Inhibition of Rumen Protozoa by Specific Inhibitors of Lysozyme and Peptidases in vitro

Defaunation studies have shown that rumen protozoa are one of the main causes of low nitrogen utilization efficiency due to their bacterivory and subsequent intraruminal cycling of microbial protein in ruminants. In genomic and transcriptomic studies, we found that rumen protozoa expressed lysozymes and peptidases at high levels. We hypothesized that specific inhibition of lysozyme and peptidases could reduce the activity and growth of rumen protozoa, which can decrease their predation of microbes and proteolysis and subsequent ammoniagenesis by rumen microbiota. To test the above hypothesis, we evaluated three specific inhibitors: imidazole (IMI), a lysozyme inhibitor; phenylmethylsulphonyl fluoride (PMSF), a serine protease inhibitor; and iodoacetamide (IOD), a cysteine protease inhibitor; both individually and in combinations, with sodium dodecyl sulfate (SDS) as a positive control. Rumen fluid was collected from two Jersey dairy cows fed either a concentrate-based dairy ration or only alfalfa hay. Each protozoa-enriched rumen fluid was incubated for 24 h with or without the aforementioned inhibitors and fed a mixture of ground wheat grain, alfalfa, and grass hays to support microbial growth. Live protozoa cells were morphologically identified and counted simultaneously at 3, 6, 12, and 24 h of incubation. Fermentation characteristics and prokaryotic composition were determined and compared at the end of the incubation. Except for IOD, all the inhibitors reduced all the nine protozoal genera identified, but to different extents, in a time-dependent manner. IOD was the least inhibitory to protozoa, but it lowered ammoniagenesis the most while not decreasing feed digestibility or concentration of volatile fatty acids (VFA). ANCOM analysis identified loss of Fibrobacter and overgrowth of Treponema, Streptococcus, and Succinivibrio in several inhibitor treatments. Functional prediction (from 16S rRNA gene amplicon sequences) using the CowPI database showed that the inhibitors decreased the relative abundance of the genes encoding amino acid metabolism, especially peptidases, and lysosome in the rumen microbiota. Overall, inhibition of protozoa resulted in alteration of prokaryotic microbiota and in vitro fermentation, and peptidases, especially cysteine-peptidase, may be targeted to improve nitrogen utilization in ruminants.