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In vitro production of gas methane by tropical grasses/Produccion in vitro de gas metano por gramineas forrajeras tropicales.

INTRODUCTION

Ruminants emit between 18 and 25% of the greenhouse gases (GHG), depending on the feeding strategy that has been established, [CH.sub.4] is the second largest contributor to this effect (1-4). Ruminant feed in tropical and subtropical regions is mainly based on the use of forage grasses whose cellulose and hemicellulose content is higher than in temperate climate grasses (5), this higher cell wall content being potentially fermented by cellulolytic bacteria species such as Ruminococcus flavefaciens, Ruminococcus albus and Fibrobacter succinogenes, which transform glucose into acetate and butyrate, whose metabolic pathway produces hydrogen ([H.sub.2]) and carbon dioxide ([CO.sub.2]), which are the main substrates for methanogenic archaea such as Methanobacterium formicicum, Methanobrevibacter ruminantium, Methanomicrobium mobile, Methanosarcina bacteri and Methanosarcina majei (6), where the highest production of [CH.sub.4] is produced by this metabolic pathway (6,7).

The production of [CO.sub.2] and [CH.sub.4] is a necessary process in ruminal biochemistry to obtain energy, this process reduces the accumulation of [H.sub.2] and pH reduction, to maintain ruminal ecology under favorable conditions (8). However, this process reduces the efficiency of energy use by the animal by 6.3% for sheep and 6.5% for cattle (9).

The use of tropical forage grasses with higher cell content and lower potentially fermentable cell wall content in ruminant feed could allow for greater energy efficiency that contributes to reducing GHG emissions for the purpose of mitigating climate change, Zheng et al (3) y Inamaga et al (4), reported that feed strategies influenced GHG emissions, also indicate that [CO.sub.2] emissions based on the production of fat-corrected milk were higher for high forage feeding strategies. Therefore, the objective of this research was to evaluate the production of total gases (GT) and [CH.sub.4] emitted by tropical fodder grasses on in vitro incubation (3,4).

MATERIALS AND METHODS

Area of study. The study was developed in the Laboratory of Animal Science of the Faculty of Agricultural Sciences, Campus IV of the University Autonomous University of Chiapas located in Huehuetan, Chiapas, Mexico and the Ruminal Microbiology and Microbial Genetics Laboratory of the Postgraduate School Livestock Program, Montecillos Campus, Texcoco, Mexico.

Treatments and chemical analysis of grasses. The treatments (pastures) evaluated were T1: Cynodon nlemfuensis; T2: Hyparrhenia rufa; T3: Megathyrsus maximus and T4: Digitaria swazilandensis; with an age of 75 days, during the month of May 2015 (average temperature of 24.81[degrees]C, relative humedad of 72.86% and 277 mm of accumulated monthly precipitation at the time of sampling, and 1438.9 mm of precipitation during the year), was obtained from a cattle ranch (El Carmen 9, in Mazatan, Chiapas) located at 14[degrees]54'23.93" N and 92[degrees]25'37.81" 0; 35 meters above sea level. With soil of the Phaeozem (Feosem) type, characterized by a high accumulation of organic matter and by being saturated at the top, the soil is mainly prairie soil, with a mollic epipedion (a relatively thick, dark, humus-rich surface horizon) and without calcium carbonate in the first meter of depth; no fertilization was carried out on the areas of the sampled forage.

The samples were dried in a drying oven at 60[degrees]C for 24 hours and ground in an ED-5 electric mill equipped with a 1 mm screen. For each of the samples, crude protein (CP) was determined by the Kjeldahl method, as well as ethereal extract (EE) and ash content after incineration of the sample in a muffle at 550[degrees] C per 4 h according to AOAC (10). Determination of the neutral detergent fiber (NDF) and acid detergent fiber (ADF) fractions according to the technique described by Van Soet et al (11).

The culture medium (Table 1) used to determine the production of total gases (GT) and methane ([CH.sub.4]), in addition to the degradation of MS, was prepared under sterile conditions and [CO.sub.2] flow. The inoculum was fresh rumen fluid (FRF) extracted at 2 h pos-prandium from a 500 kg BW bovine (F1, zebu x swiss) with rumen cannula, which received at libitum (received the first ration at 6:00 am and second at 4:00 pm) a diet based on 85% C. nlemfuensis and 15% of a concentrated feed containing 2.7 Meal of ME and 14% crude protein.

Production of [CH.sub.4]. The in vitro production of GT and [CH.sub.4] was determined in triplicate with repetition overtime of each treatment (grasses) using bottles (biodigesters) with a capacity of 2.0 L with hermetic seal, where the following mixture was added under aseptic and [CO.sub.2] flow conditions: 20 g of MS from each grass (1 g of MS for each 10 mL of medium) according to the Williams technique (12) plus 200 ml of culture medium (Table 1) each treatment was inoculated with 20 ml of FRF filtered in cotton gauze, incubated at 38[+ or -]0.5[degrees]C under [CO.sub.2] flow for 24, 48, 72 and 96 h in a thermoregulation bath. The initial total bacterial concentration was 1.35 x [10.sup.8] CFU [ml.sub.-1] based on the most probable number technique (MPN, 13) at pH 6.74. At the end of the incubation period, the production of total gases (GT) in the system was measured by moving liquids through a trap with Mariotte flasks. The displaced water was collected in a 500 ml graduated cylinder and thus the amount of GT per 20 g of fermented MS was determined.

To determine the amount of [CH.sub.4] produced in each treatment, in a second test and under the same culture conditions, times and repetitions, in the Mariotte flask traps was added a solution of NaOH (2N) with pH of 13.67 according to the technique described by Stolaroff (14); the NaOH solution reacts with [CO.sub.2] to form sodium carbonate ([Na.sub.2][CO.sub.3]) and the remaining gases released are a mixture of [CH.sub.4], [H.sub.2], [N.sub.2] and hydrogen sulphide (15). The [CO.sub.2] trap was coupled to the biodigesters using a Tygon hose (internal [PHI] 5 mm and a length of 35 cm) that was fitted with a hypodermic needle (31.8 mm) and 10 cm long). In all GT production evaluations, the results of each treatment and its respective repetition were corrected for difference with the gas production of the blank samples (200 ml of culture medium plus 20 ml of FRF).

Production of volatile fatty acids (VFA) and microbiological variables. At the end of each incubation period 5 ml of culture medium were obtained and centrifuged at 18000 G for 10 min; 2.0 ml of the supernatant was mixed 4:1 with 25% metaphosphoric acid, the vials were shaken in a Vortex and re-centrifuged at 35000 G for two minutes, the concentration of VFA was measured using a Claurus 500 gas chromatograph, using the technique and conditions described by Ley de Coss et al (16). In addition, per incubation period, 0.5 ml of culture medium was obtained from each treatment to estimate the concentration of total bacteria (BT) and cellulolytic bacteria (BC) using the MPN technique and culture media similar to those reported by Ley de Coss et al (17), which consisted to BT: 0.06 g D-(+)-glucose + 0.06 g D-cellobiose + 0.06 g starch, 30 ml clarified FR, 5.0 ml mineral solution I [6 g [K.sub.2[HPO.sub.4] in 1000 ml [H.sub.2]O], 5.0 ml mineral solution II[6 g K[H.sub.2][PO.sub.4] + 6 g K[H.sub.2][PO.sub.4] + 6 g [([NH.sub.4]).sup.2][SO.sub.4] + 12 g NaCl + 2.45 g Mg[SO.sub.4] + 1.6 g Ca[Cl.sub.2].[H.sub.2]O in 1000 ml [H.sub.2]O], 2.0 ml 8% [Na.sub.2][CO.sub.3] solution, 2 ml sulphide-cysteine solution (2.5 g L-cysteine in 15 ml NaOH (2N) + 2.5 g [Na.sub.2]S-9[H.sub.2]O dissolved in 100 ml [H.sub.2]O), 0.2 g peptone tripticase and 0.1 ml of 0.1% resazurine solution; and for BC a similar medium was used, and only the energy source (glucose+cellobiose+starch) was replaced by a strip of Whatman paper as a cellulose source (18).

Design and statistical analysis. The experimental design was completely randomized with three repetitions per treatment for each incubation period. Data on total gas production, [CH.sub.4], AGV concentration and pH of the culture medium were analyzed with the SAS GLM procedure (19), while data on BT and BC concentration were analyzed with the Kruskal-Wallis test, with the GLM procedure with data from independent ranges (Wilcoxon) and averages were compared with the Tukey test (p<0.05) with SAS.

RESULTS

The lowest total gas production was in H. rufa and D. swazilandensis, in the latter species it had lower [CH.sub.4] production, indicating higher energy production efficiency due to higher propionic acid synthesis. There was no change in BT concentration; however, in pastures with lower [CH.sub.4] synthesis there was lower BC concentration. Table 2 shows the results of the chemical composition of the grasses, showing that the crude protein content of H. rufa was less than 7%, while in C. nlemfuensis, M. maximus and D. swazilandensis it was greater than 9%. The NDF content, the lowest value was H. rufa (63.25%), while D. swazilandensis had the highest content of this compound (71.40%), with an 8.15% difference between the two species, when related to the ADF content that was similar among the four species (42.25 to 43.40%), it can be attributed that the highest content of NDF in D. swazilandensis could be due to the higher content of hemicellulose.

Total production of gases and [CH.sub.4] In all the fermented pastures, the highest proportion of gases (Table 3) was obtained in the period from 48 to 72 h, which indicates that in this period the highest activity of the bacteria to degrade the substrate was obtained. When considering the total accumulated gas production per [g.sup.-1] of dry matter fermented (DMf), it was lower for H. rufa and D. swazilandensis (p<0.05).

In the same way as GT production, the largest proportion in the production of [CH.sub.4] (Table 4) occurred in the period from 48 to 72 h, but the total accumulated production of [CH.sub.4] was similar between C. nlemfuensis, H. rufa and D. swazilandensis (p>0.05) as well as between C. nlemfuensis, H. rufa and M. maximus (p>0.05), while there was a difference between M. maximus and D. swazilandensis with lower production (p<0.05).

In relation to the percentage of [CH.sub.4] of total gas production, for the grasses H. rufa, M. maximus and D. swazilandensis represented 76.5%, while for C. nlemfuensis it was 73.9%, which indicates that the highest proportion of gas produced during fermentation corresponds to this GHG.

The total production of VFA and acetic acid production was similar in the grasses evaluated (p>0.05), while D. swazilandensis had higher production of propionic (p<0.05) and butyric acids (p<0.05). The acetic: propionic ratio showed that during the fermentation of D. swazilandensis the energy loss was lower and was related to the lower production of [CH.sub.4] obtained (Table 5).

Table 6, shows the pH of the medium during 96 h of fermentation. D. swazilandensis and M. maximus had the lowest pH at 24, 72 and 96 h of incubation, even less than 6 at 96 h.

There was no difference in BT concentration among treatments (p>0.05) during the entire incubation period and the maximum concentration, in all treatments, was [10.sup.9] cells [ml.sup.-1] of culture medium. Regarding the concentration of cellulolytic bacteria, at 24 h of incubation, the highest concentration was observed in C. nlemfuensis (p<0.05), at 48 and 72 hours there was no difference among treatments (p>0.05); while at 96 hours it was lower (p<0.05) in C. nlemfuensis and D. swazilandensis (Table 7).

DISCUSSION

Generally, grasses have a low crude protein content, with a lower nitrogen content that limits microbial activity in the rumen (20), Avellaneda et al (21), report values of 6.37 and 71.96% crude protein and NDF, respectively in Guinea grass (Panicum maximum var Mombasa), harvested at 90 days of age, similar results to those found in this study. Maximum methane production was obtained at pH 7.0 to 7.2, and may even occur in the range of 6.6 to 7.6 (3), in this study D. swazilandensis showed a lower concentration of cellulolytic bacteria, a pH below 6.5 and therefore a lower concentration of [CH.sub.4], due to the reduction of the activity of bacteria that degrade fiber by the pyruvate-lase pathway such as Ruminococcus fiavefaciens, Ruminococcus albus, Butyrivibrio fibrisolvens and Fibrobacter succinogenes (22,23) and consequently the substrates ([CO.sub.2] and [H.sub.2]) necessary in the formation of [CH.sub.4]; However, species such as Streptococcus bovis, Ruminobacter amylophilus, Succinomonas amylolytica and Selenomonas ruminantium proliferate, fermenting soluble carbohydrates and cellulose fragments to produce propionate via succinate (24), which generates a different profile in the production of VFA, producing a higher proportion of propionic acid and therefore less [CH.sub.4]. On the other hand, the ruminal fermentation of forages with a higher content of cell wall does not cause a significant decrease in pH, because the greater amount of glucose released is fermented by acetate, in this case, the released [H.sub.2] can be used as a substrate by methanogenic archaea, which is associated with higher production of [CH.sub.4] (3), as in the case of H. rufa and C. nlemfuensis, while with forages that cause low rumen pH, methanogenesis is decreased as in the case of M. maximus whose pH was less than 6.5 since 72 hours of incubation and D. swazilandensis since 24 hours.

One of the important factors affecting the production of [CH.sub.4] is the ratio of produced VFA, specifically the acetic: propionic ratio, which regulates the production and availability of [H.sub.2] and subsequent production of [CH.sub.4]; this ratio can vary from 0.9 to 4 and energy utilization is more efficient if the ratio is close to 1.0 (25). The production of [CH.sub.4] has been used as an indicator of the fermentative activity of bacteria in anaerobic fermentation processes (26) in which different groups of bacteria are involved: such as hydrolytic bacteria that fractionate polysaccharides to sugars, VFA formers and methanogenic archaea that synthesize [CH.sub.4] from [H.sub.2] and [CO.sub.2] (27,28). Acetate and butyrate originate the production of [CH.sub.4], due to the increased availability of [CO.sub.2] and [H.sub.2] for methanogenic archaea, while for propionate formation in the rumen it is considered a competitive form of [H.sub.2] uptake that causes a lower synthesis of [CH.sub.4] (29). Rumen protozoa produce [H.sub.2] as the main end-product of their metabolism and is closely associated as a substrate for methane formation by methanogenic archea. These methanogenic bacteria associated with rumen protozoa are apparently responsible for 9 to 25% of methanogenesis, but this can be reduced by around 13% when the protozoa are killed; however, this reduction occurs when the animal consumes starchy diets, which is when the protozoa generate more [H.sub.2], which is not the case when the diets are high in forage resulting in less methane formation (30). Conversely, a high proportion of acetate: propionate is related to low energy efficiency, which involves higher [CH.sub.4] production as was the case with C. nlemfuensis, H. rufa and M. maximus.

In conclusion, the tropical grasses analyzed show a high cell wall concentration, which limits their digestibility and reduces their quality as fodder; however, Digitaria swazilandensis showed a lower total production of methane and total gases, possibly due to a higher concentration of propionic acid, lower concentration of cellulolytic bacteria, a pH and a lower acetic: propionic ratio, being the most efficient in energy use.

Acknowledgements

To the National Council of Science and Technology (CONACYT) for financing the project entitled "Estimation and environmental impact of carbon sequestration in oil palm plantations (Elaeis guineensis Jacq) in the State of Chiapas", which supported the development of this research work within the guidelines of Scientific Development Projects to Address National Problems (CONACYT/PDCP[N.sub.2]013-01/216526).

INTRODUCCION

Los rumiantes emiten entre 18 y 25% de los gases que causan el efecto invernadero (GEI), dependiendo de la estrategia de alimentacion que se tenga, el [CH.sub.4] es el segundo gas que contribuye a este efecto (1-4). La alimentacion de los rumiantes en las regiones tropicales y subtropicales se basa principalmente en el uso de pastos forrajeros cuyo contenido de celulosa y de hemicelulosa, es mayor que en los pastos de clima templado (5), este mayor contenido de pared celular es potencialmente fermentado por especies de bacterias celuloliticas como Ruminococcus flavefaciens, Ruminococcus albus y Fibrobacter succinogenes, que transforman la glucosa en acetato y butirato, cuya ruta metabolica produce hidrogeno ([H.sub.2]) y bioxido de carbono ([CO.sub.2]), que son los substratos principales para las archaeas metanogenicas como Methanobacterium formicicum, Methanobrevibacter ruminantium, Methanomicrobium mobile, Methanosarcina bacteri y Methanosarcina majei (6) donde la mayor produccion de [CH.sub.4] se tiene mediante esta via metabolica (6,7)

La produccion de [CO.sub.2] y [CH.sub.4] es un proceso necesario en la bioquimica ruminal para la obtencion de energia, este proceso reduce la acumulacion de [H.sub.2] y la caida del pH, para mantener la ecologia ruminal en condiciones favorables (8). Sin embargo, este proceso reduce la eficiencia de utilizacion de la energia por el animal, 6.3% para ovinos y 6.5% para bovinos (9).

El uso de pastos forrajeros tropicales con mayor contenido celular y menor contenido de pared celular potencialmente fermentable en la alimentacion de los rumiantes, podria permitir mayor eficiencia energetica que contribuya a reducir la emision de GEI con el proposito de mitigar el cambio climatico, Zheng et al (3) y Inamaga et al (4) mencionan que las estrategias de alimentacion influyen en las emisiones de GEI, incluso, indican que las emisiones de [CO.sub.2] con base en la produccion de leche corregida en grasa fueron mayores para las estrategias de alimentacion altas en forraje, por lo anterior, el objetivo de esta investigacion fue evaluar la produccion de gases totales (GT) y [CH.sub.4] emitidos por gramineas forrajeras tropicales en incubacion in vitro (3,4).

MATERIALES Y METODOS

Area de estudio. El estudio se desarrollo en el Laboratorio de Ciencia Animal de la Facultad de Ciencias Agricolas, Campus IV tie la Universidad Autonoma de Chiapas ubicada en Huehuetan, Chiapas, Mexico, y en el Laboratorio de Microbiologia Ruminal y Genetica Microbiana del Programa en Ganaderia del Colegio de Postgraduados, Campus Montecillos, Texcoco, Mexico.

Tratamientos y analisis quimico de las gramineas. Los tratamientos (pastos) evaluados fueron T1: Cynodon nlemfuensis; T2: Hyparrhenia rufa; T3: Megathyrsus maximus y T4: Digitaria swazilandensis; con una edad al corte de 75 dias, durante el mes de mayo de 2015 (temperatura media de 24.81[grados]C, humedad relativa de 72.86% y 277 mm de precipitacion acumulada mensual al momento de muestreo, y 1438.9 mm de precipitacion en el ano), se obtuvo de un rancho ganadero (El Carmen 9, en Mazatan, Chiapas) ubicado a 14[grados]54 '23.93" N y 92[grados]25 '37.81"O; 35 msnm. Con suelo de tipo Phaeozem (Feosem), caracterizado por tener una alta acumulacion de materia organica y por estar saturados en bases en su parte superior, son suelo principalmente de pradera, con un epipedion mollico (horizonte superficial relativamente grueso, oscuro y rico en humus) y sin carbonato calcico en el primer metro de profundidad; no se realizo ningun tipo de fertilizacion en las areas de los forrajes muestreados.

Las muestras se secaron en una estufa de secado a 60[grados]C por 24 h y se molieron en un molino electrico ED-5, equipado con criba de 1 mm. A cada una de las muestras se determino, proteina cruda (PC) por el metodo Kjeldahl, asi como extracto etereo (EE) y el contenido de cenizas despues de incinerar la muestra en una mufla a 550[grados]C por 4 h segun lo descrito por la AOAC (10). La determinacion de las fracciones de fibra detergente neutro (FDN) y fibra detergente acido (FDA) de acuerdo con la tecnica descrita por Van Soet et al (11).

Medio de cultivo. El medio de cultivo (Tabla 1) utilizado para determinar la produccion de gases totales (GT) y metano ([CH.sub.4]), ademas de la degradacion de la MS fue preparado en condiciones de esterilidad y flujo de [CO.sub.2]. El inoculo fue el fluido ruminal fresco (FRF) extraido a las 2 h pos-prandium de un bovino (Fl, cebu x suizo) de 500 kg PV con canula ruminal, el cual recibio at libitum (recibio la primera racion a las 6:00 am y segunda a las 4:00 pm) una dieta a base de 85% de C. nlemfuensis y 15% de un alimento concentrado que contenia 2,7 Mcal de EM y 14% de proteina cruda.

Produccion de [CH.sub.4]. La produccion in vitro de GT y [CH.sub.4] se determino por triplicado con repeticion en el tiempo de cada tratamiento (gramineas) mediante frascos (biodigestores) con capacidad de 2.0 L con selladura hermetica, donde se adiciono bajo condiciones de asepsia y flujo de [CO.sub.2] la siguiente mezcla: 20 g de MS de cada pasto (1 g de MS por cada 10 mLde medio) segun tecnica de Williams (12) mas 200 ml de medio de cultivo (Tabla 1) cada tratamiento se inoculo con 20 ml de FRF filtrado en gasa de algodon, incubado a 38 [+ or -] 0.5[grados]C bajo flujo de [CO.sub.2] por 24, 48, 72 y 96 h en bano de termorregulacion. La concentracion inicial de bacterias totales fue de 1,35 x [10.sup.8] UFC [ml.sup.-1] con base en la tecnica de numero mas probable (NMP, 13) a pH de 6.74. Al terminar el periodo de incubacion se midio la produccion de gases totales (GT) en el sistema, mediante el desplazamiento de liquidos a traves de una trampa con frascos de Mariotte. El agua desplazada se recolecto en una probeta graduada de 500 ml y con ello se determino la cantidad de GT por los 20 g de MS fermentada.

Para determinar la cantidad de [CH.sub.4] producido en cada tratamiento, en una segunda prueba y bajo las mismas condiciones de cultivo, tiempos y repeticiones, en las trampas de frascos Mariotte se adiciono una solucion de NaOH (2N) con pH de 13.67 segun la tecnica descrita por Stolaroff (14) la solucion de NaOH reacciona con el [CO.sub.2] para formar carbonato de sodio ([Na.sub.2][CO.sub.3]) y el resto de gases liberados son una mezcla de [CH.sub.4], [H.sub.2], [N.sub.2] y acido sulfhidrico (15); la trampa de [CO.sub.2] se acoplo a los biodigestores mediante una manguera de Tygon ([FI] interno de 5 mm y una longitud de 35 cm) a la que se le coloco una aguja hipodermica (de 31.8 mm y 10 cm de largo). En todas las evaluaciones de produccion de GT, los resultados de cada tratamiento y su respectiva repeticion fueron corregidos por diferencia con la produccion de gas de las muestras blanco (200 ml de medio de cultivo mas 20 ml de FRF).

Produccion de acidos grasos volatiles (AGV) y variables microbiologicas. Al terminar cada periodo de incubacion 5 ml de medio de cultivo se obtuvieron y se centrifugaron a 18000 G por 10 min; 2.0 ml del sobrenadante se mezclaron 4:1 con acido metafosforico al 25%, los viales fueron agitados en un Vortex y se volvieron a centrifugar a 35000 G durante dos minutos, la concentracion de AGV se midio usando un Cromatografo de gases Claurus 500, usando la tecnica y condiciones descritas por Ley de Coss et al (16). Ademas, por periodo de incubacion se obtuvo 0.5 ml de medio de cultivo de cada tratamiento para estimar la concentracion de bacterias totales (BT) y bacterias celuloliticas (BC) usando la tecnica NMP y medios de cultivo similares a lo reportado por Ley de Coss et al (17) que consiste para BT en: 0.06 g de D-(+)-glucosa + 0.06 g D-celobiosa + 0.06 g de almidon, 30 ml de FR clarificado, 5.0 ml de solucion mineral I [6 g [K.sub.2[HPO.sub.4] en 1000 ml de [H.sub.2]O], 5.0 ml de solucion mineral II [6 g K[H.sub.2][PO.sub.4] + 6 g [([NH.sub.4]).sub.2][SO.sub.4] + 12 g NaCI + 2.45 g Mg[SO.sub.4] + 1,6 g Ca[Cl.sub.2]-[H.sub.2]O en 1000 ml de [H.sub.2]O], 2.0 ml de solucion al 8% de [Na.sub.2][CO.sub.3], 2 ml de solucion sulfido-cisteina (2.5 g L-cisteina en 15 mi de NaOH (2N) + 2.5 g [Na.sub.2]S-9[H.sub.2]O aforado en 100 ml de [H.sub.2]O), 0.2 g de tripticasa peptona y 0.1 ml de solucion al 0.1% de resazurina; y para las BC se utilizo un medio similar al anterior, y solo se sustituyo la fuente de energia (glucosa+celobiosa+almidon) por una tira de papel Whatman como fuente de celulosa (18).

Diseno y analisis estadistico. El diseno experimental fue completamente al azar con tres repeticiones por tratamiento por cada periodo de incubacion. Los datos de produccion total de gases, [CH.sub.4], concentracion de AGV y pH del medio de cultivo se analizaron con el procedimiento GLM de SAS (19), mientras que para los datos de concentracion de BT y BC se uso la prueba de Kruskal-Wallis, con el procedimiento GLM con datos de rangos independientes (Wilcoxon) y las medias se compararon con la prueba de Tukey (p<0.05) con SAS.

RESULTADOS

La menor produccion de gases totales fue en H. rufa y D. swazilandensis, en la ultima especie tuvo menor produccion de [CH.sub.4], indicando una mayor eficiencia en la produccion de energia debido a la mayor sintesis de acido propionico. No hubo cambios en la concentracion de BT, sin embargo, en los pastos con menor sintesis de [CH.sub.4] hubo menor concentracion de BC. La tabla 2 muestra los resultados de la composicion quimica de las gramineas, se observa que el contenido de proteina cruda de H. rufa fue inferior a 7%, mientras que en C. nlemfuensis, M. maximus y D. swazilandensis fue mayor a 9%. Con respecto al contenido de FDN, el valor mas bajo lo tuvo H. rufa (63.25%), mientras que D. swazilandensis tuvo el contenido mas alto de este compuesto (71.40%), con 8.15% de diferencia entre ambas especies, al relacionarlo con el contenido de FDA que fue similar entre las cuatro especies (42.25 a 43.40%), puede atribuirse que el mayor contenido de FDN en D. swazilandensis pudiera ser por el mayor contenido de hemicelulosa.

Produccion total de gases y [CH.sub.4] En todos los pastos fermentados, la mayor proporcion de gases (Tabla 3) se tuvo en el periodo de 48 a 72 h, lo que indica que en este periodo se tuvo la mayor actividad de las bacterias para degradar el substrato. Al considerar la produccion total de gas acumulado por [g.sup.-1] de materia seca fermentada (MSf), fue menor para H. rufa y D. swazilandensis (p<0.05).

Al igual que la produccion GT, la mayor proporcion en la produccion de [CH.sub.4] (Tabla 4) ocurrio en el periodo de 48 a 72 h, pero la produccion total acumulada de [CH.sub.4] fue similar entre C. nlemfuensis, H. rufa y D. swazilandensis (p>0.05) al igual que entre C. nlemfuensis, H. rufa y M. maximus (p>0.05), mientras que hubo diferencia entre M. maximus y D. swazilandensis con menor produccion en esta ultima (p<0.05).

En relacion con el porcentaje de [CH.sub.4] respecto a la produccion total de gas, para las gramineas H. rufa, M. maximus y D. swazilandensis represento el 76.5%, mientras que para C. nlemfuensis fue de 73.9%, lo que indica que la mayor proporcion de gas producido durante la fermentacion corresponde a este GEI.

La produccion total de AGV y la produccion de acido acetico fue similar en las gramineas evaluadas (p>0.05), mientras que D. swazilandensis tuvo mayor produccion de acido propionico (p<0.05) y acido butirico (p<0.05). La relacion acetico: propionico mostro que durante la fermentacion de D. swazilandensis la perdida de energia fue menor y se relaciono con la menor produccion de [CH.sub.4] obtenida (Tabla 5).

La tabla 6, muestra el pH del medio durante 96 h de fermentacion. D. swazilandensis y M. maximus tuvieron el menor pH a las 24, 72 y 96 h de incubacion, incluso menor de 6 a la 96 h (p<0.05), ademas estos dos pastos tuvieron el pH menor a 6.0 a la hora 96. No hubo diferencia en la concentracion de BT entre tratamientos (p>0.05) durante todo el periodo de incubacion y la concentracion maxima, en todos los tratamientos, fue de [10.sup.9] celulas [ml.sup.-1] de medio de cultivo. Respecto a la concentracion de bacterias celuloliticas, a las 24 h de incubacion, la mayor concentracion se observo en C. nlemfuensis (p<0.05), a la hora 48 y 72 no hubo diferencia entre tratamientos (p>0.05), mientras que a la hora 96 fue menor (p<0.05) en C. nlemfuensis y D. swazilandensis (Tabla 7).

DISCUSION

Generalmente las gramineas presentan un bajo contenido de proteina cruda, con menor contenido de nitrogeno que limita la actividad microbiana en el rumen (20), Avellaneda et al (21), reportan valores de 6.37 y 71.96% de proteina cruda y FDN, respectivamente en pasto Guinea (Panicum maximus var Mombasa), cosechado a 90 dias de edad, resultados similares a los encontrados en este estudio. La maxima produccion de metano se tiene a pH 7.0 a 7.2, e incluso puede producirse en el rango de 6.6 a 7.6 (3), en este estudio D. swazilandensis, mostro una menor concentracion de bacterias celuloliticas, un pH por debajo de 6.5, y por tanto una concentracion de [CH.sub.4] menor, esto debido a que se reduce la actividad de las bacterias que degradan fibra por la via piruvato-formato liasa como Ruminococcus fiavefaciens, Ruminococcus albus, Butyrivibrio fibrisolvens y Fibrobacter succinogenes (22,23) y en consecuencia los substratos ([CO.sub.2] y [H.sub.2]) necesarios en la formacion de [CH.sub.4], sin embargo, proliferan especies como Streptococcus bovis, Ruminobacter amylophilus, Succinomonas amylolytica y Selenomonas ruminantium que fermentan los carbohidratos solubles y fragmentos de celulosa para producir propionato via succinato (24), lo que da un perfil diferente en la produccion de AGV, produciendo mayor proporcion de acido propionico y por tanto menos [CH.sub.4]. Por otra parte, la fermentacion ruminal de forrajes con mayor contenido de pared celular, no causan una disminucion significativa del pH, debido a que la mayor cantidad de glucosa liberada se fermenta por la via acetato, en este caso, el [H.sub.2] liberado puede ser utilizado como substrato por las archaeas metanogenicas, que esta asociado con mayor produccion de [CH.sub.4] (3), como el caso de H. rufa y C. nlemfuensis, mientras que con forrajes que causan bajo pH ruminal, se disminuye la metanogenesis como el caso de M. maximus cuyo pH fue menor de 6.5 desde la hora 72 de incubacion y D. swazilandensis desde la hora 24.

Uno de los factores importantes que afecta la produccion de [CH.sub.4] es la relacion de AGV producidos, especificamente la relacion acetico: propionico, que regula la produccion y disponibilidad de [H.sub.2] y la subsecuente produccion de [CH.sub.4]; esta relacion puede variar entre 0.9 a 4 y la utilizacion de la energia es mas eficiente si la relacion es cercana a 1.0 (25). La produccion de [CH.sub.4] ha sido utilizado como indicador de la actividad fermentativa de bacterias en procesos de fermentacion anaerobia (26) en el que intervienen diferentes grupos de bacterias: como las hidroliticas que fraccionan los polisacaridos a azucares, las formadoras de AGV y arqueas metanogenicas que sintetizan [CH.sub.4] a partir de [H.sub.2] y [CO.sub.2] (27,28). El acetato y el butirato originan la produccion de [CH.sub.4], por la mayor disponibilidad de [CO.sub.2] y [H.sub.2] para las arqueas metanogenicas; mientras que para la formacion de propionato en el rumen se considera como una forma competitiva en la captacion de [H.sub.2] que causa una menor sintesis de [CH.sub.4] (29). Los protozoarios del rumen producen [H.sub.2] como principal producto final de su metabolismo, y este, esta estrechamente asociado como sustrato para la formacion de metano por arqueas metanogenicas. Estas bacterias metanogenicas asociadas con los protozoarios ruminales son aparentemente responsables entre 9 y 25% de la metanogenesis, pero esta puede reducirse alrededor de 13% cuando los protozoarios son eliminados; sin embargo, esta reduccion ocurre cuando el animal consume dietas ricas en almidon, que es cuando los protozoarios generan mayor cantidad de [H.sub.2], lo que no sucede cuando las dietas son altas en forraje que tiene como consecuencia una menor formacion de metano (30). Por el contrario, una alta proporcion acetato: propionato esta relacionado con baja eficiencia en la utilizacion de la energia, lo que involucra mayor produccion de [CH.sub.4] como fue el caso de C. nlemfuensis, H. rufa y M. maximus.

Las gramineas tropicales analizadas muestran una concentracion de pared celular elevada, lo cual limita su digestibilidad y reduce su calidad como forraje, sin embargo, Digitaria swazilandensis, mostro una menor produccion total de metano y gases totales, posiblemente por una mayor concentracion de acido propionico, menor concentracion de bacterias celuloliticas, un pH y una relacion acetico:propionico mas baja, siendo la de mayor eficiencia en utilizacion de la energia.

Agradecimientos

Al Consejo Nacional de Ciencia y Tecnologia (CONACYT) por el financiamiento del proyecto intitulado "Estimacion e impacto ambiental de la captura de carbono en plantaciones de palma de aceite (Elaeis guineensis Jacq) en el Estado de Chiapas" de donde se apoyo el desarrollo de este trabajo de investigacion que se encuentra dentro de los lineamientos de Proyectos de Desarrollo Cientifico para Atender Problemas Nacionales (CONACYT/PDCP[N.sub.2]013-01/216526).

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Alejandro Ley de Coss (1) Ph.D, Candido Guerra-Medina (2,3) Ph.D, Oziel Montanez-Valdez (3*) Ph.D, Francisco Guevara H (2) Ph.D, Rene Pinto R (2) Ph.D, Jose Reyes-Gutierrez (3) Ph.D.

(1) Universidad Autonoma de Chiapas, Facultad de Ciencias Agronomicas, Campus V, Carretera Ocozocoautla-Villaflores kilometro 84.5, Villaflores, Chiapas, Mexico. (2) Centro de Investigacion del Pacifico Sur, INIFAP, Carretera Tapachula - Cacahoatan Km. 18, Rosario Izapa, Tuxtla Chico, Tapachula, Chiapas, Mexico CP. 30470. (3) Universidad de Guadalajara, Centro Universitario del Sur. Grupo de Investigacion en Nutricion Animal, Ave. Enrique Arreola Silva 883, Ciudad Guzman, Jalisco. 49000. (*) Correspondence: montanez77@hotmail.com

Received: May 2017; Accepted: December 2017.

DOI: 10.21897/rmvz.1368
Table 1. Culture medium for measuring total gas production, methane and
in vitro degradation of dry matter.

Compound                        Quantity (ml) for 100 ml of
                                medium

Distilled water                 52.9
Clarified rumen liquid (1)      30.0
Mineral solution I (2)           5.0
Mineral solution II (3)          5.0
Sodium carbonate                 5.0
([Na.sub.2][CO.sub.3]), 8% (4)
Sulphide-cysteine solution (5)   2.0
Resazurin solution 0.1% (6)      0.1

(1)Clarified filtered rumen liquid filtered centrifuged at 17664 g for
15 min and sterilized 20 min at 21[degrees]C at 15 psi. (2) Contains
(in 1000 ml) 6 g [K.sub.2][HPO.sub.4] (3) Contains (in 1000 ml
[H.sub.2]O), 6 g [KH.sub.2][PO.sub.4], 6 g ([NH.sub.4])2[SO.sub.4], 12
g NaCl, 2.45 g Mg[SO.sub.4] and 1.6 g Ca[Cl.sub.2]-[H.sub.2]O. (4) 8 g
[Na.sub.2][CO.sub.3] in 100 ml [H.sub.2]O distilled. (5) 2.5 g L-
cysteine (in 15 ml 2N NaOH) + 2.5 g [Na.sub.2]S-9[H.sub.2]O (in 100 ml
[H.sub.2]O). (6) 0.1 ml resazurine in a volume of 100 ml.

Table 2. Chemical composition (%) of tropical grasses C. nlenfuensis,
H. rufa, M. maximus and D. swazilandensis at the age of 75 days.

Nutrient       C. nlemfuensis  H.rufa  M. maximus  D. swazilandensis
                                       %

CP              9.56            6.36    9.54       10.35
EE              1.85            1.25    1.92        2.35
NDF            67.24           63.25   67.25       71.40
ADF            42.56           42.25   42.25       43.40
Hemicellulose  24.68           21.00   25.00       28.00
Ashes           6.72            8.78    8.25        9.25

Table 3. Total gas production of tropical grasses C. nlemfuensis, H.
rufa, M. maximus and D. swazilandensis on in vitro incubation.

Time   C nlemfuensis  H.rufa      M. maximus         D. swazilandensis
                                  ml g [DMf.sup.-1
96      156 (b)       239 (a)      212 (a)           129 (b)
72      525 (a)       356 (c)      521 (ab)          442 (a)
48      255 (a)       242 (a)      252 (a)           254 (b)
24      170 (a)       148 (ab)     128 (b)           122 (b)
Total  1106 (a)       985.0 (b)   1113 (a)           947 (b)

Time     SEM (1)
96       17.6
72       21.4
48       42.3
24       27.7
Total    58.7

(a,b,c) Means with different letters in the same row are different
(p<0.05) (1) Standard error of mean.

Table 4. [CH.sub.4] production by period and total accumulated
[CH.sub.4] of tropical grasses C. nlemfuensis, H. rufa. M. maximus and
D. swazilandensis on in vitro incubation.

Time   C. nlemfuensis  H.rufa      M. maximus D.    swazilandensis
                                   ml [DMf.sup.-1]
96     118.5 (b)       183.3 (a)   162.6 (a)         98.9 (b)
72     373 (a)         273.8 (b)   398.8 (a)        338.8 (a)
48     195.1 (a)       185.1 (a)   192.7 (a)        192.0 (a)
24     130.1 (a)       113.4 (ab)   95.9 (b)         93.3 (b)
Total  816.7 (ab)      755.8 (ab)  852.2 (a)        723.9 (b)

Time    SEM (1)

96       21.2
72       32.1
48       16.3
24       13.5
Total   101.9

(a,b,c) Means with different letters in the same row are different
(p<0.05)

Table 5. Production of volatile fatty acids from tropical grasses C.
nlemfuensis, H. rufa, M. maximus and D. swazilandensis in vitro
incubation.

           C. nlemfuensis  H.rufa     M. maximus  D. swazilandensis
                                      mmol L-1
Acetic      74.28 (a)       73.81 (a)  73.81 (a)    64.17 (a)
Propionic   18.80 (b)       16.20 (b)  16.20 (b)    38.23 (a)
Butyric      9.43 (ab)       4.86 (b)   4.86 (b)    12.26 (a)
Total      102.43 (a)       94.87 (a)  94.87 (a)   114.60 (a)
A:P          3.95 (b)        3.50 (b)   3.5 (b)      1.67 (a)

           SEM (1)

Acetic      14.6
Propionic    9.3
Butyric      4.3
Total       33.25
A:P          0.34

(a,b,c) Means with different letters in the same row are different
(p<0.05)
(1) Standard error of mean.

Table 6. pH of the culture medium in which the tropical grasses C.
nlemfuensis, H. rufa, M. maximus and D. swazilandensis were fermented
in vitro.

Hours  C nlemfuensis  H.rufa    M. maximus  D. swazilandensis  SEM (1)
96     6.11 (ab)      6.53 (a)  5.95 (b)    5.88 (b)           0.12
72     6.74 (a)       6.64 (a)  6.09 (b)    6.24 (ab)          0.26
48     6.65 (a)       7.02 (a)  6.84 (a)    6.34 (a)           0.30
24     7.14 (a)       6.95 (a)  6.95 (a)    6.30 (b)           0.31

(a,b,c) Means with different letters in the same row are different
(p<0.05)

(1) Standard error of mean.

Table 7. Concentration of total and cellulolytic bacteria in the
culture medium in in vitro incubation.

Hours  C. nlemfuensis  H.rufa                                M. maximus
                       Total bacteria 1 x [10.sup.9]

96     11.6             6.09                                  2.13
72      7.77            4.03                                  1.26
48      5.95            3.08                                  0.94
24      5.14            2.66                                  0.77
                       Cellulolytic bacteria 1 x [10.sup.7]
96      6.74 (b)       19.80 (a)                             31.20 (a)
72      4.31 (a)       17.60 (a)                             19.90 (a)
48      2.60 (a)       14.50 (a)                             12.00 (b)
24     11.10 (a)        1.11 (b)                              1.08 (b)

Hours  D. swazilandensis  SEM (1)

96      3.53               3.14
72      2.23               3.09
48      1.71               3.08
24      1.35               3.09

96      6.50 (b)           2.70
72      4.20 (a)           2.74
48      2.50 (a)           2.24
24      1.08 (b)           2.20

(a, b, c) Means with different letters in the same row are different
(p<0.05)
(1) Standard error of mean.
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Title Annotation:ORIGINAL
Author:de Coss Alejandro, Ley; Candido, Guerra-Medina; Oziel, Montanez-Valdez; Francisco, Guevara H.; Rene,
Publication:Revista MVZ (Medicina Veterinaria y Zootecnia)
Date:Sep 1, 2018
Words:8136
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