Blood withdrawal
The following procedure applies to animals in the 33-40 inch body length range, and blood volumes in the 20-50 ml volume range. The skin of the dogfish is wiped with 70% ethanol, and the syringe needle inserted exactly on the visible midline of the animal, about one-third of the distance from cloacal opening toward the tail ( Fig. 7). We use disposable 20- or 30- ml syringes with 1.5 inch, 22-gauge needles. The needle is inserted slowly, perpendicular to the animal, until a barrier or resistance is felt. It is then drawn back very slightly and the syringe plunger pulled slowly upward. If no resistance is felt, the needle is probably off center or tilted, and should be withdrawn and reinserted. Blood will be visible almost immediately in the syringe if the needle is in the right spot (the caudal blood vessels). When the desired volume has been reached (see below), the needle is withdrawn. We remove the needle from the syringe barrel before delivering blood into heparinized Elasmobranch Ringer's solution (Cavanaugh, 1975), to speed up the process and reduce mechanical stress to the cells. The heparinized Ringer's solution and subsequently the blood-Ringers mixture are kept in a graduated cylinder immersed in a bucket containing cool water (~18-23°C) to maintain it within or close to the physiological temperature range of the dogfish during transport to and from the laboratory.
Small volumes of blood (a few ml) can be taken from smaller animals (~20 inches; growing pups) by essentially the same procedure, except using 0.5 inch, 26-gauge needles, and a much smaller V-shaped restrainer.
Tagging
The fish typically already have a single tag through their dorsal fins (applied by MBL Dept. Aquatic Resources) when we use them. After blood removal, we add a second tag (Fig. 8) before returning the animal to the holding tank. It is useful to use a red tag, because it is easily seen as the fish swim in the holding tanks and is blood-colored. We used a Mark II Regular model, with stainless steel parts and #FD-67F tags, obtained from Floy Tag Manufacturing, Inc. (Seattle, WA 98105). The gun and tags are similar to those used to tag clothing.
Tagging allows animals to be conserved and reused (by others or by us), while alerting everyone to the fact that blood has been taken. The latter is important because, potentially, blood loss could affect some types of experiments performed by other laboratories. Tagging is also vital in experiments requiring long-term maintenance and repeated use of animals (see below). For such experiments individual fish must be recognizable, and this can be accomplished through use of the following tag variables - tag color, right vs. left side of dorsal fin, and location on dorsal fin (anterior, mid, posterior).
Blood volume vs. fish size; repeated use of animals
If blood is to be drawn from a given fish only once, the volume can be relatively large. As a guide, we take 40-50 ml from animals 36-40 inches in length (nose tip to tail tip), and 20-30 ml from animals ~33-35 inches. At these volumes the fish generally resume normal swimming quickly upon return to the holding tank or sea. With respect to the possibility of repeated use of an animal, we conducted a pilot study using nine dogfish (mixed sexes, 33-39 inches) maintained in the MBL Marine Resources Center over a ten week period (during the summer of 1992). Individual fish were identified by specific tagging. Four to five blood samples were taken from each of five animals at intervals of two weeks or more and no samples were taken from three animals serving as maintenance controls. The ninth fish died of unknown causes one week after its first bleeding, but all others survived for the full period. Blood volumes were 10 ml per sample for smaller animals (33 inches) and 20 ml for larger ones (36-39inches), with one exception, a 33 inch fish from which 5 X 20 ml was taken. Thus, repeated reduced-volume bleeding of smooth dogfish maintained for long periods is feasible.