The use of oxytocin to induce oviposition in turtles is an important technique that has been in use for many years for turtle research (Ewert and Legler 1978) and as a veterinary tool to treat egg retention (Glassford and Brown 1977). Extensive reproductive data can be collected without undue injury to the turtles using oxytocin (e.g., Iverson and Smith 1993; Rowe 1995; Tucker et al. 1998). However, some turtles continue nesting attempts once their eggs have been oviposited (Iverson and Smith 1993; Tucker et al. 1995; Feldman 2007).

In the present paper, we re-examine the dosage needed to reliably induce oviposition in red-eared sliders (Trachemys scripta elegans). Oxytocin dosage has not previously been examined experimentally in turtles as it has been for swine (Mota-Rojas et al. 2005).

Methods

Many details on methods were previously presented by Tucker et al. (1995, 1998) and were largely modifications of the methods described by Ewert and Legler (1978). The current experiments were conducted during further collection of reproductive output data in turtles and used similar methods. Thus, we only discuss details not previously described. The experimental portion of the study was performed in 1999 and 2000. We also report follow-up results for turtles collected between 2000 and 2005 using the minimal dose.

We used the same apparatus to hold females once they were injected in all years of the study. This apparatus consisted of a plastic Rubbermaid brand tub. We used 1 of 3 styles differing only in capacity (i.e., 37.9, 53.0, and 68.1l) but similar in length and width (60 × 40 cm, respectively). Into each tub, we placed wire mesh fencing (mesh size = 10 × 5 cm) that had been cut to size and then bent into a platform of about the same length and width as the bottom of the tub (Fig. 1). Two perpendicular wire mesh segments about 7.5 cm tall served to elevate the platform above the tub bottom. When inserted into the tub, the platform fit snugly against the sides of the tub and kept the turtle about 7.5 cm above the bottom of the tub (Fig. 1).

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Sufficient tap water was inserted into the tub to cover the turtle to about midcarapace level. All turtles were initially injected intramuscularly in the right thigh. If re-injection was needed, turtles were then re-injected in the left thigh. We preferred intramuscular (IM) injection in contrast to intraperitoneal (IP) injection because, in general, IM is the method used in many veterinary studies (e.g., DeNardo 1996; Hoggard 2000; Mota-Rojas 2005). Moreover, it is much simpler to correctly administer even for a relatively untrained individual. We observed no signs of nerve or local tissue damage in the nearly 3000 turtles subsequently injected IM. There is no possibility of accidentally injecting an egg or the bowel if the oxytocin is given IM. Such accidents will occur when using IP.

Large active species such as the common snapping turtle (Chelydra serpentina) and smooth softshell turtle (Apalone spinifera) can be particularly difficult for IP injections. Finally, turtles with plastral hinges such as the eastern box turtle (Terrapene carolina carolina) can be easily injected in the thigh but can be difficult to inject IP without injury to the turtle or her eggs. At present there are no experimental studies comparing IP or IM methods. However, it seems that the high success rates achieved in this study with IM methods would be difficult to surpass using IP injections (but see Feldman 2007, for a different opinion).

After injection with oxytocin, the turtle was positioned on the platform and a perforated tub lid placed to prevent escape of the turtle. The turtle was then allowed 7–12 hours to lay her eggs. As eggs were laid, they fell through the mesh and into the water (Fig. 1). At the bottom of the tub they were protected from the sharp claws of the female on the platform. Newly laid eggs seemed little affected by immersion for this period of time (Tucker et al. 1997). All females were then palpated to determine if any eggs remained (see Tucker et al. 1998).

Females thought to have retained more than 2 eggs were scored as failures, whereas those that appeared to have retained 2 or fewer eggs were scored as successes. Turtles scored as failures were re-injected and returned to the tubs and re-examined 7–12 hours later to see if eggs had been laid. They were re-palpated and scored as above. All reinjected turtles that produced their remaining eggs were also scored as successes. Thus, turtles scored as successes could have received 1 or 2 injections. No turtle was injected a third time.

The oxytocin that we used in our experiments was manufactured by Phoenix Pharmaceutical, Inc. (St. Joseph, MO) and contained 20 USP units/mL. We examined dosage using T. scripta elegans in 1999 comparing 4 oxytocin dosages, 20 , 10, 5, and 2 units/kg.

In 1999, a series of sequential comparisons were made. First, we compared 20 and 10 units/kg using 28 females, 13 of which received the higher dosage and 15 the lower, all injected on 19 June 1999. Second, we compared 10 to 5 units/kg using 23 females for both the higher dosage and the lower dosage. These turtles were induced on 23 and 25 June 1999. The third comparison was made among 3 dosages (10, 5, and 2 units/kg) in turtles induced between 1 and 9 July 1999. Twelve turtles were given 10 units/kg, 12 turtles were given 5 units/kg, and 16 turtles were given 2 units/kg. Data from comparisons using similar dosages were combined for further analysis.

Between 13 May and 3 July 2000, we studied an additional 146 turtles. We continued the experiment using 2 dosages (4 units/kg and 2 units/kg) in a series of 49 turtles collected between 13 and 25 May 2000. The 4 units/kg dosage was used rather than 5 units/kg dosage from 1999 comparisons because it simplified dosage calculation. We used the 4 units/kg dosage for re-injection of turtles scored as failures after initial injections of 2 units/kg. We then used the 4 units/kg dosage for an additional 97 turtles collected between 25 May and 3 July 2000. We classified success or failure as for 1999 turtles and used the same methods as used in 1999 for re-injections. In both years, prior to release, all turtles were held for 48 hours after their last injection (Tucker et al. 1995).

Finally, we report results for all turtles induced with the final dosage (i.e., 4 units/kg) as a follow-up to the experiment. These turtles included 2669 red-eared sliders, 16 stinkpots (Sternotherus odoratus), and 24 painted turtles (Chrysemys picta). Including these allowed us to compare efficacy among species and between localities for the sliders.

All turtles were collected at nesting areas and no trapped turtles are included in this study. Collecting localities include Swan Lake in Calhoun County, Illinois, and the Stump Lake complex in Jersey County, Illinois (see Tucker 2001). Comparisons of frequency data were made with the chi square using the frequency procedure in SAS.

Results

Results for 1999 and 2000 are combined for analysis but shown separately in Table 1. The 5 dosages used differed significantly in the number of initial successes versus failures (χ² = 20.0, 4 df, p = 0.0005). These 5 dosages also differed significantly in the overall success rates following up to 2 injections when needed (χ² = 9.49, 4 df, p = 0.05). However, if the dosages less than 5 units/kg were removed, the other 3 dosages (20, 10, and 5 units) did not differ statistically in efficacy (χ² = 4.81, 2 df, p = 0.0902). Dosage used in 1999 suggested that 5 units/kg was as effective at inducing oviposition as were the higher dosage rates (Table 1). Moreover, we had more than 97% success using 5 units/kg following re-injection of failures.

Table 1. Comparison of the efficacy of different dosages (in USP units/kg of gravid mass) of oxytocin in inducing oviposition in red-eared sliders (Trachemys scripta elegans).

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In 2000, the higher dosage (4 units/kg) had a higher success rate than did the lower dosage (2 units/kg) (χ² = 7.06, 1 df, p = 0.0081). Overall success rates following re-injection of females with 4 units/kg were nearly identical between the higher and lower dosage (Table 1). The overall success rates following re-injections among the turtles given either 4 units/kg or 2 units/kg did not differ statistically (χ² = 0.102, 1 df, p = 0.7494).

In the follow-up study, we found no difference in response to 4 units/kg among the 3 species for which we had sufficient turtles (χ² = 0.1132, 2 df, p = 0.9450). Overall success rates were 94.3% for sliders, 93.8% for stinkpots, and 95.8% for painted turtles. We also found no difference in response to oxytocin between sliders collected at Swan Lake and those from the Stump Lake complex (χ² = 1.46, 1 df, p = 0.2262). Overall success rates were 93.5% and 94.7%, respectively. These success rates are similar to those we observed in the smaller experimental samples using the 4 unit/kg dose.

Discussion

Ewert and Legler (1978) recommended a dosage of 1–4 units/100 g body mass for smaller turtles and 0.5–1 unit/100 g body mass for larger turtles. They did not specify what constituted a large or small turtle. We, however, believe that the turtles we examined would likely be considered as small turtles. Thus, they recommended a dosage of 10–40 units/kg of body mass (Ewert and Legler 1978). For a brand of oxytocin containing 20 units/mL, the Ewert and Legler (1978) dosage requires 0.5 to 2 mL of solution per kilogram of body mass. Dosages in this range performed well in our experiments and resulted in 100% success in the 62 turtles we used it on.

We found that smaller dosages were equally effective as larger ones particularly once failures were re-injected (Table 1). Our study is an experimental one and we were able to statistically test the results. Moreover, we are able to report results from a large number of follow-up cases. Previous dosage recommendations (e.g., DeNardo 1996; Hoggard 2000) are based on empirical estimates of dosages. The difference in approach (experimental versus empirical) is likely the reason we found lower dosages as effective than the higher dosages recommended by other papers. Our experimental results were also arrived at using wild caught females that were physiologically ready to oviposit. Captive turtles or turtles retaining eggs may have different responses. However, our experiments give a good estimate of an initial dosage.

If the aim is to reduce oxytocin dosage as much as possible, we recommend that initial injections be made at 2 units/kg with re-injections made at 4 units/kg. Turtles that we used this dosage schedule on had an overall success rate of 92.0%. However, the relatively high failure rate in initial injections with 2 units/kg may make this lower dosage unacceptable when the objective is to return the turtle to the wild as quickly as possible. Retention in the laboratory for extended periods may affect internesting intervals (Iverson and Smith 1993). Re-injection adds at least 12 hours to the time in captivity. In studies where returning the turtle to the field quickly is important, we recommend the 4 units/kg dosage where initial success rate was high (Table 1). However, where the goal is to reach the highest success with a single dosage, then 10 units/kg may be the dosage of choice.

For our research purposes, we defined success as being able to determine clutch size with a reasonable degree of accuracy (see Tucker 2007). This requires the turtle to lay all or nearly all of her eggs with no more than 2 being retained. For veterinary treatment of egg retention success may require that all eggs be laid as the female cannot be released. In our studies females retaining eggs were released and many of these have been found laying their remaining eggs at the nesting areas. For captives, one of the higher dosages may give more satisfactory results especially where complete oviposition of eggs is essential. We had 100% success using 10 and 20 units/kg in a series of 2 injections.

Even at these higher dosages turtles that do not respond to the first 2 injections seem not to respond well or at all to subsequent injections. Hoggard (2000) reported a case that required 5 injections over a 26-day period for a captive yellowbelly slider (T. scripta scripta) to produce her entire 13 egg clutch.