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Biopsy sampling and intrusive research

 

 

Background and Rationale

Tethys policy on intrusive research

Guidelines

Skin swabbing for genetic analyses

Skin and blubber sampling for genetic, toxicological, and feeding ecology analyses

Selected literature

 

 

Background and Rationale

The remote collection of skin and blubber biopsy samples from free-ranging cetaceans has become of widespread use among scientists in recent years, given the minimal level of intrusiveness involved, as compared to sampling with lethal methods, which today is considered by many researchers as a cruel, unnecessary, and obsolete practice.

Biopsy sampling from free-ranging whales and dolphins is in fact a powerful research approach, as biopsies can provide data that in many cases cannot be obtained by other non-destructive means. Skin samples can undergo genetic analyses and these can generate for example information on social organization, kinship, mating system, individual gender and identification, movement patterns, population size, stock identity, genetic phylopatry and variability within and among populations. Moreover, the blubber portion of the samples can be used to determine contaminant levels, for various biomarker analyses and toxicological tests performed on cell cultures, and for gaining information on feeding ecology and nutritive condition through the examination of stable isotopes, fatty acids and lipid content in the blubber.

It has been suggested that biopsy sampling on individual cetaceans, if carried out responsibly, is likely to cause only low-level and short-term reactions and is not likely to produce any long-term deleterious effects. However, biopsy sampling - as any ‘intrusive’ research approach - entails risks that may be difficult to evaluate or predict. Most biopsy sampling studies involve some kind of disturbance to the animals, and a variable occurrence of ‘undesired’ events. These may include missed shots, stuck darts or broken tips remaining attached to the animals, snagging of the dart retrieval line on the animal's flukes, repeated sampling of one individual, etc. Most studies report a minority of ‘strong’ or ‘excited’ short-term reactions by the animals. These reactions are generally considered as acceptable from an ethical standpoint, and deemed as relatively irrelevant for the animals’ welfare.

However, our experience has shown that even if conducted by experienced personnel, intrusive research at sea focusing on poorly-known species or populations implies levels of risk that may be hard to assess, particularly as far as biopsy darting at small cetaceans is concerned. Under rare circumstances, potential risks or animal welfare issues may be overlooked based on lack of documented precedents or insufficient knowledge of the animals.

While avoiding even mildly intrusive research techniques may prevent accidents, it would also delay or prevent the understanding of threats that have the potential for wiping out entire cetacean populations. Non-destructive biopsy sampling often represents the most straightforward, effective and ethically-acceptable way to evaluate threats and try to counteract the disappearance of cetacean species. However, intrusive research methods need to be adopted in light of the precautionary principle, and reviewed on a regular basis according to the best available evidence. Finding the appropriate balance between individual animal welfare and the compelling need to gather information to be used for far-reaching conservation purposes may represent a challenge to the individual scientist.

 

 

Tethys policy on intrusive research

The following Table - together with the guidelines provided below - synthesizes the current policy of the Tethys Research Institute regarding intrusive research. Such position may be re-evaluated if new evidence arises, or as technology improves.

Method Target species
(Mediterranean Sea)		 Acceptable / most appropriate equipment
Time-Depth-Recorder tagging large cetaceans (fin whale, sperm whale) crossbow, pole
Time-Depth-Recorder tagging mid-size cetaceans (pilot whale, Cuvier's beaked whale, Risso's dolphin) pole
Time-Depth-Recorder tagging small cetaceans (bottlenose dolphin, common dolphin, striped dolphin) Tethys has decided to refrain from attaching TDRs to small cetaceans for the time being
Skin and blubber biopsy sampling large cetaceans (fin whale, sperm whale) crossbow
Skin and blubber biopsy sampling mid-size cetaceans (pilot whale, Cuvier's beaked whale, Risso's dolphin) biopsy pole
Skin and blubber biopsy sampling small cetaceans (bottlenose dolphin, common dolphin, striped dolphin) biopsy pole
Skin swabbing all cetacean species biopsy pole

 

Based on its experience with cetacean biopsy sampling in the Mediterranean Sea, the Tethys Research Institute proposes the following general guidelines.

The fact of providing such guidelines represents by no means an encouragement to conduct intrusive research, and is solely aimed at increasing awareness on the related problems and risks.

The guidelines are largely based on the Tethys Meeting on intrusive research methods held on 20 October 2000 at the Municipal Aquarium and Hydrobiological Station, Milan, Italy (*). Some or most guidelines are intended as commonsensical suggestions, and may be adapted on a case-by-case basis.

(*) PARTICIPANTS: Stefano Agazzi, Sabina Airoldi, Carla Almirante, Arianna Azzellino, Marta Azzolin, Giovanna Barbieri, Giovanni Bearzi, Sebastiano Bruno, Vittorio Fadda, Caterina Fortuna, Chiara Fumagalli, Nicolò Gavazzi, Maddalena Jahoda, Simone Panigada, Patrizia Patti, Giovanna Pesante, Elena Politi, Antonella Profice, Eletta Revelli, Francesco Ricciardi, Margherita Zanardelli

 

Guidelines

Sampling with lethal methods (e.g., "scientific whaling") is rejected as a cruel, unnecessary, and obsolete practice.

Some intrusive research methods may have a very low impact, but at times they may involve levels of risk that can be hard to estimate based on the available information. Therefore, intrusive research should only be considered when this represents the most effective and timely way to provide conservation data that are necessary for the protection of a given species, not obtainable otherwise.

The number of collected samples should be reduced to a minimum, and information coming from each sample should be optimized by expanding the number of possible analyses, and by taking care of all phases of the process.

Ideally, only experienced researchers and personnel should be involved in the process. Experience should be capitalized by selected people, rather than dispersed among many, in order to reduce risks for the animals, occurrence of incidents, and improper treatment of precious biological samples.

Based on its own experience, the Tethys Research Institute warns about the risks of biopsy darting at small cetaceans, a practice that Tethys has recently abandoned. On the other hand, skin swabbing, biopsy darting at large cetaceans, and biopsy sampling with a pole aimed a bowriding cetaceans are considered virtually free of risk, low-impact, and ethically acceptable if balanced by data that can be used for conservation purposes.

 

 

Skin swabbing for genetic analyses

Tethys recognizes that skin swabbing from bowriding cetaceans represents a simple and effective research method whenever blubber is not required. If carefully conducted, skin swabbing actually represents one of the least intrusive methods to obtain biological samples from free-ranging cetaceans.

Pros   Cons
Allows to perform analyses to study social organization, kinship, mating system, individual gender and identification, movement patterns, population size, stock identity, genetic phylopatry, variability within and among populations. Does not allow to perform most toxicological and feeding ecology analysis (e.g., contaminant levels, various biomarker analyses and toxicological tests performed on cell cultures, information on feeding ecology and nutritive condition through the examination of stable isotopes, fatty acids and lipid content in the blubber).
Cetaceans are sampled during bowriding, when they may expect tactlie stimuli and behavioral impact is minimal. The sample may be contaminated by foreign tissues on the swabbing sponge.
No physical lesions. Sometimes insufficent tissue is obtained, or this can be difficult to detect / remove from the sponge.
No risk of infection. Difficult to apply with cetacean species that do not bowride or stay away from the research platform.
Precision.
Low impact power transmitted to the animal.
Simple and low-cost.
Little or no ethical implications.

 

 

Skin and blubber sampling for genetic, toxicological, and feeding ecology analyses

The following Tables summmarize pros and cons, optimal sampling conditions, and equipment design / performance to mitigate the impact of biopsy sampling while increasing effectiveness, as based on Tethys own experience and on the available literature.

Pros   Cons
Allows to perform analyses to study social organization, kinship, mating system, individual gender and identification, movement patterns, population size, stock identity, genetic phylopatry, variability within and among populations. Rare occurrence of ‘undesired’ events. These may include missed shots, stuck darts or broken tips remaining attached to the animals, snagging of the dart retrieval line on the animal's flukes, repeated sampling of one individual, etc.
Also allows to perform most toxicological and feeding ecology analysis (e.g., contaminant levels, various biomarker analyses and toxicological tests performed on cell cultures, information on feeding ecology and nutritive condition through the examination of stable isotopes, fatty acids and lipid content in the blubber).
One case of mortality reported so far for biopsy darting at small cetaceans.
Behavioral impact in the mid and long-term can be difficult to ascertain.
Some behavioral impact in the short-term (due to either hit, miss, or boat manoeuvering around the animals).

Risk of infection? (no documented evidence so far).

 

Variable Mitigation strategy
Size of target species Reduce invasiveness as the size of target species decreases.
Age of target species Do not sample calves or juveniles.
Group composition Avoid sampling if calves are present in the group.
Behavior at the time of sampling Avoid sampling if the animals are socializing, mating, playing, resting, feeding, or whenever the disturbance may disrupt biologically-important activities.
Animal reactions to sampling Evaluate based on focused research with standard methods. Publish the data timely in appropriate journals.
Handling and processing of samples Subdivide the sample to enable a maximum of meaningful analyses by means of multidisciplinary work. Make sure that all the necessary equipment is in good conditions and has been properly sterilized. Chemicals / reagents must be fresh. User-friendly protocols should be available to facilitate complex procedures. All phases of handling and shipping should be programmed beforehand. Biological samples should be labelled with all necessary data (use proper ink and labels). Sample location and storage should be carefully tracked / monitored to avoid sample loss or degradation. Easy communication among team members should be ensured at all times. Labs should be checked for working efficiency and absence of contamination. Personnel / Institutional responsibilities should be defined.
Data sharing and access to information Maximize by appropriate data presentations at international conferences and timely publishing of the relevant data in peer-reviewed journals.
Sampler Should have previous experience with biopsy sampling. Should be skilled at handling and using the necessary equipment. Must have excellent aiming and timing skills. Must have appropriate field experience and knowledge of the target species, in order to evaluate the situation and do the sampling under the best possible conditions. Must adopt a precautionary approach. Must be in good psycho-physical conditions at the time of sampling. Must be aware of all the implications and potential risks. Should be willing to do the sampling based on a personal choice.
Other people on board Qualified assistants should help the sampler or be in charge of other phases of biopsy sample processing. Responsibilities and tasks on board should be clearly defined. Unexperience people should not interfere with sampling / handling operations.
Weather conditions Avoid sampling with rough sea or bad weather conditions.

 

Equipment Requirements / mitigation strategy
Dart projector Use high-quality projectors.
Use appropriate power. Adopt a precautionary approach. Evaluate power on fresh dead stranded animals if possible (Patenaude and White 1995).
Increase equipment precision. Increase aiming precision by appropriate training.
Biopsy dart Reduce dart weight to a minimum.
Reduce aerodynamic drag.
Maximize dart retrieval (ensure appropriate buoyancy, use bright colors etc.)
Make the dart solid and use materials that won't break following impact.
Avoid using dart retrieval lines that may snag on the animal's flukes / fins / bodies.
Biopsy tip Use a stop collar that is large enough as to avoid dart penetration.
Use high quality stainless steel or other high quality materials.
Reduce the total size of tissue sample to a minimum (this relates to the number and kind of lab analyses performed on the sample). Ensure minimal physical stress (best: no physical stress) to the animal.
Minimize depth of penetration, and ensure that the biopsy tip does not penetrate beyond the blubber layer.
Make sure that the biopsy tip is safely attached to the dart body.
Minimize the thickness of the biopsy tip (retention cylinder) while ensuring appropriate sturdiness to the tip.
Biopsy tip borders should be smooth where no penetration should occur (e.g., stop collar borders), and VERY SHARP where the biopsy tip enters the skin. The tip should be sharpened from the inner side of the hollow cylinder.
Several retention tools have been used, and it is unclear which ones provide the best retention. Barbed dental broaches or hooked retention needles have proved effective, as long as they have appropriate design and diameter, and they are safely attached to the biopsy tip. Retention needles should not move following penetration, and must be made of solid materials that guarantee that no parts will break apart.
Sample contamination The biopsy tip must be properly disinfected and treated with antibiotics immediately before (every) use. No contact between the biopsy tip and foreign bodies should occur after disinfection. Care should be taken to avoid contamination of the biopsy sample once taken.

 

Selected literature

Aguilar, A. and Borrell, A. 1994. Assessment of organochlorine pollutants in cetaceans by means of skin and hypodermic biopsies. pp.245-267. In: M.C. Fossi and C. Leonzio (eds.) Nondestructive biomarkers in vertebrates. Lewis Publishers, Boca Raton, FL.

Aguilar, A. and Nadal, J. 1984. Obtención de biopsias hipodérmicas de cetáceos en libertad. Invest. Pesq. 48(1):23-29.

Amos, W. and Hoelzel, A.R. 1990. DNA fingerprinting cetacean biopsy samples for individual identification. Rep. int. Whal. Commn, Special Issue 12:79-85.

Barrett-Lennard, L.G., Smith, T.G. and Ellis, G.M. 1996. A cetacean biopsy system using lightweight pneumatic darts, and its effect on the behavior of killer whales. Mar. Mamm. Sci. 12:14-27.

Bearzi, G. 2000. First report of a common dolphin (Delphinus delphis) death following penetration of a biopsy dart. J. Cetacean Res. Manage. 2(3):217-221.

Brown, M.W., Kraus, S.D. and Gaskin, D.E. 1991. Reaction of North Atlantic right whales (Eubalaena glacialis) to skin biopsy sampling for genetic and pollutant analysis. Rep. int. Whal. Commn, Special Issue 13:81-89.

Brown, M.W., Corkeron, P.J., Hale, P.T., Schultz, K.W. and Bryden, M.M. 1994. Behavioral responses of east Australian humpback whales Megaptera novaeangliae to biopsy sampling. Mar. Mamm. Sci. 10(4):391-400.

Clapham, P.J. and Mattila, D.K. 1993. Reactions of humpback whales to skin biopsy sampling on a West Indies breeding ground. Mar. Mamm. Sci. 9(4):382-391.

Cockcroft, V.G. 1994. Biopsy sampling from free-ranging bottlenose dolphins. pp. 32-33. In: G. Notarbartolo di Sciara, P.G.H. Evans and E.Politi (eds.) Methods for the study of bottlenose dolphins in the wild. European Cetacean Society Newsletter 23 (Special Issue).

Dawn Harlin, A., Würsig, B., Baker C.S., Tim, M. 1999. Skin swabbing for genetic analysis: application to dusky dolphins (Lagenorhynchus obscurus). Marine Mammal Science 15(2):409-425.

Fossi, M.C., Marsili, L., Leonzio, C., Notarbatolo di Sciara, G., Zanardelli, M. and Focardi, S. 1992. The use of non-destructive biomarker in Mediterranean cetaceans: Preliminary data on MFO activity in skin biopsy. Mar. Poll. Bull. 24:459-461.

Fossi, M.C., Lauriano, G., Simmonds, M.P. and Borsani, J.F. In press. A comment on the usefulness of biopsy techniques. Report of the Scientific Committee of the International Whaling Commission SC/52/WP13.

Fossi, M.C., Marsili, L., Neri, G., Casini, S., Bearzi, G., Politi, E., Zanardelli, M. and Panigada, S. In press. Skin biopsy of Mediterranean cetaceans for the investigation of interspecies susceptibility to xenobiotic contaminants. Mar. Environ. Res.

Gauthier, J., and Sears, R. 1999. Behavioral response of four species of balaenopterid whales to biopsy sampling. Mar. Mamm. Sci. 15(1):85-101.

Hooker, S.K., Baird, R.W., Al-Omar, S., Gowans, S. and Whitehead, H. In press. Behavioral reactions of northern bottlenose whales to biopsy and tagging procedures. Fish. Bull. US.

International Whaling Commission. 1991. Report of the ad-hoc working group on the effect of biopsy sampling on individual cetaceans. Rep. int. Whal. Commn, Special Issue 13:23-27.

Jahoda, M., Airoldi, S., Azzellino, A., Biassoni, N., Borsani, J.F., Cianfanelli, L., Lauriano, G., Notarbartolo di Sciara, G., Panigada, S., Vallini, C. and Zanardelli, M. 1996. Behavioural reactions to biopsy-darting on Mediterranean fin whales. European Research on Cetaceans 10:43-47.

Jahoda, M., Lafortuna, C.L., Almirante, C., Zanardelli, M., Panigada, S., Notarbartolo di Sciara, G. 2003. Mediterranean fin whale's (Balaenoptera physalus) response to small vessels and biopsy sampling assessed through passive tracking and timing of respiration. Mar. Mamm. Sci.

Lambertsen, R.H., Baker, C.S., Weinrich, M. and Modi, C.S. 1994. An improved whale biopsy system designed for multidisciplinary research. pp. 219-244 In: M.C. Fossi and C. Leonzio (eds.) Nondestructive biomarkers in vertebrates. Lewis Publishers, Boca Raton, FL.

Marsili, L., Fossi, M.C., Neri, G., Airoldi, S., Bearzi, G. and Panigada, S. In press. Skin biopsies for cell cultures from Mediterranean free-ranging cetaceans. European Research on Cetaceans 14.

Marsili, L., Fossi, M.C., Notarbartolo di Sciara, G., Zanardelli, M., Nani, B., Panigada, S. and Focardi, S. 1998. Relationship between organochlorine contaminants and mixed function oxidase activity in skin biopsy specimens of Mediterranean fin whales (Balaenoptera physalus). Chemosphere 37(8):1501-1510.

Natoli, A. and Hoelzel, R. In press. Genetic diversity in a Mediterranean population of the bottlenose dolphin in the context of world-wide phylogeography. European Research on Cetaceans 14.

Palsbøll, P.J. 1999. Genetic tagging: Contemporary molecular ecology. Biol. J. Linnean Soc. 68:3-22.

Palsbøll, P.J., Larsen, F. and Hansen, E.S. 1991. Sampling of skin biopsies from free-ranging large cetaceans in West Greenland: Development of new biopsy tips and bolt designs. Rep. int. Whal. Commn, Special Issue 13:71-79.

Palsbøll, P.J., Vader, A., Bakke, I. and Raafat El-Gewely, M. 1992. Determination of gender in cetaceans by the polymerase chain reaction. Can. J. Zool. 70:2166-2170.

Panigada, S., Zanardelli, M., Canese, S., and Jahoda, M. 1999. How deep can baleen whales dive? Mar. Ecol. 187:309-311.

Patenaude, N.J. and White, B.N. 1995. Skin biopsy sampling of beluga whale carcasses: Assessment of biopsy darting factors for minimal wounding and effective sample retrieval. Mar. Mamm. Sci. 11(2):163-171.

Walker, J.L., Potter, C.W. and Macko, S.A. 1999. The diets of modern and historic bottlenose dolphins populations reflected through stable isotopes. Mar. Mamm. Sci. 15(2):335-350.

Weinrich, M.T., Lambertsen, R.H., Baker, C.S., Schilling, M.R. and Belt, C.R. 1991. Behavioral responses of humpback whales (Megaptera novaeangliae) in the Southern Gulf of Maine to biopsy sampling. Rep. int. Whal. Commn, Special Issue 13:81-89.

Weinrich, M.T, Lambertsen, R.H., Belt, C.R., Schilling, M.R., Iken, J.H. and Syrjala, S.E. 1992. Behavioral responses of humpback whales Megaptera novaeangliae to biopsy procedures. Fish. Bull., U.S. 90:588-598.

Weller, D.W., Cockcroft, V.G., Würsig, B., Lynn, S.K. and Fertl, D. 1997. Behavioral responses of bottlenose dolphins to remote biopsy sampling and observations of surgical biopsy wound healing. Aquat. Mammal. 23:49-58.

Whitehead, H., Gordon, J., Mathews, E.A. and Richard, K.R. 1990. Obtaining skin samples from living sperm whales. Mar. Mamm. Sci. 6:316-326.