Joaquín Miguel Castro Montoya (Ghent University) STSM to Animal Nutrition Wageningen UR
Focus on in vitro gas production technique and the determination of methane production

Purpose of the visit
The purpose of this visit was to learn and practice all the activities related to the in vitro gas production technique (GPT) and the simultaneous determination of methane production during a 72 h incubation of substrates with rumen fluid and buffer solution. Moreover, this visit allowed the beneficiary to get advised on the treatment of the data, the interpretation of the results, and some routine activities on the care of the gas production equipment and others like calibration of it.

Description of the work carried out during the STSM
During the stay the beneficiary joined the work of Mascha Sappok a PhD student of the animal nutrition group in Wageningen University. During the five days of the STSM the main activities to which the beneficiary was dedicated were the starting and the finishing of the incubations. Two runs were set for incubation. In each run eight different units of the GPT were used. Each unit consists of 12 positions. Each position, in turn, corresponds to a 250 mL-incubation bottle in which 500 mg of substrate are incubated during 72 h. To each bottle 60 mL of buffered media are added using a dispenser; during this process the bottles are constantly flushed with CO2 to maintain the anaerobic conditions. At the end of the 72 hours fermentation process, the flasks are carefully removed from their position and samples for volatile fatty acids determination are taken into eppendorf cups containing an acidifying agent (e.g. formic acid) to immediately stop the fermentation. The pH of the incubation media after 72 h is also determined. In this stay a total of ca. 90 incubation flasks in each run were prepared containing three different substrates and eight different incubation media from pig faeces.
Before using the GPT and the equipment each of the flasks has to be calibrated for its individual volume when 60 mL of incubation medium are added. This exact volume is later needed during the analysis of the data. Furthermore, once a year it is necessary to run a calibration of the vents for each position; this calibration is usually run by using a known stable substrate. Calibrations at the Animal Nutrition Group in Wageningen University happen by using starch as substrate.
The handling of the data was also explained by Prof. John Cone. Some discussion and further explanation was also given on the interpretation of the results and the applicability of the GPT to simultaneous methane measurements in vitro.

Main results
During the STSM it was possible to learn all the process related with the gas production technique. This acquired knowledge will be very useful for the adaptation of the existing units at Animal division of The Institute for Agricultural and Fisheries Research (ILVO) in Belgium. Two units are going to be calibrated and adapted for methane monitoring.

Future collaboration with host institution.

The host institute has shown a strong will to assist and collaborate in the activities above
mentioned to be done at ILVO.

Foreseen publications/articles resulting or to result from the STSM
The adaptation of the GPT for monitoring of the methane production will finally be applied in the frame of a study being already conducted at the Laboratory of Animal Nutrition and Animal product Quality (LANUPRO) dealing with a large screening of methane mitigation additives on different substrates in an in vitro system. The results of this study are expected to be published in the future.

Joaquín Miguel Castro Montoya STSM
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