MARINE MAMMAL SCIENCE, 22(3): 683–689 (July 2006)C© 2006 by the Society for Marine MammalogyDOI: 10.1111/j.1748-7692.2006.00046.x

A REMOTE BIOPSY SYSTEM USED TO SAMPLE STELLERSEA LION (EUMETOPIAS JUBATUS) BLUBBER

LAURA K. HOBERECHT

Washington Cooperative Fish and Wildlife Research Unit,School of Aquatic and Fishery Sciences,University of Washington, Box 355020,Seattle, Washington 98195-5020, U.S.A.

E-mail: llitzky@u.washington.edu

DANIEL J. VOS

Alaska Regional Office,National Marine Fisheries Service, NOAA,

222 W. 7th Avenue, Box 43,Anchorage, Alaska 99513, U.S.A.

GLENN R. VANBLARICOM

U.S. Geological Survey, Biological Resources Division,Washington Cooperative Fish and Wildlife, Research Unit,

School of Aquatic and Fishery Sciences,University of Washington, Box 355020,Seattle, Washington 98195-5020, U.S.A.

The analysis of blubber fatty acids has become a valuable tool for studying pinnipeddiet and foraging ecology (Iverson et al. 1997, Smith et al. 1997, Bradshaw et al. 2003,Walton and Pomeroy 2003, Anderson et al. 2004, Iverson et al. 2004). In the past,blubber samples from pinnipeds were collected primarily from physically restrainedanimals or carcasses. For many species, this has made the systematic sampling ofhealthy individuals of subadult and older age classes intractable. Remote systemshave been used successfully with pinnipeds to deliver pharmaceuticals (Loughlin andSpraker 1989) and to collect skin (Gemmell and Majluf 1997, Wiig et al. 2000).To our knowledge, this technique has never before been used successfully to collectblubber from pinnipeds for fatty acid analysis. Here, we provide the description of aremote biopsy system designed to sample pinniped blubber and details regarding itssuccessful use with Steller sea lions (Eumetopias jubatus (Schreber, 1776)).

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Figure 1. The three part biopsy dart head consisting of: (1) stopper piece, (2a) long biopsytip, (2b) short biopsy tip, (3a) long retention device with washer, and (3b) short retentiondevice with washer.

Two Barnett crossbows, the Wildcat III recurve and the Rhino Sport Magnumcompound (Barnett International USA, Odessa, FL), were used to propel biopsy darts.An open-faced bow fishing reel (Game Tracker, Inc., Flushing, MI) was attached toeach crossbow and loaded with 80 lb. Spectra� fiber line by Power Pro (InnovativeTextiles, Inc., Grand Junction, CO) to tether the darts. Crossbows were chosen overpneumatic rifles for the ease of tether attachment.

The design of dart heads was based on those of Karesh et al. (1987) and Gemmelland Majluf (1997) with the syringe barrel removed and sized to screw into a standardcrossbow bolt. The heads consisted of three parts: stopper, biopsy tip, and retentiondevice (Fig. 1). The stopper prevented darts from penetrating further than the lengthof the biopsy tip on impact and were constructed of 303 stainless steel. They weredrilled with a 3.175 mm hole for tether attachment and had 8–32 threads on one endto screw into the crossbow bolt and M8 × 1.25 threads on the other end for biopsytip attachment. The end for biopsy tip attachment was drilled with a 3.302 mmdiameter hole 9.525 mm deep in which the retention device would sit. Biopsy tipscut, extracted and contained the sample and were constructed of 440C stainless steel

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heat-treated to 58–62 Rc. Tips had inner and outer diameters of 6.858 mm and 8.89mm, respectively, and a cutting edge with inner and outer angles of 18◦ and 35◦,respectively. A 35-mm-long tip was used to sample juveniles and adult females, anda 50 mm long tip was used to sample adult males. These lengths were determinedfrom average blubber thickness measurements obtained from 1,237 Steller sea lions.1

Biopsy tips had a small venting hole located 13.462 mm from the base. They wereconstructed with an interior ledge at 15.24 mm from the base with a through holeof 5.334 mm used to hold the retention device in place. Retention devices consistedof three barbed broaches of varying lengths (47 mm via Henry Schein, Palatine, IL;28 mm via Roydent Dental Products, Rochester Hills, MI) held together with epoxyin a plastic tube sized to fit into the hole in the leading end of the stopper. The threebroaches within a retention device were cut such that the first broach extended tothe end of the biopsy tip, the second broach extended to the end of the barbs on thefirst broach, and the third broach extended to the end of the barbs on the secondbroach (Fig. 1). This maximized the likelihood of retaining a sample by providingbarbs at varying angles within nearly the entire length of the biopsy tip. Retentiondevices were held in place by a #4 nylon flat washer placed under the interior ledgeof the biopsy tip. Stoppers and biopsy tips were manufactured by Wild West Guns,Inc. (Anchorage, AK) in 2002 and by Quality Manufacturing (Marysville, WA) in2003–2004. Retention devices were constructed by LKH and DJV. Equipment wastested on pinniped carcasses and the biopsy sites inspected by a veterinarian2 to insurethat no extensive trauma had occurred before use with free-ranging animals.

All parts of the biopsy head were sterilized, using an autoclave (pre-trip preparation)or disinfected with boiling water (in the field), prior to each use. Stoppers and biopsytips were reused multiple times. Biopsy tips were sharpened before each use. Retentiondevices were used only once due to dulling of barbs. After a strike was made, dartswere retrieved as quickly as possible for initial processing of samples. Total lengthof sample was recorded to the nearest 1.0 mm as well as the length of each tissuetype (skin, blubber, muscle). Skin and hair were removed from the sample and placedin dimethyl sulfoxide (DMSO). If the sample contained muscle, it was removed,wrapped in aluminum foil, and placed on ice. Blubber was placed into preweighedglass vials containing chloroform (CHCl3) with 0.01% 2,6-di-tert-butyl-4-methyl-phenol (BHT) and stored on ice while in the field. If distinct structural differenceswere apparent in the blubber, it was divided into layers. Once in the laboratory, vialscontaining blubber samples were flushed with nitrogen gas and blubber and musclesamples stored at −80◦C until further processing.

Sampling was conducted April to July 2002–2004 across the Alaskan range ofSteller sea lions. Animals on haul-outs were sampled from land, and animals inthe water and on rock outcroppings were sampled from a skiff. Wind speed wasmeasured throughout each sampling day to be sure darts would not be pushed off

1 Personal communication from K.W. Pitcher, Alaska Department of Fish and Game, Wildlife Con-servation, 525 W. 67th Ave., Anchorage, Alaska 99518, U.S.A., October 2001.

2 Personal communication from F. M. D. Gulland, The Marine Mammal Center, Marin Headlands,1065 Fort Cronkhite, Sausalito, California 94965, U.S.A., April 2002.

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Table 1. Summary of use and success of the remote biopsy system. Mode refers to whereanimals were located when sampled (land: haul-out with many animals, water: water, rock:rock with small number of animals). Percentages of tissue types obtained are based on thenumber of darts recovered.

Mode Days Strikes Recovered Skin Blubber Muscle

Land 47 289 261 (93.21%) 252 (96.55%) 247 (94.64%) 78 (29.89%)Water 3 6 5 (83.33%) 5 (100%) 5 (100%) 2 (40%)Rock 3 10 10 (100%) 9 (90%) 9 (90%) 3 (30%)

Total 53 296 276 (93.24%) 266 (96.38%) 261 (94.57%) 83 (30.07%)

track. Sampling was aborted when wind speed was greater than 13 knots. Sampleswere collected from either the shoulder or pelvic region of animals depending on theirpositioning. To achieve penetration, darts were fired 8–15 m (recurve) or 15–20 m(compound) from animals. Animals on land were carefully stalked from downwindto within 20 m and observed until an appropriate target animal was identified. Wepreferred to sample an animal without conspecifics between it and the researcherto prevent the tether from tangling. Thus, animals on the edges of the haul-outswere generally selected. In-water sampling could only occur when shallow waterwith good visibility was available. Animals were followed near-shore so researcherscould observe them beneath the surface. One animal was watched exclusively andthen sampled during a surfacing event. Animals on small rock outcroppings wereapproached from downwind slowly with a skiff. Attempts were made to remain outof the animal’s sight as long as possible and to take the shot as soon as within therange for dart penetration.

The remote biopsy system performed well, with adequate depth samples collectedfor fatty acid analysis from the majority of darts recovered (Table 1). The amountof lipid extracted from blubber samples (n = 261) ranged from 0.0008 to 0.3443g. The majority of samples were collected from land. Land-to-land sampling alsoyielded the best results for effort with an average of 6 samples collected per day anda maximum of 18 samples collected in 1 d (vs. 3 and 8 for water and rock modes,respectively). Of 296 biopsy darts that struck animals, 20 were not recovered. All ofthe losses occurred when the tether broke and the dart fell into the water. When thetether broke and the dart remained on land the sample could later be retrieved byhand. The three apparent reasons for breakage of the tether were improper alignmentof the reel, abrasions on the line caused by prior use across rocks and barnacles, andanimals running across the line and becoming momentarily entangled. It should benoted that upon entering the water the line easily came free of the tangled animalallowing the researcher to pull in the line (and often the submerged dart as well).

Four biopsy darts were recovered that did not contain any tissue (Table 1). Althoughit was not entirely clear why these darts failed, we offer two possible explanations,both relating to the barbed broaches used in retention devices. It appeared that ifa broach extended beyond the cutting edge of the biopsy tip, the end would bendon impact preventing the retention of sample. Additionally, broaches have different

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Table 2. Summary of success of the biopsy system with different sex and age classes (Ad:adult). Ad male group includes subadult males. Percentages of tissue types obtained are basedon the number of darts recovered.

Mode Strikes Recovered Skin Blubber Muscle

Juvenile 44 38 (86.36%) 35 (92.11%) 34 (89.47%) 17 (44.74%)Ad female 123 112 (91.06%) 111 (99.11%) 110 (98.21%) 49 (43.75%)Ad male 129 126 (97.67%) 120 (95.24%) 117 (92.86%) 17 (13.49%)

grades of barbs ranging from coarse to extra fine and the coarser grade barbs appearedto retain samples better than the finer grade barbs.

The biopsy system worked well on all age and sex classes sampled (Table 2). Animalsof both sexes and all age classes greater than 1 yr were targeted. Nursing females andpups were not sampled and animals of similar size to those observed nursing wereassumed to be less than 1 yr and were not sampled. The number of juvenile animalsdeemed to be greater than 1 yr found at study sites was low compared to adults. Thus,the discrepancy between numbers in animal ages sampled was due to availability, notresearcher target selection. Attempts were made to sample equal numbers of all ageand sex classes at each study site if possible. Sex of sampled animals will be confirmedvia genetic analysis of skin tissue.

The success rates of muscle collection for juveniles and females (Table 2) wereconsistent with the fact that average blubber depth measurements were used todesign dart tip length. A slightly longer tip may have been justified to increasethe number of full depth blubber samples; however, erring on the side of cautionreduces the risk of negative impact to animals and complies with current regulatoryrequirements. The low success rate of muscle collection for adult males indicates thata longer tip should have been used with this age/sex class to insure full depth blubbersamples.

Animals sampled in the water responded to being struck with the biopsy dart byswimming away rapidly. Researchers were not able to track animals after this point.Response of animals sampled on land to being struck with a dart was similar in allregions. Juveniles and adult females typically turned in the direction from whichthey were struck and then left the haul-out and entered the water. Many returnedto the haul-out within 5 min of being struck by the dart though it was not alwayspossible to track individuals once they entered the water. It is estimated that 95% ofjuveniles and 65% of adult females struck with biopsy darts left the haul-out. Adultand subadult males also tended to turn in the direction from which they were struck,but typically did not leave the haul-out site. It is estimated that 70% of subadultand adult males struck with biopsy darts did not leave the haul-out. This allowedresearchers to observe the site of dart penetration, sometimes for several hours.

Dart penetration resulted in a small circular hole in the skin that tended to bleedfor about 10 min. The amount of bleeding was affected by weather, with morebleeding observed on hot sunny days than cold rainy days. Dry animals also tendedto bleed more than wet animals. After 30–60 min it became difficult to discern thedart penetration site from other abrasions on the skin. No difference in response

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was noted as a result of sampling in the shoulder or pelvic region. Assessment oflong-term healing was not undertaken.

Animals on land responded strongly to retrieval of the tethered dart, with slowremoval causing greater disturbance than quick retrieval. The movement of thebrightly colored dart across the haul-out caused nearby animals to vocalize and moveto another area or into the water. Tidal height appeared to play a role in whetheror not animals left the haul-out and in how quickly they returned. Animals tendedto be more reluctant to leave the haul-out at low tide and tended to return to thehaul-out more quickly when the tide was rising. Sampling was not done when safeegress for the animals was not apparent.

Modifications to the biopsy system could improve the success rate and reduce usercosts. The addition of floatation material such as that used by Barrett-Lennard et al.(1996) would allow recovery of untethered darts in the water. However, there mightbe a trade-off in the accuracy or flight distance of darts with the additional material.Use of standard American threads on the tip (and subsequent threads on stopper)would allow manufacturers to use standard 0.375 in. stock as starting material whichwould greatly reduce the cost of construction.

The remote biopsy system has proven to be a highly successful technique forcollecting blubber samples from Steller sea lions. Tip lengths must be determinedon a species-specific basis to avoid injury to the animal and obtain adequate samples.Length of tips should optimally be determined by species, sex, and age of individualsto be sampled as blubber thickness differs greatly among these variables.

ACKNOWLEDGMENTS

We thank Caroline Kurle for the idea of sampling blubber remotely and the initial designof the triple prong retention device. We also thank Lowell Fritz, David Hoberecht, MichelleLander, Barbara Mahoney, Heather Smith, Ward Testa, Jim Thomason, Brogan Vos, Peg Vos,Jamie Womble, and Kate Wynne for assistance in the field. Patrick Kosbruck, Buck Laukitis,Peter Ord, Tom Walters, and Marvin Yagie safely transported us to remote field locations.The Marine Mammal Center and the Washington Department Fish and Wildlife providedpinniped carcasses for testing equipment. Advice and support was also provided by LanceBarrett-Lennard, Chad Jay, Billy Karesch, Todd Loomis, Tom Loughlin, Sally Mizroch, RodTowell, Mike Strick, and Dave Withrow. Funding for this project was provided by the StellerSea Lion Research Initiative of the National Marine Fisheries Service. Additional support wasprovided by the Washington Cooperative Fish and Wildlife Research Unit and the Schoolof Aquatic and Fishery Sciences at the University of Washington. This study was approvedby the University of Washington Institutional Animal Care and Use Committee (Protocol# 2887-08). In 2002, work was conducted under U.S. federal permit #782-1532-00 and in2003–2004 under U.S. federal permit # 1016-1651-02.

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Received: 7 June 2005Accepted: 2 November 2005