Exp. Anim. 62(1), 1–7, 2013
—Original—
Background Data for Hematological and Blood
Chemical Examinations in Juvenile Beagles
Toshiya Ishii, Hisako Hori, Makoto Ishigami, Hiroyasu Mizuguchi, and Dai Watanabe
Kannami Laboratory, Bozo Research Center Inc., 1308–125 Kuwahara-Sanbonmatsu, Kannami-cho, Tagata-gun,
Shizuoka 419-0101, Japan
Abstract: As the first step to get historical background data for physiological examinations in juvenile
dogs, hematology and blood chemistry data obtained from juvenile beagle dogs (less than 3 months
of age) used in the control group of toxicity studies conducted in our laboratory were summarized
and compared with those obtained from adult beagle dogs (6 months of age). In the hematological
examination, growth of beagle dogs was shown to be associated with increases in erythrocyte
parameters and with decreases in reticulocyte and leukocyte counts. In the blood chemical examination,
growth of beagle dogs was shown to be associated with increases in aspartate aminotransferase,
alanine aminotransferase, and creatinine and with decreases in creatine phosphokinase, glucose,
total cholesterol, and calcium. The differential leukocyte ratio showed no age relation, but the actual
count showed a tendency toward decrease. Alkaline phosphatase showed a tendency to increase
from 0 months of age to 3 months of age, but it decreased at 6 months of age. The present results
were roughly similar to those previously reported.
Key words: blood chemical examination, hematological examination, juvenile dog, safety study
Introduction
With the progress in juvenile drug development in
recent years, juvenile animal toxicity studies have come
to receive much attention as a means of examining the
potential for increased juvenile sensitivity to pharmaceuticals and for novel juvenile toxicities that are not
detected in adults [2].
In order to accurately evaluate the reaction of juvenile
animals to drugs, it is important to make good use of
historical background data on physiology, especially on
hematology and blood chemistry, because hematological
and blood chemical examinations are conducted frequently to grasp systemic or organ-specific abnormalities
in toxicity studies. However, physiological background
data in juvenile dogs are still insufficient compared with
those in juvenile rats. Although some information about
age-related changes in hematology and blood chemistry
is available in dogs [1, 3–5], there are only a few reports
that have attempted to summarize hematological and
blood chemical data in juvenile beagle dogs used as
control animals in toxicity studies conducted under controlled conditions. Therefore, as the first step to get useful historical background data in normal juvenile beagle
dogs, this study was carried out to summarize the data
on hematology and blood chemistry obtained from toxicity studies conducted in our laboratory and to compare
them with those in adult beagle dogs.
Materials and Methods
Animals
The animals used for obtaining hematological and
blood chemical background data in the present study
(Received 9 May 2012 / Accepted 3 August 2012)
Address corresponding: T. Ishii, Kannami Laboratory, Bozo Research Center Inc., 1308–125 Kuwahara-Sanbonmatsu, Kannami-cho, Tagata-gun,
Shizuoka 419-0101, Japan
©2013 Japanese Association for Laboratory Animal Science
2
T. ISHII, ET AL.
were collected from control groups in toxicity studies
conducted in our laboratory during the 6 years from 2006
to 2011. In those toxicity studies, the animals in the control group were given 0.5% w/v methylcellulose, 5% w/v
gum arabic solution (oral administration) or physiologic saline (intravenous administration), which are usually used as vehicles for drug administration. In the
present study, beagle dogs (less than 3 months of age)
obtained from different breeders [TOYO beagle, Kitayama Labes Co., Ltd. (Yamaguchi, Japan); NOSAN
beagle (present name: Narc beagle), Kitayama Labes
Co., Ltd.; and Iar beagle, Institute for Animal Reproduction (Ibaraki, Japan); 4 to 76 males and 4 to 75 females
depending on the breeder] were treated collectively in
each age group and divided into 4 age groups (0, 1, 2,
and 3 months of age), with the animals under one month
of age regarded as 0 months of age. Except for the
0-month-old group, the number of animals was expressed
as a range (Tables 1 and 2) because the number of animals used and the items examined were different among
the toxicity studies from which the data were collected.
In addition, for comparison with juvenile beagles, the
data collected from adult HRA beagles (6 months of age,
231 to 1,128 males and 220 to 1,070 females, Covance
Research Products Inc., Cumberland, VA, USA) were
also used (Tables 1 and 2).
Housing conditions
The animals were housed in an animal room environmentally controlled according to Good Laboratory Practice [temperature range, 22 ± 4°C; relative humidity
range, 55 ± 25%; air exchange, 13–15 times/h; and 12-h
lighting per day (07:00 to 19:00)]. The animals in the
0-month-old group were given Esbilac artificial milk
(Kyoritsu Seiyaku Corporation, Tokyo, Japan) 3 times
a day, those in the 1-month-old group were given a mixture of Esbilac artificial milk and pelleted diet for dogs
(DS-A, Oriental Yeast Co., Ltd., Tokyo, Japan) 3 times
a day, those in the 2-month-old group were given 150 g
of the same pelleted diet once a day, and those in the
3- and 6-month-old groups were given 300 g of the same
pelleted diet once a day.
Hematological and blood chemical examinations
Blood was collected via the cervical vein or cephalic
vein after overnight fasting and subjected to hematological examination [anticoagulant, EDTA-2K, 3.8 w/v%
sodium citrate solution for measurements of PT, APTT,
and fibrinogen; measuring instruments, ADVIA 120
Hematology System (Siemens Healthcare Diagnostics
Inc., Deerfield, IL, USA) and ACL 100 Coagulometer
(Instrumentation Laboratory, Bedford, MA, USA)]. In
addition, separated plasma was subjected to blood chemical examination [anticoagulant, heparin sodium; measuring instrument, TBA-120FR Clinical Laboratory
System (Toshiba Medical Systems Corporation, Tochigi,
Japan)].
Statistical analysis
For statistical analysis, the mean and standard deviation (SD) were calculated for each item for each age
group and compared with those in the 6-month-old
group. The data were analyzed first by F test (level of
significance 5%, unilateral) for homogeneity of variance,
and the homogeneous data were compared by the Student’s t-test (level of significance 5%, bilateral), while
the heterogeneous data were compared by the AspinWelch’s t-test (level of significance 5%, bilateral).
The studies were conducted in compliance with the
guidelines for the control and welfare of experimental
animals stipulated by the testing facility (Institutional
Animal Care and Use Committee, Bozo Research Center
Inc.).
Results and Discussion
In the hematological examination, as shown in Table
1 and Fig. 1, the values of erythrocytes, hemoglobin,
hematocrit, and MCHC increased, while those of MCV,
MCH, reticulocytes (ratio and count), and leukocytes
decreased from 0 to 2 months of age, at which point the
values became closer to those in adult dogs. Although
the differential leukocyte ratio showed no clear age-related changes, the actual count showed a tendency towards decreasing with age. The platelet count was
higher in the juvenile animals than in the adult ones, but
no clear age-related changes were observed. In addition,
prothrombin time (PT) and activated partial thromboplastin time (APTT) showed no clear age-related changes. There were no sex differences in hematology (Table
1).
In the blood chemical examination, as shown in Table
2 and Fig. 2, the values of aspartate aminotransferase
(AST), alanine aminotransferase (ALT), creatinine, total
protein, and albumin increased, while those of creatine
phosphokinase (CPK), glucose, total cholesterol, triglyc-
3
BACKGROUND DATA IN JUVENILE BEAGLES
Table 1. Hematological findings in juvenile and adult beagle dogs
Months
No. of animals* Male
Female
Erythrocyte (×104/μl)
Hemoglobin (g/dl)
Hematocrit (%)
MCV (fl)
MCH (pg)
MCHC (g/dl)
Platelet (×104/μl)
Reticulocyte ratio (%)
Reticulocyte count (×109/l)
Leukocyte (×102/μl)
Lymphocyte (%)
Neutrophil (%)
Eosinophil (%)
Basophil (%)
Monocyte (%)
LUC (%)
Lymphocyte (×102/μl)
Neutrophil (×102/μl)
Eosinophil (×102/μl)
Basophil (×102/μl)
Monocyte (×102/μl)
LUC (×102/μl)
PT (s)
APTT (s)
Fibrinogen (mg/dl)
0
1
2
3
6 (Adult)
12
12
415 ± 31c)
423 ± 25c)
10.5 ± 0.8c)
10.7 ± 0.5c)
32.9 ± 2.5c)
33.4 ± 1.4c)
79.4 ± 2.4c)
79.2 ± 2.7c)
25.3 ± 1.0c)
25.2 ± 0.7c)
31.8 ± 0.4c)
31.8 ± 0.6c)
46.7 ± 9.4c)
47.5 ± 9.0c)
4.7 ± 1.0c)
4.9 ± 1.4c)
196.3 ± 47.1c)
206.6 ± 56.3c)
208.0 ± 68.5c)
165.4 ± 70.8a)
33.2 ± 9.1
35.7 ± 10.7
52.5 ± 9.3
52.7 ± 12.5
2.7 ± 1.5
2.0 ± 0.6
0.6 ± 0.1
0.5 ± 0.2
7.8 ± 2.5
6.6 ± 2.1
3.3 ± 2.1c)
2.6 ± 1.2c)
67.0 ± 22.0c)
54.5 ± 19.9b)
112.5 ± 52.4a)
92.1 ± 58.8
5.2 ± 2.8b)
3.2 ± 1.5b)
1.1 ± 0.5b)
0.8 ± 0.5
15.6 ± 5.2c)
10.4 ± 4.1b)
6.7 ± 4.3c)
4.3 ± 3.5b)
6.2 ± 0.1b)
6.4 ± 0.3
14.0 ± 1.9
15.6 ± 1.3
–
–
4–36
6–36
470 ± 79c)
489 ± 64c)
10.8 ± 1.6c)
11.0 ± 1.0c)
33.9 ± 4.7c)
34.9 ± 3.2c)
72.5 ± 3.3c)
71.6 ± 4.1a)
23.1 ± 0.8
22.7 ± 1.1
31.9 ± 0.5c)
31.7 ± 1.3c)
57.3 ± 14.2c)
61.0 ± 18.4a)
2.9 ± 1.4
2.7 ± 2.6
129.0 ± 41.8a)
123.9 ± 102.7
192.6 ± 48.2c)
174.9 ± 54.5a)
31.7 ± 7.9
33.3 ± 8.8
56.9 ± 6.8
55.6 ± 9.1
3.0 ± 1.2a)
1.9 ± 0.6
0.5 ± 0.1
0.6 ± 0.4
7.0 ± 1.1
7.4 ± 1.1c)
0.9 ± 0.6a)
1.2 ± 1.3
60.5 ± 16.4
57.5 ± 22.9
109.8 ± 31.6c)
97.8 ± 39.0
6.1 ± 3.9
3.3 ± 1.4a)
1.0 ± 0.4b)
1.1 ± 1.1
13.3 ± 2.7c)
12.8 ± 3.8b)
1.9 ± 1.5
2.4 ± 3.2
7.0 ± 1.8a)
6.6 ± 1.3
15.1 ± 2.3
15.1 ± 3.1
–
–
46–64
44–64
544 ± 36c)
551 ± 37c)
12.3 ± 0.8c)
12.6 ± 0.7c)
37.2 ± 2.5c)
37.7 ± 2.4c)
68.4 ± 1.8c)
68.5 ± 2.0c)
22.7 ± 0.7
22.9 ± 0.7a)
33.3 ± 0.9c)
33.4 ± 0.9c)
53.0 ± 11.8c)
52.4 ± 13.7c)
1.8 ± 0.7c)
1.3 ± 0.5c)
98.7 ± 39.3c)
73.0 ± 27.5c)
138.2 ± 42.2c)
125.7 ± 34.5c)
36.4 ± 7.4
35.1 ± 6.3
53.3 ± 7.4b)
54.6 ± 5.9
1.9 ± 1.0
2.0 ± 1.2
0.5 ± 0.3
0.6 ± 0.3
7.0 ± 1.5b)
6.9 ± 1.9c)
0.8 ± 0.7a)
0.9 ± 0.8b)
48.4 ± 10.2c)
43.7 ± 13.0c)
75.5 ± 32.8
68.8 ± 20.9a)
2.5 ± 1.2
2.4 ± 1.4
0.7 ± 0.5a)
0.7 ± 0.7
9.8 ± 4.2c)
8.8 ± 4.0c)
1.2 ± 1.1b)
1.2 ± 1.5b)
6.5 ± 1.1
6.4 ± 0.3b)
15.8 ± 2.5
16.3 ± 2.4
204 ± 29b)
214 ± 26
14
14
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
6.3 ± 0.2
6.2 ± 0.1c)
15.2 ± 2.0
16.0 ± 2.3
230 ± 35
239 ± 43a)
231–866
220–822
631 ± 47
649 ± 49
14.2 ± 1.0
14.7 ± 1.1
41.1 ± 3.0
42.5 ± 3.1
65.2 ± 2.1
65.5 ± 2.2
22.5 ± 0.7
22.6 ± 0.8
34.6 ± 1.0
34.6 ± 1.0
36.5 ± 8.8
33.9 ± 7.9
0.8 ± 0.3
0.8 ± 0.3
52.1 ± 20.2
49.9 ± 20.1
112.1 ± 28.6
102.1 ± 26.0
34.3 ± 7.1
35.4 ± 7.2
56.4 ± 7.4
56.1 ± 7.3
2.0 ± 1.0
2.1 ± 1.3
0.5 ± 0.2
0.5 ± 0.3
6.3 ± 1.5
5.3 ± 1.2
0.6 ± 0.4
0.5 ± 0.3
37.9 ± 9.0
36.0 ± 8.9
65.8 ± 23.6
59.2 ± 24.1
2.2 ± 1.1
2.1 ± 1.3
0.6 ± 0.3
0.6 ± 0.3
7.3 ± 2.7
5.7 ± 2.2
0.6 ± 0.4
0.6 ± 0.7
6.4 ± 0.7
6.5 ± 1.0
15.2 ± 2.3
15.7 ± 2.5
240 ± 66
206 ± 50
Values are means ± SD. *: The number of animals is expressed as a range because the number of animals used and the
parameters examined are different among the toxicity studies from which the data were collected. a,b,c)Significantly different from the male or female animals at 6 months of age (P<0.05, P<0.01, P<0.001, respectively). –: Not applicable.
4
T. ISHII, ET AL.
Table 2. Blood chemical findings in juvenile and adult beagle dogs
Months
No. of animals*
0
Male
Female
AST (IU/l)
ALT (IU/l)
LDH (IU/l)
CPK (IU/l)
ALP (IU/l)
Total cholesterol (mg/dl)
Triglyceride (mg/dl)
Phospholipid (mg/dl)
Total bilirubin (mg/dl)
Glucose (mg/dl)
BUN (mg/dl)
Creatinine (mg/dl)
Sodium (mmol/l)
Potassium (mmol/l)
Chloride (mmol/l)
Calcium (mg/dl)
Phosphorus (mg/dl)
Total protein (g/dl)
Albumin (g/dl)
A/G
12
12
15 ± 4c)
17 ± 3c)
13 ± 2c)
14 ± 4c)
53 ± 12a)
47 ± 17
505 ± 179c)
482 ± 138c)
393 ± 62
426 ± 44a)
243 ± 40c)
248 ± 40c)
34 ± 8b)
29 ± 5a)
–
–
0.0 ± 0.0c)
0.0 ± 0.0c)
133 ± 13c)
133 ± 20c)
8 ± 3c)
7 ± 2c)
0.22 ± 0.02c)
0.23 ± 0.03c)
145 ± 1c)
146 ± 1c)
5.2 ± 0.4c)
5.5 ± 0.4c)
112 ± 2
113 ± 1a)
12.2 ± 0.2c)
12.2 ± 0.3c)
8.2 ± 0.4c)
8.2 ± 0.4c)
4.9 ± 0.3c)
4.8 ± 0.3c)
2.5 ± 0.1c)
2.5 ± 0.1c)
1.04 ± 0.10b)
1.11 ± 0.10b)
1
2
34–36
4–39
19 ± 3c)
17 ± 2c)
18 ± 6c)
17 ± 7c)
47 ± 17
45 ± 17
358 ± 80c)
363 ± 85c)
566 ± 133c)
574 ± 177c)
181 ± 32c)
176 ± 39c)
37 ± 6c)
36 ± 12c)
–
303 ± 28
0.0 ± 0.0c)
0.0 ± 0.0c)
130 ± 14c)
129 ± 16c)
4 ± 1c)
5 ± 1c)
0.22 ± 0.03c)
0.23 ± 0.03c)
145 ± 2c)
145 ± 2c)
5.0 ± 0.6b)
4.8 ± 0.6a)
109 ± 2c)
109 ± 2c)
12.0 ± 0.4c)
11.9 ± 0.5c)
8.3 ± 0.5c)
8.3 ± 0.5c)
4.9 ± 0.2c)
4.9 ± 0.3c)
2.7 ± 0.1c)
2.7 ± 0.2c)
1.18 ± 0.08b)
1.20 ± 0.11
20–76
20–75
23 ± 5c)
23 ± 4c)
24 ± 11c)
23 ± 6c)
49 ± 18
54 ± 23b)
364 ± 163b)
357 ± 113c)
661 ± 154c)
657 ± 169c)
142 ± 21c)
134 ± 23c)
28 ± 7a)
26 ± 8a)
291 ± 41
285 ± 41b)
0.0 ± 0.0c)
0.0 ± 0.0c)
116 ± 7c)
115 ± 9c)
7 ± 2c)
8 ± 2c)
0.29 ± 0.05c)
0.30 ± 0.04c)
148 ± 1a)
148 ± 1c)
4.5 ± 0.2c)
4.4 ± 0.3c)
110 ± 1c)
111 ± 2c)
11.5 ± 0.4c)
11.3 ± 0.4c)
8.2 ± 0.5c)
8.0 ± 0.6c)
5.2 ± 0.2c)
5.1 ± 0.3c)
3.0 ± 0.2c)
2.9 ± 0.2c)
1.35 ± 0.16c)
1.38 ± 0.21c)
3
5–16
5–15
26 ± 5
26 ± 5a)
22 ± 7c)
22 ± 6c)
47 ± 13
51 ± 14
–
–
747 ± 152c)
752 ± 184c)
147 ± 22b)
141 ± 16c)
26 ± 7
26 ± 11
297 ± 33
285 ± 32
0.0 ± 0.0b)
0.0 ± 0.1a)
110 ± 7c)
108 ± 6c)
9 ± 3c)
9 ± 2c)
0.36 ± 0.04c)
0.37 ± 0.06c)
147 ± 1b)
148 ± 1a)
4.6 ± 0.2a)
4.6 ± 0.3
110 ± 2a)
110 ± 2
11.3 ± 0.3c)
11.2 ± 0.2c)
7.8 ± 0.5c)
7.8 ± 0.5c)
5.3 ± 0.2c)
5.2 ± 0.2c)
3.1 ± 0.1
3.0 ± 0.2
1.34 ± 0.09c)
1.38 ± 0.11a)
6 (Adult)
725–1128
684–1070
28 ± 5
29 ± 6
31 ± 8
31 ± 8
44 ± 14
44 ± 15
233 ± 90
220 ± 76
406 ± 86
388 ± 86
131 ± 21
122 ± 21
26 ± 9
24 ± 9
281 ± 38
268 ± 37
0.1 ± 0.1
0.1 ± 0.1
101 ± 8
101 ± 7
12 ± 2
12 ± 2
0.49 ± 0.05
0.51 ± 0.06
148 ± 2
149 ± 2
4.7 ± 0.4
4.6 ± 0.4
111 ± 2
111 ± 2
11.0 ± 0.3
11.0 ± 0.3
6.4 ± 0.5
6.2 ± 0.6
5.7 ± 0.3
5.6 ± 0.3
3.0 ± 0.2
3.1 ± 0.2
1.14 ± 0.12
1.24 ± 0.15
Values are means ± SD. *: The number of animals is expressed as a range because the number of animals used and the
parameters examined are different among the toxicity studies from which the data were collected. a,b,c)Significantly different from the male or female animals at 6 months of age (P<0.05, P<0.01, P<0.001, respectively). –: Not applicable.
eride, potassium, calcium, and phosphorus decreased
age-dependently in the juvenile animals. Thereafter,
those values showed tendencies to approach the values
in the adult animals. Alkaline phosphatase (ALP) showed
an age-dependent increase from 0 to 3 months of age,
but it decreased at 6 months of age. The values of lactate
dehydrogenase (LDH), blood urea nitrogen (BUN), sodium, and chloride showed no clear age-related changes.
There were no sex differences in blood chemistry (Table
2).
In the present study, each age group included animals
obtained from different breeders and different toxicity
BACKGROUND DATA IN JUVENILE BEAGLES
5
Fig. 1.Hematological parameters showing remarkable age-related changes in beagle dogs. Open columns, males; closed columns, females. *,**,***Significantly different from animals at 6 months
of age (P<0.05, P <0.01, P <0.001, respectively).
studies as mentioned above. Although the data is not
shown in the present study, hematological and blood
chemical values in the control group seemed not to be
greatly different among breeders and toxicity studies. To
clarify this point, further analysis based on a large
amount of data is needed.
It is known that the values of hematological and blood
chemical parameters in dogs vary remarkably with
growth [5], and the results obtained in the present study
were roughly similar to those reported in previous papers
[1, 3–5]. In 2012, Rosset et al. reported age-related
changes in biochemical and hematologic variables in
borzoi and beagle puppies from birth to 8 weeks [4], and
they stressed that clinicians should be aware of temporal
changes in biochemical and hematologic values for puppies during the first 2 months of life to avoid misinterpretation of results [4]. Toxicologists also have to be
careful when they evaluate the results of hematological
and blood chemical examinations.
In conclusion, the present data will provide useful
information for evaluating the results of hematological
and blood chemical examinations in toxicity studies using juvenile beagle dogs. Hereafter, we will continue to
collect much more data on hematology and blood chemistry in juvenile beagle dogs.
6
T. ISHII, ET AL.
Fig. 2.Blood chemical parameters showing remarkable age-related changes in beagle dogs. Open
columns, males; closed columns, females. *,**,***Significantly different from animals at 6
months of age (P <0.05, P <0.01, P <0.001, respectively).
Acknowledgments
The authors would like to thank Dr. Kunio Doi, Professor Emeritus, the University of Tokyo, for reviewing this
manuscript and Mr. Daniel Dunsmore for language editing.
BACKGROUND DATA IN JUVENILE BEAGLES
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