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of Animal Pathology, Prophylaxis and Food Hygiene, University of Pisa, Via delle Piagge 2a56124 Pisa, Italy
of Animal Pathology, University of Turin, Via Leonardo da Vinci 4410095 Grugliasco (TO), Italy
A gross pathology and histological investigation was carried out on bovine target organs of anabolic substances in the Molise
Region (Italy). One hundred forty-four bovines (1224 months old, 123 males and 21 females) were included in the survey. An antemortem assessment of their behavior and clinical examination were performed. After slaughter, samples of prostate, Cowpers glands,
Bartholins glands, mammary gland, ovaries, thymus and thyroid were collected, inspected and processed for histopathology, as suggested
in the guidelines of the Italian national program for residue surveillance (PNR). Overall, 15.3% of the examined animals were classified
as suspect, 44.4% were classified as uncertain, and the remaining 40.3% were classified as negative. The most frequent lesion was
a severe thymus atrophy with fat infiltration (15.4% of males and 14.3% of females), strongly suggesting the illegal use of corticosteroids.
KEY WORDS: bovines, growth promoters, histological investigation, illegal drug treatments.
doi: 10.1292/jvms.11-0489; J. Vet. Med. Sci. 74(10): 12531259, 2012
Since the 1960s, the administration of growth promoters has been prohibited in Italy for auxinic purposes in
animals intended for human consumption. After the first
non-comprehensive laws, legislative updates were produced, in parallel with the European regulations, which led
to Decree 148/2009 [14], which implemented European
Directive 2008/97/EC [16]. In particular, at present it is
prohibited to administer to farm or aquaculture animals,
by any means whatsoever, thyrostatic substances, stilbenes,
stilbene derivatives, their salts and esters, estradiol 17 and
its ester-like derivatives, beta-agonists and substances having estrogenic (other than estradiol 17 and its ester-like
derivatives), androgenic or gestagenic action, except in the
case of regulated therapeutic or zootechnical treatments.
Among growth promoter molecules, sex steroids, beta
agonists and corticosteroids are the most frequently used.
Nevertheless, controversial growth promoter effects of
dexamethasone have been discussed in several studies [21].
Corticosteroids showed the ability to increase the external
fat thickness, carcass muscularity and dressing percentage.
Courtheyn et al. [12] reported that providing glucocorticoids
in low dosages increases feed intake and average daily gain
(ADG) and improves the feed conversion ratio. Tarantola et
al. [38] observed the lowest daily gain and the worst feed
conversion ratio in veal calves receiving a prolonged oral
low dose of dexamethasone. Corah et al. [11] reported a
greater thickness of external fat in treated steers.
*Correspondence to: Castigliego, L., Department of Animal
Pathology, Prophylaxis and Food Hygiene, University of Pisa,
Via delle Piagge 2a56124 Pisa, Italy.
e-mail: lcastigl@vet.unipi.it
2012 The Japanese Society of Veterinary Science
However, excessive exposure to sex hormones or residues of other growth promoters may lead to harmful effects
on the human endocrine glands, altering growth and puberty, and the immune system and may exert genotoxic and
carcinogenic effects [22]. In spite of strict legislation and
a national program for residue surveillance (PNR), which
specifies rules about the number of samples to be taken and
the sampling procedures, the problem of illegal treatments
with growth promoters is still extremely topical. The use of
these substances is facilitated by the fact that they cannot
be easily detected, either because of their low concentration
in biological samples, the very large number of molecules
potentially used, often modified from those indicated in the
official pharmacological protocols, or the use of so-called
cocktails. These are a mixture of molecules that act synergistically, permitting improved zootechnical results, while
lowering the relative concentration of each compound used
and thus the concentration of any residue, leading to further
difficulties in terms of analysis. However, illegal substances
used in livestock cause the occurrence of alterations, to different extents, in organs that respond to their stimulating
or inhibiting effects [1]. Indeed, over the years, studies on
this topic have suggested that the use of growth promoters can be highlighted by both macroscopic and histological changes of the genital tract organs and sex accessory
glands, in both males and females [6, 23, 24, 32], or other
organs such as the thymus [7, 28] or thyroid [37]. These
lesions persist long enough to be detected at slaughter. For
this reason, in addition to the traditional chemical analysis,
which can give unsatisfactory results [25], other auxiliary
diagnostic approaches to detect illegally treated animals are
based on anatomo-histopathological examination of target
organs [1]. Recently, biotechnological approaches [4, 33]
1254
P. IMBIMBO ET AL.
Table 1. List of organs examined and type of lesion considered
Organ
Prostate (urethra)
Prostate (glandular tissue)
Cowpers glands (ducti)
Cowpers glands (glandular tissue)
Bartholins glands (ducti)
Bartholins glands (glandular tissue)
Ovaries
Mammary gland
Thymus
Thyroid
Type of lesion
Hyperplasia, light or severe metaplasia
Hyperplasia, light or severe metaplasia; hypersecretion or cysts
Hyperplasia, light or severe metaplasia
Hyperplasia, light or severe metaplasia; hypersecretion or cysts
Hyperplasia, light or severe metaplasia
Hyperplasia, light or severe metaplasia, hypersecretion or cysts
Cysts and corpora lutea
Secreting alveoli
Atrophy of the parenchyma with fat tissue infiltration
Follicular epithelium hyperplasia
1255
82 (67.8%)
112 (91.8%)
8 (47.1%)
8 (100%)
13 (100%)
86 (62.3%)
140 (97.9%)
Uncertain
38 (31.4%)
10 (8.2%)
9 (52.9%)
0 (0%)
0 (0%)
31 (22.5%)
3 (2.1%)
Suspect
1 (0.8%)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
21 (15.2%)
0 (0%)
na
Total
2
1
4
13
8
6
1
123
123
21
21
21
144
144
The total number of animals (percentage) are reported for each group. na: not analyzed.
1256
P. IMBIMBO ET AL.
Table 3. Histological changes in target organs of males. Only animals with at least one lesion classified as uncertain are reported
N
2
4
5
6
10
11
14
15
16
17
18
19
20
21
22
23
24
25
31
32
33
34
35
40
41
43
44
47
48
49
50
51
52
54
57
59
60
63
65
66
70
72
73
74
76
77
78
79
82
85
86
87
88
89
93
96
97
98
104
106
108
110
111
112
113
115
117
119
121
124
131
135
136
137
143
Age
22
16
21
22
16
20
18
22
22
22
21
23
20
23
22
22
22
21
22
16
14
14
13
17
24
21
17
19
21
19
22
21
19
19
16
23
20
15
21
16
22
18
13
15
22
28
25
25
25
17
19
20
14
19
18
19
22
22
19
18
18
22
12
15
24
14
24
21
13
17
20
17
19
17
19
Prostate
Urethra
LM
M
H
H
H
H
H
H
H
H
LM
H
H
LM
H
H
LM
LM
H
LM
H
LM
LM
H
LM
LM
H
H
H
H
H
LM
H
LM
LM
LM
LM
H
H
H
LM
H
LM
LM
Gland
H+I
I
I
I
I
H
H
I
I
F
I
I
I+F
I
H+I
I+F
I+F
H+F
I
H+F
I
H+I
I
I
I
H+F
F
F
I
F
I
I+F
H+I
I
H
H+I
Cowpers glands
Resp
U
S
U
U
N
N
U
U
N
N
N
N
N
N
U
U
N
U
N
U
N
U
U
U
U
U
na
U
U
N
U
N
U
N
N
U
N
U
U
U
N
U
U
N
N
N
N
N
N
U
N
U
U
U
U
N
U
U
N
N
U
U
U
N
N
U
N
U
N
N
N
U
U
N
N
Ducti
Gland
I
C
I+F
I+F
H+I
Thymus
Resp
N
N
N
N
N
N
U
N
N
N
N
N
N
N
U
N
U
N
N
N
N
U
U
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
U
N
N
N
N
N
N
N
N
N
U
N
U
U
N
N
U
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
Atrophy
Mo
P
P
Mo
Mo
P
P
Mo
Mo
Mo
Mo
Mo
Mo
Mo
Mo
P
P
Mo
Mo
Mo
Mo
P
Mo
L
Mo
na
Mo
P
Mo
P
Mo
Mo
P
L
Mo
Mo
P
Mo
Mo
Mo
P
Mo
P
P
Mo
P
P
P
Resp
N
N
S
S
U
U
N
na
S
S
U
U
U
U
U
U
N
N
S
N
N
N
N
N
U
N
U
S
S
U
U
U
U
S
U
N
U
N
N
na
N
N
N
U
S
U
S
U
U
N
N
N
N
S
N
U
N
N
U
S
U
U
N
U
S
U
S
N
S
U
S
N
N
S
S
Thyroid
Resp
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
U
N
N
U
N
U
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
LM=light metaplasia; M=metaplasia; H=hyperplasia; I=hypersecretion; F=flogosis; L=light; Mo=moderate; P=prominent; N=negative;
U=uncertain; S=suspect; na=not analyzed.
1257
Fig. 1. Histological findings of a negative urethra (A) compared with epithelial metaplasia in the urethra of the only animal classified as suspect for steroids (B) (200 magnification). Staining: HE.
Fig. 2. Histological findings of a negative thymus (A) compared with an atrophic thymus classified as
suspect (B) (40 magnification). The latter shows severe atrophy of the parenchyma, lymphocyte
depletion and diffuse fat tissue infiltration. Both samples come from 17-months-old males. Staining: HE.
Table 4. Histological changes in target organs of females. Only animals with at least one lesion classified as uncertain are reported
N
1
8
12
38
102
103
114
120
124
125
129
Age
12
21
25
14
38
19
24
27
17
21
17
Bartholins glands
Ovaries
Ducti
Gland
Resp
M
M
LM
M
H+F
M
LM+I
LM+I
M
M
M
M
I
LM+I
LM+I
H+I
U
U
N
U
N
U
na
U
U
U
U
H
LM
Mammary gland
Resp
na
na
na
na
na
na
na
CL
C
CL
na
N
N
N
N
N
N
Resp
A
Sec
Sec
A+Sec
A
A
N
N
N
N
N
N
na
na
na
na
na
Thymus
Atrophy
P
Mo
P
Mo
P
Mo
Mo
Thyroid
Resp
Resp
N
N
S
U
S
U
S
N
U
N
U
N
N
N
N
N
N
N
N
N
N
na
LM=light metaplasia; M=metaplasia; H=hyperplasia; I=hypersecretion; F=flogosis; C=cysts; CL=corpus luteum; A=alveoli; Sec=secretion;
Mo=moderate; P=prominent; N=negative; U=uncertain; S=suspect; na=not analyzed.
1258
P. IMBIMBO ET AL.
30.
31.
32.
33.
34.
35.
1259