Livestock Research for Rural Development 17 (11) 2005 Guidelines to authors LRRD News

Citation of this paper

Influence of the donor animal on the in vitro gas production with the use of voided bovine faeces

Silvio Martínez Sáez, Redimio M Pedraza Olivera, Marlene León González, Cecilia E González Pérez and Guillermo Guevara Viera

Center of Studies for the Development of the Animal Production (CEDEPA)
University of Camagüey, Camagüey 74650, Cuba


The objective of this research was to assess the influence of the animal donor during the implementation of the in vitro gas production technique with the use of voided bovine  faeces. The ruminal fluid was substituted by  faeces obtained within less than 3 hours of being voided by milking cows. The samples, together with the inoculants, and the buffered mineral media, were incubated in 100 ml syringes at 39° C. The volume of gas produced was measured at different times. The data were adjusted to the equation V = a+b*(1-EXP(-c*t)). Samples of foliage of Leucaena leucocephala were evaluated. The reproducibility of the gas production when the  faeces came from only one animal to that coming from different cows was compared.

The results demonstrated that the values of a, b and c of the best adjustment to the model have an acceptable level of reproducibility when the inoculum comes from the same animal but they depend upon the donor. The preparation of a reference sample is suggested to overcome the problems of the dependence on the source of the inoculum.

Key words: Bovine faeces, donor animals, gas, in vitro, inoculum

Influencia del animal donante en la producción de gas in vitro con el uso de heces vacunas ya depuestas.


El objetivo del trabajo fue para evaluar la influencia del animal donador durante la implementación de la técnica de producción de gas in vitro con el uso de heces vacunas ya depuestas. El fluido ruminal se sustituyó por el excremento con menos de 3 horas de haber sido depuesto por las vacas en ordeño. Las muestras, junto a los inóculos y el buffer enriquecido con minerales fueron incubadas a 39° en jeringas calibradas de 100 ml. Se midió el volumen de gas producido en el tiempo. Los datos se ajustaron a la ecuación V = el a+b*(1-EXP(-c*t)). Se evaluaron muestras de follaje de Leucaena leucocephala. El reproducibilidad de la producción de gas cuando se usa el mismo animal donante se comparó con el obtenido cuando se usan vacas diferentes.

Los resultados demuestran que los valores de a, b y c de mejor ajuste mejor al modelo tienen un nivel aceptable de reproducibilidad cuando el inóculo viene del mismo animal pero que dependen del donante. Se probó la viabilidad de este método y su potencial para evaluar alimentos. Se sugiere la preparación de una muestra de referencia para mitigar los problemas debido a la dependencia del inóculo.

Palabras claves: gas in vitro, heces bovinas, variabilidad del inoculo


The serious problem of feeding thousands of human beings in developing countries could be solved by the rational use of the resources and the application of the most appropriate techniques according with the specific conditions of each area. Several researchers (Tamminga and Williams 1998; Panin 2000) coincide in pointing out the promising role of the ruminant animal as a means to obtain foods starting from local nutritional resources not directly consumed by humans. These resources include a great variety of the so-called "not traditional" foods, but their nutritive value is unfortunately not always well known, which is indispensable to guarantee their appropriate employment.

To evaluate the nutritive value of the foods that could be used by ruminants several techniques have been used. Among them, the in vitro gas production with the use of ruminal fluid as inoculum is recognized as one of the most important because of its simplicity and possibility to manage a great number of samples (Menke et al 1979; Ramachandra and Krishnamoorthy 2000). The use of faeces as source of inoculum for the evaluation of feeds have demonstrated their utility (Nguyen Van Thu 2003), with similar results to those obtained with ruminal fluid. Using faeces, the stress and possible damage to the physical integrity of the animals for extracting the ruminal fluid is avoided.

The objective of the work presented in this paper was to assess the influence of the animal donor during the implementation of the in vitro gas production technique with the use of voided bovine faeces.

Materials and Methods

Conserved samples of Leucaena leucocephala cv. Perú leaves, about 90 days regrowth, were used. The L. leucocephala was cultivated without irrigation or fertilization, in the Center for Studies of the Development of Animal Production (CEDEPA) of the University of Camagüey (located at 21°, 23' of North Latitude and 78°, 51' of West Longitude, at 104 m over the level of the sea).  The samples were dried at 65° C (AOAC 1995) in a oven with forced circulating air; after that they were milled to pass through a 1 mm sieve. At least 3 replicates of each sample were taken. The same sample of Leucaena leucocephala was used throughout.

The source of inoculum was  faeces taken from in crossbred cows (Holstein x Zebu). The animals grazed in a mixture of natural and artificial pastures and without any supplementation. They were not fed overnight the day before each experiment. The  faeces were collected from the milking area less than 3 hours after being voided (Akhter et al 1996). In all of the cases, the inoculum was carefully collected from the cemented floor a few minutes before starting the experimental. It was conserved in appropriate recipients, which were previously warmed at 39-40°C and always in an atmosphere of CO2. The suspension of  faeces was filtered through a plastic sieve (<0.06 mm) in order to separate the solid particles. The whole procedure was carried out in an atmosphere of CO2.

In vitro gas production was completed according to the procedure described by Menke and Steingass (1988). The samples, together with the inoculums and the buffered mineral media were incubated in 100 ml calibrated syringes  39° C. The volume of gas produced was measured at different times. In all of the experiments one part of  faeces was diluted in 3 parts of mineral medium (Pedraza 1998). The gas was always expressed as from a mass of 200 mg of the analyzed sample.

For the routine calculations from the direct measurements of the syringes, a worksheet of Microsoft Excel (Microsoft Corporation®, USES) was specially prepared. In this worksheet, the calculation of the volume and the graphs were also made. The parameters for the in vitro gas production were obtained using the best adjustment to the model proposed by Ørskov and McDonald (1979) [a+b*(1-EXP(-c*t))], where:

a is magnitude of the lag phase

a+b potential volume of gas that can be produced

c is rate of gas production, ml/hr

The Naway software (Chen 1995) was used for the calculation of the parameters. For the statistical processing of data the version 7 for Windows® of SYSTAT® (SPSS Inc. 1997) was used.

Results and discussion

The first of the aspects to consider when introducing a laboratory procedure is the reproducibility or repeatability, which can be considered as the similarity between two or more values of the measured magnitude obtained by one or several procedures (LeBlanc 2001; Martínez 2004).

The results of evaluating the reproducibility are shown in Table 1.

Table 1.  Influence on the parameters of the model  of in vitro gas production of L. leucocephala using  faeces from the same donor animal (different syringes).


a (ml)

B (ml)

C (ml/h)

a+b (m)



















































It is possible to appreciate (Table 1) that for a given inoculum the results of the reproducibility can be considered acceptable (CEN 1986).  On the other hand, the differences among these first volumes don't influence substantially the values of the parameters of best adjustment to the model of Ørskov and McDonald (1979), which therefore have good reproducibility.

The dynamic behavior of the in vitro gas production, as in any other microbial growing process, will depend upon the substrate, the medium and the inoculum. The substrate is what is being evaluated and the medium is always the same one, it is therefore of paramount importance to know how the inoculum (ie: other donor) influences the final result.

Table 2. Influence of the donor animal on the parameters of the model









































abc Means in columns without common letter are different at P<0.05

There were significant differences among the values of "b", "c" and of "a+b" according to the donor animal  (Table 2). These findings coincide with reports from Rymer et al (1999) and Nagadi et al (2000). They suggested that the differences could be  related with the characteristics of the animals' diet, which influences significantly the microbial composition of the rumen. It may be necessary to have a reference sample which can be used in each run (as an internal standard). This would help to eliminate the effect of the inoculum by means of the corresponding correction (Pedraza 1998, Cárdenas et al 2002).



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Received 18 May 2005; Accepted 25 September 2005; Published 1 November 2005

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