Effect of four inclusion levels of Morus alba L. cv. cubana on microbial populations and fermentative products in river buffalo (Bubalus bubalis) rumen liquid

Efecto de cuatro niveles de inclusión de Morus alba L. vc. cubana en las poblaciones microbianas y productos fermentativos en líquido ruminal de búfalos de río (Bubalus bubalis)

Niurka González,^I Juana Galindo,^I A.L. Abdalla,^II

ABSTRACT

To know by means of in vitro fermentation the effect of mulberry inclusion (M. alba Linn. cv. cubana) on the microbial populations and fermentative products in river buffalo (Bubalus bubalis) rumen liquid, four inclusion levels of M. alba Linn. cv. cubana (0, 15, 30, and 45 %) in a star grass(Cynodon nlemfuensis) basic diet were evaluated. The treatments chemical composition, gas volume and accumulated methane were determined at 4, 8, 12 and 24 h of fermentation. The sowing and count of total viable bacteria, cellulolytics, proteolytics, methanogenics and cellulolytic fungi were carried out at 0, 4 and 8 h. The protozoa count, the pH and ammonia concentration (NH₃) was made at 0, 4, 8, 12 and 24 h of incubation. A completely randomized design with factorial arrangement was applied. The treatments and the fermentation hours were the factors and four repetitions were made in time. From eight and up to 24h, all the treatments with M. alba had gas productions higher than the control (P < 0.0001), although the 30 and 45 % inclusion promoted similar amounts to each other (52.92 and 54.13 mL/g DM, respectively).The ruminal methane production was higher, when including 15 and 45 % de M. alba L. cv. cubana (P < 0.05), while the incorporation of 30% originated a volume that did not differ from control treatment. The populations of bacteria, fungi and protozoa, the pH, and the ammonia concentration were not affected with the M. alba inclusion. The obtained results allow to conclude that 15, 30 and 45 % of Morus alba cv. cubana inclusion do not exerts effect on the rumen microbial populations , pH and ammonia concentration, but when this plant is included in 15 and 45 % it increase the ruminal methane production.

Key words: fermentation, methane, microorganisms, mulberry, rumen.

RESUMEN

Para conocer mediante fermentación in vitro el efecto de la inclusión de morera (M. alba Linn. vc. cubana) en las poblaciones microbianas y  productos fermentativos en el líquido ruminal de búfalos de río (Bubalus bubalis), se evaluaron cuatro niveles de inclusión de M. alba Linn. vc. cubana (0, 15, 30, y 45 %) en una dieta base de pasto estrella (Cynodon nlemfuensis). Se determinó la composición química de los tratamientos, el volumen de gas y metano acumulado a las 4, 8, 12 y 24 h de fermentación. La siembra y el conteo de bacterias viables totales, celulolíticas, proteolíticas, metanogénicas y hongos celulolíticos se realizó a las 0, 4 y 8 h. El conteo de protozoos, el pH y la concentración de amoníaco (NH₃) se efectuó a las 0, 4, 8, 12 y 24 h de incubación. Se aplicó diseño completamente aleatorizado con arreglo factorial. Los factores fueron los tratamientos y las horas de fermentación, y se efectuaron cuatro repeticiones en tiempo. A partir de las ocho y hasta las 24 h, todos los tratamientos con M. alba tuvieron producciones de gas superiores al control (P < 0.0001), aunque la inclusión de 30 y 45 % promovió cantidades similares entre sí (52.92 y 54.13 mL/g MS, respectivamente). La producción de metano ruminal fue superior, cuando se incluyó  15 y  45 % de M. alba L. vc. cubana (P < 0.05), mientras que la incorporación de 30 %  originó un volumen que no difirió del tratamiento control. Las poblaciones de bacterias, hongos y protozoos, el pH y la concentración de amoníaco no se afectaron con la inclusión de M. alba. Los resultados obtenidos permiten concluir que  15, 30 y 45 % de inclusión de Morus alba vc. cubana no ejerce efecto en las poblaciones microbianas del rumen, pH y  concentración de amoníaco, pero cuando esta planta se incluye en 15 y 45 % incrementa la producción de metano ruminal.

Palabras clave: fermentación, metano, microorganismos, morera, rumen.

INTRODUCTION

In Cuba, for some years, it is researched about the control of the ruminal methanogenesis (Galindo et al. 2003).The same as in many countries, diet manipulation and the use of plants as part of it, is one of the used strategies for being more feasible under the current economic conditions of the country. In this sense, the mulberry (Morus alba Linn.) is highlight as forage source because of its capacity of biomass production, chemical composition (Duke 2001), high degradability and excellent results on ruminants nutrition (Kandylis et al. 2009), so that their evaluation in the control of the ruminal methanogenesis becomes necessary.

Delgado et al. (2007), when evaluating the mulberry effect on the control of ruminal metehanogenesis   founded that the 25 % inclusion of this tree reduces methane production. Taking into account this result, González et al. (2011) decided to deepen in the mulberry study and to evaluate the effect of different varieties on microbial populations, fermentative products and methane production, in in vitro conditions with river buffalo  (Bubalus bubalis) rumen liquid.

These authors selected the most promissory variety to control ruminal methanogenesis. From the evaluated varieties ( cubana, acorazonada, tigreada and indonesia), the cubana variety was the most promising to control the ruminal methanogenesis, when its inclusion in a diet based on star grass  (Cynodon nlemfuensis) was of 30%.

Given these results, it was considered to carry out studies in those that other inclusion levels of this mulberry variety were evaluated, to determine which their effects were on microbial populations, products from ruminal fermentation and among these, specifically methane production. 

The objective of this research was to evaluate, in vitro, the effect of four inclusion levels of mulberry (Morus alba Linn.) cv. cubana on microbial populations and fermentative products in river buffalo (Bubalus bubalis) rumen liquid.

MATERIALS AND METHODS

The gas in vitro production technique was applied, described by Theodorou et al. (1994) and modified by Rodríguez et al. (2008).

Animals and diet. Two adult river buffalo bulls crossbred of Bufalipso fitted with rumen cannula and average weight of 453 kg were used as donor of rumen liquor. They were allocated in individual pens, under shade and ad libitum water and feeds. Both received ad libitum star grass (Cynodon nlemfuensis) and supplementation was not applied.

Treatments. Four treatments were evaluated, which corresponded with different mulberry inclusion levels to a star grass (SG) basic diet: 1) 100 % of SG (Control), 2) SG + 15 %   M. alba L. cv. cubana, 3) SG + 30 % M. alba L. cv. cubana and 4) SG + 45 %  M. alba L. cv.
cubana.

In table 1 is showed the chemical composition of the evaluated treatments.

Experimental procedure. Samples of 0.5 g from each treatment were weighed and added to the glass bottles of 100 mL. The animals under fasting were extracted the rumen liquor through the cannula, using a vacuum pump. The material was kept in a hermetically sealed thermo to guarantee the temperature (39 °C) and anaerobiosis conditions during the transfer to the laboratory.

The rumen content of both animals was mixed and filtered through muslin. The resulting solid was added a small portion of buffering solution of Menke and Steingass(1988) and was stirred for a few seconds in a domestic blender to loosen the microorganisms adhered to the fiber. Later, the filtered material from this portion was added to the liquid fraction. The rumen liquor was kept under CO₂ atmosphere.

Each bottle was added, a mixture of 50 mL of rumen liquor and buffering solution of Menke and Steingass at a ratio of 1:3 (v/v) and they were sealed with butyl and agrafe taps. Bottles without substrate were included as blanks to correct the effect of rumen liquor on the volumes of gas produced. All bottles were randomly put in a controlled bath at 39 °C.

The gas was measured by motion of the embolus of a syringe of 10 mL, after the puncture of the tap. The gas volume and accumulated methane, the pH and ammonia concentration, were determined at 4, 8, 12, and 24 h of fermentation.

The sowing and count of total viable bacteria, cellulolytics, proteolytics, methanogenics and cellulolytic fungi were made at 0, 4 and 8 h of incubation. The protozoa count was carried out at 0, 4, 8, 12 and 24 h of fermentation.

Chemical analysis: The determination of the chemical composition of the experimental diets was carried out according to AOAC (2005). The fibrous fractions were analyzed by the procedure of Goering and van Soest (1970).The ammonia concentration was determined by the Conway (1957) method and that of methane, by gas chromatography, using a Philips PU-4400 chromatographer with capillary column of 25 m, with stationary phase DB^-1. The   FID detector was used and H₂ (1 mL. min^-1) as gas carrier. The temperature of the detector and of the injector was of 200 °C, and that of the column, was of 60 °C. One milliliter of gas from the syringe was injected. The calculations of methane concentration were performed from the equation obtained in the calibration curve: y= 0.0001x + 2.8515 (R² = 0.99).

Microbiological analysis: The Hungate (1970) culture technique in roll tubes and under strict anaerobiosis conditions was used. The sowing of total viable bacteria, cellulotytics and proteolytics were made in Caldwell and Bryant (1966) culture medium, modified by Elías (1971). In the case of proteolytics bacteria, 10% of skim milk was added, according to Galindo et al. (1984). To determine the fungi population, the Joblin (1981) culture mean was used. The methanogenic microorganisms were cultivated in the culture medium described by Anderson and Horn (1987), with mixture of hydrogen and carbon dioxide gases (60:40). For inoculations, three dilutions were used and each of them was replied three times. The counts of colonies of total viable bacteria, cellulolytics, proteolytics, methanogenics and fungi, were carried out by means of the roll tubes placement under a magnifying glass. The colonies that showed digestion halo were counted. The results were expressed in unit colony formers (ucf) for bacteria and in unit thallus formers (utf) for fungi.

The protozoa were preserved in formol at 10%. Then, they were directly counted to the optic microscope in a Neubauer camera, after tinted with a violet gentian solution at 0.01 % in glacial acid.

Experimental design and statistical analysis: A completely randomized design with factorial arrangement was used. The treatments and fermentation hours were the factors. Four repetitions were made in time.

For the results processing, a multivariate variance analysis was used. In case of find interaction between treatments and sampling hours, a split plot model was applied. The main plots were the treatments and the subplot, sampling hours. In case of not find interaction, a lineal model for the effects of treatments and sampling hours was used. Duncan test for P < 0.05 was applied when necessary (Duncan 1955). The statistical software INFOSTAT was used proposed by Di Rienzo et al. (2012).

RESULTS AND DISCUSSION

The systems of in vitro incubation have been used during decades to evaluate foods for ruminants (Stalker et al. 2013 and Muetzel et al. 2014).The in vitro gas production is one of the methods used with this purpose. This method allows evaluating the digestibility of the forages organic matter and can offer information of the degradation speed of foods (Fabio et al. 2008).

Table 2 shows the obtained results in gas production during this study. It can verify that, as the hours passes, the gas volumes for all treatments are increased. From eight hours up to 24 h of incubation, all treatments in which M. alba L. cv. cubana is included showed gas productions higher than the control treatment. The 30 and 45 % of inclusion of this mulberry variety produced similar gas quantities.

The mulberry is characterized for its high contents of easy fermentation carbohydrates, so it is expected that their participation in animals ration increases the digestibility in the rumen. This results in higher gases production. If the NDF values of the treatments under study (table 1) are observed, it can be notice that, when Morus alba L. cv. cubana is included, this indicator decrease. So, there is higher quantity of cellular content and therefore, higher ruminal degradation occurs.

The ruminal methane production was higher when 15 and 45 % M. alba L. cv. cubana was included, although the different inclusion levels of this variety did not show differences between them for this indicator (figure 1). The inclusion of 30% of cubana variety produced a methane volume that did not differ of the control, results that were similar to those obtained by González et al. (2010). 

The structural carbohydrates, as the cellulose and the hemicellulose, are fermented to a lower speed than those not structurals and produce more methane per digested substrate unit (Molano et al. 2008).This was previously showed by Galindo et al. (2003),whose explained that higher ruminal cellulolisis of the food can also increase the production of this gas. If it is taken into account that cubana variety inclusion increases the methane production for all levels, except for 30%, it could think that the inclusion of this plant in the diet favors the degradation process of the rumen cellulose.  

The inclusion of 15, 30 and 45 % of M. alba L. cv. cubana did not produce effects on populations of cellulolitic, proteolitic, methanogenesis and total viable bacteria, fungi and protozoa (table 3). These results are explained, if there are kept in mind the interrelations that are stated between the microorganisms populations in the rumen. Cheng et al. (2009) explained that the rumen microorganisms degrade the plants fibers and produce a variety of final products, in those that are included the acetate, format, lactate, ethanol, H₂ and CO₂, some of those that are used by the methanogens to produce methane.

So it could be expected that if some of the populations is affected, it would also be reflected in the methanogens `population. The protozoa counts, when not experiencing affectations with the mulberry inclusion, could explain that the methanogens numbers did not neither have variation, since the protozoa provides of a habitat for the methanogens (Hung et al. 2013).

The methane is produced by the methanogenics Archaea during the food use in the rumen (Chong et al. 2014 and Chuntrakort et al. 2014) but, according to Machmüller et al. (2003), the methanogenesis cannot always correlate with the methanogens numbers in the rumen. An experiment carried out by Kamra et al. (2006) show that while the methanogenesis is completely inhibited when there is presence of sulfonic bromoethane acid (SBE), methanogens number, quantified by means of PCR in real time, was not completely eliminated. If it considered the opinion that the methane production in the rumen is not necessarily correlated with the methanogens number, then it is logical that the increase in methane production, when 15 and 45 % of M. alba L. cv. cubana is included, was not reflected in increase in the methanogens counts for these treatments. The analysis of this result allows sharing the criteria exposed by Liu et al. (2013) regarding that the relation between methanogens and methane production is an aspect that needs further researches.

In table 4 is show that the inclusion of different M. alba L. cv. cubana levels did not produce effects on the pH and the ammonia concentration in the rumen.

It is known that if the pH in the rumen decrease, negative effect on the fiber degradation and foods intake is produce and as consequence, an acidosis occurs (Wallace et al. 2006).With the M. alba L. cv. cubana inclusion in the ration, the pH was near to the neutrality, favoring that the fiber degradation in the ruminal liquor was not affected. This could explain the higher methane productions obtained for 15 and 45% of the cubana variety inclusion regarding the control.

The higher levels of degradable protein in the rumen caused increase in the ruminal amamonia production (Dhali et al. 2006 and Saro et al. 2014).All seems that the mulberry has high composition of non degradable protein in the rumen, and this was reflected in the ammonia concentrations obtained when including 15, 30 and 45 % of the cubana variety. Liu et al. (2001) and Singh and Makkar (2002) informed moderate values of non protein nitrogen and effective degradability of crude protein of the mulberry leaves of 54.9 % and 57 %,respectively. These authors explained that this plant can be a satisfactory food for the synthesis of microbial protein in the rumen and therefore, favors higher microbial protein supply to the intestine. Yao et al. (2000) and Liu et al. (2001) explained that the mulberry can be use as protein supplement for the ruminants. The use of experimental diets in ruminants showed that the mulberry nutritive value is as high as that of some high quality foods already well-known.

The 15, 30 and 45 % of Morus alba cv. cubana inclusion have no effect on the rumen microbial populations, the pH and the ammonia concentration, but increase the ruminal methane production, when including the 15 and 45 % in the diet.

ACKNOWLEDGEMENTS

The authors appreciate the CAPES, Brazil financing, for the research development.

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Received: June 1, 2015
Accepted: July 1, 2015

Niurka González, Instituto de Ciencia Animal, Apartado Postal 24, San José de las Lajas, Mayabeque, Cuba. Email: ngonzalez@ica.co.cu