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Gender Identification by
Cloacoscopy and
Cystoscopy in Juvenile Chelonians
Albert Martínez-Silvestre, DVM, MSc, PhD, Acred AVEPA (Exotic Animals), Dipl ECZM (Herpetology)
a,
*,
Ferran Bargalló, DVM
b
, Jordi Grífols, DVM, MSc
b
Videos of endoscopic evaluation and cystoscopy evaluation accompany this
article at http://www.vetexotic.theclinics.com/
INTRODUCTION
In recent years, breeding of chelonians has become more common because of the
popularity of tortoises and turtles as pets as well as their inclusion in recovery plans
of endangered species.
1
Because of these circumstances, there is not only special in-
terest in knowing the sex of subadults but also of newborn animals.
Disclosure: The authors have nothing to disclose.
a
CRARC (Catalonian Reptile and Amphibian Rescue Center), C/Santa Clara, s/n Masquefa
08783, Spain;
b
Zoologic Badalona Veterinary Clinics, C/Conquista, 74, Badalona 08912, Spain
* Corresponding author.
E-mail address: albertmarsil@outlook.com
KEYWORDS
Cloacoscopy Cystoscopy Gender identification Chelonian Tortoise Turtle
Reproductive
KEY POINTS
Sexual dimorphism is apparent in most adult chelonians, but appears at, and not before,
maturation.
The primary method of classifying sex in hatchlings and juvenile chelonians is the direct
observation of gonads through celioscopy.
Cloacoscopic gender identification of external genitalia is not reliable because of the high
degree of misinterpretation between phallus and clitoris, especially in juveniles.
Cystoscopy is an alternative noninvasive technique (performed through natural openings)
for gender identification and is often effective for the visualization of gonads.
Further studies are necessary to verify safety and efficacy of cystoscopic gender identifi-
cation in different species of chelonians.
Vet Clin Exot Anim -(2015) -–-
http://dx.doi.org/10.1016/j.cvex.2015.04.009 vetexotic.theclinics.com
1094-9194/15/$ – see front matter Ó2015 Elsevier Inc. All rights reserved.
Sexual dimorphism is apparent in most adult chelonians. As a general guideline,
males have longer tails with distal cloaca openings, plastron concavity, differences
in size and weight, and distinctive behaviors. In some species, males have different
toenail lengths; bones or scales; eye, skin, or nose color; submandibular glands;
and even an ability to vocalize. However, all these items are secondary sexual charac-
teristics that appear at, and not before, maturation.
2
The sex of hatchlings can be identified by numerous methods. In several studies
with wild tortoises or sea turtles, some investigators have used indirect estimates
of nest sex ratios. However, because of gender differences associated with variable
thermal conditions, these indirect methods are considered imprecise.
3,4
The radioim-
munoassay of testosterone and/or estrogen is another indirect method for sex classi-
fication, but it must be validated for each species to which it is applied. To date, this
method has been successfully used for sex identification of the Arrau River turtle
(Podocnemis expansa), loggerhead sea turtle (Caretta caretta), and green sea turtle
(Chelonia mydas).
5,6
Therefore, the primary method of classifying sex remains the direct observation of
gonads in hatchlings and juveniles. Immature ovarian follicles and nonactive testes
are visible inside the coelomic cavity of even young turtles.
7
Endoscopy has been shown to be one of the main techniques for gender identifica-
tion by direct visualization of gonads.
7
Celioscopy is a minimally invasive technique
performed through the prefemoral fossa, requiring an adequate anesthetic plane.
7
In addition to gender identification, some surgical operations, such as orchiectomy
or ovarian biopsies, can be performed using this system.
7
Cystoscopy is an alternative
noninvasive technique (performed through natural openings) for gender identification
that can provide excellent visualization, without surgery, and results in a rapid recov-
ery time.
INDICATION AND CONTRAINDICATIONS FOR GENDER IDENTIFICATION BY
CLOACOSCOPY AND CYSTOSCOPY IN JUVENILE CHELONIANS
The indications and contraindications for gender identification by cloacoscopy and
cystoscopy in juvenile chelonians are presented in Table 1.
APPROACH
The technique of cloacoscopy/cystoscopy is the same for all species. Each species,
genus, or family can have particular anatomy that must be appreciated.
8
Gender iden-
tification by cloacoscopy/cystoscopy consists of indirect observation of the gonads
via a natural anatomic opening, the cloaca, and through the bladder wall.
9
For these
procedures clinicians take advantage of the transparency of membranes forming
Table 1
The most common indications and contraindications for gender identification by cloacoscopy
and cystoscopy in juvenile chelonians
Indications Contraindications
Gender identification by indirect gonad
visualization in juveniles weighing >150 g
Presence of yolk sac (it might hinder indirect
gonad visualization in hatchlings)
— Risk of mucosa damage, bladder (including
accessory bladders) and cloaca rupture
— Some species have no accessory vesicles
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´nez-Silvestre et al
2
both urinary bladder and accessory vesicles (also known as cloacal bursae or acces-
sory bladders) (Fig. 1).
8,10
Only some species of turtles have accessory vesicles. As a
general rule, many aquatic turtles have accessory vesicles, whereas they are not
present in terrestrial species.
8
Some details on the differences between species are
provided in Table 2.
Thus, in species without cloacal bursae, observation of gonads must be done by
entering the bladder. The main disadvantage of cystoscopy is the presence of uric
acid crystals and sediments that can hinder visualization.
Accessory vesicles are always close to the proctodeum and, in many species, they
are highly vascularized. Their basic functions are to provide buoyancy control and
store water that is used on land for softening the ground for nest excavation. They
are also used in stress situations (to create repulsion from predators).
8,10
They usually
consist of a transparent membrane in which no urine is stored. Accessory vesicles can
be filled and emptied quickly through the cloaca, and may therefore be used to effect
short-term changes in lung volume and, hence, buoyancy.
10
In some species the
accessory vesicular membrane lacks papillae. In the big-headed turtle (Platisternon
megacephala), although the membrane is transparent, the density of highly vascular-
ized papillae hinders the vision of gonads (Fig. 2A). In the Hilaire’s side-necked turtle
(Phrynops hilarii) accessory vesicles have a thin membrane covered with vascularized
folds (plicae) (see Fig. 2B). Other species such as the Central America wood turtle
(Rhinoclemmys pulcherrima) have a dark pigmented mucosa around the proctodeum
that makes visualization difficult or impossible.
TECHNIQUE/PROCEDURE
Preparation/Patient Positioning
Turtles are placed in ventral recumbency, and the cloaca is flushed using warmed sa-
line. This procedure allows filling of the accessory vesicles, and removes urates and
fecal material.
Cloacal lavage can be performed in conscious animals because stress urination can
be helpful for emptying the urinary bladder and accessory vesicles. In box turtles
Fig. 1. Cloacoscopic view of the entrance to the accessory vesicles using insufflation of air in
a male European pond turtle (Emys orbicularis). The transparent membrane cannot be
appreciated on this picture.
Cloacoscopy and Cystoscopy in Juvenile Chelonians 3
Table 2
Special features of the main families of continental tortoises and turtles at the time of the cloacoscopy, as well as some internal peculiarities specific to
certain species
Family Accessory Vesicles Present Accessory Vesicles Absent Common Name Details of Interest
Carettochelyidae Carettochelis insculpta — Pig-nosed turtle Multilobulated clitoris
Chelydridae Chelydra serpentina — Snapping turtle —
Macrochelys temminckii — Alligator turtle —
Emydidae Emys orbicularis/Emys trinacris — European pond turtle Two big accessory vesicles
Graptemys pseudogeographica — Common map turtle —
Trachemys scripta — Red-eared slider —
Trachemys emolli — Nicaraguan slider —
Pseudemys floridana — Coastal plain cooter —
Chrysemys picta — Painted turtle —
Mauremys leprosa — Mediterranean turtle Two big accessory vesicles
Mauremys mutica — Asian yellow pond turtle —
Mauremys sinensis — Chinese stripe-necked turtle —
Mauremys anamensis — Annam pond turtle —
Malaclemys terrapin — Diamondback terrapin —
Cuora amboinensis — Malayan box turtle One small accessory vesicle
Cuora flavomarginata — Yellow-margined box turtle One small accessory vesicle
Cyclemys dentata — Asian leaf turtle —
Terrapene carolina — Common box turtle —
—Rhinoclemmys pulcherrima Central America wood turtle Dark pigmented mucosa
Kinosternidae — Sternotherus odoratus Musk turtle —
Kinosternon scorpioides — Scorpion mud turtle —
Platysternidae Platysternon megacephalum — Big-headed turtle Papillae sieved accessory vesicles
Martı
´nez-Silvestre et al
4
Testudinidae — Testudo hermanni Herman’s tortoise —
—Testudo graeca Moorish tortoise —
—Testudo marginata Greek tortoise —
—Testudo kleinmanni Egyptian tortoise —
—Gopherus berlandieri Texas tortoise —
—Geochelone elegans Indian star tortoise —
—Chelonoidis carbonaria Red footed tortoise Large clitoris
—Chelonoidis denticulata Yellow footed tortoise Large clitoris
—Stigmochelys pardalis Leopard tortoise —
—Centrochelys sulcata African spur thigh tortoise —
—Astrochelys radiata Radiated tortoise —
—Aldabrachelys gigantea Aldabra giant tortoise —
—Geochelone chilensis Chaco tortoise —
—Manouria emys Asian giant tortoise —
Trionychidae Apalone ferox — Florida softshell turtle —
Pelodiscus sinensis — Chinese softshell turtle —
Chelidae Phrynops hilarii — Hilaire’s side-necked turtle Accessory vesicle with plicae
Pelomedusidae Pelusios subniger — East African mud turtle Two small accessory vesicles
Pelomedusa subrufa — African helmeted turtle One small accessory vesicle
Emydura subglobosa — Red-bellied short-necked turtle —
Podocnemididae Podocnemis unifilis — Yellow-spotted Amazon river
turtle
One small accessory vesicle
Cloacoscopy and Cystoscopy in Juvenile Chelonians 5
(Terrapene or Cuora), a Farabeuf abdominal retractor can be used to prevent closure
of the carapace during examination (Fig. 3).
Surgical anesthesia using inhalants is rarely required, but analgesia and tranquiliza-
tion are beneficial.
11
Anesthesia time rarely, if ever, exceeds 20 minutes. Drugs recom-
mended for this purpose are propofol, alfaxalone, or a combination of (dex)
medetomidine, ketamine, and an opiate (eg, morphine or hydromorphone).
9,11–13
Nor-
mally, both loss of consciousness and recovery from anesthesia are rapid and
uneventful.
13
In hatchlings, intravenous administration of anesthesia is impractical,
so the intramuscular route is used. Intrathecal anesthesia using lidocaine can also
be useful
14
and facilitates relaxation of hind limbs, tail, and cloaca, thereby facilitating
cloacoscopy, especially in larger species. Postoperative analgesics, like meloxicam or
tramadol, should also be considered.
12,13,15
Basic Equipment
Rigid telescope: 2.7 mm, 30, 18 cm with a 3.5-mm protective sheath
Xenon light source with light cable
Endovideo camera
Monitor
Fluid set for lactated Ringer solution infusion (1 drop every 3–4 seconds)
Special Equipment
Portable battery endoscope
Direct light source
Different diameter endoscopes can be used for this technique depending on the
size of the turtle and the cloaca opening. However, diameters of 1.9 mm to 4 mm
are generally preferred for most animals. In turtles weighing more than 200 g, the
diameter of the cloacal sphincter and the diameter of the bladder or accessory vesicle
entrance usually do not represent a physical limitation for this procedure. In very small
chelonians (eg, <20 g), the use of a small telescope (eg, 1.9-mm, rigid 30-viewing
endoscope) should be used to avoid possible size-related trauma. The endoscopic
Fig. 2. (A) Cloacoscopic view showing the high density of vascularized papillae in the proc-
todeum of a big-headed turtle (P megacephala). (B) Cloacoscopic view of vascularized folds
or plicae on the proctodeum in a side-necked turtle of the genus Phrinops.
Martı
´nez-Silvestre et al
6
equipment can be cleaned and disinfected by immersion in 2.4% glutaraldehyde
solution for 15 minutes and rinsed with sterile distilled or deionized water between
each procedure.
TECHNIQUE/PROCEDURE
Cloacoscopy
The patient is placed in ventral recumbency. The endoscope is inserted through the
cloaca, progressing cranially. One finger and thumb are placed around the vent to
act as a valve in order to control the fluid infusion and avoid the outflow of lactated
Ringer solution. With the animal in ventral recumbency, 4 structures can be seen
within the proctodeum: genitalia (ventral), urodeal folds, coprodeal entrance, and
entrances to accessory vesicles (see Figs. 1 and 5A). If cloacoscopic accessory vesic-
ular examination is performed, the endoscope easily enters the wide, natural
openings.
Sometimes air insufflation into the proctodeum allows easy access to the accessory
vesicles (Video 1) without the use of lactated Ringer solution. When air is used for the
expansion of the vesicles, urate or digestive remnants do not remain floating in front of
the endoscope, which can improve the overall image.
Access to the accessory vesicles is simple and decreases the possibility of forcing
or damaging the urethral opening. In species in which these structures exist, exam-
ining them before resorting to examination from within the urinary bladder is advisable.
Fig. 3. Positioning of an Amboina box turtle (Cuora amboinensis) for cloacoscopy using the
Farabeuf abdominal retractor. The instrument is used to prevent natural closure of the
caudal plastron hinge.
Cloacoscopy and Cystoscopy in Juvenile Chelonians 7
PHALLUS/CLITORIS IDENTIFICATION
The first image that is seen when the endoscope enters in the cloaca is the phallus or
clitoris. In females, the clitoris consists of a small structure similar in shape to a phallus
(Fig. 4A, see Video 1). In adult males, the phallus is bent anteriorly, has a differentiated
longitudinal groove (see Fig. 4B), glans (see Fig. 4C), and its color is normally bright
pink to black. In the Sicilian pond turtle (Emys trinacris) young tortoises can have a
gray phallus, more similar to a female clitoris.
16
Unlike squamates (phallic erection
by eversion), chelonian erection is caused by tumescence of 2 spongy bodies: the
corpus spongiosum/cavernosum and corpus fibrosum.
17,18
Therefore, differentiation
of phallus and clitoris can be difficult when the organ is flaccid within the cloaca,
and is almost impossible to identify in young or subadult individuals (see Fig. 4D).
17
In one study that attempted to identify gender in red-eared sliders (Trachemys
scripta elegans) by the presence of the phallus/clitoris, males were misdiagnosed as
females (100%) and females as males (38%). This rate clearly shows the degree of er-
ror associated with this technique.
19
In the author’s experience, in some species, the
clitoris is almost nonexistent, as in the big-headed turtle (P megacephala). In other
Fig. 4. (A) Cloacoscopic view of the clitoris of a mature Moorish tortoise (Testudo graeca).
Note the small structure similar to a phallus. (B) Cloacoscopic view of the longitudinal
groove (arrow) of the phallus in a male immature Moorish tortoise (T graeca). (C) Close
endoscopic view of a glans in an immature male Moorish tortoise (T graeca). (D) Cloaco-
scopic view of the phallus in a subadult male marginated tortoise (Testudo marginata).
Note the similarity with the clitoris in Fig. 4A.
Martı
´nez-Silvestre et al
8
species, such as the red footed tortoise (Chelonoidis carbonaria), the clitoris is highly
variable in size and degree of differentiation. Endocrine disruption can result in females
with a pseudophallus (with groove, glans, and corpus cavernosum), increasing the
possibilities of inaccurate assignment of gender.
20
CYSTOSCOPY
Insufflation of air can be performed in accessory vesicles but not in the urinary bladder,
for which warm (30C) lactated Ringer solution is preferred. If the urinary bladder is not
naturally distended with transparent urine, the operator needs to expand and distend
the bladder with fluid.
For access to the urinary bladder, the endoscope is directed toward the urethral
opening, which is located ventral to the entrance of the colon (see Fig. 4B). In a few
cases, the urethral opening is not clearly identified and the colon can be used as an
anatomic landmark (Video 2). The urethral opening is usually seen in a horizontal plane
in land tortoises (Fig. 5B), whereas it is seen in a vertical plane in terrapins and aquatic
turtles (see Fig. 5A). Once the endoscope is in the proximity of the colonic orifice, a
gentle caudal retraction of the endoscope allows visualization of the urethral opening.
9
Lactated Ringer solution (1 drop every 3 to 4 seconds) permits distension of the ure-
thral opening and aids entry into the bladder. Once access to the urinary bladder is
gained, the endoscope is guided dorsolaterally until the gonads can be seen. The uri-
nary bladder is a greatly distensible organ that can contain large quantities of fluid.
GONAD IDENTIFICATION
Chelonian gonads are located in the dorsocaudal coelom, lying cranioventrally to the
retrocoelomic kidneys. They are usually found in close association with the caudal
lungs and colon.
9
In juvenile turtles the immature testis is elongated to round/oval,
smooth, yellowish, with a smooth surface with superficial blood vessels, and attached
dorsally by a short mesorchium (Fig. 6). The epididymis is cream in color, convoluted,
and continues caudally as the ductus deferens.
Fig. 5. (A) Cloacoscopic view of the entrance to the accessory vesicles after sterile fluid infu-
sion in a male European pond turtle (E orbicularis). A, Urodeal folds; B, distal colon sphincter
(or entrance); C, accessory vesicles. (B) Cloacoscopic view of proctodeum and urodeum in a
Herman’s tortoise (Testudo hermanni) using sterile fluid infusion. A, Urodeum; B, distal
colon sphincter. Note the absence of accessory vesicles.
Cloacoscopy and Cystoscopy in Juvenile Chelonians 9
The immature oviduct is a straight, flat, transparent to white band located immedi-
ately dorsal and parallel to the ovary. The immature ovaries are long, irregular, semi-
transparent organs. Round and white to yellow follicles can be seen developing
throughout the organ. Previtellogenic follicles are separated from each other and do
not completely fill the parenchyma of the organ (Fig. 7).
19
In general, the identification of the gonad by indirect visualization through cystos-
copy is reliable, but sometimes is difficult and not safe (discussed later). This tech-
nique is easier in young terrestrial or giant tortoises (>150 g weight) than in young
Fig. 6. Close endoscopic view of the testis of a European pond turtle (E orbicularis) through
the transparent accessory vesicle membrane. Note the yellowish organ with small blood ves-
sels. The bigger blood vessels belong to the vesicle membrane.
Fig. 7. Cloacoscopic view of the ovary of a subadult European pond turtle (E orbicularis)
through the transparent accessory vesicle membrane. Note the white to yellow follicles
within the ovary.
Martı
´nez-Silvestre et al
10
turtles weighing less than 100 g. In hatchling land tortoises (Testudo hermanni) gender
identification by cystoscopy was accurate compared with gonadal histology.
9
COMPLICATIONS AND MANAGEMENT
In the author’s experience, there are several factors that can affect the reliability of
gender identification by cloacoscopy/cystoscopy:
1. Obesity. Fat tissue may cover and hide the gonads.
2. In offspring less than 6 months old, the presence of the internalized yolk sac greatly
complicates the identification of other coelomic structures, which makes this tech-
nique less helpful.
3. The presence of distended intestinal loops can hinder the identification of the
gonads. Thus, 3 days of starving can be helpful.
4. Infectious diseases that cause inflammation and loss of transparency of bladder
membranes can negatively affect visualization.
5. Emesis has been reported as an adverse reaction in 16% of anesthetized hatchling
Herman’s tortoises (T hermanni) undergoing cystoscopy.
9
POSTOPERATIVE CARE
Postoperative care is simple and usually does not require the use medication, with few
exceptions (eg, use of antiinflammatories because of mucosa damage). Usually, tor-
toises are eating and behaving normally after 24 hours.
CURRENT CONTROVERSIES/FUTURE CONSIDERATIONS
The order Testudines contains more than 300 species. This article shows that cysto-
scopic gender identification can be performed in the turtle families shown in Table 2.
However, a recent study performed in 30 clinically healthy immature (36–90 g) Tra-
chemys scripta shows that only 10% (3 of 30) of the animals had their gender accu-
rately identified by cystoscopy and 7 of 30 animals experienced bladder or cloacal
rupture.
21
Therefore, more studies are necessary to verify safety and efficacy of cysto-
scopic gender identification in different species. In addition, the gonads of chelonians
may vary in size and aspect seasonally, and this may complicate their identification,
especially in turtles that hibernate.
22,23
Further studies focusing on seasonal fluctua-
tions of gonadal morphology in chelonians are needed.
SUMMARY
Identifying sex in young chelonians by cloacoscopy or cystoscopy is an alternative
noninvasive technique (performed through natural openings) that is often effective
for the visualization of gonads. Moreover, some important anatomic differences exist
between different chelonians and further studies are necessary to verify safety and
efficacy of the procedures.
ACKNOWLEDGMENTS
To the veterinary technicians at the Zoologic Badalona Veterinaria, especially Simon
Lopez, as well as the technicians in the CRARC center, especially Isabel Verdaguer,
Silvia Merchan, Juan Miguel Cano, and Joaquim Soler.
Cloacoscopy and Cystoscopy in Juvenile Chelonians 11
SUPPLEMENTARY DATA
Supplementary data related to this article can be found online at http://dx.doi.org/10.
1016/j.cvex.2015.04.009.
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Cloacoscopy and Cystoscopy in Juvenile Chelonians 13