Abstract
Recently, blow (exhale) sampling has emerged as a non-invasive technique to collect information about the reproductive status or genetic makeup of individual cetaceans.1,2 However, the applicability of this method in free ranging cetaceans will benefit from controlled studies utilizing trained cetaceans. Research is ongoing with trained belugas to further develop blow sampling as a viable alternative to more invasive sampling techniques (e.g., biopsy or blood sampling) for steroid hormone and genetic analyses. For genetic analyses, field conditions necessitate fewer exhales per sample than have been previously required.1 The purpose of this study was to determine if single-exhale blow samples yield enough DNA for use in molecular gender determination in belugas. Ten single-exhale blow samples were collected from 4 belugas (2 female, 2 male) housed at the Mystic Aquarium. DNA was isolated from all 10 samples using Qiagen DNeasy blood and tissue kits with a modified protocol. A 1-hour lysis time was considered optimal, and an elution volume of 50 µl yielded DNA concentrations that were practical for downstream applications. Between 76 and 2966 ng of genomic DNA were isolated from each sample. Absorbance ratios indicated sufficient purity for use in PCR (Avg A260/A280 = 1.88). For all 4 individuals, molecular gender determination was possible with single-exhale DNA using the SRY (870 bp) and ZF genes (Y copy: 800 bp, X copy: 750 bp) by PCR amplification with beluga specific primers (94°C for 3 min; 35 cycles of 94°C for 30 sec, 55°C for 30 sec, 72°C for 30 sec; 72°C for 10 min). This study validates single-exhale blow sampling for molecular gender determination in belugas. Further research will investigate if reproductive hormone concentrations in blow correlate with levels in circulation in order to establish if blow sampling can be used to determine an unknown animal's gender and reproductive status. If so, this less invasive methodology for reproductive hormone monitoring may be especially desired for young or naïve cetaceans in zoological facilities. Blow sampling will also improve molecular and hormone research capabilities in free ranging cetaceans, especially within populations facing conservation challenges that preclude biopsy sampling.
Acknowledgements
This research is made possible by the beluga training staff at the Mystic Aquarium, especially Kristine Magao, Lindsey Nelson, and Kate McElroy. Elizabeth Baird and Christopher Card provided valuable laboratory assistance. This project is supported by the National Science Foundation Graduate Research Fellowship awarded to Justin Richard.
* Presenting author
+ Student presenter
Literature Cited
1. Frère CH, Krzyszczyk E, Patterson EM, Hunter S, Ginsburg A, Mann J. 2010. Thar she blows! A novel method for DNA collection from cetacean blow. PLOS One 5: e12299.
2. Hogg CJ, Rogers TL, Shorter A, Barton K, Miller PJO, Nowacek D. 2009. Determination of steroid hormones in whale blow: It is possible. Mar Mamm Sci 25: 605–618.