Citation: Vetere, A.; Ablondi, M.; Bigliardi, E.; Rizzi, M.; Di Ianni, F. Sex Determination in Immature Sierra Nevada Lizard (Timon nevadensis). Animals 2022, 12, 2144. https:// doi.org/10.3390/ani12162144 Academic Editor: Tom Hellebuyck Received: 23 July 2022 Accepted: 17 August 2022 Published: 21 August 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). animals Article Sex Determination in Immature Sierra Nevada Lizard (Timon nevadensis) Alessandro Vetere * , Michela Ablondi , Enrico Bigliardi, Matteo Rizzi and Francesco Di Ianni Department of Veterinary Science, University of Parma, Strada del Taglio 10, 43126 Parma, Italy * Correspondence: alessandro.vetere88@gmail.com Simple Summary: Sex determination in reptiles is frequently requested by reptile breeders, and it is a real challenge in reptiles with little or no sexual dimorphism, such as in immature subjects. Twenty- three clinically healthy young Sierra Nevada lizards (Timon nevadensis) aged between 4 and 6 months were included for sex determination using two techniques: cloacal probing and contrast radiography. Results showed that contrast radiography may have major sensitivity for sex determination compared to probing. Given the above, this technique could represent a valid and less invasive aid for sexing young lizards. Abstract: Sex determination has a fundamental role in a captive breeding context, both for commercial reasons and in relation to animal welfare itself. However, this can be particularly difficult, especially in reptiles with little or no sexual dimorphism. Twenty-three clinically healthy young Sierra Nevada lizards (Timon nevadensis) were included in this study for sex determination. The first attempt at sexing was carried out by cloacal probing. A small, buttoned probe was inserted very gently into the hemipenal pouches, and the length of the inserted part was evaluated and measured. Subsequently, for each animal, a contrast medium was administered into the cloaca, and radiography was performed within 5 min. Through probing, 11 males and 8 females were recognized. The test was, however, equivocal in four subjects. In contrast radiography, 14 males and 9 females were identified. All the animals were rechecked after 8 months through an ultrasound examination, confirming 15 of the 14 previously male sexed animals based on contrast radiography. All the animals identified as female (n = 9) by contrast radiography were confirmed. From these results, it seems that contrast radiography may have major sensitivity in sex determination compared to probing. This technique could represent a valid and less invasive aid for sexing young lizards. Keywords: sex determination; reptiles; radiology; lizards 1. Introduction The genus Timon currently comprises six species found on three different continents [1]. The Asian species Timon princeps and Timon kurdistanicus inhabit regions in Turkey, Iran and Iraq; and Timon tangitanus and Timon pater are found in Morocco, Tunisia and Algeria. Timon nevadensis and Timon lepidus are restricted to Europe [1]. Timon nevadensis are present in the south-eastern part of Spain [1]. Adults can be over 130 mm in snout–vent length (SVL) [2] and they are sexually dimorphic: the male is big compared to the female [2]. Mature males display evident prefermoral pores, whereas in females, these pores are not well developed [3]. Juveniles lack evident sexual dimorphism [2–4]. While Timon lepidus has most recently been assessed for the IUCN Red List of Threatened Species in 2008 as near threatened (NT), there are no data available for Timon nevadensis. Sex determination is frequently required in reptiles, especially for species with no evident sexual dimorphism [5]. Sex identification in lizards is usually visually performed by evaluating secondary sexual characteristics, such as the presence of well-developed femoral pores in males or the presence of brilliant colors of livery. Knowing the sex of Animals 2022, 12, 2144. https://doi.org/10.3390/ani12162144 https://www.mdpi.com/journal/animals