Contribution à l’étude du mode d’action de la levure Saccharomyces cerevisiae Sc 47 chez le ruminant: Approche thermodynamique chez la vache laitière

L'objectif de ce travail est d'apporter des éléments nouveaux de compréhension du mode d'action de la levure probiotique Saccharomyces cerevisiae, utilisée comme additif alimentaire chez la vache laitière. Pour cela et en nous appuyant sur des travaux antérieurs, nous avons attribué a priori à la levure, une aptitude particulière à consommer l'oxygène “anormalement” présent dans le rumen et en conséquence celle d'en renforcer le caractère anaérobie. Afin de conserver intactes les caractéristiques physico-chimiques qui confèrent au fermenteur rumen ses propriétés digestives particulières, nous avons choisi d'aborder cette étude par l'application de la relation de Nernst pour exprimer la pression partielle de l'O2 (log PO2) en fonction des valeurs de pH et de potentiel redox (Eh). Par le biais d'une méthode ex vivo adaptée de prélèvements et de mesures, nous avons mesuré le pH et le Eh parallèlement à la prise d'échantillons du contenu ruminal destinés aux analyses des AGV, du lactate et de l'ammoniac. Deux modèles sont choisis permettant d'abord de mettre en évidence l'effet levure sur vaches taries et ensuite de confirmer sur vaches en lactation. En présence de levure et pour un régime acidogène, le milieu ruminal devient plus réducteur créant ainsi un environnement favorable aux bactéries anaérobies strictes telles que les cellulolytiques et les utilisatrices de lactate. En abaissant le Eh, la levure favorise la transformation du lactate en propionate permettant au pH ruminal de se maintenir au dessus de 6 et évite ainsi le risque d'acidose. D'après nos résultats, la mesure du potentiel redox apparaît donc comme un paramètre important qui vient complémenter celle du pH pour aider à la compréhension du mode d'action de la levure et plus globalement de l'activité fermentaire ruminale. Elle permet partiellement d'envisager les mécanismes de captation d'énergie par les microorganismes du rumen et suggère qu'à des valeurs très basses de Eh, les bactéries cellulolytiques et utilisatrices de lactate sont les seules à pouvoir utiliser des accepteurs d'électrons faibles pour satisfaire leur besoin énergétique et augmenter les produits de fermentation utiles pour l'hôte. ABSTRACT : The objective of this study was to bring forward new arguments to gain a better insight of the mode of action of the probiotic yeast Saccharomyces cerevisiae used as a feed additive in dairy cows. Based on previous experiments, we have attributed the ability of the yeast to scavenge traces of oxygen in the rumen resulting in the strengthening of the anaerobic character of the milieu. In order to maintain the particular physico-chemical properties of the rumen, we approached this study by the use of the Nernst equation to calculate the partial pressure of oxygen (log PO2) from pH and redox potential (Eh) values. By means of an adapted ex vivo method of sampling and measurement, pH and Eh values were recorded with simultaneous sampling of ruminal fluid for the determination of VFA, lactate and ammonia concentrations. Two models were chosen to firstly gather preliminary results on dry cows and were then further confirmed on lactating animals. The supplementation of yeast in an acidotic diet increased the reducing power of the rumen thereby creating a more favourable environment to the strictly anaerobic bacteria such as the cellulolytic and lactate-utilising bacteria. By decreasing the ruminal Eh, the yeast favoured the conversion of lactate to propionate which maintained the ruminal pH above 6 thus avoiding any risk of ruminal acidosis. According to our results, the redox potential appears therefore to be an important parameter complementing pH measurements. It allows a better understanding of the fermentative activity of the rumen and helps to clarify the mode of action of the probiotique yeast. Furthermore, the Eh gives an indication of how the ruminal microflora capture their energy and suggest that at low Eh, only cellulolytic bacteria and lactate users are able to use electron acceptors issued from the fermentation process in order to satisfy their energy needs

Live yeast as a possible modulator of polyunsaturated fatty acid biohydrogenation in the rumen

In dairy cows, several studies focused on the effects of sodium bicarbonate and fibre on ruminal linoleic acid (c9c12-C18:2) biohydrogenation (BH) whereas literature is scarce about the effect of live yeast, used as a feed additive. The objective of this in vivo study was to evaluate the capacity of two dietary feed additives, sodium bicarbonate and live yeast (Strain Sc47), and hay to modulate ruminal BH and particularly conjugated linoleic acids (CLA) and trans-monoenoic acids (t-C18:1) production. Four dry dairy cows fitted with ruminal cannula, were used in a 4×4 Latin square design. They were given a control diet (CD) at a daily feeding rate of 10.4 kg of dry matter/cow supplemented with 100 g/d of sodium bicarbonate or 5 g/d of live yeast or a hay diet formulated to provide the same main fatty acids (FA) as CD during a 14-d experimental period. Ruminal pH and redox potential were measured from 1 h before feeding to 8 h after, and ruminal fluid samples were taken at 5 h after feeding for volatile fatty acids, ammonia and fatty acid determination. In addition to the in vivo experiment, an in vitro experiment was carried out to ascertain the possible mode of action of live yeast on c9c12-C18:2 BH: ruminal fluid was obtained from a donor cow fed with hay and was incubated in batch cultures over 6 h with a 6-pH buffer using starch, urea and grape seed oil as substrates. Results gathered from both experiments suggested that live yeast supplement increased the accumulation of t-C18:1 compared to sodium bicarbonate and prevented the formation of C18:0 which is usually observed when hay is added to a high concentrate diet. The accumulation of t-C18:1 observed in presence of live yeast was probably due to an inhibition of the second reduction step as a result of a more complete isomerisation of c9c12-C18:2

Effects of live yeast on the fatty acid biohydrogenation by ruminal bacteria

Addition of live yeasts in high concentrate diets for ruminants has been shown to help maintaining the ruminal pH above 6, which could enhance the microbial biohydrogenation of unsaturated dietary fatty acids. Moreover, yeasts improve the growth of Megasphera elsdenii, a bacteria which favors the trans-10 pathway of biohydrogenation. So the objective of this study was to investigate the effects of live yeasts (Saccharomyces cerevisiae) on the biohydrogenation in the rumen of dairy cows receiving a high concentrate diet without added fat. Three ruminally fistulated lactating dairy cows were given three diets based on corn silage (control, control plus 0.5g/d or control plus 5.0g/d of Saccharomyces cerevisiae NCYC SC47), according to a Latin square design. Ruminal contents were sampled and liquid and solid phases were separated with a 0.25mm metal sieve. Fatty acids profiles were obtained by gas chromatography. The two doses of yeast resulted in similar effects. Live yeast significantly decreased myristic and stearic acids proportions, and significantly increased oleic and linoleic acids proportions by 16 and 32% in the liquid and the solid phases, respectively. No significant effect was observed for other biohydrogenation intermediates, but the cis9,trans11-C18:2 tended (P = 0.154) to increase with the addition of yeasts, whereas trans10-C18:1 numerically decreased (P = 0.225). These results suggested that live yeasts affect microbial activity, lowering the extent of biohydrogenation without shifting toward the trans-10 isomers pathway

Effect of live yeast on ruminal biohydrogenation. A preliminary in vitro approach

Introduction : Ruminant dietary feed additives such as live yeasts are used on field because of their ability to induce a better diet utilisation. Even if studies on their mode of action are still going on, references are scarce about live yeast diet supplementation and ruminal biohydrogenation (BH) of dietary lipids in dairy cows. During the ruminal BH, some interesting fatty acids (FA), like conjugated linoleic acids (CLA), are synthesised by bacteria, and then could be transferred to milk. This experiment studied live yeast influence on ruminal BH with an in vitro approach. Materials and methods: Ruminal fluid was sucked out from a rumen fistulated dry dairy cow receiving a hay-based diet (57% hay, 43% concentrates) and divided in 10 flasks containing substrates (starch, hay and urea) and a buffer solution (pH 7). In 5 flasks, live Saccharomyces cerevisiae (1010 CFU/g DM, BIOSAF Sc 47, Lesaffre Feed Additives, France) was dosed at 0.15g per flask. All flasks were incubated kept from light and air in a waterbath rotary shaker at 39°C. Two not incubated control flasks without added fat were realized to determine the initial FA amount. After 6h, the incubated flasks were placed into iced water to stop microbial activity and the content was lyophilized for FA extraction and quantification by gas chromatography. Rates and efficiencies of the three reactions composing BH of C18:2 were calculated1. Data were submitted to an analysis of variance. Results and Discussion : Rates and efficiencies of the 3 steps of BH were not significantly modified by live yeast. Composition of FA of control and treated flaks did not strongly differ. The percentage C16:1+C17:0anteiso was twice higher (P0.05), including C18:2 BH intermediates, like trans-11 or trans-10 isomers of CLA and trans-C18:1. Live yeast had no effect in such conditions on the extent of C18:2 (P=0.566) or C18:3 BH (P=0.838), 51% and 54% on average disappeared during incubation, respectively. Conclusion : The fermentative substrate containing hay and the pH were favorable to a high ruminal BH. Live yeast had no effect in such conditions but this work showed that yeast had no adverse effect on BH. Because of live yeast supplementation being advised in case of high concentrate diets inducing ruminal acidosis, further studies will be carried out to investigate live yeast effect on BH in such conditions

In vivo and In Vitro Measurements of Ruminal Redox Potential : a Comparative Study

This experiment compared ruminal in vivo and in vitro conditions in which redox potential (Eh) and fermentative parameters were measured during 3 consecutive days. A rumen fistulated dry dairy cow was adapted during 13 days to a hay-based diet supplemented with 43% of concentrates. Ruminal pH and Eh were measured in vivo from feeding (0h) to 6 hours (6h) at 15 min interval on d1 and d2. On d3, ruminal fluid was sucked out and divided in 10 flasks for in vitro use. In each flask, substrates (starch, hay and urea) and a buffer solution (pH 7) were added and flasks were kept from light and air at 39° C in a waterbath rotary shaker. The pH and Eh were recorded at the start of incubation (0h) to 6 hours (6h) every 15 min. For both methods, VFA and DL-lactate contents were determined at 0h and 6h. At 0h, in vivo Eh (– 217 mV) differed (P = 0.003) from in vitro value (– 123 mV) probably because of ruminal fluid contact with air outside the rumen. After 45 min, Eh measured in rumen (– 227 mV) were not different from Eh recorded in incubated milieu (– 183 mV). After 2 h, both methods yielded similar Eh values. At 0h, total VFA and DL-lactate contents were significantly different between in vivo (60.1 and 0.03 mM, respectively) and in vitro (36.9 and 0.62 mM, respectively) methodologies, owing to the transfer of rumen fluid and the dilution by buffer for incubation purposes. At 6h, no more significant difference was observed, suggesting therefore that in vitro reflected in vivo conditions. At 6h, contents of individual VFA did not differ (49.1 mM of acetate, 10.4 mM of propionate and 9.16 mM of butyrate, on average). In conclusion, during a 6-h incubation, our in vitro experimental method offered a fermentative and reducing environment close to the rumen. Moreover, this present study put forward the capacity of ruminal microbiota to restore reducing conditions in vitro after an exogenous perturbation

The effects of a probiotic yeast on the bacterial diversity and population structure in the rumen of cattle

It has been suggested that the ability of live yeast to improve milk yield and weight gain in cattle is because the yeast stimulates bacterial activity within the rumen. However it remains unclear if this is a general stimulation of all species or a specific stimulation of certain species. Here we characterised the change in the bacterial population within the rumen of cattle fed supplemental live yeast. Three cannulated lactating cows received a daily ration (24 kg/d) of corn silage (61% of DM), concentrates (30% of DM), dehydrated alfalfa (9% of DM) and a minerals and vitamins mix (1% of DM). The effect of yeast (BIOSAF SC 47, Lesaffre Feed Additives, France; 0.5 or 5 g/d) was compared to a control (no additive) in a 3×3 Latin square design. The variation in the rumen bacterial community between treatments was assessed using Serial Analysis of V1 Ribosomal Sequence Tag (SARST-V1) and 454 pyrosequencing based on analysis of the 16S rRNA gene. Compared to the control diet supplementation of probiotic yeast maintained a healthy fermentation in the rumen of lactating cattle (higher VFA concentration [high yeast dose only], higher rumen pH, and lower Eh and lactate). These improvements were accompanied with a shift in the main fibrolytic group (Fibrobacter and Ruminococcus) and lactate utilising bacteria (Megasphaera and Selenomonas). In addition we have shown that the analysis of short V1 region of 16s rRNA gene (50–60 bp) could give as much phylogenetic information as a longer read (454 pyrosequencing of 250 bp). This study also highlights the difficulty of drawing conclusions on composition and diversity of complex microbiota because of the variation caused by the use of different methods (sequencing technology and/or analysis)

Contribution à l'étude du mode d'action de la levure Saccharomyces cerevisiae Sc 47 chez le ruminant (approche thermodynamique chez la vache laitière)

TOULOUSE-ENSAT-Documentation (315552324) / SudocSudocFranceF

Chitine ou ses dérives pour la prévention et/ou le traitement de parasitoses

The subject matter of the present invention is the use of chitin or a derivative of chitin for preventing and/or treating parasitoses, and in particular cryptosporidiosis. The present invention also pertains to a composition that comprises at least one base agent chosen from among chitin or a derivative of chitin and at least one secondary agent chosen from among an agent for stimulating immunity and an antiparasite agent, as well as the use of same for preventing and/or treating parasitoses, in particular cryptosporidiosis.La présente invention a pour objet l'utilisation de chitine ou d'un dérivé de chitine pour la prévention et / ou le traitement de parasitoses, et en particulier de la cryptosporidiose. La présente invention concerne également une composition qui comprend au moins un agent de base choisi parmi la chitine ou un dérivé de chitine et au moins un agent secondaire choisi parmi un agent pour stimuler l'immunité et un agent antiparasitaire, ainsi que son utilisation pour la prévention et/ou le traitement de parasitoses, en particulier de la cryptosporidiose

Methodology Article : Can Ruminal Reducing Power Assessed in Batch Cultures be Comparable to in Vivo Measurements?

In ruminant field of digestive research, the appeal to methods of less invasive studies and reproducing the in vivo conditions is essential. The objective of the present study was to determine whether the conditions created with the proposed in vitro batch culture was an accurate reproduction of the physico-chemical and fermentative ruminal conditions observed in vivo. Two experiments were conducted to compare ruminal reducing power measured in vitro, i.e. in batch cultures or, in vivo i.e. in live animals: dairy cows at maintenance (Experiment 1) and lactating dairy cows (Experiment 2). In Experiment 1, at the beginning of incubation period, in vitro redox potential (Eh), pH and Clark’s exponent (rH) values were significantly higher than in vivo (+42 mV, +0.25 and +1.9, respectively) whereas volatile fatty acids (VFA) contents were 2.6 fold lower on average. At the end of incubation, Eh, rH values and VFA contents were similar between both methods whereas pH still remained different. In Experiment 2, at the beginning and at the end of incubation period, in vitro Eh, pH and rH values differed significantly than in vivo. As a result, the in vitro method did not provide a tool to evaluate accurately the level of the reducing status of ruminal milieu compared with in vivo measurement. Nonetheless, it provided strong reducing conditions after 8 h of incubation with levels of rH relatively closed to those observed in vivo. In vitro batch culture could be a good alternative to in vivo trials for a screening approach from an ethic and economic point of view in ruminant field of research