LOCATION AND REMOVAL OF DESLORELIN ACETATE IMPLANTS IN FEMALE AFRICAN LIONS (PANTHERA LEO) Author(s): Anneke Moresco, D.V.M., Ph.D., Liza Dadone, V.M.D., Jason Arble, D.V.M., MSc. Dipl. A.C.V.R., Eric Klaphake, D.V.M., Dipl. A.C.Z.M., Dipl. A.B.V.P. (Avian), Dipl. A.B.V.P. (Reptile/Amphibian), and Dalen W. Agnew, D.V.M., Ph.D., Dipl. A.C.V.P. Source: Journal of Zoo and Wildlife Medicine, 45(2):397-401. 2014. Published By: American Association of Zoo Veterinarians DOI: http://dx.doi.org/10.1638/2013-0109R1.1 URL: http://www.bioone.org/doi/full/10.1638/2013-0109R1.1
BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/ terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder.
BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.
Journal of Zoo and Wildlife Medicine 45(2): 397–401, 2014 Copyright 2014 by American Association of Zoo Veterinarians
LOCATION AND REMOVAL OF DESLORELIN ACETATE IMPLANTS IN FEMALE AFRICAN LIONS (PANTHERA LEO) Anneke Moresco, D.V.M., Ph.D., Liza Dadone, V.M.D., Jason Arble, D.V.M., MSc. Dipl. A.C.V.R., Eric Klaphake, D.V.M., Dipl. A.C.Z.M., Dipl. A.B.V.P. (Avian), Dipl. A.B.V.P. (Reptile/Amphibian), and Dalen W. Agnew, D.V.M., Ph.D., Dipl. A.C.V.P.
Abstract: Contraception is necessary to manage zoo animal populations and to be able to house animals in groups without producing additional unwanted offspring. In felids and canids, an association between exposure to progestins and the occurrence of endometrial and mammary gland pathology has been documented. Therefore, the Association of Zoos and Aquariums (AZA) Wildlife Contraceptive Center recommends the use of deslorelin acetate for long-term contraception in carnivores. Return to cyclicity after deslorelin treatment has been variable; some individuals show ovarian suppression for long periods after the expected end of the deslorelin efficacy. In an attempt to reduce the time to reversal, techniques to locate and remove previous implants are being developed. This report documents the successful implementation of high-frequency ultrasonography in lions (Panthera leo) to locate and direct surgical removal of multiple deslorelin implants placed at least 2 yr previously as well as the return of follicular activity in both females at 7 months post-removal of implants. Key words: Adverse effects, carnivore, contraception, deslorelin, felid, gonadotropin releasing hormone agonist, reversal.
BRIEF COMMUNICATION Contraception is necessary to manage natural social groups in zoos without producing additional unwanted offspring. Historically progestins were commonly used as contraceptives, especially in felids1, due to their efficacy. However, synthetic progestins have been associated with significant reproductive pathology in felids and canids.6,8,9,11 Therefore, deslorelin acetate (Suprelorint, Virbac Animal Health, Fort Worth, Texas 76137, USA), plus megestrol acetate 7 days prior to and 7 days after implant insertion, is currently recommended for contraception in felids (Association of Zoos and Aquariums [AZA] Wildlife Contraception Center Recommendations: www.stlzoo.org/contraception). Deslorelin, a GnRH agonist, stimulates the pituitary, initially increasing the secretion of follicle stimulating hormone (FSH) and luteinizing hormone (LH). If large enough follicles are present, this initial stimulation phase or ‘‘flare’’ can induce ovulation and progesterone secretion. Long-term From the Reproductive Health Surveillance Program (Moresco, Agnew), Denver Zoo, 2300 Steele Street, Denver, Colorado 80205, USA (Moresco); the Cheyenne Mountain Zoo, 4250 Cheyenne Mountain Zoo Road, Colorado Springs, Colorado 80906, USA (Dadone, Klaphake) Veterinary Imaging Consultants, 2899 North Speer Boulevard, Denver, Colorado 80211, USA (Arble); and the Diagnostic Center for Population and Animal Health, Michigan State University, College of Veterinary Medicine, Lansing, Michigan 48910, USA (Agnew). Correspondence should be directed to Dr. Moresco ([email protected]).
administration of GnRH agonist reduces secretion of LH and FSH by down-regulating GnRH-receptors in pituitary gonadotrophs,7 thereby decreasing ovarian activity. Efficacy of deslorelin in domestic queens had been shown by documenting cessation of ovarian activity;10 since then, it has been successfully used in zoo felids as well.1 Deslorelin is available to AZA-accredited zoos through the AZA Wildlife Contraception Center (WCC) as a small (12 mm 3 2.3 mm), sustained release, biocompatible implant that can be placed subcutaneously through a large-gauge needle. Implants are labeled to release deslorelin over the course of at least 6 (Suprelorin Virbac, 4.7mg) or 12 (Suprelorin 12, 9.4 mg) mo, and animals are dosed by administering additional implants according to body size, species, and gender. The period of suppression of ovarian activity (anestrus) after implant placement may vary greatly among individuals.3,5,10 Duration of efficacy in zoo felids has averaged approximately twice the manufacturer-stated minimum;1 however, because of the interindividual variability and due to the paucity of reversal data in zoo carnivores, as long as contraception is needed the retreatment intervals recommended by the WCC for zoo species remain those stated by the manufacturer, representing the minimum expected duration of efficacy. One possible reason for the longer than expected duration of effect is residual deslorelin in the implants. Removing these implants would reduce residual deslorelin as a contributing factor to prolonged reversals. The challenging part of
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Figure 1. Ultrasonographic image of the subcutaneous space in a female African lion (Panthera leo) demonstrating two deslorelin acetate implants identified as the small, ovoid hypoechoic structures surrounded by hyperechoic tissue (implant 1 ¼ arrowheads; implant 2 ¼ arrows).
implant removal is locating small implants that may migrate, break, or both. Because implants were designed to be biocompatible, the need for removal had not been anticipated thus far, and only a general area of implant placement had typically been recorded. Palpation of implants is further complicated by a matrix that becomes friable shortly after placement, by the thick skin of zoo carnivores, and by copious adipose tissue in some individuals. A method to help find the implants, as well as a histologic description of the response of tissue to the implants, is reported here. Two female African lion (Panthera leo) littermates (5.5 yr) annually (from 2008–2010) received three subcutaneous deslorelin implants (9.4 mg), a total of nine implants per animal. Implants were not placed in 2011. Both females were first implanted at 15 mo of age and neither showed signs of estrus during the expected time of implant effect. Implant removal was planned to hasten return to normal ovarian activity in anticipation of a new breeding male being added to the pride. Anesthetic protocol consisted of diazepam (Qualitest Pharmaceuticals, Huntsville, Alabama 35811, USA; 10-mg tabs p.o. 0.3 mg/kg) as a preanesthetic sedative; anesthesia was induced 30 min later with medetomidine (ZooPharm Pharmacy, 3131 E. Grand Avenue #B, Laramie, Wyoming 82070-5140, USA; i.m. 0.03 mg/kg) and ketamine HCl (ZooPharm Pharmacy; i.m. 3.0 mg/kg 200 mg/ml) via pole-syringe. The lions were intubated and maintained on isoflurane
(Isoflurane USP, Patterson Company, St. Paul, Minnesota 55120, USA) and recovered in their enclosures by administering atipamezole (Antisedan, Pfizer Animal Health, Division of Pfizer Inc., New York, New York 10017, USA; i.m. 0.15 mg/kg). After implant removal, lions also received meloxicam (Cadila Healthcare Ltd., Ahmedabad 387 810, India; p.o. 0.1 mg/kg q. 24 hr 3 day) and cephalexin (Sun Pharmaceutical Industries Ltd., Acme Plaza, Andheri-Kurla Road, Andheri, Mumbai-400 059, India; 25 mg/kg p.o. q. 12 hr 7 days). The general interscapular area where the deslorelin implants had been placed was evaluated sonographically using an 18-MHz linear probe (MyLab 30 Gold, Esaote, Biosound, Genoa 16153, Italy) without clipping and only using alcohol to improve contact. Each lion also had at least one microchip transponder. The implants were identified as ovoid hypoechoic structures in the subcutaneous tissue and ranged from 9.0–11.0 mm long and 1.5–2.3 mm wide (Fig. 1). Each implant was surrounded by a thick rim of hyperechoic tissue that had a layered appearance similar to fibrous tissue. Microchip transponders were sonographically identical to the deslorelin implants but were distinguished by scanning with a microchip reader. After sonographic identification of implants and a negative reading for a microchip transponder, the area was clipped and implant location marked. After clipping, implants were palpable as small cylindrical nodules. The skin over the implants was aseptically prepared and bupivacaine (Hospira, Inc., Lake Forest, Illinois 60045, USA; Marcaine 0.5% s.c. 0.5 ml) was administered for local pain management. Implants were readily found and removed en bloc with surrounding tissue as smooth, cylindrical pieces (Fig. 2). A total of four and five tissue blocs was recovered for lions 1 and 2, respectively. Incisions were closed with a subcuticular pattern using polydioxanone 2-0 (Ethicon, Inc., Summerville, New Jersey 08876, USA). Tissues were placed in 10% normal buffered formalin and sent to the Reproductive Health Surveillance Program Pathology Laboratory (DCAHP, Michigan State University, Lansing, Michigan 48910, USA) for histopathologic evaluation. Histopathologically, the implant matrix was a crisply outlined, lightly basophilic wispy material embedded in a thin, 100–200-lm thick lamellar layer of collagen (Fig. 3). Immediately adjacent to the implant material and within the lamellar collagen, there were nodular proliferations of foamy macrophages, infrequent multinu-
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Table 1. Serum estradiol (E2) and progesterone (P4) from two female African lions (Panthera leo) on the day of deslorelin implant removal and 35 days later.
Lion 1 E2 (pg/ml) P4 (ng/ml) Lion 2 E2 (pg/ml) P4 (ng/ml)
Figure implants (Panthera minimize
2. After locating the deslorelin acetate with ultrasound in a female African lion leo), the implants were removed en bloc to breakage.
clear giant cells, and rare neutrophils as well as some areas of mineralization. Some macrophages contained vacuoles filled with basophilic material similar to the implant. Outside this dense layer were more-disorganized and loosely knit layers of collagen dissected by numerous small-caliber arterioles, veins, and capillaries. Only rare lymphocytes and plasma cells were identified in the reaction around the implants. In some areas, the crisp outlines of the implant cross-sections were disrupted, and small pools of the basophilic wispy material were surrounded by small numbers of
Figure 3. Cross-section of a deslorelin acetate implant removed en bloc from a female African lion (Panthera leo). Insert is a higher magnification of the fibrous wall surrounding the implant. A nodular aggregate of foamy macrophages and occasional neutrophils lies within a dense layer of fibrous connective tissue and an outer layer of less-packed collagen. Bar ¼ 500 lm.
Day 0
Day 35
23.2 0.8
34.2 0.9
21.9 0.6
17.4 0.4
macrophages and loose connective tissue. Tissue sections from lion 1 showed a total of 7 implant cross-sections and tissues from lion 2 showed a total of 13 cross-sections (Fig. 3). Uterus and ovaries were evaluated using a 5MHz probe and findings were typical of anestrus. Ovaries were homogenous in echogenicity and echotexture with no evidence of follicular or luteal development. The uterine wall was normal in thickness and there was no evidence of intraluminal uterine fluid. Thirty-five days later the lions were reanesthetized with the same anesthetic protocol for transport to a new exhibit; sonographic evaluation of the ovaries was not feasible at that time. Estrogen and progesterone serum concentrations were measured (Idexx, Westminster, Colorado 80234, USA) at the time of implant removal and 35 days later. Serum progesterone measurements were ,1 ng/ml for both lions at both timepoints (Table 1). Serum estradiol levels were also similar between the two lions at implant removal; levels were increased at 35 days for lion 1 but were slightly lower compared to day of implant removal for lion 2. Hormone analysis in nondomestic felids has focused on serial fecal samples because these can be acquired noninvasively. Reports on serum hormone concentrations in zoo felids remain rare, and ranges tend to be wide. Ranges reported to correspond with ovarian activity for zoo felids are very wide (10–100 pg/ml).4,12,14 Therefore, although serum estradiol levels for these lions at the time of implant removal were within ranges for ovarian activity, based on sonographic findings these lions were considered to be in anestrus. In lion 1, the sample at 35 days had higher estradiol levels, which are more suggestive of ovarian activity, while lion 2 had slightly lower levels in the sample at 35 days. Notably, 9 wk postremoval keepers reported lion 1 was the most interested of the females in the new male during the early introduction process. Serum progester-
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one measurements from both time-points were ,1 ng/ml for both lions and did not indicate the presence of a luteal tissue.13 Increased estradiol concentrations coupled with behavioral observations suggest that lion 1 may be transitioning to normal cyclicity. This is in concordance with reports that, in animals recovering from deslorelin treatment, rises in estrogen precede rises in progesterone (ovulation) by a long time.3 Seven months post-implant removal, fecal samples were collected three times weekly for four months. Fecal sample extraction and assay were performed as previously published,2 with the modification that coat-a-count kits (Siemens DPC, Los Angeles, California 90045, USA) were used. Fecal samples for both lions showed clear peaks and troughs in estrogen metabolite concentrations. Peaks were 2–4 times baseline concentrations, reaching values above 400 ng/g and occurred on average at 21.5 6 4.3 days apart. Peaks and troughs in estrogen excretion indicate a return to follicular activity. The intervals between peaks are within the range reported for interestrus periods in lions.12 The WCC now recommends placing implants in areas with thinner skin to facilitate locating them, such as in the medial aspect of the rear leg (AZA Wildlife Contraception Center Recommendations: www.stlzoo.org/contraception). The present technique greatly facilitates locating implants when they have been placed in areas with thicker skin or abundant fat, e.g., when implants are placed under physical restraint (squeeze cage) rather than under anesthesia. Assays to measure deslorelin are not widely available; therefore, there is no evidence that prolonged recovery truly is due to residual deslorelin. However, because residual deslorelin could contribute to delayed return to ovarian activity, and because removing implants is a step that can readily be performed, techniques that facilitate such removal may contribute reversal data to further assess this problem. Surgical removal of implants is not expected to cause more scarring or other problems than does the removal of melengestrol acetate (MGA) implants; however, because the redosing interval for deslorelin is shorter than for MGA, the frequency of surgery would be higher if removed each year rather than only when reversal is intended. Implants were located in the general area of placement, with less implant migration and less degradation than expected. In one lion, histopathology showed crosssections in excess of the number of implants
placed, confirming implant breakage postplacement. This increases the difficulty of accurately assessing complete implant removal as well as complicating the removal process itself. Placing tattoo ink in injection sites may improve the ability to locate and remove implants at a later date. Although implant breakage will complicate complete removal, high-frequency ultrasound, used as reported here, is a useful technique to locate previously placed deslorelin implants even if several years old and not placed in a thinskinned area. Acknowledgments: The authors would like to thank Mr. Chastain, animal care staff, and hospital technicians at the Cheyenne Mountain Zoo and Dr. Kozlowski and the Saint Louis Zoo Endocrinology Laboratory for hormone analysis.
LITERATURE CITED 1. Asa C, Boutelle S, Bauman K. AZA Wildlife Contraception Center programme for wild felids and canids. Reprod Domest. T. Anim. 2012;47(Suppl. 6): 377–380. 2. Asa CS, Bauman K, Callahan P, Bauman J, Volkman DH, Jochle, W. GnRH-agonist induction of fertile estrus with either natural mating or artificial insemination, followed by birth of pups in grey wolves (Canis lupus). Theriogenology. 2006;66:1778–1782 3. Bertschinger HJ, de Barros Vaz Guimaraes ˜ MA, Trigg TE, Human A. The use of deslorelin implants for the long-term contraception of lionesses and tigers. Wildl Res. 2008;35:525–530. 4. Briggs MB, Fithian CL, Starkey PR, Richards RE, Schramm RD, Reeves JJ. Endocrine profiles in estrus, pregnant and pseudopregnant African lions (Panthera leo) throughout the year. Proc Am Assoc Zoo Vet; 1990. p. 279–281. 5. Goericke-Pesch G, Georgiev P, Atanasov A, Albouy M, Navarro C, Wehrend, A. Treatment of queens in estrus and after estrus with a GnRH-agonist implant containing 4.7 mg deslorelin; hormonal response, duration of efficacy, and reversibility. Theriogenology. 2013;79:640–646 6. Harrenstien LA, Munson L, Seal US, American Zoo and Aquarium Association Mammary Cancer Study Group. Mammary cancer in captive wild felids and risk factors for its development: a retrospective study of the clinical behavior of 31 cases. J Zoo Wildl Med. 1996;27:468–476. 7. Loumaye E, Catt KJ. Agonist-induced regulation of pituitary receptors for gonadotropin-releasing hormone. J Biol Chem. 1983;258:12002–12009. 8. Moresco A, Munson L, Gardner IA. Naturally occurring and melengestrol acetate-associated reproductive tract lesions in zoo canids. Vet Pathol. 2009;46: 1117–1128.
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9. Munson L, Stokes JE, Harrenstien LA. Uterine cancer in zoo felids on progestin contraceptives. Vet Pathol. 1995;32:578. 10. Munson L, Bauman JE, Asa S, Jo¨chle W, Trigg TE. Efficacy of the GnRH analogue deslorelin for suppression of oestrous cycles in cats. J Reprod Fertil. 2001;57(Suppl.):269–273. 11. Munson L, Gardner IA, Mason RJ, Chassy LM, Seal US. Endometrial hyperplasia and mineralization in zoo felids treated with melengestrol acetate contraceptives. Vet Pathol. 2002;39:419–427. 12. Schmidt AM, Nadal LA, Schmidt MJ, Beamer NB. Serum concentrations of oestradiol and progesterone during the normal oestrous cycle and early
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pregnancy in the lion (Panthera leo). J Reprod Fertil. 1979;57:267–272. 13. Schramm RD, Briggs MB, Reeves JJ. Spontaneous and induced ovulation in the lion (Panthera leo) Zoo Biol. 1994;13:301–307. 14. Seal US, Plotka ED, Smith JD, Wright FH, Reindl NJ, Taylor RS, Seal MF. Immunoreactive luteinizing hormone, estradiol, progesterone, testosterone, and androstenedione levels during the breeding season and anestrus in Siberian tigers. Biol Reprod. 1985;32:361–368. Received for publication 22 May 2013
BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/ terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder.
BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.
Journal of Zoo and Wildlife Medicine 45(2): 397–401, 2014 Copyright 2014 by American Association of Zoo Veterinarians
LOCATION AND REMOVAL OF DESLORELIN ACETATE IMPLANTS IN FEMALE AFRICAN LIONS (PANTHERA LEO) Anneke Moresco, D.V.M., Ph.D., Liza Dadone, V.M.D., Jason Arble, D.V.M., MSc. Dipl. A.C.V.R., Eric Klaphake, D.V.M., Dipl. A.C.Z.M., Dipl. A.B.V.P. (Avian), Dipl. A.B.V.P. (Reptile/Amphibian), and Dalen W. Agnew, D.V.M., Ph.D., Dipl. A.C.V.P.
Abstract: Contraception is necessary to manage zoo animal populations and to be able to house animals in groups without producing additional unwanted offspring. In felids and canids, an association between exposure to progestins and the occurrence of endometrial and mammary gland pathology has been documented. Therefore, the Association of Zoos and Aquariums (AZA) Wildlife Contraceptive Center recommends the use of deslorelin acetate for long-term contraception in carnivores. Return to cyclicity after deslorelin treatment has been variable; some individuals show ovarian suppression for long periods after the expected end of the deslorelin efficacy. In an attempt to reduce the time to reversal, techniques to locate and remove previous implants are being developed. This report documents the successful implementation of high-frequency ultrasonography in lions (Panthera leo) to locate and direct surgical removal of multiple deslorelin implants placed at least 2 yr previously as well as the return of follicular activity in both females at 7 months post-removal of implants. Key words: Adverse effects, carnivore, contraception, deslorelin, felid, gonadotropin releasing hormone agonist, reversal.
BRIEF COMMUNICATION Contraception is necessary to manage natural social groups in zoos without producing additional unwanted offspring. Historically progestins were commonly used as contraceptives, especially in felids1, due to their efficacy. However, synthetic progestins have been associated with significant reproductive pathology in felids and canids.6,8,9,11 Therefore, deslorelin acetate (Suprelorint, Virbac Animal Health, Fort Worth, Texas 76137, USA), plus megestrol acetate 7 days prior to and 7 days after implant insertion, is currently recommended for contraception in felids (Association of Zoos and Aquariums [AZA] Wildlife Contraception Center Recommendations: www.stlzoo.org/contraception). Deslorelin, a GnRH agonist, stimulates the pituitary, initially increasing the secretion of follicle stimulating hormone (FSH) and luteinizing hormone (LH). If large enough follicles are present, this initial stimulation phase or ‘‘flare’’ can induce ovulation and progesterone secretion. Long-term From the Reproductive Health Surveillance Program (Moresco, Agnew), Denver Zoo, 2300 Steele Street, Denver, Colorado 80205, USA (Moresco); the Cheyenne Mountain Zoo, 4250 Cheyenne Mountain Zoo Road, Colorado Springs, Colorado 80906, USA (Dadone, Klaphake) Veterinary Imaging Consultants, 2899 North Speer Boulevard, Denver, Colorado 80211, USA (Arble); and the Diagnostic Center for Population and Animal Health, Michigan State University, College of Veterinary Medicine, Lansing, Michigan 48910, USA (Agnew). Correspondence should be directed to Dr. Moresco ([email protected]).
administration of GnRH agonist reduces secretion of LH and FSH by down-regulating GnRH-receptors in pituitary gonadotrophs,7 thereby decreasing ovarian activity. Efficacy of deslorelin in domestic queens had been shown by documenting cessation of ovarian activity;10 since then, it has been successfully used in zoo felids as well.1 Deslorelin is available to AZA-accredited zoos through the AZA Wildlife Contraception Center (WCC) as a small (12 mm 3 2.3 mm), sustained release, biocompatible implant that can be placed subcutaneously through a large-gauge needle. Implants are labeled to release deslorelin over the course of at least 6 (Suprelorin Virbac, 4.7mg) or 12 (Suprelorin 12, 9.4 mg) mo, and animals are dosed by administering additional implants according to body size, species, and gender. The period of suppression of ovarian activity (anestrus) after implant placement may vary greatly among individuals.3,5,10 Duration of efficacy in zoo felids has averaged approximately twice the manufacturer-stated minimum;1 however, because of the interindividual variability and due to the paucity of reversal data in zoo carnivores, as long as contraception is needed the retreatment intervals recommended by the WCC for zoo species remain those stated by the manufacturer, representing the minimum expected duration of efficacy. One possible reason for the longer than expected duration of effect is residual deslorelin in the implants. Removing these implants would reduce residual deslorelin as a contributing factor to prolonged reversals. The challenging part of
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Figure 1. Ultrasonographic image of the subcutaneous space in a female African lion (Panthera leo) demonstrating two deslorelin acetate implants identified as the small, ovoid hypoechoic structures surrounded by hyperechoic tissue (implant 1 ¼ arrowheads; implant 2 ¼ arrows).
implant removal is locating small implants that may migrate, break, or both. Because implants were designed to be biocompatible, the need for removal had not been anticipated thus far, and only a general area of implant placement had typically been recorded. Palpation of implants is further complicated by a matrix that becomes friable shortly after placement, by the thick skin of zoo carnivores, and by copious adipose tissue in some individuals. A method to help find the implants, as well as a histologic description of the response of tissue to the implants, is reported here. Two female African lion (Panthera leo) littermates (5.5 yr) annually (from 2008–2010) received three subcutaneous deslorelin implants (9.4 mg), a total of nine implants per animal. Implants were not placed in 2011. Both females were first implanted at 15 mo of age and neither showed signs of estrus during the expected time of implant effect. Implant removal was planned to hasten return to normal ovarian activity in anticipation of a new breeding male being added to the pride. Anesthetic protocol consisted of diazepam (Qualitest Pharmaceuticals, Huntsville, Alabama 35811, USA; 10-mg tabs p.o. 0.3 mg/kg) as a preanesthetic sedative; anesthesia was induced 30 min later with medetomidine (ZooPharm Pharmacy, 3131 E. Grand Avenue #B, Laramie, Wyoming 82070-5140, USA; i.m. 0.03 mg/kg) and ketamine HCl (ZooPharm Pharmacy; i.m. 3.0 mg/kg 200 mg/ml) via pole-syringe. The lions were intubated and maintained on isoflurane
(Isoflurane USP, Patterson Company, St. Paul, Minnesota 55120, USA) and recovered in their enclosures by administering atipamezole (Antisedan, Pfizer Animal Health, Division of Pfizer Inc., New York, New York 10017, USA; i.m. 0.15 mg/kg). After implant removal, lions also received meloxicam (Cadila Healthcare Ltd., Ahmedabad 387 810, India; p.o. 0.1 mg/kg q. 24 hr 3 day) and cephalexin (Sun Pharmaceutical Industries Ltd., Acme Plaza, Andheri-Kurla Road, Andheri, Mumbai-400 059, India; 25 mg/kg p.o. q. 12 hr 7 days). The general interscapular area where the deslorelin implants had been placed was evaluated sonographically using an 18-MHz linear probe (MyLab 30 Gold, Esaote, Biosound, Genoa 16153, Italy) without clipping and only using alcohol to improve contact. Each lion also had at least one microchip transponder. The implants were identified as ovoid hypoechoic structures in the subcutaneous tissue and ranged from 9.0–11.0 mm long and 1.5–2.3 mm wide (Fig. 1). Each implant was surrounded by a thick rim of hyperechoic tissue that had a layered appearance similar to fibrous tissue. Microchip transponders were sonographically identical to the deslorelin implants but were distinguished by scanning with a microchip reader. After sonographic identification of implants and a negative reading for a microchip transponder, the area was clipped and implant location marked. After clipping, implants were palpable as small cylindrical nodules. The skin over the implants was aseptically prepared and bupivacaine (Hospira, Inc., Lake Forest, Illinois 60045, USA; Marcaine 0.5% s.c. 0.5 ml) was administered for local pain management. Implants were readily found and removed en bloc with surrounding tissue as smooth, cylindrical pieces (Fig. 2). A total of four and five tissue blocs was recovered for lions 1 and 2, respectively. Incisions were closed with a subcuticular pattern using polydioxanone 2-0 (Ethicon, Inc., Summerville, New Jersey 08876, USA). Tissues were placed in 10% normal buffered formalin and sent to the Reproductive Health Surveillance Program Pathology Laboratory (DCAHP, Michigan State University, Lansing, Michigan 48910, USA) for histopathologic evaluation. Histopathologically, the implant matrix was a crisply outlined, lightly basophilic wispy material embedded in a thin, 100–200-lm thick lamellar layer of collagen (Fig. 3). Immediately adjacent to the implant material and within the lamellar collagen, there were nodular proliferations of foamy macrophages, infrequent multinu-
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399
Table 1. Serum estradiol (E2) and progesterone (P4) from two female African lions (Panthera leo) on the day of deslorelin implant removal and 35 days later.
Lion 1 E2 (pg/ml) P4 (ng/ml) Lion 2 E2 (pg/ml) P4 (ng/ml)
Figure implants (Panthera minimize
2. After locating the deslorelin acetate with ultrasound in a female African lion leo), the implants were removed en bloc to breakage.
clear giant cells, and rare neutrophils as well as some areas of mineralization. Some macrophages contained vacuoles filled with basophilic material similar to the implant. Outside this dense layer were more-disorganized and loosely knit layers of collagen dissected by numerous small-caliber arterioles, veins, and capillaries. Only rare lymphocytes and plasma cells were identified in the reaction around the implants. In some areas, the crisp outlines of the implant cross-sections were disrupted, and small pools of the basophilic wispy material were surrounded by small numbers of
Figure 3. Cross-section of a deslorelin acetate implant removed en bloc from a female African lion (Panthera leo). Insert is a higher magnification of the fibrous wall surrounding the implant. A nodular aggregate of foamy macrophages and occasional neutrophils lies within a dense layer of fibrous connective tissue and an outer layer of less-packed collagen. Bar ¼ 500 lm.
Day 0
Day 35
23.2 0.8
34.2 0.9
21.9 0.6
17.4 0.4
macrophages and loose connective tissue. Tissue sections from lion 1 showed a total of 7 implant cross-sections and tissues from lion 2 showed a total of 13 cross-sections (Fig. 3). Uterus and ovaries were evaluated using a 5MHz probe and findings were typical of anestrus. Ovaries were homogenous in echogenicity and echotexture with no evidence of follicular or luteal development. The uterine wall was normal in thickness and there was no evidence of intraluminal uterine fluid. Thirty-five days later the lions were reanesthetized with the same anesthetic protocol for transport to a new exhibit; sonographic evaluation of the ovaries was not feasible at that time. Estrogen and progesterone serum concentrations were measured (Idexx, Westminster, Colorado 80234, USA) at the time of implant removal and 35 days later. Serum progesterone measurements were ,1 ng/ml for both lions at both timepoints (Table 1). Serum estradiol levels were also similar between the two lions at implant removal; levels were increased at 35 days for lion 1 but were slightly lower compared to day of implant removal for lion 2. Hormone analysis in nondomestic felids has focused on serial fecal samples because these can be acquired noninvasively. Reports on serum hormone concentrations in zoo felids remain rare, and ranges tend to be wide. Ranges reported to correspond with ovarian activity for zoo felids are very wide (10–100 pg/ml).4,12,14 Therefore, although serum estradiol levels for these lions at the time of implant removal were within ranges for ovarian activity, based on sonographic findings these lions were considered to be in anestrus. In lion 1, the sample at 35 days had higher estradiol levels, which are more suggestive of ovarian activity, while lion 2 had slightly lower levels in the sample at 35 days. Notably, 9 wk postremoval keepers reported lion 1 was the most interested of the females in the new male during the early introduction process. Serum progester-
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one measurements from both time-points were ,1 ng/ml for both lions and did not indicate the presence of a luteal tissue.13 Increased estradiol concentrations coupled with behavioral observations suggest that lion 1 may be transitioning to normal cyclicity. This is in concordance with reports that, in animals recovering from deslorelin treatment, rises in estrogen precede rises in progesterone (ovulation) by a long time.3 Seven months post-implant removal, fecal samples were collected three times weekly for four months. Fecal sample extraction and assay were performed as previously published,2 with the modification that coat-a-count kits (Siemens DPC, Los Angeles, California 90045, USA) were used. Fecal samples for both lions showed clear peaks and troughs in estrogen metabolite concentrations. Peaks were 2–4 times baseline concentrations, reaching values above 400 ng/g and occurred on average at 21.5 6 4.3 days apart. Peaks and troughs in estrogen excretion indicate a return to follicular activity. The intervals between peaks are within the range reported for interestrus periods in lions.12 The WCC now recommends placing implants in areas with thinner skin to facilitate locating them, such as in the medial aspect of the rear leg (AZA Wildlife Contraception Center Recommendations: www.stlzoo.org/contraception). The present technique greatly facilitates locating implants when they have been placed in areas with thicker skin or abundant fat, e.g., when implants are placed under physical restraint (squeeze cage) rather than under anesthesia. Assays to measure deslorelin are not widely available; therefore, there is no evidence that prolonged recovery truly is due to residual deslorelin. However, because residual deslorelin could contribute to delayed return to ovarian activity, and because removing implants is a step that can readily be performed, techniques that facilitate such removal may contribute reversal data to further assess this problem. Surgical removal of implants is not expected to cause more scarring or other problems than does the removal of melengestrol acetate (MGA) implants; however, because the redosing interval for deslorelin is shorter than for MGA, the frequency of surgery would be higher if removed each year rather than only when reversal is intended. Implants were located in the general area of placement, with less implant migration and less degradation than expected. In one lion, histopathology showed crosssections in excess of the number of implants
placed, confirming implant breakage postplacement. This increases the difficulty of accurately assessing complete implant removal as well as complicating the removal process itself. Placing tattoo ink in injection sites may improve the ability to locate and remove implants at a later date. Although implant breakage will complicate complete removal, high-frequency ultrasound, used as reported here, is a useful technique to locate previously placed deslorelin implants even if several years old and not placed in a thinskinned area. Acknowledgments: The authors would like to thank Mr. Chastain, animal care staff, and hospital technicians at the Cheyenne Mountain Zoo and Dr. Kozlowski and the Saint Louis Zoo Endocrinology Laboratory for hormone analysis.
LITERATURE CITED 1. Asa C, Boutelle S, Bauman K. AZA Wildlife Contraception Center programme for wild felids and canids. Reprod Domest. T. Anim. 2012;47(Suppl. 6): 377–380. 2. Asa CS, Bauman K, Callahan P, Bauman J, Volkman DH, Jochle, W. GnRH-agonist induction of fertile estrus with either natural mating or artificial insemination, followed by birth of pups in grey wolves (Canis lupus). Theriogenology. 2006;66:1778–1782 3. Bertschinger HJ, de Barros Vaz Guimaraes ˜ MA, Trigg TE, Human A. The use of deslorelin implants for the long-term contraception of lionesses and tigers. Wildl Res. 2008;35:525–530. 4. Briggs MB, Fithian CL, Starkey PR, Richards RE, Schramm RD, Reeves JJ. Endocrine profiles in estrus, pregnant and pseudopregnant African lions (Panthera leo) throughout the year. Proc Am Assoc Zoo Vet; 1990. p. 279–281. 5. Goericke-Pesch G, Georgiev P, Atanasov A, Albouy M, Navarro C, Wehrend, A. Treatment of queens in estrus and after estrus with a GnRH-agonist implant containing 4.7 mg deslorelin; hormonal response, duration of efficacy, and reversibility. Theriogenology. 2013;79:640–646 6. Harrenstien LA, Munson L, Seal US, American Zoo and Aquarium Association Mammary Cancer Study Group. Mammary cancer in captive wild felids and risk factors for its development: a retrospective study of the clinical behavior of 31 cases. J Zoo Wildl Med. 1996;27:468–476. 7. Loumaye E, Catt KJ. Agonist-induced regulation of pituitary receptors for gonadotropin-releasing hormone. J Biol Chem. 1983;258:12002–12009. 8. Moresco A, Munson L, Gardner IA. Naturally occurring and melengestrol acetate-associated reproductive tract lesions in zoo canids. Vet Pathol. 2009;46: 1117–1128.
MORESCO ET AL.—DESLORELIN IMPLANT LOCATION AND REMOVAL IN LIONS
9. Munson L, Stokes JE, Harrenstien LA. Uterine cancer in zoo felids on progestin contraceptives. Vet Pathol. 1995;32:578. 10. Munson L, Bauman JE, Asa S, Jo¨chle W, Trigg TE. Efficacy of the GnRH analogue deslorelin for suppression of oestrous cycles in cats. J Reprod Fertil. 2001;57(Suppl.):269–273. 11. Munson L, Gardner IA, Mason RJ, Chassy LM, Seal US. Endometrial hyperplasia and mineralization in zoo felids treated with melengestrol acetate contraceptives. Vet Pathol. 2002;39:419–427. 12. Schmidt AM, Nadal LA, Schmidt MJ, Beamer NB. Serum concentrations of oestradiol and progesterone during the normal oestrous cycle and early
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pregnancy in the lion (Panthera leo). J Reprod Fertil. 1979;57:267–272. 13. Schramm RD, Briggs MB, Reeves JJ. Spontaneous and induced ovulation in the lion (Panthera leo) Zoo Biol. 1994;13:301–307. 14. Seal US, Plotka ED, Smith JD, Wright FH, Reindl NJ, Taylor RS, Seal MF. Immunoreactive luteinizing hormone, estradiol, progesterone, testosterone, and androstenedione levels during the breeding season and anestrus in Siberian tigers. Biol Reprod. 1985;32:361–368. Received for publication 22 May 2013
