The overall goal of this procedure is to perform a minimally invasive blood sample harvest from an adult zebra fish that allows repeat collections from the same individual. This is accomplished by first pulling and cutting the tips of glass micro capillary needles. In the second step, the needles are heparinized and air dried.
A needle is then inserted into the blood collection site and the blood is aspirated into the needle tip. In the final step, the blood is expelled from the needle into the appropriate receptacle for downstream analysis. Ultimately, the glucose levels of the blood samples can be measured using a handheld glucometer.
The main advantage of this technique or existing methods like tail ablation or cardiac puncture, is that this blood collection method is both minimally invasive and allows repeat draws from the semi individual zebrafish. This method facilitates the acquisition of a quick and accurate assessment of and labels in the zebrafish blood, providing insight into metabolic disorders and other blood parameters such as Turkish and pharmacokinetics of specific drugs. What treatment of interest To prepare the glass micro capillary needles for blood collection begin by pulling a one millimeter outer diameter glass capillary with a needle pull.
Then using fine scissors, cut the needle tip obliquely. The ideal tip diameter is approximately 100 to 200 microns. Next place the precut needles one at a time into the nose piece end of an aspirator tube assembly and the mouthpiece into the mouth.
Immerse the needle tip into freshly prepared heparin solution and aspirate and dispense the solution to heparinized the needle. Then air dry the heparinized needles in a 10 centimeter Petri dish for at least one hour. To perform a blood collection, place a heparinized needle into the nose piece end of the aspirator tube, and grasp the end of the aspirator tube assembly.
Then holding the nose piece end and the needle together. Use the needle tip to carefully remove the interfering scales of an anesthetized adult super fish. Next, insert the needle at a 30 to 45 degree angle along the body axis and posterior to the anus in the region of the dorsal aorta.
Taking care to avoid puncturing the gastrointestinal tract. When the needle can be felt touching the spine begin to aspirate the blood. If the needle has correctly penetrated the artery, the blood will slowly rise in a sal manner without suction.
If the blood does not rise into the tip on its own, move the needle tip gently to encourage the blood flow. When the blood begins to rise, immediately stop moving the needle and apply gentle suction to the aspirator tube. After the appropriate volume of blood has been collected, stop the suction and remove the needle.
Using soft tissue paper, press the puncture site for 10 to 20 seconds to stop the bleeding and expel the blood from the needle onto a clean piece of perfil. Then immediately transfer the fish into a clean 28 degree Celsius water tank and gently swirl the water towards the gills until the animal begins to swim. To help with the anesthesia recovery, now insert a test strip from any commercial handheld glucometer completely into the meter and directly touch the blood drop.
Observe the automatic blood draw into the strip. The blood glucose result will be displayed within five seconds. Finally, record the result and discard the test strip here.
Representative blood biochemical analyses performed after blood collection as just demonstrated, are shown in this experiment. The normal value of the hemoglobin and triglycerol did not differ significantly between the male and female SPR fish, whereas the fasting blood glucose and total cholesterol levels of the males were significantly lower than those observed for the female group. In this experiment investigating the effects of diet on blood glucose levels, normal diet fed zebrafish exhibited stable blood sugar levels throughout the entire five week study period.
The overfed zebrafish group, however, experienced high blood glucose levels as early as week one and maintained this hyperglycemic condition throughout the experiment. It is important to note that the maximum blood volume that can be collected by this method is linearly correlated with the animal's body weight. Further, when the effects of the repeated harvesting of blood samples from the same individual fish were investigated, it was observed that the collection of two microliters of blood once daily every two days, or by a single collection of five microliters per week yielded a significant decrease in hemoglobin levels of the donors one week after the harvest of a single two microliter blood sample.
However, the hemoglobin levels were observed to be only slightly below normal, suggesting two microliters per week as the ideal timing and volume of repeat zebrafish blood collection. While you are attempting this procedure, it's important to remember to insert the needle gently and into the zebrafish skin to avoid interference with the skills.
