# Validating a remote saliva collection tool for genomic analyses in free ranging dogs

## TL;DR

This study introduces a non-invasive saliva collection method for free-ranging dogs, enabling genomic research without direct handling.

## Contribution

The study presents a novel hands-off saliva collection method suitable for genomic analyses in free-ranging animals.

## Key findings

- Hands-off saliva collection yielded DNA suitable for genotyping despite lower DNA concentrations compared to direct methods.
- The hands-off method reduces sampling bias toward bold individuals in free-ranging animal studies.
- The method could be adapted for use in various species, including elusive wildlife.

## Abstract

Saliva is a well-established source of DNA for various applications due to its non-invasive collection and its provision of high-quality DNA. However, its use in wild and free-ranging animal research remains limited due to challenges in collection without direct animal handling. In this study, we developed and evaluated a hands-off saliva collection method designed for free-ranging domestic dogs (FRDs), serving as a model for non-invasive genetic sampling of wildlife. Our method utilized a funnel paired with a commercially available Performagene kit (DNA Genotek, Canada), presented to the dog in the presence of an operator. The dog was free to approach and interact with the apparatus, depositing saliva while trying to reach bait. We compared DNA yield and genotyping success from samples using this hands-off method with those collected via the manufacturer’s recommended method. We collected 461 saliva samples from 326 FRDs, performing 750 DNA extractions. Samples collected by hand yielded significantly higher DNA concentrations after the first extraction attempt (mean = 46.3 ng/µL) than those collected using the hands-off method (mean = 32.2 ng/µL). Despite lower DNA concentrations, genotyping success did not significantly differ between methods, demonstrating that the hands-off method can yield DNA suitable for genomic analyses. The hands-off saliva collection method is a viable alternative to invasive sampling, addressing ethical concerns and enabling genomic studies in wild animals. Furthermore, our method mitigates sampling bias toward bold individuals, a common limitation in behavioral and genetic studies of free-ranging animals. With minor adaptations, this method could be applied across various species, including more elusive ones, contributing to conservation genetics and behavioral ecology research.

The online version contains supplementary material available at 10.1038/s41598-025-19267-1.