Move the plunger back and forth in the syringe several times to smoothen the withdrawal and then pull on the plunger to create negative pressure in the syringe. Retract the foreskin of the anesthetized rat from the end of the penis and hold the glands between the index and thumb pulling gently. The dorsal penile vein will appear as a superficial blue cord.
With the eye of the needle pointing upward, insert the insulin syringe into the vein at an angle of 35 degrees. Once the needle has entered the vein, blood will flow into the syringe. Slowly withdraw the plunger of the syringe at a slow and steady rate to collect the desired volume.
Remove the syringe. A blood drop will form on the puncture site. Aspirate the drop to collect a few more microliters of blood in case of a non-sterile procedure.
Apply light pressure to the puncture site to stop the bleeding and wipe the area with a new alcohol wipe. Place the penis back in its neutral position. After turning off the isoflurane, monitor the rat until complete recovery and return it to its cage.
The blood volume collected in the dorsal penile vein puncture method under anesthesia was compared with the blood collected in the tail vein puncture method with and without anesthesia. The result shows that the penile vein puncture method under anesthesia and the tail vein puncture method without anesthesia yielded comparable quantities of blood.