RESEARCH WORK
In vitro fermentation kinetics of Leucaena leucocephala and Megathyrsus maximus and their mixtures, with or without energy supplementation
Xiomara Gaviria1, J. F. Naranjo2 and R. Barahona1
1Departamento de Producción Animal, Facultad de Ciencias Agrarias,
Universidad Nacional de Colombia
Calle 59A No. 63-20 Medellín, Colombia - Núcleo El Volador Medellín - Colombia.E-mail: xiomygaviria@gmail.com
2Grupo INCA-CES Universidad CES, Medellín, Colombia
ABSTRACT
In order to characterize the in vitro fermentation kinetics of Leucaena leucocephala and Megathyrsus maximus mixtures, an in vitro gas production trial was conducted in which five treatments were included: 100 % of L. leucocephala (L100), 100 % of M. maximus (G100), 100 % of supplement based on rice meal and molasses (S100), and two proportions: L23-G77 and L26-G70-S4. The forages were collected during eight months in an intensive silvopastoral system (SPSi), belonging to the Agricultural Center Cotové of the National University of Colombia. The maximum gas production varied in a range of 156 (L100) to 247 mL g-1 substratum (L26-G70-S4). The lowest gas volume at the inflection point (57,5 mL) was observed in L100, which was different from the mixtures and the supplement (p < 0,05). The disappearance of the DM at 96 h varied between 53,8 % and 66,9 %, and it was higher in L100 than in the other treatments (p < 0,05). The lowest gas production value (1,31 mL) for each gram of DM, fermented at 96 h, was observed in L100 (p < 0,05). The results suggest that the inclusion of leucaena increased the concentration of protein in the diet and reduced the NDF content, which is positive from the point of view of animal productivity. It is concluded that the utilization of higher nutritional quality forages, such as that of leucaena, modifies the fermentation profile of the diet; for which the response of the forage-grass mixtures is different from the expected one, because it depends on the individual response of each forage.
Key words: In vitro gas production, silvopastoral systems, animal productivity.
INTRODUCTION
Tropical forages commonly have high cell wall contents and low soluble carbohydrate contents (Juárez and Pell, 1999), and, in general, their conversion into products of animal origin is not very efficient (Barahona and Sánchez, 2005). Only between 10 and 35 % of the consumed energy is captured as net energy, because between 20 and 70 % of the cellulose cannot be digested by the animal. On the other hand, 12,8 g N kg-1 DM are required in the diet to guarantee the good functioning of the rumen; for which, according to the characteristics of forages in the low tropic, it is necessary to supplement with N to cover the requirements of cattle. Any feedstuff that contains more than 16 g N kg-1 is considered as protein supplement (CSIRO, 2007), and some forage legumes fulfill this requisite. For example, leucaena (Leucaena leucocephala (Lam.) de Wit) has between 25 and 35 g N kg-1 of DM (Barahona et al., 2003; Rodríguez and Fondevila, 2009; Cuartas et al., 2014b); however, due to its fiber content, it is classified as a roughage.
In order to reach higher efficiency and productivity in cattle production, silvopastoral systems have received great attention, especially those called intensive (SPSi). They show high densities of forage shrubs (more than 10 000 ha-1), such as leucaena, and the associations with improved pastures have proven to be the ones with higher perspective (Tarazona et al., 2013). In SPSi biomass productions of up to 28 t DM ha-1 year-1 have been reported (Naranjo et al., 2012), with a high protein and energy content, which has allowed a high stocking rate and a high milk and beef production per hectare. Nevertheless, although the protein intake from a SPSi is adequate for most physiological stages of ruminants, the fiber content in that diet (at least 60 % of neutral detergent fiber NDF , according to the report by Gaviria et al., 2012) could limit animal productivity if it is not efficiently degraded in the rumen.
Taking the above-explained facts into consideration, it is important to study the fermentation dynamics of the forages which compose the SPSi with leucaena, to determine the patterns of N fermentation and energy in the rumen, in order to know if they are balanced or if it is necessary to suggest adjustments or additions to the diet of the animals which graze in such system. The objective of this study was to characterize the in vitro fermentation kinetics of forage mixtures of L. leucocephala and Megathyrsus maximus, collected in silvopastoral systems.
MATERIALS AND METHODS
Evaluated substrata and bromatological characterization. The forages were collected at the Agricultural Center Cotové, village El Espinal (Santa Fé de Antioquia municipality), 74 km away from Medellín, which is located in a life zone of tropical dry forest (T-df), at a height of 540 m.a.s.l., with an average temperature of 27 ºC and a rainfall of 1 100 mm per year.
These forages came from lots of an 18-month old SPSi, formed by L. leucocephala shrubs, Guinea grass (M. maximus) and a small proportion (less than 5 % of the total biomass offer) of star grass (Cynodon plectostachyus), with an average age of 50 days of regrowth; which were grazed by Zebu steers during eight months, in an experiment of intake and selectiveness under grazing conditions (Gaviria et al., 2014). For the purpose of this trial, the evaluated forages (L. leucocephala and M. maximus) correspond to a homogeneous mixture of three subsamples collected at different moments, along such grazing period; C. plectostachyus was not included, due to its low presence in the SPSi. Additionally, a sample of a supplement based on rice meal and molasses (70 and 30 %, respectively) was supplied to only half the steers during the grazing period, in order to determine the effect of the addition of a raw material of different fermentative nature from that of the forages of the SPSi.
The bromatological analyses of the forages and the raw materials were performed on the three forage subsamples and on the supplement sample, in the laboratory of chemical and bromatological analysis of the National University, campus Medellín. The evaluated components in each of the samples, with their respective methods, are described below:
In addition, when it was necessary to estimate the nutrient requirements and the energy metabolism, the Cornell Net Carbohydrate and Protein System CNCPS (Fox et al., 1992) model was used.
Treatments. Table 1 shows the evaluated treatments. The first three corresponded to the raw materials (forages and supplement) used; while the rest was composed by mixtures that had the same proportions of raw materials as the diet consumed by the non-supplemented steers (23 % of leucaena and 77 % of Guinea grass) and by the supplemented steers (26 % of leucaena, 70 % of Guinea grass and 4 % of concentrate feed), in a grazing essay conducted by Gaviria et al. (2014).
In vitro gas production technique. The in vitro gas production was analyzed according to the technique described by Theodorou et al. (1994), with the modifications proposed by Posada et al. (2006).
Cultivation medium. A cultivation medium composed by buffer solution, solution of macrominerals, solution of microminerals, reducing solution and resazurin (Goering and Van Soest, 1970), was used.
Inoculants. At the slaughter house Central Ganadera S.A. of Medellín, rumen liquid was collected from three Holstein animals, from the Santa Rosa de Osos municipality. Each rumen liquid constituted one of the three inoculants used in the fermentation process. One hundred eight glass bottles of 110 mL, with airtight rubber stopper, were used; each one had 0,5 g of the sample corresponding to each treatment and 45 mL of cultivation medium. These bottles were placed in bain-marie at 39 ºC, with constant agitation, where 5 mL of inoculant was added to them, and during the experiment they were kept at 39 ± 1 ºC.
Readings of gas production. The pressure originated by the accumulation of gasses in the flasks was measured with a manometer Ashcroft® D1005PS Digital Pressure Gauge. The gas production readings were made at 2, 4, 6, 10, 12, 24, 36, 48, 60, 72 and 96 hours of fermentation. To convert the pressure values obtained in pounds per square inch (psi) to volume units (mL), the equation described by Posada et al. (2006) was used.
Y = -0,1375 + 0,0745x + 0,000016x2; (p < 0,0001; R2 = 0,99)
Where:
Y: gas volume (mL)
x: gas pressure (millibars)
In vitro DM degradability. The in vitro DM degradability (IVDMD) was determined by weighing the residues recovered from fermentation, after the filtration of the bottles removed at 24, 48 and 96 h after the beginning of incubation. At each of those moments, six bottles per treatment were removed from the bain-marie and transferred to a freezer (4 ºC), to stop the fermentation process. Afterwards, the content of each of the samples was filtered through a filter paper, and they were dried during 48 h at constant temperature of 65 ºC, in a forced-air oven. The filter paper with the final DM content of each bottle was weighed in an analytical balance (Pioneer OHAUS). The IVDMD was calculated by the difference between the initial DM content and the content of non-degraded DM (final DM), and was expressed as percentage of the initial DM.
Statistical analysis. To describe the dynamics of cumulative gas production in time the non-linear Gompertz model (Casas et al., 2010) was used. The curve adjustment was made in the program Curve Expert Professional 2.0.0.
y = a * exp (exp (b (c * x))
Where:
y: cumulative gas production at a time x
a > 0: maximum gas production
b > 0: difference between initial gas and the final gas at a time x
c > 0: specific rate of gas accumulation
The practical application of this model requires the conversion of the parameters a, b and c into parameters with biological meaning. They are: time at point of inflection (TPI, hours), gas at point of inflection (GPI, mL), maximum gas production rate (MGPR, mL h-1) and Lag phase (LP or microbial establishment, h). For their estimation the following formulas were used:
Where: the value of e or exp corresponds to the Euler number ≈ 2,7183.
The measured variables were analyzed with the statistical program SAS version 8.0.2. The model used was the following:
In each incubation time (24, 48 and 96 h) six replications per treatment were included. Likewise, 18 bottles with rumen liquid without substratum were used as blank, for which the total number of bottles of the experiment was 108. A completely randomized design was used, and the dependant variables were gas production and disappearance of DM. The means were compared through Tukey's test, with a significance level of 0,05.
RESULTS AND DISCUSSION
Chemical composition of the forages and the supplement. The chemical composition of the treatments is shown in table 2. The supplement contained a high fat percentage and calorific value, for which it constituted an adequate contribution of energy for fattening steers. In the field trial conducted by Gaviria et al. (2014) the steers received 0,5 kg day-1 of this supplement, and it meant an energy contribution of 1,02 Mcal of NEm day-1, or approximately 14,8 % of the requirement of NEm of a beef bovine of 400 kg of live weight; which, according to the NRC (2000), is 6,89 Mcal day-1.
The crude energy content of the two in vitro mixtures (L23-G77 and L26-G70-S4) was around 4,2 Mcal kg-1. Cuartas et al. (2014a) and Gaviria et al. (2014) reported that Zebu steers which grazed in SPSi consume at least 2,5 % of their live weight in terms of dry matter, so that steers of 300 and 400 kg would consume 31,5 and 42,0 Mcal of crude energy day-1 and 14,7 and 19,6 Mcal of metabolizable energy day-1, respectively. According to the CNCPS model, under the conditions of the evaluated SPSi a 400-kg steer requires around 12,33 Mcal of ME day-1 for its maintenance, with which it would have 7,27 Mcal of ME day-1 available for growth, which would allow a daily gain of around 500 g.
The protein of the Guinea grass was higher than that of the tropical grasses, in which a low protein percentage (≈ 7 %) and a high fiber percentage (< 60 %) are commonly reported, which contributes to a low total digestibility of the diet and a low intake by the animals (Barahona and Sánchez, 2005). The nutrient content of the Guinea grass, especially protein and fiber, can be improved through the association with leucaena, because the grass-legume interaction in silvopastoral systems allows to improve the quality of the grass, by increasing the protein content (Barahona and Sánchez, 2005). In addition, the forage availability in these systems is higher compared with that of monocrop systems, which allows an increase of the cattle production efficiency (Cuartas et al., 2014b). However, if it is compared with that of Guinea grass alone, the NDF content of the mixtures was high, especially for treatment L23-G77 (60 % of NDF), for which it is necessary to establish alternatives to improve the net energy-protein ratio in the diets consumed in SPSi based on leucaena.
Gas production. Table 3 shows the parameters obtained through the application of the Gompertz model to the in vitro fermentation data of the treatments. In all the runs of the model, the determination coefficients were higher than 0,99 and the standard error varied between 0,27 and 2,37.
The treatments showed a establishment or colonization stage of 3,36-7,61 h (p > 0,05). Although there were no significant differences, a trend of the establishment stage to be lower in the mixtures (L33-G77, L26-G70-S4) and in the supplement, was observed. The lowest GPI value (57,5 mL) was obtained in L100, which differed from the others (p < 0,05). The highest GPI values occurred between 20 and 38 hours, and coincided with the treatments that accumulated the highest gas quantity.
The maximum gas production was 156-247 mL g-1 of substratum, and treatment L100 showed the lowest value. In this sense, it has been reported that the in vitro gas production is related to the efficiency of feedstuff utilization by the rumen microorganisms.
On the other hand, the chemical composition of the forages influences the gas volume produced, the maximum gas production rate and the time in which the in vitro ruminal fermentation is reached. In this study, when comparing G100 with its mixtures (L23-G77 and L26-G70-S4), the highest gas production was obtained in the forages with lower quantity of NDF. This coincided with the highest GPI values, because, possibly, the gas production is linearly related to the degradation of the NDF; that is, the higher the content of digestible NDF is, the higher the gas production will be (Kriszan et al., 2012).
According to Fondevila et al. (2002), the high lignin contents in the forages can explain the low gas production values, as well as the fermentation rate and the degradability, because those contents cause that the potentially-digestible structural polysaccharides are less available to the access of the rumen microorganisms.
The accumulation of gas showed that the fermentation rate in the rumen was modified with the use of the mixture of forage grasses and legumes. This was also reported by Cuartas (2013), who found, after 96 h of incubation, that the mixtures of grasses and legumes showed higher degradability than the forages incubated alone, which suggests the existence of associative effects that favor the digestion of the mixtures. Likewise, Nogueira et al. (2000) reported that the substrata of higher nutritional value favor the colonization and their efficient degradation by microorganisms, as well as the increase of the fermentation rate and extension.
Machado et al. (2012) reported that the substrata with lower NDF content show higher rates of gas production, and it occurs faster. This was observed in G100, L23-G77 and L26-G70-S4, but with leucaena alone (L100). Besides the low NDF content, another factor that modulates the gas production of this forage is its content of condensed tannins, which can be high 10 % of the DM (Barahona et al., 2003), and whose presence reduces the gas production.
Disappearance of the DM. The disappearance of the DM of leucaena was higher than that of the other samples (p < 0,05), with the exception of the degradability of the supplement, at 24 hours of incubation (table 4). On the other hand, at 48 and 96 hours of incubation the degradability of the mixtures L23-G77 and L26-G70-S4 did not differ statistically from the one observed in G100 (p > 0,05).
The disappearance of DM of the mixtures was lower at all sampling hours with regards to that of treatment L100, which differs from the result obtained in the maximum gas production (table 3), as was explained above. Similar results were reported by Molina et al. (2013), who observed a lower gas production and a higher disappearance of DM in leucaena alone vs. its mixtures with Guinea grass. On the other hand, Rodríguez and Fondevila (2009), when supplementing Pennisetum purpureum with increasing levels of leucaena, obtained a positive linear relation with the gas production in the first 5 h of fermentation, just like with the digestibility of OM at 24 h. This was probably due to the contribution of the fermentable OM of leucaena. On the contrary, Sandoval et al. (2002) reported that, in in vitro fermentations, leucaena reduced the IVDMD and the gas production of leucaena-grass mixtures.
The high values of ruminal degradability of DM in the forages are indicators of a good nutritional quality, which allows the contribution of nutrients to the ruminal flora (Preston and Leng, 1990). In this study, the highest degradability was observed with the legume, which was similar to the IVDMD (61,5 %) reported by Barahona et al. (2003) for leucaena, and higher than the IVDMD (47,8 %) informed by Delgado et al. (2013) in a diet with 27 % of leucaena. The supplement used showed lower degradability than the forages, and higher gas production per unit of degraded DM. This is possibly due to its high fat content (11,6 %), which could limit its degradation.
The inclusion of leucaena and the supplement caused a decrease in the NDF content of the mixtures, with regards to the treatment with G 100 (table 2), but in turn increased the in vitro digestibility of the DM of such mixtures (table 4). Such results are not necessarily opposed to those which report that a high NDF content causes a limited availability of energy for the ruminants, because in the in vivo response additional factors should be considered such as the escape rate of the diet, which directly affects the intake. It has been observed that the voluntary intake of the leucaena-grass mixture is, at least, 1,25 times higher than that of the grass alone (Cuartas et al., 2014b; Gaviria et al., 2014), for which the results of this experiment suggest that such intake obeys rather to a higher passage rate than to an increase of the ruminal degradability.
Gas production per unit of degraded DM. The data of gas production and of the DM degradability were combined to calculate the gas production rates per unit of DM (table 5). These data suggest that when supplementing the grasses with forage trees and supplements the synthesis of microbial protein in the rumen increases. At 24, 48 and 96 h treatment L100 showed the lowest values of gas production (0,18; 0,25 and 0,27 mL, respectively) per milligram of fermented DM (p < 0,05), while the highest production was obtained with the concentrate feed.
The feedstuffs with low ratio of gas volume per quantity of degraded DM, such as leucaena (table 5), are generally more digestible (Blümmel et al., 1997), their voluntary intake by the animals is higher and they generate a lower production of methane in the rumen (Molina et al., 2013). Because the gas and the VFA produced are inversely proportional to the microbial yield, the ratio shows variations in the synthesis of microbial protein, and is an important indicator of its efficiency (Darshan et al., 2007). In general, it is possible to state that in this study the inclusion of legumes in the diet improved the fermentation conditions, which favored the synthesis of microbial protein.
CONCLUSIONS
In the diets that include leucaena-based silvopastoral systems, the presence of this legume is associated to the protein increase, while leading to a reduction in the NDF content. Both responses are positive from the point of view of animal productivity.
This study proved the high nutritional quality of leucaena and its capacity to modify the fermentation profile of the diet, by showing the lowest gas production in spite of causing the highest disappearance of DM. Nevertheless, such effect was not evident in the case of the grass-legume mixtures, for which it is not possible to predict their response from each individual forage.
RECOMMENDATIONS
It is necessary to continue with this line of research, to which the study of the initial degradation rate of DM in the rumen, the passage rate and the permanence time, should be added, in order to understand the attainment of better results regarding animal productivity, compared with the traditional diets based on grasses.
ACKNOWLEDGEMENTS
The authors thank the Ministry of Agriculture and Rural Development (MADR) for funding the Project <<Comparative analysis of beef production in steers produced in an intensive silvopastoral system and confinement>>, within which this research work was conducted; as well as the work team in CIPAV, UDEA and UNAL.
Received: March 7, 2014
Accepted: June 30, 2014
