117 DEVELOPMENT OF A NONINVASIVE, FECAL PROTEIN PREGNANCY TEST FOR POLAR BEARS

Abstract

Differentiating between pregnancy and nonpregnancy noninvasively is difficult in species that experience pseudopregnancy, including polar bears (Ursus maritimus). These bears usually breed in the spring, undergo delayed implantation until late summer or early fall, and give birth in late fall. In other species, the placental protein transthyretin (TTR) has been shown to be essential for early fetal growth, responsible for transporting thyroid hormone from the mother to the fetus during early pregnancy. Preliminary data obtained via 2D-DIGE indicated that fecal TTR is elevated during polar bear pregnancy, but further research is needed to validate its use as an accurate biomarker of pregnancy. The aim of this study was to develop a bench-side assay to characterize TTR in longitudinal fecal samples from pregnant and nonpregnant polar bears. Specific objectives were to 1) develop an effective method for total fecal protein extraction; 2) identify antibodies that cross-react with polar bear fecal TTR; 3) validate an EIA for measuring fecal TTR; and, for proof of concept, 4) compare fecal TTR concentrations among pregnant, pseudopregnant, and nonpregnant polar bears. Fecal samples (n = 205) were collected from females that produced cubs (pregnant; n = 2), did not breed but exhibited a prolonged increase in progesterone (P4; pseudopregnant; n = 2), or did not breed and did not exhibit an increase in P4 (nonpregnant; n = 2). Total protein of each sample was extracted using a modified ammonium sulfate method. Protein concentrations were quantified using the Bradford assay. Western blot was used to determine commercial antibody compatibility with polar bear fecal TTR. A sandwich EIA was optimized using a standard curve ranging from 12.5 to 400 pg of human TTR/well. A parallelism was performed using two-fold serial dilutions of pooled fecal protein. Fecal TTR concentrations were measured in duplicate and are reported as femtograms of TTR per micrograms of total protein. Mean TTR concentrations in samples collected from July–December were compared among groups. Values are reported as mean ± standard error of the mean. Total fecal protein yield was 459.38 ± 284.38 µg g^–1 of feces. Successful binding of 2 polyclonal antibodies to the 15-kDa subunit of the TTR protein was verified via western blot and indicated that fecal proteins can retain antibody-binding capacity. The parallelism exhibited strong correlation with the standard curve (R² = 0.989). Data suggest that fecal TTR is higher during late summer or early fall in pregnant bears (588.40 ± 116.52) when compared to the pseudopregnant state (111.23 ± 15.98) and the nonpregnant state (250.83 ± 34.34). This is the first EIA developed to measure fecal TTR in any species. Although preliminary, these data suggest that the fecal TTR EIA may be useful in diagnosing pregnancy in polar bears.