Review of cetacean biopsy techniques: Factors contributing to successful sample collection and physiological and behavioral impacts

Authors


Abstract

Biopsy techniques have been developed to collect skin and blubber samples through non-lethal methods. One sample can provide data on genetics, prey preferences, foraging ecology, contaminant loads, and physiological processes. The limited data available suggest that biopsy wounds heal quickly and that there are usually no discernable adverse health effects. Published accounts on factors contributing to the success of collecting biopsy samples and the behavioral impacts to cetaceans following biopsy sampling were standardized to permit statistical analysis. Several factors contribute to the success of acquiring samples; however, sampling rates do not differ significantly between delivery devices. Behavioral responses to biopsy sampling vary by species and other factors. The most predominant response for odontocetes is low, while low and moderate responses are equally prevalent for mysticetes. The use of retrieval lines may increase the occurrence of moderate and strong responses by mysticetes. These findings suggest that biopsy sampling is relatively benign, causing only minor and short-lived responses. However, most researchers do not report sufficient data to assess short- and long-term physiological and behavioral impacts. Finally, limited data suggest that biopsy sampling does not impact cetacean habitat use or distribution patterns. Yet these impacts are rarely investigated, so additional data are needed.

The population size and structure, physiology, foraging ecology, and other details of cetacean lifestyles are difficult to study because these animals spend much of their time beneath the water's surface, hidden from human observation. As humans only have limited access to these animals, mainly when they return to the water's surface to breathe, the dearth of cetacean life-history data is not surprising.

Due to the paucity of data and the necessity for understanding more about the lives of marine mammals, scientists have developed nonlethal methods for sample collection and analytical techniques to provide a wealth of information. One such method is the collection of skin and blubber biopsies that can be taken from cetaceans either when they surface to breathe or from animals that are captured and then released. The acquisition of fresh samples from free-ranging animals allows researchers to conduct tissue analyses that provide information on ecological, biological, and physiological patterns and processes. Biopsy samples collected from free-swimming cetaceans also enable researchers to compare parameters between specific individuals. These samples may also be more representative of the population than samples collected from dead or stranded animals that may be ill or emaciated.

Numerous cetacean species have been biopsied for a multitude of studies investigating genetic relationships, foraging ecology, contaminant burdens, and other physiological and biological processes (Table 1, 2). There are a wide variety of techniques that have been utilized to collect biopsies, and the optimal technique depends on many factors, including the focus of the investigation; the behavior, physiology, and morphology of the target species; and the platform from which sampling is being conducted. The decision to employ remote or manual biopsy methods is generally based on the body size and behavior of the species. For example, cetaceans that are small in body size or frequent shallow water can be biopsied via either manual or remote methods while larger cetaceans are biopsied via remote methods. Moreover, the delivery method for remote biopsy methods (e.g., pole, rifle or crossbow and the power of delivery) is dictated by the body size (e.g., small, medium, large), skin and blubber thickness, and the swimming speed of the cetacean being sampled as well as by the approach distance and maneuverability of the boat. Finally, the size of the dart or biopsy punch utilized is generally dictated by the sample that is required (e.g., skin or blubber and skin) and the depth and structure of the blubber layer.

Table 1.  Summary of studies that used biopsy methods to collect specific samples from odontocete species.
SpeciesGenetics (skin)Stable Isotopes (skin)Contaminants (blubber)Fatty Acids (blubber)Other
Beluga whale (Delphinapterus leucas)Brown Gladden et al. 1999, Hobbs et al. 2003 Andersen et al. 2001, Hobbs et al. 2003, Krahn et al. 2004Dahl et al. 2000, Krahn et al. 2004 
Long-beaked common dolphin (Delphinus capensis)Chivers et al. 2000    
Short-beaked common dolphin (Delphinus delphis)Chivers et al. 2000; Marsili et al. 2000; Bilgmann et al. 2007a, 2008; Möller et al. 2008 Fossi et al. 2000, 2003a, b; Marsili et al. 1996, 2000; Borrell et al. 2001 Fossi et al. 2000, 2003a, b (analysis of Benzo(a)pyrene monooxygenase [BPMO] activity in the skin), Kellar et al. 2009 (testosterone in blubber to assess reproductive status)
Dolphin species (Delphinid spp.)Leduc et al. 1999    
Pygmy killer whale (Feresa attenuata)Chivers et al. 2000    
Pilot whale species (Globicephala sp.)Chivers et al. 2000    
Risso's dolphin (Grampus griseus)Chivers et al. 2000, Marsili et al. 2000 Marsili et al. 2000  
Northern bottlenose whale (H. ampullatus)Hooker 1999, Gowans et al. 2000Hooker 1999, Hooker et al. 2001b Hooker 1999, Hooker et al. 2001b 
Pacific white-sided dolphin (Lagenorhynchus obliquidens)Chivers et al. 2000    
Dusky dolphin (Lagenorhynchus obscurus)Harlin et al. 1999, Chivers et al. 2000    
Northern right-whale dolphin (Lissodelphis borealis)Chivers et al. 2000    
Gray's beaked whale (Mesoplodon grayi)Gales et al. 2002    
Blainville's beaked whale (Mesoplodon densirostris)McSweeney et al. 2007    
Hector's beaked whale (Mesoplodon hectori)Gales et al. 2002    
Killer whale (O. orca)Barrett-Lennard et al. 1996; Worthy and Abend 1997; Hoelzel et al. 1998, 2002; Chivers et al. 2000; Ross et al. 2000; Pitman 2003; Baird et al. 2006; Pitman et al. 2007Worthy and Abend 1997 (note: blubber was analyzed because skin was unavailable), Herman et al. 2005; Krahn et al. 2007a, b, 2008, 2009Barrett-Lennard et al. 1996; Ross et al. 2000; Ylitalo et al. 2001; Rayne et al. 2004; Herman et al. 2005; Krahn et al. 2007a, b, 2008, 2009Barrett-Lennard et al. 1996; Worthy and Abend 1997; Herman et al. 2005, 2008; Krahn et al. 2007a, 2008 
Harbor porpoise (Phocoena phocoena)Chivers et al. 2000    
Dall's porpoise (Phocoenoides dalli)Chivers et al. 2000    
Harbor porpoise and Dall's porpoise hybrid (Phocoena phocoena and Phocoenoides dalli hybrid)Willis et al. 2004    
Sperm whale (P. macrocephalus)Winn et al. 1973, Whitehead et al. 1990, Lyrholm and Gyllensten 1998, Chivers et al. 2000, Ruiz-Cooley et al. 2004, Wise et al. 2009Ruiz-Cooley et al. 2004Taruski et al. 1975 Wise et al. 2009 (chromium levels in the skin)
False killer whale (Pseudorca crassidens)Chivers et al. 2000, 2007    
Indo-Pacific humpback dolphin (Sousa chinensis)  Leung et al. 2005  
Marine tucuxi dolphin (Sotalia fluviatilis)Cunha and Solé-Cava 2007    
Estuarine tucuxi dolphin (Guiana dophin, costero) (Sotalia guianensis)Cunha and Solé-Cava 2007    
Pantropical spotted dolphin (Stenella attenuata)Chivers et al. 2000 Borrell et al. 2004  
Spotted dolphin subspecies (Stenella attenuata subsp.)Chivers et al. 2000    
Striped dolphin (Stenella coeruleoalba)Aguilar and Nadal 1984, Chivers et al. 2000, Marsili et al. 2000 Aguilar and Nadal 1984; Focardi et al. 1992; Fossi et al. 1992; Aguilar and Borrell 1994b; Marsili and Focardi 1996; Marsili et al. 1996, 2000, 2004; Fossi et al. 2000, 2003a, b, 2004 Fossi et al. 1992, 2000, 2003a, b, 2004 (analysis of Benzo(a)pyrene monooxygenase [BPMO] activity in the skin), Fossi et al. 2008 (biomarkers in skin to assess toxicological stress), Spinsanti et al. 2008 (gene expression in skin cells after exposure to contaminants)
Atlantic spotted dolphin (Stenella frontalis)Winn et al. 1973    
Spinner dolphin (Stenella longirostris)Chivers et al. 2000, Johnston et al. 2008    
Rough-toothed dolphin (Steno bredanensis)Chivers et al. 2000    
Bottlenose dolphin (Tursiops truncatus)Chivers et al. 2000; Marsili et al. 2000; Berrow et al. 2002; Krützen et al. 2002, 2004; Parsons et al. 2003a, b; Tezanos-Pinto et al. 2009 Marsili et al. 1996, 2000; Fossi et al. 2000, 2003a, b; Berrow et al. 2002; Hansen et al. 2004; Wells et al. 2004; Litz et al. 2007; Pulster and Maruya 2008; Pulster et al. 2009Walton et al. 2007Bruce-Allen and Geraci 1985 (determine the wound healing process), Fossi et al. 2000, 2003a,b (analysis of Benzo(a)pyrene monooxygenase [BPMO] activity in the skin); Bryan et al. 2007 (trace elements in skin), Montie et al. 2007, 2008 (blubber morphology), Mollenhauer et al. 2009 (gene expression in skin cells after exposure to contaminants)
Bottlenose dolphin species (Tursiops spp.)Möller and Beheregaray 2001; Bilgmann et al. 2007a, b; Möller et al. 2008    
Cuvier's beaked whale (Ziphius cavirostris)McSweeney et al. 2007    
Table 2.  Summary of studies that used biopsy methods to collect specific samples from mysticete species.
SpeciesGenetics (skin)Stable Isotopes (skin)Contaminants (blubber)Fatty Acids (blubber)Other
Minke whale (Balaenoptera acutorostrata)Kasamatsu et al. 1991, Gauthier and Sears 1999 Gauthier et al. 1997a, b; Gauthier and Sears 1999Gauthier and Sears 1999 
Bryde's whale (Balaenoptera edeni)Chivers et al. 2000Gendron et al. 2001   
Blue whale (Balaenoptera musculus)Árnason et al. 1985, Árnason and Widegren 1989, Gauthier and Sears 1999, Chivers et al. 2000Gendron et al. 2001Gauthier et al. 1997a, b; Gauthier and Sears 1999Gauthier and Sears 1999 
Pygmy blue whale (Balaenoptera musculus brevicauda)Kato et al. 1996    
Fin whale (B. physalus)Jahoda et al. 1996, Marsili and Focardi 1996, Bérubéet al. 1998, Gauthier and Sears 1999, Chivers et al. 2000, Marsili et al. 2000Borobia et al. 1995, Gendron et al. 2001Focardi et al. 1991, 1992; Fossi et al. 1992; Marsili and Focardi 1996; Marsili et al. 1996, 1998, 2000; Gauthier et al. 1997a; Gauthier and Sears 1999; Fossi et al. 2000, 2003a, bBorobia et al. 1995, Gauthier and Sears 1999Fossi et al. 1992, 2000, 2003a, b (analysis of Benzo(a)pyrene monooxygenase [BPMO] activity in the skin)
Gray whale (E. robustus)Mathews 1986, Mathews et al. 1988 Krahn et al. 2001Krahn et al. 2001 
Southern right whale (E. australis)Kato et al. 1996, Patenaude et al. 1998, Baker et al. 1999, Malik et al. 2000, Best et al. 2005    
North Atlantic right whale (Eubalaena glacialis)Brown et al. 1991, Chivers et al. 2000, Malik et al. 2000 Brown et al. 1991, Woodley et al. 1991, Weisbrod et al. 2000  
Humpback whale (Megaptera novaeangliae)Winn et al. 1973; Mathews 1986; Mathews et al. 1988; Baker et al. 1990, 1993, 1998; Palsbøll et al. 1995, 1997; Weinrich et al. 1992; Clapham and Mattila 1993; Lambertsen et al. 1994; Medrano-González et al. 1995; Gauthier and Sears 1999; Chivers et al. 2000; Cerchio 2003; Bérubéet al. 2004; Garrigue et al. 2004; Acebes et al. 2007; Smith et al. 2006, 2009Lambertsen et al. 1994, Borobia et al. 1995Taruski et al. 1975, Lambertsen et al. 1994, Gauthier et al. 1997a, Gauthier and Sears 1999Lambertsen et al. 1994, Borobia et al. 1995, Gauthier and Sears 1999, Herman et al. 2009 

Although manual biopsy techniques (e.g., capture methods using trocars or scalpels; for examples, see Hansen and Wells 1996, Krahn et al. 2004, Wells et al. 2004) have been used on some cetaceans, researchers more often employ remote biopsy methods (pole-mounted darts or darts launched using a compound bow, crossbow, or gun, see below for references) to obtain tissue samples from free-swimming cetaceans. Indeed, the use of non-lethal projectiles to obtain both skin and blubber samples from cetaceans for scientific investigations is increasing and has been used on over 40 cetacean species worldwide (Table 1, 2), resulting in several thousand samples collected. As with many emerging technologies used for field research on large animals, research and development for marine mammal biopsy systems continue to evolve. Thus, many aspects of cetacean biopsy methods, particularly remotely delivered biopsies, have advanced considerably since the first biopsy dart was fired to collect humpback whale (Megaptera novaeangliae) tissue for cytological sexing almost 40 yr ago (Winn et al. 1973). For reviews of the history of remote biopsy techniques and a description of the equipment used see Lambertsen (1987), Mathews et al. (1988), Nishiwaki et al. (1990), Kasamatsu et al. (1991), Palsbøll et al. (1991), Aguilar and Borrell (1994a), Lambertsen et al. (1994), Patenaude and White (1995), Barrett-Lennard et al. (1996), Larsen (1998), and Krützen et al. (2002).

The present study is the first comprehensive review to examine factors that influence the success of collecting biopsy samples from free-ranging cetaceans as well as evaluate factors that influence physiological and behavioral responses for a wide range of cetacean species that have been sampled via biopsy techniques. The primary focus is remote biopsy techniques; though, some information on manual biopsy techniques is presented for comparison. The information provided can be used to improve biopsy sampling protocols and to increase the collection of suitable samples while minimizing adverse physiological and behavioral responses.

Literature Survey and Data Analysis

Studies in peer-reviewed journals and other literature sources published from 1973 through 2009 were consulted to assess factors contributing to the overall success of biopsy sampling methods, the wound healing process at the biopsy site, and the physiological and behavioral responses to biopsy sampling in cetaceans. Results and conclusions from these studies were first grouped and summarized to provide generalized qualitative information. Additionally, sampling rate (defined as the percentage of biopsy attempts that struck an animal and successfully retained a sample, following Best et al. 2005) and percentage values for a range of behavioral response levels were calculated so that results could be quantitatively compared across studies. Several steps were taken in an attempt to standardize behavioral reactions to facilitate statistical comparison across studies. First, all previously reported behavioral reactions were grouped into four distinct categories (see Table 3 for definitions). Second, percentage values for sampling rate and for each of the four behavioral response categories were calculated separately for groups of cetaceans that were from different studies or were from the same study but differed by species, differed by biopsy method used, or were sampled in different geographic regions (Table 4, 5). These values were then incorporated into statistical and graphical analyses to assess factors that influence sampling rate and behavioral responses following biopsy. All percentages were arcsine transformed prior to performing ANOVA and t-tests. In some cases, nonparametric analyses (ANOVA on ranks, Mann-Whitney rank sum test) were used when tests for normality or equal variance failed. Finally, based on the qualitative and quantitative findings of this extensive review, we identify specific biopsy techniques that provide adequate samples while minimizing disturbance to the animals and make recommendations for additional data to be systematically collected during biopsy sampling to aid in improving the technology and better assessing the impacts of these techniques.

Table 3.  Definitions of cetacean behavioral response categories.
Behavioral response categoryaDefinition
  1.  aDefinitions of categories were based on behavioral responses described by Weinrich et al. (1991, 1992). Although certain responses may be more biologically significant for some species compared to others, all responses described by studies listed in Table 4 and 5 were categorized this way in an attempt to standardize the plethora of responses depicted in the literature.

No responseAnimal continues pre-biopsy behavior
Low responseBrief and mild response (e.g., startle, immediate dive, horizontal move, increase speed, small tail flick, defecate)
Moderate responseMore forceful, but not prolonged response (e.g., porpoising, hard tail slap, breaching, trumpet blow)
Strong responseSuccession of forceful activities (e.g., flight, breaches, multiple tail slaps, numerous trumpet blows)
Table 4.  Summary of biopsy methods used, success of acquiring samples, and physiological and behavioral responses for odontocete species.
SpeciesBiopsy methodSampling rate (percentage of biopsy attempts that struck an animal and successfully retained a sample, following Best et al. 2005)Physiological response to biopsyBehavioral response to contact with biopsy deviceReference
  1.  Percentages in each behavioral response category were calculated for each study using reported data and definitions of behavioral responses based on Weinrich et al. (1991, 1992). Behavioral responses were defined as none: animal continues pre-biopsy behavior, low: brief and mild response (e.g., startle, immediate dive, horizontal move, increase speed, small tail flick, defecate), moderate: more forceful but not prolonged (e.g., porpoising, hard tail slap, breaching, trumpet blow), strong: succession of forceful activities (e.g., flight, breaches, multiple tail slaps, numerous trumpet blows). Although certain responses may be more biologically significant for some species compared to others, all responses described by studies listed in the table were categorized this way in an attempt to standardize the plethora of responses depicted in the literature.

Short-beaked common dolphin (Delphinus delphis)Dart with stainless steel dart tip (l = 20mm, dexternal= 6 mm, dinternal= 5 mm), an internal barb, and stopper delivered by variable-power pneumatic dart projector (Pneudart Model 176B). No retrieval line used.No dataDeath. Possible vertebral trauma (dart entered 40–50 mm beyond stopper). Minimal blood loss, possible vagal shock with ceased breathing and heart failure as consequence.Dolphin stopped swimming, stayed at surface with body slightly arched dorsally, followed by a head-up vertical position and sinking below surface in catatonic vertical position.Bearzi 2000
Short-beaked common dolphin (Delphinus delphis)Variable length biopsy pole (l = 1.4 m to 3.5 m) fitted with stainless steel hollow tip (l = 35 mm, d = 6 mm) with barb.95.4% (combined with their data for Tursiops spp. and at the two geographic locations)No dataFrom New South Wales: 13.6% no response, 86.4% responded (81.8% low, 4.6% moderate, 0% strong)
From South Australia: 10% no response, 90% responded (86.7% low, 3.3% moderate, 0% strong)
Note: The authors used 5 different boats and reported that smaller boats resulted in stronger responses than larger ones. The data provided in the paper are not sufficient to enable separate calculations of responses to the 5 different boats.
Bilgmann et al. 2007a
Northern bottlenose whale (Hyperoodon ampullatus)Dart with hollow stainless steel biopsy tip (l = 25 mm, d = 6 mm) and stop collar delivered by a 67-kg draw crossbow (Barnett Wildcat XL). No retrieval line used.74%No data11% no response, 89% responded (74% low, 15% moderate, 0% strong)Hooker et al. 2001a
Killer whale (Orcinus orca)Dart with a nylon stopper and stainless steel dart tip (l = 23 mm, dexternal= 6.4 mm) with a honed internal bevel and barb delivered by variable-power pneumatic dart projector (Pneudart Model 191). No retrieval line used.81.9%46 of 72 whales hit by biopsy darts were resighted after a period of one day to one year and no evidence of wound infection was seen.19% no response, 81% responded (75% low, 0% moderate, 6% strong). Level of response based on the degree of shaking and swim speed. For comparison, their “strong” is best described as prolonged low level response.Barrett-Lennard et al. 1996
Sperm whale (Physeter macrocephalus)Dart with stainless steel tip (l = 10 mm, d = 6 mm) and flange delivered by a 23 kg draw crossbow (Barnett Wildcat XL). Retrieval line used.63%No data100% responded, ranging from startle and increased speed to defecation. Data were not sufficient to estimate percentages.Whitehead et al. 1990
Sperm whale (Physeter macrocephalus)Dart with standard glass fiber arrow with four fletches and fitted with a blunt end covered with a rounded rubber head which was covered in Nylon pan-scouring material and delivered with a 23 kg draw compound bow (Barnett Safari). No retrieval line used.50%No data100% responded, ranging from startle, defecation and regurgitation to slightly raising lower jaw above the water line but without snapping. Data were not sufficient to estimate percentages.Whitehead et al. 1990
Indo-Pacific humpback dolphin (Sousa chinensis)ACC carbon fiber dart with float and dart tip (l = 25 mm) with beveled edge and three internal barbs delivered by 68 kg draw crossbow (Barnett Ranger RX-150) with red dot sight.73.5%Most of the known individuals that were hit by biopsy darts were resighted after a period of one day to 814 days. All observations at 21 days or more after sampling showed the wound to be completely healed.27% no response, 73% responded (55% low, 18% moderate, 0% strong)Jefferson and Hung 2008
Striped dolphin (Stenella coeruleoalba)Dart tip (l = 10 mm) with stopper and internal butterfly valve delivered by pneumatic rifle.80%No dataNo significant alterations of swimming pattern and no escape behavior.Aguilar and Nadal 1984
Striped dolphin (Stenella coeruleoalba)Biopsy tip mounted to 2 meter pole.No dataNo dataAll responses either none to low level (slight start).Marsili and Focardi 1996
Bottlenose dolphin (Tursiops truncatus)Dart with beveled edge tip (l = 30 mm, d = 7 mm) with one internal barb and rubber tubing for a stopper delivered by a 45 kg draw crossbow.50%Only one animal was re-sighted 19 days after biopsy. The wound left by the dart was clearly visible but appeared to be healing (white in color with darker spot at center of wound). Later re-sights in subsequent years confirmed complete healing of the biopsy site.100% responded (they characterized all reactions as moderate startle responses, but data were not sufficient to standardize the reactions and estimate percentages)Weller et al. 1997
Bottlenose dolphin (Tursiops truncatus)Surgical biopsy, l = 10 mm, d = 30–50 mm (implement not noted).100%Sixteen of the 35 dolphins were re-sighted. Re-sightings occurred sporadically between 8–476 days. Epidermis appeared to have covered wounds by 40–42 days post-biopsy but in some cases probably as early as 15–26 days. The shortest time to complete healing was 61 days (but could possibly have occurred earlier). No infections or related pathologies were ever seen.No dataWeller et al. 1997
Bottlenose dolphin (Tursiops truncatus)Dart with tip containing three internal barbs and a high-density foam collar delivered by crossbow. No retrieval line used.100%No data100% responded (62.5% low, 25% moderate, 12.5% strong)Berrow et al. 2002
Bottlenose dolphin (Tursiops truncatus)Lightweight, hollow aluminum dart with stainless steel tip (l = 17mm) and nylon stopper delivered by pneumatic dart projector (Pneudart Inc., Model 196). No retrieval line used.64% in 1998, 85% in 1999, 83% in 2000 (Higher success rates in 1999 and 2000 were attributed to newly designed stainless steel biopsy tips with a more precise internal bevel angle)Wound closed within 30 days; no visible scar after 1 year. Healing time varied by individual. Pattern and timing of healing consistent with surgical biopsy.15.6% no response, 84.4% responded (68.8% low, 15.6% moderate, 0% strong)Parsons et al. 2003a
Bottlenose dolphin (Tursiops truncatus)Stainless-steel cylindrical punch (l = 25 mm or l = 40 mm; d = 8 mm) with a compressed foam stopper delivered by a 68 kg draw crossbow (Barnett Wildcat III).No dataNo data2.5% no response, 97.5% responded (84.4% low, 10.6% moderate, 2.5% strong) Note: The authors used 2 different boats and reported that the probability of a more intense reaction increased during biopsy attempts from one of the boats. However, the data provided are not sufficient to enable separate calculations of responses to the 2 different boats.Gorgone et al. 2008
Bottlenose dolphin (Tursiops truncatus)Surgical biopsy via scalpel blade.No dataEpidermal cells bridged the incisional gap by 2 days, cuts were closed and histologically repaired but still visible as white linear marks after 7 days.No dataBruce-Allen and Geraci 1985
Bottlenose dolphin species (Tursiops spp.)Dart with hollow polycarbonate body and steel tip (l = 9 mm, dexternal= 5 mm) beveled inwards and containing three internal barbs delivered by a 0.22 caliber rifle (PAXARMS) with Pro-Point red-dot laser sight (Tasco). No retrieval line used.96.5% (Shark Bay, Western Australia site only)Repigmentation began 36 days post-biopsy; no visible scar after one year. Mean wound healing was 47.5 ± 24.2 days but this is likely an overestimate. In animals seen daily, wounds healed after approximately 23 days.4% no response, 96% responded (58.5% low, 33.7% moderate, 3.8% strong)Krützen et al. 2002
Bottlenose dolphin species (Tursiops spp.)Variable length biopsy pole (l = 1.4 m to 3.5 m) fitted with stainless steel hollow tip (l = 35 mm, d = 6 mm) with barb.95.4% (combined with their data for Delphinus delphis and at the two geographic locations)No dataFrom New South Wales: 8.6% no response, 91.4% responded (88.6% low, 2.8% moderate, 0% strong)
From South Australia: 2.9% no response, 97.1% responded (76.5% low, 20.6% moderate, 0% strong)
Note: The authors used 5 different boats and reported that smaller boats resulted in stronger responses than larger ones. However, the data provided are not sufficient to enable separate calculations of responses to the 5 different boats.
Bilgmann et al. 2007a
Table 5.  Summary of biopsy method used, success of acquiring samples, and physiological and behavioral responses for mysticete species.
SpeciesBiopsy methodSampling rate (percentage of biopsy attempts that struck an animal and successfully retained a sample, following Best et al. 2005)Physiological response to biopsyBehavioral response to contact with biopsy deviceReference
  1.  Percentages in each behavioral response category were calculated for each study using reported data and definitions of behavioral responses based on Weinrich et al. (1991, 1992). Behavioral responses were defined as none: animal continues pre-biopsy behavior, low: brief and mild response (e.g., startle, immediate dive, horizontal move, increase speed, small tail flick, defecate), moderate: more forceful but not prolonged (e.g., porpoising, hard tail slap, breaching, trumpet blow), strong: succession of forceful activities (e.g., flight, breaches, multiple tail slaps, numerous trumpet blows). Although certain responses may be more biologically significant for some species compared to others, all responses described by studies listed in the table were categorized this way in an attempt to standardize the plethora of responses depicted in the literature.

Minke whale (Balaenoptera acutorostrata)Dart with stainless steel tip and stop collar delivered by a 57 or 68 kg draw crossbow (Barnett Wildcat). No retrieval line used.100%No data16% no response, 84% responded (36% low, 44% moderate, 4% strong)Gauthier and Sears 1999
Blue whale (Balaenoptera musculus)Dart with stainless steel tip and stop collar delivered by a 57 or 68 kg draw crossbow (Barnett Wildcat). No retrieval line used.92%No data68.9% no response, 31.1% responded (24.3% low, 6.8% moderate, 0% strong)Gauthier and Sears 1999
Pygmy blue whale (Balaenoptera musculus brevicauda)Dart delivered by a modified pneumatic gun. Retrieval line used.83.3%No dataAll responses either none to low levelKato et al. 1996
Fin whale (Balaenoptera physalus)Dart with aluminum bolt and stainless steel collecting tip (1990–93: l = 20mm, d = 10mm; 1994–95: l = 40mm, d = 8mm) with internal barbs, central hook (1990–93 only), and flange (1990–93) or molded floater (1994–95) delivered by 68 kg draw crossbow (Barnett Wildcat II). No retrieval line used.No dataNo data88% no response, 12% responded (12% low, 0% moderate, 0% strong); note: paper combined hits and misses but stated that no significant difference between the two was observed).Jahoda et al. 1996
Fin whale (Balaenoptera physalus)Dart with stainless steel tip and floater delivered by a 68 kg draw crossbow (Barnett Wildcat II).No dataNo dataAll responses either none to low level (startle)Marsili and Focardi 1996
Fin whale (Balaenoptera physalus)Dart with stainless steel tip and stop collar delivered by a 57 or 68 kg draw crossbow (Barnett Wildcat). No retrieval line used.94.2%No data49.6% no response, 50.4% responded (30.1% low, 20.3% moderate, 0% strong)Gauthier and Sears 1999
Fin whale (Balaenoptera physalus)Dart with a stainless steel tip and floater delivered by a 68 kg draw crossbow (Barnett Wildcat II).No dataNo dataAll responses either none to low level (startle)Fossi et al. 2003a
Gray whale (Eschrichtius robustus)Dart with stainless steel tip (l = 10mm, d = 6mm) and closed-cell foam for a flotation collar delivered by a 11 kg draw compound bow (Darton 40). No retrieval line used.No dataNo dataAll responses minimal and transitory.Mathews 1986
Southern right whale (Eubalaena australis)Dart delivered by a modified pneumatic gun. Retrieval line used.83.3%No dataAll responses either none to low levelKato et al. 1996
Southern right whale (Eubalaena australis)Dart with polycarbonate body and custom tip (l = 20 mm, dexternal= 6.3 mm, dinternal= 4.6 mm) with a single internal barb delivered by a 0.22 caliber rifle (PAXARMS). A retrieval line was used briefly (34 of 906 attempts). Also for some samples, a different tip (dexternal= 5.5 mm, dinternal= 4.0) without a retrieval line was used.89.2% (4.0 mm untethered dart), 74.5% (4.6 mm untethered dart), 73.7% (4.6 mm tethered dart). Diameter affected the incidence of stuck darts (16.6% of stuck darts were 4.6 mm diameter; 5.1% of stuck darts were 4.0 mm diameter). Diameter affected recovering darts with a sample (61.9% of darts with sample were 4.0 mm diameter; 18.5% of stuck darts were 4.6 mm diameter.No difference in proportion of successful reproductive cycles between biopsied and unbiopsied cows. No difference in calving intervals between biopsied and unbiopsied cows.All age- and sex-classes combined: 22.4% no response, 77.6% responded (62.4% low, 15.2% moderate, 0% strong)
Males: 29.7% no response, 70.3% responded (58% low, 12.3% moderate, 0% strong)
Females without calves: 19% no response, 81% responded (64.9% low response, 16.1% moderate, 0% strong)
Male calves associated with cows: 32.1% no response, 67.9% responded (56.4% low, 11.5% moderate, 0% strong)
Female calves associated with cows: 31% no response, 69% responded (54% low, 15% moderate, 0% strong)
Cows associated with calves: 10.3% no response, 89.7% responded (71.1% low, 18.6%, 0% strong)
Best et al. 2005
Southern right whale (Eubalaena australis)Rectangular stainless steel punch with a beveled edge and internal barb delivered by a 9 m pole. Four different biopsy heads were made for calves (2) and adults (2).For calves: 79.7% For cows: 76.1%Generally, biopsy site hardly visible after biopsy, with no signs of integumentary or other trauma. The biopsy site on one neonate hemorrhaged (“thin spray of blood”), but bleeding stopped within several minutes following the biopsy and the animal's behavior returned to normal.Reported to be no greater than that from Best et al. 2005.Reeb and Best 2006
North Atlantic right whale (Eubalaena glacialis)Dart with tip (l = 30 mm, d = 7 mm) and straightened #6 cod hook delivered by either a Barnett Wildcat (45 and 68 kg draw weights), Astro-Daco (45 kg draw weight), Excalibur Wolverine (45 and 68 kg draw weights), or a Pearson compound bow (68 kg draw weight). Retrieval line used for all.61.7%No data79.5% no response, 20.5% responded (Most responses low to moderate but data were not sufficient to estimate percentages. Only two individuals demonstrated strong reactions, but both were following unusual biopsy events).Brown et al. 1991
Humpback whale (Megaptera novaeangliae)Dart tip (l = 20 mm, d = 10 mm) with internal prongs and stop collar delivered by a 68 kg draw crossbow. The dart was tethered for retrieval in 1983–1985. The same system was used in 1988–89 but added a 2 cm barb to aid in sample retention and used a floater instead of a retrieval line.87.3% (all years combined)No data1983–85: 28.3% no response, 71.7% responded (20.7% low, 46.7% moderate, 4.3% strong) 1988–89: 16.4% no response, 83.6% responded (36.1% low, 45.9% moderate, 1.6% strong). Note: these percentages are for hits and misses together.Weinrich et al. 1991
Humpback whale (Megaptera novaeangliae)Dart with a stop collar and stainless steel tip (l = 40 cm, d = 9 mm) delivered by a 68 kg draw crossbow. No retrieval line used.No dataNo data44.1% no response, 55.9% responded (22.5% low, 33.2% moderate, 0.2% strong)Clapham and Mattila 1993
Humpback whale (Megaptera novaeangliae)Dart with a tip (l = 20 mm) containing internal prongs delivered by a 68 kg draw crossbow. Retrieval line used.No dataNo data7% no response, 93% responded (26.8% low, 60.6% moderate, 5.6% strong). All strong reactions occurred when retrieval line snagged on flukes.Weinrich et al. 1992
Humpback whale (Megaptera novaeangliae)Dart with stainless steel biopsy tip (l = 20 mm, d = 10 mm) and stop collar delivered by a 57 kg draw crossbow (Barnett Panzer II). No retrieval line used.67%No data58.6% no response, 41.4% responded (23.6% low, 17.6% moderate, 0% strong)Brown et al. 1994
Humpback whale (Megaptera novaeangliae)Biopsy punch with hook and stop collar delivered by a variable-power pneumatic gun (AIRROW). Retrieval line used.95%No dataAll low to moderate level responses except for one strong response when dart was fired prematurely and entered animal at odd angle and retrieval line broke. Data were not sufficient to estimate percentages.Lambertsen et al. 1994
Humpback whale (Megaptera novaeangliae)Dart with stainless steel tip and stop collar delivered by a 57 or 68 kg draw crossbow (Barnett Wildcat). No retrieval line used.92.4%No data34.5% no response, 65.5% responded (18.4% low, 42.2% moderate, 4.9% strong)Gauthier and Sears 1999
Humpback whale (Megaptera novaeangliae)Custom made biopsy dart (samples obtained 8 × 20 mm plug of skin and blubber) delivered from a 70 kg draw crossbow (Barnett Wildcat)No dataNo data44% no response, 56% responded (43% low, 13% moderate to strong; data were not sufficient to separate reactions into the two distinct categories)Cerchio 2003

Factors Affecting the Success of Collecting Biopsy Samples

The majority of published studies that have employed biopsy techniques focus on reporting the findings of the sample analyses (see Table 1, 2), rather than reporting the rate of success of acquiring biopsy samples. From the limited data available, it appears that sampling rate (defined as the percentage of biopsy attempts that struck an animal and successfully retained a sample, following Best et al. 2005) is normally high but may vary by study, the specific methods used, and the species being sampled (Table 4, 5). For example, in studies conducted by the NOAA Southwest Fisheries Science Center from 1991 to 1999, samples were obtained from 68.4% of the darts that hit small odontocetes and 84% of all darts that contacted large odontocetes and mysticetes (Chivers et al. 2000). Likewise, a system specifically designed to sample humpback whales with a pneumatic gun achieved an impressive sampling rate of 95% (Lambertsen et al. 1994). Unfortunately, the data reported in the available literature were not sufficient to quantitatively assess how biological and physical factors influenced sampling rate.

Cetacean Behavior

Aspects of cetacean behavior that influence the success of biopsy sampling operations include swimming speed, duration of time at the water's surface, and activity state. For example, killer whales (Orcinus orca) are most readily darted when traveling at moderate speeds, because they surface in relatively predictable locations and arch their backs well above the water's surface when breathing, presenting relatively large targets (Barrett-Lennard et al. 1996). In contrast, resident killer whales foraging for fish in open water are often unpredictable in their movements and are therefore more difficult to dart. Resting killer whales can also be difficult to dart because they tend to form tight groups, surface without showing much of their backs, and consistently maintain distances of 25 m or more from the boat (Barrett-Lennard et al. 1996). Traveling animals that move in a consistent direction and spend ample time at the water's surface usually present a good biopsy opportunity (Wenzel et al. 2010), though some species may be more easily darted during other activity states. The above examples illustrate that having a good understanding of the target species’ behavior can increase the probability of successful biopsy sampling operations.

Deployment Method

From the limited data provided, it does not appear that the success of acquiring a biopsy sample once the dart has made contact with the animal is influenced by the type of delivery device, or that sampling rates of these devices differ between mysticetes and odontocetes (Fig. 1). As stated previously, though, the particular dart type and power setting on the delivery device are specific to the group of cetaceans being sampled and are thus very important factors in determining the success of obtaining biopsy samples.

Figure 1.

Sampling rate of delivery methods used on mysticetes and odontocetes. Sampling rate is defined as the percentage of biopsy attempts that struck an animal and successfully retained a sample (following Best et al. 2005). The mean percentage (+1 SE) is presented for three different delivery methods. For odontocetes (references in Table 4), the mean sampling rate was calculated separately when using a bow (crossbows and compound bows, from five studies), gun (rifles and pneumatic dart projectors, from four studies), and pole (from one study that reported one average value for two different species of dolphins each sampled in two different geographic regions, thus no error bar is presented). For mysticetes (references in Table 5), the mean sampling rate was calculated separately when using a bow (crossbows and compound bows, from four studies), gun (rifles and pneumatic dart projectors, from three studies), and pole (from one study). Sample sizes indicate the number of groups of animals used to calculate the mean. Groups were deemed independent if they were from different studies or were from the same study but differed by species, differed by biopsy method used, or were sampled in different geographic regions. Thus, sample sizes are equal to or greater than the number of studies.

Shooting Range and Angle

The researcher's ability to acquire a biopsy sample is correlated with the distance from which a dart is launched. For example, the frequency of successfully hitting animals with darts increases at closer distances (e.g., <23 m, Jefferson and Hung 2008), while the frequency of misses increases with more distant firing ranges (e.g., >15 m, Barrett-Lennard et al. 1996; >30 m, Nishiwaki et al. 1990). Though, ricochets that result in no sample collected can also occur at very close firing ranges (e.g., 15 m, Nishiwaki et al. 1990). Definitions of “close” and “far” distances vary across species. In general, biopsy samples are successfully collected from small odontocetes when darts are launched approximately 4–15 m from the target animal (Weller et al. 1997, Möller and Beheregaray 2001, Krützen et al. 2002). Yet, when biopsying larger odontocetes and mysticetes, darts are usually launched from a greater distance (approximately 5–45 m; Mathews et al. 1988; Nishiwaki et al. 1990; Whitehead et al. 1990; Kasamatsu et al. 1991; Lambertsen et al. 1994; Barrett-Lennard et al. 1996; Kato et al. 1996; Marsili and Focardi 1996; Gauthier et al. 1997a; Hoelzel et al. 1998; Larsen 1998; Marsili et al. 1998; Gauthier and Sears 1999; Ross et al. 2000; Hooker et al. 2001a, b; Ylitalo et al. 2001; Fossi et al. 2003a). The actual distance from which a dart is fired is also related to the firing device used and the weather conditions (Lambertsen et al. 1994, Chivers et al. 2000). For example, standard crossbow systems do not function well in winds greater than 12–15 kn, but the pneumatic gun and dart system described by Lambertsen et al. (1994) works successfully in wind speeds of up to 25–30 kn. When weather conditions are poor, crossbows that launch darts at higher speeds (Chivers et al. 2000) or pneumatic guns (Lambertsen et al. 1994) are better choices, as they extend the range at which samples can be obtained. The use of a red-dot laser sight increases accuracy and can also extend the operating range (Larsen 1998, Chivers et al. 2000, Krützen et al. 2002). Of course, to ensure success when using scoped guns it is also imperative that the projector/sight system is set for the range at which shots will be fired.

The ability to attain suitably large, intact samples is linked to the angle of impact as well as the location on the body where the dart strikes. For example, if the dart hits high on the back where it curves towards the dorsal ridge, the dart tends to glance off with no sample or with only a minute sample of skin (Barrett-Lennard et al. 1996). Some whales may also react more to glancing blows compared to perpendicular shots (Brown et al. 1991). The probability of obtaining a sample containing both skin and blubber increases when the angle of impact is perpendicular to the body (Brown et al. 1991, Barrett-Lennard et al. 1996, Gauthier and Sears 1999), though the angle of impact may be less critical when the dart is very sharp (Barrett-Lennard et al. 1996). Barrett-Lennard et al. (1996) also noted that when darts impacted at acute angles on killer whales, the probability that a dart would remain attached to the skin rather than bouncing free appeared to increase. Biopsy darts can also become lodged in the animal when fired directly perpendicular by a device that has its power set too high, though dart tip dimensions can also influence whether a dart sticks (e.g., see Best et al. 2005). To ensure that the dart strikes at a perpendicular angle with minimal disturbance to the animals, the best technique is to slowly approach and parallel the whales’ course (Brown et al. 1991, Clapham and Mattila 1993, Barrett-Lennard et al. 1996, Gauthier and Sears 1999).

Research Team Experience

Finally, and potentially most importantly, the experience and training of the research team are critical to the success of acquiring biopsy samples. Specifically, the success of obtaining biopsy samples increases with competency in archery/shooting and boat handling around cetaceans as well as with increased experience in biopsying cetaceans (Brown et al. 1991, Barrett-Lennard et al. 1996). Experienced researchers are more likely to strike animals in preferred zones on the body, and this will likely yield better samples with fewer traumatic wounds. Those experienced in biopsy sampling are also more likely to operate equipment safely, benefitting both target animals and crew.

Physiological Impacts and Biopsy-Site Wound Healing

Systematic assessments of the physiological effects of biopsy sampling are important to determine the potential impacts of these techniques. Studies on both marine mammal carcasses and live animals have been conducted to provide information to improve dart designs for obtaining better samples while minimizing physiological impacts.

Wound Severity and Healing Process

Experiments have been conducted on cetacean carcasses to assess the functionality and sample retention rates of different dart tips as well as evaluate the extent of tissue damage caused by biopsy darts (e.g., Palsbøll et al. 1991, Patenaude and White 1995). For example, Patenaude and White (1995) used carcasses of freshly dead belugas (Delphinapterus leucas) to determine the success of biopsy acquisition and the severity of wounds caused by biopsy darts with different combinations of biopsy tip lengths (20, 25 mm) and diameters (5, 6, 7 mm), crossbow draw weights (23, 45, 68 kg), and distances fired (1.5–15 m). Their results showed that the severity of the biopsy site wound, defined by the extent of tearing in the epidermis and dermis surrounding the puncture wound, increased with the draw weight of the crossbow (Patenaude and White 1995).

Some researchers also record physiological responses to biopsy sampling (Table 4, 5) as well as photograph the progression of wound healing in free-ranging cetaceans to assess the impacts of remote biopsy methods. In general, most sampling sites heal nearly completely following biopsy sampling via remote methods. For example, Reeb and Best (2006) reported that biopsy sites on southern right whales (Eubalaena australis) were hardly visible after biopsying took place, and there were no signs of integumentary or other trauma. Additionally, even though the biopsy site of one neonate hemorrhaged, the bleeding stopped within minutes of sampling (Reeb and Best 2006). Similarly, within a month or less of biopsy sampling, wounds on dolphins appear as small dots with no sign of infection (Weller et al. 1997, Krützen et al. 2002, Parsons et al. 2003a, Jefferson and Hung 2008). Within 50 d, the scar is barely discernable (Krützen et al. 2002, Parsons et al. 2003a). Finally, biopsy dart wounds on killer whales also heal relatively quickly.2 These wounds appear as small white dots within one day of darting, and they shrink in size and fade as the wound heals. Furthermore, no infection (e.g., swelling, discharge, etc.) of the biopsy site has been observed; and when the animals are resighted the following year, only a small depigmented spot may exist, with no evidence of permanent tissue damage (Barrett-Lennard et al. 1996, B. Hanson2).

In contrast, surgical biopsy wounds on bottlenose dolphins (Tursiops truncatus) are generally larger than remote biopsy wounds and take a longer time to heal (Weller et al. 1997). In general, it takes 15–42 d for epidermis tissue to cover these larger wounds. Yet, similar to remotely biopsied animals, wounds of animals that are surgically biopsied are either indistinguishable or slightly lighter or darker than the surrounding skin when they are resighted 60 d to 1 yr later (Weller et al. 1997). Finally, there is no indication of infection or related pathologies from surgical biopsy wounds (Weller et al. 1997).

Bruce-Allen and Geraci (1985) described the wound healing process in captive bottlenose dolphins following incisions through the epidermis and into the dermis. Their study demonstrated that cuts are histologically repaired by 7 d, but are still visible on dolphins as white linear marks. High rates of cell proliferation enable cetaceans to rapidly heal from wounds they obtain in their natural habitat. For example, bottlenose dolphins with large, open wounds, probably inflicted by sharks, heal substantially within the first month and can be completely healed within 6–7 mo (Corkeron et al. 1987, Lockyer and Morris 1990). The successful healing of these larger traumas in the wild is perhaps one of the strongest arguments to suggest that the majority of biopsy wounds will heal rapidly and that biopsy sampling will most likely not impact survival (International Whaling Commission 1991). Aguilar and Borrell (1994a) also concluded that the small wound produced by a standard biopsy dart (0.25 cm diameter) should not lead to significant physical trauma in sampled animals.

Stress Response

To date, no studies have investigated the stress response in cetaceans targeted by remote biopsy sampling methods. In comparison, a small number of researchers have investigated physiological and behavioral responses in dolphins to assess stress associated with encirclement by nets and handling, which are required during manual biopsy procedures. St. Aubin et al. (1996) found that bottlenose dolphins had elevated stress hormones (aldosterone and cortisol) following capture and handling, while most dolphins in a similar study by Ortiz and Worthy (2000) did not exhibit elevated stress hormone levels. Authors of both studies concluded that the increases in hormone levels were indicative of a mild stress response only. Another study found that bottlenose dolphin blood cells increase gene expression related to metabolism and stress, which also indicates that dolphins undergo a stress response during capture-release health assessments (Mancia et al. 2008). Finally, Esch and colleagues (2009) showed that signature whistle parameters, which may be potential indicators of stress, changed in bottlenose dolphins during brief capture-release events. However, none of the studies examined the long-term impacts of these short-lived stress responses or how physiological responses change with repeated captures of the same individual. These, as well as examining the stress response in remotely biopsied cetaceans, are important areas of future research, as the cumulative impacts of repeated capture and/or biopsy sampling (by both manual and remote methods) may be substantial.

Serious Trauma and Death

Although several thousand biopsy samples have been taken via remote biopsy techniques, the number of documented serious traumas or deaths caused by biopsy sampling is low. This may be because biopsy sampling is relatively benign, subsequent mortality or injury is unknown, or it may also be due to underreporting by researchers, who are unlikely to publish accounts of these events. The one exception is a published description of the death of a common dolphin (Delphinus delphis) following biopsy sampling (Bearzi 2000). In this report, the author claimed that the death was not a direct consequence of the biopsy wound, but rather, the result of a combination of several variables, including the malfunction of the stopper on the dart, the location on the body where the biopsy dart was embedded in the animal, the thinness of the individual's blubber layer relative to other animals in the population, handling of the animal by the sampling team after the biopsy event, and possibly a predisposition of this individual dolphin to catatonia and death during stressful events (Bearzi 2000). Although mechanical and human error played a role in this tragic event, Bearzi (2000) stated that identical methods had been used on other common dolphins with no, or only minor and temporary, behavioral responses. Thus, the author had considered the technique to be relatively noninvasive. This report demonstrates that individuals within the same species can exhibit variable responses to darting, and if assessment of body condition in the field is possible, biopsy sampling animals in poor condition should be avoided. The author also concluded that research methods should only be adopted after careful review and risk assessment and that those decisions must be reviewed on a regular basis (Bearzi 2000). For example, the Tethys Research Institute website lists pros and cons of biopsy sampling and outlines the organization's guidelines and policies on biopsy sampling, including the recent policy to cease biopsy darting small cetaceans (http://www.tethys.org/internal/biopsy.htm, accessed 27 September 2010).

Behavioral Responses to Biopsy

In addition to monitoring biopsy wounds, systematic assessments of behavioral responses to biopsy sampling are important. Researchers have occasionally monitored cetaceans during and after biopsy darting to assess the impact of the sampling equipment and protocols on behavior. Unlike monitoring the healing process of wounds, assessing behavioral responses is more subjective. A number of researchers have used video cameras to record behavioral reactions during biopsy sampling attempts (Barrett-Lennard et al. 1996, Berrow et al. 2002, C. Emmons3), and some of these cameras were attached to the firing device to enable simultaneous collection of a tissue sample and a video record of the biopsy site. This technique allows researchers to identify sampled animals, assess immediate wounds, and more accurately quantify an animal's reaction to sampling events. For example, Barrett-Lennard et al. (1996) reported very short-lived responses from killer whales that were often barely perceptible, and in some cases, only detected when reviewing the videos. In general, the immediate response of killer whales to darting consisted of a shake, usually detected by quivering of the dorsal fin, and acceleration (Barrett-Lennard et al. 1996).

A range of behavioral responses, including no perceptible response, has been observed following skin and blubber biopsy sampling of cetaceans. The majority of studies reported that most animals responded (up to 100% of individuals biopsied within a study) though some studies reported that most animals did not respond (up to 88% of individuals biopsied within a study) to contact with the biopsy device (Table 4, 5). Usually responses to darting are short-lived (0.5–3 min) and confined to the darted animal (Whitehead et al. 1990; Weinrich et al. 1991, 1992; Barrett-Lennard et al. 1996; Jahoda et al. 1996; Gauthier and Sears 1999; Berrow et al. 2002, Parsons et al. 2003a; Jefferson and Hung 2008). The vast majority of responses were classified as brief, low level reactions, consisting of a startle, immediate dive, horizontal move, increased speed, or small tail flick (Table 3, 4, 5). Interestingly, Reeb and Best (2006) noted that when southern right whales were biopsied deeply with a pole-mounted dart (11–20.5 cm long darts, depending on age-class), they did not demonstrate reactions stronger than those observed during more superficial sampling in a previous study (Best et al. 2005). Strong responses, characterized by a succession of forceful activities (e.g., flight, breaches, multiple tail slaps, numerous trumpet blows, etc.,Table 3) rarely happen, occurring in only 0% to 6% of animals biopsied in most studies (Table 4, 5). One exception is a study on bottlenose dolphins, in which 12.5% of the animals showed a strong response (Berrow et al. 2002). The high percentage is due to the fact that this study consisted of a small sample of eight dolphins, and one of the biopsied individuals demonstrated a strong response. The cause of this one individuals’ response was thought to be due to the biopsy dart striking the dorsal fin instead of the intended target site (Berrow et al. 2002). Strong responses have also been observed when biopsy tips remain lodged in the blubber of whales (Weinrich et al. 1991, 1992; Gauthier and Sears 1999) or when there is a momentary entanglement of the retrieval line on flukes (Weinrich et al. 1991, 1992). However, darts have also remained lodged in some animals for extended periods of time without mortality, infection, or behavioral changes (Clapham and Mattila 1993, Barrett-Lennard et al. 1996, Parsons et al. 2003a).

Species-Specific and Individual Variability in Behavioral Responses

A few previous reports as well as the findings from this review suggest that there are species-specific differences in behavioral reactions (e.g., between four balaenopterid species, Gauthier and Sears 1999; between odontocetes and mysticetes, Berrow et al. 2002 and Hooker et al. 2001a). From the available data, it appears that a higher proportion of odontocetes respond to biopsy sampling compared to mysticetes (P < 0.001, Fig. 2), and that the proportion of low and moderate responses varies by group as well (low responses: P < 0.001, moderate responses: P= 0.046, Fig. 2). Low and moderate responses are the predominant responses in mysticetes, and strong is the least observed response (P < 0.05, Fig. 2). For odontocetes, low is the predominant response, followed by moderate, and strong is the least observed response (P < 0.05, Fig. 2). It is also important to note that strong responses are rarely observed in either group (Fig. 2). Within a species, variable behavioral reactions to biopsy darting have been observed between age- and sex-classes (e.g., see Best et al. 2005, Fig. 3) as well as between individual animals. Finally, behavioral reactions to biopsy darting by nontarget animals have also been observed (Barrett-Lennard et al. 1996, Weller et al. 1997, Gorgone et al. 2008). As expected, the probability of a nontarget animal reacting to biopsy darting decreases with increasing distance from the target animal (Gorgone et al. 2008).

Figure 2.

Response rates of mysticetes and odontocetes following biopsy sampling. Mean percentage (+1 SE) of animals responding to biopsy and mean percentage (+1 SE) of three different response levels (low, moderate, strong; defined in Table 3) are presented. For odontocetes, the mean percentage of animals responding to biopsy was calculated from ten studies while the mean percentage of animals responding at three response levels were calculated from eight studies (references in Table 4). For mysticetes, the mean percentage of animals responding to biopsy was calculated from nine studies while the mean percentage of animals responding at three response levels were calculated from seven studies (references in Table 5). Sample sizes indicate the number of groups of animals used to calculate the mean. Groups were deemed independent if they were from different studies or were from the same study but differed by species, by biopsy method used, or were sampled in different geographic regions. Thus, sample sizes are equal to or greater than the number of studies. Significant differences between groups in the percentage of animals that responded (P < 0.001), percentage of low responses (P < 0.001), and percentage of moderate responses (P= 0.046) are noted with asterisks.

Figure 3.

Response rates of southern right whale (E. australis) age/sex classes following biopsy sampling. Percentage of animals responding and the percentage of responses at three different response levels (low, moderate, strong; defined in Table 3) were taken from one study (Best et al. 2005, Table 5). Thus, for all age/sex classes (note: male and female calves were associated with female cows during biopsy), there is only a sample size of 1 and statistical analysis of the data are not possible. There were no strong responses by any of the age/sex-classes in the study.

Variability in Behavioral Responses Attributed to Physical and Biological Factors

Differences in the type, intensity and/or frequency of behavioral responses have also been attributed to the methods and equipment used (Weinrich et al. 1991, 1992); type and size of the boat used (Bilgmann et al. 2007a, Gorgone et al. 2008); size of the biopsy dart (Gauthier and Sears 1999, Krützen et al. 2002); animal's activity prior to biopsy (Weinrich et al. 1991, 1992; Clapham and Mattila 1993; Brown et al. 1994; Hooker et al. 2001a); sex of the animal (Clapham and Mattila 1993, Brown et al. 1994, Gauthier and Sears 1999); size of the animal (Mathews 1986); whether the animal is associated with a group of conspecifics (Best et al. 2005); as well as the season, water depth and sea state (Gorgone et al. 2008). In contrast, Jefferson and Hung (2008) found that for both hits and misses, distance to the target animal had very little effect on its reaction.

Variability in Behavioral Responses Attributed to Biopsy Delivery Method

It is conceivable that the equipment and delivery method used during biopsy sampling operations contribute to the propensity of behavioral responses occurring, and possibly, the degree of the response observed. For example, retrieval lines, which can snag on animals, have been implicated in causing animals to react, and in particular, exhibit strong reactions (Weinrich et al. 1991, 1992). From the available data on mysticetes, it appears that when a retrieval line is used, moderate responses tend to be the most frequent while strong responses are the most rare (P= 0.067, Fig. 4). When no retrieval line is used, low and moderate responses are significantly greater than strong responses (P < 0.05, Fig. 4). Although there is no significant difference between the percentage of animals that respond with and without the use of a retrieval line (P= 0.614, Fig. 4), the percentage of strong responses is marginally greater when a retrieval line is used compared to when it is not (P= 0.089, Fig. 4). The percentage of moderate responses also tends to be greater when a retrieval line is used, though these findings are not significant (P= 0.131, Fig. 4). It is important to note that the power of the latter test is low due to the small sample size, so the insignificant results should be taken with caution.

Figure 4.

Response rates of mysticetes with and without the use of a retrieval line. Mean percentage (+1 SE) of animals responding with and without the use of a retrieval line are presented. For methods that utilized a retrieval line, the mean percentage of animals responding and responding at each of three levels (low, moderate, strong; defined in Table 3) was calculated from three and two studies, respectively (references in Table 5). For methods that did not utilize a retrieval line, the mean percentages of animals responding and responding at each of three levels were calculated from five studies (references in Table 5). Sample sizes indicate the number of groups of animals used to calculate the mean. Groups were deemed independent if they were from different studies or were from the same study but differed by species, by biopsy method used, or were sampled in different geographic regions. Thus, sample sizes are equal to or greater than the number of studies.

Due to the dearth of data available, the influence of the general delivery method (bow, gun, or pole) on the intensity of behavioral responses from cetaceans is equivocal (Fig. 5A, B). For odontocetes, response levels do not differ by delivery method, and for all delivery methods, the predominant response observed is low (all P < 0.05, Fig. 5A). Unfortunately, only one study reported sufficient data to assess the response of odontocetes to biopsy sampling using a pole (Bilgmann et al. 2007A). The data from this study suggest that common and bottlenose dolphins do not exhibit strong responses when a pole is used (Table 4, Fig. 5A). In contrast, it appears that response levels in mysticetes may differ by delivery method, but the sample size of one for delivery by gun precludes statistical analysis (Fig. 5B). For mysticetes that are biopsied using a bow, both low and moderate responses are equally predominant while strong responses are rare (P < 0.05, Fig. 5B). Similarly, for the one study that reported sufficient data to assess the response of mysticetes to biopsy darts delivered by gun, the predominant response was low, and no strong responses were observed (Best et al. 2005, Table 5, Fig. 5B). Finally, when bottlenose dolphins are considered separately, to eliminate species-specific variability in responses, delivery method (bow, gun, or pole) does not influence response rates or the intensity of behavioral responses. For all delivery methods, the predominant response observed is low (Fig. 6). Furthermore, as stated previously, no strong responses were observed during the one study that used a pole (Bilgmann et al. 2007a).

Figure 5.

Response rates of odontocetes (A) and mysticetes (B) by delivery method. Mean percentage (+1 SE) of animals responding to biopsy and mean percentage (+1 SE) of three different response levels (low, moderate, strong; defined in Table 3) are presented for different biopsy delivery systems. For odontocetes, the mean percentage of animals responding to biopsy delivered by bow (crossbows and compound bows), gun (rifles and pneumatic dart projectors), and pole were calculated from six, three, and one study, respectively, while the mean percentage of animals responding at three response levels were calculated from four, three, and one study, respectively (references in Table 4). For mysticetes, the mean percentage of animals responding to biopsy delivered by bow (crossbows and compound bows) and gun (rifles and pneumatic dart projectors) were calculated from eight and one study, respectively, while the mean percentage of animals responding at three response levels were calculated from six and one study, respectively (references in Table 5). Sample sizes indicate the number of groups of animals used to calculate the mean. Groups were deemed independent if they were from different studies or were from the same study but differed by species, by biopsy method used, or were sampled in different geographic regions. Thus, sample sizes are equal to or greater than the number of studies.

Figure 6.

Response rates of bottlenose dolphin (Tursiops truncatus) by delivery method. Mean percentage (+1 SE) of animals responding to biopsy and mean percentage (+1 SE) of three different response levels (low, moderate, strong; defined in Table 3) are presented for different biopsy delivery systems. The mean percentage of dolphins responding to biopsy delivered by bow (crossbow) and gun (rifles and pneumatic dart projectors) were calculated from three and two studies, respectively, while the mean percentage of animals responding at three response levels were each calculated from two studies (references in Table 4). The mean percentage of animals responding, as well as responding at three levels, to biopsy delivered by pole were calculated from one study conducted at two different geographic locations. Sample sizes indicate the number of groups of animals used to calculate the mean. Groups were deemed independent if they were from different studies or were from the same study but differed by biopsy method used or were sampled in different geographic regions. Thus, sample sizes are equal to or greater than the number of studies.

Behavioral Responses Attributed to Other Disturbances during Biopsy Sampling

Although researchers make their best effort to determine which responses are directly linked to the biopsy procedure, behavioral responses can still be influenced by other external factors, of which the researcher is unaware (Hooker et al. 2001a). It is quite difficult to identify and separate the direct effects of biopsy sampling from other man-made or natural disturbances.

For instance, disturbance from the research vessel, rather than the act of biopsy sampling, can elicit behavioral responses. Indeed, sperm whales (Physeter macrocephalus, Whitehead et al. 1990) and southern right whales (Reeb and Best 2006) have startled as the vessel approached, prior to any darting attempts. Pitman (2003) also reported that Antarctic killer whales showed little response to darting compared to the reaction caused by boat operations. Finally, Jahoda and colleagues (2003) attributed all behavioral changes during biopsy operations to close approaches by the fast-moving inflatable boat from which sampling was conducted. The study, however, was not designed to assess the impacts from biopsy darting and vessel approaches separately.

Cetaceans can also respond to darts that are fired into the water. Reactions to biopsy darts that do not make contact can range from no reaction to moderate level (e.g., startle, diving, moving away, porpoise, tail slap, Table 3) reactions (e.g., bottlenose dolphins, Weller et al. 1997, Krützen et al. 2002, Parsons et al. 2003a, Gorgone et al. 2008; bottlenose whales (Hyperoodon ampullatus), Hooker et al. 2001a; humpback whales, Clapham and Mattila 1993, Brown et al. 1994; Indo-Pacific humpback dolphins (Sousa chinensis), Jefferson and Hung 2008; sperm whales, Whitehead et al. 1990). Similarities in behavioral reactions of hit and missed animals may indicate that some observed reactions are simply due to a startle response and not necessarily due to being contacted by the biopsy dart (Clapham and Mattila 1993, Lambertsen et al. 1994, Krützen et al. 2002, Parsons et al. 2003a, Gorgone et al. 2008, Jefferson and Hung 2008). Regardless of the source of disturbance, the majority of behavioral reactions that have been reported during biopsy operations appear to be minor and are similar to those that have been observed during routine vessel approaches and whale-watching activities (e.g., see Au and Perryman 1982, Janik and Thompson 1996, Au and Green 2000, Weinrich et al. 2001, Williams et al. 2002, Noren et al. 2009, Weinrich and Corbelli 2009).

Respiratory Behavioral Observations to Assess Stress

Besides recording general behavioral observations, researchers have also recorded changes in respiration rates as an indicator of a stress response to biopsy sampling. In theory, respiration rates are a readily attainable, non-invasive, and objective method to gauge a whale's activity level or response to stimuli. However, respiration rates tend to vary across individuals and by several other factors (e.g., see Williams and Noren 2009), so this may not be the most viable method to determine whether biopsy sampling impacts cetaceans. For instance, Mathews (1986) reported that eight individual gray whales (Eschrichtius robustus) showed variable respiratory responses to biopsy sampling. Specifically, some whales showed a slight increase in the number of blows per surfacing interval and dive duration while others decreased these variables after sampling was initiated (Mathews 1986). Similarly, sperm whales both increased and decreased respiration rates following biopsy sampling, and not all changes were statistically significant (Whitehead et al. 1990). For fin whales (Balaenoptera physalus), there were no significant differences in dive time or blow interval; but surface time, blow rate, and number of blows per surfacing were significantly lower during the approach of the boat and biopsy sampling compared to both prior to and after the approach ( Jahoda et al. 2003). In contrast, for humpback whales, there were no significant changes in blow interval, number of blows per surfacing interval, surfacing interval, or dive time, but there was a significant decrease in the surface-interval to dive-time ratio post-biopsy (Weinrich et al. 1992). Similar to gray and sperm whales, individual humpback whales demonstrated variable respiratory responses to biopsy sampling (Weinrich et al. 1992). Besides demonstrating that cetaceans often exhibit inconsistent changes in respiration rates and dive variables following biopsy, none of the studies provided explanations for the biological significance of these responses. This is likely because the number of co-variates is large, yet the sample size of animals biopsied is relatively small.

Long-Term Behavioral Impacts of Biopsy Sampling

Although it is difficult to assess linkages between short-term behavioral changes and long-term impacts, the available data suggest no link between momentary changes in behavior and long-term detrimental effects to cetaceans. A number of researchers have reported that the darts may startle animals temporarily, yet they do not appear to change or disrupt the animal's behavior (Mathews 1986, Weinrich et al. 1991, Marsili and Focardi 1996, Hooker et al. 2001a, Fossi et al. 2003a). However, it is possible that when cetaceans are engaged in certain activities, their behavior will be disrupted. For example, Brown and colleagues (1994) reported that migrating humpback whales were significantly less likely to respond to successful biopsy attempts than those on the feeding grounds (Weinrich et al. 1991) or the breeding grounds (Clapham and Mattila 1993). Jahoda et al. (2003) found that fin whales cease feeding and commence traveling when approached for biopsy sampling, and Weinrich et al. (1992) found that resting humpback whales were more likely to respond to biopsy sampling than feeding whales. Unfortunately, no data on the duration of the behavioral modifications or potential linkages to long-term impacts were provided by these studies.

Interestingly, several cetaceans that have been biopsied previously do not appear to avoid vessels during subsequent biopsy sampling attempts. A group of animals can often be re-approached by a research vessel following successful biopsy sampling (e.g., bottlenose whales, Hooker et al. 2001a; humpback cow/calf pairs, Weinrich et al. 1991; Indo-Pacific humpback dolphins, Jefferson and Hung 2008; resident killer whales, Barrett-Lennard et al. 1996). Furthermore, many balaenopterid whales that have been biopsied twice within one week to five months demonstrate the same or a lesser response to the second biopsy (e.g., humpback whales, Brown et al. 1994; fin, blue [Balaenoptera musculus], and humpback whales, Gauthier and Sears 1999). In contrast, female southern right whales with calves react more strongly to repeated biopsy sampling (over periods of hours to 65 d, Best et al. 2005). Although the general trend is for no change in response or desensitization to biopsy sampling, the Best et al. (2005) study shows that sensitization to biopsy sampling can occur. It could be that certain cetacean species, or possibly specific segments of populations, are more sensitive to repeated biopsy attempts than others.

Finally, there are no indications of any long-term effects, such as avoidance of the sampling area (e.g., gray whales, Mathews 1986; sperm whales, Whitehead et al. 1990; humpback whales, Weinrich et al. 1991, Clapham and Mattila 1993; killer whales, Barrett-Lennard et al. 1996; bottlenose dolphins, Weller et al. 1997; Indo-Pacific humpback dolphins, Jefferson and Hung 2008) or adverse effects on reproductive cycles and calf survival (southern right whales, Best et al. 2005). Even though the available literature suggests that there are no long-term impacts related to biopsy sampling, it is important to note that these impacts are likely the most difficult to examine. Thus, future studies should collect data to assess both short- and long-term responses to biopsy sampling.

Conclusions

Biopsy sampling is a valuable tool used to acquire biological and physiological data from cetaceans and appears to cause relatively minor disturbance. This method can provide fresh, uncontaminated tissue suitable for concurrent genetic, fatty acid, stable isotope, and toxicological analyses that provide information on stock structure, prey preferences, and health status for each individual sampled. It is also particularly useful for directed sampling of specific individuals and for collecting a large number of samples from different individuals at one time. More importantly, according to the available literature, biopsy sampling is not likely to produce long-term behavioral alterations or result in physiological complications during wound healing, as long as experienced research teams use the appropriate equipment and techniques. However, it is important to note that the number of studies available from which to draw these conclusions is relatively low because fewer researchers report on behavioral and physiological impacts of biopsy sampling compared to reporting the results of the biopsy sample analyses. Furthermore, because researchers (or journals) may be less likely to publish failures (e.g., strong responses, severe trauma, death of an animal) during biopsy sampling operations, the available literature may also be biased to support that biopsy sampling is relatively benign. Nonetheless, most researchers have reported that biopsy sampling causes minor behavioral and physiological impacts. Thousands of individuals were sampled by all of these studies combined (see Table 4, 5). Thus, it is probable that biopsy sampling is a relatively benign method to obtain biological samples from free-ranging cetaceans. Future efforts to assess impacts of biopsy sampling could be expanded to include unpublished data included in permit reports to agencies such as the U.S. National Oceanic and Atmospheric Administration's National Marine Fisheries Service to determine if the findings presented here are consistent with the analysis of a larger data set.

Because of the value of the information obtained, the relative low risk to animals biopsied, and the ability to collect several samples, numerous researchers worldwide are currently utilizing biopsy techniques to obtain important new information on critical conservation questions. Furthermore, as new assays become available, archived and future biopsy samples will be able to provide additional information on the health of individuals and the status of populations. For example, recent studies suggest that biopsies can provide information on stress levels through skin protein analyses (Southern et al. 2002), pregnancy status through blubber progesterone analysis (Mansour et al. 2002, Kellar et al. 2006), male reproductive status through blubber testosterone analysis (Kellar et al. 2009), and individual age through the analysis of blubber fatty acids (Herman et al. 2008, 2009).

Although the advantages of obtaining samples using biopsy methods, particularly via remote methods, are many, these endeavors require adequate caution and training to ensure that animals are not harmed. For example, the NOAA NMFS Northeast Fisheries Science Center has assembled a training manual for personnel engaged in cetacean biopsy procedures (see Wenzel et al. 2010). Based on our review, we find that certain measures tend to increase the success of collecting tissue samples while minimizing disturbance to cetaceans during biopsy sampling.

First, the dart design and delivery system used must be selected to account for the biology, physiology, and behavior of the target species. Usually the length of the dart tip and strength of the delivery device are based on the body size as well as the skin and blubber thickness of the target species in order to acquire a suitable sample (either skin or skin and blubber) while preventing injury caused by penetration beyond the blubber layer. In general, lower powered delivery systems are used for small cetaceans while higher powered delivery systems are used for large cetaceans. The strength of the delivery device should also be selected according to the expected approach distance; for example, crossbows with lower draw strengths are usually used at close ranges.

Second, researchers utilizing biopsy sampling techniques must have experience with these methods. Those that practice firing biopsy darts at targets prior to initiating sampling of live animals are more likely to be successful in the field. Furthermore, success often increases with each subsequent year of biopsy sampling. It is also likely that cetaceans biopsied by experienced researchers (i.e, presumably better at operating boats near cetaceans, hitting the target animal, and sampling from the ideal body location) will have fewer physiological impacts and potentially exhibit fewer strong behavioral responses.

Third, researchers must be cognizant of the angle at which they fire biopsy darts because this can be a critical component of obtaining large samples while also minimizing disturbance. Several researchers reported that when darts hit at a perpendicular angle to the animal, the largest samples (includes blubber and skin) are excised and retained, and minimal behavioral reactions are observed.

Fourth, experienced vessel operators are paramount to the success of safely collecting biopsy samples and to minimizing disturbance. Several studies reported that slow approaches appear to minimize disturbance during biopsy sampling. Cetaceans also demonstrate less evasion when approached slowly, increasing the probability of sampling success.

Fifth, researchers should make a concerted effort to monitor and record the physiological and behavioral responses of cetaceans to biopsy sampling. Norman et al. (2004) discuss several physiological parameters that should be monitored during the capture-release, handling, and tagging of odontocetes; and these are also applicable during surgical biopsy techniques. For remote biopsy techniques, however, other methods need to be utilized. For example, Mesnick, Wenzel, and their colleagues recommended specific data to be collected during each biopsy attempt and provided examples of sampling forms in their publications (Mesnick et al. 1999, Wenzel et al. 2010). The use of video cameras, particularly those affixed to biopsy dart firing devices, allows researchers to more accurately quantify animals’ reactions to sampling events. Similarly, documenting the healing process with digital photographs of biopsy sites is important for assessing long-term impacts and providing information on the time period required for healing, which is still unknown for most cetacean species. The standardization and systematic collection of data on factors that influence the success of acquiring samples and factors that influence behavioral and physiological responses are also critical to more easily compare results across studies and to better assess the impacts of cetacean biopsy techniques so that methods can be improved to yield the best samples with minimal disturbance. It is equally important to conduct studies that assess potential long-term impacts of biopsy sampling. Finally, in order to properly assess both short- and long-term effects of biopsy sampling, it is imperative that properly designed controls be implemented into research regimes.

Footnotes

  1. 1Present address: NOAA Fisheries Alaska Fisheries Science Center, 7600 Sand Point Way N.E., Seattle, Washington 98115-6349, U.S.A.

  2. 2Personal communication from Dr. Brad Hanson, NOAA NMFS Northwest Fisheries Science Center, 2725 Montlake Boulevard East, Seattle, WA 98112, July 2008.

  3. 3Personal communication from Candice Emmons, NOAA NMFS Northwest Fisheries Science Center, 2725 Montlake Boulevard East, Seattle, WA 98112, July 2007.

Acknowledgments

We thank L. Jones for her support and encouraging us to write this manuscript. We are indebted to S. Kromann from the National Marine Mammal Laboratory Library at the NOAA Alaska Fisheries Science Center for locating many of the references required for this review. D. Janiger, Curatorial Assistant (Mammals) from the Natural History Museum of Los Angeles County, California also provided PDFs of many papers that were included in this review. Finally, we greatly appreciate T. McCosh's assistance with formatting and editing the text and tables and B. Diehl's assistance with preparing figures. This manuscript was greatly improved by comments from P. Best, C. Emmons, M. Ford, B. Hanson, A. Zerbini, E. Zolman, and an anonymous reviewer.

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