Alternative Techniques for Obtaining Blood Samples from Leatherback Turtles
ABSTRACT
We describe a low-risk, alternative technique for sampling blood from leatherback turtles (Dermochelys coriacea) using interdigitary vessels in their flippers. Because this technique allows for repeated, large-volume blood samples without requiring restraint of turtles, the technique is preferred over using the dorsal cervical sinus in almost all cases of blood sampling from leatherbacks, and also applies to other sea turtle species.
Blood sampling of wild, captive, and stranded sea turtles is fundamental to a wide variety of experimental, analytical, and health assessment applications. Specifically, analyses of blood samples have been used for population genetics studies (Dutton et al. 1999; Crim et al. 2001) and contaminant monitoring (Keller et al. 2006); to describe an individual turtle's reproductive status (Rostal et al. 1996); to assess levels of stress hormones associated with reproduction, the nesting process, and handling by humans (Gregory and Schmid 2001); to determine hemostatic processes (Soslau et al. 2004, 2005); and can be employed in field experiments of metabolism (Southwood et al. 2006; Wallace et al. 2005) and trophic ecology (Wallace et al. 2006; Seminoff et al. 2006). In addition, analyses of blood samples from stranded animals can provide information concerning the animals' physical condition, which can then be used to determine the health care necessary to rehabilitate the animal and possibly establish the cause of the stranding.
Traditionally, blood samples have been collected from the dorsal cervical sinuses (DCS) of sea turtles (Owens and Ruiz 1980). These vessels generally are convenient to access because they are superficial, the biventer and transverse cervical muscles provide consistent landmarks to facilitate easy location of the DCS (Wyneken 2001), and large volumes of blood can be obtained. For these reasons, sampling blood from the DCS is advantageous in most sea turtle species. However, sampling from the DCS of adult leatherback turtles (Dermochelys coriacea) is particularly challenging due to the animals' thick, muscular necks, the deep location of their sinuses relative to their neck surface (Wyneken 2001), and lack of repeatable, identifiable external landmarks among individuals used to locate and sample from the DCS. Furthermore, large needles (3.5–6 inches by 18 gauge) are required, and repeated samples are very difficult to obtain from the DCS in leatherbacks due to tissue swelling.
To ensure accurate location of the DCS of leatherbacks, the optimal time to sample blood would be while the animals are quiescent or motionless. Therefore, to obtain a quality blood sample from the DCS, restraint of the turtle generally is necessary. Whereas capture and restraint of other sea turtle species is regularly achieved (see Jacobson et al., http://accstr.ufl.edu/blood_chem.htm, for example), restraining leatherbacks involves significant logistical difficulties, given the massive body size of these animals (200–900 kg). Considering the challenges listed above, the DCS technique can be rather invasive and exceedingly difficult when applied to leatherbacks. Thus, alternative blood-sampling techniques for these animals would be highly useful and preferable. Here, we describe an effective, low-risk technique for obtaining blood samples from adult leatherbacks using interdigital vessels (IDV) in their flippers.
Methods
The IDV in either anterior or posterior flippers offer multiple sites for easily obtaining repeated, relatively large-volume blood samples from adult leatherback turtles. The IDVs follow the length of the phalanges and are best sampled at bifurcations located in the angles formed by the first phalanges of the digits (Fig. 1). At each bifurcation, there is a venous plexus between each pair of digits in the anterior and posterior flippers. The arrangement of the IDVs in leatherbacks (and other sea turtles) compares to that present in pinniped flippers, and the blood-sampling technique we describe here is similar to that used for pinnipeds (Bossart et al. 2001).
Citation: Chelonian Conservation and Biology 6, 1; 10.2744/1071-8443(2007)6[147:ATFOBS]2.0.CO;2
The IDVs between any pair of digits are accessible, but the first and second digits (from the tail) in the posterior flippers are probably the easiest to access. Because the IDVs are relatively superficial (ca. 1 inch or 2.5 cm deep), a 1-inch by 20–22 gauge needle (with a Vacutainer blood collection tube; Becton Dickinson, Franklin Lakes, NJ) generally is sufficient; even an extremely large leatherback can be sampled effectively using this method. After the interdigitary region of the skin has been sanitized (using a 70% ethanol solution, for example), the needle is inserted along the side of the phalanx, at an angle of approximately 20°–30° to the flipper surface, about 1 inch (ca. 2.5 cm) from the boney angle (interdigitary venous plexus) (Fig. 2). With Vacutainer tube connected, the needle is inserted parallel to the first phalanx and advanced toward the boney angle (interdigitary plexus) until blood enters the Vacutainer tube.
Citation: Chelonian Conservation and Biology 6, 1; 10.2744/1071-8443(2007)6[147:ATFOBS]2.0.CO;2
The IDVs in the anterior flippers are also parallel to the phalanges and can be located most effectively in an area up to 6 inches (ca. 15 cm) proximal or distal to the palpable joint formed by the first and second phalanges (P1 and P2). The needle is inserted through the skin in the shallow interdigital depression between the second and third digits. The needle insertion procedure is similar to that described above.
Discussion
The IDV technique is a highly effective, low-risk method for obtaining blood samples from leatherbacks. There are several distinct advantages to this IDV technique. First, repeated samples from the same turtle can be taken by using sampling sites between different digits on the same flipper or on different flippers. For example, multiple samples can be taken from a front flipper if the initial sample is taken from a distal location and subsequent samples are taken at progressively more proximal sites. Wallace et al. (2005) used the IDV technique to obtain sequential blood samples from different sites on the rear flippers of individual leatherbacks.
Second, restraint of individual nesting leatherbacks is not necessary for the IDV technique. During oviposition, for example, 1 of the posterior flippers is usually flat and motionless on the sand surface outside of the nest chamber and can be accessed easily for blood sampling. In addition, because the terrestrial gait of adult leatherbacks is characterized by a series of lunges punctuated by brief periods of no movement (Wyneken 1997), either rear flipper can be accessed for blood sampling using the IDV technique while the turtle is motionless during a resting pause on its post-nesting return to the sea. For example, Wallace et al. (2006) obtained blood samples from 18 different individual leatherbacks at Parque Nacional Marino Las Baulas, Costa Rica, during oviposition or while turtles were returning to the ocean after completion of the nesting process. Thus, the IDV technique can be used during multiple phases of the nesting process without restraining the turtle.
Third, it is possible to obtain large volumes of blood using the IDV technique. Recent studies successfully obtained samples of 5 to 20 ml using the IDV technique (Soslau et al. 2004, 2005; Wallace et al. 2005, 2006). In addition, because vasculature in flippers of other sea turtle species is similar to that in leatherback flippers, the IDV technique can be effectively applied not only to leatherbacks, but to other sea turtles species as well.
It is worth noting that recent blood biochemistry analyses on sea turtles indicate more variability in samples taken from hind limbs than from jugular veins (Jacobson et al., http://accstr.ufl.edu/blood_chem.htm). However, Wallace et al. (2005) reported that concentrations of stable isotopes in samples taken from the hind flippers were similar to those from the DCS. Thus, comparisons of biochemical analyses of blood samples from different anatomical locations should be implemented where relevant to ensure accurate interpretation of analytical results. Considering its anatomical and logistical advantages, the IDV technique is preferable to the DCS sampling technique in almost all cases where blood sampling from leatherbacks is necessary, whether on wild, captive, or stranded individuals.
Schematic diagram of phalanges and interdigitary vessels (in black) of a leatherback rear flipper.
External landmarks on leatherback rear flippers used for interdigitary vessel blood-drawing technique. The optimal needle insertion points (indicated by black arrows) are approximately 1-inch distal to the junctions of each pair of phalanges (phalanges highlighted by white dashed lines).
