Journal of Exotic Pet Medicine 34 (2020) 10 14

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Journal of Exotic Pet Medicine

journal homepage: www.elsevier.com/locate/jepm

Research

Sex determination in Mexican-beaded lizard specimens (Heloderma alvarezi

and Heloderma horridum) employing contrast radiography method
Mario Alberto Meza-Oviedo, MVZ a,*,1, Daniel Alejandro Go
mez Pizano, MVZ EMCV (FS) a,
Luis Carrillo D’Lacoste, MV EMCV (FS) , Luis Miguel Campos Guerrero, MV EMCV (MCPG) c,
b

Martha Patricia Izquierdo-Uribe, MVZ MC c, Andr
es Ernesto Ducoing-Watty, MVZ MSC PhD d
a
Department of Ethology and Wildlife, Veterinary College National Autonomous University of Mexico, M
exico City, M
exico
b
Department of Husbandry and Wellbeing, Zoofari Conservation Center, Teacalco, Morelos
c
Department of imaging, Veterinary College National Autonomous University of Mexico, M
exico City, M
exico
d
Department of distance education, Veterinary College National Autonomous University of Mexico, M
exico City, M
exico

A R T I C L E I N F O A B S T R A C T

Keywords: Background: Alternatives to gross external anatomical sex determination are often required, peculiarly in species
Echography displaying poor or absent gross sexual dimorphism, such as the beaded lizard. This study evaluates results of hem-
Follicles ipenile contrast radiography accuracy as compared to ultrasonography of the same beaded lizard (Heloderma
Hemipenes
alvarezi and Heloderma horridum). Methods: Eleven captive adult beaded lizards (4 H. alvarezi and 7 H. horridum),
Lizard
Testicles
underwent hemipenis contrast radiography, and results compared with coelomic ultrasound. Results: By ultra-
Ultrasonography sound, 2 lizards were females and 9 were males. By contrast hemipenis radiography, 2 animals were females and
9 were males. In this sample, there was a Kappa concordance of 1. Conclusions and clinical relevance: In this species,
contrast hemipenis radiography proved to be a reliable diagnostic tool for hemipenes identification, and sex deter-
mination with all test specimens. Further studies including a larger number of animals and different lizard species
are warranted.

Introduction visual sexual characteristics, making unreliable sex determination. Male

beaded lizards are reported to have longer tails than females, which
Alternative sex determination techniques are frequently required, allows a specimen to form a higher body arc and domineer its opponent
particularly in species with poor or absent gross external sexual dimor- more fiercely during male combat [5]. Such fights between males depict
phism. Reptiles are usually territorial, and male fights can lead, in the a test of dominance through a display of superior physical strength and
least pleasant scenario, to death [1]. Many saurian species lack external endurance, where the victor may have greater access to females [6].
primary or secondary sexual characteristics [2]. A specimen’s sex is Ultrasonography is an important technique for female reptile repro-
essential to conservation programs, preservation of species, and to clini- ductive status evaluation, including ovarian activity and gravid determi-
cal practice, as it relates to sex specific or related diseases (e.g., dystocia nation [7]. Ultrasonography is also an effective tool in sex
in females). Male lizards have sac-like paired hemipenes, without erec- determination. White-throated monitors (Varanus albigularis), Gila mon-
tile tissue, and no urinary function. They are placed in an inverted posi- sters (H. suspectum), beaded lizards (H. horridum), komodo dragons (Var-
tion at the base of the tail, and only 1 hemipenis at a time is used for anus komodoensis), green iguanas (Iguana iguana), and leopard gecko
copulation. Female lizards have paired ovaries and oviducts, which (Eublepharis macularius) sex has been determined by ovarian follicles
enter into the urodeum of the cloaca [3]. identification in females, and absence thereof with testes detection in
The beaded lizard name relates to the beaded appearance of their some males [2,8 10]. However, some individuals exhibited intestinal
skin. The genus Heloderma comes from Greek helos that means “studded” gas precluding identification of coelomic gonads [2,9]. In coelomic
and derma that means “skin,” because helodermatid lizards can exhibit ultrasonography, the ovaries can be detected in sexually mature female
rounded, beadlike osteoderms on dorsal surfaces of the head, limbs, lizards, as structures in the middle abdomen, positioned dorsally [11].
body, and tail [4]. The 2 species of beaded lizards (Heloderma alvarezi The ovaries are classified as previtellogenic and vitellogenic [12]; in the
and Heloderma horridum) exhibit few gender-specific external gross first of them, many anechoic small round areas called previtelline

* Corresponding author. Tel: +52 1 443 410 8070

E-mail address: mezaov@hotmail.com (M.A. Meza-Oviedo).
1
Present address: Department of Veterinary Medicine Services, Zool

ogico de Vallarta A.C., Camino el Ed
en 700, 48294, Mismaloya, Jalisco, M
exico.

https://doi.org/10.1053/j.jepm.2020.02.019
1557-5063/© 2020 Elsevier Inc. All rights reserved.
M.A. Meza-Oviedo et al. Journal of Exotic Pet Medicine 34 (2020) 10 14

follicles are observed. The vitelline follicles are detected effortlessly, contrast media infiltrated hemipenis was considered diagnostic of a
observing large (up to 2.5 cm), round and more hyperechoic structures. male. Measurements of the length and maximum diameter of the shape
Once ovulated, the spherical appearance is lost and the structures of radiographic contrast media were made, with the aid of the Exam Vue
become ovoid. As the shell is deposited, the oval structure becomes CD Viewer software (version 1.1.0.6. JPI Healthcare Solutions).
increasingly hyperechoic. In the case of males, the testes are observed as
small oval structures in the dorsal abdomen. They are more hyperechoic Ultrasonography
than the ovaries and have a homogeneous texture [11]. It can be argued
that inactive female ovaries and male gonads are difficult to localize and Ultrasonography sex determination was made via detection of ovar-
identify [7]. Nonetheless, in 1 study implemented in adult bearded drag- ian structures or testicles. SonoSite Edge (FUJIFILM) high-resolution
ons (Pogona vitticeps), between October and November (a portion of the ultrasound with linear transducer, scanning depth of 2.6 to 6 cm was
year when bearded dragons are reproductively inactive) researches use to discern the coelomic structures. Specimens were manually
could visualize the ovaries in all the females, and the testes in all the restrained in ventral recumbency on a fenestrated table. The coelom was
males [13]. Furthermore, the 2-dimensional ultrasonography has been scanned ventrally from cranial to caudal, using a longitudinal and trans-
used for testes and hemipenes description in Varanus marmoratus speci- verse grid pattern, according to the techniques formerly described
mens [14] and for sex determination in Gila monster (Heloderma suspec- [2,7,12].
tum) and beaded lizard (H. horridum) by detecting hemipenes with
positive outcomes [15]. Statistical analysis
The technique and diagnostic sensitivity of ultrasonography, contrast
radiography and computed tomography with and without contrast Descriptive analysis and confidence intervals were obtained for the
medium for sex determination (by identifying hemipenes) have been measurements of reproductive structures. Data were reported as mean,
previously published in 4 species of lizards: bearded dragons (Pogona vit- standard deviation, and range. Concordance between radiography and
ticeps), Egyptian spiny-tailed lizards (Uromastyx aegyptia), blue-tongued ultrasonography was evaluated via the Kappa coefficient for agreement,
skinks (Tiliqua scincoides), and Sudan-plated lizards (Gerrhosaurus major) using the JMP statistical package (version 14.1, SAS Inst. Inc., 2018).
[1]. Researchers reported that the accuracy of the computed tomogra-
phy with contrast medium was 100%, contrast radiography was 94.7%, Results
ultrasonography was 64.3%, and computed tomography without con-
trast medium was 63.1% [1]. Despite computed tomography with con- Animals
trast media demonstrating a 100% accuracy for gender determination in
some lizard species [1], its expense and limited availability makes it All the Mexican-beaded lizards in this study were apparently healthy
impractical for most clinicians, including this study. adult specimens and their weight range was 1.30 kg to 2.40 kg
The aim of this study was to confirm that the sexing technique by (2.87 5.29 lbs).
contrast radiography is equally accurate in beaded lizard (H. alvarezi
[=Heloderma horridum alvarezi] and H. horridum), as in other lizard spe- Ultrasonography
cies [1].
Ultrasonographic sex determination in 11 beaded lizards included 9
Materials and methods males and 2 females. All 9 males (4 H. alvarezi and 5 H. horridum) exhib-
ited mid coelomic testicles. Testicular tissue exhibited ovoid homoge-
Animals and handling nous echogenicity (Fig. 1). Testicular size ranged from 1.01 to 2.26 cm
on longitudinal axis and 0.73 to 1.19 cm on transverse axis. A mean was
Institutional Animal Care and Use Committee approval was consid- substantiated with its standard deviation, as well as confidence limits
ered nonessential, because specimens were only manually restrained, for the mean of 95% of the longitudinal and transverse axes of both tes-
and hence no invasive technique was used. The beaded lizards were ticles (Table 1).
allowed by collections from different institutions (Zoofari—Conserva- In the case of females (2 H. horridum), the follicles were located in
tion Center and Herpetarium of the Faculty of Sciences of the UNAM) the middle coelom, where the previtelline follicles oscillated between
and private-owned, all of which approved its participation in this 0.3 and 0.75 cm in diameter, observing uniformly anechoic (Fig. 2a).
research. The criteria inclusion was apparently healthy adult beaded The vitelline follicles were perceived to be more hyperechoic, compared
lizards. to the previtellines, ranging between 1.02 and 1.3 cm in diameter
Radiographic and ultrasonographic studies were performed under (Fig. 2b). A mean with its standard deviation was established for the
manual restraint. Different operators (1 for ultrasound and 2 for radio- measurement of the smaller and larger previtelline follicles, as well as
graphic contrast media) performed radiographs and ultrasonographic for the vitelline follicles (Table 2).
studies. The operators were unaware of the results of the other tech- The sex determination in males by ultrasound varied from 10 to 15
nique. The Mexican-beaded lizard is venomous and restraint was per- minutes of coelomic scanning, which was established based on the
formed by experienced operators. absence of follicular structures and the presence of paired ovoid struc-
tures consistent with testicles. In the case of females abided for approxi-
Contrast hemipenile radiography mately 5 minutes by means of coelomic scanning, which was
substantiated for the presence of follicular structures.
A radiographic study was performed on 11 captive adult beaded liz-
ards (4 H. alvarezi and 7 H. horridum), using water-soluble, nonionic, Contrast radiography
iodinated contrast medium, with low osmolality, (Iobitridol, XENETIX
300, 300 mg I/mL, Guerbet, Roissy, France), similar to that used by Di Contrast media introduced through the cloaca and distributed in at
Ianni in 2015 [1]. Contrast media was introduced into the cloaca using least 1 hemipenis was detected in the radiographic studies as spindle-
an 18G curved ball tip metal feeding tube, entering parallel to the tail shaped radiopaque structures pointing caudally from to the cloaca
and pointing caudally, until the contrast medium began to overflow. (Fig. 3). Hemipenes size ranged from 33.63 mm to 53.25 mm in length
Dose ranged between 0.5 and 1 mL per specimen. The radiographic pro- and from 5.29 mm to 9.53 mm in maximum diameter. A mean was
jections included dorsoventral, left latero-lateral, and right latero-lat- established with standard deviation, and confidence limits for the mean
eral, before and after contrast administration. Observation of at least 1 at 95% of the length and maximum hemipenis diameter (Table 3).

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M.A. Meza-Oviedo et al. Journal of Exotic Pet Medicine 34 (2020) 10 14

Fig. 1. Ultrasound image of the left testicle in a male beaded

lizard (Heloderma horridum). A. Longitudinal section (A, B =
borders of the testicle). B. Cross section (A = borders of the
testicle).

Table 1
Testicles Measurement in Heloderma Alvarezi and H. Horridum Specimens Using Ultrasound

Variable Mean Standard Deviation Range Confidence Interval for the 95% Mean N

Upper Limit Lower Limit

Left testicle (longitudinal) (cm) 1.60 0.43 (1.09 2.26) 1.32 1.88 7
Left testicle (cross) (cm) 0.91 0.14 (0.73 1.08) 0.82 1.00 7
Right testicle (longitudinal) (cm) 1.58 0.33 (1.01 2.07) 1.42 1.74 9
Right testicle (cross) (cm) 0.96 0.13 (0.80 1.19) 0.90 1.03 9

Females exhibited contrast outlining the cloacal rim, seeming as a were unable to determine the sex of 3 H. horridum specimens, due
thick radiopaque line (Fig. 4). The presence of adverse effects was not to the excessive intestinal gas precluding adequate coelomic struc-
discerned in any specimen after instillation of the contrast into the clo- tures identification. In the present study, this obstacle was nonexis-
aca. Both of the imaging techniques obtained the same sex results tent, and the sex was precisely determined in all 11 specimens.
(4 males of H. alvarezi and 5 males and 2 females of H. horridum). Excessive intestinal gas prevention may be accomplished by a fast-
Accordingly, there was a Kappa concordance of 1. ing. However, a correct fasting time in reptiles may be difficult.
Only few studies about this topic had been published. A study with
Discussion Pogona vitticeps demonstrated a median contrast transit time to the
colon was 31 hours, and longest was 72 hours [16]. Another study,
In 1996, Morris and Alberts [2] attempted ultrasonoghraphic in the same species, demonstrated contrast transit time to the cloaca
gender determination in three lizards species, including H. horridum. at >32 [17]. Species variability and biological differences aside,
They accurately obtained the sex in 2 of the species. However, they 72 hours of fasting may limit intestinal gas.

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M.A. Meza-Oviedo et al. Journal of Exotic Pet Medicine 34 (2020) 10 14

Fig. 2. Ultrasound image of follicles in a female beaded lizard

(Heloderma horridum). A. Longitudinal section of previtelline
follicles. B. Vitelline follicle (Left = Cross section; Right =
Longitudinal section; A, B, C = Edges of the follicle).

Table 2 of lizards [1]. In some specimens, only 1 hemipenis was visualized. This
Measurements of Ovarian Structures in 2 Specimens of Heloderma may be associated with feeding tube tip position for infiltration of the con-
Horridum Using Ultrasound trast media. The relativity proximity to 1 of the 2 hemipenes orifices. Cel-
Variable Mean Standard Deviation
lular debris or fluid within the hemipenis may also interfere with proper
contrast distribution. Cases according to the described in previous works
Vitelline follicle (longitudinal) (cm) 1.18 0.17 [1,20]. Gross external examination of the cloaca and saline solution flush
Vitelline follicle (cross) (cm) 1.15 0.18
Larger previtelline follicle (cm) 0.62 0.18
may facilitate contrast media infiltration.
Smaller previtelline follicle (cm) 0.36 0.08 False positives could be possible if cloacal tissues are damaged in
females. Contrast media is introduced in a deeply or traumatic method
may cause media distribution in fascial planes of the tail and resemble a
hemipenis [1]. No false positives were determined in the present study.
Temperature affects transit time as well. Body temperature of H. The feeding tube should be gently introduced to avoid cloacal trauma.
horridum, ranges from 19.9°C to 28.5°C [18]. A similar range of The small number of specimens constitutes a study limitation, as does
temperature has been observed in H. alvarezi [19]. Accordingly, a numerical gender inequality and the inclusion of only 2 species.
temperature around 28°C should keep the natural biologic function The present study supports the use of cloacal contrast radiography in
of the species. H. alvarezi and H. horridum specimens for hemipenes identification as a
Contrast hemipenis radiography was a sensitive and specific diagnos- viable alternative to other techniques used for sex determination. Based
tic tool for hemipenes identification, and hence sex determination, with on a literature search performed on the database ScienceDirect, Elsevier,
all specimens in this study. Contrast radiography proved to be a quick Wiley Online Library, JSTOR, ResearchGate, and on Mader’s Reptile
tool, as previously mentioned,[1] with an approximate span of 5 minutes Medicine and Surgery textbook [3,12], this is the first published study
per specimen. The radiographic characteristics of hemipenes in H. alvarezi that compares ultrasound for gonads detection to contrast media radiog-
and H. horridum were similar to those formerly reported in other species raphy for the hemipenes detection, both for sex determination in lizards.

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M.A. Meza-Oviedo et al. Journal of Exotic Pet Medicine 34 (2020) 10 14

Table 3
Hemipenes Measurements on Heloderma Alvarezi and H. Horridum Specimens Using Contrast Radiography

Variable Mean Standard Deviation Range Confidence Interval for the 95% Mean n

Upper Limit Lower Limit

Left hemipenis (Long) (mm) 44.21 8.06 (33.63 53.25) 39.06 49.36 7
Left hemipenis (maximum diameter) (mm) 7.28 0.89 (5.81 8.14) 6.71 7.85 7
Right hemipenis (long) (mm) 42.13 6.05 (34.64 51.51) 37.62 46.64 6
Right hemipenis (maximum diameter) (mm) 7.58 1.47 (5.29 9.53) 6.48 8.68 6

Further studies including a larger number of animals and different lizard

species are warranted.

Acknowledgments—The authors thank the Zoofari Conservation Center,

the Herpetarium of the Faculty of Sciences of the UNAM, the Biol. M.
Salmer
on-Estrada and the Biol. G. A Barona-Casta~ neda, for allowing the
access to specimens.
This research did not receive any specific grant from funding agen-
cies in the public, commercial, or not-for-profit sectors.

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