Effet des saponines de Yucca et Quillaya sur la dégradation ruminale des matières azotées de l’herbe : étude in vitro

L’effet des saponines de Yucca et/ou Quillaya sur la dégradation ruminale des matières azotées de l’herbe a été étudié in vitro. Les deux saponines ont diminué la concentration en ammoniac, beaucoup plus fortement à pH 6,8 qu’à pH 6.0, sans diminuer la disparition des matières azotées de l’herbe, mais en augmentant la fraction azotée soluble non ammoniacale, ce qui suggère une inhibition de la désamination des acides aminés résultant de la dégradation ruminale des protéines

Ruminal digestion: development of medium-term cultures of ruminal content

Introduction: Batch cultures are commonly used to study ruminal digestion because they are easy to implement. Nevertheless, most are short term studies (a few hours) that are unable to evaluate effects of dietary changes that affect the microbiota, which needs more than 24h to become apparent. So, this study aimed at evaluating persistency of microbial activities during medium-term (96h) cultures of ruminal contents. Animals, material and methods: Ruminal contents were taken from two rumen-fistulated dairy cows receiving a meadow hay based diet (58% NDF and 3% starch). One portion of the contents was used for an immediate measurement of microbial activities (disappearance of fibre, starch and nitrogen, and biohydrogenation of unsaturated fatty acids) and the second portion was incubated during 96h, with a renewal of half of the 200mL media every 12 hours with substrate and 7-pH bicarbonate buffer. This incubation simulated the rumen of a cow receiving 2 equal meals per day, containing 50% wheat bran + 10% soybeans + 40% meadow hay (51% NDF and 5% starch), and having a 6% per hour ruminal emptying, on average. Fermentation parameters (volatile fatty acids, VFA; ammonia, NH3; pH) were recorded every 12h. After 96h, microbial activities of the cultures were assayed as in the fresh rumen content during the first day. The results obtained with fresh ruminal contents on day 1 were compared to those obtained with cultures, using GLM of SYSTAT. Results and discussion: Fermentative activities were maintained during cultures: pH and total VFA concentrations remained constant. Nevertheless a decrease of acetic acid proportion (C2) and an increase of propionic acid proportion (C3) were observed, with a reduction from 3.9 to 2.7 of the C2/C3 ratio, associated with a 22% increase of butyric acid (C4) proportion. NH3 concentration decreased by about half during the first 48h and remained stable thereafter. Compared to fresh ruminal contents, 96h cultures possessed a twice lower fibre disappearance, a similar nitrogen disappearance, a twice higher starch disappearance and a 24% higher biohydrogenation extent of linoleic acid with an increase by 61% of trans-11 isomers. Nitrogen degradation was not modified by medium-term cultures, and the lower NH3 concentration could have resulted from volatilization of aqueous ammonium solution (boiling temperature: 38°C) during our 39°C incubations. Fibrolytic activity and consequently C2 concentration were not favoured by the mid-term cultures, contrary to amylolytic activity and its associated C3 concentration, most probably because of particle size of culture substrates which had been finely ground. The increase of C4 concentration was in agreement with the increase of linoleic acid biohydrogenation by trans-11 pathway, since this pathway would be mainly due to Butyrivibrio fibrisolvens (Maia et al., 2007), a fibrolytic bacteria producing C4 and preferring hemicelluloses (Dehority, 2003), which was provided by the wheat bran in our experiment Conclusion: This incubation procedure can be used to compare medium-term effects of dietary treatments on rumen microbial digestion in cultures. Such cultures do not allow a quantitative measurement of the effects but allow screening procedures and description of the nature of the effects. Further studies will investigate changes of microbiota using pyrosequencing

Dégradabilité de l’azote de tourteaux gras de colza obtenus par pressage à chaud et à froid : études in sacco et in vitro

Suite à l’autorisation d’utiliser de l’huile végétale comme carburant agricole, des éleveurs se sont équipés de presses pour extraire l’huile des graines de colza. Contrairement aux process industriels, cette méthode est réalisée sans chauffage. Le coproduit de cette extraction est un tourteau dit fermier ou gras car l’extraction de l’huile est incomplète. Ces tourteaux fermiers peuvent être utilisés en alimentation des ruminants comme source protéique ; leur valorisation nécessite la connaissance de la dégradabilité ruminale de l'azote (DTN). L'objectif de cette étude était de déterminer cette DTN par la méthode de référence in sacco, de préciser si cette DTN pouvait être déterminée in vitro par la méthode de dégradation enzymatique (DE1) avec les mêmes équations que pour les tourteaux de colza industriels, et de comparer la DTN des tourteaux fermiers à celle de tourteaux industriels expeller obtenus par cuisson et pressage. La DTN des tourteaux fermiers de colza est élevée, ce qui a comme conséquence une faible valeur PDIE et est donc une limite à leur valorisation, sauf en quantité limitée sur des rations très déficitaires en azote dégradable, type ensilage de maïs. Cette DTN peut être déterminée in vitro en appliquant les équations de correction proposées pour les tourteaux de colza. La DTN des tourteaux industriels expeller est beaucoup plus faible que celle des tourteaux fermiers, bien que supérieure à celle des tourteaux déshuilés, et est surestimée par la méthode enzymatique

Mesure in vitro de l'activité de la communauté bactérienne ruminale

Les techniques modernes de biologie moléculaire permettent une description de la micropopulation ruminale. L’étude de l’activité de cette population sur les principaux constituants de la ration est souvent réalisée in vitro. L’objectif de cette étude était de vérifier si la population bactérienne est active mais n’évolue pas pendant une courte incubation in vitro, ce qui permettrait de mettre en relation le profil et l’activité des bactéries. La communauté bactérienne mise à incuber in vitro n’a pas évolué, ni en quantité, ni en structure ou en diversité, ce qui suggère que les conditions de culture n’entraînent pas une sélection de certaines espèces au cours des 6 premières heures. Cette communauté est restée active, puisque capable de dégrader les différents substrats : matières azotées, glucides et lipides. Une mesure in vitro de l’activité microbienne est donc possible, et pourrait s’avérer intéressante à associer aux techniques de biologie moléculaire afin de mieux caractériser la communauté bactérienne d’un prélèvement

Enzymatic Study of Linoleic and Alpha-Linolenic Acids Biohydrogenation by Chloramphenicol-Treated Mixed Rumen Bacterial Species

In the rumen, dietary polyunsaturated fatty acids (PUFA) are reduced by a multistage reaction called biohydrogenation (BH). BH leads to a high proportion of saturated fat in ruminant products, but also products some potential bioactive intermediates like conjugated linoleic and linolenic acids. BH is composed of two kinds of reactions: first an isomerization of PUFA followed by reductions (two for linoleic acid, C18:2n-6; three for alpha-linolenic acid, C18:3n-3). There is little knowledge about BH enzymes as BH bacterial species are the subject of a lot of studies. Nevertheless, both aspects must be explored to control BH and enhance the fatty acids profile of ruminant products. In the present study, an alternative approach was developed to study the enzymes produced in vivo by mixed ruminal bacteria, using inactivation of bacteria by chloramphenicol, an inhibitor of protein synthesis in prokaryotes, before in vitro incubation. To study C18:2n-6 and C18:3n-3 BH several experiments were used: (1) with different incubation durations (0 to 3) to estimate average rates and efficiencies of all BH reactions, and intermediates production; and (2) with different initial quantities of PUFA (0.25 to 2 mg) to estimate Michaelis-Menten enzymatic parameters, K-m and V-max. A last experiment explored the effect of pH buffer and donor cow diet on C18:2n-6 isomerization pathways. Concerning C18:2n-6 BH, this study confirmed the high saturability of its isomerization, the inhibition of both trans11 and trans10 pathways by a low pH, and the last reduction to stearic acid as the limiting-step. Concerning C18:3n-3, its BH was faster than C18:2n-6, in particular its isomerization (V-max = 3.4 vs. 0.6 mM/h, respectively), and the limiting-step was the second reduction to t11 -C18:1. Besides, our mixed isomerases had a higher affinity for C18:2n-6 than for C18:3n-3 (K-m = 2.0 x 10(-3) vs. 4.3 x 10(-3) M, respectively), but due to their high saturability by C18:2n-6, they had a lower efficiency to isomerize C18:2n-6 than C18:3n-3, Chloramphenicol-treated ruminal fluid would be a meaningful method to study the BH enzymes activities

Image_1_Enzymatic Study of Linoleic and Alpha-Linolenic Acids Biohydrogenation by Chloramphenicol-Treated Mixed Rumen Bacterial Species.JPEG

<p>In the rumen, dietary polyunsaturated fatty acids (PUFA) are reduced by a multistage reaction called biohydrogenation (BH). BH leads to a high proportion of saturated fat in ruminant products, but also products some potential bioactive intermediates like conjugated linoleic and linolenic acids. BH is composed of two kinds of reactions: first an isomerization of PUFA followed by reductions (two for linoleic acid, C18:2n-6; three for α-linolenic acid, C18:3n-3). There is little knowledge about BH enzymes as BH bacterial species are the subject of a lot of studies. Nevertheless, both aspects must be explored to control BH and enhance the fatty acids profile of ruminant products. In the present study, an alternative approach was developed to study the enzymes produced in vivo by mixed ruminal bacteria, using inactivation of bacteria by chloramphenicol, an inhibitor of protein synthesis in prokaryotes, before in vitro incubation. To study C18:2n-6 and C18:3n-3 BH several experiments were used: (1) with different incubation durations (0 to 3) to estimate average rates and efficiencies of all BH reactions, and intermediates production; and (2) with different initial quantities of PUFA (0.25 to 2 mg) to estimate Michaelis–Menten enzymatic parameters, K_m and V_max. A last experiment explored the effect of pH buffer and donor cow diet on C18:2n-6 isomerization pathways. Concerning C18:2n-6 BH, this study confirmed the high saturability of its isomerization, the inhibition of both trans11 and trans10 pathways by a low pH, and the last reduction to stearic acid as the limiting-step. Concerning C18:3n-3, its BH was faster than C18:2n-6, in particular its isomerization (V_max = 3.4 vs. 0.6 mM/h, respectively), and the limiting-step was the second reduction to t11-C18:1. Besides, our mixed isomerases had a higher affinity for C18:2n-6 than for C18:3n-3 (K_m = 2.0 × 10^-3 vs. 4.3 × 10^-3 M, respectively), but due to their high saturability by C18:2n-6, they had a lower efficiency to isomerize C18:2n-6 than C18:3n-3. Chloramphenicol-treated ruminal fluid would be a meaningful method to study the BH enzymes activities.</p

Image_2_Enzymatic Study of Linoleic and Alpha-Linolenic Acids Biohydrogenation by Chloramphenicol-Treated Mixed Rumen Bacterial Species.JPEG

<p>In the rumen, dietary polyunsaturated fatty acids (PUFA) are reduced by a multistage reaction called biohydrogenation (BH). BH leads to a high proportion of saturated fat in ruminant products, but also products some potential bioactive intermediates like conjugated linoleic and linolenic acids. BH is composed of two kinds of reactions: first an isomerization of PUFA followed by reductions (two for linoleic acid, C18:2n-6; three for α-linolenic acid, C18:3n-3). There is little knowledge about BH enzymes as BH bacterial species are the subject of a lot of studies. Nevertheless, both aspects must be explored to control BH and enhance the fatty acids profile of ruminant products. In the present study, an alternative approach was developed to study the enzymes produced in vivo by mixed ruminal bacteria, using inactivation of bacteria by chloramphenicol, an inhibitor of protein synthesis in prokaryotes, before in vitro incubation. To study C18:2n-6 and C18:3n-3 BH several experiments were used: (1) with different incubation durations (0 to 3) to estimate average rates and efficiencies of all BH reactions, and intermediates production; and (2) with different initial quantities of PUFA (0.25 to 2 mg) to estimate Michaelis–Menten enzymatic parameters, K_m and V_max. A last experiment explored the effect of pH buffer and donor cow diet on C18:2n-6 isomerization pathways. Concerning C18:2n-6 BH, this study confirmed the high saturability of its isomerization, the inhibition of both trans11 and trans10 pathways by a low pH, and the last reduction to stearic acid as the limiting-step. Concerning C18:3n-3, its BH was faster than C18:2n-6, in particular its isomerization (V_max = 3.4 vs. 0.6 mM/h, respectively), and the limiting-step was the second reduction to t11-C18:1. Besides, our mixed isomerases had a higher affinity for C18:2n-6 than for C18:3n-3 (K_m = 2.0 × 10^-3 vs. 4.3 × 10^-3 M, respectively), but due to their high saturability by C18:2n-6, they had a lower efficiency to isomerize C18:2n-6 than C18:3n-3. Chloramphenicol-treated ruminal fluid would be a meaningful method to study the BH enzymes activities.</p

Table_2_Enzymatic Study of Linoleic and Alpha-Linolenic Acids Biohydrogenation by Chloramphenicol-Treated Mixed Rumen Bacterial Species.DOCX

<p>In the rumen, dietary polyunsaturated fatty acids (PUFA) are reduced by a multistage reaction called biohydrogenation (BH). BH leads to a high proportion of saturated fat in ruminant products, but also products some potential bioactive intermediates like conjugated linoleic and linolenic acids. BH is composed of two kinds of reactions: first an isomerization of PUFA followed by reductions (two for linoleic acid, C18:2n-6; three for α-linolenic acid, C18:3n-3). There is little knowledge about BH enzymes as BH bacterial species are the subject of a lot of studies. Nevertheless, both aspects must be explored to control BH and enhance the fatty acids profile of ruminant products. In the present study, an alternative approach was developed to study the enzymes produced in vivo by mixed ruminal bacteria, using inactivation of bacteria by chloramphenicol, an inhibitor of protein synthesis in prokaryotes, before in vitro incubation. To study C18:2n-6 and C18:3n-3 BH several experiments were used: (1) with different incubation durations (0 to 3) to estimate average rates and efficiencies of all BH reactions, and intermediates production; and (2) with different initial quantities of PUFA (0.25 to 2 mg) to estimate Michaelis–Menten enzymatic parameters, K_m and V_max. A last experiment explored the effect of pH buffer and donor cow diet on C18:2n-6 isomerization pathways. Concerning C18:2n-6 BH, this study confirmed the high saturability of its isomerization, the inhibition of both trans11 and trans10 pathways by a low pH, and the last reduction to stearic acid as the limiting-step. Concerning C18:3n-3, its BH was faster than C18:2n-6, in particular its isomerization (V_max = 3.4 vs. 0.6 mM/h, respectively), and the limiting-step was the second reduction to t11-C18:1. Besides, our mixed isomerases had a higher affinity for C18:2n-6 than for C18:3n-3 (K_m = 2.0 × 10^-3 vs. 4.3 × 10^-3 M, respectively), but due to their high saturability by C18:2n-6, they had a lower efficiency to isomerize C18:2n-6 than C18:3n-3. Chloramphenicol-treated ruminal fluid would be a meaningful method to study the BH enzymes activities.</p