Thursday, August 25, 2011

Montreal Expedition: Blood processing

New England Aquarium researchers are reporting from the Biodôme in Montréal, Quebec, where they are conducting an experiment on the stress physiology of two species of wolffish.

Warning: this post contains images of needles and fish blood samples. If you have an aversion to these images, proceed with caution.

We can get a lot of information from the blood we collected from our wolffish. Over a one month period we collected four 3mL samples from each fish (for 20 Atlantic and 20 spotted wolffish, that's 160 samples, almost a half a liter of fish blood!). We carefully calculated the amount of blood we could safely draw without compromising the health of the animals. We used an anesthetic to sedate the fish to make them easier to handle and reduce the stress of air immersion and handling.

Wolffish were sedated before blood was drawn.

We started analyzing the blood samples immediately after they were drawn. The first test we conducted was to determine the levels of hematocrit. Hematocrit is the volume of blood that is made up by red blood cells. It indicates any number of things about the overall health of the animal. We are interested in hematocrit because low levels can result if an animal is stressed. Hematocrit levels are determined by drawing blood into microcapillary tubes and centrifuging the tubes at high speeds for a short amount of time. This force compacts red blood cells at the bottom of the tube. Hematocrit is read as the volume of packed red blood cells.

Left: Microcapillary tubes before centrifuging
Right: Microcapillary tubes after centrifuging

Hope being a scientist, reading hematocrit levels.

Hematocrit is the volume of packed red blood cells as a percent of the whole blood. This sample from A. minor has a hematocrit value of 10 percent, which is relatively low for the species. Until all the data is analyzed we cannot determine whether stress is a factor in depressed hematocrit levels.

After we read the hematocrit levels, we loaded the whole blood samples into a larger, chilled centrifuge. We did this so that we could isolate the plasma, which contains hormones such as cortisol (a stress hormone released as part of the primary stress response) as well as other molecules of interest. The plasma was pipetted and divided into three aliquots, which were frozen and will be used in different tests. One test is radioactive immunoassay (RIA) analysis, which will determine the concentrations of cortisol. Another test will determine concentrations of ions and glucose.

Left: Whole blood sample before centrifuging
Right: After centrifuging
The pellet is packed red blood cells, and the supernatant is blood plasma.

After centrifuging, the plasma was pipetted with care.

-Alena Gerlek

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