Coelioscopic-Assisted Sterilization of Female Mojave Desert Tortoises ...

Coelioscopic-Assisted Sterilization of Female Mojave Desert Tortoises (Gopherus agassizii) Laila M. Proença1, DVM, MSc, PhD, Scott Fowler1,2, DVM, Stephanie Kleine1, DVM, Jane Quandt1, DVM, MS, DACVAA, DACVECC, Christine O. Mullen3, PhD, Stephen J. Divers1, BVetMed, DZooMed, DACZM, DECZM (Herpetology, Zoo Health Management), FRCVS 1. Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, University of Georgia, 501 DW Brooks Drive, Athens, GA 30602, USA 2. Current address: Blue Pearl Georgia Veterinary Specialists, 455 Abernathy Rd. NE, Sandy Springs, GA 30328, USA 3. U.S. Fish and Wildlife Service, Desert Tortoise Recovery Office, 777 E. Tahquitz Canyon Way, Suite 208, Palm Springs, CA 92262, USA Abstract:  Reproductive disorders in female tortoises are common and well documented and often require surgical access to the coelomic cavity to diagnose and correct. Coelioscopic-assisted bilateral ovariectomy (8/8, 100%) and hemiovariosalpingectomy (1/8, 12.5%) were performed in eight, presumed-healthy Mojave Desert tortoises (Gopherus agassizii) maintained at the Desert Tortoise Conservation Center in Las Vegas, Nevada. Bilateral ovariectomy was successfully conducted in all animals through a unilateral (3/8, 37.5%) or bilateral (5/8, 62.5%) prefemoral approach. Hemisalpingectomy was also performed in one tortoise (1/8, 12.5%). Significant abnormalities were observed during coelioscopy in 6/8 (75%) tortoises including ectopic and free-floating follicles, yolk coelomitis, liver disease, and oviduct and ovarian adhesions. Although non-endoscopic bilateral ovariectomy and ovariosalpingectomy have been described, they are not able to clearly visualize internal structures or pathology. Results of the eight surgeries described in this study suggest that coelioscopic-assisted ovariectomy and ovariosalpingectomy are practical and safe methods for sterilization of Mojave Desert tortoises. The magnification and optimal visualization provided by coelioscopy were considered critical for success. Key words:  Coelioscopic-Assisted, Sterilization, Tortoises, Gopherus agassizii, Ovariectomy.

Introduction Reproductive disorders in female tortoises are common and well documented and often require surgical access to the coelomic cavity to diagnose and correct (Innis and Boyer, 2002). Prefemoral coeliotomy is shorter in duration and less traumatic with faster postoperative recovery, shorter healing times, and reduced pain when compared to traditional central plastron coeliotomy (Innis et al., 2007). Soft tissue prefemoral coeliotomy without the aid of coelioscopy has been described in eastern box turtles (Terrapene carolina carolina) for ovariosalpingectomy (Minter et al., 2008). However, prefemoral coeliotomy may not provide adequate visualization of the coelomic cavity in tortoises that have smaller prefemoral fossae than aquatic species. In addition, exteriorization of the ovaries through the prefemoral coe­ liotomy of immature females is not possible (Innis et al., 2007). Excellent visualization of the coelomic cavity through the prefemoral coeliotomy can be accomplished with the aid of endoscopy. Prefemoral coelioscopic-assisted ovariectomy and ovariosalpingectomy have been described in different species of chelonians but not in medium-sized tortoises such as the Mojave Desert tortoises (Gopherus agassizii) described in this study (Innis et al., 2007; Knafo et al., 2011; Mans and Sladky, 2012). The technique was also successfully performed in chelonians with reproductive disorders including Volume 24, No. 3–4, 2014

obstructive and non-obstructive dystocias (Innis et al., 2007; Knafo et al., 2011; Mans and Sladky, 2012). The Mojave Desert tortoise is experiencing ongoing population declines due to urban development and Mycoplasma agassizii outbreaks (U.S. Fish and Wildlife Service [USFWS], 1994; Johnson et al., 2006). These displaced tortoises, in addition to abandoned and relinquished “pets” and uncontrolled backyard breeding, have resulted in many animals entering the Desert Tortoise Conservation Center (DTCC) in Las Vegas, Nevada, which is operated by the San Diego Zoo (San Diego, California) under the authority of the USFWS. Sterilization techniques, including co­ e­ lioscopic-assisted ovariectomy and phallectomy, have been previously described in large chelonians (Galapagos tortoises, Geochelone nigra) for population management (Knafo et al., 2011; Rivera et al., 2011). Given the obvious need for a safe and practical solution for population management of the Mojave Desert tortoise, a surgical sterilization feasibility study was conducted at the University of Georgia (UGA), Athens, Georgia.

Materials and Methods Animals: A group of eight, reproductively mature, female Mojave Desert tortoises, owned by the USFWS and maintained at the DTCC, was used in a prospective experimental study approved by the UGA Animal Care and Use Journal of Herpetological Medicine and Surgery   95

Committee (IACUC No. A2010 11-549-Y3-A1). The tortoises were transported to UGA and temporarily maintained in conditions approved by the Association for Assessment and Accreditation of Laboratory Animal Care. Tortoises were housed individually in 50-gal (192 L) plastic containers (90  cm  ×  70  cm  ×  45  cm), with hay as the substrate, and acclimatized to the research facilities for 7 days prior to anesthesia and surgery. Room temperature was maintained at 26.6°C (80°F). Each animal was provided with a basking heat and broad-spectrum ultraviolet light source (heating lamp, Mercury halide, LR94262, Tin-Yi Metal Manufacturing, Tainan, Taiwan) during the day. The animals were exposed to a 12:12 h light:dark photoperiod. The diet during the study consisted of soaked commercial tortoise pellets (Mazuri® Tortoise Pellets, Mazuri PMI Nutrition International, St. Louis, MO) offered daily and ad libitum water. Each tortoise was physically examined and accurate weights were recorded on the day of arrival. Unique numbers were used to identify each individual. Upon arrival, blood was collected from the jugular vein of each animal and submitted in lithium heparin to measure packed cell volume (PCV) and total solids (TS). Anesthesia: The animals were participating in a parallel anesthesia study with data to be published separately. They were fasted for 48–72 h prior to anesthesia, although access to water was maintained. They were bathed in shallow, lukewarm water for 2 h prior to the procedure to stimulate urination. Manual stimulation of the cloaca was also performed to promote bladder emptying before anesthetic induction. Oxytetracycline (Liquamycin® LA200®, 200  mg/ml, Pfizer, New York, NY) (5  mg/kg IM) and melo­ xicam (Metacam®, 5  mg/ml, Boehringer Ingelheim, Ingelheim am Rhein, Germany) (0.2 mg/kg SC) were administered preoperatively (12–24 h prior to surgery). The animals were physically restrained and induction was achieved using a combination of ketamine (Ketaset®, 100 mg/ml, Fort Dodge, Overland Park, KS) (10 mg/kg) and dexmedetomidine (Dexdomitor®, 0.5 mg/ml, Pfizer) (50 μg/kg) administered intravenously into a jugular vein. Following induction, each animal was placed in a vertical (head up) position with pressure on the site of injection to reduce hematoma formation. The tortoises were randomly assigned to receive either a combination of lidocaine (2 mg/kg, Lidocaine HCl 4%, Hospira, Lake Forest, IL) and preservative-free morphine (0.1  mg/kg, morphine sulfate, 1  mg/ml, Hospira) or an equivalent volume of saline control intrathecally after induction. Jugular vein catheterization was attempted in all animals using a 24-ga intravenous catheter. If successful, 3 ml/kg/h of lactated ringers solution (LRS) was administered intravenously. If unsuccessful, one dose of 25 ml/kg of LRS was administered subcutaneously. Each animal was intubated using an appropriately sized, uncuffed, endotracheal tube secured using a plastic mouth gag (FMG100 plastic mouth gag, PetAg, Hampshire, IL) that was taped in position. The animals were maintained on 100% oxygen for the duration of the procedure. Each tortoise was ventilated using a positive-pressure ventilator (small animal ventilator VT-5000, BAS Vetronics, Bioanalytical Systems, Abbotskerswell, Devon, U.K.) at 2 breaths per minute and at a peak inspiratory pressure of 4–10 cm H2O. If additional anesthesia was required, isoflurane 96  Journal of Herpetological Medicine and Surgery

was administered in oxygen and adjusted to patient requirements. Pulse was monitored with a Doppler ultrasonic flow detector (model 811-B, Parks Medical Electronics, Aloha, OR) placed over the carotid artery. A multiparametric monitor (LW6000 multiparametric monitor, Digicare Biomedical Technology, Boynton Beach, FL) was used to measure end-tidal capnography, esophageal temperature, and the electrocardiogram. Endoscopic equipment: A 2.7 mm (wide view), 30°, 18-cm telescope (64019BA, Karl Storz, Tuttlingen, BW, Germany), endoscopic head camera (22220055-3, Karl Storz), xenon light source (20132101-1, Karl Storz), imaging capture system (22201011U110, Karl Storz ), and 3-mm atraumatic endoscopic forceps with ratchet handle (303530N, Karl Storz) were used in this study. Two monitors were connected to the camera unit controller (SCB image 1 hub 22201020, Karl Storz) to permit coelioscopy from either side of the table and to avoid the need to move the endo­ scopy tower if a bilateral coelioscopic procedure was required. All equipment was cleaned using a neutral pH enzymatic detergent (3E-Zyme, Medisafe America LLC, Tampa, FL) and, initially, was gas-sterilized using hydrogen peroxide. If multiple surgeries were performed on the same day, the equipment was cold sterilized using 2% glutaraldehyde (Cidex®, Ethicon, Cornelia, GA) for 30 min and rinsed with sterile water before repeated use. Surgery: Each tortoise was placed onto a heated surgery table (GMRTP22E, heated water mattress, Gaymar Industries, Orchard Park, NY) maintained at 28–30°C (82.4– 86°F). Each animal was positioned in right lateral recumbency using a vacuum bean bag positioner (SN103526, Natus, Planegg, Germany). Additional elevation of the head above horizontal was undertaken to reduce the cranial movement of intrathecal medication. The hind limbs were extended caudad and taped together to allow exposure of both prefemoral fossae. The left prefemoral fossa and surrounding shell were aseptically prepared and draped using standard techniques. A 5–6 cm craniocaudal skin incision was made in the cranial prefemoral fossa using a no. 15 scalpel blade. The subcutaneous tissues were bluntly dissected and the coelomic membrane (formed by the aponeurosis of the tendinosis parts of the ventral and oblique abdominal muscles) was identified. A 3-mm incision was made in the coelomic membrane to permit insertion of the telescope and confirmation of a correct coeliotomy approach. The coelomic membrane incision was carefully extended, taking care not to damage the often voluminous and closely associated bladder nor the septum horizontale (postpulmonary septum) or lung. A ring and elastic stay retractor (Lone Star retractor, Cooper Surgical, Pleasanton, CA) were positioned to provide improved exposure of the coelom. The animal was then subsequently moved into dorsal recumbency to enable better visualization and removal of the ovaries. If a voluminous bladder was present, cystocentesis was performed after initial coeliotomy to enable optimal ovary visualization. The left ovary, consisting of variably sized follicles, was identified using the telescope. The 3-mm endoscopic Volume 24, No. 3–4, 2014

using 3-0 poliglecaprone 25 on a cutting needle using a simple horizontal mattress pattern. Total surgery time (from start of the left prefemoral incision to the end of last suture on the left or right prefemoral fossa) was recorded. Two authors performed all surgeries, one surgeon at a time (LMP and SJD).

Figure  1.  Coelioscopy-assisted ovariectomy of Mojave Desert tortoises (Gopherus agassizii). The left ovary, consisting of variably sized follicles, has been exteriorized through the incision in the left prefemoral fossa. The ring and elastic stay retractor were used to provide improved exposure of the coelom.

atraumatic forceps with ratchet handle (303530N, Karl Storz) was used to grasp the interfollicular tissue and gently exteriorize the gonad. Once the ovary was elevated to the level of the prefemoral incision, manual manipulation was used to exteriorize the organ (Fig. 1). Care was taken to assure that the entire left ovary was exteriorized from the cavity. The mesovarial vasculature was identified and ligated using medium to large stainless steel vascular clips (LT400 vas­cular clips, Ethicon Endosurgery, Cornelia, GA; 523600 Hemoclips, Teleflex, Research Triangle Park, NC) as necessary. The left ovary was transected, distal to the clips, using Metzenbaum scissors and removed before inspection of the coelom to verify hemostasis. If possible, the right ovary was visualized using the telescope via the same, left prefemoral incision. If the right gonad was visible and accessible, it was removed via the left prefemoral incision as described above. If the gonad could not be visualized or could not be exteriorized via the left prefemoral incision, the surgical site was closed and a right prefemoral approach and right ovariectomy was undertaken using the same techniques described above. The hemisalpingectomy was performed using the 3-mm endoscopic atraumatic forceps with ratchet handle to grasp the oviduct and carefully exteriorize the organ through the prefemoral incision. The vasculature of the mesovaria and mesosalpinx were ligated using the medium to large stainless steel vascular clips before transection. The oviduct was ligated at the level of the vagina and transected. After complete resection of both ovaries, the coelomic cavity was inspected to verify hemostasis before routine closure of the prefemoral fossa. Poliglecaprone 25 (MONOCRYL® Plus; Ethicon, Cornelia, GA) 3-0 suture on a tapered needle was used to close the coelomic aponeurosis and associated subcutaneous tissues in a single layer using a simple continuous pattern. The skin was closed Volume 24, No. 3–4, 2014

Postoperative care: All tortoises received postoperative hydromorphone (Hydromorphone; 2mg/ml, West-Ward, Eatontown, NJ) (0.5 mg/kg IM) for pain management. If present, the intravenous catheter was removed once the tortoise recovered and was ambulatory. The animals were kept in recovery holding pens without substrate, food, or water, for the first 12–24  h postsurgery. Each animal was given additional doses of oxytetracycline (5   mg/kg IM) and meloxicam (0.2 mg/kg SC) daily for 3 days. Incisions were inspected daily for the duration of their hospitalization at UGA (6–16 days postsurgery). Postoperative weights, PCV, and TS were measured before discharge.

Results Median preoperative weight of the eight animals was 5.96 kg (3.0–7.0 kg), median preoperative PCV was 24.5% (20–28%), and median TS was 3.75 g/dl (3.1–5 g/dl). Preoperative physical examination revealed an old plastron fracture (number 6), partial left pelvic limb amputee (number 7), and evidence of previous central plastron and left prefemoral coeliotomy (number 8). No tortoises voided urine despite manual attempts to empty the bladder, but a few animals may have urinated during the bath in lukewarm water, as fecal material was observed in the water. Bilateral ovariectomy was successfully conducted through a unilateral (3/8, 37.5%) or bilateral (5/8, 62.5%) prefemoral approach. Cystocentesis was required after initial coeliotomy in 2/8 (25%) animals. During coelioscopy, two tortoises exhibited small amounts of clear, free coelomic fluid, which was considered a normal finding. However, significant abnormalities were observed during coelioscopy in 6/8 (75%) tortoises (Fig. 2). One tortoise had an ectopic, free-floating follicle within the coelom, accompanied by free coelomic serous fluid. Two tortoises had opaque yellow fluid with lipid droplets within the coelom, consistent with yolk coelomitis. One tortoise presented with evidence of liver disease characterized by yellow discoloration and hepatomegaly. One tortoise had a large amount of free coelomic serous fluid and adhesions between the left oviduct and the body wall at the site of previous left prefemoral surgery. One tortoise presented with a large amount of free coelomic fluid with lipid droplets, the liver exhibited serosal opacity with multifocal, diffuse yellow adhesions, and extensive left ovarian adhesions to the left oviduct were observed; these findings were consistent with previous egg coelomitis. The right ovary did not have any adhesions. Due to the pathological changes, a right ovariectomy and left ovariosalpingectomy were performed as previously described (Mans and Sladky, 2012). All tortoises with coelomic abnormalities were copiously flushed with LRS before closure. Median and mean total surgery time were 112 and 100 min, respectively (81–116   min). Total surgery time of the animal that underwent bilateral ovariectomy and left hemisalpingectomy was 157  min. Only a single surgical Journal of Herpetological Medicine and Surgery   97

Figure 2.  Abnormalities observed during coelioscopy-assisted sterilization of eight female Mojave Desert tortoises (Gopherus agassizii). (a) Presence of opaque yellow fluid with lipid droplets (arrow) within the coelom, consistent with yolk coelomitis; (b) liver exhibiting serosal opacity with multifocal, diffuse yellow adhesions; (c) presence of adhesions between the left oviduct and the body wall (arrow) at the site of previous left prefemoral surgery; (d) presence of ectopic, free-floating follicle within the coelom; (e) rupture of a free-floating follicle occurred during the retrieval attempt, liberating yolk content in the coelom; and (f) presence of extensive ovarian adhesions (arrow) between the oviduct and ovary. Oviduct (O), follicle (F), ruptured follicle (RF).

98  Journal of Herpetological Medicine and Surgery

Volume 24, No. 3–4, 2014

complication was encountered in one tortoise with coelomitis. During gentle exteriorization of the left ovary, the interfollicular tissue tore and a follicle fell into the coelom. During retrieval, the follicle ruptured. The egg yolk contents were aspirated from the coelomic cavity when it was copiously flushed with lukewarm LRS. All tortoises recovered from anesthesia uneventfully. No significant blood loss was noticed. Median postoperative weight of the eight animals was 5.48 kg (2.7–6.3 kg), median postoperative PCV was 23% (17–28%), and median total solids (TS) was 3.0 g/dl (2.5–3.8 g/dl). The animal with a postoperative PCV of 17% had a preoperative PCV of 22%. All tortoises remained healthy 7 mo postoperatively.

Discussion Results for the eight surgeries described in this study suggest that coelioscopic-assisted ovariectomy and ovariosalpingectomy are practical and safe methods for sterilization in reproductively healthy and diseased tortoises. Similar to previous studies, this technique did not require the placement of an endosurgical cannula because the prefemoral incision was large enough to allow exteriorization of the ovary and introduction of the telescope and forceps through a single surgical incision (Innis et al., 2007). Although non-endoscopic bilateral techniques have been suggested for elective ovariosalpingectomy (Minter et al., 2008), it is difficult to assure the absence of reproductive disease prior to surgery based only on the physical exam. Additionally, non-endoscopic techniques may limit one’s ability to describe internal structures, or pathology and diseases might be missed during surgery. Reproductive diseases may vary greatly depending upon the underlying cause and duration of the disorder. Chelonians can be bright and alert or depressed and lethargic. Similarly, appetite can remain normal or the animal may become anorexic or hyporexic (Mans and Sladky, 2012). In the present study, just 3/8 (37.5%) tortoises had abnormal physical exam findings, with only one of them possibly related to a reproductive disorder (number 8 had evidence of a previous central plastron and left prefemoral coeliotomy; it was later confirmed to have reproductive disease at coelioscopy). Despite the unremarkable physical examinations in the remaining animals, significant abnormalities were observed during coelioscopy in 6/8 (75%) tortoises. The findings in the present study corroborate with pre­ vious publications where coelioscopic-assisted ovariectomy and ovariosalpingectomy were successfully performed in cases of reproductive disease in red-eared slider turtles (Trachemys scripta elegans) and Galapagos tortoises, where oviductal rupture, impaction, yolk coelomitis, ectopic eggs (Trachemys), severe coelomitis, and retained and ectopic eggs (Geochelone) were present. Bilateral prefemoral coeliotomy was sufficient for ovariosalpingectomy, egg removal, and coelomic lavage without any short or long-term com­ plications (Knafo et al., 2011; Mans and Sladky, 2012). Coelioscopic-assisted ovariectomy or ovariosalpingectomy were also successfully performed in 11 adult turtles belonging to five species (Trachemys scripta, Terrapene carolina, Chrysemys picta [painted turtle], Sacalia bealei [Beale’s eyed turtle], and Chinemys kwangtungensis [red-necked pond turtle]), where 5/11 (45.4%) presented with reproductive tract disease. Two (18.2%) of the 11 animals (T. carolina) Volume 24, No. 3–4, 2014

died a few days following surgery of causes unrelated to the procedure. Mean total surgery time of 100 min was longer than pre­ viously described in 12 healthy mature Galapagos tortoises (mean: 36.8 min). However, contrary to the present study, bilateral coelioscopic-assisted ovariectomies were conducted through a bilateral prefemoral approach by two surgeons, simultaneously. Nevertheless, prolonged surgery time of 100 min was recorded in one female Galapagos tortoise due to extensive reproductive disease (Knafo et al., 2011). A shorter mean surgery time of 36 min was also recorded for bilateral coelioscopic-assisted ovariectomies through a unilateral prefemoral approach in 11 turtles (Trachemys scripta, Terrapene carolina, Chrysemys picta, Sacalia bealei, and Chinemys kwangtungensis), with 5/11 (45.4%) animals presenting with reproductive tract disease (Innis et al., 2007). The present study further supports the claim that endoscopic assistance facilitates safe, effective, and minimally invasive prefemoral sterilization of chelonians (Innis et al., 2007). The coelomic lesions found in this study were unexpected and could not have been diagnosed and treated via prefemoral coeliotomy without the aid of endoscopy. Surgical complications such as follicle rupture are possible, even in healthy animals due to the size and fragility of their large follicles, and could easily be missed or more difficult to resolve without endoscopy. In the authors’ opinion some surgical conditions, including sterilization of reproductively active females, follicular stasis, dystocia, ectopic eggs or follicles, salpingitis, oviductal perforation– impaction, and egg coelomitis should not be performed under restricted visualization due to the risk of surgical complications such as rupture, hemorrhage, coelomitis, and ectopic eggs or follicles.

Conclusion Results for the eight surgeries described in this study suggest that coelioscopic-assisted ovariectomy and ovariosalpingectomy are practical and safe methods for sterilization in reproductively healthy and diseased tortoises. The magnification and optimal visualization provided by coelioscopy were considered critical for success. Acknowledgments: Supreme Pet Foods provided financial support for Dr. Proenca’s residency and also helped fund this research study. Major funding for the study was provided by Clark County, Nevada. Karl Storz Veterinary Endoscopy Inc. provided equipment support for research and development. Thanks also to Lynn Reece, Emily Garber, and Rob Miller for technical assistance.

Literature Cited Innis CJ, Boyer TH. 2002. Chelonian reproductive disorders. Vet Clinics N Am-Exotic An Pract, 5(3):555–578. Innis C, Hernandez-Divers SJ, Martinez-Jimenez D. 2007. Coelioscopic-assisted prefemoral oophorectomy in chelonians. J Am Vet Med Assoc, 230(7):1049–1052. Johnson AJ, Morafka DJ, Jacobson ER. 2006. Seroprevalence of Mycoplasma agassizii and tortoise herpesvirus in captive desert tortoises (Gopherus agassizii) from the Greater Barstow Area, Mojave Desert, California. J Arid Environ, 67(Suppl.):192–201. Journal of Herpetological Medicine and Surgery   99

Knafo SE, Divers SJ, Rivera S, Cayot LJ, Tapia-Aguilera W, Flanagan J. 2011. Sterilisation of hybrid Galapagos tortoises (Geochelone nigra) for island restoration. Part 1: endoscopic oophorectomy of females under ketamine-medetomidine anaesthesia. Vet Rec, 168(2):47. Mans C, Sladky K. 2012. Diagnosis and management of oviductal disease in three red-eared slider turtles (Trachemys scripta elegans). J Small An Pract, 53(4):234–239. Minter LJ, Landry MM, Lewbart GA. 2008. Prophylactic ovariosalpingectomy using a prefemoral approach in eastern

100  Journal of Herpetological Medicine and Surgery

box turtles (Terrapene carolina carolina). Vet Rec, 163(16): 487. Rivera S, Divers SJ, Knafe SE, Martinez P, Cayot LJ, TapiaAguilera W, Flanagan J. 2011. Sterilisation of hybrid Galapagos tortoises (Geochelone nigra) for island restoration. Part 2: phallectomy of males under intrathecal anaesthesia with lidocaine. Vet Rec, 168(2):78. United States Fish and Wildlife Service (USFWS). 1994. Desert tortoise (Mojave population): Recovery plan, U.S. Fish and Wildlife Service, Region 1, Lead Region.

Volume 24, No. 3–4, 2014