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Fall 1989
With minor blood losses, animals may be asymptomatic. With losses of approximately 10 percent or
less of the total blood volume, baroreceptor-initiated
reflexes cause release of cholinergics from the adrenal
medulla and sympathetic nerve endings. As a result,
(1) heart rate increases, (2) arteriolar beds in muscle
and skin constrict, and (3) veins and venous reservoirs
constrict. Thus arterial pressure, venous return, and
cardiac output are maintained or minimally affected.
Slower-acting compensatory mechanisms that help
replace lost volume include secretion of antidiuretic
hormone and activation of the renin-angiotensin aldosterone system.
With moderate blood losses the animal will suffer
drops in arterial pressure and cardiac output despite
compensatory mechanisms. Losses of approximately
15-20 percent of the total blood volume cause massive
cholinergic release with tachycardia and intense arteriolar constriction with further redistribution of blood
away from the gut and skin. Venous constriction
partially sustains venous return, and mobilization of
interstitial fluid to the intravascular compartment over
time restores some of the lost fluid volume. Anaerobic
glycolysis occurs due to the lack of oxygen. The
increased plasma lactate causes metabolic acidosis,
and a compensatory tachypnea ensues.
With further blood losses, drops in cardiac output,
blood pressure, and tissue perfusion become life threatening. Tissue anoxia, hypercapnia, and acidosis can
lead to widespread cell injury and irreversible tissue
damage, organ compromise, and death. With severe
blood losses, cardiac function is limited by decreased
cardiac perfusion as well as myocardial depressant
factor, which is released by the poorly perfused pancreas. Late in shock, decreased perfusion of the
medullary vasomotor center causes diminished compensatory reflexes.
Researchers should plan and execute each sampling
protocol with an appreciation for the stresses associated with blood loss to the animal and do whatever
they can to minimize the animal's reaction to the
stress. Careful planning and control of blood sampling
and all experimental variables associated with it should
not only improve the state of the animal, but also
minimize confounding influences on research data.
Dodds (1987) has four suggestions to researchers
wishing to minimize changes in blood components
while sampling blood: (1) select a more tractable
species or calm individuals, (2) precondition the animals to adapt them to handlers and procedures, (3)
use a sampling technique that minimizes changes in
blood values, and (4) note and control the use of
anesthetics, because these may change blood values.
Catheter
Fall 1989
Species
Whole Blood
Volume
(ml/kg)
%
Body
Weight
Dog
86
8.6
Cat
56 (47-66)
67"
43*.
6.7"
4.3*
Plasma
Volume
(ml/kg)
Approximate Absolute
Blood Volumes of Animals
of Described Weight
50
41 (35-52)
48"
Rat
64 (58-70)
6.4
40 (36-45)
300-g rat: 20 ml
Guinea pig
75 (67-92)
7.5
40 (35-48)
Rabbit
56 (44-70)
5.5
39(28-51)
NOTE: All values from Altman and Dittmer (1974) unless otherwise specified.
"Jain (1986).
''Breznock and Strack (1982).
Often, anesthesia can increase the stress of a sampling procedure. Wiersma reported that taking blood
samples from an atrial cannula in the rat was more
stressful when light ether was used because prolactin
levels were significantly elevated only when ether was
used (Wiersma and Kastelijn, 1985). Mattheij and van
Pijkeren (1977) observed an increase in prolactin levels
after a 45-second ether stress.
Furthermore, some anesthesias cause more stress
than others. Rats subjected to a 2.5-2.7 ml/100 g
hemorrhage over 10 minutes had a much lower survival
rate when anesthetized with intramuscular sodium
pentobarbital than with inhalant methoxyflurane (Yale
and Torhorst, 1972). Upton and Morgan (1975) found
little difference in blood parameters from the effects
of ether, pentobarbitone sodium, and fentanyl-droperidol while obtaining cardiac blood, although the use
of manual restraint significantly increased blood acidity
and raised the levels of hemoglobin, hematocrit, and
plasma protein, presumably because of stress. Lawson
and Gala (1974) observed prolonged increases in plasma
prolactin following ether and intraperitoneal injection
of sodium pentobarbitone, but no change in plasma
prolactin following ketamine or intraarterial sodium
pentobarbitone. (Refer also to "Mortality" on page 13
for references to studies on anesthesia effects in pigs.)
Rate of Bleeding
Total Blood
Volume (ml)
% Body Weight
(ml/100 g body weight)
250
17.4
6.97
300
19.5
6.51
350
21.1
6.04
400
22.3
5.58
250
2.7
39
300
2.6
40
350
2.5
42
400
2.4
43
% Total Blood
Volume Removed.
LD50
Various experiments have focused on different parameters to evaluate the effects of blood loss in humans
and animals. In the following section, discussions of
blood loss are organized according to these various
parameters. Table 4 summarizes results from the
literature on blood losses of greater than 30 percent
of total blood volume.
TABLE 4
Effects on Animals and Humans of Blood Loss Greater than 30 Percent of Total Blood Volume
Species
Volume
Removed
% Total
Blood
Volume
Results
References
Rat
25 ml/kg
40
..."
Rat
6 ml over 5 h
50
Polyethylene cannula in
caudal artery
Unaltered biodegrad. of
quinidine sulf. if vol.
replaced immed. with
bood or saline
Rat (germ-free)
2.6 ml/100 g
over 10 min
40 *
Rat (germ-free)
3.0 ml/100 g
over 60 min
47*
Rabbit
20-33 ml/kg
over 5 h
50*
Polyethylene cannula in
jugular vein
4- to 5-fold increase in
plasma levels of acid
hydrolases
Dog
> 30
20 mm Hg or greater drop
in blood pressure, pulse
> 100
Dog
46
Furneaux, 1969
Transient decrease in
plasma prothrombin,
increased activity of
aminotransferases,
phosphatases
88% mortality
Compensatory
mechanisms are
inadequate
Hemorrhagic shock
Dog
566-766 ml at
50 ml/min
over 12-16
min
43.6
('/> body
weight)
Swine
(unanesthetized)
Bled over 30
min
40
Swine
(unanesthetized)
30 ml/kg,
rapid
withdrawal
40
Human
30-40
Fall 1989
Unfortunately, the data published on these experiments do not include total blood loss and body weight,
so we cannot know what percentage of blood these
animals lost.
Furneaux (1969) observed that arterial pressure in
dogs who were bled 46 percent of their total volume
fell to 29.5 percent of the control just after the hemorrhage, rose to 45.3 percent of the control after two
hours, and then fell again as death approached. All
dogs in this study died.
According to one human study, when a drop in
blood pressure of 20 mm Hg or more corresponds with
a pulse greater than 100, blood loss is estimated to be
at least 30 percent of the total blood volume (Tovey
and Lennon, 1962).
Effects on Animals and Humans of Blood Loss Less than 30 Percent of Total Blood Volume
% Total
Blood
Volume
Rat
20 ml
30"
Rat
1-2 ml at 1
ml/7.5 min
5-11
Rat
3 ml at 1 ml/
7.5 min
Rat
Results
References
No change in plasma
corticosterone/prolactin
16"
Increased plasma
corticosterone
15"
No change in plasma
prolactin (attributed to
slow sampling method)
Rat
3 ml over 5 h
13"
Rat
1.2 ml over
20 min
6-7
Arterial catheter
Significant change in
plasma prolactin; no
change when volume
replaced by saline
Rat
(unanesthetized)
4 ml
20"
Chicken
(unanesthetized)
4 ml
Dog
(unanesthetized)
26 ml/kg
Vatner, 1974
30"
Dog
10
Furneaux, 1969
Dog
27
Furneaux, 1969
17% mortality
66% mortality
66% mortality
No signif. change in
capillary pressure to any
tissue except stomach
Vatner, 1974
"Venous removal"
Swine
Bled over 30
min; after
2 h, lost
volume
reinfused
10
20
30
Swine
15 ml/kg,
rapid
withdrawal
20
Baboon
(unanesthetized)
14 ml/kg
Human
(males)
800 ml
average
over 15.5
min
15
Fall 1989
11
Species
Volume
Removed
Ulcers
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Vasovagal Response
Mortality
Surprisingly, in studies on anesthetized pigs, a 20to 30-percent blood loss is associated with significant
mortality. In one study, a 30-percent blood loss caused
66 percent mortality (Hobler and Napodano, 1974). In
the same study, a 20-percent loss also caused 66 percent
mortality, while a 10-percent loss caused 17 percent
mortality. These results should be interpreted in the
light of comparative studies of hemorrhage in anesthetized and unanesthetized pigs. One such study
demonstrated that a 20-percent blood loss in a pentobarb-anesthetized pig has biological effects similar
to a 40-percent blood loss in a nonanesthetized pig
(Simon and Olsen, 1969). In none of the studies on
animals other than pigs was mortality a factor with
volume losses of 30 percent or less.
Biotransformation
Fall 1989
13
SAMPLING TECHNIQUES
Fall 1989
Mice
In the mouse, retroorbital sinus puncture and heart
puncture are the most common sampling techniques,
although success has been reported with other methods, including severing brachial vessels, jugular vein,
carotids, femoral vessels, and abdominal aorta, as well
as venipuncture of the caudal vena cava and tail
vessels.
Cardiac puncture in the mouse yields variable blood
volumes, and the blood is likely to be hemolyzed and
contaminated by tissue fluids (Adeghe and Cohen,
1986). Anesthesia is required for cardiac puncture
(Moreland, 1965).
Retroorbital sinus puncture can yield up to 1 ml of
venous blood in the mouse (Moreland, 1965). Adeghe
and Cohen (1986) reported that many trials are required
16
REFERENCES
Adeghe A. J. H., and J. Cohen. 1986. A better method for terminal
bleeding of mice. Lab. Anim. (London) 20:70-72.
Agrelo C. E., and J. O. Miliozzi. 1974. A technique for repeated
blood sampling with transfusion in the conscious rat. J. Pharm.
Pharmacol. 26:207-208.
Aikawa, J. K. 1950. Fluid volumes and electrolyte concentrations
in normal rabbits. Am. J. Physiol. 162:695-702.
Altman P. L., and D. S. Dittmer, eds. 1974. Biology Data Book,
2d ed., vol. 3. Bethesda, Maryland: Federation of American
Societies for Experimental Biology.
Antin, J., J. Gibbs, R. Holt, R. C. Young, and G. P. Smith. 1975.
Cholecystokinin elicits the complete behavioral sequence of
satiety in rats. J. Comp. Physiol. Psychol. 89:784-790.
Armin J., R. T. Grant, H. Pels, E. B. Reeve. 1952. The plasma,
cell and blood volumes of albino rabbits as estimated by the dye
(T1824) and 32P marked cell methods. J. Physiol. (London) 5973.
Barclay, R. J., W. J. Herbert, andT. B. Poole. 1988. The Disturbance
Index: A Behavioral Method of Assessing the Severity of Common Laboratory Procedures on Rodents. Potters Bar, UK:
Universities Federation for Animal Welfare.
Barcroft H., O. G. Edholm, J. McMichael, J. Sharpey-Schafer. and
E. P. Sharpey-Schafer. 1941. Post haemorrhagic fainting: Study
by cardiac output and forearm flow. Lancet i:489491.
Bassin R., B. C. Vladeck, S. 1. Kim, W. C. Shoemaker. 1971.
Comparison of hemodynamic responses of two experimental
shock models with clinical hemorrhage. Surgery 69(5):722-729.
Besch E. L., and B. J. Chou. 1971. Physiological responses to blood
collection methods in rats. Proc. Soc. Exp. Biol. Med. 138:10191021.
Bober, R. 1988. Drawing blood from the tail artery of a rat. Lab
Anim. 17(5):33-34.
Breazile, J. E. 1987. Physiologic basis and consequences of distress
in animals. J. Am. Vet. Med. Assoc. 191(10): 1212-1215.
Breznock. E. M., and D. Strack. 1982. Blood volume of nonsplenectomized and splenectomized cats before and after acute
hemorrhage. Am. J. Vet. Res. 43(10): 1811-1814.
Burhoe, S. O. 1940. Methods of securing blood from rats as described
in study of blood groups and their inheritance. J. Hered. 31:445448.
Burt M. E., J. Arbeit, and M. F. Brennan. 1980. Chronic arterial
and venous access in the unrestrained rat. Am. J. Physiol.
238:H599-H603.
Calligaris, L., and S. Taleisnik. 1983. Prolactin release induced by
stress and the influence of estrogen and progesterone treatments,
sex, and daily rhythm. Acta Endocrinol. (Copenhagen) 102(4): 505510.
Canadian Council on Animal Care. 1984. Guide to the Care and
Use of Experimental Animals, vol. 2. Ottawa: Canadian Council
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Volume 31, Number 4
Fall 1989
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Jain, N. C. 1986. Schalm's Veterinary Hematology, 4th ed. Philadelphia: Lea & Febiger.
Kraus, A. L. 1980. Research Methodology. P. 7 in The Laboratory
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Lestage, P., P. A. Vitte, J. P. Rolinat, R. Minot, and E. Brousolle.
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Letscher, R. M. 1987. Letter to the editor. ACLAM Newsl. 17:
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Lumsden, J. H., P. J. A. Presidente, and P. J. Quinn. 1974.
Modification of the orbital sinus bleeding technique for rabbits.
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Oliveira. G. A. Negraes, and M. A. Oliveira. 1987. Hyperosmotic
sodium salts reverse severe hemorrhagic shock: Other solutes
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May 8-9, 1987.
Fall 1989
stressful environmental stimuli, and its activation during blood sampling can rapidly modify cardiovascular
dynamics and also influence metabolic functions of
other tissues and organ systems. Blood and its various
components themselves can be altered by blood collection procedures and associated paraphernalia,
whether sampling is acute or chronic. Some of the
resulting influences may well alter the precise hematologic variable undergoing study, some represent
general systemic stress factors that seem mostly unavoidable owing to the fact that blood sampling is
inherently invasive and traumatic, some are simply
not known or recognized, and some can be catastrophic
to the experimental study. Indeed, McGuill and Rowan
poignantly point out that one attempt to develop an
arterial catheter for chronic blood sampling in rats
actually yielded "an excellent model for arterial embolism" with intestinal and renal infarctions! The
importance of such confounding influences to certain
studies, especially if they remain unrecognized and
uncontrolled, can hardly be overemphasized.
Many blood-sampling factors now are known to hold
real potential for altering hematologic and other phys19
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