COMPARATIVE ANALYSIS OF PROBING AND CONTRAST RADIOGRAPHY FOR GENDER DETERMINATION IN RETICULATED PYTHONS (Malayopython reticulatus)

Article Highlights
- Accurate gender identification supports effective breeding programs and ensures snakes' welfare.
- This study emphasizes that probing is more reliable and efficient than contrast radiography for gender determination in reticulated pythons.
- The probing method proved to have 100% accuracy in determining the gender of reticulated pythons, outperforming contrast radiography, which only achieved 75% accuracy.

Abstract
Reticulated pythons, like most snakes, do not exhibit noticeable sexual differences, which makes gender determination necessary. Probing and contrast radiography were chosen as methods to determine sex and to compare their accuracy. Six reticulated pythons with body lengths of 128-167 cm, tail lengths of 5.4-9.6 cm, and body diameters of 1.4-3.4 cm were examined twice by probing and contrast radiography. The results showed that probing had 100% accuracy, with probe lengths ranging from 1.37-3.60 cm for male snakes and 0.3-0.76 cm for female snakes. Contrast radiography had 75% accuracy, with the hemipenis appearing as a white silhouette and female snakes appearing colorless. The accuracy rates highlight the effectiveness of probing over contrast radiography for reliable gender determination in reticulated pythons

INTRODUCTION

Determining the gender of snake is vital for captive snake owners and breeders. Accurate gender determination is crucial to avoid unintended breeding and effective management of snake populations. Some owners may decide to rear several snakes in the same terrarium and wish to avoid breeding activities. Various methods are used to determine gender of snake, such as molecular techniques and morphological characteristics. Previous studies reported molecular techniques, such as quantitative PCR(Rovatsos et al., 2015).(, 2017)presented a novel molecular method of using gametologous genes for gender identification in caenophidian snake. However, a comprehensive comparison of probing and contrast radiography methods specifically for reticulated pythons has not been extensively documented. Knowledge of snake gender can also help in diagnosing diseases affecting reproductive organs(Gnudi et al., 2009). Differentiating between male and female snakes is challenging due to the relatively simple morphology, signifying the importance of gender determination(Bonnet et al., 1998).

The most common method used for snake gender determination is manual probing, which is conducted specifically to identify hemipenes(Vincent et al., 2006). This can be achieved by inserting a smooth, blunt, and thin probe into the cloaca to deviate caudally. In male snake, the probe passes through the hemipenes and reaches more deeply into the tail compared to that in female snake(Stahl, 2002). The depth of the male hemipenis and the female homologue varied between species. An alternative to the probing method is popping which is more traumatic, suitable for small snakes, and not recommended for larger snakes. Popping includes the eversion of the hemipenes outside the cloaca by rolling a finger from the tip of the tail toward the vent(Divers & Mader, 2005). Ultrasonography and contrast radiography are methods used to determine gender and evaluate breeding problems. Contrast radiography was performed by inserting a radiopaque medium into the cloaca to fill cavities, such as inverted hemipenes(Vetere et al., 2022). Ultrasound was conducted using a 3-9 MHz transcutaneous convex probe and underwater scans for gender determination have also been conducted(Mathew et al., 2015).

In regard to reproduction, snakes are either oviparous (egg-laying) or viviparous (giving birth). Examples of viviparous snakes with a placenta, include boa constrictors and green anacondas. In contrast, most vipers do not have placental connections and lay eggs that hatch before parturition (previously defined as ovoviviparous).

Pythons are common snakes kept as pets among reptile keepers and all the species are oviparous ((Booth & Schuett, 2016);(Raharjo et al., 2008)). Malayopython reticulatus, formerly known as Python reticulatus, is the longest nonvenomous snake in South and Southeast Asia, reaching a length of up to 10 m(Somaweera, 2017). It is also a native to Indonesia, where many captive breeding programs are being held nationwide. Malayopython reticulatus is still being heavily exploited because of the high demand for its skin for fashion commodities or captured alive to be sold as a pet ((Murray-Dickson et al., 2017)).

As reticulated pythons are being heavily exploited, the development of local breeders and breeding programs establishment are essential. An important aspect of a snake breeding program is an accurate gender determination. Efficient and minimally stressful gender determination methods is needed for breeders to achieve the highest success rate. Improper methods can not only slow down the breeding program rate, but also raise welfare concerns for many animals. This study aimed to compare probing and contrast radiography methods, for gender determination of reticulated pythons in terms of efficiency and accuracy.

MATERIALS AND METHODS

Materials and Tools

Materials

Reticulated pythons used in this study were selected based on availability and the willingness of owners to participate. These pythons varied in body length from 1.2 m to 1.7 m, representing a diverse range of sizes to ensure the applicability of the methods across different snake dimensions. The animals used were prepared without knowing their gender. Each snake was first subjected to manual probing, followed by contrast radiography to determine the gender. This procedure was conducted 2 times at an interval of 7 days to allow sufficient time for the contrast agent to clear from the lumens of the reproductive organs (cloaca and hemipenes). The interval was calculated from day 1 (the day of the first procedure) to day 7 (the day of the second procedure). Supporting materials used were Iopamidol (Iopamiro 300 mg/mL), KY jelly lubricant, and 70% alcohol.

Tools

Tools used in this study were a 0.07-inch diameter snake probe, digital X-ray machine, 24 G polyethylene catheter, cloth, and snake hook. This experiment was designed not to harm the experimental snakes and followed the established protocol for handling and care. All procedures were approved by the Animal Ethics Committee of the School of Veterinary Medicine and Biomedical Sciences of IPB University.

Procedures

Manual Probing

Snake was handled manually and restrained without sedation or anesthesia. During data collection, a single probe was used to handle one snake. The probe was lubed with KY jelly lubricant, placed within the vent, and directed laterally and caudally. Furthermore, the probe was gently advanced into the base of the tail toward the tip. Rotating the probe while gently advancing helps minimize trauma and allows the instrument to pass deeply into the hemipenis or shallowly into the female blind diverticulum. Both the right and left hemipenes and diverticulae were probed. Subsequently, a radiograph of the tail region was obtained,

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