Appearance of Gonads and Accessory Ducts

The gonads of small juvenile Aldabra tortoises are thin and elongate and fixed to the dorsal part of the body cavity, very close to the kidneys, adrenal glands, and lungs. The gonads and other organs (oviduct, kidney, adrenal, lung) can generally be viewed directly or sometimes through translucent peritoneal membranes such as the mesentery. Despite being attached to the dorsal coelomic wall by various membranes, gonads and reproductive tracts can change their position relative to the kidneys, adrenals, and lungs (which have more or less fixed positions), for example, when turtles are tilted from one side to the other during endoscopy.

Testes of small tortoises appear as small, transparent, thin, half-roundish sausage-like structures, bound to the kidneys by the mesorchium and with a net of fine blood vessels on the surface (Fig. 1A). With growth, testes become thicker but remain half-roundish sausage-like structures ventral to the kidneys, turning first pinkish-white and then yellowish with tubular structures visible through a thin, transparent theca containing a network of fine blood vessels (Fig. 1B) but no melanocytes. Epididymes and vas deferens in small juveniles are thin and translucent, difficult to locate, and not discernible in the photographs.

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Ovaries are attached by a transparent peritoneum to the dorsal wall of the coelomic cavity or to a membrane that separates them from the lungs. Ovaries of small tortoises appear as transparent flat sheaths ventral to the kidneys, with some oocytes and primary follicles visible (Fig. 1C). With growth, ovaries expand along the dorsal wall of the coelomic cavity ventral to the lungs and increase in thickness, and masses of previtellogenic follicles appear on the surface (Fig. 1D, E). The oviducts extend further cranially than the ovaries and are ventral or lateral to the ovaries. They often cross ventrally over the posterior part of the ovary on the way to the cloaca. Oviducts of small females are relatively thin, transparent-whitish, straight bands (Fig. 1C, D). With growth, oviducts become wider and thicker and more whitish (Fig. 1E) but still remain straight in the size classes examined during this study.

Intersex Conditions

Three tortoises of 106 (2.8%; 900, 950, and 2700 g body mass) had well-developed testes as well as ovaries and oviducts on both the left and right side of the body cavity and were classified as intersexes (Fig. 1F–I). The ovaries were comparatively thinner and smaller than in normal females of similar size (e.g., Fig. 1D). Although transparent to whitish (previtellogenic) follicles were clearly visible in the ovaries, their density appeared to be slightly reduced compared to normal females. Oviducts appeared to be comparable to those of normal females. In the tortoises classified as intersexes, the size and development stage of the testes was more or less similar to normal males of comparable size (compare Fig. 1B with Fig. 1H).

Nine juveniles with well-developed ovaries, which were classified as females, also had small patches of testicular tissue on the left side of the abdominal cavity (11% of the females, body mass 815 ± 655 g, range 263–2400 g). However, repeated endoscopy of four of those cases detected in 2000 and 2004 (initial body mass 516 ± 169 g, range 263–732 g) 4 yrs later (body mass now 6075 ± 1487 g, range 3700–7800 g) revealed that, with further growth and development, these tortoises had turned into normal females (testicular tissues no longer detectable). For this reason, all individuals showing this condition were classified as females and not considered to represent intersexes.

Sex Ratios

Of the 106 tortoises sexed endoscopically, 82 were females (77.4%), 21 were males (19.8%), and 3 were classified as intersexes (2.8%; see below). The overall male to female sex ratio of captive bred Aldabra tortoises at La Vanille was 1∶3.9.

Appearance of Gonads and Accessory Ducts

This study provides the first description and endoscopic images of testes and ovaries of hatchling and juvenile giant tortoises (family Testudinidae). Endoscopy has been successfully used before to sex captive-raised juvenile desert tortoises Gopherus agassizii with a size range of 28–1250 g body mass (Rostal et al. 1994), but this paper as well as Kuchling and López (2000) for A. yniphora did not provide endoscopic images and did not describe the morphology of gonads and accessory ducts with changing age or body size. Gonads of juvenile tortoises of other species were only recently studied: testes in juvenile A. radiata (Kuchling et al. 2012) and Geochelone platynota (Kuchling et al. 2011) have a similar morphology to that of juvenile Aldabra tortoises, but in hatchling and juvenile G. agassizii, testes appear as bright yellow, long, flat bands (Rostal et al. 1994). This demonstrates some variability in the appearance of juvenile testes in the family Testudinidae. Thus, far, only a few published papers have provided endoscopic images of juvenile testes and ovaries of species in other turtle families, which can be used as guidance to identify gonads and sex (Podocnemididae, Kuchling 2006; Cheloniidae, Wyneken et al. 2007; Trionychidae, Kuchling and Kitimasak 2009). In all those families and species, the juvenile chelonian testis can be defined by the following characteristics: it is half-round in cross-section or flat; the theca testis is always thin and translucent; and tubular structures of different size and color (transparent, white, pinkish, or yellow) or a fine net of surface vasculature is visible in close-ups (e.g., Fig. 1B, F–I)

Unfortunately, in published endoscopic images of juvenile Cuora flavomarginata (Geoemydidae, Hernandez-Divers et al. 2009), unrelated structures appear to have been misidentified and mislabeled as testis and vas deferens (Kuchling 2009), probably erroneously. It should, however, be noted that the morphological principles and concepts of the juvenile chelonian testis described above appear not to be universally accepted: Divers and Stahl (2009) re-affirmed that the juvenile “testis” of C. flavomarginata is a round and pendulous structure (not half-round or flat in cross-section) with a robust, whitish external membrane with spots of melanocytes (not thin and translucent) and without a net of fine blood vessels, and no tubular structures visible in the medulla. Veterinarians Sam Rivera of Zoo Atlanta, Charles Innis of New England Aquarium, and Eric Baitchman of Zoo New England confirmed independently that the identification of this structure as testis is accurate (Divers and Stahl 2009). This represents a completely different and novel morphological concept of a chelonian testis from that presented in the present paper and in other studies (Rostal et al. 1994; Kuchling 2006; Wyneken et al. 2007; Kuchling and Kitimasak 2009; Kuchling et al. 2011, 2012).

Intersex Conditions

An interesting finding of this study is that three tortoises were classified as intersexes. Intersex has only been found once before in a tortoise (A. yniphora, Kuchling and López 2000) but without any details having been described. Intersex conditions are better known in the more extensively studied sea turtles; Limpus et al. (2009) described an adult intersex Chelonia mydas in a Queensland population, which, as assessed by endoscopy, had ovaries with large mature-sized vitellogenic follicles, a fully developped convoluted oviduct, as well as large, mature testes in advanced spermatogenesis but no enlarged epididymes. This demonstrates that gonads of both sexes can persist side by side into adulthood and mature. Unfortunately, the authors did not publish any images of the appearance of the gonads during endoscopy. Without discussing the size and maturity status of the relevant turtles, Limpus et al. (2009) summarized that visual examination of sea turtle gonads by endoscopy in eastern Australia revealed a total of 5 intersex turtles out of 1514 Caretta caretta (0.33%), 4 intersexes out of 11,127 C. mydas (0.036%), and 3 intersexes out of 430 Eretmochelys imbricata (0.7%). Several of these intersex turtles had their gonads re-examined over periods of up to 15 yrs, but no evidence was found that they changed over time into either male or female.

A difference between the sea turtle studies and the present study is that Limpus et al. (2009) examined the gonads of mainly large juvenile, subadult, and adult sea turtles by endoscopy, whereas hatchlings (less than a year old) and small juveniles were examined in the present study. In most of the intersex sea turtles, the gonads were ovi-testes with seminiferous tubules in the medulla and scattered small previtellogenic follicles in the cortex (Limpus et al. 2009). In this study of much younger tortoises with generally less-developed gonads, the term intersex was only used if discrete male and female gonads were found side by side (Fig. 1F–I). The small areas of testicular tissue in otherwise normally developed ovaries found in 9 small Aldabra tortoises disappeared with further growth and development in the 4 that were reexamined; they had apparently developed into normal females. For this reason, these specimens were not classified as intersexes but as females. Even if large juvenile, prepubescent, pubescent, and adult intersex sea turtles may not change into either male or female (Limpus et al. 2009), at least some small juvenile tortoises seemingly can do so if one gonad type is dominant; in the case of the four small juvenile tortoises that were reexamined during this study, once their body mass had increased by about an order of magnitude, the ovaries seemed to have suppressed the further development of the testicular tissue which could no longer be identified. However, because they had not yet reached maturity, it could not be ascertained that they really developed into normally functioning females. Similarly, an A. radiata specimen that showed a small area of ovarian tissue on only one of the otherwise normally developed testes was classified as male in another study (Kuchling et al. 2012).

Despite the contrary findings of Limpus et al. (2009), there are, however, indications that the same condition—that intersexes could still become either males or females—may also apply at least for smaller juvenile sea turtles than those studied by Limpus et al. (2009). Kuchling (2004) found 5 of 41 (12.2%) intersex conditions in small juvenile, oceanic C. caretta (straight carapace length between 11.4 and 17.5 cm) washed up on beaches in Perth, Western Australia. In a publication that included maturity status and size of turtles, Limpus et al. (1994) reported that 2 of 58 (3.4%) endoscopically sexed juvenile C. caretta between 70 and 80 cm curved carapace length at the Moreton and Maroom Banks in Queensland were not clearly male or female with respect to gonad development and considered them to be intersexes but found no intersexes (0%) in 214 endoscopically examined prepubescent, pubescent, and adult C. caretta > 80 cm curved carapace length in the same population. The decreasing percentage of intersex conditions corresponding with increasing size and maturity of those C. caretta cohorts—12.2% intersexes in small oceanic C. caretta (Kuchling 2004), 3.4% in larger, neritic juveniles, and 0% in large prepubescent, pubescent, and adult C. caretta (Limpus et al. 1994)—suggests that the intersex condition could sometimes possibly be temporary in the development of some juvenile C. caretta, which may still develop into either sex at maturity. An alternative hypothesis for this phenomenon in those C. caretta cohorts (but not for the re-examined tortoises in this study) can also be that intersex conditions in C. caretta increased over recent times, for example, through environmental changes or contaminants.

However, in the three Aldabra tortoises classified as intersexes in this study and in one A. yniphora classified as intersex by Kuchling and López (2000), testes and ovaries appeared to develop more or less normally side by side, although the ovaries appeared to be slightly smaller than in normal females of similar body mass. It will be interesting to re-examine endoscopically, once they reach maturity, the gonads of those specimens to discover whether they remain intersexes for life, comparable to the green turtle specimen described by Limpus et al. (2009). As stated by these authors, the definite identification of sex in turtles remains dependent on observing eggs being laid or on the direct observation of their gonads.

Sex Ratios and Sex Determination

The La Vanille breeding program has to date produced a female-biased sex ratio, indicating that Aldabra tortoises have TSD. Important TSD parameters are the transitional range of temperatures (TRT), which is the range of temperatures in which sex ratios shift from 100% male (below the TRT) to 100% female (above the TRT), and within the TRT, the constant incubation temperature that will produce a 1∶1 sex ratio, which is referred to as the pivotal temperature (Mrosovsky and Pieau 1991). Pivotal temperatures and TRTs for sex determination can vary among species. The Aldabra tortoise breeding program produced a male to female ratio of about 1∶4, with most eggs incubated at approximately 30°C (but some occasionally also hatched from overlooked nests in the adult enclosure). This sex ratio is less female biased than that produced for A. radiata at approximately 28.9°C (Kuchling et al. 2012). It is, therefore, likely that the Aldabra tortoise has a higher pivotal temperature than A. radiata.

Because the breeding program produced males as well as females, and because a female-biased sex ratio is generally favored in turtle conservation programs (Seigel and Dodd, 2000), there does not seem to be an immediate need to change the incubation protocols in this regard. However, with more varied incubation temperatures, more rigorous incubation temperature control and monitoring, and better record keeping for individuals, it should be possible in the future to assess pivotal temperatures and TRTs for Aldabra tortoises. This information would be important to enable informed decisions regarding how to produce desired offspring sex ratios.