Cellulolytic and Butyrivibrio fibrisolvens bacteria population density , after supplementing fodder diets ( Pennisetum clandestinum )

Objective. Determine the population density of cellulolytic bacteria, Butyrivibrio fibrisolvens and the concentration of vaccenic acid, by supplementing diets consisting of kikuyu grass (Pennisetum clandestinum Hoechst. Ex Chiov.) as base ingredient, together with cassava flour and biomass (effluent from ethanol production) in rumen simulator-Rusitec. Materials and methods. Four treatments (T) were evaluated, these were composed as: T1/Control 1: 100% kikuyu grass with a total protein intake of 23.9%, T2: a mixture of 70% kikuyu grass, 20% biomass and 10% cassava flour with a total protein intake of 19.4%; T3/Control 2: 100% kikuyu grass, with a 17.8% protein intake and T4: 70% kikuyu grass, 20% biomass and 10% cassava flour with a 15.3% protein intake. One and two-way variance analysis was made and the Pearson correlation coefficient was determined. Results. An increase was observed in the population density of viable cellulolytic bacteria (CFU/ml) and B. fibrisolvens statistically significant (p<0.005) with treatment T2, in contrast to T1, T3 and T4 treatments. In addition, there was a significant increase in the concentration of vaccenic acid (mg/L) in the ruminal content in Rusitec with the same treatment (T2). Conclusions. Results obtained in this ruminal simulation study are evidence to the benefits of kikuyu grass together with cassava flour and biomass diet implementation on the growth of ruminal cellulolytic and B. fibrisolvens bacteria, as well as on the production of vaccenic acid. The study also suggests the nutritional potential that such supplements could provide to grazing bovine feeding.


INTRODUCTION
Cattle production in tropical areas like Colombia depends mainly on fodder supply from the pastures.Degradation of lignocellulosic matter contained in fodder is carried out by rumen microbial consortia, specifically by cellulolytic bacteria that take parton the degradation of structural carbohydrates to volatile fatty acids assimilated by the ruminant (1)(2)(3).
Butyrivibrio fibrisolvens is a strict ruminal anaerobe, with high cellulolytic, hemicellulolytic, proteolytic and uricolytic activity.It may represents significant portion (10-30%) of culture ruminal bacteria (4).This bacteria plays a major role in fiber degradation and it has been identified as the main bacterial species that participates in the process of biohydrogenation on unsaturated fatty acids present in diets based on fodder, grains and oils (5), which is a process that could lead to an increase in the concentration of fatty acids in milk and meat from ruminants (6,7).Some pastures available in different regions in Colombia lack adequate nutritional quality, and its production are seasonal, which reduces quality and return in the industry of milk and meat cattle (8).Previous research has suggested that supplementation of diets different industry subderivados (maize, cassava, fish oil, sunflower oil, etc.) improves use of high-fiber content and low digestibility fodder, enhance biodiversity of ruminal microorganisms and ruminal fermentation processes and, consequently, animal productivity (5,7).In the search of food supplements that improve digestion and degradation processes of fodders by bovines, diverse alternatives to pastures supplementation, mainly with fermentable and additive carbohydrates such as oils, concentrated food and grains, have been proposed.
Algunos pastizales de diferentes regiones de Colombia carecen de una adecuada calidad nutricional y su producción es estacional, reduciendo por tanto la calidad y el rendimiento de la leche y de la carne en la industria ganadera (8).Anteriores investigaciones sugieren que al suplementar diferentes dietas con subderivados de la industria (maíz, yuca, aceite de pescado, aceite de girasol, etc.) aumenta el consumo de forraje de alto contenido de fibra y baja digestibilidad, mejorando la biodiversidad de los microorganismos ruminales, y los procesos de fermentación ruminal y por consiguiente la productividad animal (5,7).En la búsqueda de suplementos alimenticios para los bovinos, con el fin de mejorar los procesos de digestión y degradación de forrajes, In 2008, and with the support of the Ministry of Agriculture and Rural Development of Colombia, the National Fuel Alcohol Program was created.One of the purposes of the program was to evaluate the production of ethanol from sugar cane and other agricultural sources such as banana, cassava and sorghum, with the objective of improving quality of fuels by biologic oxygenation and substitution of compounds such as methyl-tert-butyl ether, due to soil and underground water contamination originated in its use (9)(10)(11).Cassava (Manihot esculenta Crantz) has been considered one of the viable alternatives for ethanol production in the country, that together with corn, sugar cane and rice, are considered the most important energy sources in tropical regions worldwide, as it becomes one of the alternative crops with highest potential for ethanol production (12,13).Cassava has been widely considered a basic energetic food item in the industry of balanced food for animals, because of its protein and starch content.In another strategy, the use of cassava flour together with the biomass originated in ethanol production might constitute a promising supplementation practice for pastures in which yields are insufficient to achieve acceptable levels of production.It might also be considered an additional source of protein and energy for bovines (13).
Kikuyu grass (Pennisetum clandestinum Hoechst.Ex Chiov.) is considered a basic feeding element by specialized dairy production systems in Colombia (14).Considering that fodder quality decreases as the plant grow solder, fibrous portion increases and decreases the protein content, digestibility and availability of energy for the animal lowered, which makes the evaluation of different alternatives of energetic supplementation to be included in the diet of the animal (13), it is possible that supplementing fodder such as kikuyu, could modify the ruminal biohydrogenation process, together with the subsequent formation of the principal components of CLA: rumenic acid (cis-9,trans-11) and vaccenic acid (trans-11C18:1).
This study takes into consideration the participation of ruminal cellulolytic bacteria in fiber degradation and the participation of B. fibrisolvens in the process of biohydrogenation for CLA production as important indicators of ruminal fermentation.In this regard, the objective of this study was to determine the population density of the cellulolytic bacteria, B. fibrisolvens and the concentration of vaccenic acid, in response to the use of cassava flour and effluent biomass from ethanol production as supplements to Kikuyu grass with different concentration of protein in an artificial rumen (Rusitec).
Este estudio tiene en cuenta como indicadores importantes de la fermentación ruminal, la participación de las bacterias celulolíticas ruminales en la degradación fibrosa y de B. fibrisolvens en el proceso de biohidrogenación para la producción del CLA.En este sentido, el objetivo de este estudio fue determinar la densidad poblacional de las bacterias

MATERIALS AND METHODS
Ethical aspects of the study.This study was approved by the Ethics Committee on Animal Experimentation from the National University of Colombia, Medellín site, in accordance to Deed CEMED-027, issued on December 2nd, 2008.

Type of study.
Comparative experimental design with four treatments.

Ruminal microorganisms's inoculum used in Rusitec Fermenters.
To start the ruminal fermentation simulation (Rusitec), ruminal content from four female Holstein cows (700±25 kg live weight) was used as initial inoculum.The cows were rumen-fistulated and kept under a basal diet of kikuyu grass in the Paysandú farm, owned by the National University of Colombia, Medellín site, and located in the town of Santa Elena, at 2500 m.a.s.l, with an average temperature of 14°C (15).Rusitec is composed of eight sequential vessels (fermentation units) with a maximum capacity of 700 ml (16).In accordance to the protocols proposed by Czerkawski and Breckenridge (17), the simulated ruminal fermentation experiments were performed in the laboratory of Ruminal Biotechnology at the National University.
The experiment was started by placing, in each vessel, 200 ml of artificial saliva (pH= 8.4; 9.8g of NaHCO 3 ; 3.72g of Na 2 HPO 4 ; 0.47g of NaCl; 0.57g of KCl; 0.053g of CaCl 2 2H 2 O and 0.128g of MgCl 2 6H 2 O, of each component, per liter), 500 ml of ruminal fluid and a solid portion of ruminal content (80g).Such fractions were obtained separately after filtration through four layers of muslin, to ensure the inclusion of present microorganisms in both the liquid and solid fractions of ruminal content.The experiment was carried out at 39°C constant temperature.The simulated fermentation procedure was performed in two periods of seven days corresponding to a four-day adaptation period and a three-day measurement period (5th -7th day of simulation).
Treatments to be assessed through Rusitec.Kikuyu grass was the forage species selected to assess the effect of supplementation with cassava flour and biomass resulting from ethanol production, on cellulolytic and B. fibrisolvens bacteria, in Rusitec.Kikuyu grass was gathered in the municipality of San Pedro de los Milagros, with a 35-day regrowth age, while using nitrogen fertilization with urea (N:50-70, P2O5:45.8,K2O:18, MgO:24.75,SO4:44.85).Two types of kikuyu grass good and bad quality with contrasting nutritional quality in terms of protein content, the result of replacing both forages celulolíticas, de B. fibrisolvens y la concentración de ácido vaccénico, en respuesta a la utilización de harina de yuca y biomasa efluente de la producción de etanol, como suplementos para el pasto kikuyo con diferente concentración de proteína en un rumen artificial (Rusitec).
protein, protein by foreseeing biomass, were included to evaluate the response to biomass and cassava flour as dietary supplements.The amount of kikuyu grass, plus the addition of substrate evaluated in each vessel, was 18g (10.3-15.5g of dry matter).The grass, with a particle size of 1.5 mm, was stored in nylon bags with a pore size of 45 µm.To avoid dilution of the products resulting from the fermentation system, a dilution rate of 3.5% per hour was established.
The assessment of treatments through Rusitec was carried out as follows: On day zero, the experiment started with a test performed in the Rusitec fermenter in eight fermenting pots (2 crocks per treatment).Once the ruminal liquid and artificial saliva were poured into each crock, two nylon bags, containing 18g of the treatment to be assessed and 80g of ruminal content respectively, were introduced in the containers.This last bag was removed 48 hours after incubation initiated.During the following days (1st-7th), each bag in each fermenter was replaced by a new substrate bag every 24 hours.During the 5th, 6th and 7th days, the ruminal liquid was collected from each fermenting crock, before replacing the bag containing the substrate, in order to perform the quantification of ruminal microorganisms, the determination of vaccenic acid and the immediate pH measurement.
Two duplicates of the simulation experiment were carried out in two different moments, with a 15day interval between them, but under the same conditions.This was aimed at estimating more accurately the effect of the supplements to kikuyu on the microbial population to be evaluated.The assessed treatments (T) were: T1/Control 1: 100% kikuyu grass with a total protein intake of 23.9%; T2: a mixture of 70% kikuyu grass, 20% biomass and 10% cassava flour with a total protein intake of 19.4%; T3/Control 2: 100% kikuyu grass, with a 17.8% protein intake; and T4: 70% kikuyu grass, 20% biomass and 10% cassava flour with a 15.3% protein intake (Tables 1 and 2).
Sampling procedure.Thirty five milliliters of the ruminal content mixture collected from the four Holsteins were extracted to perform the isolation of native strains of B. fibrisolvens and determine the initial quantity of cellulolytic and B. fibrisolvens bacteria and the initial concentration of vaccenic acid, contained in the initial inoculum.
After the adaptation period to the system, during the three days of sampling, 2 aliquots of ruminal content were extracted from each fermenter, every 24 hours: 15 ml were employed to measure the pH and complete the procedures of cellulolytic bacteria quantification; 20 ml, which were stored at -20°C, were later used for DNA extraction and B. fibrisolvens real-time qPCR quantification.In addition, to measure vaccenic fatty acid through gas chromatography, for this process were took between 0.15 to 0.20 g sample, 4.0 ml of 0.5 N NaOH was added in methanol and placed in a water bath at 90°C, the system was allowed to reflux for 7 minutes elapsed time reflux, 5 ml of buffer F3 was added to 12% methanol by the top of the condenser and allowed to reflux for 2 minutes.Subsequently 4.0 ml of N-heptane was added to the top of the condenser and was refluxed for 1 minute, cooled and 100 ml of saturated NaCl to ensure the extraction of the compounds separated in two phases solution is added.
The organic phase was removed with a Pasteur pipette into a test tube and added anhydrous sodium sulfate to remove moisture was used, in a column: DB-23, capillary of 50.0 m, inside diameter 0.25 mm, film thickness of 1.4 µm, detector temperature of 280°C and injector temperature of 250°C, hydrogen injection (carrier gas): 40 m/seg, H2 = 45 ml/min flow detector and 450 ml/min airflow (Agilent 6890 Series Gas Chromatograph, Wilmington, DE).

V i a b l e c e l l u l o l y t i c b a c t e r i a ( V C B ) quantification.
Serial dilutions (until 10 -6 ) in triplicate, using solvent (phosphate and mineral salts, resazurin, calcium carbonate and L-cysteine-HCL) were carried out using 1ml of the sample extracted from the initial inoculum and each fermenter, during sample period.One milliliter from dilutions 10 -3 to 10 -6 was extracted and added to solid pre-reduced media prepared by means of Roll-tube technique (18).These culture media contained rumen fluid clarified at 40%, cellobiose at 0.1% and yeast extract at 0.25%, together with other components recommended by Grubb and Dehoroty (19).In order to prepare the solid medium film in culture tubes, agar was added to a final concentration of 1.5% and the culture was grown in anaerobic conditions, by inoculating the hermetically closed sample, through the rubber lid of the tube and El experimento de simulación se llevó a cabo por duplicado en dos momentos diferentes, con un intervalo de 15 días entre ellos, pero bajo las mismas condiciones.Esto tuvo como objetivo estimar con más exactitud el efecto de los suplementos del kikuyo sobre la población microbiana a evaluar.Los tratamientos evaluados (T) fueron: T1/Control 1: 100% pasto kikuyo con un aporte proteico total de 23.9%; T2: una mezcla de 70% pasto kikuyo, 20% biomasa y 10% harina de yuca con un aporte proteico total de 19.4%; T3/Control 2: 100% pasto kikuyo, con un 17.8% de aporte proteico; y T4: 70% pasto kikuyo, 20% biomasa y 10% harina de yuca con un 15.3% de aporte proteico (Tablas 1 y 2).
using a syringe containing N 2 .The colony count was performed after 72 hours of incubation at 39°C and the results were given in terms of Colony Forming Units (CFU/ml) (18).qPCR of B. fibrisolvens.The standard curve for the quantification of B. fibrisolvens through qPCR was performed with the reference strain ATCC #19171, stored in axenic culture (10 8 cells/ ml).The number of cells in the culture after 24 hours was calculated at a wavelength of 640 nm.
One milliliter was extracted from the culture for centrifugation and DNA extraction with DNeasy® Blood & Tissue commercial kit (QIAGEN, MD, USA).
The serial dilution (10 -1 -10 -6 ) was performed for the DNA extracted from the reference strain.Such dilutions were used to perform the specific standard curve for the qPCR of B. fibrisolvens in the samples of rumen obtained from the Rusitec.The curve was generated by considering the values of the cycle threshold (Ct), observed in 35 cycles, versus the logarithm of the number of cells/ml, starting with the six dilutions of pure culture DNA of well-known concentration.
The qPCR protocol followed in this study was validated by the analysis of two internal controls, genomic DNA from the reference strain ATCC #19171 and DNA from the reference strain ATCC #19171 added to 1 ml of ruminal content and subjected to extraction protocol.The amplification of both controls was compared in separate and multiple reactions, to determine the efficiency and specificity of the method and to discard the presence of inhibitors in the ruminal content samples.Genomic DNA extraction was performed from the samples stored at -20°C, collected from the initial inoculum and from each fermenter in the Rusitec during the sampling period.Quality and concentration of the extracted DNA was assessed using NanoDrop ND1000 Spectrophotomer (Thermo Scientific, Wilmington, DE) and 80 ng from the DNA extracted from each sample were analyzed.
The qPCR was performed in Applied Biosystems 7500 Fast Real-Time PCR system (Applied Biosystems, CA, USA).The amplification reactions were carried out in a final volume of 25µl that contained 12.5 µl of SYBR ® Green (QIAGEN, MD, USA) and 0.5 µM of each primer for the ARNr 16S gene amplification.After a 15 minute denaturation at 95°C, the amplification and quantification program was repeated 35 times (30s at 94°C; 30s at 55ºC and 30s at 72°C), followed by the fusion curve analysis (46°C-95ºC, with temperature increase of 3°C per minute and continuous measurement of fluorescence).
The Ct value was calculated automatically by the equipment and the amplification of
sample was considered negative when the Ct value was lower than 8 and higher than 35, or when an amplification curve was not obtained.
The amplification reaction was repeated for all samples with Ct values higher to 35.Each amplification protocol included a negative control (without template DNA), six positive controls in triplicate corresponding to the know concentration DNA (from the standard curve) and the DNA from the internal controls.
The results were reported considering the number of cells/ml.Q-PCR Efficiency (E) was calculated from the slope during the exponential phase, in each cycle according to the equation E=10

Population density of viable cellulolytic bacteria and pH in the in Rusitec.
The amount of CFU/ml obtained from the initial inoculum, used to start the ruminal fermentation simulator was 7.5x10 5 CFU/ml.The analysis the CFU/ml amount of cellulolytic bacteria, among the different treatments, showed statistically significant differences (p=0.03).The growth of microbial population during the 5th and the 6th day of the sampling period in treatments T1, T2 and T4 on average were of 19.23x10 5 CFU/ ml, in contrast to the population of cellulolytic bacteria in T3, whose recount mean was lower in relation to the other treatments (1.5x10 5 CFU/m lon average) on the 6th day of measurement.When the evaluation of the population growth during the 7th day of the sampling period was made, a higher (p=0.0001)population of viable La calidad y concentración del ADN extraído de cada muestra se evaluaron utilizando el Espectrofotómetro NanoDrop ND1000 (Thermo Scientific, Wilmington, DE) y para el análisis de cada muestra se utilizaron 80 ng del ADN extraído.
Considering cellulolytic bacteria population in T3, growth in the population was evident, but there were not statistically significant differences in comparison with the other treatments.A statistically significant decrease (p=0.003) in the population was evident, in contrast to treatments T1 and T4 on the 7th day of measurement (Figure 1).Statistically significant differences on the days of population density measurement were evident when the paired comparison, between the collective average recounts of both viable cellulolytic and B. fibrisolvens bacteria, was made.Statistically significant differences were evident in the population density of both viable cellulolytic and B. fibrisolvens from the 7th day, in contrast to the 5th and 6th days of measurement, when the average recount was lower than the one found on the 7th day.The pH of the ruminal content in the different Rusitec fermenters did not change significatively (p=0.165)during the three sampling days, however, a statistically significant decrease in the pH observed in the Rusitec was evident (~pH 6.8), in relation to the pH value that the ruminal flow initial inoculum presented.
Considerando la población de bacterias celulolíticas en T3, el crecimiento poblacional fue evidente, pero no hubo diferencias estadísticamente significativas en comparación con los otros tratamientos.En el 7° día de medición, se encontró una disminución estadísticamente significativa (p=0.003)en la población, en contraste con los tratamientos T1 y T4 (Figura 1 The population density of B. fibrisolvens obtained from qPCR initial inoculum was 4.94x10 6 cells/ ml.The density of B. fibrisolvens, during the three sampling days was 9.19x10 6 cells/ml (media±ESM).Statistically significant differences were not found in the population density of B. fibrisolvens on the fifth and sixth days of sampling with T1 and T4, where population remained constant, whereas a decrease in the population without statistically significant differences was observed in T2.In T3, the population density increased significantly (p=0.045)within a range of 5.2x10 4 a 1.25x10 6 cells/ml.However, when the analysis of the population density of B. fibrisolvens during the 7th day was carried out, a statistically significant increase was observable (p=0.001) in T2 (1.18x10 6 cells/ml).The supplementation of kikuyu grass (19.4% protein) with 20% biomass and 10% cassava flour caused a significant surge in the population of B. fibrisolvens in contrast to the other treatments (Figure 2).
The concentration of vaccenic acid obtained from the initial inoculum was 15 mg/L.Statistically significant differences in the concentration of vaccenic acid in the ruminal content from the Rusitec, during the sampling period, for treatments T1, T3 and T4, were not found.However, in T2, there was a statistically significant increase (p=0.001) in the concentration of this acid.Also, a positive correlation between the amount of cells/ml of B. fibrisolvens and the concentration of vaccenic acid, was evident (r=86%, p=0.0038).In other words, the vaccenic acid concentration was directly proportional to the population density of B. fibrisolvens in the ruminal content during the Rusitec simulation.

DISCUSSION
In this study, it was observed that population density of cellulolytic bacteria and specifically B. fibrisolvens, increased significantly in response to the supplementation of kikuyu grass (19.4% protein) with cassava flour and biomass effluent from ethanol production.It may be due to the starch and protein input in this treatment, contrary to other treatment where the percentage of starch and protein were lower (13) which may have stimulated the amilolytic and proteolytic activity of this type of bacteria (4,11).Likewise, research carried out by Wanapat et al (13) reports that cassava chip and other forms of cassava root can be successfully fermented with yeast (Saccharomyces cereviceae) to obtain a final product with high Crude protein and a relatively high profile of amino acids.It was found that cassava could replace soybean meal completely and was beneficial to cattle in terms of the efficiency of rumen fermentation, microbial protein synthesis, nitrogen retention and nutrient digestibility.Nevertheless, the results from this study in treatments T3 and T4 were completely opposite to results in T2, which may suggest that the positive response to supplementation in T2, relies on the nutritional quality of kikuyu grass.Studies carried out by Garcia et al (20) assessed the effect of supplementation with biomass obtained as a byproduct from ethanol production by replacing different percentages of protein, of contrasting nutritional quality (high, mid, low) in three kikuyu grass fodders, with protein from biomass.After the evaluation of the fractions (dry matter, neutral detergent fiber and acid detergent fiber) researchers observed that supplementation had a significant positive effect (p<0.01) on the fermentation parameters evaluated with the
En estudios realizados por García y colaboradores (20) se evaluó el efecto de la suplementación con biomasa, obtenida como un subproducto del etanol, reemplazando diferentes porcentajes de proteína en tres forrajes de pasto kikuyo, de calidad nutritiva contrastante (alta, media, baja) por proteína procedente de la biomasa.Después de la evaluación de las fracciones (materia seca, fibra detergente neutra y fibra detergente ácida), los investigadores observaron que la suplementación había tenido un efecto positivo (p<0.01)sobre los parámetros de fermentación evaluados con el pasto de alta calidad, en contraste con los resultados obtenidos con la suplementación del pasto de media y baja calidad.Esto sugiere que la respuesta positiva a la suplementación con biomasa se relaciona con la calidad del pasto kikuyo, lo cual puede también high-quality grass, in contrast to the results obtained with the supplementation of mid and low-quality kikuyu grass.This suggests that the positive response to supplementation with biomass is related with the nutritional quality of kikuyu grass, which may also influence the population of cellulolytic and B. fibrisolvens bacteria observed in this study in T2, which included the supplementation of high-quality grass with biomass and cassava flour.
The genus Butirivibrio may be divided in several groups corresponding to three different biotypes, in accordance to its capacity to produce some fatty acids.Such fact classifies them as lactic and succinic acid producers, instead of acetate and butyrate producers (6,21,22).Mrazek et al (23), identified that the Pseudobutyrivibrio ruminis and Butyrivibrio proteoclasticum isolations found in bovine rumen, produce succinate and propanoate, but they do not produce butyric and acetate, in contrast to the type strain for B. fibrisolvens, which produces butyric and lactic acids and formic and succinic acids at a lower quantity.The species of this genus belong to the cluster XIVa of the Subphylum Clostridium and take part in important functions of the ruminal ecosystem, including proteolysis process for fiber degradation and fatty acids biohydrogenation (13,21,24).They also have the ability to hydrogenate linoleic to vaccenic acid, through enzymatic activity, linoleic acid isomerase and linoleic acid reductase, conferring specificity to the use of linoleic acid for conjugated linoleic acid formation (6).Previous research has showed that the synthesis and concentration of CLA in milk and meat from bovines may be enhanced or inhibited by factors such as the microbial composition of the rumen, animal breed, bromatologic composition of pastures and supplementation with other food rich in protein, lipids and fiber and even with some geographical conditions, such as altitude (3,12,13).
In this study, a significant increase in the vaccenic acid concentration was detected in T2 in Rusitec, this is a monounsaturated acid trans that predominates in the fat fraction of the products of ruminal origin and it originates as intermediate product, during the microbial biohydrogenation of unsaturated fatty acids (22).Its concentration is directly related with the diet supplied to animals, which is rich in linoleic, alphalinoleic acid, protein and fiber and requires active participation of the ruminal microorganisms in charge of such process, as it is the case for ruminal bacteria B. fibrisolvens (6,7,13,24).Although during this study a direct quantification of CLA production was not carried out, due to the use of a ruminal simulation system in vitro, instead of meat and influenciar la población de bacterias celulolíticas y B. fibrisolvens observada en este estudio en T2, incluyendo la suplementación de pasto de alta calidad con biomasa y harina de yuca.
En este estudio, en Rusitec, se detectó en T2, un incremento considerable de la concentración del ácido vaccénico; éste es un ácido monoinsaturado trans que predomina en la fracción grasosa de los productos de origen ruminal y origina un producto intermedio durante la biohidrogenación microbiana de ácidos grasos insaturados (22).Su concentración se relaciona directamente con la dieta suministrada a los animales, la cual es rica en ácidos linoleicos y alfalinoléico, proteína y fibra y requiere activa participación de los microorganismos ruminales encargados de dicho proceso, como es el caso de la bacteria ruminal B. fibrisolvens (6,7,13,24).Aunque durante este estudio no se realizó una cuantificación directa de la producción del CLA (debido a la utilización del sistema de simulación ruminal in vitro) en carne y productos lácteos, fue posible observar que la concentración de ácido vaccénico en Rusitec, fue directamente proporcional a la densidad poblacional de B. fibrisolvens conforme a la suplementación del pasto kikuyo con harina dairy products, it was possible to observe that the vaccenic acid concentration in Rusitec was directly proportional to the population density of B. fibrisolvens under the supplementation of kikuyu grass with cassava flour and biomass.These results suggest that the application of this type of supplementation in the field may cause a significant increase in the CLA concentration in milk and meat from bovines, if the role of vaccenic acid as one of its main originators is considered (6,24).
In a parallel study to this research, it was observed that the supplementation of kikuyu grass with cassava flour and biomass (T2) had an statistically significant effect on some digestibility indicators, such as the degradation of dry and organic matter and the production of ammonia and did not have any significant effect on the degradation of the cell wall, raw protein and gas production (13,20).The results from this study, regarding the significant increase of viable cellulolytic and B. fibrisolvens bacteria population density, as well as the vaccenic acid concentration in response to the supplementation of kikuyu grass with cassava flour and biomass, together with the results obtained by García et al (20), in regard of the ruminal digestibility indicators, suggest that this type of diet could enhance significatively the metabolic capacity of ruminal microorganisms and, consequently improve the energetic balance of bovines, which would result in an increase of the nutritional quality of products derived from cattle, as it is the case of milk and meat (6).It has been reported that cassava is a basic energetic food in the industry of balanced food for animals without any relevance to it being flour, flake or granular type (13).Cassava is a highly energetic compound, due to its content of dry matter, cellulose, hemicellulose, protein, starch and other polysaccharides easily fermentable (9,13).Likewise, biomass (protein-rich yeast) effluent from ethanol production, also presents high content of dry matter, protein, lignin, cellulose and hemicelluloses (9,13).
In conlusions the results from this study, although gathered from an in vitro system, suggest the nutritional potential of cassava flour and biomass effluent from ethanol production, as supplements in grazing bovines feeding.However, additional field research is required to assess the effect of supplementation with cassava flour and biomass in the diet for bovines, and to analyze important indicators such as diet palatability, weight gain, milk production, as well as the in situ effect on the population of ruminal microorganisms, including the characterization of cellulolytic and B. fibrisolvens bacteria, that together, may be useful to determine whether these additives can be used in the formulation of diets for cattle.
de yuca y biomasa.Estos resultados sugieren que la aplicación de este tipo de suplementación en el campo puede causar un aumento considerable en la concentración de CLA de la leche y carne de los bovinos, si se tiene en cuenta la función del ácido vaccénico como una de las principales precursores (6,24).
Para concluir, los resultados de este estudio, aunque recogidos de un sistema in vitro, indican el potencial nutricional de la harina de yuca y de la biomasa residual de la producción del etanol, como suplementos en la alimentación de bovinos de pastoreo.Sin embargo, para evaluar el efecto de la suplementación con harina de yuca y biomasa en la dieta del ganado, se requiere una investigación de campo adicional para analizar los indicadores importantes, tales como la apetencia de la dieta, aumento de peso, producción de leche, así como el efecto in situ sobre la población de microorganismos ruminales, incluyendo la caracterización de las bacterias celulolíticas y B. fibrisolvens, que en conjunto, puede ser útil para determinar si estos aditivos se pueden utilizar en la formulación de dietas para el ganado.

Figure 1 .
Figure 1.Population density of viable cellulolytic bacteria (VCB) according to diet and sampling time (one measurement per fermenter).Results are given as means ± standard deviation of the determinations obtained over the sampling period in the Rusitec.A P-value of <0.05 was considered statistically significant.* p<0.05, ** p<0.01 and *** p<0.001.

Figure 2 .
Figure 2. Population density of B. fibrisolvens according to diet and sampling time (one measurement per fermenter).Results are given as means ± standard deviation of the determinations obtained over the sampling period in the Rusitec.A P-value of <0.05 was considered statistically significant.

Table 1 .
Chemical composition of the treatments evaluated in Rusitec.
CP: Crude protein, EE: Ether extract, NDF: Neutral detergent fiber; ADF: Acid detergent fiber, ADL: Acid detergent lignin, A: Ashes, OM: Organic matter.*Nutritional quality in terms of protein content,the result of replacing both forages protein, protein by foreseeing biomass.

Table 2 .
Chemical composition of cassava flour and biomass product of the production ethanol.
primers.High efficiency was observed in the PCR amplification of the analyzed samples, from 80 ng of genomic DNA, with efficiency values within the range of 1.76 to 2, and high linearity between the results obtained in the slope of each sample (Pearson correlation coefficient, r>0.9).