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Contents

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Author contributions
  9. References

Concentrations of 17β-oestradiol (E2), testosterone (T), 5α-dihydrotestosterone, prolactin (PRL) and relaxin (RLN) were determined in peripheral blood serum or plasma and prostatic secretion of 77 physically healthy intact male dogs (19 Rhodesian Ridgebacks/RR, 58 dogs of other breeds, 1–9 years of age). Furthermore, the concentrations of acid phosphatase in prostatic secretion and canine prostate-specific esterase (CPSE) were measured in blood plasma. All dogs were submitted to a complete breeding soundness examination, including B-mode sonography. Prostatic volume was larger, and blood plasma levels of CPSE were higher in ageing dogs and in dogs with benign prostatic hyperplasia (BPH) compared with young dogs and dogs with normal prostate. Furthermore, a higher E2/T ratio was found in dogs with BPH. Despite missing significant differences in PRL concentrations, the slight increases in PRL concentrations in the prostatic secretion observed both with increasing age and in dogs with BPH and the observed correlations between concentrations of PRL and testicular steroids may possibly indicate a role of PRL in the pathogenesis of canine BPH. Serum RLN concentrations were at similar level in all dogs. Regarding breed differences, an appreciably larger prostatic volume and higher concentration of CPSE were verified in RR than in other pure-bred dogs, confirming our suspicion of a premature enlargement of the prostate gland, which may result from a genetic disposition for BPH in this breed.


Introduction

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Author contributions
  9. References

Prostatic integrity and function are known to directly depend on testicular testosterone and oestrogen secretion. Benign prostatic hyperplasia (BPH) is the most common prostatic disorder in ageing intact male dogs. It is characterized by hyperplasia and also hypertrophy of prostatic epithelial cells. To our knowledge, no breed predisposition for BPH has been reported so far. According to clinical observations, prostatic enlargement in Rhodesian Ridgeback (RR) dogs seems to occur at an earlier age than in other breeds (Wolf, unpublished observations). Dihydrotestosterone (DHT) is accepted as key hormone in stimulating enlargement of the canine prostate gland, but the pathogenesis of BPH is not completely understood. Prolactin (PRL) appears to be involved in prostate development, growth and differentiation and displayed independent hypertrophic effects on the prostate in experimental models (Syms et al. 1985). Immunoreactive relaxin (RLN) of prostatic origin has been detected in seminal plasma of men, but its role in male reproduction is still debated (Essig et al. 1982). Therefore, measurement of concentrations of PRL and RLN in peripheral blood serum or seminal plasma may be of scientific and clinical relevance in dogs. In this study, concentrations of 17β-oestradiol, testosterone and DHT as well as of PRL and RLN were determined in peripheral blood serum or plasma and in prostatic secretion. Furthermore, the concentrations of acid phosphatase in prostatic secretion and canine prostate-specific esterase in peripheral blood plasma were measured.

Materials and Methods

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Author contributions
  9. References

A total of 77 physically healthy intact male dogs including 10 Beagles [1–7 years (years) of age, 12.3–21.8 kg body weight] and 67 dogs from private owners [19 Rhodesian Ridgebacks (RR), 48 dogs of a total of 15 other breeds, 1–9 years of age, 8–83 kg body weight] were included in the study. The 19 RR belonged to 16 kennels. Except for one father and two sons and two brothers from another genetic strain, the dogs were not closely related. Serum concentrations of thyroid-stimulating hormone (TSH) and thyroxine (T4) were measured in all dogs to exclude hypothyroidism and a related thyrotropin-releasing hormone (TRH)-induced elevation of PRL in blood serum.

The Beagles were kept in groups of two in covered outdoor kennels provided with two shelter huts each. All dogs were kept under natural light conditions and were fed with commercial food. Water was available ad libitum. Animal housing, care and experimentation complied with animal welfare regulations in Germany (Permit no. AZ 42502-05-09A669).

The dogs were grouped according to age (≤2 years, n = 27; >2–4 years, n = 25; >4 years, n = 25), prostate gland status (normal, n = 40; BPH, n = 37) and breed [RR, n = 19, 1–9 years of age, 33–45.7 kg body weight; other pure-bred dogs with corresponding body weight (30.8–45.3 kg), n = 37, 1–9 years of age]. All dogs were submitted to a complete breeding soundness examination including semen collection and evaluation as well as B-mode ultrasonography of the testes, epididymides and prostate gland. The prostate gland status was assessed upon findings raised by rectal palpation and sonography as well as haemospermia and classified according to the related literature (Ruel et al. 1998). Prostatic length, width and height were measured three times on transverse, and sagittal ultrasound images and mean values were calculated. Prostatic volume was estimated using the formula for volume of an ellipsoid body (volume = length × width × height × 0.523).

Semen collection was performed by digital manipulation in the presence of an oestrous teaser bitch, and the sperm-rich fraction and the prostatic secretion were separated.

Blood samples were collected between 10.00 and 14.00 a.m. in each dog. Blood serum and plasma were separated by centrifugation at 1700 × g for 10 min within 20 min of collection and divided into split samples according to the number of evaluated parameters. The latter and a corresponding number of prostatic secretion split samples were stored at −20°C until assayed.

Prolactin (PRL) concentrations were determined with a previously validated heterologous radioimmunoassay (RIA) (Okkens et al. 1985). Assay sensitivity was 0.8 ng/ml. Intra- and interassay coefficients of variance (CVs) were 3.5% and 11.5%, respectively.

Relaxin (RLN) was analysed using a modified species-specific enzyme immunoassay (EIA) (Einspanier et al. 2002) with a sensitivity of 0.03 ng/ml and intra- and interassay CVs of 8.7% and 12%, respectively. Concentrations of oestradiol (E2) were determined by a previously validated EIA using a secondary antibody-coating technique and horseradish peroxidase as enzyme label (Meyer et al. 1990). The minimal detectable concentration was 3.0 pg/ml. For 5α-dihydrotestosterone (DHT) analysis, a competitive EIA [Demeditec 5α Dihydrotestosterone (DHT) ELISA DE2330; Demeditec Diagnostics GmbH, Kiel, Germany] was used according to the instructions of the manufacturer. Assay sensitivity was 6.0 pg/ml, and intra- and interassay CVs were ≤11.4% and ≤12.1%, respectively. Testosterone (T) concentrations were analysed by a competitive EIA (Hoffmann and Meyer 1987), with an assay sensitivity of 10.0 pg/ml.

Acid phosphatase (AP) was measured using a commercial assay (ACP Saure Phosphatase®; Roche Diagnostics GmbH, Mannheim, Germany). Assay sensitivity was 0.5 U/l, and intra- and interassay CVs were 1% and 3.5%, respectively. Canine prostate-specific esterase (CPSE) concentrations were determined by means of a commercial enzyme-linked immunosorbent assay (ELISA) (Odelis®;CPSE, Virbac, Bad Oldesloe, Germany) with a sensitivity of 0.39 ng/ml. Intra- and interassay CVs were 3.1% and 6.6%, respectively.

For statistical evaluation, the sas® program (SAS Institute Inc., Cary, NC, USA) was employed. The descriptive statistic was performed by univariate procedure. Wilcoxon's signed-rank test for independent samples was used to compare the differences in the various parameters between groups. Results are presented as mean (±SD). Correlation analysis was performed by Spearman's rank correlation test. p < 0.05 was considered significant.

Results

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Author contributions
  9. References

Mean prostatic volume differed in relation to prostate gland status (normal 24.6 ± 15.6 cm3 vs BPH 59.8 ± 46.9 cm3), age (≤2 years 26.0 ± 17.3 cm3 vs >4 years 61.2 ± 55.5 cm3) and breed (RR 61.4 ± 26.2 cm3 vs other breeds 34.6 ± 20.4 cm3) (p < 0.05) (Fig. 1). Positive correlations were found between prostatic volume and age (R = 0.51, p < 0.0001), CPSE (R = 0.61, p < 0.0001) as well as prostatic fluid concentrations of T (R = 0.39, p < 0.001) and E2 (R = 0.31, p < 0.01).

image

Figure 1. Prostate volume (mean ± SD) with regard to prostatic health status, age and breed of the dogs. Values with different letters within groups differ significantly (p < 0.05)

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No differences were found in blood serum concentrations of T and DHT between any of the constituted groups, whereas in prostatic secretion, mean T values were higher in dogs with BPH (85.7 ± 75.3 pg/ml) than in dogs with normal prostate (52.4 ± 39.7 pg/ml) (p < 0.05). Highest prostatic fluid concentrations of DHT and E2 were measured in dogs >4 years (DHT 412.2 ± 181.1 pg/ml; E2 12.3 ± 7.9 pg/ml) differing significantly from those of younger dogs (DHT ≤ 2 years 291.7 ± 166.0 pg/ml, >2–4 years 313.8 ± 162.7 pg/ml; E2 ≤ 2 years 7.9 ± 4.0 pg/ml, >2–4 years 9.0 ± 5.7 pg/ml) (p < 0.05). The mean E2/T ratio in blood was higher in dogs with BPH (1 : 94) than in dogs with normal prostate gland (1 : 78) (p < 0.05).

No differences were observed in mean PRL concentrations either in blood serum or in prostatic secretion in relation to age (≤2 years 5.0 ± 1.4 ng/ml and 2.8 ± 1.2 ng/ml; >2–4 years 5.1 ± 1.4 ng/ml and 2.8 ± 1.0 ng/ml; >4 years 4.8 ± 1.4 ng/ml and 3.2 ± 1.2 ng/ml) and prostate gland status (normal 5.0 ± 1.3 ng/ml and 2.8 ± 1.1 ng/ml; BPH 4.9 ± 1.5 ng/ml and 3.1 ± 1.2 ng/ml). However, in RR, mean PRL values in blood serum, but not in prostatic fluid, were significantly higher than in the other breeds (blood serum 6.0 ± 1.4 ng/ml vs 4.9 ± 1.3 ng/ml, p < 0.05; prostatic fluid 3.0 ± 1.1 ng/ml vs 3.2 ± 1.1 ng/ml) (Fig. 2). No correlation was found between PRL concentrations in blood serum and prostatic secretion.

image

Figure 2. Prolactin concentration (mean ± SD) in blood serum with regard to prostatic health status, age and breed of the dogs. Values with different letters within groups differ significantly (p < 0.05)

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Mean RLN concentrations were similar in all groups (age ≤ 2 years 0.65 ± 0.51 ng/ml, >2–4 years 0.72 ± 0.57 ng/ml, >4 years 0.72 ± 0.44 ng/ml; normal prostate gland 0.64 ± 0.48 ng/ml, BPH 0.76 ± 0.52 ng/ml; RR 0.57 ± 0.49 ng/ml, other breeds 0.60 ± 0.35 ng/ml). In prostatic fluid, RLN was at undetectable level. Mean CPSE blood concentration differed according to prostate gland status (normal 415.6 ± 698.3 ng/ml vs BPH 998.9 ± 776.8 ng/ml; p < 0.05), age (≤2 years 429.1 ± 681.1 ng/ml vs >4 years 1005.7 ± 799.9 ng/ml; p < 0.05) and breed (RR 1206.2 ± 834.0 ng/ml vs other breeds 470.0 ± 657.6 ng/ml; p < 0.05) (Fig. 3). Mean values of prostatic fluid AP increased slightly with age (≤2 years 1053.0 ± 835.3 U/l; >2–4 years 1262.7 ± 840.9 U/l; >4 years 1515.6 ± 1047.4 U/l) and were significantly lower in dogs with normal prostate (924.2 ± 618.3 U/l) compared to dogs with BPH (1633.4 ± 1038.0 U/l) (p < 0.05).

image

Figure 3. CPSE concentration (mean ± SD) in blood plasma with regard to prostatic health status, age and breed of the dogs. Values with different letters within groups differ significantly (p < 0.05)

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There was a weak but significant correlation between blood concentrations of PRL and DHT (R = 0.24, p < 0.05) as well as T (R = 0.26, p < 0.05). In prostatic fluid, PRL values were significantly correlated with concentrations of T (R = 0.37, p < 0.01), DHT (R = 0.52, p < 0.0001), E2 (R = 0.42, p < 0.001) and AP (R = 0.46, p < 0.0001).

Discussion

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Author contributions
  9. References

Prostatic volume was larger, and blood plasma levels of CPSE were higher in ageing dogs and dogs with BPH compared with young dogs and dogs with normal prostate. Similarly, there were higher E2/T ratio in dogs with BPH than in normal dogs. Concentrations of T and E2 in blood and prostatic secretion were similar to previous reports (Lange et al. 2001), whereas median DHT concentrations were approximately ten times higher, probably resulting from using different hormone assays as the ratio between the DHT concentrations in peripheral blood and prostatic secretion is almost identical.

A slight increase in PRL concentrations in prostatic secretion and a slight decrease in PRL concentrations in blood serum were observed both with advancing age and in dogs with BPH. It is plausible that this may relate to a possible role of PRL in the pathogenesis of canine BPH in elderly dogs. This proposition is supported by the positive correlations between blood serum concentrations of PRL, DHT and T and most notably between prostatic fluid concentrations of PRL and T, DHT, E2 and AP. The ability of PRL to induce abnormal prostatic growth was indicated by increased prostate-specific expression of PRL in transgenic mice (Kindblom et al. 2003). Prolactin is synthesized and secreted by lactotroph cells in the anterior pituitary gland. The expression of PRL and its receptor has been demonstrated in the human prostate (Nevalainen et al. 1997), and PRL might also act as local growth factor besides the classical endocrine route. Further studies are needed to investigate the source of PRL in the prostatic secretion and to explain the missing correlation of the PRL concentrations in blood serum and prostatic fluid. The higher PRL concentrations in blood serum of RR dogs compared with dogs of other breeds with identical body weight range indicate potential breed differences in pituitary PRL secretion as described by Urhausen et al. (2009).

Regarding blood serum RLN concentrations, no relation to the prostate gland status was obvious. High RLN immunoreactivity has been described in canine hypertrophic prostate gland tissue only in connection with a perineal hernia, indicating RLN to be a marker for connective tissue weakening, whereas little or no RLN was detected in normal and atrophic canine prostatic tissue (Niebauer et al. 2005). Furthermore, in our study, the third ejaculate fraction representing almost pure prostatic secretion was analysed, whereas in a study on RLN content in human ejaculates, seminal plasma derived from centrifuged whole semen (Essig et al. 1982). The age-related elevation of selected prostate gland marker concentrations may reflect the onset of BPH development with age. Regarding breed differences, an appreciably larger prostatic volume and higher concentration of CPSE were verified in RR as compared with the other large pure-bred dogs, confirming our suspicion of a premature enlargement of the prostate gland, which may result from a genetic disposition for BPH in this breed.

Conflicts of interest

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Author contributions
  9. References

None of the authors have any conflicts of interest to declare.

Author contributions

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Author contributions
  9. References

KW carried out the experiments and wrote the manuscript. HK was involved in the experiments. CU was involved in the experiments and was the assistant supervisor. MP conducted the T, DHT, TSH, T4 and CPSE analyses. RM conducted the AP analyses. SK was involved in the planning and supervision of the study with special regard to animal welfare requirements. AE conducted the E2 and RLN analyses. CHYO conducted the PRL analyses. AGA was responsible for the organization and supervision of experiments and manuscript revision.

References

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conflicts of interest
  8. Author contributions
  9. References