EVALUATION OF A DEHYDROGENASE ASSAY BASED ON TETRAZOLIUM REDUCTION FOR RAPID IN VITRO ESTIMATION OF FERMENTATION ACTIVITY IN RUMEN CONTENTS

Abstract

Rumen contents obtained from fistulated steers fed brome–alfalfa hay was used to evaluate a dehydrogenase assay based on the reduction of 2,3,5-triphenyltetrazolium chloride (TTC) to triphenylformazan (TPF) for rapid in vitro estimation of fermentation activity. Maximum absorbance of TPF occurred at 485 nm over the pH range 3–10. TPF formed from TTC was stable to atmospheric O2. Absorbance decreased if TPF was exposed to light. In the presence of excess TTC, production of TPF in strained rumen fluid (SRF) at 39 °C was linear with elapsed incubation times up to at least 5 min. Dehydrogenase activity was abolished in the presence of air. It was significantly increased if the CO2 gas phase above SRF was replaced with N2 or H2, owing, at least in part, to an increase in the pH of the fermentation. The dehydrogenase activity of SRF was maximal at pH 7.8–8.0. Fractionation of SRF by differential centrifugation showed that about 50% of the dehydrogenase activity was associated with a protozoal fraction sedimenting at 188 × g, and 42% with a bacterial fraction sedimenting at 1,475 × g. Neither the cell-free supernatant obtained by centrifuging SRF at 21,500 × g, nor boiled SRF, nor the hay fed to the animals showed dehydrogenase activity. Whole rumen contents (WRC) showed a mean dehydrogenase activity that was 58% higher than that of SRF. The dehydrogenase activity of SRF was increased in the presence of substrates representing major plant carbohydrates, the greatest stimulatory effect being shown by ground brome–alfalfa hay. When rumen contents were sampled at the same time by stomach tube and through the fistula, the mean dehydrogenase activity of SRF prepared from the stomach tube samples was 137% higher than that of SRF prepared from the fistula samples. In an animal fed at 12-h intervals, the dehydrogenase activity of SRF rose within 2 h after feeding, declined for the next 6 h, and showed a second peak at 9 h. Activity then declined to the pre-feeding level. The dehydrogenase activity of WRC showed a similar pattern but the peaks of activity were less pronounced. It was concluded that dehydrogenase activity based on TTC reduction provides a simple and rapid means of estimating the fermentation activity of rumen contents which may reflect the availability of energy substrates to the microbiota.