ORIGINAL ARTICLE
Biological effect of tannins from four tropical tree species on in vitro ruminal fermentation indicators
Efecto biológico de taninos de cuatro especies arbóreas tropicales en indicadores de la fermentación ruminal in vitro
R. Rodríguez,I Niurca González,I J. Alonso,I Yasmila Hernández,I Yolaine Medina,I
]]> IInstituto de Ciencia Animal, Apartado Postal 24, San José de las Lajas, Mayabeque, Cuba.
ABSTRACT
In order to estimate the in vitro biological effect of tannins within moringa (Moringa oleifeira cv. Super Genius), mulberry (Morus alba Linn cv. Cubana), trichanthera (Trichanthera gigantea) and leucaena (Leucaena leucocephala cv. Perú) foliage meals on ruminal fermentation indicators, the in vitro gas production was measured and the kinetics parameters were estimated according to Gompertz model, the in vitro degradability of the neutral detergent fiber (NDF) and the nitrogen. The biological effect was estimated as the quotient between the value of each indicator when the tannins are inactive and active, expressed as increase percent. A random block design was applied, with four incubations as repelicates and the biological effect of tannins as treatment. At the beginning of fermentation (4h), the trichanthera tannins had little effect on gas production (P < 0.05). At 12h, the mulberry and moringa tannins showed the lowest biological effects, while those of leucaena and trichanthera the highest (P < 0.01). After 24 h of incubation, the moringa and leucaena tannins had similar effects and lower to the trichanthera tannins (P <0.001). From 48 h, the performance of the biological effect of tannins was trichanthera> leucaena> moringa> mulberry (P <0.001). There was only moderate biological effect on the kinetic parameters for leucaena and trichanthera tannins (P <0.001). Only the trichanthera tannins showed effect on the organic matter degradability (12%) and NDF (17.3%, P <0.01).The leucaena tannins had moderate biological effect in the NH3 concentration (11.3 %). The highest effects were observed for trichanthera tannins (29.4%, P <0.001). It is concluded that trichanthera tannins showed higher biological effect on the analyzed ruminal fermentation indicators, while in leucaena tannins were more moderate; those of moringa and mulberry did not influence on the nutritional value of them.
Key words: Moringa oleifera, Trichanthera gigantea, Morus alba, Leucaena leucocephala, tannins.
RESUMEN
Para estimar el efecto biológico in vitro de los taninos de harinas de follaje de moringa (Moringa oleifeira vc. Super Genius), morera (Morus alba Linn vc. Cubana), trichanthera (Trichanthera gigantea) y leucaena (Leucaena leucocephala vc. Perú) en indicadores de la fermentación ruminal, se midió la producción de gas in vitro y se estimaron los parámetros cinéticos según modelo de Gompertz, la degradabilidad in vitro de la fibra neutro detergente (FND) y del nitrógeno. El efecto biológico se estimó como el cociente entre el valor de cada indicador cuando los taninos están inactivos y activos, expresado como por ciento de incremento. Se aplicó diseño de bloques al azar, con las cuatro incubaciones realizadas como réplicas y el efecto biológico de los taninos como tratamiento. Al inicio de la fermentación (4 h), los taninos de trichanthera tuvieron poco efecto en la producción de gas (P < 0.05). A las 12 h, los taninos de morera y moringa mostraron los menores efectos biológicos, mientras que los de leucaena y trichanthera los mayores (P < 0.01). Luego de 24 h de incubación, los taninos de moringa y leucaena tuvieron efectos similares e inferiores a los taninos de trichanthera (P< 0.001). A partir de las 48 h, el comportamiento del efecto biológico de los taninos fue trichanthera > leucaena > moringa > morera (P < 0.001). Solo hubo efecto biológico moderado en los parámetros cinéticos para los taninos de leucaena y trichanthera (P < 0.001). Solo los taninos de trichanthera mostraron efecto en la degradabilidad de la materia orgánica (12 %) y la FND (17.3 %, P< 0.01). Los taninos de leucaena tuvieron efecto biológico moderado en la concentración de NH3 (11.3 %). Los mayores efectos se observaron para los taninos de trichanthera (29.4 %, P< 0.001). Se concluye que los taninos de trichanthera mostraron mayor efecto biológico en los indicadores de la fermentación ruminal analizados, mientras que en los taninos de leucaena fueron más moderados; los de moringa y morera no influyeron en el valor nutritivo de ellas.
Palabras clave: Moringa oleifera, Trichanthera gigantea, Morus alba, Leucaena leucocephala, taninos.
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INTRODUCTION
Shrubs are every time more frequent in the feeding systems of ruminants in the tropical and subtropical region (Melesse 2012). The tree species are important due to their high protein content, their contribution with easy fermentation carbohydrates and fiber of better degradability, as well as their positive effect on the use of nitrogen (N) within the rumen, elements which allow to increase the productivity of animals fed with grasses (Rubanza et al. 2007). However, these plants also contain secondary metabolites that may decrease their nutritional value and even affect the animal which intake them. Among the secondary compounds that are more frequently in the shrubs plants are tannins, which can be beneficial or harmful depending on their concentration, nature and reactivity (Alexander et al. 2008).
The in vitro gas production technique allows quantifying the effect of these secondary compounds on the ruminal microbial fermentation, by incubating the substrates to evaluate, alone or in the presence of polyethyleneglycol (PEG), chemical compound of high affinity by tannins. The ability of PEG to join and inactivate the activity of these secondary compounds, without affecting the ruminal microbial activity (Makkar et al. 1995), is a simple and reproducible tool to quantify the effect of these compounds, not only on gas production, but in other fermentation indicators and nutritional value of substrates (Rodriguez et al. 2014a).
This technique allows estimating the magnitude of the effect of these secondary metabolites on the ruminal fermentation of plants which contain them, without having to characterize the chemical nature of them nor determine their concentration (Rodriguez et al. 2014a). The objective of this study was to estimate the in vitro biological effect (BE) of tannins within moringa (Moringa oleifeira cv. Super Genius), mulberry (Morus alba Linn cv. Cubana), trichanthera (Trichanthera gigantea) and leucaena (Leucaena leucocephala cv. Peru) foliage meal on ruminal fermentation indicators.
MATERIALS AND METHODS
Plant material. The foliage meals of tropical tree species were evaluated: moringa (M. oleifera L, cv. Super Genius), mulberry (M. alba Linn cv. Cubana), trichanthera (T. gigantea Humbolt et Bonpland, Ness) and leucaena (L. leucocephala cv. Perú). The four species were collected during October, 2012, in experimental areas from the Instituto de Ciencia Animal (ICA), located in San José de las Lajas, Cuba. Mainly young leaves and stems were collected from random plants. Every plant used in sampling had several years of establishment in a typical red ferrallitic soil (Hernández et al. 1999), without irrigation and fertilization, except those of M. oleifeira that had only ten months of establishment.
The plant material was collected, at a rate of around 200 g of fresh matter per each tree until obtaining, around, 2 kg from each species. All the collected material was dried for 72 h, inside a forced air oven, with a regulated temperature (60 ºC).Later, it was grounded in a hammer mill, until reaching a particle size of 1 mm. Later, it was properly stored in sealed nylon bags.
Experimental procedure. The in vitro technique of gas production in glass bottles, described by Theodorou et al. (1994), was applied. An amount of 1.0 g of dry matter (DM) of each shrub was incubated in bottles of 100 mL, in a culture medium (Menke and Steingass 2015) and an inoculum of ruminal microorganisms, in a proportion of 0.20 regarding the total volume of incubation (80 mL). The evaluated substrates were incubated alone or with 0.5 g of PEG to inactivate tannins. In each case, four bottles (repetitions) were incubated. Four control bottles were also incubated without substrate to determine the gas contribution of the microbial inoculum.
]]> The inoculum used was the ruminal content of two cows with cannula in the rumen, fed ad libitum with grasses forage and free access to water and mineral salts. The ruminal content of each animal was collected before offering the morning food. Later, it was kept in closed thermos until getting to the lab, where it was filtered with several layers of gauze and the two inocula were mixed in equal proportions. During the process, the temperature of the inoculum was 39 ± 1 ºC. The anaerobiosis conditions were kept through continuous flow of CO2. The bottles were sealed and incubated in a bath at a controlled temperature (39 ºC).That moment was considered as the starting time of incubation.Gas production was measured at 2,4, 6, 8, 10, 12, 16, 20, 24, 36, 48, 72 and 96 h using a manometer HD8804, connected to a pressure calibrator TP804 (DELTA OHM, Italy).After each measuring, the gas was released until the external pressures were equal to the internal pressure of the bottles. The volume of gas was estimated using the pressure data of a previously established equation of linear regression (Rodríguez et al.2013).
The volume of gas was expressed by grams of incubated organic matter (incOM). In order to estimate the kinetics of gas production, the single-phased model of Gompertz was used (parameter A; asymptote when t= ∞; mL g-1 incOM):
Y = A*Exp (−B*Exp (-C*t))
Besides, the maximum speed of gas production was estimated (Vmax; mL g-1 incOM h-1), when substituting Tvmax( value of the inflexion point of the sigmoidal model) in the first derivative of the model.
At the end of incubation (hour 96), the bottles were opened and its content was filtered through nylon bags. A sample of the filtrate was preserved to subsequently determine the NH3 concentration.The bags with the fermentation residues were dried for 72 h in a forced air oven, with a regulated temperature (60 ºC). The IVDNDF was determined using the gravimeter method as the difference between the neutral detergent fiber (NDF) within the incubated substrate and the solid residue of fermentation (96 h), divided by the NDF incubated in each bottle, respectively (Blümmel et al. 1997).
Metabolizable energy (ME) and the digestibility of organic matter (DOM) of the evaluated substrates, at 24 h, were estimated through the equations proposed by Menke et al. (1979):
ME (MJ kg-1 DM) = 2.20 + 0.136 GP24h + 0.057 CP
DOM (%) = 14.88 + 0.889 PG24h + 0.45 CP
Where,
]]> GP24h is the volume of gas produced at 24 h (mL• 200 g-1 incubated DM)CP is the crude protein expressed in percent
Chemical analysis. The DM, organic matter (OM) and CP were determined according to AOAC (1995).The NDF was obtained by the procedure described by van Soest et al. (1991).In addition the content of DM and NDF was determined in the solid residues of fermentation. The analyses of ammonia were carried out according to Conway (1957).
Estimation of BE of tannins. It is known as the tannins BE the extent in which these compounds can affect a ruminal fermentation indicator of substrates (Rodriguez et al. 2014a). The BE is estimated as the quotient between the value of the indicator, when tannins are inactive (incubated substrates in the presence of PEG) and when are active (substrates incubated alone) (Makkar et al. 1995).
In this research, the tannins BE of shrubs was estimated for each of the indicators that were selected and expressed as the increase (%) of these, when inactivating tannins with PEG, regarding to the values obtained for the plant with all its active tannins (Makkar et al. 1993), according to the equation:
BE (%) = [(Measured indicator (In presence of PEG)/Measured indicator (In absence of PEG))X100]-100
The BE in the indicators gas production, gas production potential, maximum speed of in vitro gas production, the NDF and the OM degradability, ME and ammonia concentration was evaluated.
Statistical analysis. A random blocks experimental design was used, in which the four incubations performed in the time (replications) were considered as blocks and the tannins biological effect of the evaluated tree species in each fermentation indicator as treatment. The results were analyzed by ANOVA by means of the InfoStat statistical package (Di Rienzo et al. 2010). When differences (P < 0.05) were found, treatment means were compared using the multiple range test of Duncan (1955).
RESULTS AND DISCUSSION
]]> The results of chemical composition, gas production, kinetic parameters, chemical and gravimetric indicators of fermentation of the four shrubs were previously reported (Rodriguez et al. 2014b). In this study only the biological effects of tannins of these plants are informed on the previously reported indicators.Table 1 shows the results of tannins BE of shrubs evaluated in the gas production and two kinetic indicators of the in vitro fermentation. At the start of the fermentation (hour 4), the highest effects on gas production were observed in leucaena and moringa
(P <0.05), although the moringa effect not differ from the mulberry. The trichanthera tannins had lower effect, and also did not differ from those of mulberry. However, at 8 h of incubation all tannins showed similar biological effect (P> 0.05). At 12 h of incubation, the mulberry and moringa tannins showed the lower BE, while those of leucaena and trichanthera produced the highest (P <0.01).
At 24h of incubation is not observed BE of mulberry tannins on the in vitro gas production. The moringa and leucaena tannins had similar and lower effects than those of trichanthera (P <0.001). From 48 hours and until the end of the incubation, mulberry tannins either had BE on gas production. The BE of tannins was the following: trichanthera> leucaena> moringa> mulberry (P <0.001).
The reduction of the biological effect in time for the moringa, leucaena and mulberry tannins could be related with the presence of hydrolysable tannins. The hydrolysable tannins could be degraded by the action of ruminal microorganisms during the first incubation hours, which would explain the BE decrease in time (Rodriguez et al. 2015). The increase in time of the magnitude of the BE of trichanthera tannins may indicate that the tannins in this shrub are linked to the insoluble fraction of plant material.
Respect to the BE in the evaluated kinetic parameters, mulberry tannins do not affect the potential of gas in vitro production of this shrub, while those of moringa had lower effect on this parameter. A moderate BE for trichanthera and leucaena tannins, which did not differ between them (P <0.001) was appreciated. Similarly, mulberry tannins do not affect the gas production Vmax. The effect of moringa tannins on this parameter was moderate and the higher BE were observed for leucaena and trichanthera tannins. The latter species showed the highest effects (P <0.001).
There is available information about the tannins content of the four shrubs species evaluated (Scull 2004, Garcia et al. 2008, Bakhashwain et al. 2010 and Pedraza et al. 2013). However, it is little known about their effects on ruminal fermentation. Getachew et al. (2002) studied the gas production of 39 tropical shrub species, when incubate them with and without PEG.
These authors observed a positive correlation (r = 0.76; P <0.001) between the BE on gas production and the tannins content of the samples. However, the tannins BE on gas production not only depend on the concentration of these compounds but also of their chemical structure and reactivity (Bueno et al. 2008 and Rodriguez et al. 2015)
The tannins BE of the evaluated shrubs on the OM and NDF degradability, as well as on the estimated ME, is show in figure 1. Only the trichanthera tannins showed effect on these indicators (P <0.01), although this BE was moderate in the three indicators (11-17%). The increase in the available energy by the increase of degradability and ME, when inactivating the trichanthera tannins, can lead to increases in the final products of fermentation (short-chain fatty acid and gas) (table 1), and also in the microbial biomass synthesis. Both processes are inversely related (Blummel et al. 1997).
Figure 2 shows the tannins BE of species evaluated on the NH3 concentration at 96 h of incubation, as an indicator of the effect of these secondary metabolites on the nitrogenous metabolism. The mulberry and moringa tannins do not have BE on the NH3 concentration, while in leucaena tannins the BE was moderate. The highest effects were observed for trichanthera tannins (P <0.001).
]]> The higher affinity of tannins by the PEG causes, in presence of this compound, the availability of such proteins is increased which would otherwise remain joined to these secondary compounds as strong complex tannins: proteins and protected from the action of microbial proteases (Mtui et al. 2009). Therefore, the PEG inclusion favors the ruminal proteolysis (Getachew et al. 2000), although the BE magnitude will depend on the source of tannins which is evaluate (Rodriguez et al. 2014a) and the proteins protection degree that these metabolites achieve in natural conditions.Tannins can join to proteins and carbohydrates and have a significant effect on their fermentation in the rumen, when limiting their use by ruminal microorganisms (McAllister et al. 1994 and Makkar et al. 1995), to interfere in the microbial adherence to food particles or directly affect their microbial populations or enzymatic complexes (Bakhashwain et al. 2010). These effects depend not only on the concentration of tannin in the plant, but also from the reactivity of these compounds, that depends on their chemical nature (Bueno et al. 2008 and Rodriguez et al. 2015). It is known that the same tannins concentration from different sources can produce effects of different magnitude (Bueno et al. 2008).
In this study, the highest BE on the different monitored indicators corresponded to trichanthera tannins. Scull (2004) found moderate concentrations of these compounds in this species. However, the BE of trichanthera tannins does not justify the low nutritional value attributed to this species, probably because this could be the result of the combined effect of its high fiber content (Rodriguez et al. 2014b), lignin and BE of their tannins on the digestibility (Ammar et al. 2005), without taking into account the anti-nutritional effect of other secondary compounds in this shrub (Scull 2004).
Leucaena also showed moderates BE, but higher than the moringa and mulberry tannins in gas production (96 h), gas production potential, maximum speed and proteins degradation (NH3 release to the incubation medium). The moderates BE of leucaena coincide with previous results on the nature and reactivity of the tannins which this legume species contains (Rodriguez et al. 2013).
The low BE of moringa and mulberry tannins coincide with the low concentration and the reported activity for these compounds in these species (Garcia et al. 2008 and Bakhashwain et al. 2010).
The obtained results of tannins BE on gas production, degradability of OM and NDF, ME and NH3 concentration allow to concluded that the trichanthera tannins showed the highest BE on the ruminal fermentation indicators analyzed, while those of leucaena showed more moderate effects. The moringa and mulberry tannins did not influence on the nutritional value of these shrubs.
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Received: July 9, 2015
Accepted: March 16, 2016
R. Rodríguez, Instituto de Ciencia Animal, Apartado Postal 24, San José de las Lajas, Mayabeque, Cuba. Email: rrodriguez@ica.co.cu
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