OROTIC ACID AND NON-PROTEIN NITROGEN OVERLOAD IN SHEEP

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OROTIC ACID AND NON-PROTEIN NITROGEN

OVERLOAD IN SHEEP

Lionel Bueno

To cite this version:

OROTIC ACID

AND

NON-PROTEIN NITROGEN OVERLOAD

IN

SHEEP

L. BUENO

Laboratoire de

_{Physio%gie-Pharmacodynamie -}

Eco% Nationale Vétérinaire, 23 chemin des Capel%s

-31076 Toulouse Cedex, France

Résumé

ACIDE OROTIQUE ET SURCHARGE EN AZOTE NON PROTEIQUE CHEZ LE MOUTON. - Les effets de l’administration directe d’urée

(0,5

g/kg)

dans le rumen sur la concentration du sang en

ammoniaque,

la motricité du réticulo-rumen et le comportement alimentaire sont

_envisagés

en

présence

ou non d’acide

orotique

ou d’acide

_acétique.

L’acide

_acétique

et l’acide

orotique

à la dose de

0,2

g/kg

de

poids

vif ne modifient pas la concentration maximale du rumen en ammonia-que mais celle-ci diminue

rapidement

dans le cas de l’acide

_orotique.

Elle est de 162 ± 21 mg

NH!-N/100

ml au bout de 2 heures au lieu de 229 + 31 mg

_NH

₃

_-N/100

ml.

_{Parallèlement,}

l’hyper-ammoniémie est réduite de 47% par rapport aux valeurs témoins.

L’addition d’urée exerce une action inhibitrice vis-à-vis des contractions

_primaires

et secondaires du rumen. Cet

effet, qui

persiste

120 min en

_présence

d’acide

_acétique,

n’excède pas 90 min dans le cas de l’acide

_orotique.

Enfin,

la

quantité

de foin

consommée,

qui

est diminuée de moitié dans le cas de l’addition d’urée dans le rumen, n’est réduite

_que

de

_40,7%

en

_présence

d’acide acéti-que. L’anorexie est

_supprimée

dans le cas de l’acide

_orotique.

The

_efficiency

of

_dietary

urea as a source of

nitrogen

in the ruminant is limited

_by

the rate at which urea is

_hydrolyzed

in the rumen, the

resulting

ammonia

being

produced

more

rapidly

than the

microorganisms

can utilize it for amino-acid and

_protein

synthesis

(Repp

et

a/.,

1955). Both

_nitrogen

assimilation and pro-tein

_{synthesis by}

the microflora are

_impaired

by

the

increased

rumen

_pH,

the

_high

blood ammonia

_being

correlated with reticulo-rumen

paralysis

and

_probably

anorexia (Huber and

Cook, 1972 ;

Webb

et al., 1972 ;

Wilson

et al.,

1975).

An usual

approach

to limit the

high

rumen

ammonia concentration is to reduce the rate of

urea

_hydrolysis

in the rumen

_{by physical}

pro-cedures such as

_coating.

Another solution to the

_problem

of the

_rapid

rate of ammonia release from urea could be the use of urease

inhibitors such as

_hydroxamic

acid (Streeter et

a/.,

1969).

An alternative

_approach

could be the increase in the rate of

nitrogen

assimilation

by

the

_{microorganisms}

such as addition of low

weight

carbohydrates

(Thivend et

_aL,

1977).

Orotic

acid,

a

compound

found in the milk

(Hallanger

et

aL,

1953),

is known to protect

the liver

_{against hepatotoxic}

agents in rats

(Gordonoff and

Schneeberger,

1959). Further-more, an intravenous

injection

of

DL-lysine

orotate has been found able to prevent

ammonia

_toxicity

in cirrhotic

(Caroli

et

al.,

1965). Orotic acid takes

_origin

from

aspartic

acid and

carbamyl-phosphate,

a

substance

involved in the

synthesis

of

pyrimidic

nucleoti-des such as

uridylic

acid,

a _potent precursor of uridine

_triphosphate

(UTP) which

_requires

free ammonia to be converted in

_cytidine

triphos-phate

(CTP). In

_addition,

_{protein synthesis}

is stimulated

by

the transformation of CTP into nucleic acids

(Schwietzer,

1956 ;

Naono and

Gros,

1960).

The purpose of the

study reported

herein

was to determine the effect of orotic acid on

the rumen ammonia

production

subsequent

to

non

_protein

_nitrogen

overload. A

_companion

study compared

the effect of acetic acid to eli-minate the role of

pH

changes.

Both

reticulo-rumen

_motility

and food intake were used as

criteria of the influence of orotic

acid

in

pre-venting

the deleterious effect of ammonia

toxi-city.

Methods

Four adult Lacaune ewes,

weighing

40-45

kg

and fitted with _permanent rumen cannula

and an

_{indwelling jugular}

catheter were

_placed

in metabolism cages. The animals received

hay

in excess _every

_morning

at 9 :00 a.m., the

daily

amount of food intake

_being

evaluated

by

weighing

the refusal. At

weekly

intervals,

and for 3 consecutives

_weeks,

food distribution

was

preceded

by

ruminal administration of

urea (solution at 200

g/I ;

0.4

g/kg

of

body

weight).

In two similar series for each animal

urea was

_accompanied

by

orotic

(monohy-drate salt) or acetic acid administration at a

,dose

of 0.2

_g/kg.

In each case, urea and

ammonia concentrations were determined

before and

during

7 hours after urea, or urea

and orotic or acetic acid was added into the rumen, from blood

samples

taken from the

jugular

vein and the rumen fluid obtained from

the ventral sac

_using

a

_polyvinyl

tube introdu-ced via the rumen cannula.

Two combined Technicon

_{autoanalyzers}

with sodium borate for

dialysis

(lmler et

al.,

1972) were used for the

_dosages.

Urea

con-centration was determined

by

its color

reac-tion with

diacetyl

monoxime in the presence of thiosemicarbazide measured at 520 nm

accor-ding

to Marsh et al. 119651. Ammonia

concen-tration of the blood and the rumen fluid was

deve-loped

in the _presence of

_phenol

and sodium

hypochlorite

and measured at 625 nm

accor-ding

to Horn and

Squire

(1967). To prevent

post-sampling

ammonium

formation,

the

sam-ples

were taken onto sodium

_heparinate

(5%)

and

_{centrifugated}

at + 4°C. The supernatant

was introduced into the

autoanalyzer

starting

within 10 min of collection.

The

_cyclical

contractions of the rumen were

recorded

_during

the whole

_experimental

ses-sion

_by

means of rubber-air filled balloons

pla-ced into the dorsal sac of the rumen and

con-nected to a _pressure transducer. The fre-quency and

amplitude

of the rumen contrac-tions were determined

_directly

from the

recor-dings

obtained on a

_Physiograph

(E’°

Narco,

Houston). The nature of contractions

_(primary

and

_secondary)

was taken into account

accor-ding

to Ruckebusch and Tomov

(1973)

and their mean

_frequency

was calculated from 5-min

_samples

recorded from 60 to 90 min after urea administration.

Results

Ammonia

_toxicity

The ruminal administration of urea alone

was

_accompanied

in 9 of the 12 trials

_by

tor-por, salivation and muscle tremor

_lasting

from 60 to 120 min. In cases,

approximately

1 mole of acetic acid (5% v/v) per 0.5 mole N admi-nistered was

_{given intraruminally}

after 120 min to facilitate recovery. Both food intake and rumination were absent

during

about 5

hrs,

i.e. from 9:00 a.m. to 14:00 p.m.

With the mixture of acetic or orotic acid and

urea

_{respectively,}

3 and 2 cases

_only

for 24 trials of ammonia

_toxicity

were observed. The

signs

lasted less than 60 min and were obser-ved

only

in the first serie of trials.

Ammonia and urea levels

On a

_{hay regimen,}

the mean rumen N-ammonia level was 11.4 + 2.1

mg/100

ml

(n

= ₃₆₎ with _an

average

pH

of 6.49 ± 0.21 at 9:00 a.m. The ammonia

peak

in the rumen of animals

_receiving

urea reached 248 ± 34

mg/100

ml about one hour after adminis-tration. This effect

persisted

for an additional administration of acetic or orotic

acid,

but a

rapid

decrease in orotic acid was recorded : the rumen

_NH

₃

_N

level was 2 hours later

equal

A maximal

_pH

value of the rumen (7.96 + 0.41) was also observed at the end of the first hour after urea ruminal administration. Such an increase was not found when urea

adminis-tration was combined with that of acetic or

orotic acid.

Furthermore,

variations in the

pH

value did not exceed 0.4 unit (see

Fig.

1) for orotic acid.

The blood level of

_NH

₃

_-N

which varied from 50 to 100

_!g/100

ml was

nearly

threefold increased 90 min after the ruminal administra-tion. Such an increase also occurred in the presence of acetic or orotic acid

_(Fig.

2) but

the mean ammonia level was half that of

con-trol values with urea 2 hrs later for orotic acid

from 5 to 7 hrs later for acetic acid.

The blood level of urea

_{progressively}

increa-sed

_during

the 7 hrs

following

urea

administra-tion and

subsequent

hepatic ureogenesis.

This

phenomenon

was

_{significantly higher}

(P < 0.01) after a ruminal administration of

urea and orotic acid (33.1 ± 4.2

_mg/100

ml)

than after urea (21.3 ± 4.2

mg/100

ml) or urea

and acetic acid (19.7 ± 2.6

_mg/100

mi).

Rumen

motility

Both

frequency

and

amplitude

of the

pri-mary rumen contractions were

_strongly

redu-ced from 8-12 min after the ruminal urea admi-nistration

_during

90 to 120 min

_(Fig.

3). A simi-lar

_phenomenon

was recorded for the

secon-dary

rumen contractions (Table 11.

_{). _}

The effect of urea on

_motility

was

_impaired

by

acetic and

_by

orotic acid. In both cases, the

motility

pattern recorded 90 min after ruminal administration was not

_{significantly}

different from that observed in the control

_period.

The effect of orotic acid was more

_pronounced

than that of acetic acid as

_judged

_by

an earlier recovery in

amplitude

and

_frequency

ot the ruminal contractions. The

amplitude

expres-sed as a _percentage of the control values was

about 92 at 60 min after ruminal administration of orotic acid and

_only

68 for acetic acid.

Feeding

behaviour

The mean

_daily

food intake (1 050 ± 118 g of

_dry

matter - DM) was reduced

_by

51.3 and 22.5 %

respectively

the first and second

day

following

ruminal administration of urea. The reduction was recorded

_{only during}

24 hrs and did not exceed 40.7% for an additional admi-nistration of acetic acid (Table 2). The

ano-rexia

subsequent

to ruminal administration of

urea was

prevented by

orotic acid. The

figure

4 shows that the effect was consistent for the

different trials in each animal. The amount of food intake was halved

_by

urea addition in

sheep

n°2 and additional orotic acid resulted in a

_slight

increase of food intake. Food intake

The time spent

ruminating

per 24 hrs

(510 + 24 min/24 hrs) was lowered

_by

18% after ruminal administration of urea (421 ± 36 min/24 hrs). There were no

_significant

changes

in the presence of either acetic or

oro-tic acid.

Discussion

The toxic effects of

_high

ammonia level

fol-lowing

the ruminal administration of 210 mg

N/kg

in the form of urea were similar to those

previously

reported

in _sheep

_by

Bartik et al.

(1971) and

_by

Chalmers and White (1969). The concomitant inhibition of rumen

_motility

is related to the level of ammonia in blood and the rumen

fluid,

the rumen wall

being

sensitive to both free

_NH

₃

and

_NH

₄

_*

_regardless

of the

pH

values (Bueno

et al.,

1977).

The examination of the toxic effects of a

non-protein nitrogen

overload indicates that at

least three steps are

involved ;

(i) the increases ruminal ammonia

_production

induces that of the

pH

which in turn increases the

absorption

of free

_NH

₃

(Webb

et al., 1972) ;

(ii) the eleva-ted

_portal

blood

ammonia increase cannot be counterbalanced

by

the

_{hepatic ureo-synthesis}

inducing

(iii) an increase in

_peripheral

blood ammonia which acts on the central nervous

system and thus reduces food intake. All the trials

_reported

here show a beneficial effect of orotic acid.

_{Many explanations}

of this

improvement

may be

given :

(i) the acid

func-tion,

although

weaker than that of acetic

_acid,

interacts with alkalinization of the rumen fluid

by ammonia ;

(ii) its transformation into

cyti-dine

triphosphate

(CTP)

_requires

the

incorpo-ration of free

_NH

₃

_;

(iii) its

absorption

through

the rumen wall stimulates the

_hepatic

decrease of

_N-NH

₃

in the rumen fluid in

_sheep

receiving

both urea and orotic acid suggests a

faster assimilation of

_NH

₃

_by

the

microorga-nisms in the presence of orotic acid. A

_striking

feature is

presented by

the increase of ureoge-nesis in the case of orotic acid addition.

The

_anorexigenic

effect of urea even for a

ruminal administration indicates that olfaction and taste are not

_necessarily

involved as

sug-gested

Bolduau et al. (1971 That orotic acid

was able to _prevent any reduction in the amount of food intake

_despite

the inhibition of

motility,

strengthened

the

_hypothesis

that

hyperammoniemia

is a

_major

factor of

ano-rexia related to urea.

It must be also noted that the

ability

of

oro-tic acid in

_reducing

urea

_toxicity

in ruminants could be modified in the case of

repeated

administration,

a

_phenomenon

which has been considered in the rat

_by

Novikoff et al.

(1966). On the other

hand,

the increased assi-milation of N-ammonia

by microorganisms

(unpublished

results) would be

_expected

to

lead to a _greater

_nitrogen

retention

during

non-protein

N overload.

It seems that the presence of orotic acid increases the

_ureogenesis

of the liver even

though

that the blood ammonia level was

lower (ammonia reduced ruminal

absorption)

than that observed with urea alone. Two other

explanations

can be

suggested according

to recent

unpublished

results : orotic acid treat-ment reduces the renal elimination of urea

and/or the

_high

level or

portal

blood ammonia observed with urea alone inhibits the

_hepatic

ureogenesis.

Acknowledgements

Thanks are due to V. Doulou for his skillful assistance.

Accepted

for

_publication

_July

7 th 1978.

Summary

Experiments

were conducted in

_sheep

to

_investigate

the

protective

effect of orotic acid versus

acetic acid (0.2

_{g/kg body weight) against}

the effects of the administration of urea

_{(0.5 g/kg)}

into the rumen. Blood ammonia

levels,

motility

of the reticulo rumen and food intake were measured. The

_quantity

of ammonia

nitrogen produced

in the rumen was

_only

162 + 21 mg

NH

3

-N/100

ml 2 hours after the urea and orotic acid addition instead of 229 + 31 mg

NH

3

-N

for urea and acetic acid. The blood ammonia level was reduced

by

47% when orotic acid was used instead of acetic acid.

The

_long-lasting

inhibition of the contractions of the reticulo-rumen

by

urea was restricted to 120 and 80 min

by

acetic acid and orotic acid

_{respectively.}

The amount of food intake which was hal-ved

_by

urea was

_only

reduced

_by

40.7% when both acetic acid and urea were administered. Reduction in the amount of food intake due to urea was

_{suppressed by}

the administration of

oro-tic acid.

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