo dell’alto dosaggio di allil-sulfide, cinnamaldeide, limonene, e monensin (-32%, -12%, -43%, -18%, rispetto al controllo). Solo gli alti dosaggi di allil- sulfide e limonene hanno ridotto significativamente anche la proporzione di CH4 (-18% e -12% rispetto al controllo). I risultati più promettenti sono stati osservati per la cinnamaldeide, che ha depresso la produzione CH4 senza influenzare negativamente gli altri parametri fermentativi. Il quinto e ultimo contributo sperimentale ha valutato la possibilità di ridurre la produzione in vitro di gas e CH4 attraverso la combinazione di un diretto inibitore del CH4 (cloroformio) con due inibitori indiretti: i) i nitrati, che agiscono come accettori di idrogeno e ii) le saponine, dei noti agenti antiprotozoari. L’esperimento è stato condotto utilizzando un sistema di simulazione ruminale a flusso semi-continuo (RUSITEC®). Una dieta base è stata incubata singolarmente (dieta 1) o addizionata con 31.5 g/ kg di nitrati (dieta 2) o con 50 g/kg saponine ottenute dall’estratto d’edera (dieta 3). Queste tre diete sono state incubate senza o con l’aggiunta di cloroformio (2 µL/L), ottenendo un totale di 6 trattamenti testati. Ogni trattamento è stato incubato in quattro bottiglioni, per un totale di 24 bottiglioni. L’intera incubazione è durata 21 giorni. I risultati mostrano che la produzione totale di gas (in media 2.56 l/d) non è stata influenzata dalla dieta o dalla presenza del cloroformio. Quando utilizzati singolarmente, cloroformio, nitrati e saponine riducono la produzione giornaliera di CH4 del 96, 66 e 22%, rispettivamente. Nonostante ciò, quando il cloroformio era combinato con inibitori indiretti, non si è evidenziato nessun effetto addizionale sulla riduzione di CH4 prodotto. Concludendo, si può quindi affermare che nessun effetto sinergico è emerso tra l’inibitore diretto di CH4 e i due inibitori indiretti.

Com o objetivo de estudar o uso de inóculo de fezes em substituição do conteúdo ruminal de bubalinos na técnica in vitro de produção de gases, este presente trabalho comparou as avaliações realizadas em três ensaios. Foram utilizados três bubalinos da raça Mediterrâneo, machos, adultos, castrados, fistulados no rúmen com média de peso vivo de 450 (± 18,7) kg. Estes animais receberam uma dieta basal, composta de silagem de milho (70%) e concentrado (30%). Estes animais foram os doadores dos 2 tipos de inóculos, que foram conteúdo ruminal (CR) e fezes. O primeiro ensaio realizado foi com alimentos concentrados: grão de milho, farelo de soja, farelo de trigo e farelo de algodão; o segundo ensaio foi com leguminosas forrageiras: alfafa (Medicago sativa L. ), estilosantes pioneiro (Stylosanthes macrocephala cv. Pioneiro), soja perene (Neonotonia wightii) e leucena (Leucaena leucocephala); e o terceiro e último ensaio foi realizado com gramíneas forrageiras: capim braquiarão (Brachiaria brizantha cv. Marandu), capim buffel (Cenchrus ciliaris L. cv. Biloela), estrela africana (Cynodon plectostachyus) e capim mombaça (Panicum maximum Jacq. cv. Mombaça). Os valores médios obtidos de produção potencial de gases em cada ensaio foram menores (P<0,05) para as amostras fermentadas com inóculos de fezes que com conteúdo ruminal, sendo respectivamente, para concentrados (140,23 e 194,08 mL.g-1MS), gramíneas (161,99 e 230,25 mL.g-1MS) e leguminosas (141,78 e 170,70 mL.gm-1MS). Conclui-se que inóculos de fezes não apresentam condições satisfatórias para substituição do inóculum com conteúdo ruminal para uso na técnica de produção in vitro de gases.
In order to study the use of faeces as inoculum as alternative for buffalo rumen contents in the in vitro gas production technique, the present work evaluations of three tests. We used three Mediterranean buffalos, male, adult, neutered, fistulated in the rumen, with an average live weight of 450 (± 18.7) kg. These animals received a basal diet composed of corn silage (70%) and concentrated (30%). These buffalo were the donors of the two types of inocula, rumen content (CR) and faeces. The first test was carried out with concentrate foods: corn grain, soybean meal, wheat bran and cottonseed meal, the second test was with legumes: alfalfa (Medicago sativa L. ), Pioneiro estilo (Stylosanthes macrocephala cv. Pioneiro) , perennial soybean (Neonotonia wightii) and leucaena (Leucaena leucocephala) and the third and last test was carried out with grasses: Marandu grass (Brachiaria brizantha cv. Marandu), buffel grass (Cenchrus ciliaris L. cv. Biloela), African Star grass (Cynodon plectostachyus) and Mombasa grass (Panicum maximum Jacq. cv. Mombasa). The average values of potential production of gas in each test were lower (P<0.05) for samples fermented with an inoculum of faeces with rumen contents, being respectively, for concentrates (140.23 and 194.08 mL.g-1MS), grasses (161.99 and 230.25 mL.g-1MS) and legumes (141.78 and 170.70 mL.g-1MS). It follows that fecal inoculum unsatisfactory condition for replacing the inoculum with rumen technique for use in the in vitro production of gases technique.

Based on flow assurance studies a Pipe-in-Pipe solution was selected to assure the flow for a gas pipeline connecting the well 4-ESS-138 to FPSO Cidade de Vito´ria located in Canapu field. The flow assurance studies also define the value of 0.8W/m2K for the overall heat transfer coefficient (OHTC) in order to prevent hydrate formation. The temperature of 87°C on the wellhead should be preserved to permit the proper gas flow in 20km PIP length between wellhead and FPSO. Canapu field is located in the offshore area of Brazil (Espirito Santo state) in water depths of 1608m. This paper presents the main aspects related to the detailed design, thermo-mechanical requirements, materials specifications, functional qualification tests performed on materials and on pipe-in-pipe systems to satisfy installation constraints defined by the reel-lay method and operational issues.

Articular cartilage is the load bearing soft tissue of diarthrodial joints, and mechanical loading maintains the integrity of the tissue. The predominant extracellular matrix constituents, proteoglycans and collagen, allow cartilage to support the high compressive and tensile loads experienced in diurnal loading. Our laboratory has been successful in cultivating engineered cartilage constructs with a compressive equilibrium modulus and glycosaminglycan (GAG) content near native values [1, 2]. Many approaches to cultivating engineered cartilage have been limited by low collagen production in vitro, an impediment for attaining native functional load-bearing properties [3].

Numerous in vitro studies have demonstrated that chondrocytes react to mechanical stimuli. Compression of chondrocytes in an agarose gel induces disruption of the actin cytoskeleton [1]. In addition, chondrogenic gene expression decreases for chondrocytes cultured in monolayer, while inhibition of actin polymerization causes an increase in type II collagen and GAG production [2]. Despite such extensive in vitro investigations, the mechanisms by which chondrocytes actively respond to mechanical loading are not well understood. Simple hyperelastic and viscoelastic computational cell models have previously been used to model chondrocytes. However, such passive models ignore the key biomechanisms by which cells sense and react to external loads, and hence offer limited insight or predictive capability [3].

Introduction : Le plasma atmosphérique froid est un gaz ionisé produit à pression atmosphérique. Plusieurs applications médicales sont étudiées, notamment la cicatrisation des plaies chroniques et l’effet antimicrobien. En effet, le traitement par plasma permet de générer de nombreuses espèces réactives de l’oxygène et de l’azote. L’application d’un tel traitement in-vitro sur des cellules eucaryotes a montré de nombreux effets cellulaires tel que l’apoptose. Les applications dans le domaine de l’oncologie ont par conséquent été étudiées. Objectif : L’objectif de cette revue systématique est d’analyser l’utilisation du plasma atmosphérique froid en oncologie ainsi que les méthodologies (lignées cellulaires ciblées, paramètres physiques, thérapies directes ou indirectes) mises en oeuvre jusqu’à ce jour. Matériels et méthodes : Les bases de données Pubmed, ICTRP et Google Scholar ont été explorées jusqu’au 17/01/2017 afin de recenser les études traitant de l’utilisation du plasma en oncologie, que ce soit des études in-vitro, in-vivo ou des essais cliniques. Résultats : 150 articles originaux ont été inclus. Les Jets de plasma sont les systèmes de production de plasma les plus utilisés (73,3%). L’hélium est le gaz le plus utilisé (34%) suivi par l’air (28%) et l’argon (19,3%). Les études sont principalement in-vitro (94%). L’application directe du plasma est la plus représentée (84,2%). Les lignées cellulaires ciblées sont la plupart dérivées de lignées cancéreuses humaines (82%), en particulier des lignées issues de cancer du cerveau (16,6%). Conclusions : Cette étude met en évidence la multiplicité de moyens de production et d’applications clinques du plasma atmosphérique froid en oncologie. Alors que certains dispositifs peuvent être utilisés directement sur les patients, d’autres ouvrent la voie au développement de nouveaux produits pharmaceutiques qui pourraient être produits à échelle industrielle. L’utilisation clinique du plasma nécessite la mise au point de protocoles fiables et standardisés afin de déterminer le plasma le plus adapté à chaque type de cancers et d’envisager son association avec les traitements conventionnels.

Abstract For an open pipe pile installed on the North Sea to serve as the foundation for an offshore gas production platform the capacity was determined in various ways. First the Cone Penetration Test data obtained at the location were used to simulate the vibro driving process. Then the monitoring data obtained during vibro driving were used adjust the soil model used for the initial simulation and to derive the pile capacity. Next the monitoring data for the Beginning of Restrike and the End of Impact Driving were used to assess the capacity yet again. The results as well as the soil models used for the simulations and the capacity estimates are presented to demonstrate that there is no need to drive a pile to its final penetration with an impact hammer to assess its bearing capacity, which when adopted in the industry will greatly reduce pile driving duration and thus cost.

Abstract For an open pipe pile installed on the North Sea to serve as the foundation for an offshore gas production platform the capacity was determined in various ways. First the Cone Penetration Test data obtained at the location were used to simulate the vibro driving process. Then the monitoring data obtained during vibro driving were used adjust the soil model used for the initial simulation and to derive the pile capacity. Next the monitoring data for the Beginning of Restrike and the End of Impact Driving were used to assess the capacity yet again. The results as well as the soil models used for the simulations and the capacity estimates are presented to demonstrate that there is no need to drive a pile to its final penetration with an impact hammer to assess its bearing capacity, which when adopted in the industry will greatly reduce pile driving duration and thus cost.

Hepatitis E virus (HEV) is a member of the Hepeviridae family capable of infecting humans producing a range of symptoms from mild disease to kidney failure. Epidemiological evidence suggests that hepatitis E genotype III and IV cases may be associated with the consumption of undercooked pork meat, offal and processed products such as sausages [1]. A study carried out by the Animal Health and Veterinary Laboratories Agency (AHVLA), found hepatitis E virus contamination in the UK pork production chain and that 10% of a small sample of retail pork sausages were contaminated with the virus [2]. Furthermore, studies have confirmed the presence of HEV in the food chain and the foodborne transmission of Hepatitis E virus to humans [reviewed in 5]. Likewise, Scottish shellfish at retail [6] have also been found positive for HEV viral nucleic acid and some preliminary studies indicate that the virus is also detectable in soft fruits (L Scobie; unpublished data). There are current misunderstandings in what this data represents, and these studies have raised further questions concerning the infectivity of the virus, the processing of these foods by industry and the cooking and/or preparation by caterers and consumers. There are significant gaps in the knowledge around viral infectivity, in particular the nature of the preparation of food matrices to isolate the virus, and also with respect to a consistent and suitable assay for confirming infectivity [1,3]. Currently, there is no suitable test for infectivity, and, in addition, we have no knowledge if specific food items would be detrimental to cells when assessing the presence of infectious virus in vitro. The FSA finalised a comprehensive critical review on the approaches to assess the infectivity of the HEV virus which is published [3] recommending that a cell culture based method should be developed for use with food. In order to proceed with the development of an infectivity culture method, there is a requirement to assess if food matrices are detrimental to cell culture cell survival. Other issues that may have affected the ability to develop a consistent method are the length of time the virally contaminated sample is exposed to the cells and the concentration of the virus present. In most cases, the sample is only exposed to the cells for around 1 hour and it has been shown that if the concentration is less that 1x103 copies then infection is not established [3,5,10,11].

Rugose wood is a complex disease of grapevines, characterized by modification of the woody cylinder of affected vines. The control of rugose wood is based on the production of healthy propagation material. Detection of rugose wood in grapevines is difficult and expensive: budwood from tested plants is grafted onto sensitive Vitis indicators and the appearance of symptoms is monitored for 3 years. The etiology of rugose wood is complex and has not yet been elucidated. Several elongated clostero-like viruses are consistently found in affected vines; one of them, grapevine virus A (GVA), is closely associated with Kober stem grooving, a component of the rugose wood complex. GVA has a single-stranded RNA genome of 7349 nucleotides, excluding a polyA tail at the 3' terminus. The GVA genome includes five open reading frames (ORFs 1-5). ORF 4, which encodes for the coat protein of GVA, is the only ORF for which the function was determined experimentally. The original objectives of this research were: 1- To produce antisera to the structural and non-structural proteins of GVA and GVB and to use these antibodies to establish an effective detection method. 2- Develop full length infectious cDNA clones of GVA and GVB. 3- Study the roll of GVA and GVB in the etiology of the grapevine rugose wood disease. 4- Determine the function of Trichovirus (now called Vitivirus) encoded genes in the virus life cycle. Each of the ORFs 2, 3, 4 and 5 genes of GVA were cloned and expressed in E. coli and used to produce antisera. Both the CP (ORF 4) and the putative MP (ORF 3) were detected with their corresponding antisera in-GVA infected N. benthamiana and grapevine. The MP was first detected at an early stage of the infection, 6-12 h after inoculation, and the CP 2-3 days after inoculation. The MP could be detected in GVA-infected grapevines that tested negative for CP, both with CP antiserum and with a commercially available ELISA kit. Antisera to ORF 2 and 5 encoded proteins could react with the recombinant proteins but failed to detect both proteins in GVA infected plants. A full-length cDNA clone of grapevine virus A (GVA) was constructed downstream from the bacteriophage T7 RNA polymerase promoter. Capped in vitro transcribed RNA was infectious in N. benthamiana and N. clevelandii plants. Symptoms induced by the RNA transcripts or by the parental virus were indistinguishable. The infectivity of the in vitro-transcribed RNA was confirmed by serological detection of the virus coat and movement proteins and by observation of virions by electron microscopy. The full-length clone was modified to include a gus reporter gene and gus activity was detected in inoculated and systemic leaves of infected plants. Studies of GVA mutants suggests that the coat protein (ORF 4) is essential for cell to cell movement, the putative movement protein (ORF 3) indeed functions as a movement protein and that ORF 2 is not required for virus replication, cell to cell or systemic movement. Attempts to infect grapevines by in-vitro transcripts, by inoculation of cDNA construct in which the virus is derived by the CaMV 35S promoter or by approach grafting with infected N. benthamiana, have so far failed. Studies of the subcellular distribution of GFP fusion with each of ORF 2, 3 and 4 encoded protein showed that the CP fusion protein accumulated as a soluble cytoplasmatic protein. The ORF 2 fusion protein accumulated in cytoplasmatic aggregates. The MP-GFP fusion protein accumulated in a large number of small aggregates in the cytoplasm and could not move from cell to cell. However, in conditions that allowed movement of the fusion protein from cell to cell (expression by a PVX vector or in young immature leaves) the protein did not form cytoplasmatic aggregates but accumulated in the plasmodesmata.

This proposal is focused on examination of two plant interactions: parasitic with Orobanche, and symbiosis with arbuscular mycorrhiza fungi (AMF), and the involvement of a newly define plant hormones, strigolactones (SLs), in these plant interactions. In addition to strigolactones role in regulation of above-ground plant architecture, they are also known to be secreted from roots, and to be a signal for seed germination of the parasitic plants Orobanche. Moreover, secreted strigolactones were recognized as inducers of AMFhyphae branching. The present work was aimed at Generation of RNAi mutants of both tomato and Medicago, targeting multiple genes that may be involved in strigolactone production, carotenoid biosynthesis pathway, Pi signaling or other metabolic pathways, and hence affect AMF colonization and/or Orobanche resistance. Following the newly formed and existing RNAi mutants were examined for AMF colonization and Orobanche resistance. At the first phase of this project Orobanche seed germination assays and AMF colonization were examined in intact plants. These assays were shown to be effective and resulted with enhancement of Orobanche seed germination and AMF colonization in WT tomato plants, whereas roots of strigolactones impaired lines did not result with Orobanche seed germination and mycorrhiza colonization. Unexpectedly, root organ cultures (ROC) that were produced from the same wild type (WT) and mutant lines did not induce the Orobanche seed germination and AMFhyphal branching. This implies that under in vitro conditions ROC cultures are missing an important component for induction of Orobanche seed germination and AMFhyphal branching. In another line of experiments we have tested transgenic lines of Medicagotruncatula for AMFhuyphal branching and Orobanche seed germination assays. These lines included lines silenced for a GRAS transcription factor (RNAi 1845), an NBS-LRR type resistance gene (RNAi 1847), a kinase (RNAi 2403) and a protein of unknown function (RNAi 2417). In all cases, five independent transgenic root lines showed altered AMFphenotypes with reduced or aberrant colonization patterns. Following, we transformed tomato plants with the M. truncatulaTC 127050 PhosphoinositidekinaseRNAi construct. Transgenic lines that contained GUS constructs were used as control. All transgenic lines showed reduced level of Orobanche seed germination, masking any strigoalctones-specific effect. The research demonstrated that SLs production may not be examined in ROC –based bioassays. It was shown by the 3 independent assays employed in this project that none of the recognized characters of SLs may be reflected in these bioassays. However, when the whole plant root exudates were examined, SLs activity in root exudates was demonstrated. Hence, it can be concluded that the presence of an intact shoot, and possibly, shoot factors, may be necessary for production of SLs in roots. Another point of interest that rises from these results is that the presence of SLs is not necessary for AMF completion of life cycle. Hence, it may be concluded that SLs are important for AMFhyphal branching, before symbiosis, but not essential for AMF colonization and life cycle completion under ROC system conditions.