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.
Highlights
Ruminants depend on the diverse rumen microbial assembly, comprising bacteria, archaea, protozoa, and fungi, for their survival and growth and the production of animal products
Known specific inhibitors of lysozyme, cysteine peptidases, serine peptidases, and metallopeptidases were screened using a monoculture of E. caudatum, and IMI at 100 mmol/L, phenylmethylsulphonyl fluoride (PMSF) at 3 mmol/L, and IOD at 0.5 mmol/L were found effective in inhibiting E. caudatum and lowering ammonia concentration without decreasing feed digestion or fermentation (Park et al, 2019)
Rumen protozoa depend on live bacteria for nutrients essential for their survival and growth (Park et al, 2017), and their digestive enzymes including lysozyme and peptidases are required to digest the engulf bacteria (Bonhomme, 1990; Morgavi et al, 1996)
Summary
Ruminants depend on the diverse rumen microbial assembly, comprising bacteria, archaea, protozoa, and fungi, for their survival and growth and the production of animal products (beef, lamb, milk, and wool). 70% of the dietary nitrogen (primarily as protein) is hydrolyzed in the rumen to oligopeptides and free amino acids, both of which are fermented to short-chain fatty acid (SCFA) and ammonia. Some of the ammonia nitrogen (NH3-N) is used as the nitrogen sources for ruminal microbes, primarily bacteria, to synthesize cellular proteins, which are the major direct nitrogen source for the host animals (Storm et al, 1983; Leng and Nolan, 1984; Hackmann and Firkins, 2015), but a large portion of the microbial cells (about 24% of the total ruminal bacteria daily) are engulfed by ruminal protozoa (Hespell et al, 1997), and approximately 50% of the engulfed bacterial proteins is hydrolyzed by protozoa to form oligopeptides and free amino acids (Jouany, 1996), which can be fermented back to SCFA and ammonia. Ruminal protozoa mediate intraruminal recycling of microbial protein and decrease the ruminal outflow of microbial proteins
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