Full text of DOI:10.1258/002367700780387714

Laboratory Animals
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A refined method for sequential blood sampling by tail incision in rats
Marc Fluttert, Sergiu Dalm and Melly S. Oitzl
Lab Anim 2000 34: 372
DOI: 10.1258/002367700780387714
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A refined method for sequential blood sampling
by tail incision in rats
Marc Fluttert, Sergiu Dalm & Melly S. Oitzl
Leiden/Amsterdam
Centre for Drug Research, Division of Medical Pharmacology, PO Box 9503, 2300
RA Leiden, The Netherlands
Summary
Levels of endogenous or administered substances can be estimated by blood sampling. This
allows an evaluation of the relationship between clinical signs, physiological parameters,
pharmacological treatments and behaviour of the animal. We show that blood samples can be
taken occasionally as well as sequentially by means of a small incision at the end of the rats'
tails. Up to 300 III of blood can be collected within 90 s. The advantages of this method are:
(i) anaesthesia and surgery or restraint of the animal are not necessarYj [iiI the procedure
can be considered stress-free as indicated by the low, basal levels of the stress hormone
corticosterone, even with frequent sequential blood sampling over 3 hj and (iii) it can be used
for longitudinal studies allowing intra-individual comparisons over months and even years.
Blood samples collected via an intravenous catheter and, at the same time, by our tail incision
method resulted in comparable amounts of corticosterone. Moreover, we consider the tail
incision method for rats to be 'animal-friendly' and a real alternative to other conventionally
used blood sampling techniques.
Keywords Blood sampling; tail incision methodj ratj corticosteronej stress environmentj
animal welfare
research concerns hormones involved in the
regulation of the stress system and behaviour
(de Kloet et al. 1998, 1999). In the past, we
took blood samples from rats via an intra-
venously implanted catheter (jugular vein).
This allowed us to measure the levels of
There is an increasing demand for techniques
that really do allow easy, fast, reliable,
repeated and 'animal-friendly' collection of
blood. Methods for blood removal from
laboratory animals have been reviewed by
the BVA/FRAME/RSPCA/UFAW Joint
Working Group on Refinement (Morton et al. hormones like corticosterone and ACTH in
the blood under resting or activated states,
e.g. in the home cage, in novel environments
or related to emotional challenges (van
Eekelen et al. 1991, 1995). Even if this proved
to be an excellent blood sampling technique,
we experienced several shortcomings
1993). Most of these methods are unne-
cessarily stressful for the animal, simply
because of the anaesthesia or restraint and
the discomfort associated with a particular
technique. For instance, restraining animals
is one of the experimental techniques fre-
quently used in stress research to elevate
blood corticosterone levels, which reflect the
activation of the stress system (McEwen &
Sapolsky 1995). The main topic of our
(1) When only one or two blood samples
were required, rats had to undergo
surgery and were thereafter housed
singly to prevent them gnawing off each
other's cannulae. Single housing of
social animals like the rat for longer
Correspondence to: Dr M. S. Oitzl
E-mail: m.oitzl@lacdr.leidenuniv.nl
© Laboratory Animals Ltd. Laboratory Animals (2000) 34. 372-378
Accepted 31 May 2000
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Blood sampling by tail incision in rats
373
than a few days alters the stress-respon-
sive system (Brain & Benton 1979).
(2) Blood samples could be collected safely
for about 1-2 weeks, but there were
always some failures. Thus, longitudinal
experimental designs over months or
years were not possible.
(3) Senescent rats are sensitive to surgery
(Claassen 1994).
(4) Other methods described more recently
temperature (21°q and humidity (50-60%)
controlled rooms of the animal house at the
Sylvius Laboratory, University of Leiden, The
Netherlands. They were housed singly the
evening before the blood sampling. Sequen-
tial blood sampling over 3 h was used (time
points are given in Fig 2) and the amount of
corticosterone was estimated. To establish
the effect of the environment, rats (n = 9)
were placed into a novel cage or into their
as refined methods for blood sampling in home cage during sampling. Two blood
rats, like tail-cuts (Liu et a1. 1996:
amputation of parts of the tail; Zeller
et a1. 1998: blood sampling from the
sublingual vein; van Herck et a1. 1991,
1998: orbital puncture) are not suitable
for repeated blood collection in relation
to behaviour, require anaesthesia and
activate the corticosteroid stress system.
For blood collection by decapitation at
several time points, large groups of
animals have to be used.
samples were collected with an interval of
60 min from a separate group of rats housed
in their home cage In = 6). Under isoflurane
anaesthesia rats (n=5) were equipped with a
cannula in the jugular vein (Steffens 1969)
and blood samples were collected from both
the jugular vein and the tail incision. About
10min after the onset of anaesthesia, one
blood sample was taken. Thereafter, 1mg/kg
corticosterone (in PEG 300) was injected
subcutaneously and blood samples were col-
lected. This was part of an experiment where
we tested which concentration of exogenous
corticosterone would result in plasma levels
comparable with the ones induced by
novelty. Furthermore, glucose concentration
and haematocrit values were measured in the
blood collected by tail incision (300 Ill) in a
separate group of rats In = 5; Wistar, 300 g
bodyweight) at the Central Laboratory Ani-
mal Institute at the University of Utrecht,
The Netherlands. All experiments were per-
formed during the light period, between
08:00 and 12:00h when basal resting corti-
costerone levels of rats are low.
Consequently, we developed a procedure that
was not hampered by any of these factors
During the last few years we have fre-
quently used and have refined the technique
of blood sampling by means of a small inci-
sion at the end of a rat's tail. Additionally, we
have shown that levels of plasma cortico-
sterone found in blood samples taken from a
jugular vein cannula were comparable with
those taken from a tail incision of the same
animal. That the tail incision can be con-
sidered to be stress-free is expressed by the
continuous low values of corticosterone from
rats observed in their home cage. In contrast,
simply placing a rat into a novel cage (a pro-
cedure, which is performed weekly in the
animal house) increases corticosterone
Animal care procedures were conducted in
accordance with the EC Council Directive of
November 1986 (86/609/EEq. All experi-
ments were approved by the Animal Experi-
ment Committee of the University of Leiden,
The Netherlands.
levels, indicating a stress response.
Materials and methods
Animals and experiments
Determination of plasma corticosterone
Data of the present study are derived from
male Wistar (200-250 g bodyweight; Charles
River, The Netherlands). Animals were
Blood samples were centrifuged (3000rpm for
10min at 4°q. The plasma was stored
at -20°C. Plasma corticosterone was deter-
housed in pairs with free access to food (2122 mined using an antiserum raised in sheep
SMR-A 10mm, 0.9 mRADi Hope Farms, against corti costerone- 21-hemisuccinate
Woerden, The Netherlands) and water and a bovine serum albumin (generous gift of Dr
12h light/dark cycle (lights on at 08:00 h) in Th. Benraad, University of Nijmegen, The
Laboratory Animals (2000) 34
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374
Fluttert, Dalm & Oitzl
Netherlands) as described previously (Veld-
huis et a1. 19821using a 1:10 ether extraction
to avoid interference of other plasma com-
ponents. Detection limit was O.ll-lgjdl; intra-
assay variation 8% and inter-assay variation
was 10%.
counter (model K-1000, Sysmex, Ijsselstein,
The Netherlands).
Blood sampling procedure
This procedure via the tail incision is depic-
ted in Fig 1.
Handling the animals before blood
sampling
Determination of other parameters in
the blood
If several blood samples are required, the
experimenters handle the rats for 4-5 days
before the collection of blood begins. During
the daily 2 min of handling, the experimental
situation should be mimicked and rehearsed:
place the rat on a towel; fold the towel and
create a hole; allow the animal to explore the
towel and a somewhat limited space formed
by hands (Fig 1AI; stroke gently along the
For glucose measurement, blood samples
were centrifuged (3000 rpm for 10min at
4°C), stored at -20°C and colorimetrically
analysed (Vitallab200, Starn BV,Epe, The
Netherlands). For estimation of the haema-
tocrit Icells per volume %) blood was col-
lected in EDTA coated tubes, stored at 4°C
and analysed the same day in a blood cell
Fig 1 Blood collected by tail incision. (A) the rat is loosely held in a towel; (8) holding the rat's tail with one
hand and performing the incision; (C) stroking the tail gently results in blood droplets at the incision; (D)
collection of blood in a capillary
Laboratory Animals (2000) 34
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Blood sampling by tail incision in rats
375
rat's tail; press the end of the tail with two
fingers.
collection of 300 IIIof blood takes no more
than 90 s from the opening of the lid of the
cage until the returning of the rat to its home
cage.
Blood sampling
In one day, several blood samples can be
collected from the same incision. Stroking
over the incision gently with a hydrophilic
gauze sponge dipped in saline re-opens it.
Then, the tail has to be dried and the pro-
cedure of stroking the tail and collecting the
blood droplets can be repeated. If longer time
intervals (i.e. several days or months) occur
between blood samples, new incisions have
to be made, 1 to 3 mm away from the last
incision, towards the base of the tail. Occa-
sional or single blood samples can be taken
without prior handling, since the whole
blood collection procedure doesn't take long.
The experimental procedure is most easily
run with two persons: one pays attention to
the animal, the other takes the blood sam-
pIes. Blood samples can be taken from group-
or singly-housed animals, in their home cage
as well as before and after performing beha-
vioural tasks. Interference with cage mates
can be excluded by housing the animals
singly overnight. This is also preferable, if
more than one blood sample is collected
within several hours.
Procedure
The rat is removed from its cage, placed on a
towel on a table and held gently in place (Fig
lA). Rats keep quiet in this small and dark-
surrounding. The rat should be restrained
only if the experiment requires such a pro-
cedure. Figure lB shows how to make the
incision. Hold the end of the tail, fixed
between two fingers, onto the table. Take a
single edge razor blade and make a small,
about 2 mm long, diagonal incision 15mm
from the end of the tail. Do it slowly. This
incision will allow the collection of blood
from the dorsal tail vein, which runs from
the base to the tip of the tail. While making
the incision, increase the pressure of the
fingers on the tail above the incision. Gen-
erally, no avoidance response like tail flick-
Results
Blood samples collected with an interval of
60 min contained low amounts of the steroid
(Ilgjdl: time of incision: 1.31± 0.2; 60 min
later: 1.15±0.2). Animals were placed into
their home cage after blood sampling. Placing
an animal into a novel cage (i.e. clean cage
with fresh bedding) resulted in elevated
levels of corticosterone which were 8-10
times higher and returned to basal resting
levels 120min after novelty exposure (Fig2;
one way analysis of variance: F(l,812L841
P = 0.002). Corticosterone levels from ani-
mals in their home cage remained low
« 2llgjdll. Even frequent sampling with an
ing is observed. A drop of blood forms at the interval of 20-30 min only slightly elevated
corticosterone levels. Comparable con-
centrations of corticosterone were found in
blood from the jugular vein and the tail (Fig3;
site of the incision. Take an EDTA-coated
cup (Microvette CB 300, Sarstedt, Niim-
brecht, Germany) or capillary tube suitable
for blood collection. Figure lC: Stroke gently Pearson correlation coefficient I = 0.9871.
Blood glucose concentration was
6.96 ± 0.97 mmolj1 (or 125.39± 17.4 mgj
100ml) and the haematocrit was
with thumb, index and middle finger of your
other hand from the base of the tail towards
the incision. Again, a drop of blood is formed
and can be collected. Avoid squeezing the tail
because it is (il painful for the animal and (ii)
counteractive to the blood collection proce-
dure as it induces vasoconstriction and may
affect the quality of the sample. Repeat this
47.02±O.86%.
Discussion
Collecting blood by means of a tail incision is
procedure until the required amount of blood easy to learn and most of all, it is animal
I
is collected (Fig 1D). Without further stimu-
lation of the tail, bleeding stops and the rat
can be placed back into its home cage. The
friendly'. Other methods reviewed by the
BVAjFRAMEjRSPCAjUFAW Joint Working
Group on Refinement (Morton et al. 1993)
Laboratory Animals (2000) 34
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Fluttert, Dalm & Oitzl
376
25
vein (Hem et a1. 1998) or sublingual vein
puncture (Zeller et a1. 1998) cannot be used
in behavioural studies. Both ether anaes-
thesia (orbital puncture) and restraint acti-
vate the stress system (van Herck et a1. 1991,
de Kloet et a1. 1998) and therefore interfere
with ongoing processes of learning and
memory (de Kloet et a1. 19991.Furthermore,
all these methods exclude frequent blood
sampling which causes much distress for an
animal. As we have shown, even blood
sampling via intravenous cannulae can be
replaced by the tail incision method.
Blood glucose concentration and haemato-
crit values give a first indication for the
quality of the blood collected by tail incision.
The haematocrit (48%) is comparable with
values reported by van Herck (1999)-hae-
matocrit tail vein 49%, orbital puncture
50%. The concentration of plasma glucose in
our study (125mg/100 ml) is in the normal
range (e.g. Besch & Chou 1971: glucose con-
centration was lowest in blood obtained by
~
20
15
:>.
'"
~
e
••
li 10
u
:e
8
5
o
40
60
90
-60
20
120
180
Time In minutes
I • home cage
0 novel cage
Fig 2 Effect of cage-environment
on corticosterone
levels. Sixty minutes after the tail incision rats were
placed either into a novel cage where they remained
during the following
180 min (n = 5; open bars) or
taken out shortly and returned to their home cage
(n = 4; black bars). Blood samples were taken 20, 40,
60, 90, 120 and 180 min thereafter. Note that
exposure to a novel cage increases corticosterone
levels over a long period of time
Corticosterone 119J dl
cardiac puncture-122mg/100
ml, and high-
40
est in blood from ether-anaesthetized rats-
165mg/100ml). Of course, studies compar-
ing different blood sampling techniques are
required to establish parameters regarding
the quality of the collected blood.
c
30
Gi
>
:;; 20
'5
III
., to
=
The intense handling procedures preceding
the blood sampling are most relevant in
behavioural studies and if sequential blood
samples are required. However, rats do not
experience distress by taking repeated blood
samples from the tail using this incision
method, as proven by the low corticosterone
levels. Moreover, with this method, occa-
sional and small volume samples can be
taken without (extensive Iprior handling as it
takes less than 30 s from opening the lid of
the cage to the collection of 100/-11of blood. It
can be easily combined with weighing the
rat, and the method can be used in various rat
strains, from youth to senescence [Gomez
et a1. 1996, Workel et a1. 2000).
Taken together, the tail incision method
provides a series of advantages over other
blood sampling techniques. First, anaes-
thesia, surgery and restraint of the animal are
not necessary. Second, the procedure can be
considered stress-free as indicated by the low,
basal levels of the stress hormone cortico-
10
40
20
30
Tall Incision
Fig 3 Comparable corticosterone levels are mea-
sured in blood collected from the jugular vein and tail
incision (r = 0.987)
and still frequently used, incorporate a series
of unnecessarily aversive conditions for the
animals. For example, in order to insert a
needle into the tail vein of the rat, it has first
to be immobilized, e.g. in a restraining tube,
and furthermore, the environment or the tail
itself is heated up [Furuhama & Onodera
1983, Conybeare et a1. 1988, Frank et a1.
1991). Cutting off the tip of the tail, even
under anaesthesia, leaves it prone to inflam-
mation, is unnecessarily painful and requires
intense post-surgery care (Verbaeys et a1.
1995, Liu et a1. 1996). Orbital puncture (van
Herck et a1. 1998), foot bleeding (Snitily et a1.
1991), blood collection from the saphenous
Laboratory Animals (2000) 34
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Blood sampling by tail incision in rats
377
Brain P, Benton D (19791 The interpretation of
sterone even with frequent sequential blood
sampling over 3 h (Fig2). Concentrations of
corticosterone between 1 and 5 J.!gd/l are
considered as low basal levels. Third, blood
sampling by tail incision can be used for
longitudinal studies allowing intra-indivi-
dual comparisons over months and even
years. In addition, the present study indicates
the importance of the animals' home during
and after sampling. Placing the animal in a
novel cage, a simple procedure which hap-
pens weekly in the animal house and often
before experiments begin, elevates the corti-
costerone levels. Restraint of the animal can
boost corticosterone levels even more (Oitzl
et 01. 1995).In pharmacological studies one
should be aware of the activation of the stress
(and other physiological) systems as these
might interfere with other measurements
(O'Neill & Kaufmann 1990, Sarlis 1991,
Morton et 01. 1993).
physiological correlates of differential housing in
laboratory animals. Life Sciences 24, 99-116
Claassen V (19941 Neglected factors in pharmacology
and neuroscience research. In: Techniques in the
Behavioral and Neural Sciences, Vol. 12 (Huston
JP, ed). Amsterdam: Elsevier, pp 382-421
Conybeare G, Leslie GB, Angles K, Barrett RJ, Luke JS,
Gask DR (19881 An improved simple technique for
the collection of blood samples from rats and mice.
Laboratory Animals 22, 177-82
de Kloet ER, Vreugdenhil E, Oitzl MS, Jods M (19981
Brain corticosteroid receptor balance in health and
disease. Endocrine Reviews 19, 269-301
de Kloet ER, Oitzl MS, Jods M (19991 Stress and
cognition: are corticosteroids good or bad guys?
Trends in Neuroscience 22, 422-6
Durschlag M, Wurbel H, Stauffacher M, Von Holst
(1996) Repeated blood collection on the laboratory
mouse by tail incision-modification of an old
technique. Physiology & Behavior 60, 1565-8
Frank P, Schoenhart GL, Burton E (1991) A method
for rapid and frequent blood collection from the rat
tail vein. Tournai of Pharmacological Methods 26,
233-8
D
The refined tail method for blood sampling
in rats as well as a similar procedure for mice
(Durschlag et 01. 1996) is used regularly at
our institute. Over recent years, both meth-
ods have been incorporated in the course
given by the University of Leiden and Rot-
terdam. The Netherlands to acquire License
Category C, i.e. persons responsible for
directing animal experiments (see FELASA
recommendations: Wilson et 01. 1995).We
consider blood sampling by tail incision to be
a validated animal-friendly alternative to
other conventionally used methods and
techniques for collecting blood from labora-
tory rats.
Furuhama K, Onodera TA (1983) A Simple technique
for repeated blood collection from the tail vein of
the rat. TournaI of Toxicological Science 8, 161-3
Gomez F, Lahmame A, de Kloet ER, Amario A (19961
Hypothalamic·pituitary-adrenal
response to
chronic stress in five inbred rat strains: differential
responses are mainly located at the adrenocortical
level. Neuroendocrinology
63, 327-37
Hem A, Smith AJ, Solberg P (1998) Saphenous vein
puncture for blood sampling of the mouse, rat,
hamster, gerbil, guineapig, ferret and mink.
Laboratory Animals 32, 364-8
Liu JY, Diaz TG, Vadgama Tv. Henry JP (19961 Tail
sectioning: a rapid and simple method for repeated
blood sampling of the rat for corticosterone deter-
mination. Laboratory Animal Science 46, 243-5
McEwen BS, Sapolsky RM (19951 Stress and cognitive
function. Current Opinion in Neurobiology 5,
205-16
Acknowledgments
This study was supported by
Morton DB, Abbot D, Barclay R, Close BS, Ewbank R,
Gask D, Heath M, Mattic S, Poole T, Seamer J,
Southee J, Thompson A, Trussell B, West C,
Jennings M (19931 Removal of blood from labora-
tory mammals and birds, First report of the
BVA/FRAME/RSPCAjUFAW Joint Working Group
on Refinement. Laboratory Animals 27, 1-22
Oitzl MS, van Haarst AD, Sutanto W, de Kloet ER
(1995) Corticosterone, brain mineralocorticoid
receptors (MRs) and the activity of the hypotha-
NWO grant 554-545. We thank Alexander Holzhauer
for arranging the photographs, Toon Hesp, Ron
Timmermans (Central Laboratory Animal Institute,
University of Utrecht) and Jaap Goekoop (Psychiatric
Hospital Endegeest, Leiden) for analysing the haema-
tocrit and blood glucose concentrations.
References
lamic-pituitary-adrenal
(HPAI axis: the Lewis rat as
Besch EL, Chou BJ (1971) Physiological responses to
blood collection methods in rats. Proceedings of
the Society for Experimental Biology and Medicine
138, 1019-21
an example of increased central MR capacity and a
hyporesponsive HPA axis. psychoneuroendocri-
nology 20, 655-75
Laboratory Animals (2000) 34
Downloaded from lan.sagepub.com at OAKLAND UNIV on October 18, 2014

Fluttert, Dalm & Oitzl
378
van Herck H (1999) Orbital Puncture: A Non-
O'Neill PJ, Kaufmann LN (1990) Effects of indwelling
arterial catheters or physical restraint on food
consumption and growth patterns of rats: advan-
tages of non-invasive blood pressure techniques.
Laboratory Animal Science 40, 641-2
Sarlis NJ 119911Chronic blood sampling techniques in
stress experiments in the rat-a mini review.
Animal Technology 42,51-9
Terminal Blood Sampling Technique in Rats
IThesisl. The Netherlands: University of
Utrecht
Veldhuis HD, van Koppen C, van Ittersum R, de Kloet
ER (1982) Specificity of adrenal steroid receptor
system in rat hippocampus. Endocrinology 110,
374-80
Snitily MU, Gentry MJ, Mellencamp MA, Preheim LC
(19911A simple method for collection of blood from
the rat foot. Laboratory Animal Science 41, 285-7
Steffens AB (1969) A method for frequent sampling of
blood and continuous infusion of fluids in the rat
Verbaeys A, Ringoir S, van Maele G, Lameire N
119951Influence of feeding, blood sampling method
and type of anesthesia on renal function in the
normal laboratory rat. Urological Research 22,
377-82
Wilson MS, Berge E, Maess J, Mahouy G, Natoff 1,
Nevclainen T, van Zutphen LFM, Zanelly P 119951
without disturbing the animal. PhYSiology
Behavior 4, 833-6
&
FELASA recommendations
on the education and
van Eekelen JA, Rots NY, Sutanto W, de Kloet ER
(19911 The effect of aging on stress responsiveness
and central corticosteroid receptors in the Brown
Norway rat. Neurobiology of Aging 13, 159-70
van Eekelen JA, Oitzl MS, de Kloet ER (1995) Adreno-
cortical hyporesponsiveness and glucocorticoid
feedback resistance in old male Brown Norway rats.
TournaI of Gerontology: Biological Sciences 50, B83-9
training of persons working with laboratory ani-
mals: categories A and C. Laboratory Animals 29,
121-31
Workel JO, Oitzl MS, Fluttert M, Lesscher H,
de Kloet ER (20001 Persistent effects of maternal
deprivation during life: hypotha1amic-pituitary-
adrenal axis activity and reactivity of Brown
Norway rats. Journal of Neuroendocrinology
(in pressl
Zeller W, Weber H, Panoussis B, Barge T, Bergmann
(19981 Refinement of blood sampling from the
sublingual vein of rats. Laboratory Animals 32,
369-76
van Herck H, Baumans V, de Boer SF, van derGugten
J, van Woerkom AB, Beynen AC (1991) Endocrine
stress response in rats subjected to singular orbital
puncture while under diethyl-ether anaesthesia.
Laboratory Animals 25, 325-9
van Herck H, Baumans V, Brandt CJWM, Hesp APM,
Sturkenboom JH, van Lith HA, van Tintelen G,
Beynen AC (19981 Orbital sinus blood sampling in
rats as performed by different animal technicians:
the influence of technique and expertise. Labora-
tory Animals 32, 377-86
R
Laboratory Animals (2000) 34
Downloaded from lan.sagepub.com at OAKLAND UNIV on October 18, 2014