Dataset: seal_and_prey_fatty_acids
Deployment: ASET_seals_and_prey

Methods below are from Bromaghin et al. (2013):

Seal samples
From 2007 to 2008, harbor seals were captured at 3 sites in the San Juan Islands of Washington State (Padilla Bay, Vendovi Island, and Bird Rocks), and at a fourth site in the adjacent Gulf Islands of British Columbia (Belle Chain Islets). Seals were captured in salmon landing nets, restrained, and processed following Jeffries et al. (1993). The left side of the pelvic region was shaved with a razor, rinsed with isopropyl alcohol, scrubbed with Betadine, and again rinsed with isopropyl alcohol. A complete cross-section of blubber from skin to muscle was collected with a sterile 6-mm biopsy punch. The biopsy site was then filled with antiseptic cream and left open to drain. Samples were placed immediately in chloroform with 0.01% butylated hydroxytoluene in glass vials with Teflon lids, placed on ice while in the field, and subsequently stored frozen at –80 degrees C until analysis.

Prey samples
Fish and cephalopod species that are known to be consumed by harbor seals were sampled. Some adult salmon samples were obtained from seafood processors and staff of the NOAA Northwest Fisheries Science Center (NWFSC). The lab ID numbers of the NWFSC samples begin with "SOW" (sockeye), "PKW" (pink), and "03AC" (blackmouth chinook). Other prey were captured throughout the study area between June and December of 2008 using a variety of gear (including hook and line, longline, and trawl). Samples were obtained from the following species: black (Sebastes melanops), yellowtail (S. flavidus), copper, and Puget Sound (S. emphaeus) rockfish; Chinook, chum (Oncorhynchus keta), coho (O. kisutch), sockeye (O. nerka), and pink (O. gorbuscha) salmon; Pacific herring, walleye pollock; Pacific sand lance (Ammodytes hexapterus); northern anchovy (Engraulis mordax); shiner perch (Cymatogaster aggregata); plainfin midshipman (Porichthys notatus); spiny dogfish (Squalus acanthias); opalescent inshore squid (Loligo opalescens); kelp greenling (Hexagrammos decagrammus); Pacific staghorn sculpin (Leptocottus armatus); and starry flounder (Platichthys stellatus). Prey samples were placed in airtight plastic bags and stored at -80 degrees C.

In the lab, specimens were given unique ID numbers, partially thawed, weighed and measured, and homogenized with a mechanical blender. The smallest speicmens were homogenized with a mortar and pestle. Stomach contents were not removed from prey specimens, to mimic ingestion by predators. Samples of 5-10 g of homogenate were placed in scintillation vials and stored in a -80 degree C freezer, then shipped on dry ice to the Applied Sciences, Engineering, and Technology (ASET) Laboratory at the University of Alaska, Anchorage.

Fatty acid extraction
All samples were processed at ASET through the use of a Dionex ASE 200 automated solvent extraction system (Thermo Fisher Scientific, Waltham, Massachusetts). The total body mass, percent fat composition, and fat mass of prey specimens were obtained. Total mass data were not available for mature Chinook, sockeye, and pink salmon obtained from the Northwest Fisheries Science Center; therefore, an approximate mean mass for these prey classes (e.g., Quinn, 2005) was used in calculation of fat mass.

Extracted lipids were dissolved in hexane to a concentration of 100 mg/mL, hydrolyzed by a base-catalyzed reaction with potassium hydroxide, and then esterified to form fatty acid methyl esters (FAMEs) by reaction with boron trifluoride in methanol. Each sample was spiked with a C21:0 internal standard (25 ug/mL) and separated on a Hewlett-Packard 5890 gas chromatograph (GC) with flame ionization detector (FID) (Hewlett-Packard Co., Palo Alto, California), using a 60-m J&W DB-23 column (Agilent Technologies, Inc., Santa Clara, California) with a 0.25-mm inside diameter and 0.25-um cyanopropyl polysiloxane film. Signal data were collected and analyzed with Agilent GC Chemstation software.

Supelco 37-Component FAME Mix (Catalog No. 47885-U; Sigma-Aldrich Co., St. Louis, Missouri) was used as a continuing calibration verification (CCV) to verify both the retention times and recovery values. This CCV also contained 25 ug/mL of a C21:0 internal standard, which is required to meet a tolerance of no greater than +/- 20% of actual value. Analyte identity was verified further by mass spectrometry through the use of a Varian CP3800 GC (Agilent Technologies, Inc.) with a Varian Saturn 2200 ion trap mass spectrometer with a scan range of 50–400 mass-to-charge ratios (m/z). Additionally, a National Institute of Standards and Technology 1946 international standard was used to externally verify the method and the quality of recoveries.

(See ASET's Fatty Acid Methyl Ester(FAME) Analysis SOP, also available on the ASET website for more on their methodologies.)