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. 2012 Jun;93(3):172–178. doi: 10.1111/j.1365-2613.2012.00814.x

Adverse effects of the anabolic steroid, boldenone undecylenate, on reproductive functions of male rabbits

Samah S Oda *, Ibrahim M El-Ashmawy †
PMCID: PMC3385914  PMID: 22583130

Summary

This study was conducted to evaluate the adverse effects of the anabolic steroid, boldenone undecylenate (BOL) on reproductive functions of male rabbits. Thirty white New Zealand mature male rabbits were divided into three groups (10 rabbits 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 months. BOL had no significant effect on the bwt and bwt gain. Testes and epididymis weights were decreased significantly in the BOL-treated groups. BOL caused significant reduction in serum testosterone level, seminal volume, sperm motility, and sperm count. No abnormalities were detected in the sperm morphology of the BOL-treated groups. Histopathological alterations in the testes and epididymis were marked in the group C rabbits. These results indicate that administration of BOL exerts a significant harmful effect on the reproductive functions of male rabbits.

Keywords: anabolic steroids, boldenone, histopathology, rabbits, testes

Anabolic androgenic steroids (AAS) are synthetic derivatives of the male testosterone hormone that have been modified to improve their anabolic rather than androgenic activity (Shahidi 2001). The anabolic effects of AAS promote protein synthesis, muscle growth and erythropoiesis (Mottram & George 2000). Hence, AAS are used to enhance strength and durability of canine, equine and human athletes (Teale & Houghton 1991; Schänzer & Donike 1992; Schänzer 1996). Boldenone (BOL) is an anabolic steroid that differs from testosterone only by one double bond at the 1- position (Stolker et al. 2007) (Figure 1). It is used mainly as undecylenate ester by bodybuilders and is administered illegally to racing horses. However, it is used as a growth promotor on farms improving the growth and feed conversion of cattle; it may be abused to achieve more efficient meat production (Gryglik et al. 2010). In developing countries with rapid growth of population, like Egypt, the demand for edible protein exceeds the supply and the gap is expanded. Meat from animals, including from rabbits, provides a valuable and palatable source of protein. We found BOL to be used heavily in Egypt, not only in the field of animal production, but also by athletes and bodybuilders. BOL increases muscle size owing to promotion of positive nitrogen balance by stimulating protein production and reducing protein destruction, as well as causing retention of body water, nitrogen, sodium, potassium and calcium ions (Forbes 1985; Mooradian et al. 1987).

Figure 1.

Figure 1

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Structure of Boldenone and Testosterone.

Like other androgenic steroids, BOL is classified by the International Agency for Research on Cancer (IARC) in class 2A (growth promotors – steroids), as a probable human carcinogen (e.g. prostate and liver tumours), with a carcinogenicity index higher than that of other androgens, such as nandrolone, stanozolol and testosterone and is thus a banned substance (IARC Monograph 1987; De Brabander et al. 2004). Despite these restrictions, AAS are easily obtained. The abuse of AAS can lead to serious and irreversible organ damage (Maravelias et al. 2005). Among the most common adverse effects of AAS that have been described are reduced fertility (Dohle et al. 2003), hypertension (Ferenchick 1990), atherosclerosis (Cohen et al. 1988), blood clotting (Parssinen & Seppala 2002), hepatic neoplasms and carcinoma (Velazquez & Alter 2004), tendon damage (Battista et al. 2003), psychiatric and behavioural disorders (Clark & Henderson 2003).

There have been relatively few studies which have investigated the detrimental effects of BOL administration on male function. These have explored their role as growth promotors on testis; bulbourethral glands and prostates of veal calves (Groot & Biolatti 2004; Cannizzo et al. 2007), on reproductive function of stallions (Squires et al. 1982; Squires & Mckinnon 1987; Garcia et al. 1987) and on reproductive performance of male rabbits (Thabet et al. 2010). Hence, this study was performed to determine the effects of high-dose administration of BOL on body weight (bwt), reproductive organ weight, semen characteristics, serum testosterone levels and histopathological features of the reproductive organs of mature male rabbits.

Materials and methods

Animals

Thirty white New Zealand mature male rabbits, 9–9.5 months of age, were housed in metal cages. Fed pelleted commercial feed (Ibex Co., Cairo, Egypt) and water was supplied without restriction. Rabbits in all groups received humane care in compliance with the animal care guidelines of the National Institute of Health, and the local ethical committee approved this study.

Experimental design

Rabbits were divided into three groups (10 rabbits each). Group A rabbits served as control group and received 0.25 ml sesame oil/kg bwt. Group B received 4.4 mg/kg bwt boldenone undecylenate 5% oily solution (Equi-gan®; Lab Tornel, Co., Mexico). Group C rabbits received 8.8 mg/kg bwt boldenone undecylenate. All groups were injected intramuscularly twice weekly for 2 months. The doses of BOL were calculated according to Paget and Barnes (1964).

Evaluated parameters

Body weight and weight gain

Rabbits were ranked by restricted randomization procedures that approximately equalized the initial bwts among the different groups. They 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, epididymis and prostate glands were removed, grossly examined and weighed. The index weight (I.W.) of each organ was calculated by Matousek (1969) I.W. = organ weight (g)/100 × body weight (g).

Semen collection and sperm characteristics

Ejaculates were collected from each male rabbit prior to the treatment, after one month of treatment and at the end of the experiment with a rabbit artificial vagina. Each buck was conditioned to react with the artificial vagina as described by Breddman et al. (1964). Each male was allowed a false mounting for teasing prior to the actual mounting. The semen was evaluated immediately after collection for the following criteria:

  • Semen volume

    The volume of ejaculate in ml was measured to the nearest 0.1 ml using a graduated collecting tube.

  • Sperm motility

    Immediately following collection of a semen sample, a small drop was taken with a capillary pipette and placed 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 according to Bearden and Fuquay (1980).

  • Sperm concentration

    Sperm cells were counted using a haemocytometer to determine sperm concentration according to Bearden and Fuquay (1980). An aqueous solution of eosin 2% was used as diluent that kills sperm, so that counting can be accomplished. Eosin, stains sperm heads, so that they are easier to count. The semen was pulled to the mark 1.0 of the pipette, and the pipette was filled with the diluent. The number of sperm in five squares was multiplied by 10.000 to obtain the number per ml.

  • Sperm abnormalities

    A total of 300 sperm was counted on each slide under light microscope at ×400 magnification, and the percentages of morphologically abnormal spermatozoa (detached head and coiled tail) were recorded according to Evans and Maxwell (1987).

Determination of serum testosterone levels

Blood was collected from the ear vein of each rabbit before euthanasia. Serum was separated for assessment of the total serum testosterone according to Demetriou (1987) using solid-phase radioimmunoassay (RIA) kits. This assay was based on presence of a testosterone-specific antibody immobilized to the wall of the polypropylene tube.

Histopathological studies

At the end of the experiment, rabbits were necropsied. Testes, epididymis and prostate glands were collected, weighed (as outlined above) and fixed rapidly in 10% neutral-buffered formalin for at least 24 h. The fixed specimens were processed through the conventional paraffin-embedding technique (Culling 1983), sectioned at 5 μm and stained with Mayer’s haematoxylin and eosin (HE).

Statistical analysis

Results were analysed statistically by one-way analysis of variance followed by Duncan’s multiple range test (SAS 2001). Data are presented as means plus or minus the standard error. The minimum level of significance was set at P ≤ 0.05.

Results

Body weight and body weight gain

The initial bwt of all groups was equalized approximately. Treatment with BOL had no significant effect on the final bwt and the bwt gain of the treated groups compared with the control group (Table 1).

Table 1.

Effect of BOL on bwt and bwt gain of male rabbits

Parameters
Groups Initial bwt (g) Final bwt (g) Bwt gain (g)
A 3400 ± 81.65a 3227 ± 350a −173 ± 432a
B 3567 ± 347a 3505 ± 214a −61.5 ± 134a
C 3600 ± 743a 3578 ± 152a −22 ± 610a
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All values are expressed as mean ± SE. Values with different letters at the same raw are significantly different at P ≤ 0.05 (anova) with Duncan’s multiple range test. A = control, B = 4.4 mg/kg bwt BOL treated, C = 8.8 mg/kg bwt BOL treated.

Reproductive organs weights and serum testosterone level

Table 2 shows that the index weight of the testes and epididymis was decreased significantly (P ≤ 0.05) in BOL-treated groups compared with the control group. This reduction was marked in the group C. No significant changes were found in the index weight of the prostates. Moreover, there was a significant reduction (P ≤ 0.05) in the serum testosterone level in the groups B and C compared with the control group. This reduction was prominent in the group C (Table 2).

Table 2.

Effect of BOL on reproductive organs weights and serum testosterone levels of male rabbits

Parameters
Groups Testes I.W. Epididymes I.W. Prostate I.W. Testosterone (ng/ml)
A 0.18 ± 0.03a 0.09 ± 0.01a 0.09 ± 0.02a 3.5 ± 0.52a
B 0.14 ± 0.01b 0.06 ± 0.01b 0.09 ± 0.01a 2.8 ± 0.25b
C 0.11 ± 0.01b 0.04 ± 0.01c 0.10 ± 0.00a 1.8 ± 0.27c
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All values are expressed as mean ± SE. Values with different letters at the same column are significantly different at P ≤ 0.05 (anova) with 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 mg/kg bwt BOL treated.

Semen analysis and sperm characteristics

The sperm characteristics of the treated groups were not changed at the first two time points of semen collection compared with the control group (Table 3). At the end of the experiment, ejaculate volume was significantly reduced (P ≤ 0.05) in both of groups B and C. Group C showed a significant reduction (P ≤ 0.05) in the sperm motility and the sperm count compared with the control group. No significant abnormalities in the sperm morphology were found in all treated groups compared with the control group (Table 3).

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
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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 = control, B = 4.4 mg/kg bwt BOL treated, C = 8.8 mg/kg bwt BOL treated.

Histopathology

Histopathological findings of testes, epididymis and prostate gland were evaluated under light microscopy. Incidence and severity of lesions in BOL-treated groups are summarized in (Table 4).

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
Organ/Lesion Absent (−) Mild (+) Moderate (++) Severe (+++) Absent (−) Mild (+) Moderate (++) Severe (+++)
Testes
 Small disorganized tubules with thickened irregular basement membrane 1 3 6 0 0 0 2 8
 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
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*

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.

Testes

Testes of the control mature rabbits had normal histoarchitecture, and were composed of uniform, well-organized seminiferous tubules with complete spermatogenesis and interstitial connective tissue (Figure 2a). Testes of group B rabbits showed degenerative changes that were characterized by small, disorganized seminiferous tubules with irregular basement membrane and decreased spermatogenesis. Moreover, there was vacuolar degeneration of the germinal epithelium and Sertoli cells. Lumina of the majority of seminiferous tubules contained sloughed germinal epithelial cells and giant cell formations (Figure 2b). Some tubules showed coagulative necrosis with hyalinized luminal contents. Testicular sections of group C rabbits exhibited marked small-sized, disorganized seminiferous tubules with marked thickened hyalinized basement membrane (Figure 2c,d). Vacuolation of spermatogonia and Sertoli cells was seen. There was obvious cessation of spermatogenesis: the majority of seminiferous tubules had single or double cell layers. Also, some tubules had sloughed germinal epithelial cells within their lumina. In the interstitium, there was marked thickening due to increased by fibrous connective tissue.

Figure 2.

Figure 2

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Photomicrograph of rabbit testis stained with HE: (a) Normal testis histo-architecture of a control rabbit. (Bar = 100 μm). (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 μm). (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 seminiferous tubules had single or double cell layers. (Bar = 100 μm). (d) Higher magnification of (c) showing that, small-sized seminiferous tubules with thickened hyalinized basement membrane had vacuolated germinal epithelium (arrows) (Bar the = 50 μm).

Epididymis

Control mature rabbits showed normal epididymal histological architecture with normal sperm density (Figure 3a,b). In group B rabbits some of the epididymal ductules were empty of mature spermatozoa, and others had low density of spermatozoa and sloughed germ cells in their lumina (Figure 3c,d). Epididymal ductules of group C rabbits were free from mature spermatozoa, and some cauda epididymal ductules contained sloughed germ cells (Figure 3e,f).

Figure 3.

Figure 3

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Photomicrograph of rabbit epididymis stained with HE. (Bar = 100 μm): 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).

Prostate gland

The prostate of the control rabbits was histologically normal (Figure 4a). No detectable changes were noticed in both treated groups apart from some moderate tubular dilatation (Figure 4b,c).

Figure 4.

Figure 4

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Photomicrograph of rabbit prostate stained with HE: (a) Prostate of control rabbit with normal histological structure. (Bar = 100 μm). (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 μm).

Discussion

Few papers have studied the effect of high dose BOL treatment on male reproductive function. Consequently, this study was performed to evaluate the effects of BOL on bwt, bwt gain, reproductive organ weight, serum testosterone level, semen analysis and sperm characteristics and histopathology of reproductive organs of mature male rabbits. Our study revealed that treatment with BOL had no significant effect on the final bwt and the bwt gain of the treated groups compared with the control group. Similar results have been reported in horses (Maher et al. 1983), in female rats (Howe & Morello 1985) and in veal calves (Cannizzo et al. 2007). The index weight of testes and epididymes was decreased significantly in the BOL-treated groups, particularly in group C compared with the control group. This result was parallel with the significant reduction in serum testosterone level in these groups compared with the control group. This is consistent with the previous finding that BOL has a detrimental effect on spermatogenesis and testis size, associated with a decrease in testis weight and the number of developing germ cells (Groot & Biolatti 2004; Cannizzo et al. 2007). In contrast no significant changes were found in the index weight of the prostates. Concerning semen quality, at the end of the experiment ejaculate volume, sperm motility and sperm count of BOL-treated rabbits showed a significant reduction, particularly in group C. These results were similar to those reported in stallions by Squires et al. (1982). However, no significant changes were detected in sperm abnormalities. Parallel to these findings, the testes of BOL-treated rabbits exhibited different histopathological changes which were more marked in group C. These changes manifested as shrunken, disorganized seminiferous tubules with marked thickened hyalinized basement membrane, and vacuolation of spermatogonia and Sertoli cells. Also, there was obvious cessation of spermatogenesis. The majority of seminiferous tubules had single or double cell layers. Also, some tubules had sloughed germinal epithelial cells 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 generations of germ cells in seminiferous tubules. Consequently, low level of intratesticular testosterone may lead to detachment of germ cells from seminiferous epithelium and may initiate germ cell apoptosis and subsequent male infertility (Blanco-Rodriguez & Martinez-Garcia 1998). Exogenous treatment with testosterone or AAS such as BOL are followed by suppression of both gonadotropin-releasing hormone production by the hypothalamus and luteinizing hormone production by pituitary gland and consequently lead to suppression of testicular testosterone production (Dohle et al. 2003). The testicular lesions were similar to those described by Cannizzo et al. (2007). The epididymal lesions reflected the cessation of spermatogenesis particularly in group C. The prostatic lesions were limited except for some moderate tubular dilatation that may be due to hypersecretion; however, there was no significant increase in the index weight of prostates. Similar findings were described by Groot and Biolatti (2004) who found that BOL induced hypersecretion, hyperplasia and cyst formation in the prostate and bulbourethral gland, with reduced spermatogenesis and enhanced degeneration of testicular germinal epithelium. The literature reports that both hypersecretion (Dabadie 1984; Grandmontagne, 1986; Chaubeau & Grandmontagne, 1990) and degeneration of germinal epithelium (Godfrey et al. 1989) could be the consequence of the pharmacological action of androgenic steroids. Thus in conclusion, this study revealed that AAS, and in particular BOL significant had no major effect on bwt gain but induced a deleterious effect on fertility of male rabbits.

Acknowledgments

The authors gratefully thank Dr Mahmoud M. A. Elmaghraby professor of Animal Breeding and Production, Department of Animal Husbandry & Animal Wealth Development, Faculty of Veterinary Medicine, Alexandria University, Egypt, for performing the statistical analysis.

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