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Tropical and subtropical agroecosystems

versión On-line ISSN 1870-0462

Trop. subtrop. agroecosyt vol.14 no.3 Mérida sep./dic. 2011




Precision of an equation to estimate dry matter degradability of Clitoria ternatea


Precisión de una ecuación para estimar la degradabilidad de la materia seca de Clitoria ternatea


Rafael Macedo-Barragán1*, Nora Isela López-Poblete1, Victalina Arredondo-Ruiz2 and Rosario Rodríguez-Ramírez3


1 Facultad de Medicina Veterinaria y Zootecnia, Universidad de Colima, Km. 40 Autopista Colima-Manzanillo, Tecomán, Colima, México, C.P. 28100, Tel (312)3229407, E-mail:

2 Private Practice, Valle de las Flores 93, Colonia Real del Valle, Tecomán, Colima, México, C.P. 28140, E-mail:

3 Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias, Campo Experimental Valle de Tecomán, Km. 35 Autopista Colima-Manzanillo, Tecomán, Colima, México, C.P. 28100, E-mail:

* Corresponding author


Submitted May 17, 2010
Accepted August 08, 2011
Revised received June 15, 2011



The objective of this study was to evaluate the precision of an equation developed to estimate the dry matter digestibility (DMD) of alfalfa (Medicago sativa) when used on the tropical legume Clitoria (Clitora ternatea). Acid detergent fiber from Clitoria was determined and its DMD estimated using the equation: %DMD = 88.9 - (0.779 x %ADF), and subsequently in situ and in vitro degradability (DMDE) of the mentioned legume was determined. Mathematical, in situ and in vitro estimates were compared by ANOVA as a completely randomized design and precision of the equation as DMDE estimator of Clitoria hay was determined if P>0.05. Mathematical DMD estimates were not different (P>0.05) to those obtained by in vitro method, and both were significantly different (PO.05) and lower than in situ estimates. It can be concluded that the equation initially developed to estimate the DMD of alfalfa was a closer estimator of in vitro DMDE of Clitoria, whereas underestimated the in situ parameter by 8.6%.

Key words: Tropical legumes; in vitro degradability; in situ degradability.



El objetivo del presente trabajo fue evaluar la precisión de una ecuación desarrollada para estimar la digestibilidad de la materia seca (DMS) de la alfalfa (Medicago sativa), al ser utilizada en la leguminosa tropical Clitoria {Clitora ternatea). Se determinó el contenido de fibra detergente ácido de la Clitoria y se calculó su DMS utilizando la ecuación: %DMS = 88.9 - (0.779 X %FDA) para posteriormente determinarse la degradabilidad (DEMS) in situ e in vitro de la mencionada leguminosa. Los estimados obtenidos por los tres métodos fueron comparados por ANOVA usando un diseño completamente al azar y se consideró como criterio para declarar que la ecuación cumplió con precisión como estimador de la degradabilidad si P>0.05. Los estimados matemáticos de la DMS fueron similares (P>0.05) a los obtenidos por el método in vitro y ambos fueron significativamente diferentes (P<0.05) y menores a los obtenidos por el método in situ. Se concluye que la ecuación desarrollada para estimar la DMS de la alfalfa resultó un estimador preciso de la DEMS de la Clitoria obtenida por el método in vitro, en tanto subestimó en un 8.6% la DEMS obtenida por el método in situ.

Palabras clave: Leguminosas tropicales; degradabilidad in vitro; degradabilidad in situ.



Forage quality is an expression of the potential of livestock to produce meat, milk, and other products from forage through the utilization of its available nutrients. The level of animal production is controlled nutritionally by the daily intake of digestible nutrients and by the efficiency with which such nutrients can be metabolized and used for body processes. Both quality and amount of digested nutrients available per unit time are important to the production of specific animal products. Thus, forage quality may be defined as the type and amount of digestible nutrients available to the animal per unit time (Barnes and Marten 1979; Bochi-Bmmetal. 1999).

An accurate and precise assessment of quality before forage is fed to animals will have a marked effect on the economic feasibility. Measurement of forage quality becomes increasingly more important as one intensifies the production system (Barnes and Marten 1979). Several methods are used to estimate ruminal digestibility of dry matter, between which in tropical countries, the most frequently used methods are the in situ (nylon bag) technique (Harris et al. 1967) and the in vitro method of Tilley and Terry (1963).

In situ method has achieved the widest use and is routinely used for studying effects of the rumen environment. However, this method requires a large number of nylon bags to be ruminally incubated for each feed sample and, in turn, a substantial amount of human work (Olaisen et al. 2003). In vitro Tilley and Terry technique is accepted as the most appropriate and utilized laboratory methodology to estimate the digestibility of feedstuffs for ruminants, and has been used extensively because of a high degree of correlation to in vivo digestibility (Marten and Barnes 1980). Over the years, the technique has been modified to improve the precision of in vitro digestibility dry matter estimates as well as improve labor and time efficiency of assays (Holden 1999). In addition, both in situ and in vitro determinations are expensive, rendering these techniques impractical for routine analyses (De Figueiredo et al., 2000; Giraldo et al., 2007).

Therefore, a need exists for less expensive, time-efficient and animal friendly techniques, in particular for routine analysis, within which mathematical approaches have been proposed. In this context, the aim of this study was to evaluate the precision of one equation developed to estimate the dry matter digestibility (DMD) of alfalfa (Medicago sativa), when was used on Clitoria (Clitora ternatea), one of the most promissory legumes for tropical and subtropical regions.




The study was conducted at the Laboratory of Nutrition, University of Guadalajara located in Ciudad Guzman, Jalisco, México at 19°42'10" north latitude and 113°27'45" west longitude. The site has a warm climate with an average temperature of 20.2°C and 732 mm of annual average rainfall.


Experimental procedure

Acid detergent fiber from 30 samples of ground early flowering Clitoria hay, were determined by the method of Goering and van Soest (1970), and used to calculated Clitoria DMD according to the equation proposed to estimate DMD of alfalfa by Linn and Martin (1989) in which, %DMD = 88.9 - (0.779 x %ADF).

According to the procedure proposed by Harris et al. (1967) to estimate in situ dry matter degradability (DMDE), 30 nylon bags, 52 μm pore size; 5 x 10 cm (33 mg/cm2 ratio of sample size to bag surface area) containing 5 g of Clitoria hay were incubated for 48 h in the rumen of a mature Holstein cow fed corn silage, alfalfa hay and Clitoria hay and fitted with ruminal cannulae. After removal, bags were hand-rinse until colorless, dried at 60°C in a forced-air oven for 48 h, and the residual material weighed.

Finally, in vitro DMDE was estimated, using the first stage of the Tilley and Terry technique (Tilley and Terry, 1963). Thirty samples of 0.5 g of Clitoria hay were placed into 100 ml plastic test tubes and incubated at 39°C with 10 ml of cow ruminal fluid and 40 ml of McDougal's artificial saliva buffer (McDougal 1948). After 48 h, tubes were removed from the in vitro bath, hand-rinsed until colorless, dried at 60°C in a forced-air oven for 48 h, and the residual material weighed.


Statistical Analyses

Mathematical DMD estimates were compared with those DMDE obtained by in situ and in vitro methods by ANOVA as a completely randomized design using the general linear model procedure of SAS (1989). Significant differences were determined using Tukey test and equation precision as DMDE estimator of Clitoria was declared if P>0.05. In addition a Pearson Correlation test was performed to set a relationship between estimators.



Average ADF content of Clitoria hay was 35.74 ± 5.06% with minimum and maximum values of 29.06 and 47.07% respectively. Results presented in Table 1 show that mathematical DMD estimates were not different (P>0.05) to those obtained by in vitro method; however, both were significantly different (PO.05) and lower than in situ estimates. Mathematical method underestimated the in situ parameter by 8.6%, while the in vitro technique underestimated it by 11.71% (Table 1).


DMD estimates obtained by mathematical method and DMDE obtained by in vitro and in situ techniques showed no significant correlations (P>0.05), being positive among in situ and in vitro techniques and negative among mathematical and the previous mentioned techniques (Table 2).


In situ, mathematical and in vitro Clitoria digestibility and degradability estimates obtained in the present study (66.32, 61.06 and 59.37% respectively) were higher than the in vivo value (56.9%) found by Juma et al. (2006). Variations among both studies results could be explained by differences in the method of estimation and in the ADF content of Clitoria samples. Some authors report that 48 h in situ and in vitro incubations of many feedstuffs overestimate in vivo digestibility and when the incubation period is reduced to 24 h, the in vitro technique accurately predict the in vivo digestibility (Holden, 1999; Damiran et al. 2008). Oba and Allen (2005) point out that 24 to 30 h of incubation is appropriate to evaluate feedstuff digestibility. On the other hand, Juma et al. (2006) reported an ADF content of 47.4% versus 35.74% obtained in this work. In agreement with the equation %DMD = 88.9 - (0.779 X %ADF) a lower ADF content results in a higher DMD estimate. Another study found an intermediate value of 60.40% (Ratan et al. 1982).

As happened in the present study, Varel and Kreikermeier (1995) and Torres et al. (2009) found that in situ method consistently provided a greater extent of digestion of legumes and grasses than the in vitro method. In vitro results can be affected by several factors such as grinding size, distribution of particle size in the sample (Judkins et al. 1990; Damiran et al. 2008) and smaller concentration of inoculum (Varel and Kreikermeier 1995).

Also, adaptation and development of new procedures to determine in vitro digestibility of feeds constitute an additional source of variation. The traditional Tilley and Terry method to determine the in vitro DMD of forages has been compared with new methods such DaisyII system with inconsistently results. Whereas Damiran et al. (2008) found that Daisy11 in vitro DMD estimates were greater than traditional Tilley and Terry estimates, Holden (1999) reported that the method of analysis did not affect in vitro DMD of alfalfa hay. Different values for the conventional in vitro method and DaisyII technique seems to be related to incubation environment. In the traditional method sample particles are in direct contact with the inoculum and the buffer solution, the DaisyII system used filter bags placed into incubation jars (Spanguero et al. 2003). Nevertheless, authors concur that Daisy11 system allows simultaneous incubation of a large number of samples compared with fermentation batch, giving advantages in terms of labor consumed and costs per determination.

Contrary to the results of the present study, Giraldo et al. (2007) found that in vitro DMD estimates of forages using the DaisyII system were significantly higher than those obtained using the in situ nylon-bag method. In that study differences were attributed to the size pore of the bags and to the sample size to bag surface area ratio (SS:SA). Despite studies that showed that a reduction of in SS:SA from 54 to 16 mg/cm2 resulted in a dramatic increase of DM disappearance (Mehrez and 0rskov, 1977) and recommended SS:SA values ranging from 10 to 20 mg/cm2 (Vanzant et al., 1998), the value used in this study (33 mg/cm2) did not inhibit in situ DMD. Mehrez and Ørskov (1977) suggest that the inhibition of digestion in large SS:SA ratios was a result of inadequate mixing and removal of the digestion end products from bags. On the other hand, an appropriate pore size allowed sufficient influx of digestive microorganism, permitting adequate efflux of digestion end products, and minimizing influx of ruminal digest residues and efflux of small sample particles (Vanzant et al, 1998; Foster et al, 2007). While some evidence suggests that pore size ranging from 40 to 60 um introduces large variation in measurements of digestion in situ (Nocek, 1985; Vanzant et al, 1998). In this study, standard error of in situ estimates and hence variability, was smaller than those obtained by mathematical and in vitro methods. Pore size used (52 um) was similar to the one (53 um) most reported in the literature (Vanzant et al, 1998).

Other factors that influence in s/Ya-derived estimates of rumen degradation are bag material, sample processing, host animal, animal diet, feeding level and frequency, bag insertion, removal procedures, location of bags within the rumen and containment procedures for the bags, rinsing procedures, microbial correction and incubation times (Huntington and Givens 1995; Vanzant et al., 1998; Spanguero et al, 2003).

Due to technique-derived variability, some studies found significant correlations between in situ and in vitro digestibility estimates (Judkins et al. 1990; Kamalak et al. 2005). Others indicated that in situ and in vitro digestibility showed a positive and significant correlation only after 48 h of incubation (Ceballos et al. 2008), and as it was observed in the present study, Silveira et al. (2009) did not find significant correlations.

It can be concluded that equation %DMD = 88.9 -(0.779 x %ADF) developed to estimate the DMD of alfalfa was a closer estimator of in vitro degradability of Clitoria, whereas underestimated the in situ parameter by 8.6%. This equation could be used as an easier alternative reference of Clitoria DMDE. However, present results should be cautiously applied because of capability of the equation to predict in vitro data depend upon ADF content of Clitoria.



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