Contraceptive effect and potential side-effects of deslorelin acetate implants in rats (Rattus norvegicus): Preliminary observations. Abstract.

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1 Article Contraceptive effect and potential side-effects of deslorelin acetate implants in rats (Rattus norvegicus): Preliminary observations Claire Grosset, Stijn Peters, Franck Peron, Joëlle Figuéra, Christelle Navarro Abstract During the last ten years, numerous species have been treated with deslorelin implants to induce contraception. The aims of the study were 1) to assess contraceptive efficacy of 4.7 mg subcutaneous deslorelin implants in rats, 2) to determine the latency of contraceptive effect, and 3) to determine potential side effects. Three experimental females were implanted and their estrous cycle was studied by vaginal smear. Two weeks after implantation, a male whose fertility was previously assessed with a control female, was introduced into their cage. No female conceived during the 4 mo following implantation. Additionally, 38 pet rats were recruited from clients in practice to test for potential side effects, including 6 males and 32 females with a mean age of 14 mo. Local reaction and transient weight gain during the first 2 wk, as well as behavioral changes were recorded. According to this pilot study, deslorelin implant could be used as a contraceptive method in female rats. The latency period is about 2 wk. Nevertheless, it might be possible to refine the treatment further using hormonal measurements. The duration of contraceptive effect is to be determined in an upcoming study. Résumé Au cours des dix dernières années, de nombreuses espèces ont été traitées avec des implants de deslorelin à des fins contraceptives. Les objectifs de la présente étude étaient 1) déterminer l efficacité contraceptive d implants sous-cutanés de 4,7 mg de deslorelin chez le rat; 2) déterminer la latence de l effet contraceptif; et 3) déterminer les effets secondaires potentiels. Trois femelles d expérimentation ont reçu des implants et leur cycle œstral a été étudié via des frottis vaginaux. Deux semaines après l implantation, un mâle dont la fertilité avait préalablement été évaluée avec une femelle témoin, a été introduit dans la cage. Aucune des femelles n a conçu au cours des 4 mois qui ont suivi la mise en place des implants. De plus, 38 rats (6 mâles et 32 femelles; âge moyen 14 mois) servant d animaux de compagnie ont été recrutés auprès de clients d une pratique vétérinaire afin de vérifier les effets secondaires potentiels. Une réaction localisée et un gain de poids temporaire durant les 2 premières semaines, ainsi que des changements de comportement ont été notés. Suite à cette étude pilote, des implants de deslorelin pourraient être utilisés comme méthode contraceptive chez des rats femelles. La période de latence est d environ 2 semaines. Néanmoins, il pourrait être possible de raffiner le traitement en utilisant des dosages hormonaux. La durée de l effet contraceptif est à être déterminée dans une étude ultérieure. (Traduit par Docteur Serge Messier) Introduction Rats are becoming very popular as companion pets. They are prolific rodents, with puberty occurring from 4 wk of age (1). Their estrus cycle lasts for 4 to 5 d (2) and may be followed by examination of vaginal smears (2,3), which have demonstrated a good correlation with blood levels of ovarian steroid hormones (4). Gestation lasts 21 to 23 d, and there can be 13 pups per litter (1). Therefore, contraception is often requested by owners that have a couple of rats in a single cage. However, considering the price and the risk of spaying, many owners decline surgery. Thus, there is a need for a less invasive method of contraception in rats. Gonadotropin-releasing hormone (GnRH) agonists, such as deslorelin, act primarily on the anterior pituitary, inducing a transient early rise in gonadotropin release. With continued use, GnRH agonists cause pituitary desensitization or downregulation, which leads to suppressed circulating levels of gonadotropins and sex hormones (5). This inhibition has been reported to be fully reversible on discontinuation of the drug (6 9). Long-lasting GnRH agonist formulations have been tested in rats (8,10,11). No side effects have been reported so far in rats (8). In a preliminary study the contraceptive efficacy of a 4.7-mg deslorelin acetate implant in rats was assessed and the latency of the effect was determined. In addition, potential side effects were evaluated in a larger population. Our hypotheses were that the implant would induce contraception in female rats, that the latency would be approximately 1 mo, and side effects would include local inflammation and/or metritis. Companion Exotic Animal Medicine and Surgery Service, University of Davis, California, USA (Grosset); Dierenziekenhuis Eindhoven, Marconilaan 26, 5621 AA Eindhoven, The Netherlands (Peters); Laboratoire d Ethologie et Cognition Comparées, Université Paris Ouest, Nanterre, France (Peron); Virbac France, Carros, France (Figuéra); Medical Department, Virbac SA, Carros, France (Navarro). Address all correspondence to Dr. Claire Grosset; telephone: ; claire.grosset@gmail.com Dr. Grosset s current address is 2110 Camino Court, Davis, California 95616, USA. Received April 10, Accepted August 16, ;76: The Canadian Journal of Veterinary Research 209

2 Figure 1. Typical vaginal smears from a rat before implantation, showing the stages of the estrus cycle. [1a proestrus, 1b estrus 1c diestrus] Diff Quick stain; original magnification Materials and methods The preliminary study was conducted between January 2007 and January 2012 according to the Guide to the Care and Use of Experimental Animals (12) of the Canadian Council on Animal Care. Five 3-month-old rats (1 male and 4 female) were purchased from a pet store as representative pet rats. During a 1-mo acclimation period they were weighed once a week and received prophylactic antiparasitic treatment with imidacloprid and moxidectin (Advocate; Bayer, Puteaux Cedex, France). The females were kept in a wire cage 1-m wide and 50-cm deep; the male was housed alone in a wire cage 60-cm wide and 40-cm deep. The animals were fed rat pellets (Rongis Rat et Souris, Compagnie des Pet Foods, Gallardon, France) and vegetables. Water was provided ad libitum in bottles. All the rats were exposed to natural light. Each animal was handled daily for approximately 1 mo before vaginal smears were first taken. Implants containing 4.7 mg of deslorelin acetate (Suprelorin; Virbac, Carros, France, used off-label) were manufactured by a proprietary method that involved extrusion of deslorelin within a lipid matrix. The implants were surgically implanted in 3 of the female rats at a veterinary practice using the following procedure. The rats were anesthetized with isoflurane (Baxter, Maurepas Cedex, France) in oxygen administered with the use of a face mask and were placed on a heating pad. The interscapular site of implantation was shaved and the skin surgically prepared. The implant was inserted subcutaneously with the standard device furnished by Virbac. The skin was closed with surgical glue (Vet Bond; 3M, Cergy-Pontoise, France) or with polyglactin 910 suture material (Vicryl 3-0; Ethicon, Auneau, France). Meloxicam (Metacam; Boehringer Ingelheim, Reims, France), 0.3 mg/kg body weight (BW), was injected subcutaneously at the end of the procedure. During recovery, oxygen was delivered with the use of a face mask, and the heating pad was kept on. Upon full recovery the rats were placed in a quiet cage and monitored carefully. For 2 wk thereafter they were kept isolated from the male. A control female did not receive an implant and was put in the same cage as the male. For the next 7 mo the contraceptive efficacy of the implant was assessed by the absence of litters and by the pattern of vaginal 210 The Canadian Journal of Veterinary Research smears. Smears were taken from the 3 experimental females twice a day for 2 wk before and after implantation in order to follow the estrus cycle. After the male was put in the females cage, smears were taken once a week for 2 wk and then once a month. To obtain a vaginal smear, a wet cotton swab was used as described by Allmann-Iselin (13). Care was taken not to insert the swab too deeply, because inadvertent cervix stimulation can initiate pseudopregnancy (2) lasting 12 to 14 d (14). Slides were dried before being stained with Diff Quick (Dade Behring, Düdingen, Swizerland). Cytologic examination with a light microscope at 100x and 400x magnification was done within 24 h after sample collection. The stages of the estrus cycle were identified by the same observer (C.G.), using the criteria described by Hubscher et al (2). The rats level of activity and food intake were evaluated daily by the same observer (C.G.) and scored according to a semiquantitative system composed of the following categories: normal (normal behavior and level of alertness), slight depression (decreased spontaneous movement and locomotion), lethargy (decreased reaction to gentle stimulation), and severe depression (absence of reaction to gentle stimulation). Each rat was weighed and examined by the same observer (C.G.) once a week throughout the study period. To test for potential side effects 38 pet rats (6 male and 32 female) were recruited from in-practice clients between 2007 and 2010 and received an implant. The rats ages ranged between 4 and 34 mo, with a mean of 14 mo. The owners were properly informed about the study and gave their written agreement to contribute. Some wanted to test medical contraception, whereas others just wanted to contribute to the study. The owners were asked about their pets appetite, attitude toward the owner, and estrus behavior, especially mounting behavior and lordosis when stroked by the owner. Every animal recheck included a complete physical examination and a vaginal smear. Sixteen female rats with a mean age of 20 mo had presented with a mammary tumor. Nine rats that had presented with chronic respiratory disease received occasional antibiotic treatment during the study. If death occurred during the study, the cause and date of death were recorded, necropsy was strongly recommended to the owner and was done by a pathologist. 2000;64:0 00

3 Figure 2. Typical vaginal smear from a rat 2 wk after receiving a 4.7 mg implant of deslorelin acetate, showing cornified cells. Diff Quick stain; original magnification Figure 4. Dermatitis caused by pruritus cranial to the surgical wound, which had been closed with surgical glue. Results In the 3 experimental female rats the estrus cycles before implantation had a classic duration of 4 d (Figure 1). Metestrus and diestrus stages could not be determined precisely: both were therefore recorded as diestrus stage. No pseudopregnancy was observed after sampling before implantation. The surgical implantations were uneventful except that in 1 rat the initial implantation was unsuccessful because the implant was not placed far enough from the incision and extruded through it. The implant was replaced by means of sterile forceps and tissue glue while the rat was still under anesthesia. The total duration of the procedure in the entire group of 41 rats receiving an implant was approximately 5 min each. The skin was closed with surgical glue in 12 of the 41 rats and with suture material in the other 29. No postsurgical complications were recorded. 2000;64:0 00 Figure 3. Typical diestrus-like vaginal smear from a rat in the period from 3 wk after implantation through the next 7 mo, showing a heavy population of leukocytes, some nucleated cells, and occasional cornified cells. Diff Quick stain; original magnification The latency period was monitored by means of the vaginal smears. After implantation, the smears showed that estrus became more and more frequent. By 2 wk the cyclic pattern had stopped and every vaginal smear showed cornified cells (Figure 2). The male was placed with the 3 females of the experimental group at this point to confirm the contraceptive effect. Male fertility had been assured by the fact that 1 litter had been produced with the control female 1 wk beforehand. The latency period was therefore determined to be approximately 2 wk. After 3 wk and for the next 7 mo, every treated female showed a diestrus-like vaginal smear: a heavy population of leukocytes, some nucleated cells, and occasional cornified cells (Figure 3). None of the 3 experimental females gave birth during the 7 mo after implantation. None of the pet rats that were in contact with a male had conceived by a maximum of 10 mo after implantation. The contraceptive efficacy of the deslorelin implant was therefore proven both indirectly and directly. Neither behavioral changes nor changes in food intake were detected after implantation in the 3 experimental rats. Moreover, none died during the next 7 mo. The rats had a mean weight increase of 21% (19% to 23%) during the 1st week after implantation; however, this was transitory. Unfortunately, all the pet rats could not be rechecked 1 wk after implantation, but some owners reported a weight gain. Of the 41 females receiving implants 2 showed signs of pruritus cranial to the surgical wound that led to dermatitis (Figure 4). In both cases the wound had been closed with surgical glue. With daily application of povidone iodine (Betadine; Meda Pharma, Paris, France) the dermatitis resolved within 1 wk. Two of the pet rats, which had received their implants at the ages of 4 mo and 2 y, respectively, had become less active and less aggressive 2 wk after implantation, according to their owner. Neutrophils were detected in the vaginal smear taken 5 mo after implantation from a rat that had received the implant at 9 mo of age; the rat was surgically neutered, and histologic examination of the uterus did not reveal any abnormality. Another female, which had received the The Canadian Journal of Veterinary Research 211

4 Table I. Causes of death among rats that received a 4.7 mg deslorelin acetate implant for contraception Number Mean age at Mean time after Cause of death of rats death (mo) implantation (mo) Respiratory distress Neurologic disorder a Unknown Total a From an inner ear abscess resulting from chronic respiratory disease. implant at the age of 13 mo, was surgically neutered when metritis developed 6 mo after implantation. During the study period 10 of the rats that received an implant died, at an average age of 25 mo. Seven had been more than 1 y old at the time of implantation. The causes of death are recorded in Table I. In 9 cases death was attributed to a chronic respiratory disease that had caused an inner ear abscess (n = 5) or severe respiratory distress (n = 4). Two sisters had received an implant at 1 y of age and showed neurologic signs approximately 13 mo after implantation. Before implantation 1 had had a mammary tumor removed. A lipoma developed 8 mo after implantation in the other sister. The owner declined necropsy after euthanasia. Discussion The fertility of the female rat decreases naturally after 1 y. This is why only young females were included in this study. Secondary osteopenia can develop in rats neutered before 4 mo of age (15,16); therefore, implantation was done after 4 mo of age. Deslorelin implants have shown contraceptive effects in birds (17), domestic mammals such as dogs (9,18), cats (19,20), and ferrets (21,22), and zoo and wild mammals, including sea otters (Enhydra lutris) (6), cheetahs (Acinonys jubatus) (6,23,24), lions (Panthera leo) (23), leopards (Panthera pardus) (23), African wild dogs (Lycaon pictus) (23), fennec foxes (Vulpes zerda) (6), female tammar wallabies (Macropus eugenii) (25), and wild boars (Sus scrofa) (26). However, it is possible that some pituitary GnRH receptors are quite specific. Chicken GnRH-II was shown to inhibit reproductive hormones in captive male green iguanas (Iguana iguana), whereas mammalian GnRH was not effective (27). In male tammar wallabies (28) and in bulls (29), plasma testosterone concentrations remained within the normal range after the insertion of a deslorelin implant. In marmoset monkeys (Callithrix jacchus) (30) and in red deer (Cervus elaphus) (31), testosterone levels were unchanged or even increased after treatment with buserelin. In rats, GnRH agonists have already been tested (8,10,11). After 1 injection of long-lasting leuprolide acetate (Lupron depot; Abbott Japan, Pharma Products Group, Tokyo, Japan), 3 mg/kg, plasma luteinizing hormone levels of rats decreased after 7 d and remained below the basal level for 28 d (8,32). Long-term administration of 100 ng of a GnRH agonist twice a week induced a progressive decrease of testicular, prostatic, and seminal vesicular weight in rats after 2 to 8 wk of treatment, with a maximal inhibitory effect after 12 wk (10). In the present study, deslorelin implants stopped the estrus cycle and produced a contraceptive effect for at least 7 mo in rats. In the current study the latency period before contraceptive effect was found to be approximately 2 wk in rats. In dogs the plasma testosterone concentration decreased to less than 1 ng/ml within 6 to 25 d after insertion of a 6-mg implant (9). In female ferrets the signs of estrus stopped spontaneously 2 wk after insertion of a 4.7-mg deslorelin implant (22). In our study the vaginal smears indicated that during the first 2 wk after implantation estrus became more and more frequent. This pattern has been described in rats during the transition period at the beginning of estropause (33). This stage was attributed to a transient early rise in gonadotropin release induced by the implant. At 2 wk every vaginal smear in our study showed cornified cells, which indicated a constant estrus condition, as has been described during rat estropause (33). After 3 wk the smears in our study showed a pattern similar to that of diestrus, as has been described after ovariectomy in rats (2,33). Depending on species, body weight, and implant size, the duration of the contraceptive effect of a deslorelin 4.7-mg implant is highly variable (9). The effect lasts between 6 and 27 mo in bitches (9), whereas it lasts between 17 and 27 mo in female ferrets (22). In rats the exact duration has not been established and requires further study. Among the rats in our study, none showed signs of estrus in the 7 mo of follow-up by vaginal smear after implantation, and according to information about the pet rats the contraceptive effect may last for at least 10 mo. Reversibility of the effect has not yet been documented in rats. The temporary weight gain in the rats in our study could raise the question of aborted pregnancy. Some bitches can get pregnant if mated during the estrus induced after implantation, and they abort around day 40 of gestation (34). In every rat in our study the temporary weight gain took place during the first 2 wk after implantation (i.e., before contact with the male). After the 2nd week no weight increase was observed. The low number of animals did not enable us to study interindividual variability. Nevertheless, according to this preliminary study it would be safe for owners to place their male and female rats together 2 wk after implantation. It might be possible to further refine recommendations with measurement of hormone levels in the blood, a more reliable method of determining the latency period. A follow-up study to do this is planned. The reported side effects among the rats in this study included dermatitis due to pruritus during the 1st week, weight gain, and behavior changes. Weight gain and behavior changes are not unusual after neutering (35). The pruritus occurred only among the animals in which the surgical wound was closed with surgical glue. However, the number of animals is too low to draw any conclusion regarding the potential role of surgical glue in this problem. Side effects associated with deslorelin implants in dogs have included local reaction and pyometra in bitches that had endometrial hyperplasia before implantation (18). Whether or not the metritis observed in this study in 1 rat was due to the implant needs to be assessed. In the 2 sister rats that had neurologic signs 13 mo after implantation a pre-existing hypophyseal tumor could be suspected since a mammary tumor was removed from 1 sister before implantation and a lipoma was removed from the other sister 8 m after implantation. Fibroadenomas are known to be induced by prolactin in rats (36 39). A case report indicated a possible growth-stimulating effect of longacting leuprorelin on a pre-existing hypophyseal tumor in a human 212 The Canadian Journal of Veterinary Research 2000;64:0 00

5 (40). Pituitary tumors are common in rats: a pituitary adenoma developed in 25% (41) to 84% (42) of control populations of rats that did not receive an implant. Whether or not the implant could have stimulated the tumors needs to be assessed. Unfortunately, necropsy was declined for the sister rats in our study. In the 3 other rats which showed neurologic signs, it was considered unlikely that the symptoms were related to the implant. The average age at death of rats free of nonspecific pathogens is reported to be 20 to 22 mo (41). In our study the average age at death was 25 mo. The implant does not seem to shorten life expectancy in rats, but a study in a controlled environment would be necessary for proof. In conclusion, deslorelin implants can be recommended for use in young female rats from the age of 4 mo for induction of contraception within 2 wk of implantation. However, the duration of action has not yet been determined in this species. 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