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

Abstract

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.

The Effects of Artificially Dosed Adult Rumen Contents on Abomasum Transcriptome and Associated Microbial Community Structure in Calves

“…Collectively, the reduced expression of LYZ enzymes may imply that most artificially dosed microorganisms from the donor cow colonized in the rumen successfully, instead of being digested in the abomasum. On the other hand, lower lysozymes could reduce the activity and growth of rumen protozoa, which are one of the main causes of inefficient nitrogen use in cattle [ 60 ]. Dosing may be a potentially meaningful approach to improve the feed efficiency in cattle at an early age.…”

Practical strategies to mitigate ruminant greenhouse gas emissions

“…Methanogens attached to the cilia of enteric protozoa can be indirectly removed if protozoa are removed from the rumen 104 . Although protozoa removal can reduce up to 42 percent of methane emissions in cattle 105 , defaunation also disturbs beneficial microorganisms, impairing digestion and animal performance [106][107][108][109][110] . Despite being among the oldest proposed enteric methane mitigation strategies, defaunation requires complex experimental procedures (i.e.…”

“…Despite being among the oldest proposed enteric methane mitigation strategies, defaunation requires complex experimental procedures (i.e. isolation of newborns, or fistulating adults) and no practical and sustainable method is available 104,107,108,111 .…”

— Invited Review — Ruminal ciliates as modulators of the rumen microbiome

“…However, further investigation is required. In vitro studies utilizing both ciliate monocultures and rumen fluid inocula have shown that restraining protease activities, while not limited to ruminal ciliates, could enhance NUE without adverse effects on ruminal fermentation and digestion [ 89 , 90 ]. Approaches such as selecting target enzymes specific to ruminal ciliates, screening feed additives with anti-protozoal properties, and adjusting rumen passage rates are also worthy of consideration.…”