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Monitoring the reproductive characteristics of a species can complement existing conservation strategies by understanding the mechanisms underlying demography. However, methodology to determine important aspects of female reproductive biology is often absent in monitoring programs for large mammals. Protein biomarkers may be a useful tool to detect physiological changes that are indicative of reproductive state. This study aimed to identify protein biomarkers of reproductive status in serum collected from free-ranging female brown bears (Ursus arctos) in Alberta, Canada, from 2001 to 2018. We hypothesized that the expression of proteins related to reproduction in addition to energetics and stress can be used to answer specific management-focused questions: (i) identify when a female is pregnant, (ii) detect if a female is lactating, (iii) determine age of sexual maturity (i.e. primiparity) and (iv) assess female fertility (i.e. reproduction rate). Furthermore, we investigated if silver spoon effects (favourable early life conditions provide fitness benefits through adulthood) could be determined using protein expression. A target panel of 19 proteins with established relationships to physiological function was measured by peptide-based analysis using liquid chromatography and multiple reaction monitoring mass spectrometry and their differential expression was evaluated using a Wilcoxon signed-rank test. We found biomarkers of pregnancy (apolipoprotein B-100 and afamin), lactation (apolipoprotein B-100 and alpha-2-macroglobulin) and sexual maturity (corticosteroid-binding globulin), but there were no statistically significant relationships with protein expression and fertility. The expression of proteins related to reproduction (afamin) and energetics (vitamin-D binding protein) was associated with the nutritional quality of the individual’s present habitat rather than their early life habitat. This study highlights potential biomarkers of reproductive status and provides additional methods for monitoring physiological function in wildlife to inform conservation.

Large carnivores play critical roles in the maintenance and function of natural ecosystems; however, the populations of many of these species are in decline across the globe. Therefore, there is an urgent need to develop novel techniques that can be used as sensitive conservation tools to detect new threats to the health of individual animals well in advance of population-level effects. Our study aimed to determine the expression of proteins related to energetics, reproduction and stress in the skin of grizzly bears (Ursus arctos) using a liquid chromatography and multiple reaction monitoring mass spectrometry assay. We hypothesized that a suite of target proteins could be measured using this technique and that the expression of these proteins would be associated with biological (sex, age, sample location on body) and environmental (geographic area, season, sample year) variables. Small skin biopsies were collected from free-ranging grizzly bears in Alberta, Canada, from 2013 to 2019 (n = 136 samples from 111 individuals). Over 700 proteins were detected in the skin of grizzly bears, 19 of which were chosen as targets because of their established roles in physiological function. Generalized linear mixed model analysis was used for each target protein. Results indicate that sample year influenced the majority of proteins, suggesting that physiological changes may be driven in part by responses to changes in the environment. Season influenced the expression of proteins related to energetics, reproduction and stress, all of which were lower during fall compared to early spring. The expression of proteins related to energetics and stress varied by geographic area, while the majority of proteins that were affected by biological attributes (age class, sex and age class by sex interaction) were related to reproduction and stress. This study provides a novel method by which scientists and managers can further assess and monitor physiological function in wildlife.