6 Evaluation of reproductive status using near infrared spectroscopy in an endangered anuran

Follicular developmental stage is a vital physiological parameter that informs management decisions surrounding breeding programs in several industries, including agriculture, medical research, and conservation. Anuran (i.e. frog or toad) conservation programs are one area that require immediate attention because of the ongoing amphibian extinction crisis. Traditional hormone monitoring methods have proven useful for tracking the reproductive status of many female vertebrate species but are especially difficult to conduct in amphibian species due to the ample time and extensive knowledge needed to validate assays with low hormone sensitivity. Although ultrasonography is effective for assessing follicular development, image interpretation requires species-specific knowledge, and grading scales may not translate well across species or genera. Near infrared spectroscopy (NIRS) systems are portable and cost-effective, and have the advantage of requiring little expertise to interpret results once a model has been calibrated. As opposed to most single-purpose assays, NIRS is a rapid and noninvasive biophotonic tool that captures a suite of biochemical information from a single spectral signature. Because of the porous nature of amphibian skin, which resembles a fully functioning endocrine gland, it is possible to apply NIRS in vivo. The goal of this study was to detect and discriminate follicular developmental stage in endangered Anaxyrus houstonensis, a model anuran species, using NIRS validated with ultrasound imaging. NIR spectra (N = 240) were collected in triplicate from the cloacal region of females (N = 80) at the Fort Worth Zoo during their breeding season. The reflectance spectra for each female were categorised according to their ultrasound grade designation, which ranged from 0 to 3. These designations were split into two groups: a low grade (grades 0–1, no or low follicular development) and a high grade (grades 2–3, advanced follicular development). Balanced datasets of spectra representing each ultrasound grade category were used to perform multivariate analysis in the near infrared range (700–2500 nm). Principal component and linear discriminant analyses (PCA-LDA) were conducted to reduce the dimensionality of the dataset, detect outliers, and create a prediction model to assess reproductive state. PCA-LDA correctly classified the reproductive state of female toads with an accuracy ≥80% for the training and cross-validation datasets and ≥70% in the test set. This demonstrates the capability of NIRS for assessing reproductive status in the current amphibian model. Fast and effective evaluation of reproductive states is critical to amphibian breeding programs, because only individuals with sufficiently advanced follicular stages should be selected to receive hormones to induce ovulation and spawning. Additional spectral data are needed to increase the accuracy of the prediction model, as it would be beneficial to delineate between ultrasound grades for targeted hormone therapy to advance follicular development from earlier stages.