Evaluation of Inhibitors of Lysozyme and Peptidases as New Approaches to

Control Growth of Rumen Protozoa

Thesis

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science
in the Graduate School of The Ohio State University

By
Chongwu Yang, M. A.
Graduate Program in Animal Sciences

The Ohio State University

2017

Thesis Committee:
Zhongtang Yu, Advisor
Jeffrey Firkins
Alejandro Relling
Copyright by
Chongwu Yang
2017
 Abstract

Rumen ciliates are the only predators in the rumen. They engulf ruminal bacteria

and other microbes and use the microbial protein as a source of nitrogen and other

nutrients after digesting the ingested bacteria using digestive enzymes contained in

lysosomes. Both genes and mRNAs coding for lysozyme and peptidases including

serine peptidase, metallopeptidase, and cysteine peptidase were found in Entodinium

caudatum (E. caudatum), which represents the one of most predominant ruminal

ciliates (Williams and Coleman, 1992). Lysozyme is a small and stable enzyme that

lyses bacterial cells by breaking bacterial cell walls. Enzymatically, lysozyme

hydrolyzes the 1,4-beta linkages between N-acetylmuramic acid and N-acetyl D-

glucosamine residues in peptidoglycan. Serine peptidase, metallopeptidase, and

cysteine peptidase are proteases that cleave specific peptide bonds of proteins. By

degrading microbial proteins in the rumen, rumen ciliates drive intra-ruminal nitrogen

recycling, which decreases nitrogen utilization efficiency in ruminant animals. It was

hypothesized that inhibition of lysozyme and peptidases could inhibit growth of rumen

E. caudatum, and thereby potentially improve nitrogen utilization by ruminants.

Experiments were designed to determine the effects of different chemical

inhibitors of lysozyme and peptidases using E. caudatum as a model protozoan species.

In the first in vitro experiment (chapter 3), two inhibitors each of lysozyme, serine

peptidase, metallopeptidase, and cysteine peptidase at varying concentrations were

ii
tested for their inhibition to E. caudatum in vitro. The results of the first in vitro

experiment (chapter 3) show that all the tested inhibitors including imidazole,

phenylmethylsulfonyl fluoride (PMSF), Pefabloc®, phosphoramidon disodium salt,

bestatin, and iodoacetamide reduced (p < 0. 01) counts of E. caudatum at 24 and 48 h

incubation in vitro. The increased doses of each inhibitor all caused linearly (p < 0.01),

quadratic (p < 0.01) and cubic (p < 0.01) decrease of E. caudatum counts. Based on the

results of the first in vitro experiment (chapter 3), imidazole at 100mM, PMSF at 3 mM,

and iodoacetamide at 0.5 mM were selected for further evaluation. These inhibitors

were also evaluated in vitro using E. caudatum cultures both individually and in two

and three-way incubations. We examined the effects of these inhibitors on E. caudatum

abundance, culture pH, ammonia concentration, the concentrations of total volatile fatty

acids (VFA) and profiles, dry matter digestibility (DMD), and neutral detergent fiber

digestibility (NDFD). Effects on diversity and structure of bacteria and archaea were

investigated using amplicon sequencing analysis of 16S rRNA gene. All the three

inhibitors, both alone and in combination decreased E. caudatum counts (p < 0.01) and

ammonia concentration (p < 0.01), and the two- and three-way combinations were more

effectively than the inhibitors individually for lowering ammonia concentration (p <

0.05). No differences (p > 0.05) were observed for total VFA concentrations, DMD,

and NDFD among control and the treatments. However, the inhibitors affected the

relative abundances of some rumen microbiota at phylum and genus levels. These

results indicate that the inhibitors can effectively inhibit E. caudatum without adversely

affecting feed digestion or fermentation even though they changed the bacterial

populations in the cultures.

iii
 Dedication

Dedicated to the students at The Ohio State University

iv
 Acknowledgments

I would like to thank my advisor Dr. Zhongtang Yu first. I am so grateful for

his guidance and patience. Dr. Yu always encourages me to think critically and never

blamed me even I made simple and stupid mistakes. He taught me to think more

critically and creatively and do experiments more independently. With his

encouragement and mentoring, I gradually become more confidence doing experiments

and grow my knowledge base and skill sets.

Second, I appreciate the help of my committee members. I also thank Dr. Firkins

for allowing me to do my E. caudatum experiments in his lab. And from the beginning

when I took his Macronutrient Metabolism and Ruminant Nutrition courses, I learned

a lot from. I also want to thank Dr. Relling for his patience and concerns for my thesis

and encouragement. I have learned a lot from his Endocrinology and Ruminant

Nutrition classes.

I would like to thank Tansol Park, who taught me the procedures of growing

Entodinium caudatum cultures, conducting E. caudatum experiments and helped me to

analyze my data. I would like to thank Dr. Lingling Wang for training me in the lab and

helping me to complete the metagenomic analysis. She is patient and always corrects

my mistakes. I would like to thank Bethany Denton for her help with VFA analysis.

v
 I would also like to thank the entire lab of Dr. Yu and Dr. Firkins for all their

help with my experiments and data analysis.

Finally, I thank my father, Zaibin Yang, and my mother, Yuzhen Wang, in

Shandong Agricultural University, for their supports and encouragements.

vi
 Vita

May 2011…………………………………No.1 high school, Taian, Shandong

June 2015…………………………………B.S., Biotechnique, Department of Life

Science, Shandong Agricultural

University

Aug 2015 to present……………………..Graduate Research Associate,

Department of Animal Sciences, The

Ohio State University

Publications

C. W. Yang, Y. Cao, A. L. Mu, Y. Li, Z. B. Yang. 2014. Effects of Star Anise, Salvia

Miltiorrhiza and Ginger Root on Laying Performance, Antioxidant Status and

Egg Quality of Laying Hens. Poult. Sci. 93 (E-Suppl. 1): 134 (Abstr.)

Fields of Study

Major Field: Animal Sciences

vii
 Table of Contents

Abstract ...................................................................................................................................... ii

Dedication ................................................................................................................................. iv

Acknowledgments...................................................................................................................... v

Vita........................................................................................................................................... vii

Publications .............................................................................................................................. vii

Fields of Study ......................................................................................................................... vii

Table of Contents .................................................................................................................... viii

List of Tables ............................................................................................................................ xi

List of Figures ......................................................................................................................... xiii

List of Abbreviations .............................................................................................................. xiv

Chapter 1: Introduction ............................................................................................................. 1

Chapter 2: Review of Literature................................................................................................ 3

Rumen ciliate protozoa .......................................................................................................... 3

The efficiency of nitrogen utilization ..................................................................................... 5

Efficiency of microbial protein synthesis .............................................................................. 7

Digestive enzymes ................................................................................................................. 8

Inhibitors of rumen protozoa ................................................................................................ 11

viii
Chapter 3: In vitro Experiments to Test the Effects of Inhibitors of Lysozyme and

Peptidases on E. caudatum Growth.......................................................................................... 13

Abstract ................................................................................................................................ 13

Introduction .......................................................................................................................... 14

Materials and Methods ......................................................................................................... 15

Inhibitors selection................................................................................................... 15

SP medium ............................................................................................................... 16

Preparation of Entodinium Caudatum culture ........................................................ 17

Evaluation of the inhibitors in vitro ......................................................................... 17

Statistical analysis .................................................................................................... 18

Results .................................................................................................................................. 18

Discussion ............................................................................................................................ 19

Chapter 4: Effects of Selected Inhibitors of Lysozyme and Peptidases on Entodinium

caudatum, Fermentation and Prokaryotic Community in vitro ................................................ 23

Abstract ................................................................................................................................ 23

Introduction .......................................................................................................................... 24

Materials and Methods ......................................................................................................... 26

Medium, protozoal culture, feed, and in vitro incubation ........................................ 26

Treatments with inhibitors ....................................................................................... 26

Fermentation analysis .............................................................................................. 26

DNA extraction and metagenomic analysis of microbiota ...................................... 27

Statistical Analysis ................................................................................................... 28

Results .................................................................................................................................. 29

Inhibition of E. caudatum and effects on fermentation characteristics .................... 29

Effect on the Microbiota in the in vitro E. caudatum cultures ................................. 30

ix
 Discussion ............................................................................................................................ 32

Effect of the inhibitors on feed substrate digestion and fermentation characteristics

of the E. caudatum culture ....................................................................................... 32

Effect of the inhibitors on the microbiota of the E. caudatum culture ..................... 37

Chapter 5: Conclusion............................................................................................................. 52

References ................................................................................................................................ 54

x
 List of Tables

Table 3.1 E. caudatum counts in the presence of imidazole, PMSF, Pefabloc®,

bestatin, iodoacetamide and PDS at 24h and 48h .................................................... 21

Table 4.1 Effects of imidazole, PMSF, iodoacetamide and their combination on E.

caudatum counts at 24, 48 and 72h in vitro fermentation ........................................ 41

Table 4.2 Effects of imidazole, PMSF, iodoacetamide and their combinations on pH,

NH3N concentration, DM digestibility, and neutral detergent fiber (NDF) digestibility

in vitro fermentation ................................................................................................ 42

Table 4.3 Effect of imidazole, PMSF, iodoacetamide and their combination on

volatile fatty acids (VFA) concentrations (mM) in vitro fermentation cultures ...... 43

Table 4.4 Effect of imidazole, PMSF, iodoacetamide, and their combinations on

relative abundance of bacteria and archaea in the in vitro E. caudatum cultures when

treated with the inhibitors for 72h ............................................................................ 45

Table 4.5 Effect of imidazole, PMSF, iodoacetamide, and their combinations on

relative abundance of bacterial phyla in the in vitro E. caudatum cultures when treated

with the inhibitors for 72h ........................................................................................ 46

xi
Table 4.6 Effect of imidazole, PMSF, iodoacetamide, and their combinations on

relative abundance of bacterial genera in the in vitro E. caudatum cultures when

treated with the inhibitors for 72h ............................................................................ 48

xii
 List of Figures

Figure 4.1. Principal coordinate analysis of bacterial communities of the in vitro E.

caudatum cultures after treatment with the inhibitors for 72 h. C, control; Imi,

imidazole at 100 mM, PMSF, phenylmethylsulphonyl fluoride at 3 mM; Iodo,

iodoacetamide at 0.5 mM; Imi+PMSF, combination of 100 mM imidazole and 3 mM

PMSF; Imi+Iodo, combination of 100 mM imidazole and 0.5 mM iodoacetamide;

PMSF+Iodo, combination of 3 mM PMSF and 0.5 mM iodoacetamide;

Imi+PMSF+Iodo, combination of 100 mM imidazole, 3 mM PMSF, and 3 mM

iodoacetamide .......................................................................................................... 50

Figure 4.2 Relative abundance (represented by % of total sequences) of the major

bacterial phyla identified in the in vitro E. caudatum cultures. Imi, imidazole at 100

mM; PMSF, phenylmethylsulphonyl fluoride at 3 mM; Iodo, Iodoacetamide at 0.5

mM; Imi+PMSF, combination of 100 mM imidazole and 3 mM PMSF; Imi+Iodo,

combination of 100 mM imidazole and 0.5 mM iodoacetamide; PMSF+Iodo,

combination of 3 mM PMSF and 0.5 mM iodoacetamide; IPI, combination of 100

mM imidazole, 3 mM PMSF, and 3 mM iodoacetamide ......................................... 51

xiii
 List of Abbreviations

Ammonia ..............................................................................................................NH3

Branched chain fatty acids ................................................................................ BCVFA

Crude protein .......................................................................................................CP

Dry matter digestibility ........................................................................................DMD

Dry matte .............................................................................................................DM

Efficiency of microbial protein synthesis ............................................................EMPS

Fluid-associated bacteria ......................................................................................FAB

Gas Chromatography ...........................................................................................GC

Microbial crude protein ........................................................................................MCP

Neutral detergent fiber .........................................................................................NDF

Neutral detergent fiber digestibility .....................................................................NDFD

Nitrogen utilization efficiency .............................................................................NUE

Nitrogen ...............................................................................................................N

Operational Taxonomic Units ..............................................................................OTU

Organic matter .....................................................................................................OM

xiv
Organic matter digestibility .................................................................................OMD

Particle-associated bacteria ..................................................................................PAB

Phenylmethylsulphonyl fluoride ..........................................................................PMSF

Phosphoramidon disodium salt ............................................................................PDS

Principal coordinates analysis ............................................................................... PCoA

Quantitative Insights Into Microbial Ecology ......................................................Qiime

Rumen degradable protein ...................................................................................RDP

Rumen undegradable protein ...............................................................................RUP

Short-chain fatty acids .......................................................................................... SCFA

Volatile fatty acids ...............................................................................................VFA

xv
 Chapter 1: Introduction

The rumen is the microbial habitat with abundant bacteria, archaea, viruses,

fungi, and protozoa. Nutrient degradation including hydrolysis of lignocellulosic feeds

and degradation of protein are accomplished by the collective degradative activities of

rumen microorganisms (Santra and Karim, 2002). Cellulolytic bacteria, amylolytic

bacteria, and fungi together determine the rate and extent of polysaccharide degradation,

while rumen protozoa play a minor role in feed digestion (Similarities, 2014; Lee and

Ha, 2000); Mendoza et al., 2014). This has been demonstrated in previous studies that

compared feed digestibility in faunated (protozoa-carrying) and defaunated (protozoa-

free) sheep. Firstly, rumen protozoa promote methane production due to their

production of methanogenesis substrates and positive association with methanogens.

Secondly, and more importantly, rumen protozoa prey on rumen bacteria and mediate

wasteful intra-ruminal dietary nitrogen recycling (Jouany and Ushida, 1999). Therefore,

rumen protozoa are believed to be primarily responsible for low nitrogen utilization

efficiency (NUE) and excessive nitrogen excretion from ruminant animals. In addition,

it is a long-standing interest to improve nitrogen utilization efficiency by inhibiting the

growth of rumen protozoa, rather than by defaunation, using inhibitors.

Previous studies have evaluated various protozoal inhibitors, such as yucca

saponin (Wang et al., 1999), tea saponin (Hu et al., 2005), and essential oils (Patra and

Yu., 2012; Patra et al., 2015), for their efficacy to inhibit rumen protozoa. However,

none of these are specific inhibiters, and often adverse effects on feed intake or
1
digestions are reported for these plant derivatives. There is a need to identify inhibitors

that can specifically control rumen protozoa with little or no adverse effect on rumen

function or ruminal microbiota. it was hypothesized that the unique digestive enzymes

of rumen protozoa, such as lysosomal lysozyme and peptidases, could inhibit growth

of E. caudatum. In this study, this hypothesis was tested by evaluating inhibitors of

lysozyme and three families (serine peptidase, metallopeptidase, and cysteine

peptidases) for their ability to inhibit Entodinium caudatum, one of the most

predominant and bacterivorous protozoan species in the rumen. Promising inhibitors

for each type of the enzymes were further investigated for their efficacy and their effects

on feed digestion, fermentation, ammonia concentration, and relative abundance of

rumen microbiota. The results were promising, and the inhibitors of those lysosomal

enzymes may have potential to be used in control rumen protozoa to achieve improved

nitrogen utilization efficiency.

2
 Chapter 2: Review of Literature

Rumen ciliate protozoa

The quantification of protozoal biomass has been hampered by a suitable

chemical marker (Firkins et al.,1998), however, the most predominant protozoa are

ciliates at a density of approximately 104 to 106 cell counts per ml rumen fluid (Clarke,

1977). Ruminal ciliates are either oligotrichs or holotrichs, and their populations and

proportions are primarily determined by the composition and the physical

characteristics of diet as well as feeding frequency (Jouany, 1989). Holotrichs, with

cilia covering their entire body, primarily utilize soluble carbohydrates including starch

and sugar, whereas oligotrich ciliates with cilia found only in the mouth region,

consume and ferment insoluble carbohydrates such cellulose and hemicellulose

(Williams, 1986). Oligotrichs represent more than 95% of the total ciliate community,

whereas holotrichs only account for less than 5% of the ciliate population in the rumen.

However, due to their large cellular size, holotrichs can account for nearly 40% of the

protozoal biomass (Williams, 1986). E. caudatum is one of the most predominant

protozoan species in the rumen, and it shares similar morphological appearance and

fermentative characteristics with other oligotrich ciliates on the rumen. E. caudatum

population increased in ruminants when fed high amount of insoluble carbohydrates

(Belecki et al., 2016).

3
 In addition to utilizing sources of carbohydrates from diets to obtain energy,

rumen protozoa engulf and digest bacteria by lytic enzymes enclosed in lysosomes for

their core protein sources (Coleman and Laurie, 1974). A single protozoan cell in the

rumen is able to take up 102-104 bacteria per hour (Coleman, 1975). The rate of such

engulfment can be affected by several factors including starvation, and the engulfment

can be both selective and in-selective (Coleman, 1986). Coleman and Sandford (1979)

found that one of the most predominant rumen protozoa, E. caudatum, ingests nearly

all bacteria without any selection while other species like Epidinium caudatum, selects

certain bacteria of rumen origin in in vitro experiments. And the amounts of protozoa

consumed per protozoa is greater in vitro studies than in vivo studies. During

engulfment, bacteria are engulfed and digested by protozoa rapidly, and the uptake of

bacteria increased with the increasing initial concentration of bacteria in the culture

(Coleman, 1964). Uptake of bacteria by protozoa decreases nitrogen utilization

efficiency (NUE) and efficiency of microbial protein synthesis (EMPS) in the rumen.

This has been demonstrated in several studies where defaunation, complete elimination

of protozoa from the rumen, decrease ammonia concentration in the rumen (Koenig et

al., 2000; Newbold et al., 2015). The importance of the rumen protozoa to the health of

adult ruminants and growth of young ruminants has been debated since 1929 (Beck,

1929; Williams and Coleman, 1992). Some studies (Koenig et al., 2000) found that

defaunation decreases ammonia concentration, indicating better dietary nitrogen

utilization efficiency (NUE). However, despite reduced protozoa decrease intra-

ruminal N recycle and elevated efficiency of microbial protein synthesis (EMPS) after

feeding dietary fat (Firkins, 1996), benefits of defaunation are still remaining

controversy. Defaunation of rumen decreases cellulose degradation rate, ammonia

concentration, methane production, increase bacterial density but depress fiber

4
digestibility. However, studies of defaunation are mostly with nonlactating or low-

producing animals (Hristov and Jouany, 2005). And suppression of rumen protozoa is

a better way which increase efficiency of microbial protein synthesis (EMPS) without

the negative impact of complete defaunation (Firkins et al., 2006). It was hypothesized

that inhibiting rumen ciliates growth, rather than eliminating them, might be beneficial

for nitrogen utilization efficiency (NUE) and avoiding decreasing fiber digestibility.

Entodinium caudatum is widely distributed in rumen fluids and its single cell

was isolated from rumen as pure culture for in vitro experiment (Dehority, 2010). The

optimum temperature for growth of Entodinium caudatum in the rumen is about 39oC

(Williams and Coleman, 1992). Dehority (2005) found that E. caudatum is sensitive to

rumen pH as other species of rumen ciliates. Entodinium caudatum would increase

when pH rose from 5.5 to 6.0 and decrease as pH dropped to approximately 5.4 and

eventually disappeared after exposed at pH 5.4 for an extended period of time (Dehority,

2005).

The efficiency of nitrogen utilization

The efficiency of nitrogen utilization (NUE) in the rumen is the key factor in

ruminants in determining animal production and environmental impact (Calsamiglia et

al., 2010) Ruminal ammonia concentration is a predicator of efficiency of dietary

nitrogen conversion into microbial nitrogen (Firkins et al.,2007). Ruminants do not

utilize dietary nitrogen as efficiently as non-ruminants, and more than half of dietary N

is converted to ammonia, and large portion of ammonia (Calsamiglial et al., 2010).

Ammonia is recycled back to the rumen from urea and utilized by rumen

microorganisms. However, when ammonia is produced too rapidly in rumen which

5
exceeds the utilization by rumen microorganism, large portion of ammonia is excreted

in urea and feces (Firkins, 2005; Ishler, 2004). When ammonia is released into the

atmosphere, it reacts with other gases including sulfuric acid to form ammonium sulfate,

which is harmful to the air quality (Firkins, 2005; Ishler, 2004). Under low dietary

protein conditions, cattle are more likely to utilize nitrogen more efficiently especially

for high production animals (Calsamiglial et al., 2010). However, the average NUE

among ruminants is only around 25%, and low NUE not only increases production cost

but also leads to environment pollution with excreted nireogen. Higher ammonia

concentration in the rumen appears to be associated with lower NUE (Bach et al., 2005).

Thus, ruminal ammonia concentration is one of the indicators for predicting NUE

(Newbold et al., 2015).

There have been several comprehensive reviews (Bach et al., 2005; Calsamiglia

et al., 2010; Jouany, 1996) on the relationship among rumen protozoa, bacteria, and

NUE. This relationship was extended to defaunation and ammonia concentration in

many studies and was corroborated in a recent meta-analysis (Newbold et al., 2015).

Because rumen bacteria provide 50-80% of total absorbable protein for ruminants

(Storm and Orskov, 1983), the bacterial population in the rumen is one of the most

important factors affecting NUE. Rumen protozoa, including E. caudatum, reduce NUE

by converting bacterial N to ammonia after engulfing rumen bacteria at a rate of 20-

3000 bacteria per protozoan per hour (Coleman and Laurie, 1977; Bach et al., 2005;

Virtanen, 1969). Another explanation of low NUE attributed to protozoa is that

protozoa pass out of the rumen relatively slower than bacteria and therefore a relatively

smaller proportion of protozoal protein can be utilized by ruminants (Williams and

Coleman, 1992).The importance of rumen protozoa to ruminant health and the

6
development of ruminants has been debated since Becker (1929) first identified that

ruminant animals could survive and live normally after defaunated. However,

defaunation showed numerical positive influences on rumen function such as higher

bacterial concentration, lower methane emission, increased microbial N flow, higher

efficiency of microbial protein synthesis (EMPS), although several adverse effects were

noted after defaunation such as lower organic matter digestibility (OMD) and neutral

detergent fiber digestibility (NDFD).

Efficiency of microbial protein synthesis

Achieving efficient animal production and better animal performance requires

attention to optimize the efficiency of microbial protein synthesis (EMPS) and NUE

(Isher, 2004). Synthesis of microbial protein, expressed as grams of microbial N per

unit of rumen available energy, in the rumen is the result of rumen microorganism

metabolism. When protein requirement exceeds synthesis of protein caused by low

EMPS especially for lactating cows, production and protein content of milk and meat

can be reduced (Koenig et al., 2000; Reynolds and Kristensen, 2008).

The efficiency of microbial protein synthesis has been discussed in relation to

the NUE. Bach et al. (2005) concluded that NUE in the rumen peaked around 69%

when EMPS is 29 g of bacterial N/kg of fermented organic matter implying that high

NUE can be achieved by optimizing EMPS. Rumen protozoa prefer engulfing bacteria

and utilize the microbial protein as the primary protein source over using amino acids

or protein directly from feeds (Bach et al., 2005; Sakanari et al., 1989). The role of

microbial protein synthesis and degradation played by protozoa in the rumen remain

unclear. The flow of protozoa from rumen to duodenum is low because of the
7
sequestration of protozoa on feed particles and the rumen epithelium (Hook et al.,2017).

Sok et al. (2017) found that approximately 16.5% of microbial crude protein (MCP)

arising from protozoal protozoa reach to duodenum, whereas 33% of MCP is from

fluid-associated bacteria (FAB) and 50% particle-associated bacteria (PAB) by using

rigorous screening and quantification approaches ). Rumen Protozoa are not essential

to the animal survival, defaunation (removal of protozoa) has been used to optimize

rumen function and animal performance in research (Becker, 1929; Williams and

Coleman, 1992). A meta-analysis done by Newbold et al. (2015) summarized and

evaluated previous studies. The value of EMPS from different studies in defaunated

animals differed; however, low ammonia concentration and high microbial protein flow

are the most consistent of the effects after removing protozoa (Demeyerc and Van

Nevel, 1979). The increase in overall EMPS observed in defaunated animals highlights

the fact that elimination of rumen protozoa has positive effects on rumen function,

animal performance as well as milk and meat production (Newbold et al., 2015).

However, previous studies of defaunation are primarily for low-producing cows and

non-lactating cows, and the contribution of protozoal protein to microbial protein under

different dietary conditions for high-producing cows is not known (Firkins et al., 2007).

Digestive enzymes

Although ciliates possess the ability to utilize dietary carbohydrates, it is well

established that ciliates engulf bacteria and use the microbial protein as their major

protein sources (Coleman and Laurie, 1974). Digestive enzymes, such as lysozyme and

proteases, are found in protozoa (McKerrow, 1993). Rumen protozoa, especially

oligotrichs, are very bacterivorous and can engulf large numbers of rumen bacteria,

8
exerting a significant effect on NUE and EMPS. Two types of enzymes, lysozyme and

peptidase produced by protozoa, are contained in lysosomes. Lysosomes are organelles

in the cytosol of rumen protozoa containing digestive enzymes, including lysozymes,

peptidases, glycoside hydrolases, lipase, etc. The mechanism or process of engulfment

of bacteria by rumen protozoa is not well understood, but it is thought to be similar as

that of environmental ciliates. After the first step of adherence of microbe to protozoa,

the cell membrane of the protozoa at the oral area is invaginated, and a phagosome is

successfully formed. Upon fusion with a lysosome, a phagolysosome forms (Williams

and Coleman, 1992). Bacteria in the phagolysosome are thought to be lysed by the

digestive enzymes, including lysozyme and peptidase and lipase. Previous studies

showed that hydrolytic enzymes, including lysozymes and several types of peptidases

derived from lysosome, are released from protozoa in the suspension of in vitro

incubation, and some bacterial debris could be seen after adding protozoa, suggesting

that protozoa produce lysozymes and peptidase that can digest bacteria after engulfment

(Muller, 1972; Coleman and Laurie, 1974; Ling, 1990). Direct evidence of lysozyme,

peptidases, glycoside hydrolases, and lipase was obtained from a recent genomic and

transcriptomic studies (unpublished data of our lab).

Regarding the effects of lysozyme effects on bacterial digestion, some old

literature reported degradation of bacterial cell wall by hydrolyzing the beta-1, 4-

linkage between N-acetylmuramic acid and N-acetyl-D-glucosamine residues of

peptidoglycan, the major compound of bacterial cell wall（Ghuysen and Strominger,

1966). Coleman and Laurie (1974) isolated lysozyme released from three Epidinium

species from the rumen. Bukharin and Nemtseva (2000) determined that some bacterial

strains were resistant to lysozymic activity and survived from protozoa in vitro.

9
 Proteases are another type of digestive enzymes produced by rumen protozoa.

They can be classified into a number of types including serine peptidase,

metallopeptidase, and cysteine peptidase (Coleman and Laurie, 1974; Muller, 1972).

Brock et al. (1982) confirmed the presence of serine peptidase, metallopeptidase, and

cysteine protease by adding their inhibitors to protozoal cultural supernatant in-vitro.

Serine peptidase in the rumen is found ubiquitous in protozoa after engulfing bacteria.

Serine peptidase hydrolyzed the peptide bonds of protein or peptides containing special

serine residues (Hedstom, 2002). The mechanism of serine protease involves catalyzing

the hydrolysis of ester or amide (Wong and Whitesides, 1994). Serine protease genes

are highly expressed in protozoa and have been successfully isolated from rumen

protozoa in previous studies (Sakanari et al., 1989). Metallopeptidases were isolated

from protozoa and act as important enzymes (Rawling and Barret, 1995).

Metallopeptidases probably aid digestion of bacteria by affecting the cell surface and

extracellular matrix (Baker, 2002). Metal ions are needed to bind the substrate to the

active site of metallopeptidase and catalyze hydrolysis of side chains of peptides.

Cysteine protease plays a vital role in the digestion of rumen protozoa by catalyzing a

nucleophilic cysteine thiol in catalytic triad formed by the cysteine and histidine

residues of protein (Grzonka et al., 2001). Cysteine protease has been described as the

major protease in rumen protozoa that contributes to more than half of endogenous

proteolytic activity (Barret et al., 1977). Cysteine peptidases have been successfully

isolated from bacteria, plants as well as rumen protozoa (Otto and Schirmeister, 1997).

Activities of protease produced by protozoa are affected by pH and temperature in a

similar fashion as other enzymes. Despite the impact on protozoal digestion, the roles

of protease in rumen protozoa survival remain unclear.

10
Inhibitors of rumen protozoa

In any event, although defaunation appeared to show benefits in rumen

functions and animal performance, it is noteworthy to observe that previous studies

showed adverse effects, including decreased OM digestibility. Besides the adverse

effects of defaunation, many chemicals added to eliminate ciliates are toxic or harmful

to ruminants. Therefore, identifying inhibitors to control ruminal protozoa, instead of

eliminating them, may minimize the adverse effects mentioned above of defaunation.

Natural and synthesized inhibitors have been tested both in vitro and in vivo to

determine their effects on rumen protozoal growth (Firkins et al., 2005). However, these

protozoa inhibitors, such as yucca saponin (Wang et al., 1999), tea tannin (Hu et al.,

2005) and essential oils (Patra and Yu, 2012; Patra and Yu, 2015), also show adverse

effects on rumen functions, decrease feed intake, and broadly impact ruminal bacteria.

Considering the important role played by digestive enzymes produced by protozoa,

specific inhibitors of lysozymes and peptidases may be explored to control rumen

protozoa. Lysozyme inhibitors, imidazole and indole-3 propionic, bind to the center of

the hydrophobic core of lysozymes, changing the conformation of the active site (Swan,

1972). Imidazole and its derivatives form the charge-transfer type between them and

the indole moiety of the tryptophan residues present in the active sites of enzymes

(Shinitzky et al., 1966). Imidazole and its derivatives are effective inhibitors, and

previous studies have shown that imidazole and its derivative imidazole, inhibit the

lysis of Micrococcus Lysodeikticus by lysozyme at a concentration of 0.01-0.4 M

(Shinitzky et al., 1966). Two serine esterases, phenylmethylsulphonyl fluoride (PMSF)

and Pefabloc®, with a potential of selective sulfonylation of serine peptidase, were first

11
found by Gold and Fahreny (1963). Aside from their role in inhibiting chymotrypsin-

like serine protease, it is well known that PMSF and Pefabloc® inhibit serine protease

purified from rumen ciliates (Brock et al., 1982). Phosphoramidon sodium salt (PDS)

and bestatin, as cell membrane surface metallopeptidase inhibitors, can effectively

inhibit thermolysin and zinc metallopeptidase (Baker, 2002; Kitagishi and Hiromi,

1984). However, the inhibitory effect of PDS and bestatin on metallopeptidase purified

from rumen ciliates has not yet been confirmed. Because cysteine peptidase is

responsible for 60 to 70% of endogenous proteolytic activity in rumen ciliates,

inhibition of cysteine peptidase can achieve effective inhibition of ruminal protozoa.

Because iodoacetamide can successfully and quickly alkylate cysteine residues to block

cysteine peptidase, iodoacetamide has been used for cysteine residue protector and as a

reagent of cysteine modification since the early 1930’s (Chalker et al., 2009).

Previous studies have tested the effects of indo-3 propionic, imidazole, PMSF,

Pefabloc®, except PDS and bestatin, in enzymes purified from rumen ciliates. However,

no previous studies have ever tested their effects on ciliates and bacteria growth. I

hypothesized that inhibition of these digestive enzymes will effectively inhibit cell

counts of ruminal E. caudatum. This hypothesis was tested in this thesis research using

E. caudatum as the model rumen protozoa in vitro. The effects of the inhibitors on feed

digestion, ammonia concentration, pH, VFA concentration, and bacterial and archaeal

communities were also examined.

12
 Chapter 3: In vitro Experiments to Test the Effects of Inhibitors of Lysozyme

and Peptidases on E. caudatum Growth

Abstract

Nitrogen utilization inefficiency not only increases feed costs and decreases

animal production, but also causes air and soil pollution by increasing ammonia output

from excreted urine and manure. Rumen microbes possess the ability to utilize dietary

nitrogen to synthesize microbial proteins, which contribute up to more than half of the

total absorbable proteins of ruminants. Rumen ciliates, with their abilities to engulf and

digest bacteria, were assumed to be critical for decreasing nitrogen utilization efficiency

(NUE). I hypothesized that rumen protozoa could be inhibited by inhibitors of lysozyme

and peptidases and decease ammonia genesis. The first objective of the in vitro

experiment was to identify promising inhibitors of lysozyme, cysteine peptidase, serine

peptidase, and metallopeptidase, each at three concentrations, can effectively inhibit the

growth of Entodinium caudatum. One or two specific inhibitors each enzyme type was

tested at one selected concentrations. The experiment was in vitro using a single-species

of the protozoal culture of E. caudatum in SP medium. In order to exclude the

influences of solvents of a few inhibitors, same volumes of solvents were added to each

treatment. All cultures were incubated at 39oC, and the protozoa abundance was

enumerated by microscopic counting at 24 and 48 h post incubation. All six inhibitors

(imidazole,
13
phenylmethylsulphonyl fluoride, Pefabloc®, bestatin, iodoacetamide, phosphoramidon

disodium salt) decreased (p < 0.01) protozoa abundance at very low concentration.

Some of these inhibitors may be further evaluated for their efficacy and effect on rumen

fermentation and other microbes.

Introduction

Dietary protein is the most expensive feed ingredient of food animals, including

both ruminant and non-ruminant animals. Besides affecting the profitability of animal

producers, nitrogen utilization efficiency (NUE) also determines the amount nitrogen

discharged into the environment. Low NUE both increases livestock production cost

but also cause pollution of the environment (Reynolds and Kristensen, 2008). However,

NUE in ruminants is much lower and variable than in non-ruminants because of the

microbial metabolism in the rumen (Kohn et al., 2005; Calsamiglia et al., 2010). Up to

60% of the ingested dietary protein is metabolized by rumen microbiota and become

microbial protein. Microbial protein is the major direct nitrogen source, contributing to

60-70% of total degradable protein that ruminants need. Rumen ciliates play an

important role in low NUE because they engulf various ruminal bacteria and digest

their cellular protein into oligo peptides and free amino acids, both of which can be

rapidly fermented by some ruminal bacteria (Bach et al., 2005; Virtanen, 1969),

especially hypo-ammonia producing bacteria (HAB), into ammonia and short-chain

fatty acids (SCFA) (Koenig et al., 2000). Although protozoa make up nearly half

proportion of the rumen microbial biomass low producing ruminants, the flow of rumen

protozoa from reticulo-rumen to duodenum is quite low (Harrison et al., Leng et al.,

1980; Hook et al., 2017). Therefore, rumen protozoa are the primary cause of wasteful

14
intra-ruminal nitrogen recycling. Although there are exceptions, defaunation decreases

rumen ammonia concentration and increased NUE (Newbold et al., 2015). Although

lower ammonia concentration has been confirmed in defaunated sheep (Newbold et al.,

2015), adverse effects including decreased NDF digestibility and duodenal fatty acids

flow have also been observed. Besides, chemicals used for defaunation are toxic.

Therefore, controlling protozoal growth instead of killing them totally may minimize

the adverse effects caused by defaunation.

Numerous previous studies have evaluated different approaches in controlling

the populations of ruminal protozoa, including plant secondary metabolites and lipids,

none of them could specifically and consistently control the population of rumen

ciliates (Hu et al., 2005; Patra et al., 2012; Patra and Yu, 2012; Patra and Yu, 2015;

Wang et al., 1999). Because lysozymes and peptidases are necessary to digest engulfed

bacteria, both Gram-positive and Gram-negative bacteria, inhibition of these two types

of enzymes can specifically inhibit protozoal growth. In this in vitro experiment, six

inhibitors for lysozyme, cysteine peptidase, serine peptidase, and metallopeptidase

were tested using E. caudatum in vitro. I hypothesized that E. caudatum growth could

be inhibited by the inhibitors. The objective of the experiment was to determine the

effect of lysozyme and peptidases, each at 3 doses, on inhibiting growth of E. caudatum.

Materials and Methods

Inhibitors selection

A literature search was conducted with the key words “lysozyme” and

“inhibitors” and “protozoa” and “peptidase” or “protease” and “inhibitors” and

15
“protozoa” using PubMed and found no published studies. Then literatures were

searched again by not including the keyword “protozoa” and a few inhibitors were

found that have been used in enzymological studies. After evaluating their inhibitory

efficacy in the literature and commercial availability, the following six different

inhibitors were selected for primary screening: imidazole (a lysozyme inhibitor),

phenylmethylsulphonyl fluoride (a serine peptidase inhibitor), Pefabloc® (a serine

peptidase inhibitor), phosphoramidon disodium salt (a metallopeptidase inhibitor),

bestatin (a metallopeptidase inhibitor), and iodoacetamide (a cysteine peptidase

inhibitor). To identify the effects of different doses of promising inhibitors, each of the

inhibitors was tested at low, medium and high doses with 3 replicates per dose:

imidazole at 13.5, 27, 54 mg/ml; phenylmethylsulphonyl fluoride at 0.2, 0.4, and 0.8

mg/ml; Pefabloc® at 0.5, 1, and 2 mg/ml; phosphoramidon disodium salt at 0.006, 0.03,

and 0.12 μg/ml; bestatin at 0.01, 0.05, and 0.1 mg/ml; iodoacetamide at 0.018, 0.09,

and 0.45 μg/ml. These concentrations were selected based on the effective

concentrations in enzymological reactions, with the low and high concentration being

below and above the enzymatically effective concentrations, respectively. Each

concentration was tested in vitro in three replicates. A control containing no inhibitor

was also included in parallel in three replicates. A separate stock solution was prepared

for each inhibitor. Phenylmethylsulphonyl fluoride is not water soluble, so it was

dissolved in absolute ethanol. To rule out the solvent effect, the same volume of the

ethanol was added to the control.

SP medium

16
 The SP media (Dehority, 1993) was prepared and used to test the growth of

Entodinium caudatum prior to the experiment. The composition of the SP medium (g/l)

was as follows: mineral mix SP-1 (20 g/l K2HPO4), mineral mix SP-2 (16 g/l KHPO4,

4 g/l NaCl, 0.212 g/l CaCl2`H2O, 0.154 g/l MgSO4), clarified rumen fluid, 6% (w/v)

NaHCO3, 3% (w/v) cysteine` HCl, 0.1% resazurin and dH2O. SP medium was

autoclaved at 121oC for 20 min and let cool down to room temperature after preparation.

SP media was kept for overnight at 39oC to check for contamination and oxidation.

Preparation of Entodinium Caudatum culture

The rumen ciliate E. caudatum culture, containing no other rumen protozoa,

was activated from frozen stock by incubation in 39oC with SP media. The E. caudatum

was cultured in glass bottles under CO2 atmosphere, with each tube sealed with a rubber

stopper. The cultures of E. caudatum were fed 0.12 ml feed suspension per 10 ml

culture daily. The feed contained (g/l): 0.3 g ground wheat, 0.1 g ground grass hay, and

0.1 g ground alfalfa. The culture volume was enlarged by adding an equal volume of

fresh SP media every 3 to 4 days of incubation at 39oC. Counts of Entodinium caudatum

were checked before each dilution.

Evaluation of the inhibitors in vitro

The E. caudatum culture prepared above was inoculated into SP medium

containing individual inhibitor at a preselected concentration in autoclaved 16×150 mm

culture tubes. Each tube contained 2.4 ml SP media and 0.6 ml E. caudatum culture.

The headspace of the tubes was O2-free. The tubes were incubated at 39oC for 24 and

17
48 h and were fed 0.12 ml protozoal feed daily. One subsample (0.3 ml) was collected

from each tube at 24 and 48 h of incubation, fixed with a 1 ml formalin fixative (50%

formalin and glycerol) containing 10 μl brilliant green (catalog B67456), and save for

microscopic counting. The growth of E. caudatum was estimated by microscopic

counting at 100x magnification (Dehority, 1993) using a counting chamber (Hausser

Scientific, catalog #3800).

Statistical analysis

Data were analyzed using the MIXED model procedure of SAS (SAS Institute

Inc., Cary, NC v9.4): Yijk = μ + Di + Hj + DHij + eijk, continuous variable, μ is the overall

E. caudatum population mean, Di is the fixed effects of the ith inhibitors dose (i = 1, 2,

3, 4), Hj is the fixed effect of jth hour (j = 1, 2), and eijk is the residual error. DHij is the

fixed effect of the interaction of ith inhibitor doses and jth hour. Orthogonal polynomial

contrasts were used to analyze the linear, quadratic, and cubic effects of the increasing

doses of each inhibitor on E. caudatum counts.

Results

The increased doses of each inhibitor all caused linear (p < 0.01), quadratic (p

< 0.01) and cubic (p < 0.01) decrease of E. caudatum counts (Table 3.1). There was no

significant interaction for inhibitor doses of imidazole (p = 0.33), PMSF (p = 0.82),

Pefabloc® (p = 0.06), bestatin (p = 0.80) by time except phosphoramidon disodium salt

Compared to the control without inhibitors, the highest doses (i.e., 54.46 mg/ml

imidazole, 0.80 mg/ml phenylmethylsulphonyl fluoride, 0.45 mg/ml iodoacetamide, 2.0

18
mg/ml Pefabloc®, 0.10 mg/ml bestatin, and 0.12 mg/ml phosphoramidon disodium salt)

decreased in E. cell counts by 96.9%, 95.9%, 94.9%, 95.9%, 75.4%, and 80.6%,

respectively at 24 h and by 97.9%, 96.8%, 94.7%, 96.8%, 82.1%, and 82.1%

respectively, at 48 h (calculated in Table 3.1). Thus, imidazole, phenylmethylsulphonyl

fluoride, Pefabloc®, and iodoacetamide could inhibit E. caudatum more effectively and

be used as specific inhibitors.

Discussion

All the digestive enzymes of rumen ciliates are contained inside of lysosomes

to avoid self-destruction of cellular components (Khanna and Yadav, 2004). Lysozyme

degrades the peptidoglycan of the cell wall of bacteria, while serine peptidase,

metallopeptidase, and cysteine are the major enzymes that hydrolyze proteins

(McKerrow, 1993; Muller 1972; Coleman and Laurie 1974; Ling, 1990). As

hypothesized, inhibition of the lysozyme, peptidases, and peptidases (cysteine, serine,

or metallo types) all substantially (by greater than 80% and up to 97%) inhibited the

growth of E. caudatum. These results indicate that E. caudatum depends on these

digestive enzymes for the acquisition of essential nutrients and that engulfed ruminal

bacteria and other microbes are the sources of these essential nutrients.

The tested inhibitors inhibited E. caudatum cell counts to different degrees. The

lysozyme inhibitor, imidazole, inhibited E. caudatum the most, followed by the

inhibitors of cysteine peptidase (iodoacetamide) and serine peptidase (Pefbloc® and

phenylmethylsulphonyl fluoride). The lysozyme inhibitor had probably prevented E,

caudatum from lysing the engulfed bacteria and thus accessing the cellular proteins,

whereas inhibitors of cysteine peptidase and serine peptidase probably did not prevent

19
bacterial lysis but inhibited hydrolysis of the bacterial proteins. Phosphoramidon

disodium salt and bestatin, both of which are metalloprotease inhibitors, were less

effective than the other inhibitors. Metallopeptidase is mainly responsible for digesting

the proteins in cell surface (Baker, 2002), and the results might suggest that

metallopeptidases are less important in digesting engulfed bacteria compared with

lysozyme, serine peptidase, and cysteine peptidase.

Different inhibitors can potentially affect other aspects of the in vitro

fermentation and microbes. In the first in vitro experiment, the goal was to screen

available inhibitors for further evaluation and thus no other analysis was performed. By

comparison of inhibitory effects and cost, imidazole, PMSF, and iodoacetamide were

selected for further evaluation in Chapter 4.

20
 Treatment 2 Concentration P value
SEM 3
Imi，mg/ml 0 13.5 27 54 Linear Quadratic Cubic
24h 28456 3493 2038 873 412 < 0.01 < 0.01 < 0.01
48h 27656 2038 1164 582 118 < 0.01 < 0.01 < 0.01
PMSF，mg/ml 0 0.2 0.4 0.8
24h 28456 6696 2620 1164 445 < 0.01 < 0.01 < 0.01
48h 27656 5822 2038 873 145 < 0.01 < 0.01 < 0.01
pefabloc®，mg/ml 0 0.5 1 2
24h 28456 3493 1747 1164 394 < 0.01 < 0.01 < 0.01
48h 27656 1456 873 873 168 < 0.01 < 0.01 < 0.01
Bet，mg/ml 0 0.01 0.05 0.1
21

24h 28456 11062 10771 6987 708 < 0.01 < 0.01 < 0.01
48h 27656 9607 9174 4949 539 < 0.01 < 0.01 < 0.01
Iodo，mg/ml 0 0.018 0.09 0.45
24h 28456 5822 2620 1456 504 < 0.01 < 0.01 < 0.01
48h 27656 6987 1747 1456 118 < 0.01 < 0.01 < 0.01
PDS，mg/ml 0 0.006 0.03 0.12
24h 28456 9024 6697 5531 385 < 0.01 < 0.01 < 0.01
48h 27656 11936 10189 4949 291 < 0.01 < 0.01 < 0.01
Table 3.1 E. caudatum counts in the presence of imidazole, PMSF, Pefabloc®, bestatin, iodoacetamide and PDS at 24h and 48h1
(Continued)
21
 Table 3.1: Continued

Data were analyzed using dose levels of 0, 13.5, 27, and 54 mg/ml for imidazole; 0, 0.2, 0.4 and 0.8 mg/ml for PMSF; 0, 0.5, 1, and 2

mg/ml for Pefabloc®; 0, 0.01, 0.05, and 0.1 mg/ml for bestatin; 0, 0.018, 0.09, and 0.45 mg/ml for iodoacetamide; 0, 0.006, 0.03 and

0.12 mg/ml for phosphoramidon disodium salt.

1
imidazole dose x time interaction (p = 0.33); PMSF dose x time interaction (p = 0.82); Pefabloc dose x time interaction (p = 0.06);

bestatin dose x time interaction (p = 0.80); iodo dose x time interaction was (p = 0.05); PDS dose x time interaction (p < 0 .01).

2
Imi, imidazole; PMSF, phenylmethylsulphonyl fluordie; pef, Pefabloc®; bet, bestatin; iodo, iodoacetamide; PDS, phosphoramidon

disodium salt.
22

3
SEM, the standard error of the mean

22
 Chapter 4: Effects of Selected Inhibitors of Lysozyme and Peptidases

on Entodinium caudatum, Fermentation and Prokaryotic Community in vitro

Abstract

Bacteria, protozoa, and fungi cohabitate the rumen and play important roles in

affecting NUE. Entodinium caudatum, by engulfing and digesting bacteria, can affect

ammonia concentration, VFA concentration and composition, DM and NDF

digestibility and pH. The experiment (chapter 3) helped identify three inhibitors of

(imidazole, phenylmethylsulphonyl fluoride, and iodoacetamide) that can dramatically

inhibit E. caudatum in vitro. The objective of this study was to further evaluate the

efficiency of the three selected inhibitors, both individually and in two- and three-way

combinations, for their ability to inhibit growth of E. caudatum and effects on feed

digestibility, VFA concentration, ammonia concentration, and pH in vitro experiment.

The effects of the inhibitors on the microbiota in the E. caudatum cultures were also

examined by amplicon sequencing of 16S rRNA genes. Imidazole (at 100 mM),

phenylmethylsulphonyl fluoride (at 3 mM), and iodoacetamide (at 0.5 mM) all

substantially inhibited E. caudatum at 24, 48, and 72h. The two- and three-

combinations of these inhibitors tended to inhibit E. caudatum to a greater extent than

the inhibitors individually. The inhibitors had little effect on culture pH. Ammonia

concentrations were dramatically decreased (p < 0.01) by all the inhibitors and their
23
combinations. Total VFA concentrations and molar proportion of individual VFA

remained not affected except isovalerate (p = 0.01). The inhibitors did not decrease DM

digestibility or NDF digestibility. However, the inhibitors affected the microbiota at

phylum and genus ranks, with the relative abundance of Bacteroidetes being decreased

while that of Firmicutes increased. The above effects were also noted for the genera

Prevotella and Streptococcus. Proteobacteia was increased by imidazole and its

combination with phenylmethylsulphonyl fluoride. The inhibitors decreased or tended

to decrease the relative abundance of Ruminobacter. These results indicate that the

inhibitors can effectively inhibit E. caudatum without adversely affecting feed

degradation or fermentation even though they changed the bacterial populations in the

cultures.

Introduction

Nitrogen sources from the feed of ruminants are primarily used for microbial

growth and protein synthesis (Storm and Orskov, 1983). Microbial protein synthesized

in the rumen contributes to 40-90% of the total protein that dietary cows need (Leng

and Nolan, 1984). Rumen ciliates, such as Entodinium caudatum, engulf bacteria,

digest them by digestive enzymes such as lysozymes and peptidase, utilize a portion of

the digested/degraded bacterial protein to synthesize their own protein, but most of the

digested bacterial protein is further degraded to ammonia and short-chain fatty acids

(SCFA). Only a small portion of the resultant ammonia can be used in microbial protein

synthesis, resulting in decreased nitrogen utilization efficiency (Bach et al., 2005). This

is largely attributable to the wasteful intra-ruminal nitrogen cycling caused by rumen

protozoa.

24
 Numerous studies have evaluated different approaches to controlling the

population of rumen protozoa. However, some natural inhibitors such as saponins and

essential oil tend to inhibit the growth of protozoa, but few of these inhibitors at any of

the tested doses affect concentrations of ammonia (Patra et al., 2012; Patra and Yu

2015), indicating that NUE may not be effectively improved. By searching for previous

literatures, one of the most predominant rumen ciliate species, Entodinium caudatum,

produces lysozymes and various peptidases including serine peptidases,

metalloprotease, and cysteine at a high level (Morgravi et al., 1996; Swan, 1972;

Sakanari et al., 1989; Rawlings and Barret, 1995). Because lysozyme is required for

lysing bacteria engulfed by rumen protozoa, and peptidases are responsible for

proteolysis, I hypothesized that inhibition of these two types of enzymes will provide a

new approach to control rumen protozoa.

In first in vitro experiment, it was found that inhibitors of these digestive

enzymes did inhibit E. caudatum in vitro substantially. The objective of this experiment

was to evaluate if the inhibitors of each enzyme have interaction in inhibiting E.

caudatum. It was hypothesized that the selected specific inhibitors (imidazole, PMSF,

iodoacetamide), both alone and in two- and three-way combinations, can efficiently

inhibit E. caudatum, decrease ammonia concentration without affecting volatile fatty

acids (VFA) concentration, dry matter digestibility (DMD), Neutral detergent fiber

digestibility (NDFD). I also hypothesized that inhibitors could also affect bacterial and

archaeal populations.

25
Materials and Methods

Medium, protozoal culture, feed, and in vitro incubation

The medium (SP medium), protozoal culture (E. caudatum), protozoal feed, and

in vitro incubation were essentially the same as described in Chapter 3, with the

following exceptions: The in vitro culture volume was increased to 10 ml each, with 2

ml E. caudatum inoculum 7.755 ml of SP medium.

Treatments with inhibitors

A total 8 treatments, including one control that received no inhibitor, were used

to systematically evaluate one inhibitor each for lysozyme (imidazole, at 100 mM),

serine peptidase (phenylmethylsulphonyl fluoride (PMSF) at 3 mM), and cysteine

peptidase (iodoacetamide at 0.5 mM), both individually and in two- and three-way

combinations (Table 4.1). Each treatment had six replicates. All the treatment cultures

were incubated for 72 h with daily feeding. At 0, 24, 48 and 72 h, 0.3 ml subsample

each was collected to enumerate E. caudatum cell counts under a microscope. At 72 h,

the end of incubation, pH was recorded.

Fermentation analysis

At the end of the 72h incubation, two milliliters of each culture were collected

into a microtube and centrifuged at 16,000 x g at 4oC for 10 minutes to collect the

supernatant for analysis of VFA using GC (gas chromatography) (Pantoja et al., 1994)

and of ammonia using a colorimetric method (Chaney and Marbach, 1962), while the

pellets were preserved at -80oC for DNA extraction. The supernatants for VFA analysis
26
were mixed with one volume of 33% metaphosphoric acid first, filtered, and stored in

GC vials at 4oC. Supernatant for ammonia analysis was stored at -20oC. The remaining

cultures (8 ml) were strained through 50 μm Ankom bags and dried at 55 oC for two

days to determine dry matter digestibility (DMD), After DMD analysis, the filter bags

were processed to determine the NDF (Van Soest et al., 1991).

DNA extraction and metagenomic analysis of microbiota

Genomic DNA was extracted from pelletes by using the method RBB+C (Yu

and Morrison, 2004). The DNA quality was checked by agarose gel and quantified by

Quant-iT™ dsDNA Assay Kit (Thermo Fisher Scientific). The DNA was further

diluted to 5 ng/ul and sent to the Moleular Cell Imaging Center at The Ohio State

University (Wooster, OH) to undergo Illumina Miseq sequencing following the 2 × 300

paired-end protocol. The V4-V5 hypervariable region (primers set 515F and 806R) of

the 16S rRNA gene was amplified.

The sequencing data were analyzed using Quantitative Insights Into Microbial

Ecology (Qiime) (Caporaso et al., 2010) as described previously (Kigerl et al., 2016).

Bases with a quality score lower than 25 were removed, then the paired reads were

joined by the fastq-join algorithm (Erik and Aronesty, 2011). Clean sequences shorter

than 252 bp were removed. Chimera sequences were identified using ChimeraSlayer

(Haas et al., 2011) and filtered out. Species-equivalent operational taxonomic units

(OTU) were clustered with Qiime using the pick-open-reference method against the

Silva_119_release reference sequences at 97 % similarity using the uclust algorithm

(Edgar, 2010). Minor OTUs, those each represented by less than 0.5% of total

sequences, were filtered out (Bokulich et al., 2010). The alpha diversity measurements
27
(including Chao1 estimate, Simpson and Shannon indices) were calculated by Qiime.

To compare the similarity between the microbial community between samples, the

distance matrix between the samples was calculated by the weighted-Unifrac algorithm

and PCoA plot was drawn based on the matrix thereby.

Statistical Analysis

The data on E. caudatum counts were analyzed using the Mixed model

procedure of SAS (SAS Institute Inc., Cary, NC v9.4): Yijk = μ + Ti + Hj + TiHj + eijk, μ

is the overall E. caudatum population mean, Ti is the fixed effect of the treatments (j =

1, 2, 3, 4, 5, 6, 7, 8), Hj is the fixed effect of jth hour (k = 1, 2, 3), and eijk is the residual

error. TiHj is the fixed effect of the interaction of the ith inhibitor type and the jth hour.

Data were also analyzed among fermentative characteristics (pH, ammonia

concentration, VFA concentration, DMD, and NDFD) and relative abundances of

individual microbial taxa for the 72 hour time point using SAS 9.4. Data of the alpha

diversity measurements (including Chao1 estimate, Simpson and Shannon indices)

after calculating by Qiime were also analyed using SAS 9.4. Significant differences of

E. caudatum counts, fermentative characteristics, relative abundances of microbial taxa

were analyzed using Duncan’s multiple range tests. Significance was declared at p ≤

0.05, and tendency at 0.05 < p < 0.10.

28
Results

Inhibition of E. caudatum and effects on fermentation characteristics

There was significant interaction for treatments (p < 0.01) by time. All the three

inhibitors, both alone and in combination, decreased (p < 0.01) E. caudatum counts

after 24 h, 48 h and 72 h in vitro incubation compared with those of control (Table 4.1).

Although there was significant effect of inhibitors on inhibiting E. caudatum growth (p

< 0.01) except PMSF+Iodo treatment (p = 0.17) from 24 h to 48 h in vitro fermentation,

E. caudatum seems to have no further decrease from 48 h to 72 h (p > 0.10). E.

caudatum counts with supplementation of individual inhibitor and the two-way

combinations did not show differences (p > 0.10) at any of the three incubation time

(24, 48, 72 h), while E. caudatum counts were decreased significantly at 48 h compared

with E. caudatum counts at 24 h (p < 0.05). The three-way combinations also showed

the greatest decrease in E. caudatum counts numerically. The two combinations of

PMSF with imidazole or iodoacetamide reduce E. caudatum significantly (p < 0.05)

compared with PMSF alone, indicating only PMSF show additive effects.

Compared with the control, imidazole and its two- and three-way combinations

increased (p < 0.01) the culture pH at the end of the in vitro incubation, but the increase

was only less than 0.4 pH unit (Table 4.2). All the inhibitors, both individually and in

combinations, decreased (p < 0.01) ammonia concentrations compared with the control

(Table 4.2). Imidazole decreased NH3-N the most, but PMSF the least decrease

numerically. The three-way combination of inhibitor decreased ammonia concentration

the greatest, by 86.1%. There were no inhibitory effects on DMD and NDFD (p > 0.05)

between the control and all the treatments (Table 4.3). No difference in DMD and

NDFD was seen either among the inhibitors, including their combinations.
29
 All treatments with the inhibitors, both individually and in combinations, had

similar concentrations of acetate (p = 0.31), propionate (p = 0.35), butyrate (p = 0.46),

valerate (p =0.18), and total volatile fatty acids (p = 0.54) as the control (Table 4.3).

There was only a trend of increased isovalerate concentration (p = 0.09) and acetate to

propionate ratio (p = 0.07) in the treatments compared to with the control. However,

PMSF, iodoacetamide, imi + PMSF, imi + iodo, and PMSF+iodo resulted in significant

increase (p = 0.01) in isobutyrate concentration.

Effect on the Microbiota in the in vitro E. caudatum cultures

The proportions of bacteria and archaea, as represented by % of sequence

assigned to each domain, were affected by several of the inhibitors (Table 4.4). Overall,

the proportion of total bacteria was significantly increased (p < 0.01), whereas that of

archaea was decreased (p = 0.02) by all the inhibitors except imidazole and imidazole

plus phenylmethylsulfonyl fluoride, both of which tended to decrease archaeal

proportion.

Principle coordinates analysis (PCoA) of the microbiota based on taxonomic

assignments of the sequences showed that imidazole, iodoacetamide,

phenylmethylsulphonyl fluoride, and the combination of all the three inhibitors resulted

in clusters that were separated from that of the control along PC1, which explains

greater than 64% of total variation (Figure 4.1). However, all the binary combinations

of the three inhibitors did not result in clear separation. The treatments were not clearly

separated along PC2 that explains 11.64% of the variation.

30
 Fourteen phyla of bacterial were detected by the 16S rRNA gene sequences

(Table 4.5). Only four phyla were each represented by greater than 1% of total

sequences. All the inhibitors except PMSF significantly decreased (p < 0.01) the

relative abundance of Bacteroidetes but increased (p < 0.01) that of Firmicutes (PMSF

and its combination with imidazole tended to increase Firmicutes) (Figure 4.2).

Imidazole, PMSF, and their combinations tended to increase (p = 0.01) the relative

abundance of Proteobacteria, whereas the other treatments tended to decrease (p = 0.01)

the relative abundance of this phylum. Synergistetes, the fourth largest phylum, was not

affected by imidazole but was decreased (p < 0.01) by the other inhibitors and their

combinations. The minor phyla were also affected (p < 0.01), but only to small extents.

Euryarchaeota was the only archaeal phylum detected in the cultures, and its relative

abundance was deceased (p = 0.02) significantly by PMSF, iodoacetamide, imidazole

plus iodoacetamide, and PMSF plus iodoacetamide, and the combination of all the three

inhibitors (Table 4.5).

In total 33 genera of bacteria and 2 genera of archaea were detected in the E.

caudatum cultures. Only eight genera of bacteria were each represented by greater than

1% of the total sequences in at least one of the treatment (Table 4.6). In the control

cultures, Prevotella was the most predominant genus, followed by Ruminobacter. All

the inhibitors and combinations, except PMSF alone, decreased (p < 0.01) the relative

abundance of Prevotella, but the opposite was true for the Streptococcus (p < 0.01).

The relative abundance of Ruminobacter was decreased (p = 0.04) significantly (by

imidazole plus iodoacetamide, PMSF plus iodoacetamide, and the three-way

combination) or tended to be decreased (by the three inhibitors individually or by

imidazole plus PMSF). Of other minor genera, most of the inhibitors and their

31
combinations changed their relative abundance (p < 0.01), which remained less than 1%

of the total sequence.

Discussion

The purpose of this study was to evaluate inhibitors of enzymes that are needed

by E. caudatum to lyse and digest the engulfed bacteria. We focused on their effect on

feed substrate digestion, fermentation, and microbiota because they are all important to

rumen functions.

Effect of the inhibitors on feed substrate digestion and fermentation characteristics

of the E. caudatum culture

Utilization of nutrients is dependent on the collective activities of rumen

bacteria, archaea, fungi, and protozoa. Defaunation improves rumen fermentation by

increasing nitrogen and energy utilization efficiency, but it can cause some adverse

effects such as low digestibility of organic matter (OM), milk lactose, and body weight

gain (Koenig et al., 2000; Newbold et al., 2015). The inhibitory effects of imidazole,

PMSF, and iodoacetamide and their combinations on E. caudatum were in agreement

with many other inhibitors described previously such as tea saponin (Hu et al., 2005)

and steroidal saponins from Yucca schidifera (Patra et al., 2012; Wang et al., 2000).

However, compared to the control, all the tested inhibitors decreased E. caudatum to a

much greater magnitude than the plant extracts, by 89.63% by imidazole (Imi, an

inhibitor of lysozyme), 78.28% by phenylmethylsulphonyl fluoride (PMSF, an inhibitor

of serine peptidase), 88.65% by iodoacetaminde (Iodo, an inhibitor of cysteine

32
peptidase), 89.63% by Imi+PMSF, 88.65% by Imi+Iodo, 86.89% by PMSF+Iodo, and

92.17% by Imi+PMSF+Iodo after 72 h incubation. These results showed that the

selected inhibitors are very effective in inhibiting or controlling the growth of E.

caudatum. Consistent with the results of the first in vitro experiment, the lysozyme

inhibitor is the most inhibitory. No significant differences between inhibitors alone and

combinations except PMSF suggest that any of the three types of enzymes is essential

for E. caudatum to obtain the nutrients required for its survival and any of these

enzymes can be a target to inhibit and control E. caudatum. These inhibitors may be

able to inhibit the growth of other types of rumen protozoa, but further experiments are

needed to test that possibility.

Among major rumen bacteria, Gram-positive bacteria such as Ruminococcus,

Butyrivibrio, and Pseudobutyrivibrio, and Gram-negative bacteria including

Fibrobacter and Prevotella are recognized as fibrolytic bacterial species, probably

playing a major role in fiber degradation in the rumen (Koike and Kobayashi, 2009).

Previous studies have also suggested that Ruminococcus albus and R. flavefaciens are

particularly important cellulolytic bacteria (Pope, 2011; Mitsumori and Sun, 2008). In

the present study, DMD and NDFD were not decreased (p > 0.05) by any of the

inhibitors, either individually or in combination, indicating that the inhibition of the

lysozyme and peptidases by the test inhibitors did not adverse inhibited fiber

degradation. Among the rumen protozoa, Epidinium, Polyplastron, and Eudiplodinium,

all of which belong to the family Ophyroscolecidae, are cellulolytic, while Entodinium

is only hemicellulolytic (Takenaka et al., 2004). Compared to Epidinium, Entodinium

is less capable of xylan degradation (Williams and Coleman, 1992). The lack of

depression of DMD and NDFD when E. caudatum was inhibited suggests that the

33
contribution of Entodinium to fiber digestion is minimal or can be compensated by other

microbes in the in vitro cultures. It should be noted that these inhibitors do not directly

inhibit fiber degradation by rumen protozoa because they only inhibit bacterial cell lysis

and protein hydrolysis. Given that defaunation can decrease fiber digestibility (Arakaki

et al., 1995), inhibition of rumen protozoa by these inhibitors may alleviate that adverse

effect on fiber digestion.

Rumen pH is a general indicator of feed quality and digestibility, saliva

secretion, VFA concentration, and ammonia concentration. Normal ruminal pH varies

between 5.5 to 7.3 and many rumen microbes may lose their vigor when pH is below

5.5 (Dehority, 2005). Ruminal pH is an important factor for sustaining a normal internal

environment of the rumen, and low pH usually causes subacute ruminal acidosis

(Ghorbani et al.,2000). Besides, catalytic activities of enzymes produced by rumen

microorganisms decrease rapidly when pH values fall below 6.0 reducing ENU. The

optimal ruminal pH for rumen E. caudatum growth is between 5.6 to 6.9, while the

optimal ruminal pH for cellulolytic bacteria to grow is from 6.1 to 6.9. Therefore, we

monitored the pH of the in vitro E. caudatum cultures to assess the effect of the

inhibitors on fermentation.

The average pH of the in vitro E. caudatum cultures ranged from 6.68 to 7.06

in this study, which lies in the normal pH range for rumen microbiota. The values were

relative higher (0.4 pH unit or less) compared with those reported in other in vitro

studies (Patra et al., 2012; Patra and Yu, 2015; Patra and Yu, 2012). Because VFA

concentration is the major factor determining the pH (Khafipour and Krause, 2009;

Krause and Oetzel, 2015), the reason for the relatively high pH is that the E. caudatum

cultures had lower VFA concentration (discussed below) than those reported in those

34
in vitro ruminal cultures. Among the treatments, imidazole and its two- and three-way

combinations resulted in higher pH. The result could be attributed to imidazole, but the

reason is not known.

Ammonia is an intermediate of ruminal nitrogen metabolism, and its

concentration is determined by the rate of ammonia genesis (mainly from amino acid

fermentation and urea hydrolysis) and rate of assimilation into microbial protein

(Busquet, et al., 2005). Therefore, a high ammonia concentration indicates low EMPS

and a low ammonia concentration accompanied with defaunation reflects improved

ENU (Newbold, et al., 2015). Consistent with the results of decreased abundance of E.

caudatum and defaunation studies (reviewed by Newbold et al., 2015), the ammonia

concentrations were much lower in the E. caudatum treated with all the inhibitors, both

individually and in combination. These results indicate that all the three inhibitors

decreased deamination from amino acids. Theoretically, both decreased protein

hydrolysis and subsequent amino acid fermentation can decrease ammonia

concentration in vitro. Because the inhibitors used in the present study inhibit lysis of

bacterial cells (the lysozyme inhibitor imidazole) or protein hydrolysis (the inhibitor of

serine peptidase PMSF and the inhibitor of cysteine peptidase iodoacetamide), the

observed low ammonia concentration in the E. caudatum cultures resulted from

decreased protein hydrolysis. The results suggest that inhibition of the lysozyme and

peptidases, at least cysteine peptidases and serine peptidases, can potentially improve

ENU in ruminants.

The concentrations of total VFA and individual VFAs were lower in the E.

caudatum cultures, irrespective the addition of the inhibitors, than those observed in in

vitro rumen fermentation cultures (Patra et al., 2012; Patra and Yu, 2015; Patra and Yu,

35
2012). This might be explained by the low density of microbes of and the limited

amount of feed added to the E. caudatum cultures. Except for the concentration of

isobutyrate, a BCVFA, the inhibitors, individually or in combinations, did not affect

the VFA concentrations. This is consistent with the lack of effect on the digestibility of

DM and NDF. The acetate: propionate ratio was high in all the E. caudatum cultures,

including the control culture. The acetate : propionate ratio was increased numerically

by imidazole, PMSF, and their combination. The combination of imidazole and

iodoacetamide also tended to increase the acetate: propionate ratio numerically. High

acetate : propionate ratio is generally associated with high fiber diet. The feed fed to

the E. caudatum cultures contained high fiber and low starch to slow down growth of

the bacteria. The high acetate : propionate observed in all the E. caudatum cultures was

probably attributed to the high fiber fed. The microbiota in the E. caudatum was

probably very different from that in the rumen or in vitro ruminal cultures, and such

microbiota difference could also be attributed to the high acetate : propionate ratio.

However, most of the inhibitors and their combinations increased the concentration of

isobutyric acid. Isobutyric acid can be produced in rumen by oxidative deamination and

decarboxylation of valine (Daniel and Eldon, 1986). It remains to be determined if the

increase of isobutyrate is derived from enhanced oxidative deamination and

decarboxylation of valine. Valerate is produced mainly from fermentation of

carbohydrates and from amino acids such as proline (Daniel and Eldon, 1986). The lack

of changes of valerate suggests that supplementation of the inhibitors might not alter

the metabolism of carbohydrate and proline that leads to valerate production. Previous

study has found that supplementation of isoacids could increase milk production,

microbial synthesis and enhance the growth of cellulolytic and hemicellulolytic

microorganisms in the rumen (Daniel and Eldon, 1986). The increase of isobutyrate, as
36
an isoacid, might facilitate protein synthesis and fiber digestion. Further experiments

are needed to analyze the effects of the tested inhibitors on microbial protein synthesis

and fiber digestion.

Effect of the inhibitors on the microbiota of the E. caudatum culture

Rumen protozoa were thought to have association with and prey on select

bacterial and archaeal populations based on results from studies using defaunated sheep

(Mosoni et al., 2011; Morgavi et al., 2006; Xia et al., 2014). I hypothesized that the

inhibitors could affect the bacterial and archaeal populations, directly or indirectly, of

the E. caudatum cultures. Therefore, I analyzed the microbiota of the E. caudatum

cultures. The inhibitors decreased the relative abundance of archaea, possibly due to

decreased hydrogen production when E. caudatum was inhibited. Alternatively, the

inhibitors could directly inhibit archaea, which can be tested using cultures of rumen

methanogens.

Based on the PCoA analysis of sequence data, all the inhibitors and their

combination, except PMSF and iodoacetamide, affected the overall microbiota.

Interestingly, the three inhibitors and their three-way combination resulted in clear

separation of the microbiota from the control, but the two-way combinations did not.

The large variations among the replicates of imidazole plus PMSF and imidazole pus

iodoacetamide are also difficult to explain.

Imidazole and the combinations containing imidazole increased the relative

abundance of Firmicutes, a bacterial phylum containing primarily Gram-positive

bacteria, which are more susceptible to lysozyme. One possible explanation is that
37
inhibition of the lysozyme protected members of Firmicutes, from digestion lysozyme

produced by E. caudatum or other members of the cultures. This speculation is

corroborated by the finding that consumption of lysozyme-rich milk decreased the

relative abundance of Firmicutes in the feces of piglets (Maga et al., 2012). However,

PMSF, an inhibitor of serine peptidase, also decreased the relative abundance of

Firmicutes. The relative abundance of Bacteroidetes, the major Gram-negative phylum

in the rumen and the E. caudatum cultures, showed the opposite trend as Firmicutes. It

is not known if this is because of the increase in relative abundance of Firmicutes. The

decrease of Bacteroidetes and the increase of Firmicutes, except in the presence of

PMSF, could increase the ratio of Firmicutes : Bacteroidetes. Jami et al. (2014)

demonstrated that Firmicutes : Bacteroidetes ratio was strongly positively correlated

with daily milk fat yield in an in-vivo study. Therefore, the results suggest that

supplementation of inhibitors except PMSF might have a potential to increase milk fat

yield in vivo. Proteobacteria was represented in greater proportion in the E. caudatum

cultures than in the rumen or in vitro rumen cultures. It is not known how this phylum

became more adapted to the conditions of the E. caudatum cultures than those of the

rumen. Proteobacteria was increased by imidazole and its combination with PMSF.

Some genera of Proteobacteria such as genus Desulfovribrio show a correlation with

milk-fat yield in vivo experiment (Jami et al., 2014). The increase of Proteobacteria

might increase milk-fat yield, and real-time quantitative PCR method is needed to

quantify some of the proteobacterial genera. Again, further in vivo experiment and real-

time quantitative PCR method are needed to investigate if and how these inhibitors

affect members of Proteobacteria. It should be cautioned that change in relative

abundance of one phylum can change that of other phyla. More quantitative methods,

38
such as real-time phylum-specific PCR, are needed to accurately assess the effects of

the inhibitors on the main bacterial phyla.

The distribution patterns of Prevotella, a Gram-negative genus of Bacteroidetes,

and of Streptococcus, a Gram-positive genus of Firmicutes, resemble those of

Bacteroidetes and Firmicutes, suggesting that the effects to those two phyla were

primarily owning to the effects on these two genera. The increase in relative abundance

of Streptococcus could be due to the decreased predation after E. caudatum was

inhibited by the inhibitors. However, the lack of effect of PMSF on this genus does not

support this premise. Previous studies have shown that Streptococcus bovis produces

lactic acid, which reduces rumen pH (Otto, 1984). However, pH in this study didn’t

show any decrease compared with the control. One probable explanation is that some

lactating-using bacteria might have increased as well and increased consumption of

lactic acid. Unfortunately, we did not analyze lactic acid concentration in the E.

caudatum cultures. Quantitative analysis of Streptococcus using qPCR and lactic acid

concentration is needed in future studies evaluating these inhibitors.

Prevotella plays significant roles in metabolism of proteins (Similarities, 2014).

Although uncharacterized Prevotella sometimes predominate in the rumen (Firkins and

Yu, 2015), the reduced relative abundance of Prevotella suggests that the

supplementation of inhibitors might have affected rumen protein metabolism.

Prevotella can degrade dietary protein and produce ammonia. It is not known if the

decreased relative abundance of Prevotella also contributed to the decreased ammonia

observed in the treatment cultures. Butyrivibrio species in rumen are major butyrate-

producing bacteria and are involved in butyrate production (Similarities, 2014). The

supplementation of inhibitors had mixed effects on the relative abundance of

39
Butyrivibrio, null effect from PMSF, iodoacetamide, and the three-way combination

but decrease by imidazole and all the two-way combinations. Because the butyrate

concentration was not significant changed, other butyrate-producing species were

probably present and they might have compensated the decrease in Butyrivibrio by

some of the inhibitors. Other minor genera, represented by less than 1% of the total

sequences, were also affected by the inhibitors, differently for different inhibitors.

Again, because changes in their relative abundance may not accurately reflect their

actual changes, specific real-time PCR is needed to determine the effects of these

inhibitors on these genera of bacteria that may be important to feed digestion and

fermentation.

40
 Treatments2 Control Imi3 PMSF Iodo Imi+PMSF Imi+Iodo PMSF+Iodo Imi+PMSF+Iodo SEM3 p value

24h 17030a 3057c 4585b 2402cd 2183cd 1965cd 1747d 1528d 334 <0.01

48h 9316a 1019bc 2038b 1019bc 1019bc 1019bc 1164bc 728c 370 <0.01

72h 7423a 728c 1601b 873bc 728bc 873bc 1019bc 582bc 250 <0.01

Table 4.1 Effects of imidazole, PMSF, iodoacetamide and their combination on E. caudatum counts at 24, 48 and 72h in vitro
incubation1
41

1
Count treatments x time interactions were insignificant (p < 0.01)

2
Imi, imidazole at 100 mM; PMSF, phenylmethylsulphonyl fluoride at 3 mM; Iodo, iodoacetamide at 0.5 mM; Imi+PMSF, combination
of 100 mM imidazole and 3 mM PMSF; Imi+Iodo, combination of 100 mM imidazole and 0.5 mM iodoacetamide; PMSF+Iodo,
combination of 3 mM PMSF and 0.5mM iodoacetamide; Imi+PMSF+Iodo, combination of 100 mM imidazole, 3 mM PMSF, and 0.5
mM iodoacetamide.

3
SEM, the standard error of the mean.

a-d
Means followed by different superscripts in a row differ significantly (p < 0.05).

41
 Treatments 1 Control Imi PMSF Iodo Imi+PMSF Imi+Iodo PMSF+Iodo Imi+PMSF+Iodo SEM3 p value

pH 2 6.71c 7.06a 6.73c 6.71c 7.02ab 6.98b 6.68c 6.99b 0.02 <0.01

NH3 concentration 2 74.82a 19.51d 46.34b 32.94c 29.43c 18.22d 21.13d 10.42e 3.16 <0.01

DM digestibility 2 43.01 43.92 44.83 43.62 44.59 44.22 45.13 45.43 2.13 0.99

NDF digestibility 2 32.88 33.77 33.94 34.12 33.41 34.82 34.29 34.65 2.78 0.99

Table 4.2 Effects of imidazole, PMSF, iodoacetamide and their combination on pH, NH3N concentration, DM digestibility, and neutral
detergent fiber (NDF) digestibility in the in vitro fermentation.
42

1
Imi, imidazole at 100 mM; PMSF, phenylmethylsulphonyl fluoride at 3 mM; Iodo, Iodoacetamide at 0.5 mM; Imi+PMSF, combination
of 100 mM imidazole and 3 mM PMSF; Imi+Iodo, combination of 100 mM imidazole and 0.5 mM iodoacetamide; PMSF+Iodo,
combination of 3 mM PMSF and 0.5 mM iodoacetamide; Imi+PMSF+Iodo, combination of 100 mM imidazole, 3 mM PMSF, and 3 mM
iodoacetamide.

2
pH, NH3 concentration (mg N/L), and digestibility (%) of DM and NDF were determined at the end of 72 h in vitro incubation.

3
SEM, the standard error of the mean.

a-c
Means followed by different superscripts in a row differ significantly (p < 0.05).
42
 Treatments1 Control Imi PMSF Iodo Imi+PMSF Imi+Iodo PMSF+Iodo Imi+PMSF+Iodo SEM3 p value
Acetate 16.22 16.95 15.99 17.07 14.73 15.45 18.43 16.81 1.30 0.31
Propionate 3.37 2.40 2.69 3.57 2.81 2.64 3.57 3.19 0.33 0.35
Isobutyrate 0.56c 0.91bc 2.21a 1.90a 1.31ab 1.59ab 1.72ab 0.90bc 0.30 <0.01
Butyrate 1.67 1.27 1.44 1.61 1.50 1.72 1.01 1.20 0.25 0.46
Isovalerate 0.40 0.88 0.49 1.27 1.80 1.05 0.81 1.40 0.21 0.09
Valerate 0.25 0.20 0.27 0.41 0.40 0.61 0.42 0.78 0.15 0.18
Total VFA 22.48 22.62 23.09 25.83 22.54 23.07 25.96 24.29 4.55 0.54
A:P 2 4.81 7.06 5.94 4.78 5.24 5.85 5.16 5.27 0.67 0.07
Table 4.3 Effect of imidazole, PMSF, iodoacetamide and their combinations on volatile fatty acids (VFA) concentrations (mM) in the in
43

vitro E. caudatum cultures.

1
Imi, imidazole at 100 mM; PMSF, phenylmethylsulphonyl fluoride at 3 mM; Iodo, Iodoacetamide at 0.5 mM; Imi+PMSF, combination
of 100 mM imidazole and 3 mM PMSF; Imi+Iodo, combination of 100 mM imidazole and 0.5 mM iodoacetamide; PMSF+Iodo,
combination of 3 mM PMSF and 0.5 mM iodoacetamide; Imi+PMSF+Iodo, combination of 100 mM imidazole, 3 mM PMSF, and 3 mM
iodoacetamide.

(Continued)

43
 (Table 4.3 Continued)

2
A : P，acetate to propionate ratio.

3
SEM, the standard error of the mean.

a-c
Means followed by different superscripts in a row differ significantly (p < 0.05).
44

44
 Domain Control Imi PMSF Iodo Imi+PMSF Imi+Iodo PMSF+Iodo IPI SEM 2 p value

Bacteria 98.28d 99.06abc 99.14abc 98.96bc 99.23abc 99.36ab 99.49a 98.83c 0.05 0.08

Archaea 0.79a 0.56ab 0.14b 0.13b 0.48ab 0.30b 0.26b 0.28b 0.05 0.02

Unassigned 0.95a 0.38b 0.71a 0.91a 0.29b 0.34b 0.25b 0.89a 0.03 <0.01

Table 4.4 Effect of imidazole, PMSF, iodoacetamide, and their combinations on relative abundance of bacteria and archaea in the in vitro
E. caudatum cultures when treated with the inhibitors for 72h 1.
45

1
Imi, imidazole at 100 mM; PMSF, phenylmethylsulphonyl fluoride at 3 mM; Iodo, Iodoacetamide at 0.5 mM; Imi+PMSF, combination
of 100 mM imidazole and 3 mM PMSF; Imi+Iodo, combination of 100 mM imidazole and 0.5 mM iodoacetamide; PMSF+Iodo,
combination of 3 mM PMSF and 0.5 mM iodoacetamide; IPI, combination of 100 mM imidazole, 3 mM PMSF, and 3 mM
iodoacetamide.

2
SEM, the standard error of the mean.

a-c
Means followed by different superscripts in a row differ significantly (p < 0.05).

45
 Phylum Control Imi PMSF Iodo Imi+PMSF Imi+Iodo PMSF+Iodo IPI SEM2 p value
Bacteroidetes 47.54a 5.73c 46.20a 28.16b 4.60c 7.11c 6.08c 23.03b 1.18 <0.01
abc ab abc c a c c bc
Proteobacteria 37.78 55.87 36.11 28.40 56.74 30.55 18.80 32.88 2.12 0.01
d bcd cd bc bcd ab a bc
Firmicutes 8.17 34.11 14.24 40.81 35.71 59.82 72.97 41.77 2.71 <0.01
Synergistetes 2.89a 2.73a 0.63b 0.86b 1.63b 1.25b 0.95b 0.67b 0.10 <0.01
a b b b b b b b
Verrucomicrobia 0.82 0.33 0.07 0.18 0.24 0.26 0.33 0.13 0.03 <0.01
b c a c c c c c
Fibrobacteres 0.47 0.00 0.81 0.20 0.00 0.00 0.00 0.06 0.02 <0.01
Spirochaetes 0.22b 0.10b 0.87a 0.17b 0.10b 0.13b 0.14b 0.16b 0.03 <0.01
a b ab ab b ab ab b
Chloroflexi 0.21 0.06 0.10 0.11 0.08 0.11 0.09 0.03 0.01 0.17
a bc c bc bc b bc bc
Lentisphaerae 0.14 0.03 0.02 0.05 0.04 0.08 0.07 0.06 0.01 <0.01
c b ab b b b b b
Planctomycetes 0.06 0.01 0.03 0.02 0.01 0.01 0.01 0.00 0.01 0.02
c a b ab ab a ab ab
Elusimicrobia 0.01 0.01 0.00 0.01 0.01 0.01 0.01 0.01 <0.01 0.11
a a a a a a a a
Actinobacteria 0.01 0.04 0.05 0.00 0.01 0.01 0.01 0.00 0.01 0.18
a a a a a a a a
Cyanobacteria 0.00 0.03 0.00 0.01 0.04 0.04 0.03 0.05 0.01 0.62
a ab b b ab b b b
Euryarchaeota 0.78 0.56 0.14 0.13 0.48 0.30 0.26 0.28 0.04 0.02
46

Table 4.5 Effect of imidazole, PMSF, iodoacetamide, and their combinations on relative abundance of bacterial phyla in the in vitro E.
caudatum cultures when treated with the inhibitors for 72h 1.

1
Imi, imidazole at 100 mM; PMSF, phenylmethylsulphonyl fluoride at 3 mM; Iodo, Iodoacetamide at 0.5 mM; Imi+PMSF, combination
of 100 mM imidazole and 3 mM PMSF; Imi+Iodo, combination of 100 mM imidazole and 0.5 mM iodoacetamide; PMSF+Iodo,
（Continued）
46
 (Table 4.5 Continued)

combination of 3 mM PMSF and 0.5 mM iodoacetamide; IPI, combination of 100 mM imidazole, 3 mM PMSF, and 3 mM
iodoacetamide.

2
SEM, the standard error of the mean.

a-d
Means followed by different superscripts in a row differ significantly (p < 0.05)
47

47
 Genus Control Imi PMSF Iodo Imi+PMSF Imi+Iodo PMSF+Iodo IPI SEM2 p value
Prevotella 34.66a 1.18c 38.50a 23.32b 2.06c 3.50c 2.06c 19.40b 1.09 <0.01
Streptococcus 0.09d 25.07c 0.69d 35.57bc 31.24bc 56.00ab 69.53a 37.93bc 2.71 <0.01
Butyrivibrio 0.16a 0.05b 0.30a 0.22a 0.05b 0.08b 0.06b 0.20a 0.02 <0.01
Ruminococcus 0.64a 0.33a 3.06a 0.62b 0.77b 0.76b 0.38b 0.36b 0.18 0.05
gL7AE11 0.00b 1.96a 0.00b 0.00b 0.00b 0.01b 0.00b 0.00b 0.07 <0.01
p-75-a5 0.02b 2.17a 0.00b 0.02b 0.03b 0.03b 0.07b 0.00 0.09 <0.01
Treponema 0.11b 0.05b 0.84a 0.17b 0.05b 0.06b 0.09b 0.11b 0.03 <0.01
Pyramidobacter 1.31a 0.32b 0.15b 0.25b 0.22b 0.28b 0.39b 0.18b 0.04 <0.01
Fibrobacter 0.47c 0.00c 0.81a 0.20c 0.00c 0.00c 0.00c 0.06c 0.02 <0.01
Ruminobacter 21.44ab 8.94abcd 7.71bcd 19.23abc 11.06abcd 5.10cd 3.00d 23.57a 1.35 0.04
Succinivibrio 0.18b 3.38a 0.31b 0.14b 3.02ab 0.72ab 0.06b 2.01ab 0.27 0.05
Table 4.6 Effect of imidazole, PMSF, iodoacetamide, and their combinations on relative abundance of bacterial genera in the in vitro E.
48

caudatum cultures when treated with the inhibitors for 72h1.

1
Imi, imidazole at 100 mM; PMSF, phenylmethylsulphonyl fluoride at 3 mM; Iodo, Iodoacetamide at 0.5 mM; Imi+PMSF, combination
of 100 mM imidazole and 3 mM PMSF; Imi+Iodo, combination of 100 mM imidazole and 0.5 mM iodoacetamide; PMSF+Iodo,

(Continued)

48
 (Table 4.6 Continued)

combination of 3 mM PMSF and 0.5 mM iodoacetamide; IPI, combination of 100 mM imidazole, 3 mM PMSF, and 3 mM
iodoacetamide.

2
SEM, the standard error of the mean.

a-d
Means followed by different superscripts in a row differ significantly (p < 0.05).
49

49
 Score plot

0.4

0.3

0.2
PC2 (11.638%)

0.1

-0.1

-0.2

-0.3
-0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5

PC1 (64.724%)

Figure 4.1 Principal coordinate analysis of bacterial communities of the in vitro

E. caudatum cultures after treatment with the inhibitors for 72 h. C, control; Imi,
imidazole at 100 mM, PMSF, phenylmethylsulphonyl fluoride at 3 mM; Iodo,
iodoacetamide at 0.5 mM; Imi+PMSF, combination of 100 mM imidazole and 3
mM PMSF; Imi+Iodo, combination of 100 mM imidazole and 0.5 mM
iodoacetamide; PMSF+Iodo, combination of 3 mM PMSF and 0.5 mM
iodoacetamide; Imi+PMSF+Iodo, combination of 100 mM imidazole, 3 mM
PMSF, and 3 mM iodoacetamide.

50
 100
90
Relative abundance (% 80
70
60
50
40
30
20
10
0

Treatment

Figure 4.2 Relative abundance (represented by % of total sequences) of the major

bacterial phyla identified in the in vitro E. caudatum cultures.

Imi, imidazole at 100 mM; PMSF, phenylmethylsulphonyl fluoride at 3 mM; Iodo,

Iodoacetamide at 0.5 mM; Imi+PMSF, combination of 100 mM imidazole and 3 mM

PMSF; Imi+Iodo, combination of 100 mM imidazole and 0.5 mM iodoacetamide;

PMSF+Iodo, combination of 3 mM PMSF and 0.5 mM iodoacetamide; IPI,

combination of 100 mM imidazole, 3 mM PMSF, and 3 mM iodoacetamide.

51
 Chapter 5: Conclusion

All the inhibitors of lysozyme and peptidases tested substantially inhibited E

caudatum, the most dominant species of rumen protozoa. Evidently, E. caudatum

depends on these digestive enzymes to digest the engulfed bacteria for essential

nutrients. The concurrent decrease in ammonia concentration suggests that these

inhibitors decreased degradation of proteins, most likely microbial protein, and or

ammonia genesis. Because they did not adversely affect the digestibility of DM and

NDF or fermentation characteristics, these inhibitors may hold potential to be used to

control rumen protozoa and improve nitrogen utilization efficiency. However, these

inhibitors affected the microbiota by decreasing the phylum Bacteroidetes and its genus

Prevotella and increasing the phylum Firmicutes including its genus Streptococcus.

Further research using quantitative analysis such as real-time PCR is needed to

determine the effect on individual genera and phyla of bacteria. The E. caudatum

cultures used in this study appeared to have very different microbiota than that of the

rumen. Studies using in vitro rumen cultures inoculated from fresh rumen fluid should

also be conducted to assess the effect of these inhibitors on the rumen microbes

typically present in the rumen, including other protozoa. Of course, in vivo experiments

are needed to further verify these inhibitors, but toxicity information needs to be

obtained first, either from the literature or from animal experiments. Furthermore, the

52
E. caudatum culture used in this study had not been exposed to the inhibitors before.

Thus, research using long-term exposure to these inhibitors is also needed to examine

if E. caudatum can become adapted to the inhibitors.

53
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