Molecular vs. endoscopic sexing of domestic pigeons (Columba livia domestica): accuracy, sample type, and practical recommendations for bird sexing

Abstract

The domestic pigeon ( Columba livia domestica ) lacks distinct sexual dimorphism, making sex determination based on external morphology unreliable. This species can serve as a model for evaluating avian sex determination methods and identifying minimally invasive and effective techniques for monomorphic birds. This study aimed to compare several sexing approaches and assess their reliability. Twenty-five deceased pigeons were examined. Sex was initially determined by celioscopy, followed by necropsy and direct visualization of the gonads. During necropsy, feather samples, oral swabs, and blood clots were collected. DNA was extracted from each sample type, and polymerase chain reaction (PCR) amplification was performed for molecular sex determination. Molecular sexing correctly determined sex in 100% of cases (95% CI: 86.7%–100.0%), whereas necropsy accurately identified sex in 84.0% (95% CI: 65.3%–93.6%) of cases and celioscopy in 72.0% (95% CI: 52.4%–85.7%). Juvenile pigeons with small or underdeveloped gonads could only be accurately sexed using molecular methods. Among the sample types, blood clots showed the highest amplification success (100%; 95% CI: 86.7%–100.0%) and DNA concentration (181.68 ± 138.83 ng/μL), followed by oral swabs (88.0%; 95% CI: 70.0%–95.8%; 84.69 ± 79.85 ng/μL) and feathers (80.0%; 95% CI: 60.9%–91.1%; 9.54 ± 11.07 ng/μL). Blood clot samples provided the highest success rate for molecular sexing; however, oral swabs represent a practical and minimally invasive alternative. Molecular sexing using oral swabs is therefore a reliable method for sex determination in pigeons, including newly hatched chicks, and represents a valuable tool for breeders and avian practitioners.