Effects of pH and Concentrations of Linoleic and Linolenic Acids on Extent and Intermediates of Ruminal Biohydrogenation in Vitro

Three experiments were conducted by in vitro incubations in ruminal fluid to investigate the effects of pH and amounts of linoleic and linolenic acids on the extent of their biohydrogenation, the proportions of conjugated linoleic acid (CLA) and trans-C18:1 as intermediates, and the ratio trans-10:trans-11 intermediates. The effects of pH and amount of linoleic acid were investigated in kinetic studies, and effects of the amount of linolenic acid were studied with 6-h incubations. With identical initial amounts of linoleic acid, its disappearance declined when the mean pH during incubation was under 6.0 compared with a mean pH over 6.5, and when the amount of linolenic acid increased from 10 to 180 mg/160-ml flask, suggesting an inhibition of the isomerization step of the biohydrogenation. Low pH decreased the ratio of trans-10:trans-11 intermediates. With initial amounts of linoleic acid increasing from 100 to 300 mg, the percentage of linoleic acid disappearance declined, but the amount that disappeared increased, without modification of the trans-10:trans-11 ratio, suggesting a maximal capacity of isomerization rather than an inhibition. Moreover, increasing initial linoleic acid resulted in high amounts of trans-C18:1 and an increase of C18:0 that was a linear function of time, suggesting a maximal capacity for the second reduction step of biohydrogenation. High amounts of initial linolenic acid did not affect the amounts of CLA, trans-C18:1, or the ratio trans-10:trans-11. Based on these experiments, a ruminal pH near neutrality with high amount of dietary linoleic acid should modulate the reactions of biohydrogenation in a way that supports CLA and trans-11C18:1 in the rumen

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 Vitro Versus in Situ Ruminal Biohydrogenation of Unsaturated Fatty Acids from a Raw or Extruded Mixture of Ground Canola Seed/Canola Meal

Raw or extruded blends of ground canola seeds and canola meal were used to compare in vitro and in situ lag times and rates of disappearance due to ruminal biohydrogenation of unsaturated fatty acids. The in situ study resulted in higher lag times for biohydrogenation for polyunsaturated fatty acids and lower rates of biohydrogenation of unsaturated fatty acids than the in vitro study, so the in situ biohydrogenation of polyunsaturated fatty acids was not complete at 24 h of incubation. With both methods, rates of biohydrogenation of polyunsaturated fatty acids were higher than for cis-9C18:1. Extrusion did not affect the rate of biohydrogenation of cis-9C18:1, but resulted in higher rates of biohydrogenation of polyunsaturated fatty acids with higher proportions of trans intermediates of biohydrogenation at 4 h of incubation in vitro and at 8 h of incubation in situ. These results suggest that extrusion affects the isomerization of polyunsaturated fatty acids, rather than the hydrogenation steps. In conclusion, in vitro and in situ methods can both show differences of ruminal metabolism of unsaturated fatty acids due to processing, but the methods provide very different estimates of the rates of disappearance due to biohydrogenation

A new method to measure the redox potential (Eh) in rabbit caecum: relationship with pH and fermentation pattern

[EN] This study aimed to assess the anaerobic status of the caecal biotope in the rabbit through the measurement of its redox potential (Eh). Since the caecal content has a high viscosity, the duration of the Eh measurement is high (10 to 20 min) and two methods were compared in 10 week- old rabbits: in vivo vs. post-mortem. In addition, Eh, pH and temperature of the caecal digesta were analysed according to caecotrophy and three periods in the day (soft faeces production: 08:00-10:00 h and 12:00-14:00 h; hard faeces production: 17:00-19:00 h) were compared, using 34 rabbits aged 65 d and weighing 2.3 kg. Caecal Eh decreased 2 min after measurement began, and then stabilised from 20 min onwards (from -152 to 221 mV, P<0.001), in contrast to caecal pH which remained constant over time. Mean values for Eh (at 20 min) and pH were - 219 mV and 6.2 respectively, and did not change according to method or collection period. Only the caecal temperature was 2°C higher (P<0.001) for the in vivo (39°C) than for the post-mortem (37°C) method. Average caecal dry matter and total volatile fatty acid were on average 22 % and 106 mmol/L, and were affected neither by the method nor by the collection period. Caecal Eh was negatively correlated to caecal pH (R²=0.22; P=0.006, n=34), but not to other biotope traits. The Eh measurement in rabbit caecal content could be performed with a minimum recommended duration of 20 min, under anaesthesia or post-mortem. We confi rmed that the rabbit caecal ecosystem is highly anaerobic.This work was partly funded by a grant from Lesaffre Feed Additives (France)Kimsé, M.; Monteils, V.; Bayourthe, C.; Gidenne, T. (2009). A new method to measure the redox potential (Eh) in rabbit caecum: relationship with pH and fermentation pattern. World Rabbit Science. 17(2):63-70. https://doi.org/10.4995/wrs.2009.659637017

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)

In situ ruminal biohydrogenation of fatty acids from extruded soybeans: effects of dietary adaptation and of mixing with lecithin or wheat straw

Kinetics and intermediates of biohydrogenation of fatty acids were investigated in situ using extruded soybeans, a blend of extruded soybeans and lecithin (99:1), or a blend of extruded soybeans plus wheat straw (66:34). Two dry dairy cows received successively a diet with added palmitic acid and a diet with added extruded soybeans, and assays were completed after a 3-week adaptation to each diet. Adaptation of the cows to dietary polyunsaturated fatty acids suppressed the lag time before the beginning of biohydrogenation. Adaptation of cows, and mixing straw with soybeans, increased the rate of biohydrogenation of C18:2 and C18:3, resulting in less C18:2 and C18:3, and more trans C18:1 and C18:0 in the in situ bags. Lecithin did not affect the kinetics of biohydrogenation or the profile of fatty acids in the in situ bags. Differences in the rate of biohydrogenation, and profile of residual fatty acids in the bags were observed between the two cows. Even with a mixture of soybeans and straw in cows receiving dietary polyunsaturated fatty acids, biohydrogenation was slower and resulted in higher proportions of trans-C18:1 than expected from results of literature in vivo. Resultsshow that the biohydrogenation in situ is slow, highly dependent on experimental conditions, and that the use of several cows, adapted to the test fat source before the assay is initiated, is necessary in order to obtain a reliable estimate of kinetics parameters

Analyse comparée des écosystèmes digestifs du rumen de la vache et du caecum du lapin

Dans cette revue nous avons synthétisé les données obtenues dans notre équipe et celles de la bibliographie afin de contribuer à une meilleure connaissance de l’écologie des communautés bactériennes et archées des fermenteurs digestifs des mammifères herbivores. L’analyse a porté sur la comparaison des deux principales stratégies digestives rencontrées chez les mammifères herbivores actuels : un fermenteur en position proximale, le rumen, et un fermenteur en position distale, le caecum. Parmi les espèces d’intérêt agronomique,la vache et le lapin on été choisis comme animaux modèles. Après avoir rappelé les caractéristiques anatomiques et physicochimiques de ces fermenteurs digestifs, nous avons analysé les spécificités de leurs communautés procaryotiques liées à l’hôte, la variabilité individuelle, la structuration spatiale (inter- et intra- fermenteurs digestifs) et la dynamique temporelle (journalière et hebdomadaire) avec ou sans perturbation nutritionnelle induite