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24 Jul 2020

Gas production technique (GPT) for nutritional valorisation in ruminants

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Gas Production Technique (GPT) allows to classify different raw materials according to the rhythm and extent of its ruminal degradation. It is relatively simple, its cost is relatively low and the sample sizes are small. This article give the theroretical foundations of the technique, explains its applications, and discusses its benefits and limitations.

In ruminants, food is fermented in the rumen before gastric digestion. The final products of such fermentation greatly contribute to fulfil the energy and protein requirements of the animal. Therefore, the characterization and quantification of these products is key to the nutritional valorisation of feedstuff.

To determine ruminal degradation and-or synthesis of microbial protein in vivo, it is necessary to have animal with fistulas practiced in the rumen, abomasum, and duodenum. Keeping fistulated animals is not only costly and laborious, but also poses ethical issues, and presents legal limitations. Such inconveniences have contributed to the development of jn vitro techniques that try to simulate ruminal fermentation.

The ruminal degradation of feed is a complex process that implies interactions between ruminal microorganisms (bacteria, protozoa, fungi and methanogenic archaea) and the host animal. These characteristics complicate the in vitro simulation.

The main factors to consider in in vitro systems are temperature, pH, buffer capacity, anaerobiosis, and the supply of nitrogen and essential nutrients for ruminal microorganisms.

In vitro techniques of nutritional valorisation: Gas Production Technique (GPT).

The simplest in vitro systems consist of vials or bottles in which the dry matter to be valorised is incubated with buffered ruminal fluid, in anaerobiosis, at 39 °C, and at pH close to 7.0. This system is used to determine in vitro digestibility of forages according to the method by Tilley and Terry (1963). The method consists of incubating the sample in buffered ruminal fluid for 48 h, followed by incubation with pepsin in an acid medium,

In the past years, the technique o gas production (GPT) has become increasingly popular. This consist of incubating a sample of buffered ruminal fluid and measuring, at different intervals, the production of gas during fermentation: This system is based on the premise that gas production is directly related to the quantity of organic matter being fermented (Menke et al., 1979).

What does the gas production technique (GPT) measure?

Figure 1. Stereochemical reactions of ruminal fermentation of hexoses (Hungate, 1966).

Rumen microorganisms obtain energy for growth from the fermentation of carbohydrates, mainly starch an cell-wall carbohydrates.
Carbohydrates are broken into monomers (hexoses and pentoses), which are fermented down to volatile fatty acids (VAFs), CO₂ and hydrogen (Fig. 1).
Ruminal methanogenic archaea use CO₂ and hydrogen to form CH₄, hence CO₂ and CH₄ are the main gases produced on the rumen.
VFAs are absorbed and utilized by the host as a source of energy and as precursors in the synthesis of other compounds. Gases are expelled through eructation.
The gases generated during the fermentation are the ones measured in GPT, although gas is also generated by the bicarbonate buffer used in the culture medium.
As shown in Figure 1, the production of acetic and butyric acids produces CO₂ and hydrogen. However, the production of propionic acid does not only generate gas but also consumes hydrogen.

TPG only measures the total quantity of gas produced, therefore it does not give any information on the type of fermentation (VFAs profile) occurring. If such information is needed, then it will be necessary to obtain a sample of the liquid from the vials and analyse its concentration in VFAs.

Methodology and interpretation of the data in gas production technique (GPT).

TPG requires a small amount of sample (generally milled at 1 mm), ruminal fluid, and an anaerobic culture medium that does not limit the growth of ruminal microorganisms. The methodology is relatively simple (Fig. 2).

Figure 2. Methodology of gas production technique (GPT).

Weighing of the sample

The sample is weighed in vials. Then, a determined amount of the mix of ruminal fluid and culture medium is added to the sample (peristaltic pump is used for precise dosage). The amount of sample, ruminal fluid and culture medium vary between labs. However, the most common protocol uses 0.5 g of sample, 10 ml of ruminal fluid and 40 ml of culture medium.

Process temperature

A fundamental aspect of the process in to keep the temperature constant at 39 ºC, and constant anaerobiosis conditions. Ruminal microorganisms are very sensitive to thermal shock and oxygen.

Incubation time

After inoculation with the combination of ruminal fluid and culture medium, vials are hermetically closed and placed in a 39 ºC incubator. They are kept in these conditions during, at least, 72 hours to ensure the potential biodegradability of the sample is reached.

The amount of gas produced is measured at different intervals, shorter at the beginning of incubation (every 2-3 hours), beginning to widen after 12 hours of incubation (every 5-6 hours during day 1 and, later, every 12 to 24 hours).

Measuring gas production

Measurement of gas production can be performed manually (Fig. 2) using a manometer and a calibrated syringe (Theodorou et al., 1994), allowing to obtain a value of pressure and volume at each sampling time. A regression equation is fitted to relate the values of volume against pressure, so that it could later be used to estimate the precise volume of gas produced based on the values of pressure measured with the manometer.

In automatised systems, the pressure in the vials is registered in a computer. The tube stoppers that seal the vials have an electro valve that opens when the pressure inside the vial reaches a threshold, releasing the gas produced. These systems require less operators and give a larger number of measures than manual systems. However, the price of the equipment is high.

Filtration

At the end of the incubation, the content of the vials is filtered through porous-bottom crucibles. The sediments are used to determine, gravimetrically, the proportion of dry matter that has not been degraded.

Determination of organic matter (OM)

Afterwards, the OM content of the filtered residues is determined. The potential degradability is calculated based on the value of OM calculated for the sample (since ashes do not contribute to the production of gas).

A particularly important point is to reproduce in vitro the individual variability present in vivo. Generally, four vials per sample are incubated, which are filled with ruminal fluid from different animals. Alternatively, the incubation is repeated in four consecutive weeks, using a mix of ruminal fluid from different animal, in order to obtain 4 replicates per sample.

Vials without samples (whites)

Additionally, it is necessary to incubate vials without simples (whites), which only have a mix of ruminal fluid and culture medium. The gas production in these vials, which is the gas generated in the fermentation of the inoculum of ruminal fluid, is used to correct the gas production data measured in the vials with sample.

Sample origin

Ruminal fluids can be obtained from fistulated animals, although it can also be obtained in the abattoir. Laboratories routinely using this technique have fistulated animals, since the characteristic of the inoculum is one of the main factors affecting GPT results.

It is fundamental that the donor animals receive always the same diet, an also that the extraction of ruminal fluids is performed every day at the same time, generally before the first meal of the day. Furthermore, it is convenient to incubate a pair of standards to overrule the possibility of incubation problems.

Registering gas volume

The volume of gas registered at each time is added to the volume previously produced, building the curves of cumulative production (Fig. 3).

Figure 3. Cumulative gas production curves for different ray materials.

As shown in Figure 3, the most degradable raw materials (barley grain) do not only produce more gas (ml gas/g sample) compared with less degradable raw materials (straw and olive pomace). They also have a higher rate of gas production per unit of time. This is an advantage of GPT over other in vitro techniques to measure digestibility, because it does not only allow to measure the extent of the degradation, but also its rhythm.

Mathematical models to represent profiles of gas production

Profiles of gas production can be described using different mathematical models. To choose the model it is necessary to consider the goodness of fit to the experimental data, as well as the biological meaning of the obtained parameters.

Exponential models are the ones more frequently used. They are based on first order kinetics, assuming a constant fractional rhythm of fermentation for all the fractions of the sample.

Using automatic systems to measure gas production, it is possible to obtain a higher number of measurements. Then, the data could be fitted with multiphasic models, which allow to differentiate between the fractions that are degraded fast from the ones that degrade at slower rate (Willliams, 2000). However, its biological interpretation is more complicated.

Orskov and macdonal equation Equation 1.

Calculating the average rhythm of gas production

These parameters allow to calculate the average rhythm of gas production, which is the rhythm of production of gas between the beginning of incubation and the time taken to reach half of the potential gas production.

This parameter has more biological meaning than the fractional rhythm of gas production (c in Equation 1), since it indicates the degradation rhythm of the samples during the initial time of incubation. This period is usually similar to the retention time of the ingesta in the rumen, especially with raw materials with a low to medium content in neutral detergent fibre (NDF).

The average rhythm of gas production for the raw materials shown in figures 3 and 4 were 9.13, 5.31, 3.62. 4.27 ml/h for barley, oat hay, barley hay and olive pomace, respectively. This indicated that barley was fermented more quickly (between 1.7 and 2.5 times) than the rest of the analysed raw materials.

Figure 4. Average rhythm of gas production for barley grain, oat hay, barley straw and olive pomace.

The parameters estimated by modelling the data can be used to estimate the effective degradability of organic matter (EDOM), for a given time of permanence of the ingesta in the rumen (Equation 2).

EDOM calculation Equation 2.

Values of k_p of 0.04, 0.06 and 0.08 are frequently used. They correspond to animals with low, medium, and high levels of ingestion, respectively. These values would give information on the ruminal degradability of a given raw material, when administered to animals in different productive situation.

Applications and limitation of GPT

GPT allows to classify different raw materials according to the rhythm and extent of its ruminal degradation. The obtained values are fundamentally related to their energy supply.
The information on the degradation of raw materials allows to adequately combine them in diets, in order to maximise microbial growth in the rumen and synchronising the release of energy and protein.
GPT is also especially useful to valorise new raw materials, especially if they are incubated simultaneously with other two or three raw materials of known nutritional value.
GPT presents many advantages, such as its relative simplicity, the small size of the sample required, relatively low cost, and the possibility of performing simultaneous tests of numerous samples.
It should be considered that the results of this technique only give information on the ruminal degradation of the sample, and not on the digestion on the rest of the digestive tract.
Furthermore, gas is only one of the final products of ruminal fermentation, and GPT does not give any information on the production of VFAs, protein degradation, or microbial growth.
The combination of this technique with a short incubation periods (17- 24 h), during which the production of VFAs is measured, allows to obtain information on the ruminant fermentation profile of the analysed raw material.