Observing the action of phagocytosis in rumen protozoa through the utilization of fluorescent latex beads

Abstract

Protozoa comprise 10 to 50% of the rumen microbial biomass and contribute to fiber digestion and pH stability of the rumen, but they promote breakdown of protein from ingested feed or bacterial cells, which decreases efficiency of feed conversion into animal product. Protozoa take in feed by phagocytosis. We hypothesized that carboxylate-modified blue fluorescent latex beads could be used to assess rates of feed and bacteria uptake by protozoal cultures. We conducted an experiment to determine the optimum bead concentration to add to cultures and to assess differences between fed and unfed cells. Treatments were: 1.05 x 107 and 1.05 x 108 beads/ mL. We added 58 µL of each treatment to samples of fed and unfed 10-mL cultures and then incubated the samples for 1 min, 20 min, 1 hr and 24 hr. We fixed the samples with formalin and observed a 40-µL sample under a fluorescence microscope. The number of cells that consumed beads was counted out of 100 random cells. We discovered that the cells were in fact taking up the beads and that the number of cells that contained beads increased at a decreasing rate until a plateau as the incubation times increased. Through statistical analysis, we determined that saturation was reached by 3 hours, and the incubation time at which bead uptake was at one-half saturation was approximately 20 minutes. The total bead uptake was higher for the higher bead concentration and increased when cells were fed. Therefore, feeding triggers uptake of feed and carboxylated beads that mimic bacterial size and cell wall chemistry. With this valuable information, we can proceed with additional research on the phagosome-lysosome digestion process that drives protozoal nutrient acquisition and growth.