Biomarkers of reproductive health in wildlife and techniques for their assessment

Historically, reproductive health in wildlife species has been evaluated primarily via immunoassay detection of fecal and urinary steroid hormone metabolites. This combination of sample type, biomarker category, and assay has been preferred for decades due to the ease of assessing reproductive health through the evaluation of stable compounds in easily collected biological samples using a cost-effective method. Increasingly, beginning with high performance liquid chromatography (HPLC) and more recently with convergence chromatography and ultra HPLC coupled with mass spectrometry (MS), wildlife studies are incorporating more sensitive and specific high-throughput technologies for the assessment of not only steroid hormone metabolites but proteins as well. Of note, a comprehensive health evaluation requires the measurement of biological readouts that modulate reproduction such as: glucocorticoids, leptin, insulin, thyroid hormones, melatonin, the microbiome, and markers of inflammation. Emerging modulatory biomarkers of reproductive health include acute phase proteins, microRNAs, and reactive oxygen species. Several of these biomarkers require application of newer technologies such as LC-MS/MS and sequencing, which demonstrates the need for the field of wildlife reproductive biology to diversify from its reliance on immunoassays. Importantly, endocrine disrupting chemicals adversely affect many aspects of reproductive function and evaluation of these compounds requires high throughput technology such as LC-MS/MS. The application of sequencing, particularly Next Generation Sequencing of bulk RNA (RNA-Seq) and single cell RNA-Seq, is uncommon in studies of wildlife reproductive health. However, as the cost of these methods decreases and consortiums of wildlife researchers band together to raise funds in support of studies using these technologies, their use will become more routine. Future research should focus on integration of known biomarkers of related systems into comprehensive reproductive assessments and the development of new biomarkers which are sensitive, precise, and employ non-invasive methodologies for the assessment of reproductive health of wildlife species.