Materials and Methods

Animals—Fifty-eight hatchling Chinese box turtles (Cuora flavomarginata) produced by a captive breeding program in New York as part of the IUCN Turtle Survival Alliance chelonian conservation program were used in the study. Following completion of the study, turtles were returned to the breeder and subsequently distributed to other breeding programs in the United States.

For the present study, turtles were individually marked by means of a carapace notching system, placed in separate plastic cups, and transported overnight in foam boxes to the University of Georgia. At the university, turtles were physically examined and placed in 4 plastic temporary housing containers measuring 75 × 45 cm. Each container was filled to a depth of 0.5 cm with aged tap water and placed on a 10° slope to create wet and dry areas. The room was maintained at 24°C (75°F), but temperature was increased to 29°C (84°F) with broad-spectrum mercury halide incandescent lamps for 12 hours each day. Turtles were fed a complete pelleted ration.^a

Anesthesia—Food was withheld for 24 hours prior to anesthesia and surgery. Turtles were transported in groups of 15 to the preoperative area, which was maintained at a temperature of 24°C. Turtles were weighed and randomly assigned to a general anesthesia or local anesthesia group. For turtles in the general anesthesia group, anesthesia was induced with a mixture of morphine^b (1.5 mg/kg [0.68 mg/lb]), ketamine^c (10 mg/kg [4.54 mg/lb]), and medetomidine^d (0.1 mg/kg [0.045 mg/lb]). Drugs were diluted 1:10 with sterile water, combined into a single syringe, and delivered by means of IM injection with a 27-gauge needle in the pectoralis major muscle of the left pectoral limb. Following injection of the morphine-ketamine-medetomidine mixture, turtles were placed in an incubator^e maintained at 29°C for 30 minutes prior to surgery. Turtles were not intubated or instrumented.

For turtles in the local anesthesia group, a local anesthetic mixture was made by combining lidocaine^f (0.50 mL of a 20 mg/mL solution), sodium bicarbonate^g (0.12 mL of an 84 mg/mL solution), and sterile water (19.38 mL) to produce a solution with a lidocaine concentration of 0.5 mg/mL. Lidocaine (1 mg/kg [0.45 mg/lb]) was injected SC into the left prefemoral area 5 minutes before surgery.

Endoscopy—Endoscopy was performed with a 1.9- or 2.7-mm rigid 30°-viewing telescope housed within a protective sheath.^h Equipment was sterilized by immersion in 2.4% alkaline glutaraldehyde solutionⁱ for 15 minutes and was rinsed with sterile water before use.

Turtles were manually restrained in right lateral recumbency with the left pelvic limb retracted caudad to expose the left prefemoral fossa (Figure 1). The surgery site was prepared with chlorhexidine,^j and a 2- to 3-mm-long craniocaudal skin incision was made with a No. 15 scalpel blade in the center of the prefemoral fossa. Small straight mosquito hemostats inserted through the incision were directed craniad and gently advanced until the coelomic aponeurosis was penetrated. The sheathed telescope was then inserted into the coelom. A 12-mL syringe containing sterile lactated Ringer's solution was attached to a port on the sheath, and 1 to 3 mL of Ringer's solution was used to insufflate the coelomic cavity. The telescope was directed toward the dorsocaudal region of the coelom to inspect the reproductive system. Gonads were identified as testes, ovaries, or unknown, and gross appearance of the reproductive system was recorded as normal or abnormal. Ease and speed of endoscopic sex identification were scored on a scale from 1 to 4, where 1 = excellent visualization obtained without any difficulties, requiring < 1 minute to identify sex; 2 = good visualization obtained with minor difficulties, requiring 1 to < 3 minutes to identify sex; 3 = poor visualization obtained with major difficulties, requiring 3 to < 10 minutes to identify sex; and 4 = impossible to identify sex, and procedure discontinued after 10 minutes. Quality of anesthesia during surgery was also scored on a scale from 1 to 4, where 1 = excellent anesthesia without any movement, 2 = good anesthesia with 1 or 2 spontaneous movements, 3 = fair anesthesia with 3 to 5 spontaneous movements, and 4 = poor anesthesia with > 5 spontaneous movements or constant struggling.

View Full Size

Photograph illustrating positioning and restraint of a Chinese box turtle undergoing prefemoral coelioscopy.

Citation: Journal of the American Veterinary Medical Association 234, 6; 10.2460/javma.234.6.800

• Download Figure
• Download figure as PowerPoint slide

At the end of the procedure, excess fluid was aspirated from the coelomic cavity. The skin incision was closed with a liquid tissue adhesive.^k

Recovery and postoperative care—Following endoscopic sex identification, all turtles were given a single dose of meloxicam^l (0.2 mg/kg [0.09 mg/lb], IM in the crureus muscle of the right pelvic limb). In turtles that underwent general anesthesia, anesthetic effects were reversed with atipamezole^m (0.5 mg/ kg [0.23 mg/lb], IM in the pectoralis major of the right pectoral limb) and naloxoneⁿ (0.2 mg/kg, IM in the crureus muscle of the left pelvic limb). Recovery time was defined as the period from administration of reversal agents to first spontaneous ambulation.

Turtles were monitored constantly until recovery and 10, 30, and 60 minutes after they were returned to their holding enclosures. Turtles were reexamined the day after surgery and transported back to the Turtle Survival Alliance. Body weight was measured 7 and 14 days after surgery.

Statistical analysis—Endoscopy scores, anesthesia scores, recovery times, and body weights were summarized (Table 1). Data were analyzed for normality by use of Shapiro-Wilk tests. Data for body weight and weight gain were normally distributed, and values were compared between anesthesia groups by means of Student t tests. Recovery times and endoscopy and anesthesia scores were not normally distributed and were compared between groups by means of Wilcoxon signed-rank tests. All analyses were performed with standard software.^o Values of P < 0.05 were considered significant.

Results

Carapace length prior to surgery ranged from 41 to 70 mm, and body weight prior to surgery ranged from 12 to 58 g. In all 58 hatchlings, the gonads were located and identified as testes or ovaries (Figure 2). Four of the 58 hatchlings were identified as males, and 54 were identified as females. The reproductive system was recorded as grossly normal in 57 of the 58 hatchlings. One female appeared to have a mass or cyst within or adhered to the oviduct and closely associated with but not involving the ovary (Figure 3). In most turtles, the left liver lobe, stomach, small intestine, large intestine, left lung, left kidney, and left adrenal gland could be seen in addition to the reproductive tract.

View Full Size

Endoscopic appearance of the reproductive tract in male (a, b, and c) and female (d, e, and f) hatchling Chinese box turtles. a—Endoscopic appearance of the testis (T). Notice the spatial relationship of the testis to the kidney (K), caudal lung lobe (L), and colon (C). b—Close-up view of the testis illustrating the smooth surface and surface vasculature. c—Close-up view of the vas deferens (V) coursing caudad to the testis (T). Notice the spatial relationship of the vas deferens to the kidney (K) and colon (C). d—Endoscopic appearance of the ovary (O) and oviduct (D). Notice their spatial relationship to the kidney (K), adrenal gland (A), and colon (C). e—Closeup view of the ovary illustrating the follicular appearance. f—Close-up view of the oviduct (D) illustrating its spatial relationship to the ovary (O) and kidney (K).

Citation: Journal of the American Veterinary Medical Association 234, 6; 10.2460/javma.234.6.800

• Download Figure
• Download figure as PowerPoint slide

View Full Size

Endoscopic appearance of the reproductive tract in a female hatchling Chinese box turtle with a mass (M) or cyst within or adhered to the oviduct (D) and closely associated with but not involving the ovary (arrow) or colon (C). a—Overview of the mass. b—Close-up view of the caudal aspect of the mass.

Citation: Journal of the American Veterinary Medical Association 234, 6; 10.2460/javma.234.6.800

• Download Figure
• Download figure as PowerPoint slide

For turtles that underwent general anesthesia, median recovery time was 6 minutes (range, 0 to 21 minutes). Endoscopy scores, body weight, and weight gain were not significantly different between groups (Table 1). However, anesthesia scores were significantly (P < 0.001) worse for turtles that received local anesthesia alone, compared with turtles that underwent general anesthesia. Two of the 29 (7%) turtles that underwent general anesthesia vomited after naloxone and atipamezole were administered. In 2 of the 58 (3%) turtles, the skin incision required resealing with additional tissue adhesive within 30 minutes after recovery. All wounds healed without further complications.

Discussion

Results of the present study suggested that use of a rigid endoscope following insufflation with lactated Ringer's solution was a safe and effective method for visualizing the gonads and identifying the sex of hatchling Chinese box turtles. No anesthetic complications were encountered in turtles anesthetized with a combination of ketamine, medetomidine, and morphine, and anesthesia scores were significantly better for turtles that underwent general anesthesia than for turtles that received only local anesthesia. In turtles that received local anesthesia, movement and struggling were generally not seen in association with the initial skin incision but rather during penetration of the coelomic cavity and manipulation of the telescope within the cavity. Assuming that movement and struggling were primarily responses to discomfort or pain, we conclude that local anesthesia with lidocaine provided inadequate visceral analgesia during coelioscopy and cannot be recommended.

All turtles that were anesthetized in the present study recovered with spontaneous ambulation within 21 minutes after administration of reversal agents. This was shorter than recovery times reported for loggerhead sea turtles (Caretta caretta) anesthetized with propofol.²³ Because naloxone antagonizes the analgesic properties of opiates, we believed that administration of meloxicam was necessary for postoperative analgesia. Vomiting has been documented as an adverse effect of atipamezole administration and was observed in 2 of 29 (7%) turtles in the present study.²⁴ Free-ranging aquatic animals should not be released or permitted access to water until fully recovered, and all turtles in the study were behaving normally within 2 hours after surgery.

In the present study, we did not detect any significant difference in weight gain 7 and 14 days after surgery between the 2 groups. Ideally, we would have evaluated body weight periodically up to 8 weeks after surgery, as wound healing is typically not complete until this time. However, this was not possible because many of the turtles were shipped to other institutions before this time. Although hatchlings used in the present study were small, requiring that drugs be diluted prior to use, our findings suggest that previous concerns regarding the safety, efficacy, and adverse effects of general anesthesia in turtle hatchlings are unjustified. Given the poor anesthesia scores for turtles that received local anesthesia alone, the rapid and reliable reversibility of medetomidine and morphine following administration of atipamezole and naloxone, and the lack of differences between groups in regard to postoperative weight gain, we conclude that general anesthesia should be used for all turtles undergoing coelioscopy. In clinical practice, conditions that preclude the use of general anesthesia would also preclude the use of coelioscopy.

The insufflation technique used in the present study was simple and inexpensive and did not require a source of compressed CO₂ gas or the use of an insufflator. Injection of sterile lactated Ringer's solution created a fluid working space within the coelom and provided excellent visualization. Subjectively, it also allowed a more extensive examination of the reproductive tract, particularly of the vas deferens and oviduct, and provided better image quality than coelioscopy without insufflation.^11,25 Previous authors have expressed concerns regarding the use of insufflation in aquatic species because of the potential for abnormal buoyancy secondary to residual gas in the coelom.²³ However, lactated Ringer's solution has a specific gravity of approximately 1.005 and an osmolarity comparable to that of reptile plasma, is 800 times as dense as air, and can safely be used within the coelom without affecting buoyancy. Care must be taken to avoid overdistension of the coelom, which could prevent venous return or cause pressure-related tissue damage. We found that this could be prevented by maintaining only a loose seal between the sheathed telescope and skin, allowing excess fluid to simply drain out of the incision. Prior to skin closure, excess coelomic fluid can be aspirated or simply allowed to drain by rotating the turtle until the incision becomes dependent.

The nature of the oviductal mass observed in 1 female turtle in the present study was not determined. Although the turtle did not have any clinical signs attributable to the mass, we recommended that it not be released or used for captive breeding. Endoscopic biopsy can be used to obtain a histologic diagnosis in larger animals but is likely to be associated with greater risks in hatchlings.^15,26 Ultrasonography can be used to evaluate the reproductive tract of turtles,²⁷ but in our experience, coelioscopy was preferred for differentiating between testes and ovaries in immature animals.