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Contribution of ciliate protozoa to the rumen fermentation and nutrition of the ruminant

Ankrah, Peter, Ph.D.

The Ohio State University, 1989

UMI 300 N. Zceb Rd. Ann Arbor, MI 48106

CONTRIBUTION OF CILIATE PROTOZOA TO THE RUMEN FERMENTATION AND

NUTRITION OF THE RUMINANT

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

Peter Ankrah, B.Sc., M.Sc.

««**«*«

The Ohio State University

1989

Dissertation Committee: Approved by

S. C. Loerch

B. A. Dehority

W. L. Shockey

J. L. Firkins Advisor Department of Animal Science i

Dedicated to my father, the late J. 0. Ankrah and my two aons,

Anthony and Peter

11 ACKNOWLEDGMENTS

I would like to express my sincere appreciation to my advisors,

Drs. Steven C. Loerch and B. A. Dehority, for their encouragement, guidance, patience, support and great interest throughout the course of my studies and during preparation of thi3 thesis. I would also like to thank Dr. Jack H. Cline for the part he played in getting me over to the U.S.A. for the Ph.D. program and for his relentless ef fort in getting my family to join me.

I thank Drs. W. L. Shockey and J. L. Firkins for serving on my committee and for their useful suggestions and contributions to this thesis.

Thanks is extended to Bev Fisher for her expertise in the typing of thi3 thesis.

My appreciation is extended to Fay for her assistance in caring for the experimental animals. The laboratory assistance by Peggy,

Pat, Diane and Tony is greatly appreciated. Thanks also go to my fellow graduate students in Animal and Dairy Science departments for formal and informal discussions and for their encouragement.

My deepest appreciation is extended to my parents, Jonathan 0.

Ankrah and Grace K. Simpson for their love, care, support and en couragement throughout my education. Dad, I wish you had lived to see your dream come true. You saw in me what I did not think possible.

H i Finally, I am extremely grateful to my wife Juliana and two sons, Anthony and Peter for their love, constant support, patience, understanding and enduring the rough times with me.

iv VITA

April 22, 1954 . . . Born - Acora, Ghana.

1978 ...... B.Sc. (Hons.), Agriculture, Animal Science, University of Ghana, Ghana.

1979 -.. 1980...... Teaching Assistant, Department of Animal Sci ence, University of Ghana.

1983 ..... • ...... M.Sc., Agriculture, Animal Science, Universi ty of Sydney, Australia.

1983 - 1984...... Agricultural Projects Officer, Ghana Invest ment Center, Ghana.

1984 - 1985...... Graduate Teaching Assistant, Department of Animal Science, The Ohio State University, Columbus, Ohio.

1986 - 1988...... Graduate Research Associate, Department of Animal Science, The Ohio State University, Wooster, Ohio.

Publications

Williams, G. E. S. and P. Ankrah. 1979. Evaluation of heat-treated full-fat soybean seeds as protein source for broiler chicks. Proceedings of the Tenth Animal Science Symposium, Ghana Animal Science Association 10:49-51.

Ankrah, P., S. C. Loerch and B. A. Dehority. 1987. Occurrence of 2- aminoethylphosphonic acid in feeds, rumen bacteria and duodenal digesta from defaunated sheep. Proceedings of the Nineteenth Biennial Conference on Rumen Function, Chicago, IL., Nov. 17-19, 19i38. (Abstr.).

Ankrah, P., S. C. Loerch and B. A. Dehority. 1988. Occurrence of 2- aminoethylphosphonic acid in feeds, rumen bacteria and duodenal digesta from defaunated sheep. J. Anim. Sci. (In press)..

v Ankrah, P., S. C. Loerch and B. A. Dehority. 1988. Effects of di etary protein source and defaunation on lamb performance. J. Anim. Sci. 66(Suppl. l):497-498.

Ankrah, P., S. C. Loerch, B. A. Dehority and K. A. Kampraan. 1988. Effects of defaunation on lamb performance and ruminal nutrient digestion in steers, tin preparation for submission to J. Anim. Sci.).

FIELDS OF STUDY

Major Field: Animal Science - Ruminant Nutrition

vi TABLE OF CONTENTS

ACKNOWLEDGEMENTS ...... ill

VITA ...... v

LIST OF TABLES...... x

LIST OF FIGURES...... xii

CHAPTER PAGE

I- 1

INTRODUCTION...... 1

LITERATURE REVIEW ...... 3

Rumen Microbial Markers ...... 3 Alkyl-phosphonates and their Derivatives. . . . 4 Physical and Chemical Properties of AEP .... 5 Synthesis and Metabolism of Alkyl-phosphonates. 6 Distribution of AEP in Biological Material. . . 9 Laboratory Methods for Analyzing AEP..... 10 Defaunation Methods ...... 12 Effects of Defaunation on: Animal Performance ...... 15 Nutrient Digestion ...... 17 Rumen and Blood Metabolites...... 19 Rumen Bacterial Population...... 22 Escape of Protozoal Protein from the Rumen. . . 25

LITERATURE CITED...... 31

II. OCCURRENCE OF 2-AMIN0ETHYLPH0SPH0NIC ACID IN FEEDS, RUMINAL BACTERIA AND DUODENAL DIGESTA FROM DEFAUNATED SHEEP...... 44

INTRODUCTION...... 44

EXPERIMENTAL PROCEDURE...... 45

Apparatus...... 45 HPLC Procedures ...... 45 Reagents...... 46

vii Linearity, Precision and 2-Aminoethylphosphonic Acid Recovery...... 47 Acid Hydrolysis Procedures...... 48 Organic Phosphorus Analysis ...... 48 Isolation of Mixed Ruminal Bacteria and Protozoa...... 50 Pure Cultures of RumenBacteria ...... 51 In Vitro AEP Fermentation...... 51

RESULTS AND DISCUSSION...... 53

LITERATURE CITED...... 68

III. EFFECTS OF DIETARY PROTEIN SOURCE AND DEFAUNATION ON LAMB PERFORMANCE...... 72

INTRODUCTION...... 72

MATERIALS AND METHODS ...... 74

Experimental Animals andDiets ...... 74 Defaunation and Refaunation Procedures...... 76 Microbial Assays...... 78 Experimental Design ...... 78

RESULTS AND DISCUSSION...... 79

LITERATURE CITED...... 89

IV. DERIVATION OF A SELECTIVE CONTAINER TO STUDY THE PHENOMENA OF PROTOZOAL SEQUESTRATION, MIGRATION AND LYSIS IN THE RUMEN...... 93

INTRODUCTION...... 93

MATERIALS AND METHODS ...... 94

Study A: Evaluation of the Selective Container Design...... 97 Study B: Investigation of the Phenomena of Protozoa Sequestration, Migration and Lysis in the Rumen...... 100 Experiment 1...... 100 Experiments 2 - 6 ...... 101 Experimental Design ...... 101

vlli RESULTS AND DISCUSSION...... 101

Experiment 1...... 101 Experiment 2...... 105 Experiment 3...... 108 Experiment 4...... 112 Experiment 5...... 113 Experiment 6...... 119

LITERATURE CITED...... 124

V. SUMMARY...... 127

APPENDICES...... 129

A. Data relative to Chapter.4 ...... 130 B. Data relative to Chapter 4...... 131 C. Data relative to Chapter 4...... 132

BIBLIOGRAPHY...... 134

ix LIST OF TABLES

Table Page

1 Recovery of AEP added to ruminal fluid ...... 56

2 Extent of in vitro fermentation of AEP by mixed ruminal bacteria...... 57

3 Concentration of AEP in pure strains of ruminal bacteria and in mixed bacteria and protozoa isolated from sheep . 61

4 Concentration of AEP in feedstuffs and in duodenal digesta and ruminal fluid from faunated and defaunated sheep...... 62

5 Concentration of AEP in feed and mixed ruminal bacterial hydrolysates as determined by HPLC and organic phosphorus (P) methods ...... 65

6 Composition of diets for lamb performance trial...... 75

7 Effects of dietary protein level on performance and rumen microbial concentrations of refaunated lambs . . . 80

8 Main effects of defaunation on lamb performance and rumen microbial concentrations ...... 82

9 Main effects of protein source on lamb performance and rumen microbial concentrations ...... 84

10 Effects of dietary protein source and defaunation on lamb performance and rumen microbial concentrations. . . 85

11 Summary of procedural variable for experiments 2, 3, 4, 5 and 6 ...... 102

12 pH and protozoal concentrations in ruminal contents at 0 and 24 h postfeeding and in containers after 24 h incubation in the rumen (Exp. 1 ) ...... 103

13 pH and protozoal concentrations in ruminal contents and containers at 0, .67 and 4 h postfeeding (Exp. 2). . . . 106

14 pH and protozoal concentrations in ruminal contents and containers at 6, 24 and 24.67 h postfeeding (Exp. 3) . . 109 x 15 pH and protozoal concentrations in ruminal contents and containers at 67 and 24 h postfeeding (Exp. 4) ...... 114

16 pH and protozoal concentrations in ruminal contents and containers at 0 and 6 h postfeeding (Exp. 5 ) ...... 117

17 pH and protozoal concentrations in ruminal contents and containers at 24 and 48 h postfeeding (Exp. 6) ...... 120

xi LIST OF FIGURES

Figure Page

1 2-Aminoethylphosphonic acid (AEP) concentration vs peak area as determined by the HPLC technique...... 55

2 Effect of incubation time on in vitro fermentation of 2-Aminoethylphosphonic acid (AEP) by mixed ruminal bacteria...... 53

3 Components used in the selective container ...... 95

4 Assembled containers ready for incubation in the rumen . 96

5 pH and protozoal concentrations in ruminal contents and containers at 0, .67 and 4 h postfeeding (Exp. 2). . . . 107

6 pH and protozoal concentrations in ruminal contents and containers at 6, 24 and 24.67 h postfeeding (Exp. 3) • • 110

7 pH and protozoal concentrations in ruminal contents and containers at 6 and 24 h postfeeding (Exp. 4)...... 115

8 pH and protozoal concentrations in ruminal contents and containers at 0 and 6 h postfeeding (Exp. 5 ) ...... 118

9 pH and protozoal concentrations in ruminal contents and containers at 24 and 48 h postfeeding (Exp. 6) ...... 121

xli CHAPTER I

INTRODUCTION

Rumen microorganisms provide ruminant animals with the ability to attack and convert plant structural carbohydrates, most of which contain B-configuration linkages not normally hydrolyzed by the con stitutive enzymes of the animal, into available energy and to ulti mately convert non-protein nitrogen into animal protein. Bacteria, ciliate protozoa and in some oases fungi, are the main microorganisms which inhabit the rumen. The protozoa biomass may approximately equal that of bacteria and comprise up to 80£ of the total volume occupied by microorganisms In the rumen under certain circumstances, such as feeding a restricted grain diet (Leng and Nolan, 1984). How ever, their role in the rumen fermentation and contribution to the nutrition and overall performance of the host animal is not well understood.

Research efforts aimed at establishing the function or role of ciliate protozoa in the animal host have increased in the past 30 years. Investigations of metabolic activities of the protozoa have shown that they are essentially similar to those of bacteria, I.e., the use of carbohydrates as energy sources and formation of acidic fermentation products and cell materials which are utilized by the

1 host animal (Kurihara et al,, 1978)* However*, results of the perfor mance by ruminant animals with and without ciliate protozoa have been contradictory. Bryant and Small (1960) and Eadie (1962) found no differences in performance between faunated and defaunated ruminants, while other researchers reported small or substantial differences in performance between the two groups (Abou Akkada and El Shazly, 1965;

Bird and Leng, 1978a; and Veira et al., 1983).

The research reported in this thesis, is directed toward an un derstanding of the role of ciliate protozoa in the ruminant. The literature review covers rumen microbial markers and in particular 2-

Aminoethylphosphonic acid (AEP) as a protozoal marker, followed by defaunation methods and effects of defaunation on animal performance, nutrient digestion, rumen and blood metabolites and population of bacteria in the rumen. Lastly, a brief account is given on the es cape of protozoal protein from the rumen to the small intestine. 3

LITERATDRE REVIEW

A) Rumen Microbial Markers

Estimation of protein requirements of ruminant animals requires some information on the amount of non-protein nitrogen needed for optimal microbial protein synthesis and on the amounts of bacterial, protozoal, dietary and endogenous proteins that become available in

the abomasum for subsequent digestion and absorption in the small

intestine.

Marker techniques have been used to investigate the source of

the nitrogenous compounds that enter the duodenum. By measuring the concentration in whole digesta of a marker which is specific to, and of known concentration in the component, the proportion of that com ponent in the digesta can be estimated. Markers that have been used

for estimating proportions of microbial protein entering the duodenum include nucleic acids, measured as total RNA and DNA (McAllan and

Smith, 1969; Coelho da Silva et al., 1972), DNA (Czerkawski, 1976),

RNA (Smith and McAllan, 1970), microbial incorporation of isotopic labels such as 35s (Beever et al., 1974), (Mathison and Milligan,

1971; Salter et al., 1979), 32p (Bucholtz and Bergen, 1973) and vari ous amino acids such as lysine, proline, methionine and leucine (Ely et al., 1967; Potter et al., 1971). The contribution of bacteria in the microbial pool entering the duodenum has been estimated using 4

2,6-diaminopimelie acid (Weller et al., 1958; Hogan and Weston, 1971;

Hutton et al., 1971; Amos and Evans, 1976). 2-Aminoethylphosphonic

acid (AEP), an alkyl-phosphonate, has been proposed as a marker for

measuring the protozoal component in the microbial protein (Abou

Akkada et al., 1968). However, various researchers have raised ques

tions concerning the U3e of AEP as a marker for rumen protozoa (Abou

Akkada et al., 1968; Czerkawski, 1974; Ling and Buttery, 1978; Dufva

et al., 1982; Cockburn and Williams, 1984). In their review on meth

ods for determining, and factors affecting, rumen microbial protein

synthesis, Stern and Hoover (1979) discussed the advantages and dis

advantages of microbial markers. The validity of any marker tech

nique to estimate microbial protein in the gastrointestinal digesta

is difficult to establish because there is no absolutely accurate

method for quantitating amounts of microbial protein in vivo

(Theurer, 1982).

Alkyl-phosphonatea and their Derivatives.

Alkyl-phosphonates are organophosphorus compounds containing

direct carbon-phosphorus (C-P) bonds that are resistant to acid hy

drolysis. 2-Aminoethylphosphonic acid, sometimes referred to as

Ciliatine, was the first natural C-P compound detected and wa3 iso

lated from rumen ciliate protozoa (Horiguchi and Kandatsu, 1959) and

sea anemones (Kittredge et al., 1962). Subsequently, it was reported

to be the major component of naturally occurring alkylphosphonates

(Horiguchi, 1971). Although several synthetic C-P compounds exist, 5 very few of them have been shown to occur in biological material, and

the naturally occurring forms are believed to be precursors, meta

bolic products or derivatives of AEP. Apart from AEP, the other

naturally occurring alkyl-phosphonates include: (a) 2-Amino-3-

phosphonopropionic acid (Phosphonoalanine) which was isolated from

the Zoanthid, Zoanthus sociatus and Tetrahymena pyriformis species,

and proposed as a likely precursor of AEP (Kittredge and Hughes,

1964), (b) Phosphonoacetaldehyde, which was isolated as an intermedi

ate metabolite of AEP breakdown by bacteria (La Nauze and Rosenberg,

1968; Cook et al., 1978; Wackett et al., 1987) and (c) N-methyl de

rivatives of AEP which consist of 2-Methylaminoethylphosphonic acid

(N-methyl AEP), 2-Diraethylaminoethylphosphonic acid (N, N-diraethyl

AEP) and 2-Triraethylaminoethylphosphonic acid (N, N, N-trimethyl AEP)

isolated from the sea anemone (Kittredge et al., 1967).

Physical and Chemical Properties of AEP.

2-Aminoethylphosphonic acid is a colorless, odorless, crystal

line solid with a melting point of 250-299°C (Horiguchi, 1971). It is soluble in water but insoluble in usual organic solvents.

The chemical formula for AEP is H2NCH2CH2-P-0 (0H)2- It is a ninhydrin-positive compound with a molecular weight of 125.07. The

C-P linkage in AEP is not attacked by phosphatase (Abou Akkada et al., 1968) and it is resistant to drastic hydrolytic procedures with strong mineral acids or alkalis. No liberation of inorganic phos phate was observed upon hydrolytic treatment of AEP with 6N HC1 at 110°C for 48 h (Kittredge et al., 1962). However, digestion of AEP with 60% HCIO4 or a mixture of concentrated HNO3 , HgSOi} and HClOn has been reported to break the C-P bond (Horiguchi, 1971; Kirkpatrick and

Bishop, 1971; Czerkawski, 1974). In solution, AEP produces no re sponse to reagents which react with phosphate (Horiguchi, 1971).

Synthesis and Metabolism of Alkyl-pho3phonate3 .

The biochemical role of Alkyl-phosphonates and the mechanisms of their synthesis by some living organisms is still not known. It is, however, known that organisms which do not synthesize AEP may readily incorporate it into their tissues and this may account for the detec tion of AEP in some Crustacea, mammals and other organisms (Kittredge and Roberts, 1969). 2-Amino-3-phosphonopropionic acid has been pro posed as a likely precursor of AEP because the biosynthetic route to many amines is through the decarboxylation of the corresponding amino acid (Kittredge and Roberts, 1969). The rapid incorporation of 32p_ labelled phosphate ion into AEP has been demonstrated with sea anemo nes, hydra, snails and slugs (Liang and Rosenberg, 1968).

The metabolism of AEP is important if AEP is to be used as a ciliate protozoal marker. It is critical that any AEP from lysed protozoa or other sources must be degraded completely in the rumen. Information on the degradability of AEP by rumen microorganisms is very limited. Cockburn (1982), as reported by Cockburn and Wil liams (1984), pointed out that AEP was not degraded by rumen microor ganisms in vitro. In a subsequent study, Cockburn and Williams

(1984), showed that pure AEP infused into the rumen of both faunated and defaunated steers disappeared more rapidly than did PEG infused at the same time, and AEP disappearance could not be correlated with an increase in AEP content of the microbial population. They there fore concluded that since the rate of disappearance of AEP was faster than would be accounted for by digesta flow alone, it was probably degraded, absorbed across or metabolized in the rumen wall. There is adequate evidence to show that pure AEP can be degraded by non- ruminal bacteria. Cook et al. (1978), isolated bacteria from sewage and soil which are capable of using different Alkylphosphonates as a phosphorus source. One of the isolates, identified as Pseudomonas putida, grew with AEP as its sole carbon, nitrogen and phosphorus source and released nearly all of the organic phosphorus as orthophosphate and 70$ of the AEP nitrogen as ammonium. Biological cleavage of the C-P bond has also been reported in several species of bacteria (Zeleznick et al., 1963; Harkness, 1966; Rosenberg and La

Nauze, 1967; Alam and Bishop, 1969; Wackett et al., 1987). The min eralization of phosphonates is therefore widespread among bacteria. 8

The mechanisms by which bacteria catabolize the C-P bond have been established. Catabolism of phosphonates involves a transamina tion reaction which produces the intermediate compound: phosphonoacetaldehyde (La Nauze and Rosenberg, 1967; Roberts et al.,

1968; Kittredge and Roberts, 1969)* This intermediate compound was first thought to undergo spontaneous decomposition to form acetaldehyde and inorganic phosphorus (De Koning, 1966; Roberts et al., 1968;

Kittredge and Roberts, 1969), but there is now available evidence indicating that this last step of breaking the C-P bond is carried out enzymatically (La Nauze and Rosenberg, 1968; Cook et al., 1978;

Wackett et al., 1987). This enzyme was subsequently identified in

Baccilus cereus by La Nauze et al. (1970); they suggested 2- phosphonoacetaldehyde pho3phonohydrolase as the systemic name and phosphonatase as the trivial name. Cook et al. (1978) also indicated that cell-free extracts of Pseudomonas putida contained an inducible enzyme system that required pyruvate and pyridoxal phosphate to re lease orthophosphate from AEP, and acetaldehyde was tentatively iden tified as a second product. La Nauze et al. (1970), summarized the mechanism by which AEP is broken down by bacteria in the following equation:

Pyruvate Alanine H2O

AEP ^ — .]> 2-phosphonoacetaldehyde*^^ Acetaldehyde + Pi

pyridoxal phosphate

(transaminase enzyme) (Phosphonatase enzyme) 9

Distribution of AEP in Biological Material

Since Abou Akkada et al. (1968) proposed the use of AEP as a rumen ciliate protozoal marker, subsequent reports of the validity of

AEP as a marker for ciliate protozoal-N have been contradictory.

While some researchers have reported the unique presence of AEP in protozoa and its absence in feeds and rumen bacteria (Abou Akkada et al., 1968; El Shazly et al., 1975; Dufva et al., 1982), others have detected substantial quantities in rumen bacteria (Czerkawski, 197*1;

Ling and Buttery, 1978; Cockburn and Williams, 1984) and in feed

(Ling and Buttery, 1978; Cockburn, 1982 as cited by Cockburn and Wil liams, 1984). Still others (Rahnema and Theurer, 1986) could not detect AEP in acid hydrolysates of rumen protozoa or abomasal digesta.

In addition to the conflicting reports on the validity of AEP as a protozoal-N marker, there are considerable variations in the con centrations of AEP reported within mixed rumen bacteria and mixed rumen ciliate protozoa. It has been shown that AEP concentration varies among species of protozoa (Abou Akkada et al., 1968; Whitelaw et al., 1983) and between small and large mixed rumen bacteria

(Czerkawski, 1974), and diet influences their proportions (Hungate,

1966). The concentrations of AEP in Isotricha species and Entodinium species obtained by Whitelaw et al. (1983), are in reverse order and considerably lower (26.4 and 37*8 mg/g N, respectively) than the val ues of 233*1 and 56.2 mg/g N, respectively, reported by Abou Akkada et al. (1968). AEP concentration in rumen bacteria has been reported to be in the range of 80-362 yg/g DM (Czerkawski, 1974), 30-680 yg/g

DM (Ling and Buttery, 1978) and 1,245-1443 yg/g DM (Cockburn and Wil liams, 1984). The reported concentrations for mixed rumen ciliate protozoa are 315 yg/g DM (Czerkawski, 1974) and 3,292-4,301 yg/g DM

(Cockburn and Williams, 1984).

The ubiquitous nature of AEP in organisms such as protozoa, Coe- lenterates, Molluscs, Echinoderms, Arthropods, Vertebrates, Schizomy- cophytes and Thallophytes was reported by Horiguchi (1971)* Detect able quantities of AEP have also been found in goat liver (Kandatsu and Horiguchi, 1965) bovine brain (Shimizu et al., 1965), myco bacteria (Sarraa et al., 1970) bile of bovine (Tamari and Kametaka,

1973), bovine milk (Tamari and Kandatsu, 1985) and human tissues

(Alhadeff and Daves, 1971).

Laboratory Methods for Analyzing AEP

Various methods for the determination of AEP in biological mate rial have been described. Using the inert property of the C-P bond to acid hydrolysis, AEP has been determined indirectly by measuring the difference between total phosphorus formed on combustion and in organic phosphorus formed on acid hydrolysis (Quin, 1965; Abou Akkada et al., 1968), or by separation of AEP from inorganic phosphorus af ter acid hydrolysis, using ion exchange chromatography and determin ing organic phosphorus after combusting the C-P bond of AEP (Czerkawski, 1971*; Cockburn and Williams, 1984). Apart from the pos sibility of phosphorus contamination of the apparatus and the tedious nature of these indirect techniques, they may not be sensitive enough to detect very small amounts of AEP in biological material. It is, therefore, imperative to use highly concentrated hydrolysates in or der to detect AEP when these indirect techniques are used. In addi tion, the latter procedure requires the collection of fractions from large chromatographic columns.

Success with the use of direct methods for measuring AEP con centrations has been variable. Cockburn and Williams (1982) and

Rahneraa and Theurer (1986) were not successful in assaying AEP in biological hydrolysates using the ion exchange column of an automatic amino acid analyzer with a ninhydrin detector. On the other hand,

Ling and Buttery (1978) were able to assay AEP using a similar tech nique. AEP has also been determined by sequential acetylation

(acetic anhydride-acetic acid) and methylation (diazomethane) of acid hydrolysates of tissues or tissue extracts to produce dimethylacetamidoethylphosphonate, which is then subjected to preparative thin-layer chromatography prior to analysis of the de rivative using gas-liquid chromatography (GC) (Alhadeff and Daves,

1971)• Apart from the numerous steps involved in this procedure, the authors pointed out that the sensitivity of the technique is drasti cally reduced if the preliminary thin-layer chromatographic separa 12 tion is omitted. For example, they were able to detect AEP in human brain using this technique, but it was not possible when the prelimi nary thin layer chromatographic separation was omitted. Karlsson

(1970) reported the separation of trimethylsilyl (TMS) derivative of

AEP from those of phosphate analogues by GC and identification of the

TMS derivative by Mass Spectrometry.

Quin (1965), proposed that in principle, 31p nuclear magnetic resonance spectroscopy might be useful for analyzing AEP because a chemical shift of 31p attached to one or more carbons is different

(negative) from that of 3Ip attached only to oxygen or nitrogen in phosphates. However, the high concentrations of AEP requirement of this method prevents its utilization in the screening of biological extracts.

B) Defaunation Methods

The process of completely removing the population of ciliate protozoa (fauna) from the rumen is known as defaunation. Several techniques have been used to defaunate ruminant animals with varying effectiveness. Some of these techniques are quite harsh on the ani mals, and their effects on the population of other rumen microor ganisms is not yet known.

Defaunated animals have been obtained by isolating the young at birth (Abou Akkada and El Shazly, 1964). However, this technique is time consuming and requires special facilities and feeding practices in order to avoid refaunation through contact with faunated animals. 13

Reduction of rumen pH by feeding milk, unrestricted high concentrate diet or addition of HC1 after starvation, has also been used to defaunate animals. Protozoa do not survive extended exposure to acidi ties outside the pH range of 5-5 to 8.0 (Hungate, 1966). A procedure in which the rumen contents are completely removed and the inside of

the rumen is washed repeatedly with water and physiological saline was developed by Eadie and Oxford (1957). A portion of the rumen

contents are heated to 50°C for 15 minutes in order to kill the pro

tozoa, and the contents are returned to the rumen. Although the ani mals were kept isolated, Entodinioraorphs reappeared in the rumen af

ter several weeks. Detergents have also been used to defaunate ani mals. Abou Akkada et al. (1968) introduced the use of dioctyl sodium

sulphosuccinate (DSS) as an effective defaunating agent for cattle, and Dawson and Kemp (1969) successfully used it to defaunate sheep.

In his studies on the defaunation of the ovine rumen using DSS, Orpin

(1977) reported that the minimum concentration of DSS necessary to kill 100£ of the ciliate protozoa in washed suspensions was about 30 times lower than that required to remove the ciliate protozoa com pletely from the rumen (33•4 pg/ml vs 1000-2000 yg/ml). He pointed out that DSS interacted with the particulate material in the rumen, as suggested earlier by Wright and Curtis (1976), and in this state it was not effective for killing the protozoa. Sufficient DSS must therefore be added to the rumen to saturate the particulate material completely, and to provide sufficient DSS free in the supernatant 14 fluid to kill the protozoa present. Decreasing the amount of par ticulate matter in the rumen by starving the animal allows higher concentration of DSS in the liquid phase. Nonyl phenyl ethoxylate

(Teric GN9) is another detergent which was reported by Australian researchers as an effective defaunating agent for both sheep and cat tle (Bird and Leng, 1978a; Bird et al., 1979).

The mechanisms by which detergents kill ciliate protozoa in the rumen is not completely understood. Orpin (1977) reported that en- todinioraorphid protozoa immediately contracted their cilia and ceased movement in the presence of toxic or subtoxic DSS concentrations.

Within 1-10 min the cilia became detached, were extruded from the cell, and the cell contents were leaked via the gullet or anus, re sulting in a partially 'ghosted' cell. No leakage occurred through the cuticle, indicating that DSS damage probably occurred at the mem branes lining the gullet and anus. However, with the Isotrichidae, large area3 of the cilia and underlying fibrous network were some times sloughed from the cell prior to leakage of cell contents and leakage did not occur at any fixed point of the cell, but rather at different points in different cells (Orpin, 1977).

Animals treated with defaunating agents invariably suffer from loss of appetite post-treatment and some may die from possible causes such as rurainitis and nephritis, resulting in electrolyte imbalance and dehydration as well as pulmonary congestion (Lovelock et al.,

1982). 15

Effect3 of Defaunation on: a) Animal Performance

Studies conducted with faunated and defaunated animals have shown that the absence of protozoa did not cause any ill effects to the animals (Bryant and Small, 1960; Eadie, 1962). However, Pounden and Hibbs (1950) observed that defaunated calve3 fed limited quanti ties of milk and alfalfa hay appeared to have rougher hair coats and their abdomens appeared to be deeper than the faunated calves on similar diets. This 'pot-bellied' condition was not apparent in other defaunated calves which received grain (Pounden and Hibbs,

1948) or had access to lawn pasture (Pounden and Hibbs, 1949). The number of animals used in these studies were too low to demonstrate the effect of the absence of protozoa on animal health and appearance.

The gross effects of protozoa on the nutrition of ruminants can be gauged by their effects on growth rate and feed intake (Veira,

1986). Studies on the effects of the presence or absence of rumen ciliate protozoa on growth rate have been contradictory. Early re searchers showed that faunated and defaunated animals grew at the same rate (Becker and Everett, 1930; Bryant and Small, I960; Eadie,

1962). Subsequent studies on the effect of protozoa on the growth of ruminants have shown that growth rate in faunated animals is either higher (Abou Akkada and El Shazly, 1964; Christiansen et al., 1965;

Borhami et al., 1967), lower (Bird and Leng, 1978a, 1984) or similar (Pounden and Hibbs, 1950; Eadie, 1962; Chalmers et al., 1968; Eadie and Gill, 1971; Williams and Dinusson, 1973) to that of defaunated animals. Leng and Nolan (1984) suggested that the contradictory re sponse of defaunation on performance of ruminants may be due to the diet, physiological state and age of the experimental animals used.

Bird and Leng (1978a) studied the effects of defaunation on growth of weaned mixed sex Hereford calves (average weight 180 kg) fed low- protein high-energy diets and they found no differences in growth rate between faunated and defaunated cattle fed the basal diet. Ad dition of a very low level of by-pass protein, however, resulted in a

43# increase in growth rate for the defaunated group. They suggested that the absence of protozoa may have enhanced both the availability of protein at the duodenum and provided a relatively constant protein to energy ratio. Similar studies with mixed sex Merino x Border

Leicester lambs, weighing an average of 16 kg live weight and fed a basal diet of oaten chaff and sugar with two levels of urea sup plementation, showed that defaunated lambs gained live weight at a higher rate (9%, P<0.06) and grew 37% more wool (P<0.01) than lambs with large populations of protozoa in their rumen (Bird and Leng,

1984).

There is substantial evidence to indicate that feed intake is similar between faunated and defaunated animals (Christiansen et al.,

1965; Chalmers et al., 1968; Bird and Leng, 1978a, 1978b, 1984; Bird 17 et al., 1979; Rowe et al., 1985) and therefore feed conversions for the two groups of animals depend on their rates of growth.

In their studies on the effects of rumen ciliate protozoa on digestion in the stomach of sheep, Veira et al. (1983) concluded that defaunation is likely to be beneficial only when diets high in energy and adequate in rumen fermentable nitrogen but low in by-pass pro

teins are fed to ruminants with large requirements for amino acids

(e.g., young rapidly growing animals), b) Hutrient Digestion

Ever since the discovery of ciliate protozoa in the rumen (Gruby and Delafond, 1843), their role in the digestion of feed in the rumi nant has been of great interest to scientists. There is adequate evidence and general agreement that rumen ciliate protozoa, like bac teria, possess active systems for hydrolyzing both complex and simple carbohydrates, proteins and lipids (Oxford, 1951; Hungate, 1955; Abou

Akkada and Howard, 1962; Bailey and Clarke, 1963; Prins and Van

Hoven, 1977). It is generally believed that Isotrichidae and smaller species of Entodinia are involved in the utilization of soluble sug ars and starch granules while the rest of the entodinioraorphs ingest plant particles and may therefore contribute to the degradation of plant cell walls. 18

In hia review on ruraen microbes and digestion of plant cell

walls, Demeyer (1981) reported that rumen ciliate protozoa are re

sponsible for about 30 to 40J6 of total rumen microbial fiber diges

tion. Amos and Akin (1978) reported that the contribution of ciliate

protozoa to in vitro digestion of orchard grass and bermuda grass was

20 and 1256, respectively. Kayouli et al. (1984) also observed that

defaunation significantly lowered the rate of straw digestion from

nylon bags incubated in the rumen of sheep.

Experiments with faunated and defaunated animals appear to indi

cate that there are no clear effects on total tract nutrient digest

ibilities. Punia et al. (1987) reported reductions in apparent di

gestibilities of organic matter (0M), acid detergent fiber (ADF) and

neutral detergent fiber (NDF) by animals with reduced fauna compared

to those with normal fauna. Klopfensteln et al. (1966) observed

greater dry matter (DM) digestion by faunated sheep for two of the

three rations used in their studies. Dietary differences can there

fore have an effect on the results obtained. Similar results were reported by Conrad et al. (1950), Yoder et al. (1966), Kurihara et al. (1978), Jouany and Senaud (1979) and Veira et al. (1983)- On the other hand, Luther et al. (1966) found no significant differences between faunated and defaunated lambs in apparent digestibilities of

DM, 0M, crude protein or gross energy of either high or low concen trate rations. However, total tract digestibilities do not give an indication of the site of digestion. Ushida et al. (1986) observed 19 that although defaunation reduced rumen OM digestion, the digestibil ity in the whole tract was not affected. They hypothesized that de faunation caused a shift in digestion from the rumen to the intes tines. Similar observations were made by Lindsay and Hogan (1972) and Knight et al, (1978). In contrast, Rowe et al. (1985) did not observe statistically significant differences in apparent digestion of OM or starch in the forestoraachs of sheep as a result of defauna tion but they observed a significant decrease in apparent digestion of OM in the whole digestive tract as a result of defaunation.

Eadie and Hobson (1962) reported that in the defaunated animal, bacterial numbers increase markedly and the functions of the protozoa appear to be taken over by the bacteria. This will be discussed in depth in the section dealing with effects of defaunation on rumen bacterial population, c) Rumen and Blood Metabolites

In studies that have compared the concentration of metabolites in the rumen of faunated and defaunated animals, the most consistent effect has been a decrease in rumen NH3 concentrations as a result of defaunation. Males and Purser (1970) reported values for rumen NH3 N levels of defaunated animals as a percentage of those in control fau nated animals obtained by several researchers: 47? by Abou Akkada and El Shazly (1964), 52% by Christiansen et al. (1965), 39-50? by

Klopfenstein et al. (1966) and 49-50? by Chalmers et al. (1968).

Abou Akkada and El Shazly (1965) reported higher concentrations of reducing sugars, NH3 and volatile fatty acid (VFA) production in the rumen fluid of faunated lambs and higher blood NH3, urea and non protein N in the unfaunated lambs. Males and Purser (1970) pointed out that NH3 concentrations in the rumen of defaunated animals are lower than in faunated animals as a result of greater bacterial con centrations, thus causing greater NH3 utilization i.e., the NH3 lev els are higher in faunated animals as a result of an inability to effectively utilize NH3 rather than as a result of excessive NH3 production. Another suggestion is that of Coleman (1975) who pointed out that the higher rumen NH3 concentrations observed in faunated animals may be due to protozoal digestion of rumen bacteria, result ing in the release of NH3.

Although high ruminal concentrations of VFA have often been as sociated with faunated animals (Abou Akkada, 1964; Abou Akkada and El

Shazly, 1964; Christiansen et al., 1965; Eadie and Gill, 1971; Cole man, 1980), there is a great deal of disagreement on the effect of defaunation on the relative proportions of individual VFA in the ru men. Abou Akkada and El Shazly (1964), Christiansen et al. (1965),

Luther et al. (1966), Williams and Dinusson (1973) and Kurihara et al. (1978) obtained a higher acetate/propionate ratio as a result of defaunation; however, Youssef and Allen (1968), Males and Purser

(1970), Leng (1976), and Demeyer and Van Nevel (1979) obtained an opposite effect. Klopfenstein et al. (1966), Luther et al. (1966),

Males and Purser (1970), Grummer et al. (1983) and Ushida et al. 21

(1986) observed a marked deerease in the level of butyrate due to defaunation. In contrast, Bird and Leng (1978a) and Rowe et al.

(1981) obtained significantly higher butyrate concentration in the rumen of defaunated animals compared to that of the faunated group.

Lindsay and Hogan (1972) and Rowe et al. (1985) observed no signifi cant changes in the molar proportions of individual VFA due to defau nation. It is possible that these differences in pattern of VFA con centration in response to defaunation may also be related to dietary differences and the types of bacteria present in the rumen after de faunation and not just the absence or presence of protozoa per se.

Conrad et al. (1958), Christiansen et al. (1965), Youssef and

Allen (1968), Males and Purser (1970) and Grummer et al. (1983) ob tained higher ruminal pH values in faunated animals compared to those in the defaunated group. Higher ruminal pH associated with faunated animals may be due to protozoal uptake of readily fermentable sugars and starch, thereby removing them from immediate fermentation by bac teria coupled with the reduced numbers of bacteria in the rumen of faunated animals (Veira et al., 1983).

In contrast to the finding by Eadie and Gill (1971) that no sig nificant difference exists in methane production between faunated and defaunated animals, Whitelaw et al. (1984) observed a 1*7 fold in crease in methane production in faunated animals compared to the de faunated group. 22

d) Rumen Bacterial Population

Imai and Ogimoto (1978) observed that bacteria are attached to

the external surface of protozoa. Based on microscopic techniques,

they estimated that there were about 10 to 20 bacteria per 200 um2 of

the surface of a protozoa cell. Some of the bacteria appeared

trapped among the cilia and grew around the body of the ciliates.

Rumen bacteria have also been observed in the digestive vacuoles

within the protozoal cell (Gutierrez and Hungate, 1957y Gutierrez,

1958; Wallis and Coleman, 1967; Stern et al., 1977).

The major source of nitrogenous compounds for the synthesis of

protozoal protein and nucleic acids has been reported to be bacteria which are engulfed and digested in the vesicles of the protozoal cy

toplasm (Coleman, 1964; Coleman and Hall, 1969; Onodera and Kandatsu,

1969). Gutierrez and Hungate (1957) and Clarke and Hungate (1966) pointed out that protozoa cannot be cultured successfully in the ab sence of viable bacteria. Coleman and Hall (1969) observed that En- todinium caudatum grown in vitro engulfed any particle small enough to pass down the esophagus and took up a mixture of bacteria from the medium in the proportion in which they were present, showing no pref erence or dislike for any bacteria. When jS. coli was used a3 a test bacterium, the maximum rate of uptake was 200 bacteria per protozoon per minute (Coleman, 1964). Subsequent work by Coleman and Sandford

(1979) suggested that the high rate of uptake of E. coli by cultured

Entodinlum caudatum is not typical and that values of 10-40 bacteria per protozoon per hour for the engulfment of rumen bacteria are more realistic. Coleman (1980) reported that electron microscopic studies showed that bacterial cells were digested by protozoa with a steady loss of cell contents until only the lipopolysaccharide membranes of the cell envelope remained. The undigested remnants of the bacteria were then extruded through the anus. In order to determine if the engulfment of an inert particle was injurious to protozoa, Coleman

(1975) added polystyrene latex beads (1*3 pm in diameter) to a cul ture of Entodinium caudatum which was then maintained normally with twice weekly dilution with fresh medium and the occasional addition of fresh beads. Immediately after the addition of the beads, the protozoa appeared to be completely filled with them, although they still lived and divided normally. He further pointed out that clus ters of beads could be seen attached to the protozoa in the region of the anus during the first few days, suggesting that the protozoa were able to rid themselves of the undigestible beads via the anus.

In view of the foregoing evidence on bacteria engulfment by pro tozoa, it is not surprising that there is general agreement in the literature that defaunation of animals results in a marked increase in bacterial population, due to the disappearance of predation and nutritional competition between protozoa and bacteria (Eadie and Hob son, 1962; Eadie and Gill, 1971; Kurihara et al., 1978; Kayouli et al., 1984; Newbold et al., 1986; Veira, 1986). However, Jouany et al. (1981) did not observe an increase in total bacteria numbers in 24

defaunated sheep. Bacterial concentration in faunated animals have

been shown to decrease by 38-56? (Eadie and Gill, 1971), 62? (Kuriha-

ra et al., 1978), 71? (Rowe et al., 1981, 1985) and 94? (Newbold et

al., 1986) when compared to those of defaunated animals fed the same

diet. In addition to reducing the total bacterial counts in the ru

men, protozoa have also been reported in some cases to change the

type of bacteria in the rumen. Rowe et al. (1985) observed similar

morphological types of bacteria in both faunated and defaunated ani

mals but small cocci, rod or vibrio-shaped organisms predominated in

defaunated animals while a higher proportion of large oval bacteria

occurred in the refaunated group. Kurihara et al. (1978) reported

that cellulolytic bacteria formed a major part of the flora of fau

nated sheep fed a pure diet, while amylolytic bacteria were the major

group in the defaunated sheep. They hypothesized that a combination of competition for energy (starch) and physical removal of the bac

teria with the starch granules could thus lead to the low numbers of amylolytic bacteria found in the faunated sheep. On the other hand, the majority of protozoa are non-cellulolytic and so would be expec ted neither to compete with cellulolytic bacteria for energy nor to remove them physically from cellulose fibers. Abou Akkada and El

Shazly (1964) reported a greater number of Oscillospira, large oval organisms and actively motile flagellates in the rumen of protozoa- free lambs than In faunated lambs. Eadie (1962) also found that a population of flagellate protozoa developed in the rumen when ciliate protozoa disappeared.

Coleman (1975), summarized the role of rumen ciliate protozoa by- reporting that protozoa engulf about 1% of the bacteria in the rumen/ rain in animals fed high concentrate diets and approximately half of the material is retained by the protozoa and the other half is re leased into the medium principally in the form of free amino acids.

These amino acids then provide a substrate for further bacterial growth and fermentation to VFA and ammonia, which results in loss of amino acids to the host animal.

C. Escape of Protozoal Protein from the Rumen

It was initially speculated that ciliate protozoa contributed significantly to the protein nutrition of the host because of their large mass in the rumen, their higher lysine content and greater di gestibility when compared to bacteria (McNaught et al., 1954). Early researchers assumed that all fractions in the rumen contents passed into the small intestine together and therefore the proportion of protozoal nitrogen in rumen contents was taken as a measure of its importance to the protein nutrition of the host animal (Ibrahim and

Ingalls, 1972). However, based on measurements of carbon and nitro gen recoveries and passage of polyethylene glycol (PEG) out of the rumen, Hungate et al. (1971) concluded that only half of rumen nitro gen escaped from the rumen with the marker. They reported a PEG turnover rate of 2.27 total rumen volumes per day, microbial turnover rate of 1.25 volumes per day and protozoal turnover of 0.69 volumes

per day. This indicated that protozoa left the rumen at a slower

rate than bacteria. Weller and Gray (1954) observed that the con

centrations of protozoa in the omasal digests of slaughtered sheep

were much lower than in the rumen; they speculated that this was due

to destruction of the organisms in the omasum. Bird et al. (1978b)

showed that the density of protozoa in omasal digests was 8-10? of

that in the rumen. Abe and Kumeno (1973) studied the effects of

fluid turnover on protozoal populations in vitro and they pointed out

that in the rumen, protozoal concentrations were maintained despite a much greater fluid turnover rate than in their in vitro systems.

They therefore suggested that this may be due to a lower rate of re moval of protozoa than of fluid. This suggestion is in agreement with that of Hungate et al. (1971)» and Weller and Pilgrim (1974).

The latter authors estimated that protozoa actually pass out of the rumen at 20? or less of the fluid rate based on direct protozoa counts in rumen fluid and rumen effluent obtained from continuously fed sheep. Their results indicated that protozoal nitrogen leaving the rumen amounted to 2? or less of the dietary nitrogen intake.

Potter and Dehority (1973) indicated that fluid turnover is slower in animals fed once or twice per day. This suggests even less passage of dietary nitrogen under once or twice a day feeding and therefore less protozoal nitrogen leaving the rumen. Punia et al. (1987) noted that protozoa constituted 2% of the microbial nitrogen flow in ani mals with reduced fauna and 24 to 27? in animals with normal fauna.

The observation that ciliate protozoa are flushed from the rumen at a greatly reduced rate than bacteria has led researchers to re

evaluate the role of protozoa in the protein nutrition of the rumi nant animal and to speculate on the mechanisms by which the protozoa are selectively retained in the rumen. It has been suggested that protozoa avoid escaping from the rumen by sequestering; however, ex perimental evidence to support this hypothesis is still limited.

Based on reports of generation times of protozoa in the rumen (6-48 h), Demeyer (1981) suggested that protozoa can only maintain them selves in the rumen when a •matrix' for sequestration is present.

Hungate (1966) speculated that some protozoa may maintain their num bers in the rumen by attaching to slower-moving components of diges ts. Orpin and Letcher (1978) demonstrated the attachment of Iso- trichidae to plant particles. Bauchop and Clarice (1976) also demon strated that Epldinium attach firmly to feed particles within the rumen; Clarke (1977) predicted that this attachment may apply to other Entodinioraorphs such as Ophryoscolex. The diurnal variations observed in the numbers of Isotrichidae in the rumen also strengthen the sequestration hypothesis. Isotrichidae begin to increase in num bers at the time of feeding, or just before or after feeding, reach ing the maximum at feeding or in many cases within 1 or 2 h after feeding and then decrease abruptly to the pre-feeding level (Purser, 1961). Purser (1961) attributed this behavior of Isotrichidae to sequestration among feed particles and/or papillae on the rumen wall.

The phenomenon of protozoal sequestration has been demonstrated in vitro in bags of feed suspended in a liquid medium (Nakamura and

Kurihara, 1978; Czerkawski and Breckenridge, 1979). Abe et al.

(1981) observed a thick protozoal mass that could be seen with the naked eye on the wall of the reticulum of steers slaughtered after overnight starvation. The mass occurred among cells surrounded by folds in the shape of a beehive and it was easily disengaged by vig orous vibration of the reticulum wall. These same authors pointed out that microscopic observation indicated that the mass consisted of

53$ Isotricha, 25? Dasytricha and 22? small Entodlniomorphs. The authors therefore concluded that Isotrichidae would ordinarily se quester on the wall of the reticulum, with subsequent migration into the rumen for a few hours after feeding. Chemical stimulus in re sponse to the diet has been suggested as a possible cause for the migration of Isotrichidae into the rumen fluid at the time of feed ing. However, Abe et al. (1983) pointed out that the act of ingest ing feed and the contractions of the reticulum during feed intake in cattle or during anticipation of feed in sheep and goats may also be involved.

Potter and Dehority (1973) reported that if an animal is starved for one day, protozoan numbers decrease by 80? and fluid turnover rate is negligible. However, infusion of low or high level of sol uble substrate (starch, glucose and casein) into the rumen maintained

68 and 9**56 of the original protozoal numbers, respectively. They further pointed out that, because the high level of soluble substrate was able to maintain the total rumen protozoal population when turn over was minimal, substrate could be the main factor controlling the protozoal population at low feed levels. Because turnover rate was almost zero after starvation of the animals, the disappearance of protozoa after starvation must have been the result of cell lysis and not passage from the rumen. Leng et al. (1986) also noted that the generation time of Isotrichidae indicated that they were extensively retained in the rumen and that most protozoa do not leave the rumen in digesta and therefore eventually die and are degraded in the ru men. Ffoulkes and Leng (1988) indicated that only 26? of the proto zoa in the rumen entered the lower digestive tract and the remaining

76% lysed. If this is true, then the wasteful recycling of protozoa protein in the rumen will be a loss of both protein and energy to the host animal.

Based on the foregoing, three main studies were conducted. The objectives of the first study were: 1) to develop a simplified, sen sitive and direct method for analyzing AEP using reversed-phase HPLC,

i 2) to determine the degradability of AEP by mixed ruminal bacteria in vitro and 3) to determine the occurrence of AEP in feeds, ruminal microorganisms and gastrointestinal contents. The second study was conducted to determine the effects on performance of faunated and defaunated lambs fed diets supplemented with protein sources of low

(fish meal) or high (soybean meal) rumen degradability. The third

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OCCURRENCE OF 2-AMINOETHYLPHOSPHONIC ACID IN FEEDS, RUMINAL BACTERIA

AND DUODENAL DIGESTA FROM DEFAUNATED SHEEP

INTRODUCTION

2-Aminoethylphosphonlc acid (AEP), sometimes referred to as

Ciliatine, is a naturally occuring organophosphorus compound contain ing a C-P bond that is resistant to acid hydrolysis. 2-

Aminoethylphosphonic acid was first isolated in ruminal ciliate pro tozoa by Horiguchi and Kandatsu (1959) and, subsequently, was pro posed as a ciliate protozoal-N marker by Abou Akkada et al. (1968).

However, reports on the validity of AEP as a marker for ciliate protozoal-N are contradictory. Although some researchers have reported the unique presence of AEP in ciliate protozoa and Its absence in feeds and ruminal bacteria (Abou Akkada et al., 1968; El Shazly et al., 1975; Dufva et al., 1982), others have found substantial quanti ties in ruminal bacteria (Czerkawski, 1974; Ling and Buttery, 1978;

Cockburn and Williams, 1984) and in feed (Ling and Buttery, 1978;

Cockburn, 1982). Still others (Rahnema and Theurer, 1986) could not detect AEP In acid hydrolysates of ruminal protozoa or abomasal digesta. The discrepancies In the literature on the occurrence of

AEP in nature may be the result of inadequate methods for analysis of this compound. The objectives of this study were: 1) to de velop a simplified, sensitive and direct method of analyzing for 44 45

AEP, using reversed-phase HPLC, 2) to determine the degradability of

AEP by mixed ruminal bacteria in vitro, and 3) to determine the oc

currence of AEP in feeds, ruminal microorganisms and gastrointestinal

contents.

EXPERIMENTAL PROCEDURE

Apparatus

A Beckman Gradient HPLC System^ (Model 334) consisting of two

solvent delivery pumps^ (Model 110B), a system organizer^ (Model

210A), a controller^ (Model 421A), a fluorescence detector^ (Model

157), an integrator (Model 427)^ and a reversed-phase analytical col umn (250 x 4.5 i.d.) packed with C-18 ultrasphere 5 pm spherical 80A pore was used.

HPLC Procedures

The procedure was a modification of the HPLC method developed by

Lindroth and Moper (1979) for the determination of subpicomole

amounts of amino acids. One hundred-fifty microliter3 of orthop-

thalaldehyde (OPA) derivatizing reagent was added to 90-pl of sample

(acid hydrolysates or AEP standards2) and mixed well. A 20-yl Injec

tion of the mixture wa3 made after a precise 1 min incubation.

The solvent gradient started at 45% methanol and 55% buffer; .1

Beckman Instruments, Inc., San Ramon, CA).

2Aldrich Chem. Co., Milwaukee, WI. 46 min after injection, the percentage methanol was increased by 3?/min up to 75? methanol within 10 min. The gradient remained at 75? methanol for 11.9 min, by which time the AEP and all the amino acids had eluted. At the end of the run, initial conditions were restored by decreasing the methanol to its initial value of 45? within 4 min

(i.e. 7.5?/min), and maintaining it for 9 min at this gradient to allow equilibration of the column before injecting the next sample.

Total run time including equilibration was 35 min. Flow rate was constant at 1 ml/min and column temperature was ambient.

Reagents. The solvents used were HPLC grade methanol and a com bination of phosphate and citrate buffer. The buffer solution was prepared by mixing 1 liter each of .1 H raonosodium phosphate and .1 M disodium phosphate plus two to four drops of pentachlorophenol solu tion (.125 g pentachlorophenol in 25 ml ethanol). Five hundred mil liliters of the phosphate buffer were then added to 2 liters of .1 M trisodium citrate and titrated to pH 6.2 with dilute sulphuric acid before degassing and adding 62.5 ml HPLC grade tetrahydrofuran.

The OPA derivatizing reagent was prepared by dissolving 200 mg

OPA in 2.5 ml HPLC grade methanol. Mercaptoethanol (200 pi) was add ed and the solution was made to 25 ml with 1 M potassium borate.

Brij-35 solution^ (.5 ml) was added and the solution was mixed gently

^Fisher Scientific Company, Fairlawn, NJ. 47 and stored in a dark brown bottle under refrigeration for at least 1 d before use. To keep the reagent reduced, 20 yl raercaptoethanol was added every 3 to 4 d. The reagent was usable for at least 1 mo if it was refrigerated (4°C).

Linearity, Precision and 2-Aminoethylphosphonic Acid Recovery.

Four identical gradient runs were performed for AEP standard solu tions ranging in concentration from .0002 pg/ml to 18.76 pg/ml in order to determine the linear range, precision and limit of detection of AEP. Fluorescence response was measured using peak area. Linear regression procedures (Steel and Torrie, I960) were used to determine the correlation coefficient for AEP peak area vs concentration of AEP injected. Ruminal contents from faunated ruminally fistulated Tar- ghee sheep fed a pelleted diet (45? corn cobs, 35% alfalfa, 13.1% oats, 5% molasses, .4% urea and 1.5% raineral-vitarain mix) were strained through two layers of cheese cloth and 50 ml aliquots of the resulting ruminal fluid were spiked with 0 , 400, 800, 2,000 or 4,000 pg AEP standard. Duplicate samples of ruminal fluid containing each

AEP level were lyophilized prior to acid hydrolysis and subsequent assay for AEP. The AEP recovery data were analyzed by AN0VA pro cedures (Steel and Torrie, 1960) for a completely randomized design with four levels of AEP addition and duplicate samples for each level tested. The statistical model contained effects due to level of AEP addition. 48

Acid Hydroly3i3 Procedures. Feedstuffs, mixed and pure strains

of ruminal bacteria, mixed ruminal protozoa, ruminal fluid and duode

nal digesta from faunated and defaunated sheep were each hydrolyzed

in sealed 100 ml serum vials with 6N HC1 in the presence of 20 pi of

mercaptoethanol for 24 h at 110°C under nitrogen. The hydrolysate

was filtered through Whatman No. 541 filter paper and the residue was

washed several times with .1N HC1 into the filtrate. The filtrate

was evaporated to dryness under reduced pressure at 50°C in a rotary

evaporator and made up to the desired volume with deionized water.

The sample was then filtered through a .45 pm filter prior to AEP

analysis on the HPLC.

Organic phosphorus analysis. The quantitation of AEP by HPLC

was validated by comparing AEP measurement by HPLC with a modifica

tion of the AEP organic phosphorus assay of Abou Akkada et al.

(1968). The AEP phosphorus was determined in feed and bacteria by measuring the difference between total phosphorus formed on combus

tion and inorganic phosphorus formed on acid hydrolysis. Attempts to

determine AEP phosphorus in a small amount of feed (1.5 g) were not

successful due to the relatively small amount of AEP phosphorus com

pared to inorganic phosphorus. Consequently, larger amounts of feed

were U3ed in order to concentrate the AEP. In addition, inorganic

phosphorus in the hydrolysate was removed using cation exchange res

in. Approximately 20 g of feed and 1.2 g mixed ruminal bacteria were

hydrolyzed separately in 6n HC1, filtered, partially dried and 49 brought to a 100 ml volume with deionized water. A strongly acidic cation exchange resin (Dowex 50W-xM 200 to 400 mesh, H+ form) was placed on ashless Whatman No. M2 filter paper in a Buchner funnel and washed with deionized water using suction until the filtrate became neutral, indicating that any excess acid had been removed. The hy drolysate wa3 poured onto the resin bed and washed repeatedly with deionized water to flush any material not adsorbed by the resin into the filtrate. This filtrate was found to contain only a minute trace of ninhydrin-positive material. The resin bed was then washed with a

50% NHijOH solution until all of the ninhydrin positive material had been eluted, as determined by spotting successive portions of the eluate on filter paper, drying and spraying with .25% (w/v) ninhydrin in 95% ethanol and redrying at 65°C for 2 to 5 rain. Ammonia was re moved from the eluate by boiling and the eluate was brought to a vol ume of 100 ml with deionized water.

Samples of the eluate were U3ed for the determination of AEP concentration by the HPLC technique and by organic phosphorus analy sis. A sample of the eluate was also re-hydrolyzed with 6n HC1 and analyzed for inorganic phosphorus. Total phosphorus in the eluate was determined after oxidizing' three replicates of 10 ml eluate with

5 ml HNO3 and 2.5 ml HCIQ4 for M h at an initial temperature of

100°C, rising gradually to a final temperature of 200°C. Phosphorus was then determined using raolybdovanadate reagent (A0AC, 1975). In organic phosphorus in the eluate and re-hydrolyzed eluate were also 50

determined using this phosphorus assay. Precaution was taken to

avoid exogenous phosphorus contamination by steeping all glassware

used for the phosphorus analysis in weak HC1 solution overnight prior

to thorough rinsing with deionized water.

Isolation of mixed ruminal bacteria and protozoa. Mixed bac

teria were obtained from ruminal contents of a defaunated sheep. The

sheep was defaunated by adding dioctyl sodium sulphosuccinate (3 g/d

for 3 consecutive days) into the rumen via the ruminal cannula.

Starting the second day after initial dosing with dioctyl sodium sul

phosuccinate, the sheep was infused every other day with a high sub

strate casein hydrolysate (20 g corn starch, 40 g sucrose and 20 g

casein hydrolysate in 300 ml of water) for seven days. The defau

nated sheep was housed in a separate room to prevent possible pro

tozoal inoculation from faunated animals. The defaunation procedure was successful in maintaining the sheep protozoa-free as determined

by microscopic examination. Approximately one month after defaunat-

ing the sheep, ruminal contents were collected and strained through

two layers of cheese cloth and the fluid was centrifuged at 150 x g

for 10 min. The supernatant was then centrifuged at 20,000 x g for

15 min. The resulting supernatant was discarded and the bacterial

pellet was washed three times by resuspension in water and recentri

fuging. The washed bacterial pellet was lyophilized, ground and re-

drled in preparation for acid hydrolysis. To obtain protozoal iso

lates, ruminal contents of faunated sheep were squeezed through two layers of cheesecloth to remove large feed particles. The resulting

fluid was then diluted 1:1 (v/v) with phosphate buffer (pH 6.5) and

incubated for 24 h at 39°C in separatory funnels equipped with Bunsen

valves. The feed-free, off-white mass at the bottom of the funnels

was collected, diluted with its own volume of water and centrifuged

at 150 x g for 15 min to isolate the protozoa. The resulting proto

zoal sediment was washed 3 times by resuspension in water and centri

fuging. Microscopic examination of a sample of the protozoal pellet

showed that it was free of feed particles. The isolated protozoa were lyophilized, ground and redried in preparation for acid hydrolysis.

Pure cultures of rumen bacteria. Cultures of Bacteroides suc-

cinogene3 B21a, Ruminococcus flavefaciens Bla, Bacteroides ruminlcola

H2b, Butyrivibrio fibrisolvens H1Qb, H4a and H17c, Lachnospira multi-

parus D15d and Streptococcus bovis ARD-5d (Dehority, 1963, 1966,

1969, 1975) were analyzed for AEP content. The bacterial cell3 were

treated as described above for mixed cultures.

In vitro AEP fermentation. Rate and extent of AEP degradation

by mixed ruminal bacteria were determined in vitro using inoculum

from the rumen of a defaunated sheep. The inoculum was prepared by

straining ruminal contents through one layer of cheesecloth to remove

large feed particles. Microscopic examination of a sample of the

Inoculum showed that It was free of ciliate protozoa. 52

Extent of AEP degradation by mixed ruminal bacteria was deter mined in vitro using 0 (blank), 240 or 720 pg AEP per tube. There were three replicates and two incubation times (0 and 24 h) for each level of AEP. Each tube contained .5 g substrate (the corn cob- alfalfa based sheep diet previously described), 20 ml McDougall's artificial saliva (McDougall, 1948), 10 ml ruminal fluid as inoculum and the appropriate level of AEP. The levels of AEP used for this study were chosen to approximate concentrations previously reported

(El Shazly et al., 1975). Immediately after addition of AEP and in oculum, the tubes were flushed with CO2 , stoppered with Bunsen valve stoppers and incubated at 39°C. Tubes were swirled every few hours.

Fermentation in the tubes was stopped by adding 1 ml of 5% HgCls per tube. Contents of each tube were then made to a volume of 50 ml with deionized water, mixed well by shaking and centrifuged at 20,000 x g for 10 rain to remove bacteria and feed particles. The resulting su pernatant was analyzed for AEP in duplicate, using the HPLC procedure a3 previously described. For each level of AEP used, AEP dis appearance was calculated as the difference between AEP recovered at

0 and 24 h of incubation. The AEP disappearance data were analyzed by ANOVA procedures (Steel and Torrie, 1960). The statistical model contained effects due to initial level of AEP.

The rate of AEP disappearance was determined in a similar manner with incubation times of 0, 3, 6, 9, 12 and 24 h. For each incuba tion time, duplicate tubes containing 0 (blank) or 1,130 pg AEP were 53 used. Subsequent to determining the extent of AEP fermentation, AEP concentration in ruminal fluid from faunated sheep as determined in our laboratory by the HPLC procedure was approximately 50 yg/ml.

Level of AEP addition to determine rate of AEP fermentation was cho sen to approximate this concentration. A total of 24 in vitro tubes were used. The rate of AEP disappearance was calculated as the slope of the regression of the natural logarithm of % residual AEP against time.

RESULTS AND DISCUSSION

The nature of the chemical reaction involved in the derivatiza- tion of AEP with OPA under alkaline conditions in the presence of a reducing agent and subsequent detection of the isoindole product by fluorimetry is similar to that reported for amino acids by Lindroth and Mopper (1979). Several solvent gradients were tried but the gra dient reported was the most successful in eluting AEP from a standard

t amino acid mixture (AA-S-18, Sigma Chemical Co.) and protein hydroly sates. Under the HPLC conditions reported, AEP eluted as a single symetrical peak at 9-4 min and by 22 min into the run, all the amino acids in the standard amino acid mixture and protein hydrolysate had eluted. The elution time of AEP is short compared to values reported for other direct methods of analyzing AEP. Using automatic amino acid analyzers, AEP elution times of 27 to 28 min (Ibrahim et al.,

1970; Mackie, 1973), 52 min (Ling and Buttery, 1978), 50 min

(Cockburn and Williams, 1982), 33 min (Dufva et al., 1982) and 93 min 54

(Rahneraa and Theurer, 1986) have been reported. The HPLC method is, therefore, more rapid than the direct methods presently available for analyzing AEP quantitatively.

The standard curve of AEP concentrations, as measured by peak areas, was linear from .0019 yg/ifll (15 nM) to 12.5070 yg/ml (100 yM).

The correlation coefficient in this linear range was .99 (Figure 1).

The HPLC technique was highly sensitive, with an AEP detection limit of .0019 yg/ml. The fluorimetric procedure is known to be about one hundred times more sensitive than colorimetric procedures such as ninhydrin (Roth, 1971). The mean coefficient of variation for AEP concentration in the linear range was 2.1 with a range of .6 to 5.8?.

The percentage recovery of AEP added to ruminal fluid of faunated sheep was 97.4 to 98.7 (Table 1). The spiked levels of AEP were cho sen based on preliminary analysis which showed that 50 ml of ruminal fluid of faunated sheep contained approximately 2,000 yg AEP. The

AEP recoveries did not differ (P>.05) due to amount of AEP added to the sample.

In order for AEP to be valid as a ruminal ciliate protozoal marker to determine protozoal-N contribution to total protein in the duodenum, any AEP from lysed protozoa or other sources must be de graded completely In the rumen. Information on the degradability of

AEP by ruminal microorganisms is very limited. Data regarding extent and rate of AEP fermented in vitro (Table 2 and Figure 2) demonstrate the ability of ruminal microorganisms to degrade AEP. When Initial IUE . 2-AMINOETHYLPHOSPHONICFIGURE 1. (AEP) ACID CONCENTRATION VSPEAK AREA AS DETERMINED PEAK AREA (1X10 100 30- 40- 50- 70- 80- 60- 90- BY THE HPLC TECHNIQUE AEP CONCENTRATION (1X10 2 /zG/ML) 2 CONCENTRATIONAEP (1X10 5 1000 750 Ln Ln TABLE 1. RECOVERY OF AEPa ADDED TO RUMINAL FLUID

Spiked levels AEP of AEP, pg recovery, SD

400 98.7 2.2

800 97.4 .5

2,000 97.9 .5

4,000 98.6 .7

a 2-aminoethylphosphonic acid.

b Each value is the mean of two replicates. 57

TABLE 2. EXTENT OF IN VITRO FERMENTATION OF AEPa BY MIXED

RUMINAL BACTERIA*1

AEP (yg/ml) at:

0 h 24 h AEP fermented, % SD

5.40 0 100 0.0

15.33 .64 95.8° 1.09

a 2-aminoethylphosphonic acid.

b Each value is the mean of three replicates.

c The percentage of AEP fermented was lower (P<.01) when Initial AEP concentration was 15.33 yg/ml compared to 5.4 yg/ml. 100

80-

60-

u. 40-

2 0 -

0 3 6 9 12 15 18 21 24 27

INCUBATION TIME, H

FIGURE 2. EFFECT OF INCUBATION TIME ON IN VITRO FERMENTATION OF 2-AMINOETHYLPHOSPHONIC ACID (AEP) BY MIXED RUMINAL BACTERIA

Ln oo (0 h) AEP concentration was 5.40 ug/ml, 100£ of the AEP was fermented by 24 h of incubation. During the 24 h incubation, 95.8J of the AEP was fermented when the initial AEP concentration was 15.33 Mg/ral, which was lower (P<.01) than for the incubations with an initial AEP concentration of 5.4 ng/ml. In contrast, Cockburn (1982), as re ported by Cockburn and Williams (1984), suggested that AEP was not degraded in vitro. In a subsequent study, Cockburn and Williams

(1984) showed that pure AEP infused into the rumen of both faunated and defaunated steers disappeared more rapidly than did polyethylene glycol infused at the same time, and its disappearance could not be correlated with an increase in AEP content of the microbial popula tion. They, therefore, concluded that because the rate of disappear ance was faster than would be accounted for by digesta flow alone,

AEP was probably degraded, absorbed or metabolized in the ruminal wall. Although Cockburn and Williams (1984) could not correlate AEP disappearance in the rumen with an increase in the AEP content of the microbial population, this does not necessarily mean that ruminal microorganisms are not capable of degrading AEP because the AEP may have been metabolized to other products rather than being directly

incorporated by the microbes.

The metabolism of AEP is not unique to ruminal bacteria. Cook et al. (1978) isolated from sewage and soil bacteria which were capa ble of using different alkylphosphonates as phosphorus sources. One of the isolates, identified as Pseudomonas putlda, grew with AEP as 60 its sole carbon, nitrogen and phosphorus source and released nearly all of the organic phosphorus as orthophosphate and 72% of the AEP nitrogen as ammonium. Biological cleavage of the C-P bond has also been reported in several species of bacteria (Zeleznick et al., 1963;

Harkness, 1966; Rosenberg and La Nauze, 1967; Alam and Bishop, 1969).

The rate of disappearance of AEP (Figure 2) from 3 to 12 h of incuba tion was relatively slower (2.63%/h) compared to that from 12 to 24 h

(10.07%/h). The overall rate of AEP disappearance from 3 to 24 h wa3

7.28%/h. The lag in AEP fermentation rate from 3 to 12 h could be due to an increase in bacterial numbers in the in vitro tubes after

12 h and(or) a 3hift in bacterial species which favored fermentation of AEP.

The ability of mixed rurainal bacteria to metabolize AEP was en couraging for its use as a protozoal-N marker. However, AEP was not only detected in ruminal ciliate protozoa but substantial quantities were also found in mixed and pure strains of ruminal bacteria, some feedstuffs, duodenal digesta and ruminal fluid from defaunated 3heep

(Tables 3 and 4). Concentration of AEP in mixed ruminal ciliate pro tozoa (98.6% Ophryoscolicidae and 1.4% Xsotrichidae) was approximate ly three time3 the concentration in mixed ruminal bacteria. Except for the cellulolytic bacteria B. succinogenes and R. flavefaciens, which- had lower concentrations of AEP, all other bacterial strains examined appeared to have similar amounts of AEP. The AEP concentra tion in the mixed ruminal bacteria was slightly higher than any of TABLE 3. CONCENTRATION OF AEPa IN PURE STRAINS OF

RUMINAL BACTERIA AND IN MIXED BACTERIA AND

PROTOZOA ISOLATED FROM SHEEP

AEP concentration

Sample yg/g DMb SD

Mixed ruminal protozoa0 304 438

Mixed ruminal bacteria 1,383 90

B. succlnogenes B21a 733 8

R. flavefaciens Bla 937 23

B. ruminicola H2b 1,156 59

B. fibrisolvens HlOb 1,120 5

B. fibrisolvens H4a 1,039 37

B. fibrisolvens H17c 1,166 4

L. multiparus D15d 1,158 8

S. bovis ARD-5d 1,012 4

a 2-aminoethylphosphonic acid.

b Each value is the mean of two replicates.

G 98.6? Ophryoscolecidae and 1.4£ Isotrichidae. 62

TABLE 4. CONCENTRATION OF AEPa IN FEEDSTUFFS AND IN DUODENAL DIGESTA AND RUMINAL FLUID FROM FAUNATED AND DE- FAUNATED SHEEP

Sample AEP concentration SD

----- yg/g D M ---

Corn 154 1.4

Oats 263 11.3

Corn cobs 30 .0

Wheat straw 25 1.4

Alfalfa hay 191 4.2

Orchardgrass hay 150 7.1

Duodenal digesta from defaunated sheep 90 5.0

Duodenal digesta from faunated sheep 540 11.7

Ruminal fluid from defaunated sheep 30 .6

Ruminal fluid from faunated sheep 55 5.2 a 2-aminoethylphosphonic acid. b Each value is the mean of two replicates. 63 the isolated bacterial strains examined. This could possibly be due to higher concentrations of AEP in one or more strains of ruminal bacteria not examined in this study.

The concentration of AEP in duodenal digesta of faunated sheep

(Table 4) was six times that in the duodenal digesta from defaunated sheep fed the same diet, but the concentration in strained ruminal fluid of faunated sheep was only approximately twice that in defau nated sheep. 2-Aminoethylphosphonic acid concentration appeared to be highest for oats, followed closely by corn and alfalfa and or chardgrass hay. Corn cobs and wheat straw had the lowest concentra tions of AEP.

The occurrence of AEP in feed and bacterial hydrolysates was confirmed by analyzing these samples using the indirect AEP organic phosphorus procedure (Abou Akkada et al., 1968). Prior to determin ing AEP-phosphorus in feed and bacterial hydrolysates, duplicate sam ples of known amounts of pure AEP were subjected to similar condi tions used for total phosphorus analysis and then analyzed for inor ganic phosphorus in order to determine the % recovery of the total phosphorus in AEP. Average AEP phosphorus recovery was 98.2%. Re hydrolyzed eluates of both feed and bacteria were both found to con tain similar amounts of inorganic phosphorus as their original elu ates, confirming that the organic phosphorus (carbon-phosphorus bond) in the eluate was inert to acid hydrolysis. Amounts of AEP found in feed and bacterial hydrolysates a3 determined by the two methods are shown in Table 5. Quantification of AEP did not differ due to method of analysis. 2-Arainoethylphosphonic acid has been reported to be the major component of natural phosphonates (Horiguchi, 1971). Although other phosphonates have been reported in a few organisms such as

Tetrahyraena and Zoanthid, none have been found in feed or bacteria

(Kittredge and Roberts, 1969). It is therefore assumed that the or ganic phosphorus in the feed and bacteria hydrolysates in the present study originated from AEP. The close agreement between the HPLC method and the organic phosphorus method confirms this assumption.

The detection of AEP in feed and bacteria in this study is con trary to reports by Abou Akkada et al. (1968), El Shazly et al.

(1975) and Dufva et al. (1982). Reports of the presence of AEP in feed and(or) ruminal bacteria is, however, not unique to this study.

Using an automatic amino acid analyzer with a ninhydrin detector,

Ling and Buttery (1978) detected substantial quantities of AEP not only in isolated ruminal protozoa but also in mixed ruminal bacteria and dietary material. This was confirmed by Cockburn and Williams

(1984), who used the organic phosphorus technique to assay AEP and reported its presence in mixed ruminal bacteria but not in any of the feed ingredients used in their study. However, preliminary work by

Cockburn (1982), as reported by Cockburn and Williams (1984), indi cated that low concentrations of AEP were present in hay, fish meal, soybean meal and molassed peat. Czerkawski (1974) also reported sig nificant amounts of AEP in mixed ruminal bacteria, with small ruminal 65

TABLE 5. CONCENTRATION OF AEPa IN FEED AND MIXED RUMINAL BAC

TERIAL HYDROLYSATES AS DETERMINED BY HPLC AND ORGANIC

PHOSPHORUS (P) METHODS

Method of analysis

Sample HPLC Organic P SE

AEP concentration, pg/mlb

Feed hydrolysate 23.^6 22.21 .51

Mixed ruminal ■=rCO

bacterial hydrolysate 18.69 17.52 •

a 2-arninoethylpho3phonic acid.

b Each value is the mean of three replicates. 66 bacteria having higher amounts of AEP (362 pg/g DM) than mixed rumi nal protozoa (315 pg/g DM). The concentration of AEP in mixed rumi nal bacteria obtained in our study (1,383 pg/g DM) is consistent with the range of 1,245 to 1,443 pg/g DM reported by Cockburn and Williams

(1984) but higher than the range of 30 to 680 pg/g DM reported by

Ling and Buttery (1978) and 80 to 362 pg/g DM reported by Czerkawski

(1974). The concentration of AEP in mixed ruminal protozoa (4,304 pg/g DM) determined by HPLC is in close agreement with the range of

3,292 to 4,301 pg/g DM reported by Cockburn and Williams (1984) but higher than the values reported by Czerkawski (1974).

The wide variation in reported concentrations of AEP in bacteria and protozoa could be due to the various methods used for the assay of AEP, to the relative proportions of the different strains and spe cies of bacteria and protozoa in their respective mixtures and to the type of diet fed to the animals from which the samples were taken.

2-Aminoethylphosphonic acid concentration varies among species of protozoa (Abou Akkada et al., 1968; Whitelaw et al., 1983) and be tween small and large mixed ruminal bacteria (Czerkawski, 1974) and diet influences their proportions (Hungate, 1966) and chemical com position (Meyer et al., 1967). The detection of substantial amounts of AEP in both mixed and pure strains of ruminal bacteria suggests that AEP may be synthesized by these bacteria.

The amount of AEP in duodenal digesta from defaunated sheep (90 pg/g DM) in this study is lower than the range of 120 to 200 Pg/g DM reported by Cockburn and Williams C198H), who pointed out that the presence of AEP in duodenal digesta from defaunated steers confirms

its presence in ruminal bacteria.

The ubiquitous nature of AEP in biological tissues such as pro tozoa, Coelenterates, Molluscs, Echinoderms, Arthropods, Vertebrates,

Schizoraycophytes and Thallophyte3 was reported by Horiguchi (1971).

Detectable amounts of AEP have also been found in goat liver (Kandat- su and Horiguchi, 1965), bovine brain (Shimizu et al., 1965), myco bacteria (Sarraa et al., 1970), bile of the bovine (Tamari and

Kametaka, 1973), bovine milk (Tamari and Kandatsu, 1985) and human tissues (Alhadeff and Daves, 1971). Because AEP has been detected in many biological materials, it is not unusual that AEP wa3 found in substantial quantities in some feedstuffs and ruminal bacteria.

It is concluded that the HPLC technique is a sensitive, rapid and direct method for quantitating AEP. Although AEP can be degraded by mixed ruminal bacteria, its presence in feed and ruminal bacteria precludes its usefulness as a marker for ruminal ciliate protozoa. LITERATURE CITED

Abou Akkada, A. R., D. A. Messmer, L. R. Fina and E. E. Bartley. 1968. Distribution of 2-aminoethylphosphonic acid in some rumi nal microorganisms. J. Dairy Sci. 51:78.

Alam, A. U. and S. H. Bishop. 1969. Growth of Escherichia coli on some organophosphonic acids. Can. J. Microbiol. 15:1043*

Alhadeff, J. A. and G. D. Daves, Jr. 1971. 2-aminoethylphosphonic acid: Distribution in human tissues. Biochira. Biophys. Acta 21(4:211.

AOAC. 1975. Official Methods of Analysis (12th Ed.). Association of Official Analytical Chemists. Washington, DC.

Cockburn, J. E. 1982. The measurement of protozoal protein in the digestive tract of ruminants. M. Phil. Thesis, University of Reading. England.

Cockburn, J. E. and A. P. Williams. 1982. Some problems associated with the analysis of 2-aminoethylphosphonic acid using automated ion-exchange chromatography. J. Chroraatogr. 249:103

Cockburn, J. E. and A. P. Williams. 1984. The simultaneous estima tion of the amounts of protozoal, bacterial and dietary nitrogen entering the duodenum of steers. Br. J. Nutr. 51:111.

Cook, A. M., C. G. Daughton and M. Alexander. 1978. Phosphonate utilization by bacteria. J. Bacteriol. 133:85.

Czerkawski, J. W. 1974. Methods for determining 2-6-diaminopimelic acid and 2-aminoethylphosphonlc acid in gut contents. J. Sci. Food Agric. 25:45.

Dehority, B. A. 1963. Isolation and characterization of several cellulolytic bacteria from in vitro rumen fermentations. J. Dairy Sci. 46:217-

Dehority, B. A. 1966. Characterization of several bovine rumen bac teria isolated with a xylan medium. J. Bacteriol. 91:1724.

Dehority, B. A. 1969. Pectin-fermenting bacteria isolated from the bovine rumen. J. Bacteriol. 99s189-

Dehority, B. A. 1975. Characterization studies on rumen bacteria Isolated from Alaskan reindeer (Ranglfer tarandus L.). In: 68 69

Proceedings of the 1st International Reindeer and Caribou Sympo sium, Fairbanks, Alaska. Biological Papers of the University of Alaska, Special Report no. 1. p. 228.

Dufva, G. S., E. E. Bartley, M. J. Arambel, S. J. Galitzer and A. D. Dayton. 1982. Content of 2-aminoethylphosphonic acid in feeds, bacteria and protozoa and its role a3 a ruminal protozoal marker. J. Anim. Sci. 54:837.

El Shazly, K., A. M. Nour and A. R. Abou Akkada. 1975. A method for determining 2-aminoethane phosphonic acid in ruminal contents. Analyst 100:263.

Harkness, D. R. 1966. Bacterial growth on aminoalkylphosphonic acids. J. Bacteriol. 92:623.

Horiguchi, M. 1971. Natural carbon-phosphorus compounds. In: An alytical chemistry of phosphorus compounds. M. Holman, Ed., pp 703. John Wiley and Sons,London.

Horiguchi, M. and M. Kandatsu. 1959. Isolation of 2-aminoethane- phosponic acid from rumen protozoa. Nature 184:901.

Hungate, R. E. 1966. The Rumen and Its Microbes. Academic Press, New York.

Ibrahim, E. A., J. R. Ingalls and D. B. Bragg. 1970. Separation and identification of amino acids present in ruminal microorganisms. Can. J. Anim. Sci. 50:397 (as corrected in Can. J. Anim. Sci. 53:761).

Kandatsu, M. and M. Horiguchi. 1965.The occurrence of Ciliatine (2-aminoethylphosphonic acid)- in the goat liver. Agric. Biol. Chem. 29:781.

Kittredge, J. S. and E. Roberts. 1969. A carbon-phosphorus bond in nature. Science 164:37*

Lindroth, P. and K. Mopper. 1979. High performance liquid chroma tographic determination of subploomole amounts of amino acids by precolumn fluorescence derivatizatlon with O-phthaldialdehyde. Anal. Chem. 51:1667.

Ling, J. R. and P. J. Buttery. 1978. The simultaneous use of ribo nucleic acid, 35s, 2,6-diaminopimelio acid and 2-aminoethylphosphonic acid as markers of microbial nitrogen entering the duodenum of sheep. Br. J. Nutr. 39:165. 70

Mackie, R. I. 1973. Column chromatography of 2-aminoethylphosphonic acid. J. Dairy Sci. 56:939*

McDougall, E. I. 1948. Studies on ruminant saliva. 1. The com position and output of 3heepfs saliva. Biochem. J. 43:99.

Meyer, R. M., E. E. Bartley, C. W. Deyoe and V. F. Colenbrander. 1967. Feed Processing. 1. Ration effects on ruminal microbial protein synthesis and amino acid composition. J. Dairy Sci. 50:1327.

Rahnema, S. H. and B. Theurer. 1986. Comparison of various amino acids for estimation of microbial nitrogen in digesta. J. Anim. Sci. 63:603.

Rosenberg, H. and J. M. La Nauze. 1967. The metabolism of phospho nates by microorganisms. The transport of aminoethylphosphonic acid in Bacillus cereus. Biochim. Biophys. Acta 141:79-

Roth, M. 1971. Fluorescence reaction for amino acids. Anal. Chem. 43:880.

Sarma, G. R., V. Chandramouli and T. A. Venkitasubramanian. 1970. Occurrence of phosphonolipids in mycobacteria. Biochim. Bio phys. Acta 218:561.

Shimizu, H., Y. Kakimoto and I. Sano. 1965. Isolation and identi fication of 2-aminoethylphosphonic acid from bovine brain. Na ture 207:1197.

Steel, R. G. D. and J. H. Torrie. 1960. Principles and Procedures of Statistics. McGraw-Hill Book Co., New York.

Tamari, M. and M. Kametaka. 1973. Isolation of Ciliatine (2-arainoethylphosphonic acid) from the bile of the bovine. Agric. Biol. Chem. 37:933.

Tamari, M. and M. Kandatsu. 1985. Isolation and identification of Ciliatine (2-aminoethylphosphonic acid) from acid soluble frac tion of skim milk powder. Nagasaki Daigaku Kyoikugakubu Shizen Kagaku Kenkyu Hokoku 36:95.

Whitelaw, F. G„, L. A. Bruce, J. M. Eadie and W. J. Shand. 1983. 2- aminoathylphosphonic acid concentrations in some ruminal ciliate protozoa. Appl. Environ. Microbiol. 46:951. 71

Zeleznick, L. D., T. C. Myers and E. B. Titchener. 1963. Growth of Escherichia ooll on methyl- and ethylphosphonic acids. Biochim. Biophys. Acta 78:546. CHAPTER III

EFFECTS OF DIETARY PROTEIN SOURCE AND DEFAUNATION ON LAMB PERFORMANCE

INTRODUCTION

Although ciliate protozoa were discovered in the rumen nearly one and a half centuries ago (Gruby and Delafond, 1843), their role in the ruminant animal is still not well understood. There is gen eral agreement and adequate evidence to indicate that rumen ciliate protozoa possess active systems for hydrolyzing both complex and sim ple carbohydrates, proteins and lipids (Oxford, 1951; Hungate, 1955;

Abou Akkada and Howard, 1962; Bailey and Clarke, 1963; Prins and Van

Hoven, 1977). Despite these findings, reports of studies on the ef fects of the presence or absence of rumen ciliate protozoa on general performance of ruminants have been contradictory. Early researchers found no differences in performance of ruminants due to faunation or defaunation of the rumen (Becker and Everett, 1930; Bryant and Small,

1960; Eadle, 1962) while subsequent researchers observed either small or substantial differences in growth rate, feed efficiency, fermenta tion and fermentation characteristics (Abou Akkada and El Shazly,

1964, 1965; Christiansen et al., 1965; Klopfenstein et al., 1966;

Bird and Leng, 1978; Veira et al., 1983). Bird and Leng (1978) stud ied the effects of defaunation on growth of cattle fed low protein- high energy diets and observed no differences in growth rate between

72 73 faunated and defaunated cattle. Addition of a very low level of pro tein of low rumen degradability, however, resulted in 43? increase in growth rate for the protozoa free group. They suggested that the absence of protozoa may have enhanced both the availability of pro tein at the duodenum and provided a relatively constant protein to energy ratio. Veira et al. (1983) concluded that defaunation is likely to be beneficial only when diets high in energy and adequate in rumen fermentable nitrogen but low in proteins of low rumen de gradability are fed to ruminants with a large requirement for amino acids.

Because protozoa are capable of engulfing proteins of low rumen degradability, that may otherwise be directly digested and absorbed in the small intestine of the ruminant animal, it is possible that defaunation may have a positive impact on performance of young rumi nants fed high-energy rations supplemented with protein sources of low rumen degradability, provided the protein level in the diet is below their requirements for optimal growth. It was, therefore, hy pothesized that defaunated lambs fed a diet supplemented with protein of low rumen degradability (e.g., fish meal (FM)) would perform bet ter than faunated lambs on similar diets because of the absence of protozoa, which would enhance the passage of the rumen undegradable protein to the duodenum for more efficient utilization by the lambs.

It was also hypothesized that lambs fed a diet supplemented with pro tein of low rumen degradability would perform better than those fed a 74 similar basal diet supplemented with protein of high rumen de gradability (e.g., soybean meal (SBM)).

This study was conducted to determine the effects on performance of faunated and defaunated lambs fed diets supplemented with protein sources of low (FM) or high (SBM) rumen degradability.

MATERIALS AND METHODS

Experimental Animals and Piet3

Thirty mixed sex (20 ewes and 10 wethers) crossbred Targhee lambs weighing 23-27 kg were drenched with an anti-helminthic drug and also inoculated against Clostridium perfringes as a prophylactic measure against enterotoxemia prior to starting the experiment. All lambs were defaunated with dioctylsodium sulphosuccinate (DSS) and allotted by weight and sex to five groups of six lambs, such that each group had 4 females and 2 males. Each group wa3 then allotted randomly to one of five treatment combinations: (a) defaunated, FM-

9-5% CP; (b) defaunated, SBM-9.5% CP; (c) refaunated, FM-9.5%CP; (d) refaunated, SBM-9.5% CP and (e) refaunated, SBM-12% CP, which acted as positi/e control. Diets were isocaloric and isonitrogenous (9.5%

CP) except for the positive control diet, which was 12% crude pro tein. All diets were pelleted and were composed primarily of corn, corn cobs and corn starch (Table 6). TABLE 6. COMPOSITION OF DIETS FOB LAMB PERFORMANCE TRIAL

Dietary treatments

SBM-12%CPa SBM-9.5% CPb FM-9.5% CPb Item (Positive Control)

% DM basis

Ingredients

Corn, ground 37.00 37.00 37.00

Corn cobs 30.00 30.00 30.00

Corn starch 17.69 22.38 25.67

Soybean meal 12.90 8.10

Fish meal 6.02

Urea 0.30 0.30 0.30

Ammonium chloride 0.27 0.27 0.27

Dicalcium phosphate 0.97 0.67

Limestone 0.91 0.82 0.28

Zinc oxide 0.01 0.01 0.01

Selenium (201 mg Se/kg) 0.05 0.05 0.05

Trace mineral salt0 0.29 0.29 0.29

Vitamin E (99 IU/g) 0.08 0.08 0.08

Vitamin A (30,000 IU/g) 0.01 0.01 0.01

Vitamin D (3,000 IU/g) 0.02 0.02 0.02

Chemical analysis

Crude protein 12.12 9.61 9-52

Neutral detergent fiber 38.57 36.69 33-22

Acid detergent fiber 15.77 15.38 19.98

a SBM constituted 56% of the crude protein in the diet. b SBM and FM constituted 99% of the crude protein in the diets. 0 The trace mineral salt contained 98% NaCli .35% 2n, .28% Mn, .175% Fe, .035% Cu, .007% I and .007% Co. 76

All lambs in each group were paired and housed in pens such that

the total liveweight of animals in each pen was similar. The defau

nated lambs were housed in an isolated room adjacent to the main

barn.

As a result of restriction imposed by the availability of space

and animals, it was not possible to have positive controls for both

sources of protein and for both defaunated and refaunated lambs.

Crude protein content of the dietary treatments (9.5% CP) was below

the NRC (1985) requirements (11£ CP).

Lambs were offered their respective diets daily, ad libitum and

water was available at all times. Feed intake of lambs in each pen

was measured daily and lambs were weighed weekly. Feed offered and

feed refusals were sampled daily at the time of feeding, bulked sepa

rately and subsampled at the end of each week for dry matter (DM) determination. Samples of each diet were taken for the determination of crude protein by Kjeldahl procedure and for acid detergent fiber

(ADF) by the method of Goering and Van Soest (1970). Neutral deter gent fiber (NDF) was determined by the amylase procedure of Robertson and Van Soest (1977)-

Defaunation and Refaunation Procedures

One day prior to starting the defaunation procedure, all lambs were fed half their ration (500 g). Feed was then withheld for the 3 d defaunation period. Lambs were not fed during defaunation because

the presence of particulate matter in the rumen reduces the effective 77 concentration of dioctylsodium sulphosuccinate (DSS) due to its ad herence to feed particles (Orpin, 1977). Three doses of 3 g DSS/dose

(3 g DSS dissolved in 60 ml of boiling water and allowed to cool) of

DSS were administered to each lamb on consecutive days through a polyethylene tube inserted down the esophagus. On the second and third days, 300 ml of a sterile aqueous solution containing 20 g corn starch + ^0 g sucrose + 20 g casein were infused into the rumen of each lamb via the esophagus 2 h postdosing and every other day until they recovered from the defaunation treatment and their feed intakes were similar to previous intakes before the DSS dosage. This period varied between 5 and 7 d. The infusate was used to sustain the bac terial population in the rumen when the animals were off feed and to help the animals recover after the dosing procedure by stimulating feed intake. Care was taken to avoid accidental contamination of the defaunated lambs with protozoa by attending to the defaunated animals first and also by washing hands thoroughly with soap and changing clothes when moving from the animal room harboring the refaunated lambs to the adjacent room where the defaunated lambs were housed.

All animals were placed on their various dietary regimens start ing from the last day of DSS dosage. Rumen contents from defaunated lambs were examined 2 wk after the final dosage of DSS and found to be free of protozoa. On the same day, lambs in the faunated treat ment group were refaunated by inoculating the rumen (via polyethylene tube inserted down the esophagus) with 300 ml of rumen fluid obtained from a nominally fistulated steer having mixed rumen fauna. Faunated lambs were first defaunated before refaunation so that any side ef fects of the defaunating agent, such as effects on rumen bacteria, if any, would be similar for lambs in both faunated and refaunated treatment groups. Refaunated lambs were allowed 10 d for the estab lishment of protozoa in the rumen before the 56-d growth trial was

Initiated. In all, the total time lambs were allowed to adjust to their dietary regimen before initiation of the trial was 24 d.

Microbial Assays

Rumen samples were obtained from all lambs by stomach tube on d

1 and 56 to determine protozoal concentrations. Using roll tubes, viable bacteria counts were measured on the d-56 rumen contents. In addition to the above sampling times, rumen contents from the defau nated lambs were obtained on d 28 for microscopic examination in or der to confirm the absence of protozoa. Rumen contents (10 ml) were mixed with an equal volume of 50% formalin (1:2 dilution of commer cial 37% formaldehyde solution), stained with brilliant green dye, diluted with 30% glycerol as required and counted in a Sedgwick- rafter counting chamber.

Experimental Design

The experiment was a completely randomized design with a 2 x 2 factorial arrangement of treatments. The main effects were protein source (SBM and FM) and faunation status (defaunated and refaunated lambs). There were 3 replicates per treatment combination and 2 ani mals per replicate (pen). Treatment differences were analyzed sta tistically using ANOVA procedures (Snedecor and Cochran, 1968).

Analysis of variance procdures were also used to determine if responses to the positive control treatment (SBM-12% CP) for refau nated lambs differed from those of refaunated lambs fed the SBM-9.5%

CP diet.

RESULTS AMD DISCUSSION

The defaunating agent and the procedure used for defaunation were very effective in completely removing all protozoa from the ru men of the lambs and getting the lambs back to their normal appetite in a short time. All the defaunated animals remained protozoa-free for the duration of the 56-d trial (Tables 8 and 10).

Data regarding dietary protein level (SBM-12% CP and SBM-9.5%

CP) on performance of refaunated lambs (Table 7) indicated that lambs fed the positive control diet had 76% greater rate of gain than those fed SBM-9.5% CP diets (201 g/d vs 114 g/d, respectively; P<,05).

Feed conversion (g DMI/g live-weight gain) tended to improve with the higher level of dietary protein supplementation but this was not sig nificant (P>0.05) due to a higher dry matter intake by animals fed the positive control diet (P<0.05). Gordon (1980) pointed out that increased protein intake stimulates feed intake. Clay and Satter

(1979) also obtained higher dry matter intakes with increased dietary TABLE 7. EFFECTS OF DIETARY SOYBEAN HEAL (SBH) LEVEL OH PERFORMANCE AND RUMEN MICROBIAL CONCENTRATIONS OF REFAUNATED LAMBS

Protein level

SBM-12J CP SEM-9-5J CP Item (Positive control) SE

No. of lambs 6 G

Initial ut, kg 2B.2 25.B 1.3

DM feed intake, g/d 1331 1081 49.83

Cain, g/d 201 11i| 20.9b

Feed/gain, g/g 6.7 10.3 1.43

D-1 Rumen protozoal concentration x 10b, no./ml 2.37 2.12 0.861

D-56 Rumen protozoal concentration x 10^, no./ml 3.24 H.11 0.707

D-56 Rumen bacterial concentration x 10?, no./g 35.95 29.45 4.948 a DM intake of lambs fed SBM-12J CP differed from those fed SBM-9.5J CP (P<.05). b Average daily gain of lambs fed SBM-12J CP differed from those fed SBM-9.5J CP (P<.05). 81 protein level in early lactating dairy cows fed four levels of soy bean meal. The higher growth rate of lambs fed the positive control diet indicated that 9.5% dietary CP was below the requirements for maximum growth for the lambs used in this trial. Level of protein in the diet did not appear to have any effect on protozoal and bacterial concentrations in the rumen.

Defaunation did not affect (P>.05) feed intake or feed conver sion (Table 8). Similar feed intakes between faunated and defaunated animals have also been reported by other investigators (Christiansen et al., 1965; Chalmers et al., 1968; Bird and Leng, 1978, 1984; Bird et al., 1979; Rowe et al., 1985). Defaunation also did not affect growth rate (P>.05). This observation is in agreement with the re sults of Pounden and Hibbs (1950); Eadie (1962); Chalmers et al.

(1968); Eadie and Gill (1971) and Williams and Dinusson (1973). It is, however, contrary to the findings of Abou Akkada and El Shazly

(1964); Christiansen et al. (1965); Klopfenstein et al. (1966) and

Borhami et al. (1967). Leng and Nolan (1984) suggested that the con tradictory response of defaunation on performance of ruminants may be due to the diet and the physiological states and ages of the experi mental animals used. Bird and Leng (1984) observed a 9% higher growth rate in lambs as a result of defaunation. In their study, however, they used relatively younger lambs having an average liveweight of 16 kg compared to the average liveweight of 25 kg used in this experiment. Due to the absence of any interaction between 82

TABLE 8. MAIN EFFECTS OF DEFAUNATION ON LAMB PERFORMANCE AND RUMEN MICROBIAL CONCENTRATIONS

Item Refaunated Defaunated SE

Na. of lambe 12 12

Initial wt, kg 25.5 24.9 .49

DM feed Intake, g/d 1160 1087 49.9

Gain, g/d 169 108 18.9

Feed/gain, g/g 0.0 0.9 .7

D-1 Rumen protozoal concentration x 10®, no./ml 2.3 O .32a

D-56 Rumen protozoal concentration x 10®, no./ml 4.1 0 ,43a

D-56 Rumen bacterial concentration x 10^, no./g 30.9 29.0 2.60 a Troatmont offoot3 differed (P<.01). 83 protein source and protozoa status of the lambs in this study (Table

10) it is likely that, even if the ciliate protozoa engulfed some of the bypass protein (fish meal) in the rumen, it was not sufficient to cause a reduction in growth rate of the refaunated lambs.

Lambs fed the FM supplemented diet had higher (P<.05) dry matter intakes and improved feed conversions (Table 9) compared to those fed the SBM supplemented diet. Bird and Leng (1978) reported that sup plementation of a protein pellet of low rumen degradability (mixture of soybean meal, cottonseed meal and fish meal) in a diet for cattle resulted in an increase in feed intake. In the present trial, growth rate of lambs fed the FM supplemented diet was 69% higher (PC.01) than those fed the SBM supplemented diet (224 g/d vs 133 g/d, respec tively). This may be due to the low rumen degradability of protein in fish meal which may result in higher flow of protein to the duode num. The fraction of protein in SBM which is undegraded in the rumen is about 25% but that in FM is about 78% (NRC, 1985). The relatively poor feed conversion obtained for refaunated lambs fed the SBM-9-5%

CP diet (Table 10) was due to a lower liveweight gain obtained for lamb3 in one of the three pens on this diet (71 g/d compared to the average of 135 g/d for the other two pens). Rectal temperature, feed intake and visual observation of the two lambs in this pen suggested that they were normal. The reason for the reduction in liveweight gain is therefore unclear. 84

TABLE 9. MAIM EFFECTS OF PROTEIN SOURCE ON LAMB PERFORMANCE AND RUMEN MICROBIAL CONCENTRATIONS

Item Soybean meal Fiah meal SE

No. of lamb3 12 12

Initial wt, kg 25.1 25.3 .49

DM feed intake, g/d 1042 1205 49.9a

Gain, g/d 133 224 10.9b

Feed/gain, g/g 8.5 5.5 • 7a

D-l Rumen protozoal concentration x 10b, no./ml 1.06 1.22 .32

D-56 Rumen protozoal concentration x 1Qb, no./ml 2.05 2.03 .43

D-56 Rumen bacterial concentration x IQ^, no./g 29.73 30.10 2.60

a Treatment effeota differed (P<.05). b Treatment oiTccta dlffcrod (P<.01). TABLE 10. EFFECTS OF DIETAHY PROTEIN SOURCE AND DEFAUNATIOH ON LAMB PERFORMANCE AND RUMEN MICROBIAL CONCEN TRATIONS

Refaunated Defaunated

Protein source

Item SBMa FMb SBM FM SE

No. of lambs 6 6 6 6

Initial wt, kg 25.8 25.3 24.4 25.4 -7

DM feed Intake, g/d 1081 1239 1002 1171 70.5C

Gain, g/d 114 223 151 225 26. Bd

Feed/gain, g/g 10.3 5.8 G.G 5.2 1.05

D-1 Rumen protozoal concentration x 10®, no./ml 2.1 2.4 0 0 .45e

D-56 Rumen protozoal concentration x 10®, no./ml 4.1 4.1 0 0 .6le

D-56 Rumen bacterial concentration x 10?, no./g 29.5 32.3 30.0 27.9 3.67 a Soybean meal. b Fishmeal. c DM intake and feed conversion of lambs fed SBM differed from those fed FM (P<.05). d Average daily gain of lambs fed SBM differed from those fed FM (P<.01). e Rumen protozoal concentration of defaunated lambs differed from refaunated lambs (P<.01). 86

Bacterial concentrations in the rumen appeared to be unaffected by protein source (Table 9)* Contrary to general agreement that defaunation of animals results in a marked increase in bacterial popu lation due to the reduction of predation and nutritional competition between protozoa and bacteria (Eadie and Hobson, 1962; Eadie and

Gill, 1971; Kurihara et al., 1978; Kayouli et al., 1984; Newbold et al., 1986; Veira, 1986), no differences (P>.05) were found in bac terial concentrations between the defaunated and refaunated lambs

(Tables 8 and 10). The reason for this observation is not clear but it is possible that the stomach tubing technique used to obtain rumen contents in this study was a factor. Rumen samples obtained by this technique usually results in contamination of the samples with saliva which would affect the determination of bacterial concentration.

Jouany et al. (1981), also observed no differences in bacterial numbers between faunated and defaunated sheep. They, however, re ported that the distribution of the different size-type categories of bacteria varied. Compared to a protozoa-free rumen, they observed that the number of small bacteria (<3 yim) increased with Polyplastron multiveslculatum and Isotrlcha prostoma inoculations and decreased with the inoculation of Entodinium species. The number of average sized bacteria (3-9.5 ym) was always higher in faunated than in de faunated sheep, except for sheep which were inoculated with Entodini um and Isotricha pro3toma species. The number of large bacteria (9.5 87

- 18.5 ym) decreased in the faunated sheep, except for sheep inocu lated with Isotrlcha species. The largest bacteria (18.5 - 25 ym) were more numerous in the faunated sheep and showed a maximum with sheep inoculated with an Entodinium and Isotricha combination. They attributed the alteration in the number of different kinds of bac teria after inoculation of protozoa into the rumen to selective pre dation by the inoculated ciliates, or competition for nutrients or both and they concluded that the increase in the number of the larg est bacteria was likely to be due to the fact that they cannot be ingested by Isotricha which select their prey (Gutierrez, 1958) or by

Entodinium species because of their size and shape (Coleman, 1964).

Similar changes in type of bacteria in the rumen after defaunation have been reported (Eadie and Hobson, 1962; Abou Akkada and El Shaz- ly, 1964; Kurihara et al., 1978; Coleman and Sandford, 1979; Rowe et al., 1985). In the present study, the composition of protozoa in the rumen of the lambs fed SBM-1226 CP was made up of 99.6526 Entodiniinae,

0.3226 Diplodinilnae and 0.0326 Isotrichidae. The corresponding values for lambs fed SBM-9.5% CP were 99.13%, 0.84% and 0.03% while those for lambs fed the FM-9.5% CP diet were 99.80%, 0.17% and 0 .03%, re spectively. Bird et al. (1979) pointed out that removal of protozoa results in changes in the ecological system in the rumen making it difficult to define the mechanisms responsible for the observed ef fects of the defaunated state. 88

In conclusion, defaunation did not affect the performance of growing lambs or rumen bacterial concentrations, but supplementing fish meal resulted in improved lamb performance compared to lambs fed supplemental soybean meal. LITERATURE CITED

Abou Akkada, A. R. and K. El Shazly. 1964. Effects of absence of ciliate protozoa from the rumen on microbial activity and growth of lambs. Appl. Microbiol. 12:384.

Abou Akkada, A. R. and B. H. Howard. 1962.The biochemistry of ru men protozoa. 5. The nitrogen metabolism of Entodinium. Bio- chera. J. 82:313*

Bailey, R. W. and R. T. Clarke. 1963. Carbohydrase activity of ru men Entodinium species from sheep on a starch-free diet. Nature (London) 198:787.

Becker, E. R. and R. C. Everett. 1930. Comparative growths of nor mal and infusoria-free lambs. Amer. J. Hyg. 11:362.

Bird, S. H., M. K. Hill and R. A. Leng. 1979. The effects of de faunation of the rumen on the growth of lambs on low-protein high-energy diets. Brit. J. Nutr. 42:81.

Bird, S. H. and R. A. Leng. 1978. The effects of defaunation of the rumen on the growth of cattle on low-protein high-energy diets. Brit. J. Nutr. 40:163.

Bird, S. H. and R. A. Leng. 1984. Further studies on the effects of the presence or absence of protozoa in the rumen on liveweight and wool growth of sheep. Brit. J. Nutr. 52:607.

Bryant, M. P. and N. Small. 1960. Observations on the ruminal mi croorganisms of isolated and Inoculated calves. J. Dairy Sci. 43:654.

Chalmers, M. I., J. Davidson, J. M. Eadie and J. C. Gill. 1968. Some comparisons of performance of lambs with and without rumen ciliate protozoa. Proc. Nutr. Soc. 27:29A. 89 90

Christiansen, W. C., R. Kawashima and W. Burroughs. 1965. Influence of protozoa upon rumen acid production and liveweight gains in lambs. J. Anim. Sci. 24:730.

Clay, A. B. and L. D. Satter. 1979. Milk production response to dietary protein and methionine hydroxy-analog. J. Dairy Sci. 62(Suppl. 1):75.

Coleman, G. S. 1964. The metabolism of Escherichia coli and other bacteria by Entodinium caudatum. J. Gen. Microbiol. 37:209.

Coleman, G. S. and D. C. Sandford. 1979* The engulfraents and diges tion of mixed rumen bacteria and individual bacterial species by single and mixed species of rumen ciliate protozoa grown in vivo. J. Agric. Sci. (Camb.) 92:729.

Eadie, J. M. 1962. The development of rumen microbial populations in lambs and calves under various conditions of management. J. Gen. Microbiol. 29:563.

Eadie, J. M. and J. C. Gill. 1971. The effects of the absence of rumen ciliate protozoa on growing lambs fed on a roughage- concentrate diet. Brit. J. Nutr. 26:155.

Eadie, J. M. and P. N. Hobson. 1962. Effect of the presence or ab sence of rumen ciliate protozoa on the total rumen bacterial count in lambs. Nature (London) 193:503.

Goering, H. K. and P. J. Van Soest. 1970. Forage fibre analyses: Apparatus, Reagents, Procedures and Some Applications. Agric. Handbook No. 379, ARS, USDA. Washington, D.C.

Gordon, F. J. 1980. Feed Input-milk output relationships in the spring-calving dairy cow. p 15. In: Recent Advances in Animal Nutrition. W. Haresign, ed. Butterworths, London.

Gruby, D. and H. M. 0. Delafond. 1843. Recherches sur de3 animal cules se devellopant en grand nombre dans L'estomac et dans les intestins pendant la digestion des animaux herbivores et carni vores. Compte rendu hetsdomadaire des seances de l'Arcadamie des sciences (Paris) 17:1304.

Gutierrez, J. 1958. Observations of bacterial feeding by the rumen ciliate Isotricha prostoma. J. Protozoology, 5:122.

Hungate, R. E. 1955. Mutualistic intestinal protozoa. In: Bio chemistry and Physiology of Protozoa. S. H. Hutner and L. A. Lwoff, ed. Academic Press, New York. 91

Jouany, J. P., B. Zainab, J. Senaud, C. A. Groliere, J. Grain and P. Thivend. 1981. Role of the rumen ciliate protozoa Polyplastron multivesiculatum, Entodinium sp. and Isotricha prostoma in the digestion of a mixed diet in sheep. Beprod. Nutr. Dev. 21:871.

Kayouli, C., D. I. Demeyer, C. J. Van Never and R. Dendooren. 1984. Effects of defaunation on straw digestion in sacco and on parti cle retention in the rumen. Anim. Fd, Sci. Technol. 10:165.

Klopfenstein, T. J., D. B. Purser and W. J. Tyznik. 1966. Effects of defaunation on feed digestibility, rumen metabolism and blood metabolites. J. Anira. Sci. 25:765.

Kurihara, Y., T. Takechi and F. Shibata. 1978. Relationship between bacteria and ciliate protozoa in the rumen of sheep fed a puri fied diet. J. Agric. Sci. (Camb.) 90:373.

Leng, R. A. and J. V. Nolan. 1984. Nitrogen metabolism in the ru men. J. Dairy Sci. 67:1072.

National Research Council. 1985. Ruminant Nitrogen Usage. National Academy Press. Washington, D.C.

Newbold, C. J., D. G. Chamberlain and A. G. Williams. 1986. The effects of defaunation on the metabolism of lactic acid in the rumen. J. Sci. Fd. Agric. 37:1083.

Orpin, C. G. 1977. Studies on the defaunation of the ovine rumen using dioctyl sodium sulphosuccinate. J. Appl. Bacteriol. 43:309.

Oxford, A. E. 1951. The conversion of certain soluble sugars to a glucosan by holotrich ciliates In the rumen of sheep. J. Gen. Microbiol. 5:83.

Pounden, W. D. and J. W. Hibbs. 1950. The development of calves raised without protozoa and certain other characteristic rumen microorganisms. J. Dairy Sci. 33:639.

Prins, R. A. and W. Van Hoven. 1977. Carbohydrate fermentation by the rumen ciliate Isotricha prostoma. Prostistologica 13:549.

Robertson, J. B. and P. J. Van Soest. 1977* Paper presented at 69th meeting of the American Society of Animal Science, University of Wisconsin, Madison. July 23-27. 92

Rowe, J. B., A. Davies and A. W. J. Broome. 1985. Quantitative ef fects of defaunation on rumenfermentation and digestion in sheep. Brit. J. Nutr. 54:105.

Snedecor, G. W. and W. G. Cochran. 1980. Statistical Methods (7th Ed.). Iowa State Univ. Press, Ames.

Veira, D. M. 1986. The role of ciliate protozoa in nutrition of the ruminant. J. Anira. Sci. 63:1547.

Veira, D. M., M. Ivan and P. Y. Jui. 1983. Rumen ciliate protozoa: Effects on digestion in the stomach of sheep. J. Dairy Sci. 66:1015.

Williams, P. P. and W. E. Dinusson. 1973. Rutninal volatile fatty acid concentrations and weight gains of calves reared with and without ruminal ciliated protozoa. J. Anlm. Sci. 36:588. CHAPTER IV

DERIVATION OF A SELECTIVE CONTAINER TO STUDY THE PHENOMENA OF PROTO

ZOAL SEQUESTRATION, MIGRATION AND LYSIS IN THE RUMEN.

INTRODUCTION

The observation that ciliate protozoa wash out of the rumen at a greatly reduced rate compared to bacteria (Hungate et al., 1971;

Weller and Pilgrim, 1974; Punia et al., 1987), has revived the inter est of researchers to evaluate the role of protozoa in the protein nutrition of the ruminant animal. It also raises speculation on the mechanisms by which protozoa are selectively retained in the rumen.

The suggestion ha3 been made that protozoa avoid passing out of the rumen by sequestering, but experimental evidence to support this hy pothesis is limited. Based on reports of generation times of pro tozoa in the rumen, Demeyer (1981) suggested that protozoa can only maintain themselves in the rumen when a matrix for sequestration is present. Hungate (1966) speculated that some protozoa may maintain their numbers in the rumen by attaching to slower-moving components of digests. Bauchop and Clarke (1976) and Orpin and Letcher (1978) reported on the ability of protozoa to attach to plant particles within the rumen. Abe et al. (1981) observed a thick protozoal mass on the wall of the reticulum of steers slaughtered after overnight starvation and thus suggested that Iaotrichidae would ordinarily se quester on the wall of the reticulum with subsequent migration into 93 the rumen for a few hours after feeding. The diurnal variations ob served in the numbers of Isotrichidae in the rumen (Purser, 1961;

Warner, 1962; Clarke, 1965; Dehority and Mattos, 1978; Abe et al.,

1981; Dehority and Tirabasso, 1987) strengthens the sequestration hypothesis.

If a selective container that is permeable to rumen bacteria but not protozoa could be developed, it should be possible to study the phenomenon of protozoa sequestration in the rumen by simultaneously monitoring protozoa numbers inside and outside the container when it is filled with ruminal contents and placed in the rumen. Because the container would not provide any niche for sequestration of protozoa, differences in protozoa numbers between the container and the rumen could provide evidence of protozoa sequestration and migration. The objectives of this study were: a) to develop selective containers which when placed in the rumen would be permeable to bacteria but not protozoa and b) to use these containers to Investigate the phenomena of protozoa sequestration, migration and lysis in the rumen.

MATERIALS AND METHODS

The selective container consisted of a plastic^ cylinder (7x6 x 7 cm; 6.5 cm i.d.) with screw-on caps at both ends and a spout at the side of the cylinder as illustrated in Figures 3 and M. The con tainer was similar to that developed by Fina et al. (1962). Three

1 Enfield Plastics Ltd., England. y

FIGURE 3. Components used in the selective container. From left to right in order of assembly: screw on cap, 2 mm pore fiberglass window screen, glass wool, neoprene sealing ring, 20 ym pore stainless steel wire, 10 ym pore nylon mesh, neoprene sealing ring, tapered sealing ring and container with an exposed end. ^ FIGURE 4. Assembled containers ready for incubation in the rumen. O' screens were used. The Inner screen was a twill nylon mesh2 with 10

vm pores and the middle screen was a stainless steel wire2 with 20 pin

pores. The outer screen was a fiber glass window screen with 2 mm

pores. The middle and outer screens were U3ed to protect the finer

inner screen from rupture and to prevent clogging of the inner screen with particulate matter. The containers were assembled by placing

the screens and sealing rings into the caps as shown in Figure 3,

starting from left to right. The caps were then tightly screwed on

to the plastic cylinder. A fairly rigid rubber tubing (9mm, i.d.) was connected to the spout of the container and the other end of the

tubing was inserted through a hole created in the rubber cap of the

rumen cannula. The length of the tubing was such that when the con

tainer was placed in the rumen, it rested on the bottom of the rumen

cavity. In addition, the container was weighted by tying a metal

weight around the spout to ensure that it remained at the bottom.

Study A: Evaluation of the Selective Container Design

In a preliminary study with inner screens having 10, 15 or 20 pm

pores, protozoa were observed in the containers after 8 h incubation

in the rumen. Because most of the rumen ciliate protozoa species are

greater than 20 pm in width (Hungate, 1966) it was surprising to find

2Tetko Inc., New York. both large and small protozoa in the containers when the different screen sizes were used. Although not quantified, the size and num bers of protozoa in the container with the 10 pm screen were greatly reduced compared to the screens with larger pores. To determine if the protozoa found in the containers were entering through the screens or through a leak around the threads of the cylinders of the containers, the screens were replaced with polyethene material that had no pores and the containers were incubated in the rumen for 8 h.

The contents of the containers were devoid of protozoa after the 8 h incubation. It was implied that the protozoa entered the containers via the pores in the screens. Microscopic observation of rumen con tents placed on a 15 urn screen indicated that the protozoa forced their way through the holes in the screens. Because the numbers and sizes of protozoa observed in the container with 10 pm inner screen were greatly reduced compared to the other screen sizes, this screen was used as the inner screen and the 20 pm and 2 mm screens were used as the middle and outer screens respectively, in all subsequent studies.

In another preliminary study, two containers were filled with rumen contents from a fistulated steer fed a high concentrate diet and the containers were incubated in the rumen of the steer. Micro scopic examination revealed that most of the protozoa in the con tainers were dead after 8 h. pH measurements of contents in the ru men and containers after the incubation showed that the pH in the 99 containers was lower (5.1) as compared to that in the rumen (6.0).

Thus, the environmental conditions in the rumen were not in equi librium with those in the containers. Consequently, an in vitro study was carried out to determine the time required for pH equili bration across the screens of the containers. Four containers were each filled with 150 ml buffer solution (pH 7) and immersed in four beakers containing acetic acid (pH 3.82 to 4.24). Two of the beakers were agitated using a shaker. Changes in pH in the containers and beakers were monitored over a 24 h period. Data from this study

(Appendices A and B) indicate that even after 24 h, equilibration was not complete between the. solution in the containers and that in the beakers. Agitation of the beakers did not seem to have any effect on the rate of equilibration across the screens of the containers. In a follow-up study, two containers were filled with 1.3818 mg/ml of polyethylene glycol (PEG) solution and incubated in the rumen for 6 h in order to determine the rate of fluid turnover in the containers.

Data from this study (Appendix C) indicate that fluid turnover in the containers was 3.42 to 4.16/d. Although the fluid turnover rate was high, it did not seem to be fast enough to bring about equilibration of conditions in the rumen and the containers.

The problem of protozoal death in the container after 8 h in cubation in the rumen wa3 partially overcome by changing the diet of the steer from a high concentrate diet to a restricted all-hay (first cutting alfalfa) diet (5.4 kg/d). The containers were then filled 100 with rumen contents from the steer immediately after feeding and in cubated in the rumen for 24 h. Although the pH in the containers was slightly lower than that in the rumen (6.07 vs 6.37, respectively), viability of the protozoa in the containers wa3 not markedly af fected. Therefore, in all subsequent studies, the steer was fed a restricted all hay diet.

Study B: Investigation of the phenomena of protozoa sequestration,

migration and ly3is in the rumen

Experiment 1

In order to determine the proportion of protozoa in the rumen which move into the containers during a 24 h incubation, rumen con tents were obtained from a rutninally cannulated steer just before feeding (0 h), and protozoa counts (Dehority, 1984) and pH were mea sured. The steer was then fed 5.4 kg alfalfa hay. Two containers, each containing 10 g ground alfalfa hay and 100 ml water, were as sembled as previously described and placed in the rumen during the time the steer was eating. After 24 h incubation, the containers were removed from the rumen and a 3ainple of rumen contents was ob tained. pH and protozoal counts were then performed on the rumen sample and the contents in the containers. The protozoal concentra tions in the rumen at 0 h and 24 h were averaged and the concentra tions in the containers were expressed as a proportion of the average concentration in the rumen. 101

Experiments 2 to 6

In Experiments 2, 3, 4, 5 and 6, the steer was fed 5.4 kg alfal fa hay once per day at 0800 h. It took approximately .67 h for the steer to consume the ration. Two containers were used simultaneously in each experiment and each container was filled with rumen contents

(150 ml) prior to being incubated in the rumen for a specified period of time (Table 11). Each experiment was repeated twice. pH and pro tozoal counts were measured on all samples.

Experimental Design

The experimental designs for Experiments 2, 3> 4, 5 and 6 were randomized complete blocks with repetitions as blocks. Data from these experiments were analyzed by ANOVA, with treatment means com pared by the Multiple Comparison Method of Least Significant Dif ference protected by a significant (P<.10) F value (Steel and Torrie,

I960).

RESULTS AND DISCUSSION

Experiment 1

This study was done to determine the proportion of protozoa in the rumen which move inito the containers during a 24 h incubation.

The selective containers used in the studies were quite effective in preventing protozoa from moving in or out of the containers. Follow ing a 24-h incubation, protozoal concentration of non-inoculated con tainers was only 3% of that in the rumen (Table 12). Ophryosco- lecidae constituted 6756 of the protozoa found in the containers and 102

TABLE 11. SUMMARY OP PROCEDURAL VARIABLES FOR EXPERIMENTS 2, 3, 5 AND 6

Time postfeeding, ha

Container filled with ruminal Ruminal Container Feeding Experiment contents measurements measurements schedule

2 .67 0 Ix/d .67 .67 4 4

3 6 6 6 1x/d 24 24.67 24.67

4 6 6 6 Ix/d 24 24

5 0 0 0 Ix/d 6 6

6 24 24 24 Fed on d 48 48 1 only a 0 and 24 h indicates samples were taken immediately before feeding. 103

TABLE 12. pH AND PROTOZOAL CONCENTRATIONS IN RUMINAL CONTENTS AT 0 AND 24 H POSTFEEDING AND IN CONTAINERS AFTER 24 H INCUBATION IN THE RUMEN (EXP. 1)

Proportion Rumen Rumen Containera in container,13 Item 0 h 24 h 24 h % pH 7.10 7.08 6.33 - Total protozoal x 1oVml 19.20 20.64 .60 3.0 Ophryoscolecidae x 10^/ml 17-44 19.36 .40 2.2 laotrlchidae x loVral 1.76 1.28 .20 13.2 a Mean of two replicates. 13 Calculated as % of average in rumen at 0 and 24 h. 104 the remaining 3356 was made up of Isotrichidae. The proportion of protozoa which moved into the containers is possibly a slight over estimation because of growth and multiplication in the containers.

It is interesting to note that the concentration of Ophryoscolecidae in the containers was only 2.2% of that in the rumen, while the con centration of Isotrichidae in the containers was 13.2^ of that found in the rumen (Table 12).

Normal protozoa sizes in the rumen are greater than the 10 ym mesh inner screens used in the containers (Hungate, 1966). However, similar observations have been reported by Jouany and Senaud (1979),

Lindberg et al., (1984) and Meyer and Mackie (1986). Based on pre liminary studies with the selective containers used in the present studies, we suggest that the protozoa force their way through the pores of the screen. Jouany and Senaud (1979) suggested that pore sizes less than 10 ym are necessary to exclude protozoa completely.

However, screen sizes less than 10 ym could also lead to a consider able reduction in pH due to the combined effect of rapid fermentation in the containers and a limited fluid exchange with the surrounding rumen contents (Lindberg et al., 1984). In the present experiments, although pH in the containers was lower than that in the surrounding rumen contents (Table 12, 13, 14, 15 and 17), they were apparently not reduced enough to result in protozoal lysis. 105

Experiment 2

Experiment 2 was designed to compare protozoal concentrations from prefeeding to 4 h postfeeding in the rumen and in the con tainers. Data from this experiment (Table 13 and Figure 5) indicated that the total rumen protozoal concentration increased (P<.05) 40 rain postfeeding and decreased (P<-05) back to prefeeding levels at 4 h postfeeding. This increase in total protozoa concentration in the rumen at feeding time and the sharp drop in concentration 4 h post feeding was primarily due to changes in the concentration of

Isotrichidae.

The concentration of Ophryoscolecidae in rumen contents wa3 fairly constant at all sampling times (Table 13 and Figure 5). Thus, little or no sequestration occurred and that their passage out of the rumen was minimal during this period. This observation is in agree ment with that of Clarke (1965), Warner (1966), Hungate et al. (1971) and Weller and Pilgrim (1974). The latter authors estimated that protozoa actually pass out of the rumen at 20% or less of the fluid rate based on direct protozoa counts in the rumen fluid and rumen effluent obtained from continuously fed sheep. Their results Indi cated that protozoal nitrogen leaving the rumen amounted to 2% or less of the dietary nitrogen intake. Potter and Dehority (1973) in dicated that fluid turnover is slower in animals fed once or twice daily. This suggests even les3 passage of dietary nitrogen under once or twice a day feeding and therefore less protozoal nitrogen 106

TABLE 13. pH AND PROTOZOAL CONCENTRATIONS IN RUMINAL CONTENTS AND CONTAINERS AT 0, .67 AND 4 H POSTFEEDING (EXP. 2)

Rumen and Rumen container Rumen Container Item 0 h .67 h 4 h 4 h SE pH 6.90a 6.l49at> 6.51ab 6.01b . 12 Total protozoal x 10Vml 11.96a I8.52b 11.32a 15.38° .66 Ophryoscolecidae x 10^/inl 11.08 10.92 10.MO 10.18 .55 Isotrichidae x 1oVml .88a 7.62b .92a 5.20° .29 a,b,c Means in the same row with different superscripts differ (P<.05). IUE . l n poool ocnrtos n uia cnet and contents ruminal in concentrations pll protozoal and 5. FIGURE PROTOZOAL CONCENTRATION^! 0 /ML) 20 8 7 5 6 4 0 0 oties t , 6 ad hpsfeig Ep 2) (Exp. postfeeding h 4 and .67 0, at containers container filled container 1 TIMEAFTER FEEDING, H 2 2 o... •- 4 proclcde container Ophryoscolecidae, .oOphryoscolecidae, rumen rumen .oOphryoscolecidae, 3 3 oa pooo, container protozoa, Total Total protozoa, rumen rumen protozoa, Total k Isotrichidae,container Isotrichidae,rumen Isotrichidae,rumen 4 o * ° rumen ° rumen 4 container 5 54 107 6 6 108 leaving the rumen. Similar observations were reported by Bird and

Leng (1978) and Punia et al. (1987).

From prefeeding to 40 min postfeeding, Isotrichidae concentra tion in the rumen increased by 9*7 fold (P<.05), and then dropped to prefeeding level3 4 h postfeeding (Table 13 and Figure 5). Although the concentration of Isotrichidae in the containers 4 h postfeeding was lower (P<.05) than the starting concentration at 40 min postfeed ing, this decrease was only 3256 compared to an 88% decrease in Iso trichidae concentration in the rumen during this time period. If it is assumed that lysis and multiplication of Isotrichidae in the rumen was similar to that in the containers, then the difference at 4 h postfeeding must be attributed to dilution effect of water, passage out of the rumen, sequestration or a combination of these factors, all of which were absent in the containers. Although there was a trend for pH in the containers at 4 h to be lower than that in the rumen, pH in the containers wa3 above 6.0 (Table 13 and Figure 5).

Experiment 3

Based on the results of Experiment 2, Experiment 3 was designed to deterimine if the increase in Isotrichidae concentration observed in the rumen 40 min postfeeding also occurs in the containers. In

Experiment 3> ruminal pH increased (P<.05) from 6 to 24 h postfeeding and then decreased C P< - 05) at 40 min post feeding on the second day

(Table 14 and Figure 6). At 40 min postfeeding on the second day, pH was lower (P<.05) in the container than in the rumen, but remained 109

TABLE 14. pH AND PROTOZOAL CONCENTRATIONS IN RUMINAL CONTENTS AND CONTAINERS AT 6, 24 AND 24.67 H POSTFEEDING (EXP. 3)

Rumen and container Rumen Rumen Container Item 6 h 24 h 24.67 h 24.67 h SE pH 6.32a 7.08b 6.35a 6.02° .03 Total protozoal x 10Vral 9.36a 14.48b 20.68° 13-12b .79 Ophryoscolecidae x 10^/ml 8.60 13.16 13-32 12.56 .98 Isotrichidae x 10Vml .76ac 1.32° 7.36b .56a .44 a»b>° Means in the same row with different superscripts differ (P<.05). IUE . Hadpoool ocnrtos n uia cnet and contents ruminal In concentrations protozoal and pH 6. FIGURE i PROTOZOAL CONCENTRATION^!OyML) D- 22 20 18 16 10 12 14 0 2 4 8 6 ■

- 0 0 oties t , 4 n 2.7 psfeig Ep 3) (Exp. postfeeding h 24.67 and 24 6, at containers o * Isotrichidae,container A ----- ** Ophryoscolecidae.container • •“ o-■ • -o Ophryoscolecidae.rumen o Isotrichidae,rumen oa protozoa,container Total “ °Total protozoa,rum en en protozoa,rum °Total 6 container filled container u u i m TIMEAFTER FEEDING, H C j L i c m i u l m u ' " 12 12 1 • —> ■— —j • •— ■*——>■ - — —— | 18 18 ° rumen ° rumen * container 24 24 Fed 110 Ill above 6.0. The concentration of total protozoa in the ruraen in creased (P<.05) from 6 to 24 h postfeeding and wa3 increased further by 40 rain postfeeding on the second day (Table 14 and Figure 6).

Concentration of total protozoa in the container at 40 rain postfeed ing on the second day was 37% lower (P<.05) than the concentration in the rumen at this time, and wa3 similar to the concentration in the ruraen at 24 h postfeeding. There were no differences (P>.05) among the concentrations of Ophryoscolecldae in the rumen at 24 h postfeed ing and in the ruraen and container at 40 min postfeeding on the sec ond day. This again suggests that sequestration of Ophryoscolecldae is very small or non-existent. The increase in concentration of to tal protozoa in the rumen 40 min postfeeding was due to a 5.6 fold increase (P<.05) in the concentration of Isotrichidae. In contrast,

Isotrichidae concentrations did not increase in the containers during this time period. The increase in concentration of Isotrichidae in the rumen was thus attributed to migration of sequestered Isotrichi dae into the ruraen digesta.

Isotrichidae concentrations in the containers did not follow the pattern of fluctuation which occurred in the rumen, i.e., they did not decrease at 4 h postfeeding (Exp. 2), and did not increase at 40 min postfeeding (Exp. 3). Thus, it appears that ruminal fluctuations of Isotrichidae were due to sequestration and migration. The results of Experiment 2- and 3 are in agreement with reports that Isotrichidae begin to increase In numbers just before or at feeding time, reaching 112 maximum concentrations at feeding or within 1 to 2 h postfeeding

(Purser, 1961; Warner, 1962} Clarke, 1965; Dehority and Mattos, 1978;

Abe et al., 1981; Dehority and Tirabasso, 1987). Abe et al. (1981) observed a 4-fold increase in numbers of Isotrichidae within 1 h af ter the commencement of feeding followed by a rapid decrease to pre feeding levels. Murphy et al. (1985) also observed a 10-fold in crease in Isotrichidae concentration 2 h after feeding, returning to prefeeding levels 5 to 6 h later. Cheraotactic response to soluble sugars in the diet has been suggested as a possible cause for the migration of Isotrichidae into the rumen contents at the time of feeding (Orpin and Letcher, 1978; Murphy et al.,1985). Abe et al.

(1983) also suggested that the act of ingesting feed and the contrac tions of the reticulum during eating or the anticipation of feed may also be involved. Nakamura and Kurihara (1978) also provided in vi tro evidence for protozoal migration and sequestration or attachment to particulate matter. Using an agitated, continuous in vitro fermentation system, with the substrate added in nylon bags, protozoa rapidly migrated from a previously incubated bag to a new bag added to the in vitro system. The concentration of protozoa in both bags were similar within 2 h and less than 10^ of the numbers in the bags occurred in the free fluid.

Experiment 4

Protozoal concentrations in the rumen are at their lowest ap proximately 6-8 h postfeeding (Purser and Moir, 1959; Warner, 1966; 113

Potter and Dehority, 1973). Experiment 4 was designed to determine if rumen protozoal concentrations increase from 6 to 24 h postfeeding as a result of multiplication and whether these changes, if any, also occur in the containers. This experiment differs from Experiment 3 in that the container contents were counted at 24 h. In this experi ment ruminal pH increased from 6.49 at 6 h postfeeding to a high value of 7-01 at 24 h postfeeding (Table 15 and Figure 7). Although pH in the containers at 24 h postfeeding was lower than that in the rumen and the initial pH at 6 h postfeeding, it was apparently not low enough to cause death of protozoa in the containers. Both the total protozoal and Ophryoscolecidae concentrations increased (P<.05) in the container and the rumen from 6 to 24 h postfeeding; however, no significant increase in Isotrichidae concentration was observed.

The increase in concentration of total protozoa and Ophryoscolecidae was presumably due to multiplication, because a similar increase oc curred in both the rumen and containers. Isotrichidae concentration in the rumen was low at 6 h postfeeding and remained low in both the rumen and containers at 24 h postfeeding, although their concentra tion tended to increase slightly in the rumen 24 h postfeeding, prob ably as a result of migration into the rumen.

Experiment 5

Experiment 5 was designed to determine if the decline in pro tozoal concentrations 6 h postfeeding is due to lysis, the dilution 114

TABLE 15. pH AND PROTOZOAL CONCENTRATIONS IN RUMINAL CONTENTS AND CONTAINERS AT 6 AND 24 H POSTFEEDING (EXP. 4)

Rumen and container Rumen Container Item 6 h 24 h 24 h SE pH 6.49a 7.01b 6.11° .01 Total protozoal x loVml 8.88a 12.60b 12.l6b .60 Ophryoscolecldae x loVml 8.24a 11.32b 11.38b .42 Isotrichidae x loVml .64 1.28 • 78 .19 a»bi° Means in the same row with different superscripts differ (PC.05). x CL IUE . Hadpoool ocnrtos nrmnl otns and contents ruminal in concentrations protozoal and pH 7. FIGURE PROTOZOAL CONCENTRATION(X1 O'*/UL) 14 12 10 8 4 6 0 2 0 0 oties t ad 4h otedn (x. 4) (Exp. postfeeding h 24 and 6 at containers ■ rumen • -o Ophryoscolecidae, Ophryoscolecidae,container •■ — * Isotrichidae,container isotrichidae, rumen rumen isotrichidae, Total protozoa, rumen rumen protozoa, Total oa protozoa,container Total 6 6 container filled container TIMEAFTER FEEDING, H 12 12 * container 0 rumen rumen 18 18 24 24 115 116

effects of feed and water and(or) passage out of the rumen. pH val

ues followed the general trend of being highest just before feeding

and then decreased (P<.05) both in the containers and the rumen 6 h

post feeding (Table 16 and Figure 8). There was no change in Iso

trichidae concentration in the rumen or containers between 0 h

(prefeeding) and 6 h postfeeding; however, total protozoal and

Ophryoscolecidae concentrations In the rumen and the containers de

creased (P<.05) from 0 to 6 h postfeeding. These decreases in total

protozoal and Ophryoscolecidae concentrations were greater (P<.05) in

the rumen than in the containers. The decrease in concentration of

total protozoa and Ophryoscolecidae in the containers was probably

the result of cell lysis. Ophryoscolecidae are not known to migrate.

Also, because the containers prevented passage, and counts of proto zoa in the containers at 6 h were intermediate between counts in the rumen at 0 and 6 h, it appears that about 5036 of this decrease in concentration was due to dilution and passage and 50? was due to lysis.

The diurnal fluctuation in total protozoal and Ophryoscolecidae concentration in the rumen, observed in Experiments 4 and 5 was simi lar to that reported by Warner (1966) and Potter and Dehority (1973).

The latter authors attributed the fall in protozoa numbers 1-6 h postfeeding to dilution by feed, saliva, drinking water and passage of digests from the rumen, but the Increase In protozoal numbers at 117

TABLE 16. pH AND PROTOZOAL CONCENTRATIONS IN RUMINAL CONTENTS AND CONTAINERS AT 0 AND 6 H POSTFEEDING (EXP. 5)

Rumen and container Rumen Container Item O h 6 h 6 h SE

PH 7.15a 6.50b 6.63° .02 Total protozoal x loVml 20.88a 13-88b 17-32° .38 Ophryoscolecidae x loVml 19.24a 12.80b 15-98° .27 Isotrichidae x loVml 1.64 1.08 1.3*1 .19 a>b>° Means in the same row with different superscripts differ (P<.05). 118

8.000

0 ------° rumen ------4 container

7. 500-

x Q_ 7. 000-

container filled and steer fed 6.000 0 2 4 6 R 22 20 o X 18 x" o 16

TIME AFTER FEEDING, H FIGURE 8. pH and protozoal concentrations in ruminal contents and containers at 0 and 6 h post feeding (Exp. 5) 119

6-24 h postfeeding via3 attributed to protozoal cell division. Al though no change in Isotrichidae concentration was observed between 0 and 6 h postfeeding, migration probably occurred after feeding and the Isotrichidae had sequestered by 6 h.

Experiment 6

The objective of experiment 6 was to determine whether protozoa would lyse in the rumen when feed wa3 withheld from the host animal for 24 h. The concentration of total protozoa and Ophryoscolecldae in both the ruraen and the containers decreased sharply following a 1- d withholding of feed from the steer (Table 17 and Figure 9). This indicated that Ophryoscolecidae lysed in the rumen when substrate was lacking. The Isotrichidae concentrations in the rumen were greater

(P<.05) at 48 h than at 24 h or in the container at 48 h. This may have been due to migration of sequestered Isotrichidae into the rumen digesta (Dehority and Tirabasso, 1987).

Total protozoal concentrations decreased by 82 and 79? in the ruraen and containers, respectively, between 24 and 48 h postfeeding.

The corresponding values for Ophryoscolecidae were 86? and 81?, re spectively. Potter and Dehority (1973) also observed that protozoal numbers decreased by 80? and fluid turnover rate was negligible when sheep were starved for one day. However, ruminal infusion of low or high levels of soluble substrate (starch, glucose and casein) main tained 68? and 94? of the original protozoal numbers respectively. 120

TABLE 17. pH AND PROTOZOAL CONCENTRATIONS IN RUMINAL CONTENTS AND CONTAINERS AT 24 AND 48 H POSTFEEDING (EXP. 6)

Ruraen and container Rumen Container Item 24 h 48 h 48 h SE

PH 7 .16a 7.8813 7.10a .02 Total protozoal x loVml 19.28a 3.48b 4.12b 2.43 Ophryoscolecidae x ID1*/ml 17.04a 2.12b 3.32b 2.38 Isotrichidae x loVml 1.47a 2.24b 1.36a .12 a »b Means in the same row with different superscripts differ (PC.05). 121

8.000

container filled 7. 500-

£ 7. 000-

° rum en * container Fed 6.000 0 24 48

5 20

18- o 16- 2 °Total protozoa.rumen O 14- •*Total protozoa.container !< cz 12- i— 2 10- •° Ophryoscolecidae,rumen UJ o ■* Ophryoscolecidae.container X 8 - o o------o isotrichidae,rumen o 6 - *------a Isotrichidae,container < o 4 - M o I— 2 o cz 0 Cl 0 2 4 TIME AFTER FEEDING, H FIGURE 9. pH and protozoal concentrations in ruminal contents and containers at 24 and 48 h postfeeding (Exp. 6) 122

These authors concluded that, because the high level of soluble sub

strate was able to maintain the protozoal population when turnover was minimal, substrate could be the major factor controlling the pro

tozoal population at low feed levels. They also concluded that the

disappearance of protozoa after starvation must have been the result

of cell lysis and not passage out of the rumen. Because passage out

of the rumen cannot occur for protozoa in the containers, the sharp

decrease (P<.05) in protozoal concentrations in both the rumen and

containers at U8 h (Table 17 and Figure 9) must have been due to ly

sis. This situation would lead to wasteful recycling of protozoal

protein in the rumen and subsequent loss of both protein and energy

to the host animal.

It was interpreted from results of these experiments that the observed diurnal fluctuations in rumen Isotrichidae concentrations are primarily due to sequestration and migration. Ophryoscolecidae do not seem to exhibit the phenomena of sequestration and migration to any significant extent. When animals were fed a restricted intake diet once a day, about 50% of the decrease In Ophryoscolecidae con centration immediately after feeding resulted from dilution effects and passage out of the ruraen. The remaining 50% of the decrease ap peared to be from cell lysis. The results also suggest that de creases in rumen Ophryoscolecidae concentration during periods of substrate restriction are probably due to lysis, with only minimal passage out of the rumen. 123

The selective container may be useful in future investigations to determine the contribution of mixed or known rumen ciliate proto zoa species to feed degradation in situ. LITERATURE CITED

Abe, M., T. Iriki, N. Tobe and H. Shibui. 1981. Sequestration of holotrich protozoa in the reticulo-rumen of cattle. Appl. En viron. Microbiol. 41:758.

Abe, M., Y. Suzuki, H. Okano and T. Iriki. 1983. Specific differen ces in fluctuation pattern of holotrich concentration in the ruraen of cattle, goat and 3heep. Jpn. J. Zootechnol. Sci. 54:457.

Bauchop, T. and R. T. Clarke. 1976. Attachment of the ciliate Epidiniura crawley to plant fragments in sheep ruraen. Appl. En viron. Microbiol. 32:417.

Bird, S. H. and R. A. Leng. 1978. Ruminal protozoa and growth in lambs. Proc. Austr. Soc. Anira. Prod. 12:137.

Clarke, R. T. J. 1965. Diurnal variations in the number of ciliate protozoa in cattle. N.Z. J. Agric. Res. 8:1.

Dehority, B. A. 1984. Evaluation of subsampling and fixation pro cedures used for counting rumen protozoa. Appl. Environ. Micro biol. 48:182.

Dehority, B. A. and W. R. S. Mattos. 1978. Diurnal changes and ef fect of ration on concentrations of the rumen ciliate Charon ventriculi. Appl. Environ. Microbiol. 36:953*

Dehority, B. A. and P.A. Tirabasso. 1987* Factors affecting the migration and numbers of rumen protozoa in the family Iso trichidae. Proceedings of the Nineteenth Biennial Conference on Rumen Function, Chicago, IL, Nov. 17-19. 19:40 (Abstr.).

Demeyer, D. I. 1981. Ruraen microbes and digestion of plant cell walls. Agric. Environ. 6:295.

Fina, L. R., C. L. Keith and E. E. Bartley. 1962. Modified In vivo artificial rumen (VIVAR) techniques. J. Anlm. Sci. 21:930.

Hungate, R. E. 1966. The Rumen and its Microbes. Academic Press, New York.

Hungate, R. E., J. Reichl and R. Prins. 1971. Parameters of rumen fermentation in a continuously fed sheep: evidence of a micro bial rumination pool. Appl. Microbiol. 22:1104.

124 125

Jouany, J. P. and J. Senaud. 1979- Role of rumen protozoa In the digestion of food cellulosic materials. Ann. Rech. Vet. 10:261.

Leng, R. A., D. Dellow and G. Waghorn. 1986. Dynamics of large pro tozoa in the rumen of cattle fed on diets of freshly cut grass. Brit. J. Nutr. 56:455.

Lindberg, J. E., A. Kaspersson and P. Ciszuk. 1984. Studies on pH, number of protozoa and microbial ATP concentrations in rumen- incubated nylon bags with different pore sizes. J. Agric. Sci. 102:501.

Meyer, J. H. F. and Mackie, R. I. 1986. Microbiological evaluation of the intrarurainal in 3acculus digestion technique. Appl. En viron. Microbiol. 51:622.

Murphy, M. R., P. E. Drone, Jr. and S. T. Woodford. 1985. Factors stimulating migration of holotrich protozoa into the rumen. Appl. Environ. Microbiol. 49:1329.

Nakamura, F. and Y. Kurihara. 1978. Maintenance of a certain rumen protozoal population in a continuous in vitro fermentation sys tem. Appl. Environ. Microbiol. 35:500.

Orpin, C. G. and A. J. Letcher. 1978. Some factors controlling the attachment of rumen holotrich protozoa Isotricha intestinalls and JT. prostoma to plant particles in vitro. J. Gen. Microbiol. 106:33.

Potter, E. L. and B. A. Dehority. 1973- Effects of changes in feed level, starvation and level of feed after starvation upon the concentration of rumen protozoa in the ovine. Appl. Microbiol. 26:692.

Punia, B. S., J. Leibholz and G. J. Faichney. 1987. The role of rumen protozoa in the utilization of paspalum (paspalura dilata- tum) hay by cattle. Brit. J. Nutr. 57:395.

Purser, D. B. 1961. A diurnal cycle for holotrich protozoa of the rumen. Nature 190:831.

Purser, D. B. and R. J. Moir. 1959- Ruminal flora studies in the sheep.IX. The effect of pH on the ciliate population of the rumen in vivo. Aust. J. Agr. Res. 10:555.

Steel, R. G. D. and J. H. Torrie. 1960. Principles and Procedures of Statistics. McGraw-Hill Book Co., New York. 126

Warner, A. C. I. 1962. Some factors influencing the rumen microbial population. J. Gen. Microbiol. 28:129-

Warner, A. C. I. 1966. Diurnal changes in the concentration of microorganisms in the rumens of sheep fed limited diets once daily. J. Gen. Microbiol. 45:213*

Weller, R. A. and A. F. Pilgrim. 1974. Passage of protozoa and volatile fatty acids from the rumen of the sheep and from a con tinuous in vitro fermentation system. Brit. J. Nutr. 32:341. CHAPTER V

SUMMARY

A quantitative method of analysis for 2-Aminoethylphosphonic acid (AEP) was developed using reversed-phase HPLC. The detection limit for AEP was 15 nM and the detector response (peak area) was linear for AEP levels ranging from 15 nM to 100 pM with a correlation coefficient of .99. Mean recovery of AEP added to strained ruminal fluid from faunated 3heep wa3 98.2%. When AEP was added to a fermen tation mixture at a concentration of 22.6 yg/ml, 78% disappeared dur ing a 21! h incubation. 2-Aminoethylphosphonic acid was readily de tected in preparations of mixed ruminal ciliate protozoa and in mixed and pure strains of ruminal bacteria, feedstuffs, and ruminal fluid and duodenal digesta from defaunated sheep. The occurrence of AEP in feed and bacterial hydrolysates was confirmed by both HPLC and or ganic phosphorus analyses. Because the occurrence of AEP was not found to be limited to ruminal ciliate protozoa, it is of little use as a marker for protozoal passage out of the rumen.

In a second study, a 56-d growth trial was conducted to deter mine the effects of defaunation and protein source on lamb perfor mance. A 2 x 2 factorial arrangement of treatments was used to com pare faunation vs defaunation and soybean meal vs fish meal. These protein supplements were added to achieve 9.5% dietary protein. An

127 128

additional positive control treatment (faunated lambs fed supplemen

tal soybean meal at 12% protein) was also included. Lambs fed the

positive control diet had greater (P<.05) gains than faunated lambs

fed soybean meal at 9.5% protein (201 vs 114 g/d, respectively) in

dicating 9.5% protein was below the requirement for maximum growth.

Lambs fed fish meal (compared to those fed soybean meal) had greater

gains (224 V3 133 g/d), greater dry matter intakes (1205 vs 1042 g/d)

and improved feed conversion (5.5 V3 8.5 kg/kg), respectively

(P<.05). Performance was not affected by defaunation and no protein

source x protozoa status interactions existed (P>.05).

In the third study, selective containers which when placed in

the ruraen would be permeable to bacteria and soluble substrate but not protozoa were developed. These containers were used to investi gate the phenomena of protozoa sequestration, migration and lysis in

the rumen. These investigations suggested that the diurnal fluctua tions in rumen Isotrichidae concentrations are predominantly due to sequestration and migration. Ophryoscolecldae did not exhibit se questration and migration to any significant extent. The results also suggest that decreases in Ophryoscolecidae concentration during periods of substrate restriction are probably due to lysis in the rumen and passage out of the rumen is limited. APPENDICES 130

APPENDIX A. pH CHANGES WITH TIME IN BEAKERS (CONTAINING ACETIC ACID) AND IN CONTAINERS (CONTAINING BUFFER SOLUTION). A) NO AGITATION OF BEAKERS

Time after immersion of pHa in: containers in beakers (H) Beakers Containers

0 4.22 7.06 .5 4.59 6.94 1 4.69 6.82 2 4.83 6.58 3 4.92 6.52 4 4.95 6.38 5 4.98 6.21 6 4.99 6.11 7 5.03 5.99 8 5.02 5.92 24 5.10 5.52 a Mean of two replicates. 131

APPENDIX B. pH CHANGES WITH TIME IN BEAKERS (CONTAINING ACETIC ACID) AND IN CONTAINERS (CONTAINING BUFFER SOLUTION). B) BEAKERS AGITATED

Time after immersion of pHa in: containers in beakers (H) Beakers Containers

0 3.83 7.04 .5 4.28 7.00 1 4.45 6.96 2 4.63 6.78 3 4.75 6.65 4 4.81 6.56 5 4.87 6.43 6 4.92 6.31 7 4.95 6.21 8 4.96 6.05 24 5.05 5.69 a Mean of two replicates. 132

APPENDIX C. RATE OF FLUID TURNOVER IN SELECTIVE CONTAINERS PLACED IN RUMEN

Standard PEG concentrations, mg/ml Optical Densitya

0 0 .0024 .13 .0071 .32 .0188 .58 .0235 .87 .047 1.20

Samples

PEG concentration, 0 h .925 Container 1, PEG concentration, 6 h .45 Container 2, PEG concentration, 6 h .39 a Mean of two replicates.

Regression equation for standard curve:

Y = 0.0977 + 25.4453X

r = 0.9753 Y = O.D. X = PEG concentration, mg/ml Dilution factor for PEG concentration at 0 h = XL X JL2. = 42.5 2 2

PEG concentration at 0 h = 1.3818 mg/ml.

Dilution factor for PEG concentration at 6 h (container 1) = 42.5 PEG concentration at 6 h (container 1) s 0.5884 mg/ml.

Dilution factor for PEG concentration at 6 h (container 2) = 42.5 PEG concentration at 6 h (container 2) = 0.4882 mg/ml.

Regression equation of Ln PEG concentration vs time (Container 1): Y = 0.3234 - 0.1423X r = -1 Y = ln PEG conc. X - time (h)

Turnover of PEG in container 1/d = 3.42

Regression equation of Ln PEG concentration vs time (Container 2): Y = 0.3234 - 0.1734X

r = -1 Y = ln PEG conc. X = time (h)

Turnover of PEG in container-2/d = 4.16 BIBLIOGRAPHY

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INFORMATION to USERS the Most Advanced Technology Has Been Used to Photo­ Graph and Reproduce This Manuscript from the Microfilm Master

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