A comparison of alternative sources of inocula in an in vitro digestibility technique

Original

article

A

_comparison

of alternative

sources

of inocula

in

an

in vitro

_{digestibility}

technique

AES Borba

JMC

Ramalho Ribeiro

1

Departamento

de Ciëncas

_Agrarias

da Universidade dos

_Açores,

9700

Angra

do Heroismo,

Açores;

2

_Departamento

_de

Nutriçâo

da

_Estaçâo

Zootécnica Nacional, 2000 Vale de Santarém,

Portugal

(Received

16 March 1994;

accepted

20

_{April 1995)}

Summary —

In this paper we make the

_comparison

between sources of inocula for an in vitro

digestibility

method based on that of

_Tilley

and

_{Terry (1963)}

as modified

_by

Alexander and McGowan

(1966).

The best results for the

prediction

of the in vivo

digestibility

were obtained

using

the traditional method of rumen _liquor from fistulated

sheep.

However, with the two alternative methods, one _using rumen _liquor from

slaughtered

cattle, and another, a

_sheep

faeces

suspension

as inocula sources, we

obtained results

_{significantly}

correlated with in vivo

_{digestibility}

at the 5% level,

although

with lower

cor-relation coefficients. The method that uses rumen

_liquor

from

_slaughtered

cattle is, in our

_opinion,

a valid alternative to the traditional method.

in vivo

_{digestibility}

/ in vitro

digestibility

/ sources of inocula / alternative methods

Résumé —

_Comparaison

de sources alternatives d’inoculum pour la mesure de la

_{digestibilité}

in vitro. Dans cet essai, nous avons

_comparé

l’effet de trois sources _{d’inoculum pour la} détermination

de la

_{digestibilité in}

vitro

_{par la}

méthode de

_{Tilley et Terry}

₍₁₉₆₃₎ modifiée par Alexander et McGowan

(1966).

Les sources d’inoculum étaient :

du jus

de rumen de moutons fistulés

(méthode

tradition-nelle), du

jus

de rumen de vaches abattues,

prélevé

à l’abattoir

(méthode

de Nikolic et ai,

1987)

et des fèces de moutons

(méthode proposée

par El Shaer et al, 1987). Au total, 24 échantillons de

gra-minées ont été

employés:

avoine, ray-grass d’Italie, ray-grass

anglais

et mais, à trois stades de

déve-loppement,

en vert et

_{après ensilage.}

Le coefficient de

_{digestibilité}

in vivo des

_{fourrages (tableau 1)}

a

été déterminé sur six moutons adultes, mâles, alimentés ad libitum

pendant

une

période

de 21

jours,

14 _jours

_{d’adaptation}

et

_{7 jours}

de mesure. Les meilleurs résultats pour la

prévision

de la

digestibilité

in vivo furent obtenus avec la méthode traditionnelle, avec

du jus

de rumen

prélevé

sur des moutons

fistulés. Des liaisons

_{significatives (niveau}

5

%)

avec la

_{digestibilité}

in vivo ont aussi été obtenues

avec les méthodes alternatives, mais les R2

_{sont plus}

faibles (tableau 111). La méthode _qui utilise du jus

de rumen de vaches abattues est

cependant

une bonne alternative.

INTRODUCTION

Digestibility

is the most

_important

_biological

measure of a

_forage.

It can be measured in

vivo,

but this method is

laborious,

consumes

large

amounts of

_forage

and

_requires

a

rel-atively large

number of animals.

Therefore,

several

_laboratory

methods have been

pro-posed

for this estimation. The in vitro meth-ods

_attempt

to simulate the natural

rumi-nant

_digestive

processes. The method of

Tilley

and

_{Terry (1963)}

modified

_by

Alexan-der and McGowan

₍₁₉₆₆₎

can be consid-ered the most

usual,

presenting

however,

one

_{disadvantage,}

in that it

_requires

fresh inocula from

_permanently

fistulated animals. With the

_objective

of

_avoiding

this

obstacle,

several authors have

proposed

alternative methods. Until

_recently

these methods were

mainly

chemical,

_physical

and

_enzymatic,

in which the

cellulolytic

function of the rumen

liquor

is substituted

_by

an

_enzymatic

prepa-ration. More

_recently,

however,

other

labo-ratory techniques

have been

_proposed.

A modified

_{two-stage technique}

is based on

the classic

_{procedure (Tilley}

and

_Terry,

1963),

but

_employing

bovine rumen fluid from a

_{slaughterhouse (Nikolid}

et

al,

1987),

and the method

_{proposed by}

El Shaer et al

(1987)

based on the use of faecal

microor-ganisms

contained in a filtered

_suspension

of

_sheep

faeces. The microbial methods have clear

advantages

over the

enzymatic

techniques

in the

_prediction

of the in vivo

digestibility

(Omed

et

al,

1989).

Our

_objective

is to determine if the alter-native sources of inocula can substitute for the traditional source

_{(rumen liquor}

from fis-tulated

_sheep)

in the determination of in vitro

digestibility by

the method of

_Tilley

and

_Terry

(1963)

modified

by

Alexander and McGowan

_(1966),

and _{therefore go}

_beyond

the inconvenience of the method: the need for fistulated animals. The use of alterna-tive sources of inocula make a

_significant

contribution to the welfare of the

_laboratory

animals,

and

_simplify

the method because it

is much easier to obtain

_sheep

faeces or rumen

_liquor

from

_slaughtered

cattle than

rumen

_{liquor from}

fistulated

_sheep.

MATERIALS AND METHODS

Forage

samples of 24

graminaceae (oat,

Italian

ryegrass, perennial ryegrass and

_maize)

in three

stages of

growth,

fresh and ensiled, with a known chemical _compositon and in vivo

digestibility

were

employed.

The

forages

were harvested with a

precision-chop

machine and

_{chopping length}

was

15 mm.

_{Approximately}

1.5 tonnes of each fresh

forage

was frozen at -15 °C, and the other portion

(1.5 tonnes)

was ensiled. The silos were

opened

after 60

days

and the

silage

was frozen at-15 °C. Six adult

Romney-Marsh

male

sheep

per

treat-ment, with _{body weights} of

approximately

15 5

kgO.75,

were used for the in vivo

_{digestibility}

deter-minations. The

_{digestibility}

trials lasted 21

day:

a

_{14-day adaptation period}

and a

_7-day

collec-tion

period.

The animals were fed ad libitum twice

daily,

at 9 00 and 17 00 hours. The

_quantities

of feed offered were the

previous days

consump-tion amounts

_plus

10%. _{The ranges of} in vivo

_dry

matter

_{digestibilities}

are shown in table I.

For the laboratory method,

forage samples

were dried at 65 °C for 48 hours and milled

through

a screen of 1.0 mm.

The reference method used was that

_proposed

by

Tilley

and

Terry (1963)

and modified

by

Alexander and McGowan

_(1966):

0.5 g of each

sample was

_weighed

in _triplicate and incubated for 48 hours with a mixture of 40 ml of

_McDougall

(1948)

buffer solution and 10 ml of inocula satu-rated with

C0

2

.

At the end of the first fermentation

stage, microbial

_activity

was _stopped with 2.2N HCI until a _pH of 1.2 was achieved. This was fol-lowed

_by

an incubation _period of 48 hours with 50 ml of a solution of acid pepsin. The residue obtained after filtration in a

_G

₂

crucible was dried at 105 °C and then ashed at 550 °C.

This method was

_employed

with rumen

_liquor

obtained from three rumen fistulated adult

Rom-ney-Marsh sheep

that were fed ad libitum a stan-dard diet of

_{mid-quality hay}

and 200 g of

com-pound

feed _per

_day

_{(table II).} We also used the inocula source

_{reported by}

Nikolid et al

(1987);

rumen contents were removed from six

_healthy

cattle

_immediately

after the

_slaughter,

and stored in _thermolagged containers. After _straining

through

four

layers

of gauze, the rumen

liquor

was mixed with

_{McDougall’s}

buffer solution, in

porportion

4:1, and saturated with

_C0

₂

_-

Since

previous dietary history

was _unknown, the use

of a

_large

number of ’donor animals’ reduced the

variability of the inocula microbial _activity. The other method used was that

_{proposed by}

El Shaer et al

_(1987):

the source of inocula is not

rumen _liquor but

_sheep

faeces. A 50 g

sample

of wet

_sheep

faeces were collected within 1 hour

of

voiding

from three

sheep

that were maintained

on a _{diet of average}

_{quality hay}

_{and 200 g of}

compound

feed

(table II).

The faeces were

mac-erated and mixed with 50 ml of

McDougall’s

buffer

solution, which had previously been saturated with

_C0

₂

_.

The mixture was filtered and then made

up to 300 ml with buffer solution, and the

pH

was

adjusted

to 6.8.

A _simple linear

regression

and paired t-tests

were _{used to compare the relationship} between in

vitro and in vivo

_{digestibilities.}

The data were

sub-jected

to

_analysis

of variance

(Anova)

based on

the results of the _dry matter

_{digestibility.}

Com-parisons

were made for the three _stages of

growth,

and two forms of conservation and were

tested for the interaction between

species

and

growth,

species and conservation, and

growth

and conservation. The

_{multiple comparison}

of the means were made

using

the t-test

_(Steel

and

Torrie,

1980).

RESULTS

The

equations

of

_regression

between the in vitro

_{digestibility}

of

_dry

matter and the in vivo determinations for all

_forages

are

pre-sented in table III and for fresh and ensiled

forages separately

in table IV.

The

_{relationships}

between in vitro

digestibility

determined

_by

the method with

sheep

rumen

_liquor,

with

_slaughtered

cat-tle rumen

_liquor

and with

sheep faeces,

and the in vivo

_dry

matter

_{digestibilities,}

respec-tively,

are shown in

_figures

1,

2 and 3. The variance

_analysis

of the results of the

dry

matter

_{digestibility}

obtained

by

in

vivo and in vitro methods for the three

stages

of

_growth

and two forms of

conser-vation are

_presented

in table V.

DISCUSSION

Based on the coefficients of determination

(R

2

),

we can assume that

_satisfactory

esti-mates of the in vivo

_{digestibility}

values were

obtained

_by

all three methods.

_{Judging by}

the residual standard

deviation,

the tradi-tional method gave the most

_precise

esti-mate. We verified that the alternative meth-ods to the

_Tilley

and

_{Terry (1963),}

modified

by

Alexander and McGowan

_(1966),

with

rumen

_liquor

from fistulated

sheep

gave results

_{significantly}

correlated

_(P

_{= 0.05)}

with in vivo

_{digestibility.}

The method that used rumen

_liquor

from

_slaughtered

cattle

was

_highly

correlated with the in vivo

_organic

matter

_{digestibility (rvalue}

=

_0.969)

and

cor-roborated the

_findings

of El Shaer et al

(1987),

who observed an rvalue of 0.98 for the correlation between the results of the in vivo

_dry

matter

_{digestibility}

and the results of the

sheep

faeces method.

The method that used the rumen

_liquor

from

_slaughtered

_{cattle gave} an R2 lower than that of the traditional

_Tilley

and

_Terry

(1963),

modified

_by

Alexander and McGowan

_(1966),

in the

_prediction

of the in vivo

_{digestibility (table V).}

The

_greatest

advantage

of the method

_using

rumen

_liquor

from

_slaughtered

animals was the

avoid-ance of fistulated animals as the inocula

source and the ease and low cost of the

procedure.

The results of the method that used the faeces

_suspension

as the source of inocula had the lower

_relationship

with the in vivo

digestibility

and a

_higher

standard devi-ation

(table V).

This

_technique

is,

in our

opin-ion,

not recommended for the

_prediction

of

in vivo

_{digestibility}

for this reason and because the results are much

_higher

than those determined

by

the in vivo method

(table V).

This occurs with the methods that used faeces

_{microorganisms}

because of the difference between the rumen and the faecal microbial

_population

and their

_ability

to break down cell walls.

The

_{regression equations}

between the in vitro

_{digestibility}

of

_dry

matter and the in

vivo determinations show that the method that used the

_sheep

rumen

_liquor

obtained

a

_higher

R

z

with the fresh

_forages

than with ensiled

_{forages (table IV).}

The method that

used the rumen

_{liquor from slaugtered}

for-ages but did not

_give

a

_significant

correlation

at the level 5% from the ensiled

_{forages (fig}

2).

The method that used

_sheep

faeces also gave results

significantly

correlated with in

vivo

_{digestibility}

from fresh and ensiled

for-ages. The

relationships

obtained were

gen-erally

better for fresh

_forage

than for ensiled

forage. Vanderhaege

and Biston

₍₁₉₈₇₎

and Omed et al

₍₁₉₈₉₎

obtained the same

rela-tionship

with a

_{pepsin-cellulase}

method in a

trial that

compared

faeces

_{liquor-pepsin,}

rumen

_{liquor pepsin}

and

_{pepsin-cellulase,}

three

_techniques

for the

_prediction

of the

dry

matter

_{digestibility.}

Barber et al

_(1984),

using

a rumen

_{liquor-pepsin}

method,

reported

a

_higher

Rzwith

_silages

than with fresh

_forages;

Aufrere

_(1982),

with a

pepsin-cellulase

method,

obtained rvalues of 0.94 and

0.96,

respectively,

for fresh grass and ensiled

_forages.

Our results should be confirmed with the

repetition

of the trial and the utilization of a

blind test

_population

of

_forages.

_Clary

et al

(1988) reported

that when a

_multiple

donor

approach

is combined with the use of

stan-dard reference

_forages,

the most stable and reliable in vitro

_digestion

values should be obtained. The use of control

_samples

in

each series is thus necessary to

_permit

com-parison

between

_laboratory

results

_(Aufr6re

and

Michalet-Doreau,

1988).

CONCLUSION

The

_{two-stage procedure}

for the determi-nation of the in vitro

digestibility

described

by

Tilley

and

_{Terry (1963),}

and modified

_by

Alexander and McGowan

_(1966),

with

rumen

_liquor

from fistulated

_sheep

as inoc-ula gave the best results for the

prediction

of the in vivo

_{digestibility.}

However,

we also had

_good

results with the same method when the source of inocula was rumen

_liquor

from

_slaughtered

cattle. The method that used faeces

_suspension

as inocula had the lowest correlation with the in vivo

digestibil-ity

and a

_higher

standard deviation.

ACKNOWLEDGMENTS

We would like to thank

Eng

2

J Vacas de Carvalho

for

_help

with statistic

_analysis

and

_Eng’

Lurdes Enes,

Eng’

JP Barreiros and Dr DL Hostetter for the revision of the text.

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