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Vitamin B12 levels in ewe colostrum and milk and in
lamb serum
Jj Ramos, T Saez, Jp Bueso, Mc Sanz, Arnaud Fernandez
To cite this version:
Short
note
Vitamin
B
12
levels in ewe
colostrum
and milk and in lamb
serum
JJ Ramos
T Saez
JP
Bueso
MC Sanz
A Fernandez
1
Dpto
dePatología
Animal, Facultad de Veterinaria,ClMiguel
Ser!ef, ,177, 50013 -Zaragoza;
2Centro de Sanidad Animaly
Campañas
Ganaderas,Zaragoza, Spain
(Received
10 November 1993; accepted 4February
1994)Summary ―
VitaminB!2
was measured in samples of normal ovine colostrum and milk and in seraof lambs and ewes taken at various stages of lactation, from
lambing
up to 16 d. Colostrumobtained within 24 h of
lambing
containedhigh
concentrations of vitaminB!2,
but within a few daysthe levels fell. The lambs received
high
levels of vitaminB!2
from their dams’ colostrum but aparallel
decrease of vitaminB
i2
levels in lambs’ serum and ewes’ milk wassubsequently
observed.Milk vitamin
B
i2
wasanalysed
by themicrobiological
method(Lactobacillus
leichmannii, ATCC7830)
and a radioisotope dilution (RID) technique. Milk vitaminB!2
concentrations were greaterwith the
microbiological technique compared
with the RID method. Theradioassay
method has been shown to be as reliable as themicrobiological
method formeasuring
vitaminB
i2
in ovine milkexcept for very low levels.
vitamin
B
12
/ colostrum I milk Isheep
Résumé ― Concentrations en vitamine
B!2
dans le colostrum et le lait de brebis et dans lesérum des agneaux. Les concentrations en vitamine
B
12
ont été mesurées dans le colostrum, le lait et le sérum de 10 brebis ainsi que dans le sérum des agneaux depuis lejour
del’agnelage
jusqu’à
16 j après la mise bas. Le colostrum obtenu 24 haprès l’agnelage
contenait uneconcentration élevée de vitamine
8!2
mais celle-ci diminuait rapidement. Les agneaux recevaient du colostrum maternel une grande quantité de vitamineB
12
;
celle-ci diminuait parallèlement dans lesérum des agneaux et dans le lait de brebis. Les 2 méthodes utilisées pour doser la vitamine
8
12
(méthode microbiologique
ettechnique
de dilutionradioisotopique)
ont donné des résultats semblables dans le lait sauf pour des concentrations faibles de vitamineB
12
-vitamine
B
12
/ colostrum / lait / mouton*
INTRODUCTION
Vitamin
B
i2
(cyanocobalamin)
is anessen-tial nutrient
occurring
inmilk,
which acts as anintegral part
of various enzymes in thebody.
Itsdeficiency
affects red-blood-cellmaturation,
reducesnitrogen
retention and limits the concentration in blood and liver. VitaminB
12
deficiency
in ruminants is due toinsufficient
dietary
intake ofcobalt,
which isrequired by
the rumen bacteria for the syn-thesis of this vitamin. Inlambs,
until therumen is
functional,
theonly
vitaminB
i2
supply
is their mother’s milk. It is believed that the concentration of vitaminB!2
in the milk is a reliable index of cobaltsufficiency
in ruminants
(Annenkov, 1982).
However there is littlepublished
information about the concentration of vitaminB!2
in normalewe colostrum and milk.
Microbiological
assays,using
Lacto-bacillus leichmanii or othermicroorganims,
havelong
been the mostfrequently
used methods forquantitative
determination of vitaminB
12
in milk. Thedevelopment
ofradioisotope
dilution(RID)
assays resulted in a trend towards theserapid
and less tedioustechniques.
With humanmilk,
themicrobiological
methods forassaying
vitaminB!2
havelargely
beenreplaced by
radio-assay methods(Thomas
ef al,
1979),
whichwere also
adapted
toveterinary
medicine.A
comparative study
of vitaminB
i2
levels
has been made in the liver and sera ofsheep
measuredby microbiological
andradioassay
methods(Millar
andPenrose,
1980;
Mohammed andLamand, 1985;
Schultz, 1987)
but no evaluation has been made of these methods whenapplied
to theanalysis
of vitaminB
i2
in ewes’ milk. This paper comparesmicrobiological
andradioassay
methods to establish whether the RIDtechnique
may be used in therapid
determination of vitamin
B
i2
in ewecolostrum and milk. In
addition,
it deals with thedynamics
of vitaminB
12
incolostrum,
milk and serum from
sheep
and in lambs’serum
during
the first 16 d afterlambing.
MATERIALS AND METHODS
Ten Castellana ewes
aged
2-6 yr and their 10 0lambs were used for this
study,
with 5 other lambs which did not receive colostrum. Blood, colostrumand milk
samples
were obtainedimmediately
after
lambing,
at 24 and 48 h and thereafter at 4, 8 and 16 d, except for thecolostrum-deprived
lambs, which were
only sampled
twice, newborn and at 24 h. In addition, milksamples
from otherewes were also used
during
the first month post partum for the correlationstudy.
The
jugular
vein was used foracquisition
ofsamples.
Sera for vitaminB
iz
determinationswere
separated
within 2 h of collection and were stored at -20°C for nolonger
than 1 month beforeanalysis.
Samples
of colostrum and milk were obtainedby
manualexpression
from normallactating
ewes. None of the donors werereceiving
treatment withvitamin
B
i2
or antibiotics.The milk was extracted as described
by
Gre-gory(1954),
to liberate the vitamin from its carrier. Thesample,
onceprepared,
waskept
at -20°Cuntil its determination.
Microbiological
assay wasperformed
as describedby
Hansen and Hauschildt(1974),
using
L leichmannii 313(ATCC- 7830).
Cyanocobalamin
was used as a reference stan-dard in a concentration range of 1-40pg/ml.
Diluted
sample
or standard was added to 5 mlmedium
(Difco
VitaminB
i2
assaymedium)
and the contents of the tubes wereadjusted
to 10 ml l with distilled water. Beforeadding
the assayorganism, the tubes were sterilized by autoclaving.
After incubation at 37°C for 24 h,
growth
of theassay
organism
was measured asturbidity
at 540 nmusing
a Perkin-Elmer Lambda 5spec-trophotometer. The
samples
and standards wereassayed in triplicate.
The Solid Phase No-Boil Dualcount kit, from
Diagnostic
ProductsCorporation,
was used forvitamin
B
iz
measurement in serum and milk. Thiswas considered as an ideal commercial product for the determination of this vitamin in ovine serum
in a
comparative study (Schultz, 1987).
Purifiedwere used for the RID method, and the proce-dures recommended
by
the manufacturers were used. Allsamples
and standards were assayed induplicate. Radioactivity
due to[
57
C
O]
was deter-mined with an LKB-1282Compugamma
counter.Statistics for
intra-assay precision
werecal-culated for each of 3
samples
from the results of10 estimates in a duplicate assay. For inter-assay
precision,
statistics were calculated using milksamples of 3 different concentrations of vitamin
B!2
and based on 5 estimates.RESULTS
Vitamin
B
t2
levels in serum, colostrum and milksamples
of ewes and serumlambs,
taken from
lambing day
up to 16d after,
determined
by
both methods are shown in table I. The lambs that received colostrum at24 h of life
registered significantly
(P
<0.001) higher
levels(3.38
± 0.54ng/ml)
for vitaminB!2
than those that had nocolostrum intake
(1.18
± 0.48ng/ml)
andwere fed on milk substitutes. For newborn
lambs,
the same levels were 1.05 ± 0.43ng/ml
and 1.01 ± 0.45ng/ml, respectively.
Comparison
of the vitaminB
12
levels foundusing
themicrobiological
and the RID assay kit gave anequation
of theregres-sion line
(y=
0.852x+2.387)
and acorre-lation coefficient of 0.75
(P
<0.001
Both
analytical
methods formeasuring
vitaminB!2
in 40 milksamples
had a coefficient ofvariation < 15%.
The
intra-assay precision
for ovine milksamples
was between 4.7 and 9.0% for themicrobiological
method and 7.6 and 7.8% for RID assay kit. Theinter-assay precision
ranged
between 7.8 and 9.5% for the first method and between 7.4 and 18% for the second(table II).
DISCUSSION
The vitamin
B
i2
in serum from ewesremained above the
adequate
range for adultsheep (Fisher
andMacPherson,
1990)
and
significant changes
associated withlambing
or lactation were not detected. Inlambs,
colostrumingestion produced
anotable increase of vitamin
B
12
serum levels(table I),
which decreasedprogressively
dur-ing
thefollowing days, falling
even below the levelregistered
on the firstday
oflife,
and on
day
16 the lowest level wasregis-tered. The rise in serum vitamin
B!2
vita-mins. In the same way, the lambs that
received colostrum at 24 h
registered
sig-nificantly (P < 0.001)
higher
levels than those that had no colostrum intake. Serumvitamin
B
12
concentration in lambsresponds
rapidly
toingestion
and thus serum deter-mination is not agood
indicator ofdeficiency
(Millar and
Penrose, 1980;
Sutherland,
1980).
The vitamin has no functional role in
serum and
blood,
which maysimply
act asa
transport
mechanism(McMurray
etal,
1985)
and as a store(Suttle,
1986)
of it.According
toHalpin
andCaple (1982),
colostrumprovides
the essential source of vitaminB
12
to the newborn lamb after birth. Under cobalt sufficientconditions,
the lambsstart to
produce
their own vitaminB!2
from about 1 month of age, whenthey
become ruminants. The vitaminB
i2
requirement
forsheep
is about 11 pgdaily (Marston, 1970).
In
preruminants
cobalamin is arequired
nutrient,
although
therequirement
for lambs is notprecisely
known.Milk vitamin
B
12
concentrationsregis-tered a notable decrease
during
the first24-48 h after
lambing,
and after the 8thday
provides
little vitaminB!2
to the lambs(table
I).
Similardynamic
results were observedin human
samples;
the colostrum obtained within 48 h ofdelivery
containedhigh
con-centrations of vitamin
B
12
by
within a fewdays
the levels declined(Thomas
etal,
1979;
Samson andMcClelland,
1980).
Our results are different in theirdynamics
fromthose
registered by
Wohltef al (1981)
for milk from Dorsetsheep,
in which nosignif-icant differences were observed between
day
0(5.5 ng/ml), day
5(5.1
ng/ml)
orday
28
(6.2 ng/ml),
but levels of vitaminB
12
inraw
sheeps’
milk weresimilar,
after the 8thday,
to thosereported by
Williams et al(1976) (9.8 ng/ml),
Scherz and Kloos(1981)
(5.1
ng/g),
and Scott andBishop (1986) (6.4
ng/ml).
The method’s
precision
wasacceptable
(<
10%)
except
in one case where low lev-els of vitaminB
i2
concentrations were found with the RID method(table II).
The radio-assay method gave results that correlatedwell
(P
< 0.001 )
with those obtained with the L leichmanniimethod,
but this method gavehigher
values. Themajor
discrepancy
between the methodscompared
was that themicrobiological
method gavehigher
results with
samples
of low vitaminB
i2
con-centration. It has been
suggested
thatdeoxyribosides (Gregory, 1954)
andthymi-dine
(Gullberg,
1973)
may stimulate thegrowth
of the assayorganism
in the deter-mination of vitaminB
12
in human milk.How-ever, in a further
study
of theability
of these and other metabolites to stimulate thegrowth
of the assayorganism,
none of these substances stimulatedgrowth
(Samson
andMcClelland,
1980).
We do not know which factor or factors
may cause the differences obtained
between
methods,
although
the RID assay kit contains apurified hog
intrinsic factor toeliminate
problems
withnon-specific
binding
proteins
and should bindonly
true vitaminre-commended the use of a
purified
intrinsic factor formeasuring
true vitaminB!2
content in food for the RID assays.The RID method has been shown to be
as reliable as the
microbiological
method formeasuring
vitaminB
i2
in ovine milkexcept
at very low levels. It is faster thanthe
microbiological
method and is notsub-ject
toproblems
ofsample
contamination.ACKNOWLEDGMENTS
We are
grateful
to the ’Centre de Sanidad Ani-mal yCampanas
Ganadera‘ for the facilities andhelp with
analysis
and theDiputaci6n
General deAragon
for the financial support.REFERENCES
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