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Contents

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

The purpose of this study was to define (i) the interval between treatment and sterility, and (ii) semen quality in male dogs administered a 4.7-mg deslorelin implant. Six healthy, adult dogs of various breeds and body weights were implanted with deslorelin (Suprelorin, Virbac) and followed every 2 weeks with semen and blood collections. Semen quality remained stable or even improved during the first month following treatment and then showed a progressive decline until the end of the study, except for sperm morphology, which was unaffected by the treatment. Complete sterility was achieved on post-treatment days 70, 84, 60, 23, 51 and 40 for dogs 1 to 6, respectively. The 4.7 mg deslorelin implant caused a significant (p < 0.05) decrease in serum testosterone as well as sperm motility. Our results (i) confirm the efficacy of deslorelin in causing reversible sterility in male dogs, (ii) confirm and provide details about endocrine and seminal parameters involved in this process and (iii) contribute to define the interval between treatment and achievement of complete sterility. Practitioners should be aware that such interval may be longer than 2 months in some cases, and that fertility may actually be increased during the first 2–4 weeks post-treatment.


Introduction

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

The control of pet overpopulation remains a key issue in many countries of the world. While in developing countries, the approach to this problem has been generally more radical and irreversible (surgical gonadectomy), in Western countries the search for non-surgical and/or reversible techniques for gonadectomy has produced several options for treatment such as the use of GnRH agonists and antagonists or the development of vaccines against gonadotropin-releasing hormone (GnRH), luteinizing hormone (LH) and its receptors, sperm antigens or oocyte zona pellucida (reviewed by Kutzler and Wood, 2006; Ball et al. 2006; Walker et al. 2007; Garcìa Romero et al. 2012). The GnRH agonist deslorelin was developed towards the end of last century in Australia and has been marketed in Europe for a few years with the indications of control of aggressiveness and induction of reversible sterility in male dogs. Despite its well-known efficacy on the induction of reversible sterility because of a temporary pituitary block caused by a prolonged positive feedback on the pituitary, little is known about the interval between administration of deslorelin and complete sterility in male dogs. The drug leaflet (Suprelorin, Virbac, Carros, France) warns owners to not put male dogs treated with a deslorelin implant together with oestrous bitches during the first 6 weeks after implantation. Trigg et al. (2006) reported that sterility was achieved by day 48 post-treatment, while histologically Junaidi et al. (2007, 2009) observed complete atrophy of seminiferous epithelium in the testicles of 90% of treated dogs at 41-day post-treatment. In the study of Junaidi et al. (2007), histological exams were performed at 41- and 101-day post-treatment; therefore, it was impossible to know when (during the period 42–100 days after deslorelin administration) the remaining 10% of dogs would achieve complete sterility. Based on official data published by the European Medicines Agency (EMEA 2012), dogs treated with a 4.7 mg implant deslorelin show a reduction of serum testosterone (T) below 0.4 ng/ml by 22–33 days and a lack of semen production by 6-week post-treatment.

In small animal practice, the request of induction of non-surgical sterility is increasing in dogs, and owners of animals treated with a non-surgical sterilant always want to know how long it will take for their dogs to become sterile after treatment, in order to be safely placed with oestrous bitches. In our clinical practice, we had noticed cases of dogs still capable of ejaculating semen with acceptable motility and morphology beyond 1 month after implantation with deslorelin. Therefore, our study was designed to answer the following questions in dogs treated with a-4.7 mg implant of deslorelin: (i) effect on semen quality and (ii) interval for a dog to become sterile.

Materials and Methods

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

Six privately owned dogs of various breeds and body weights (an 18-month old, 14.2 kg bw Tibetan terrier; an 8½-year old, 7.8 kg bw Yorkshire terrier; an 8-year old, 11.3 kg bw mongrel; 3 Dobermans of 2½, 6 and 6 year of age and 40.2 ± 2.4 kg bw) were used for this study. Dogs were referred to the Veterinary Teaching Hospital of the University of Padova (Italy), with the request to control aggressiveness and/or fertility. The following protocol was used for each dog on the first day of the study (Day 0): (i) collection of data related to signalment, general/reproductive history and general physical/reproductive exam; (ii) collection of a blood sample for haematology, serum biochemistry and assay of basal serum T concentration; (iii) collection and evaluation of semen; (iv) intravenous injection of 50-mcg gonadorelin GnRH (Fertagyl; Intervet Peschiera Borromeo, Italy); (v) second blood collection 60 min later to assay post-GnRH serum T concentration; (vi) administration of one 4.7-mg implant of deslorelin (Suprelorin; Virbac) between the shoulder blades. Owners were instructed to bring back their dogs for a single blood and semen collection every 2 weeks until the observation of two consecutive semen collections in which the dog could be considered infertile. Semen collections were performed routinely without the presence of a bitch in heat, and collection was continued for at least 5 min even if no semen was being produced. In the event of aspermia, a urine sample was collected and the urine sediment analysed for presence of spermatozoa, to rule out retrograde ejaculation. Progressive motility was evaluated under light microscopy at 10X, and morphology of 100 spermatozoa was assessed on a Diff Quik-stained semen smear under light microscopy at 100X. The number of sperm cells was counted using a Bürker chamber. The study ended when complete sterility was achieved, based on presence of <10 million of progressively motile sperms (PMS) and semen volume <0.5 ml.

Blood samples were collected in plain and EDTA vacutainer tubes. Following haematology and serum biochemistry, serum samples were aliquoted and stored frozen. Serum T was assayed using a chemiluminescence system (Immulite; Medical System, Genova, Italy) previously validated in our laboratory for the dog (data not shown).

Statistical analysis: results were analysed using analysis of variance for repeated measures, grouping data in the following classes of distance (or periods) from the day of treatment (Day 0): period 1 = day 0; period 2 = day 9–17; period 3 = day 23–32; period 4 = day 37–47; period 5 = day 51–60; period 6 = day 64–75; period 7 = day 84–89. Classes of distance from day 0 were considered as the independent factor. Pearson's correlation coefficients (considered significant when >0.4) were used to investigate correlation between treatment and fertility parameters.

Results

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

General clinical as well as endocrine and fertility parameters were within normal limits for all dogs at the onset of the study. Attempts at collecting semen in dog 4 were unsuccessful at the time of the first visit despite the use of a bitch in heat. This dog was subsequently collected at his home, where he was relaxed and gave a good semen sample 2 weeks after implantation. He was aspermic from the third visit onwards. As this was an aggressive dog, GnRH stimulation was not performed on him; therefore, only basal T values are available for this dog.

Initially, semen motility increased (dogs 1, 2, 3 and 6) or remained stable (dog 4) and subsequently decreased from day 23–32 onwards in all dogs (Fig. 1); average percentage of PMS was 65 ± 14%, 80 ± 2%, 65 ± 14%, 56 ± 13%, 32 ± 15% and 10 ± 10% during periods 1 through 6, respectively. Total number of sperms showed a decrease from day 9–17 onwards (dogs 3 and 6) or was initially stable and showed a peak at day 23–32 before decreasing progressively (dogs 1, 2, 5); average total number of sperm was 620 ± 160, 550 ± 120, 750 ± 60, 300 ± 50, 100 ± 20 and 0 millions during periods 1 through 6, respectively (Fig. 2). Semen volume showed an increase until day 23–32 (dogs 1, 2, 3 and 6) followed by a gradual and continuous decrease, while it decreased progressively throughout the study period in dog no 5; average semen volume was 6.0 ± 1.8, 5.8 ± 1.4, 5.2 ± 2.0, 2.2 ± 1.4, 1.0 ± 0.6 and 0 cc during periods 1 through 6, respectively (Fig. 3). Semen morphology was unaffected by the treatment; percentage of abnormal sperms fluctuated between 12% and 10% throughout the entire study. Serum T showed an increase at 9-17 days followed by a decrease (dogs no 1, 2 and 5) or a progressive decrease (dogs no 3, 4 and 6) throughout the study periods; average serum T concentration was 5.3 ± 1.6, 4.5 ± 1.2, 2.5 ± 0.6, 0.9 ± 0.3, 0.7 ± 0.3 and <0.2 ng/ml during periods 1 through 6, respectively (Fig. 4). The GnRH stimulation test was performed in 5/6 dogs; in all 5 tested dogs, post-stimulation serum T concentrations more than doubled with respect to pre-treatment values. In dog no 3, basal serum T concentration was very high, 13.1 ng/ml, and post-GnRH serum T was >16 ng/ml (beyond the upper limit of assay sensitivity). The treatment caused a significant (p < 0.05) decrease in serum T as well as sperm motility.

image

Figure 1. Average percentage semen motility in five dogs prior to and following treatment with a 4.7 mg implant of deslorelin (Suprelorin, Virbac). Time refers to intervals from the day of treatment (Day 0): period 1 = day 0; period 2 = day 9–17; period 3 = day 23–32; period 4 = day 37–47; period 5 = day 51–60; period 6 = day 64–75; period 7 = day 84–89

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image

Figure 2. Average number of total spermatozoa in five dogs prior to and following treatment with a 4.7 mg implant of deslorelin (Suprelorin, Virbac). Time refers to intervals from the day of treatment (Day 0): period 1 = day 0; period 2 = day 9–17; period 3 = day 23–32; period 4 = day 37–47; period 5 = day 51–60; period 6 = day 64–75; period 7 = day 84–89

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image

Figure 3. Average semen volume in five dogs prior to and following treatment with a 4.7 mg implant of deslorelin (Suprelorin, Virbac). Time refers to intervals from the day of treatment (Day 0): period 1 = day 0; period 2 = day 9–17; period 3 = day 23–32; period 4 = day 37–47; period 5 = day 51–60; period 6 = day 64–75; period 7 = day 84–89

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image

Figure 4. Average serum testosterone concentration in six dogs prior to and following treatment with a 4.7 mg implant of deslorelin (Suprelorin, Virbac). Time refers to intervals from the day of treatment (Day 0): period 1 = day 0; period 2 = day 9–17; period 3 = day 23–32; period 4 = day 37–47; period 5 = day 51–60; period 6 = day 64–75; period 7 = day 84–89

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Complete sterility was achieved on post-treatment days 70, 84, 60, 23, 51 and 40 for dogs 1 through 6, respectively. All dogs were still considered fertile at their previous biweekly check based on per cent motility and total sperm count (Table 1). On average, sterility was achieved at 54 ± 21 days post-treatment.

Table 1. Dog identification and age, days post-treatment (post-Tx), semen volume,% progressive motile sperm (PMS) and total number of spermatozoa in the ejaculate (t-count) at the last semen collection in which the dog was considered fertile, and at the first semen collection in which the dog was considered sterile, following administration of a-4.7 mg implant of deslorelin
Dog IDAge (year, month)Days post-Tx (semen vol., PMS, t-count)Days post-Tx (semen vol., PMS, t-count)
11, 659 (3.0 cc, 60%, 247 millions) = fertile70 (0.3 cc, 2%, 76.8 millions) = sterile
28, 670 (0.5 cc, 50%, 82.0 millions) = fertile84 (0.05 cc, 50%, 7.8 millions) = sterile
38, 041 (2.0 cc, 70%, 18.4 millions) = fertile60 (0.05 cc, 30%, 3.5 millions) = sterile
42, 69 (7.0 cc, 80%, 1512 millions) = fertile23 (0 cc) =sterile
56, 044 (0.2 cc, 60%, 67 millions) = fertile51 (0 cc) = sterile
66, 028 (6.0 cc, 80%, 376 millions) = fertile40 (0.03 cc, 50%, 0.96 millions) = sterile

Discussion

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

The endocrine profile of male dogs treated with a 4.7 mg deslorelin implant has been well described. However, there is paucity of data on semen quality and timing of onset of sterility after treatment. Following deslorelin treatment, serum T shows an acute increase starting 60 min after implantation and lasting for approximately 6 days, after which it becomes undetectable during the second or third week post-treatment (Trigg et al. 2001, 2006; Romagnoli et al. 2005; Junaidi et al. 2007). The timing of onset of complete sterility is reported to vary between 36 and 48 days (Trigg et al. 2006; Junaidi et al. 2007; EMEA 2012), and semen parameters are reported to show a decrease by 28–35 days post-implantation (Junaidi et al. 2007). The results of our study confirm previous observations on serum T following deslorelin treatment and provide new information on semen quality and interval to sterility in treated dogs; semen quality may not be negatively affected initially; and some parameters may actually be improved in some animals. In fact, semen motility was unaffected by the treatment during the first month, and the total number of spermatozoa increased from approximately 300 and 600 to 2400 and 950 millions in dogs no 1 and no 2, respectively. Our results on semen quality are not in accordance with those of Junaidi et al. (2007) who only observed a decrease in semen quality after day 28 post-treatment. However, in their study, post-treatment semen quality was not checked prior to day 28, which may explain why a change in semen quality parameters (and perhaps even improvement) was not observed (Junaidi et al. 2007). During the second and third month of our study, a progressive decline of most seminal parameters occurred. Sperm morphology was unaffected throughout the entire study, but all dogs became eventually aspermic. Aspermia is probably due to atrophy of seminiferous tubules occurring in 90% of deslorelin treated dogs at 41-day post-treatment (Junaidi et al. 2007).

This is the first report detailing changes in canine semen quality after administration of a 4.7 mg deslorelin implant. Although the number of dogs in our study was not large, it is important to underline that onset of sterility following treatment with deslorelin may occur later than previously expected. Dogs no 1 and 2 in our study were still fertile at 59- and 70-day post-treatment, respectively, and may have remained fertile even longer as their subsequent semen evaluation was performed at 70- and 84-day post-treatment, respectively. Furthermore, semen quality does not start to decrease immediately, but rather remains stable for the first month or may even improve in some cases. When advising owners of dogs treated with deslorelin, small animal clinicians should warn them that their animals might remain fertile for over 2 months and that semen fertility of treated dogs might actually improve for the first few weeks. Our results confirm that a deslorelin implant can reliably cause a down regulation of the hypothalamic pituitary axis leading to a progressive decline of seminal parameters and eventually to an arrest of semen production. Because of its initial stimulating action, the use of deslorelin (perhaps as a short-acting formulation or placed in a location from where it can be easily removed after a few weeks) might be investigated as a mean to improve semen quality in subfertile dogs.

Acknowledgements

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

This study was financed with the University of Padova Grant no 60A08-5851/12.

Author contributions

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

S. Romagnoli contributed to the study design and wrote the paper; A. Siminica performed semen collections and evaluations and took care of all other practical aspect of the project; C. Stelletta contributed to the study design and performed the statistical analysis; C. Milani, H. Sontas and A. Mollo contributed to the study design and helped with semen collection and evaluation.

References

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