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Int. J. Exp. Path.

(2012), 93, 172–178

ORIGINAL ARTICLE

Adverse effects of the anabolic steroid, boldenone undecylenate,


on reproductive functions of male rabbits
Samah S. Oda* and Ibrahim M. El-Ashmawy 
*Departments of Pathology and  Pharmacology, Faculty of Veterinary Medicine, Alexandria University, Edfina-Rashid-Behera, Egypt

INTERNATIONAL Summary
JOURNAL OF This study was conducted to evaluate the adverse effects of the anabolic steroid,
EXPERIMENTAL boldenone undecylenate (BOL) on reproductive functions of male rabbits. Thirty
white New Zealand mature male rabbits were divided into three groups (10 rabbits
PATHOLOGY
each). Group A rabbits served as a control group. Group B rabbits received
4.4 mg ⁄ kg body weight (bwt) BOL 5% oily solution. Group C rabbits received
8.8 mg ⁄ kg bwt BOL. Rabbits were injected intramuscularly twice weekly for two
doi: 10.1111/j.1365-2613.2012.00814.x
months. BOL had no significant effect on the bwt and bwt gain. Testes and epididy-
mis weights were decreased significantly in the BOL-treated groups. BOL caused sig-
Received for publication: 12 July 2011
Accepted for publication: 11 February nificant reduction in serum testosterone level, seminal volume, sperm motility, and
2012 sperm count. No abnormalities were detected in the sperm morphology of the BOL-
Correspondence: treated groups. Histopathological alterations in the testes and epididymis were mark-
Samah Shehata Oda ed in the group C rabbits. These results indicate that administration of BOL exerts a
Lecturer of Pathology
significant harmful effect on the reproductive functions of male rabbits.
Department of Pathology
Faculty of Veterinary Medicine
Alexandria University
PO Box 22758
Edfina-Rashid-Behera
Egypt
Tel.: +20 106503624
Fax: +20 452960450 Keywords
E-mail: samahoda@yahoo.com anabolic steroids, boldenone, histopathology, rabbits, testes

Anabolic androgenic steroids (AAS) are synthetic derivatives atable source of protein. We found BOL to be used heavily
of the male testosterone hormone that have been modified in Egypt, not only in the field of animal production, but also
to improve their anabolic rather than androgenic activity by athletes and bodybuilders. BOL increases muscle size
(Shahidi 2001). The anabolic effects of AAS promote protein owing to promotion of positive nitrogen balance by stimu-
synthesis, muscle growth and erythropoiesis (Mottram & lating protein production and reducing protein destruction,
George 2000). Hence, AAS are used to enhance strength and as well as causing retention of body water, nitrogen, sodium,
durability of canine, equine and human athletes (Teale & potassium and calcium ions (Forbes 1985; Mooradian et al.
Houghton 1991; Schänzer & Donike 1992; Schänzer 1996). 1987).
Boldenone (BOL) is an anabolic steroid that differs from tes- Like other androgenic steroids, BOL is classified by the
tosterone only by one double bond at the 1- position (Stol- International Agency for Research on Cancer (IARC) in class
ker et al. 2007) (Figure 1). It is used mainly as undecylenate 2A (growth promotors – steroids), as a probable human car-
ester by bodybuilders and is administered illegally to racing cinogen (e.g. prostate and liver tumours), with a carcinoge-
horses. However, it is used as a growth promotor on farms nicity index higher than that of other androgens, such as
improving the growth and feed conversion of cattle; it nandrolone, stanozolol and testosterone and is thus a
may be abused to achieve more efficient meat production banned substance (IARC Monograph 1987; De Brabander
(Gryglik et al. 2010). In developing countries with rapid et al. 2004). Despite these restrictions, AAS are easily
growth of population, like Egypt, the demand for edible pro- obtained. The abuse of AAS can lead to serious and irrevers-
tein exceeds the supply and the gap is expanded. Meat from ible organ damage (Maravelias et al. 2005). Among the most
animals, including from rabbits, provides a valuable and pal- common adverse effects of AAS that have been described are

 2012 The Authors.


172 International Journal of Experimental Pathology  2012 International Journal of Experimental Pathology
Boldenone undecylenate and male rabbits 173

were then weighed weekly until the end of the experiment.


Final bwt was recorded, and weight gain was calculated.

Reproductive organs weights. All rabbits were killed at the


end of the experiment. After dissection, the testes, epididy-
mis and prostate glands were removed, grossly examined
Figure 1 Structure of Boldenone and Testosterone. and weighed. The index weight (I.W.) of each organ was
calculated by Matousek (1969) I.W. = organ weight
(g) ⁄ 100 · body weight (g).
reduced fertility (Dohle et al. 2003), hypertension (Ferenchick
1990), atherosclerosis (Cohen et al. 1988), blood clotting
(Parssinen & Seppala 2002), hepatic neoplasms and carci- Semen collection and sperm characteristics
noma (Velazquez & Alter 2004), tendon damage (Battista
Ejaculates were collected from each male rabbit prior to the
et al. 2003), psychiatric and behavioural disorders (Clark &
treatment, after one month of treatment and at the end of
Henderson 2003).
the experiment with a rabbit artificial vagina. Each buck
There have been relatively few studies which have investi-
was conditioned to react with the artificial vagina as
gated the detrimental effects of BOL administration on male
described by Breddman et al. (1964). Each male was
function. These have explored their role as growth promo-
allowed a false mounting for teasing prior to the actual
tors on testis; bulbourethral glands and prostates of veal
mounting. The semen was evaluated immediately after col-
calves (Groot & Biolatti 2004; Cannizzo et al. 2007), on
lection for the following criteria:
reproductive function of stallions (Squires et al. 1982;
Squires & Mckinnon 1987; Garcia et al. 1987) and on • Semen volume
reproductive performance of male rabbits (Thabet et al. The volume of ejaculate in ml was measured to the near-
2010). Hence, this study was performed to determine the est 0.1 ml using a graduated collecting tube.
effects of high-dose administration of BOL on body weight • Sperm motility
(bwt), reproductive organ weight, semen characteristics, Immediately following collection of a semen sample, a
serum testosterone levels and histopathological features of small drop was taken with a capillary pipette and placed
the reproductive organs of mature male rabbits. over a warm clean glass slide. A cover slip was placed over
the semen droplet, and the percent of motile spermatozoa
was microscopically estimated at ·400 magnification accord-
Materials and methods
ing to Bearden and Fuquay (1980).
Animals • Sperm concentration
Sperm cells were counted using a haemocytometer to
Thirty white New Zealand mature male rabbits, 9– determine sperm concentration according to Bearden and
9.5 months of age, were housed in metal cages. Fed pelleted Fuquay (1980). An aqueous solution of eosin 2% was used
commercial feed (Ibex Co., Cairo, Egypt) and water was as diluent that kills sperm, so that counting can be accom-
supplied without restriction. Rabbits in all groups received plished. Eosin, stains sperm heads, so that they are easier to
humane care in compliance with the animal care guidelines count. The semen was pulled to the mark 1.0 of the pipette,
of the National Institute of Health, and the local ethical and the pipette was filled with the diluent. The number of
committee approved this study. sperm in five squares was multiplied by 10.000 to obtain the
number per ml.
Experimental design • Sperm abnormalities
A total of 300 sperm was counted on each slide under
Rabbits were divided into three groups (10 rabbits each). light microscope at ·400 magnification, and the percentages
Group A rabbits served as control group and received 0.25 ml of morphologically abnormal spermatozoa (detached head
sesame oil ⁄ kg bwt. Group B received 4.4 mg ⁄ kg bwt bolde- and coiled tail) were recorded according to Evans and Max-
none undecylenate 5% oily solution (Equi-gan; Lab Tornel, well (1987).
Co., Mexico). Group C rabbits received 8.8 mg ⁄ kg bwt bolde-
none undecylenate. All groups were injected intramuscularly
twice weekly for 2 months. The doses of BOL were calculated Determination of serum testosterone levels
according to Paget and Barnes (1964).
Blood was collected from the ear vein of each rabbit before
euthanasia. Serum was separated for assessment of the total
Evaluated parameters serum testosterone according to Demetriou (1987) using
Body weight and weight gain. Rabbits were ranked by solid-phase radioimmunoassay (RIA) kits. This assay was
restricted randomization procedures that approximately based on presence of a testosterone-specific antibody immo-
equalized the initial bwts among the different groups. They bilized to the wall of the polypropylene tube.

International Journal of Experimental Pathology 2012, 93, 172–178


174 S. S. Oda & I. M. El-Ashmawy

Histopathological studies Results


At the end of the experiment, rabbits were necropsied. Tes-
tes, epididymis and prostate glands were collected, weighed
Body weight and body weight gain
(as outlined above) and fixed rapidly in 10% neutral-buf- The initial bwt of all groups was equalized approximately.
fered formalin for at least 24 h. The fixed specimens were Treatment with BOL had no significant effect on the final
processed through the conventional paraffin-embedding tech- bwt and the bwt gain of the treated groups compared with
nique (Culling 1983), sectioned at 5 lm and stained with the control group (Table 1).
Mayer’s haematoxylin and eosin (HE).

Reproductive organs weights and serum testosterone level


Statistical analysis
Table 2 shows that the index weight of the testes and
Results were analysed statistically by one-way analysis of var- epididymis was decreased significantly (P £ 0.05) in BOL-
iance followed by Duncan’s multiple range test (SAS 2001). treated groups compared with the control group. This reduc-
Data are presented as means plus or minus the standard tion was marked in the group C. No significant changes
error. The minimum level of significance was set at P £ 0.05. were found in the index weight of the prostates. Moreover,
there was a significant reduction (P £ 0.05) in the serum
Table 1 Effect of BOL on bwt and bwt gain of male rabbits testosterone level in the groups B and C compared with the
control group. This reduction was prominent in the group C
Parameters (Table 2).

Groups Initial bwt (g) Final bwt (g) Bwt gain (g)
Semen analysis and sperm characteristics
A 3400 ± 81.65a 3227 ± 350a )173 ± 432a
B 3567 ± 347a 3505 ± 214a )61.5 ± 134a The sperm characteristics of the treated groups were not
C 3600 ± 743a 3578 ± 152a )22 ± 610a changed at the first two time points of semen collection
All values are expressed as mean ± SE. Values with different letters compared with the control group (Table 3). At the end of
at the same raw are significantly different at P £ 0.05 (anova) with the experiment, ejaculate volume was significantly reduced
Duncan’s multiple range test. A = control, B = 4.4 mg ⁄ kg bwt BOL (P £ 0.05) in both of groups B and C. Group C showed a
treated, C = 8.8 mg ⁄ kg bwt BOL treated. significant reduction (P £ 0.05) in the sperm motility and
the sperm count compared with the control group. No sig-
nificant abnormalities in the sperm morphology were found
Table 2 Effect of BOL on reproductive organs weights and in all treated groups compared with the control group
serum testosterone levels of male rabbits
(Table 3).
Parameters
Histopathology
Epididymes Prostate Testosterone
Groups Testes I.W. I.W. I.W. (ng ⁄ ml) Histopathological findings of testes, epididymis and prostate
A 0.18 ± 0.03a 0.09 ± 0.01a 0.09 ± 0.02a 3.5 ± 0.52a gland were evaluated under light microscopy. Incidence and
B 0.14 ± 0.01b 0.06 ± 0.01b 0.09 ± 0.01a 2.8 ± 0.25b severity of lesions in BOL-treated groups are summarized in
C 0.11 ± 0.01b 0.04 ± 0.01c 0.10 ± 0.00a 1.8 ± 0.27c (Table 4).
All values are expressed as mean ± SE. Values with different letters at
the same column are significantly different at P £ 0.05 (anova) with Testes
Duncan’s multiple range test. I.W. = organ weight (g) ⁄ 100 · body
weight (g). A = control, B = 4.4 mg ⁄ kg bwt BOL treated, C = 8.8 Testes of the control mature rabbits had normal histoarchi-
mg ⁄ kg bwt BOL treated. tecture, and were composed of uniform, well-organized

Table 3 Effect of BOL on sperm characteristics of male rabbits

Parameters

Ejaculate volume (V, ml) Motility (M, %) Sperm count (C, ·106 ⁄ ml) Sperm abnormality (Ab, %)

Groups V1 V2 V3 M1 M2 M3 C1 C2 C3 Ab1 Ab2 Ab3

A 0.55 ± 0.05a 0.53 ± 0.1a 0.54 ± 0.1a 96.8 ± 2.4a 98.3 ± 2.4a 96.8 ± 2.4a 572 ± 302a 774 ± 442a 615 ± 175a 8.8 ± 1.0a 9.0 ± 0.8a 9.0 ± 0.8a
B 0.54 ± 0.29a 0.52 ± 0.1a 0.33 ± 0.1b 98.3 ± 2.4a 75 ± 20.4a 73.3 ± 18.4ab 523 ± 120a 588 ± 305a 466 ± 183a 8.0 ± 1.4a 9.0 ± 2.9a 10.3 ± 1.7a
C 0.53 ± 0.05a 0.43 ± 0.1a 0.25 ± 0.1b 98.3 ± 2.4a 66.8 ± 31.2a 65 ± 29.4b 552 ± 163a 365 ± 158a 45.8 ± 14.1b 8.3 ± 1.3a 9.0 ± 1.6a 10.0 ± 2.2a

All values are expressed as mean ± SE. Values with different letters on the same row are significantly different at P £ 0.05 (anova) with Duncan’s multiple range test. A = con-
trol, B = 4.4 mg ⁄ kg bwt BOL treated, C = 8.8 mg ⁄ kg bwt BOL treated.

International Journal of Experimental Pathology 2012, 93, 172–178


Boldenone undecylenate and male rabbits 175

Table 4 Incidence and severity of histopathological lesions in the testes, epididymis and prostate glands of BOL-treated groups;
B = 4.4 mg ⁄ kg bwt BOL treated, C = 8.8 mg ⁄ kg bwt BOL treated

Incidence* and Severity  of Histopathological Lesions

Group B Group C

Absent Mild Moderate Severe Absent Mild Moderate Severe


Organ ⁄ Lesion ()) (+) (++) (+++) ()) (+) (++) (+++)

Testes
Small disorganized tubules with thickened 1 3 6 0 0 0 2 8
irregular basement membrane
Vacuolation of germ cells and Sertoli cells 3 1 3 3 0 0 4 6
Sloughing germ cells 0 2 3 5 0 1 3 6
Giant cell formations 3 1 6 0 5 3 2 0
Reduced spermatogenesis 0 1 5 4 0 0 1 9
Coagulative necrosis of tubules 6 4 0 0 3 7 0 0
Interstitial fibrosis 8 2 0 0 0 2 2 6
Epididymis
Sloughing germ cells 0 2 4 4 0 2 3 5
Low sperm density 0 1 5 4 0 0 1 9
Prostate
Tubular dilatation 8 0 2 0 6 0 4 0
*Number of rabbits with lesions per total examined (10 rabbits per group).
 
Severity of lesions was graded by estimating the percentage area affected in the entire section. Lesion scoring: ()) absence of the lesion = 0%,
(+) mild = 5–25%, (++) moderate = 26–50% and (+++) severe ‡50% of the examined tissue sections.

seminiferous tubules with complete spermatogenesis and Moreover, there was vacuolar degeneration of the germinal
interstitial connective tissue (Figure 2a). Testes of group B epithelium and Sertoli cells. Lumina of the majority of
rabbits showed degenerative changes that were character- seminiferous tubules contained sloughed germinal epithelial
ized by small, disorganized seminiferous tubules with irreg- cells and giant cell formations (Figure 2b). Some tubules
ular basement membrane and decreased spermatogenesis. showed coagulative necrosis with hyalinized luminal con-

(a) (b)

(c) (d)

Figure 2 Photomicrograph of rabbit testis stained with HE: (a) Normal testis histo-architecture of a control rabbit. (Bar = 100 lm).
(b) Shrunken, buckled, disorganized seminiferous tubules, vacuolation (arrows) and sloughing of the germinal epithelium with giant
cell formations (arrowheads) in the lumen of seminiferous tubules of a rabbit that received 4.4 mg ⁄ kg bwt BOL 5%. (Bar = 50 lm).
(c) Small-sized seminiferous tubules with marked thickened hyalinized basement membrane, vacuolation (arrows) and sloughing of
the germinal epithelium in the lumen of seminiferous tubules of a rabbit that received 8.8 mg BOL 5% ⁄ kg bwt; the majority of semi-
niferous tubules had single or double cell layers. (Bar = 100 lm). (d) Higher magnification of (c) showing that, small-sized seminifer-
ous tubules with thickened hyalinized basement membrane had vacuolated germinal epithelium (arrows) (Bar the = 50 lm).

International Journal of Experimental Pathology 2012, 93, 172–178


176 S. S. Oda & I. M. El-Ashmawy

(a) (b)

(c) (d)

(e) (f)

Figure 3 Photomicrograph of rabbit epididymis stained with HE. (Bar = 100 lm): normal histological structure with normal sperm
density of caput epididymis (a) and (b) cauda epididymis of a control rabbit. Caput epididymis (c), cauda epididymis (d) of a rabbit
that received 4.4 mg ⁄ kg bwt BOL 5% had low density of spermatozoa and sloughed germ cells in their lumina. Caput epididymis
(e), cauda epididymis (f) of a rabbit that received 8.8 mg BOL 5% ⁄ kg bwt: epididymal ductules were free from mature spermatozoa
and some cauda epididymal ductules contained sloughed germ cells (star).

tents. Testicular sections of group C rabbits exhibited


Prostate gland
marked small-sized, disorganized seminiferous tubules with
marked thickened hyalinized basement membrane (Fig- The prostate of the control rabbits was histologically normal
ure 2c,d). Vacuolation of spermatogonia and Sertoli cells (Figure 4a). No detectable changes were noticed in both
was seen. There was obvious cessation of spermatogenesis: treated groups apart from some moderate tubular dilatation
the majority of seminiferous tubules had single or double (Figure 4b,c).
cell layers. Also, some tubules had sloughed germinal epi-
thelial cells within their lumina. In the interstitium, there
was marked thickening due to increased by fibrous connec-
Discussion
tive tissue. Few papers have studied the effect of high dose BOL treat-
ment on male reproductive function. Consequently, this study
was performed to evaluate the effects of BOL on bwt, bwt
Epididymis
gain, reproductive organ weight, serum testosterone level,
Control mature rabbits showed normal epididymal histo- semen analysis and sperm characteristics and histopathology
logical architecture with normal sperm density (Figure 3a,b). of reproductive organs of mature male rabbits. Our study
In group B rabbits some of the epididymal ductules were revealed that treatment with BOL had no significant effect on
empty of mature spermatozoa, and others had low density the final bwt and the bwt gain of the treated groups compared
of spermatozoa and sloughed germ cells in their lumina (Fig- with the control group. Similar results have been reported in
ure 3c,d). Epididymal ductules of group C rabbits were free horses (Maher et al. 1983), in female rats (Howe & Morello
from mature spermatozoa, and some cauda epididymal duc- 1985) and in veal calves (Cannizzo et al. 2007). The index
tules contained sloughed germ cells (Figure 3e,f). weight of testes and epididymes was decreased significantly in

International Journal of Experimental Pathology 2012, 93, 172–178


Boldenone undecylenate and male rabbits 177

(a) (b)

(c)

Figure 4 Photomicrograph of rabbit prostate stained with HE: (a) Prostate of control rabbit with normal histological structure.
(Bar = 100 lm). (b) Prostate of a rabbit that received 4.4 mg ⁄ kg bwt BOL 5%: moderate tubular dilatation (Bar = 300). (c) Prostate
of a rabbit that received 8.8 mg BOL 5% ⁄ kg bwt: moderate tubular dilatation (Bar = 100 lm).

the BOL-treated groups, particularly in group C compared treatment with testosterone or AAS such as BOL are followed
with the control group. This result was parallel with the sig- by suppression of both gonadotropin-releasing hormone pro-
nificant reduction in serum testosterone level in these groups duction by the hypothalamus and luteinizing hormone pro-
compared with the control group. This is consistent with the duction by pituitary gland and consequently lead to
previous finding that BOL has a detrimental effect on sper- suppression of testicular testosterone production (Dohle et al.
matogenesis and testis size, associated with a decrease in testis 2003). The testicular lesions were similar to those described
weight and the number of developing germ cells (Groot & Bi- by Cannizzo et al. (2007). The epididymal lesions reflected
olatti 2004; Cannizzo et al. 2007). In contrast no significant the cessation of spermatogenesis particularly in group C. The
changes were found in the index weight of the prostates. Con- prostatic lesions were limited except for some moderate tubu-
cerning semen quality, at the end of the experiment ejaculate lar dilatation that may be due to hypersecretion; however,
volume, sperm motility and sperm count of BOL-treated rab- there was no significant increase in the index weight of pros-
bits showed a significant reduction, particularly in group C. tates. Similar findings were described by Groot and Biolatti
These results were similar to those reported in stallions by (2004) who found that BOL induced hypersecretion, hyper-
Squires et al. (1982). However, no significant changes were plasia and cyst formation in the prostate and bulbourethral
detected in sperm abnormalities. Parallel to these findings, the gland, with reduced spermatogenesis and enhanced degenera-
testes of BOL-treated rabbits exhibited different histopatho- tion of testicular germinal epithelium. The literature reports
logical changes which were more marked in group C. These that both hypersecretion (Dabadie 1984; Grandmontagne,
changes manifested as shrunken, disorganized seminiferous 1986; Chaubeau & Grandmontagne, 1990) and degeneration
tubules with marked thickened hyalinized basement mem- of germinal epithelium (Godfrey et al. 1989) could be the con-
brane, and vacuolation of spermatogonia and Sertoli cells. sequence of the pharmacological action of androgenic ste-
Also, there was obvious cessation of spermatogenesis. The roids. Thus in conclusion, this study revealed that AAS, and
majority of seminiferous tubules had single or double cell lay- in particular BOL significant had no major effect on bwt gain
ers. Also, some tubules had sloughed germinal epithelial cells but induced a deleterious effect on fertility of male rabbits.
within their lumina. These findings may be attributed to
decreased serum testosterone levels in BOL-treated groups.
Testosterone is essential for the attachment of different gener-
Acknowledgements
ations of germ cells in seminiferous tubules. Consequently, The authors gratefully thank Dr Mahmoud M. A. Elmagh-
low level of intratesticular testosterone may lead to detach- raby professor of Animal Breeding and Production, Depart-
ment of germ cells from seminiferous epithelium and may ini- ment of Animal Husbandry & Animal Wealth Development,
tiate germ cell apoptosis and subsequent male infertility Faculty of Veterinary Medicine, Alexandria University,
(Blanco-Rodriguez & Martinez-Garcia 1998). Exogenous Egypt, for performing the statistical analysis.

International Journal of Experimental Pathology 2012, 93, 172–178


178 S. S. Oda & I. M. El-Ashmawy

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