Steroid hormone analysis in blow (respiratory vapor) may provide a minimally-invasive way to assess the reproductive status of cetaceans in aquaria or the wild, but biological validation of the method is needed to allow for the interpretation of hormone measurements in blow samples.1,2 Utilizing samples collected from trained belugas (Delphinapterus leucas), the aims of this project were to validate an assay for progesterone in beluga blow, determine if progesterone concentrations in blow correlated with those in matching blood samples, and to evaluate the ability to assess the pregnancy status and ovarian function of the beluga using blow progesterone concentrations. Utilizing pooled female blow samples, an enzyme immunoassay for progesterone was validated for use with beluga blow samples following extraction with diethyl ether. The binding of a serially diluted pool of ether extracted female blow samples was parallel to the standard curve (F(1,6) = 0.05, p = 0.83). The recovery of progesterone from spiked blow sample pools was 102 ± 17% (y = 1.06x – 5.62, R2 = 0.97). The slope of the regression line was not significantly different from 1 (95% CI [0.74, 1.37]), demonstrating accuracy. The extraction efficiency for blow ranged from 100% to 117%, with a mean of 110%. Progesterone concentrations in 64 matching blood and blow samples from 11 females were positively correlated (F(1,62) = 94.0, p < 0.0001). A total of 167 samples collected from 12 females of various reproductive stages were assayed for progesterone. Using mixed models to account for repeated sampling from the same individuals, progesterone concentrations in blow were shown to vary significantly by reproductive status; pregnant females (410.6 ± 87.8 pg/ml) and females in the luteal phase of the estrous cycle (339.5 ± 51.0 pg/ml) had higher blow progesterone concentrations than non-pregnant females without a corpus luteum (242.5 ± 27.3 pg/ml). A longitudinal change in blow progesterone was observed that corresponded with a change in pregnancy status or ovarian function in three females that were pregnant and one female with a non-conceptive estrous cycle during the study period. While additional sampling will be required to fully develop the diagnostic value of this method, only pregnant belugas had progesterone concentrations in blow > 420 pg/ml. In concert with testosterone measurements, blow sampling may ultimately be used to detect maturity, seasonality, pregnancy status, or ovulation in belugas in aquaria, or wild belugas that are temporarily stranded in areas such as the Cook Inlet, Alaska, providing a minimally invasive way to identify the reproductive status of an individual.
This research is made possible by the beluga training and veterinary staffs at the Mystic Aquarium (especially Kristine Magao, Kate McElroy, Carey Richard, Allison Tuttle and Allyson McNaughton), Georgia Aquarium (especially Cara Field and Tonya Clauss), SeaWorld San Antonio (especially Shannon Bond, Carley Lindgren, Kristen Werner, Steve Aibel, Kristie McCann, and Sherry Dickerson), and Shedd Aquarium (especially Lisa Takaki, Maris Muzzy and Frank Oliaro). Special thanks to Mandy Keogh and Laura Thompson for their guidance and to Renee Bakker and Crysania Brady for laboratory assistance. This project was supported by funding from the Marine Mammal Commission, the Sea Research Foundation, SeaWorld and Busch Gardens Reproductive Research Center, and the University of Rhode Island Enhancement of Graduate Research Award. This material is based upon work supported by the National Science Foundation Graduate Research Fellowship.
* Presenting author
+ Student presenter
1. Hogg CJ, Rogers TL, Shorter A, Barton K, Miller PJO, Nowacek D. Determination of steroid hormones in whale blow: it is possible. Mar Mamm Sci. 2009;25:605–618.
2. Hunt KE, Rolland RM, Kraus SD. Detection of steroid and thyroid hormones via immunoassay of North Atlantic right whale (Eubalaena glacialis) respiratory vapor. Mar Mamm Sci. 2014;30:796–809.