Evaluation of different methods to improve the utilization of roughage fed to sheep

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Evaluation of Different Methods to od.

Utilization of Roughage Fed to Sheep

A thesis submitted to the

University of Adelaide

for the degree of

Master of Applied Science (Agriculture)

Peng Hai Hong

Department of Animal Science The University of Adelaide

January 1995 by

6.f.{c

¿/BR c ()

i: lt UNly

I

Table of Contents List of Íigures

List of tables Summary

Acknowledgements

Chapter 1. Introduction and Literature Review

Introduction Literature review

l. Plant aspects that influence lignocellulose utilization by ruminants

1.1. Plant cell

wall

1.1.1. Chemical properties of the plant cell walls 1.1.2. Physical properties of plant cell walls 1.1.3. Degradation of plant cell

walls

2. Animal aspects that influence the utilization of roughage

2.1. Mastication of ligonocellulose during ingestion and rumination 2.2. Rumen micro-organisms

2,2.I.

Rumen bacteria

2.2.I.1. Cellulolytic

bacteria 2.2.L2.

Amylolytic

bacteria 2.2.I.3 .

koteolytic

bacteria 2.2.2. Rumen protozoa 2.2.3. Rumen

fungi

2.3.Enzymes involved in cell

wall

degradation

2.3.L

Cellulases 2.3.2. Xylanases

2.4. Influence of various factors on rumen micro-organisms 2.4.L. Diurnal variation of population

2.4.2. Nutrient

limitation

2.4.3. Effects

of

diet 2.4.4.

Effect

of rumen

pH

3. Methods to improve feed utilization

3.1. Chemical treatment of fibrous plant materials

VI VII

vrII x

1

1

2

2 2 3

4 4

6 7

7

I

8 9 10 10 11

t2

12 13 13

t4 r4

15 16 16 T6

II

3.2. supplying the nutritional requirements of rumen

microflora

to ensure maximum microbial activity and microbial growth

yield

3.2.1.

l,evel

of feeding

3.2.2. Ratio of concentrate to roughage 3.2.3. Nitrogen and sulphur

3.2.4. Supplementation of microbial feeds

33. Modifying

microbial populations 3.3.1.

Addition

of antibiotics

3.3.2. Elimination of rumen protozoa

3.3.3.

Addition

of protein degradation protectors 3.3.4.

Addition

of methane

inhibitors

3.3.5.

Addition of buffer

substances

4. Genetic engineering of rumen bacteria to optimise rumen fermentation

4.1. Potential objectives

for

improving ruminant production by genetic engineering of rumen bacteria

5. Conclusions

t7 l7

18 18 20

2t 2I

23 25 25 26

26

27 28

Chapter 2. Effects of difïerent supplements on the digestibility and Nitrogen balance of sheep fed low quality roughage

³⁰

2.l.Introduction

30

2.2.

Materials

32

2.2.I. Sheep

32

2.2,2. Feeds and

feeding

32

2.2.3.

Chemicals

32

2.3.

Methods

33

2.3.1,

Dry

matter content of feed and

faeces

33 2,3.2.

Acid

detergent fibre content of feed and

faeces

33 2.3.3, Nitrogen content of feed, faeces and

urine

³³

2.3.4. Energy

content

34

2.3.5. In vivodigestibility

34

2.3,6. Nitrogen

balance

35

2.3.7. Statistical

analysis

35

2.4.

Results

36

2.4.1. Feed intake and

composition

36

2.4.2. InvívodigestibilityandNbalance

36

2.5. Discussion and

conclusions

39

III

Chapter 3. Effects of yeast supplement on the digestibility and ruminal ammonia concentration of sheep fed low quality roughage 4I

3.1.

Introduction 4I

3.2.

Materials

42

3.2.L

Sheep and experimental

design

42

3.2.2. Feeds and

feeding

42

3.3.

Methods

43

3.3.1. Feed faecal

analysis

43

3.3.2. In vivodigestibility

43

3.3.3. Invitrodrymatterdigestibility

43

3.3.3.1. Sample

preparation

43

3.3,3.2. Rumen

fluid

and

buffer

43

3.3.3.3.

Incubation 4

3.3.3.4.

Dry

matter

digestibility 4

3.3.4, Ruminal ammonia

concentration 4

3.3.5. Statistical

analysis

⁴⁵

3.4.

Results

45

3.5. Discussion and

conclusions

⁴⁷

Chapter 4. Effects of Avoparcin supplement on the performance of

sheep fed low quality roughage

⁵⁰

4.l.Introduction

50

4.2.

Materials

⁵¹

4.2.I. Sheep 5l

4.2.2. Feeds and

feeding

⁵¹

4.3.

Methods

52

4.3.1.

Body

weight

changes

52

4.3.2. Ruminal protozoa

number

⁵²

4.3.3. Blood plasma urea N

concentration

⁵²

4.3.4. Blood plasma glucose

concentration

⁵³

4.4. Statistical

analysis

⁵³

4.5.

Results

54

4.5.1. Effects on body weight

change Y

4.5.2. Effects on concentration of plasma

glucose

54

4.53.

Effects on concentration of plasma urea

N Y

4.5.4. Effects on the density of protozoa

number

55

4.6. Discussion and

conclusions

57

IV

Chapter 5. Studies of novel rumen bacteria Clostridium chørtøtabidum

on fïbre digestion

⁶⁰

S.l.Introduction

⁶⁰

5.2.

Materials

⁶¹

5.2.1.Chemicals 6l

5.2.2. Bacterial strains and plasmid pCel

1

⁶¹

5.2.3. Cultural

medium

⁶²

5.2.4.

Buffer

and

solutions

⁶³

5.2.5. Sheep and

feed

⁶⁵

5.3.

Methods

⁶⁵

5.3.1. Medium

preparation

⁶⁵

5.3.2. Collection of rumen

samples 6

5.3.3. Cultivation of rumen

bacteriaforcounting

^arrd

isolation 6

5.3.4. Counting total and

cellulolytic

bacteria ^and isolation of bacteria

producing orange

colonies 6

5.3.5. Chromosomal

DNA extraction

⁶¹

5.3.6.

Dgestion

of chromosomal

DNA

and gel

electrophoresis

⁶⁷

5.3.7. Southern

transfer

⁶⁸

5.3.S.

Oligo-labelling

^{of a}

DNA probe

⁶⁸

5.3.9. Prehybridization and

hybridization

⁶⁸

5.3.10, Metabolic test and analysis of

VFA

products

of

C. straín

pl

⁶⁹

5.3.11. Sample preparation and inoculation

for invítrodigestibility

⁶⁹

5.3.12. Invitrodrymatterdigestibility

^7O

5.3.l3.Inoculation

and monitoring the survival of

C.chartatabidum

⁷⁰

5.3.14.

Insacco digestibility of lucerne

chaff

⁷⁰

5.3.15. Statistical

analysis

⁷²

5.4.

Results

⁷²

5.4.1. Elfect of diets on total and

cellulolytic

bacteria in rumen and

presence

of C.chnnatabidum

⁷²

5,4.2. Homology between C.chartalabidumand C.

strainpl

⁷³

5.4.3. Metabolic test and fermentation products analysis

of

C. strain

pl

⁸⁰

5.4.4.

Effect

of monoculture

of

C.clnrtalabidann, C.

strainpl

and R. albus or co-culture with mixed rumen bacteria on oaten chaff digestion

invitro

⁸²

5.4.5.

Survival of C.clnrtatabidurninvivo A

5.4.6. Effect of introducing C. chartatabiduminto rumen

oîinsacco

digestibility of lucerne

chaff

⁸⁵

5.5. Discussion and

conclusions

⁸⁶

V

5.5.1. Effect of diets on total and

cellulolytic

bacteria in rumen and presence

of

C.clnnanbidum

⁸⁶

5.5.2. Homology between C.chartatabídum ard C. straín

pI

⁸⁶

5,5.3. Metabolic test and fermentation products analysis of C. strøin

pI

⁸⁷

5.5.4.

Effect

of monoculture

of C.clnrtarabidum,

C. strain

pl

and R. albus or co-culture with mixed rirmen bacteria on oaten chaff

digestioninvitro

88

5.5.5. Survival

of C.chørtatabiduminvívo

⁸⁸

5.5.6. Effect of introducing ^C.

clnrtatabiduminto

rumen on.insacco

digestibility of lucerne

chaff

⁸⁹

Chapter 6 General discussion, conclusions and future studies

6.1. Genenal discussion and conclusions

6.2. Future studies

References

90

90 94

95

VI

List of figures

Figure 5.1. Cell and spore morphology

of

C. clwrta.tabidum and C.

strainpl

Figure 5.2. Gram-stain

of C.clnrtatabidwn

and C.

strainpl

Figure 5.3. Orange colonies formed by C.chartatabidumand C.

strainpl

Figure 5.4. Clear zones formed

by

C.chørtatabidum and C. strain

pl

Figure 5.5. Growing

of

C. strain

pl

on modifìed M2

with

cellulose and xylan

as sole carbon-source

Figure 5.6. Homology test

of

C.chartatabídum attdC,

straínpI

by probing

with pCell DNA

Figure 5.7. Homology test

of

C.chartatabidum and C.

straínpI

by probing

with DNA

of

C.clnnanbidum

74

75

76 77

78

19

79

VII

List of tables

Table

2.LFeedintake

of sheep fed supplemented roughage diets

Table 2.2.Feed composition of two supplemented roughage diets fed to sheep Table 2.3.

In

vivo

digestibility

^and N balance of sheep fed supplemented roughage

diets

Table 3.1.

In vivo, Invitro

digestibility and ruminal ammonia concentration

of

roughage diet

with

or without yeast supplement in sheep Table 3.2. Feed composition

Table 3.3. Composition of yeast culture (Yea-sacc) Table 4.1. Feed and supplements

Table

4.2.Effect

of Avoparcin on body weight change of sheep

Table

4.3.Effect

of Avoparcin on glucose concentration in blood plasma Table

. .Elfectof

Avoparcin on concentration of plasma urea

N

Table 4.5. Effects Avoparcin on protozoal population

Table 5. 1. Effect of diets on density of total and

cellulolytic

bacteria in the rumen Table 5.2. Distingui shing characteristics of cellulolyti c c ln stridia

Table 5.3. Metabolic test of C.

strainpl

Table 5.4.

Digestibility

of oaten chaff by monoculture or co-cultured bacteria Table 5.5. Density

of

C . chartatabidum

in in

vivo conditions after inoculation Table 5.6. Effect of introducingC.chnrta.tabidum on insacco

digestibility of

lucerne

chaff

38 37 37

M

47 47 52 55 55

%

56 73 80

8l

83

u

85

VIII

Summary

The

effects

of different

supplements on

digestibility

and

N

balance

of

sheep

fed

an oaten

chaff

based

diet

were studied.

No differences in in vivo digestibility

and

N

balance

of

sheep supplemented

with

either lucerne

chaff or

lucerne

chaff

^plus oaten grain were observed. However, both groups

of

sheep had a

positive N

balance.

This

compares with other studies in which a negative N balance was obtained

with

sheep fed oaten chaff supplemented with urea. This suggested that supplementation of a roughage diet with highly digestible feed or readily fermentable feed can improve the efhciency

of

nitrogen utilization in sheep.

The

effects

of

yeast

culture

supplementation

on ín vivo, ín vítro digestibility

and ammoniaconcentrationinsheepfedanoatenchaff baseddietwere studied. There were no

significant

differences

in in vivo

and ín

vitro dígestibility

between treatment and

control

groups. However, the treatment group

of

sheep had a

slightly lower ruminal ammonia concentration than the control group. This may indicate that

improved microbial growth was obtained after yeast culture supplementation.

The

effects

of Avoparcin

supplementation on body

weight,

plasma glucose, plasma urea N and ruminal protozoa were studied. No significant differences in plasma glucose concentration, plasma urea N concentration and protozoal number were found between treatment and controls.

However

sheep

with Avoparcin

supplementation tended to maintain ^a higher body weight than the control group.

The contribution

of

Clostrídíumchartatabidum,

ahighly cellulolytic

rumen bacterium, to fibre digestion was studied. Its

ability

to degrade oaten chaff

in vitro

was compared

with

Ruminococcus

albus,

Clostridium

\ü16

(isolated during

this

study) or co-culture

with mixed rumen bacteria. The results

demonstrated

a synergistic effect

among

IX

ruminal bacteria on fibre digestion. The

survival of

C. chartatabidum after introduction into the rumen was studied. The results showed that this bacterium could be maintained at around 106 cells per

ml of

rumen

fluid

over a three month period. The effect

of C.

clnrtalabidumon in

sacco

digestibility of lucerne chaff was studied. The

results indicated that the

initial (2l

hours) rate

of

digestion

of

neutral detergent

fibre (NDF)

digestion was

significantly

improved.

No significant

differences

in NDF digestibility

were observed at 48 hour

and72

hours. No differences

in

dry

matter(DM) digestibility

were obtained

during

the experimental periods.

DNA-DNA hybridization,

metabolic tests and fermentation product analyses were used

to identify

the

new isolate,

as a previously undescribed ruminal species of Clostrídium.

X

Acknowledgements

I

would

like

to thank the

following

^people

for

their assistance and support

during

the course of this thesis:

Drs Jim Gallagher and John Brooker, to whom

I

am greatly indebted

for

guidance and encouragement

during

the supervision

of this project

and

for help

and constructive criticism of this thesis;

Dr

Helena W'ard,

for

her guidance during rumen

microbiology

and molecular

biology

studies, the provision of plasmid

DNA

pCel 1 and

helpful

criticism of the thesis;

Dr David Taplin, for his assistance with feed energy analysis;

Dr Peter Clifton,

for

the help of plasma urea N analysis;

Dr

Andrew Butcher,

for

the analysis

of fermentation

products

of

the

newly

isolated Clostridum;

Drs Cindy Bottema and

Andrew

Thomson,

for their

valuable suggestions during this study;

All

the people

in Dr Brooker's laboratory, the staff

and students

of Department of

Animal Science,

for

their help and friendship, and

particularly Mrs

J. Prosser and

Mr

R. Connolly and

Mr

^H.

Bozyk;

My

sincerestthanks to

my

parents, Peng Cheng and Hwang Mei-Rong,

for

their love andencouragement;

Finally,I

would

like

to acknowledge the

Australia-China

Sheep Research Project

for

financial support.

XI

XI

Statement

This thesis contains no material which has been accepted

for

the award

of

any other degree

or diploma in any University

^and

to the best of my knowledge and belief

contains no material previously published or

written by

another person, excePt where due reference

is

made

in

the

text. I

consent

to this

thesis being made available

for

photocopying and loan.

Peng Hai Hong

30/L/9s

.l