The issue of repeated blood collection methods needs to be addressed, since the majority of studies on rats require them. It is important to take into account that a sampling method can be very efficient in certain context, but become unsuitable for another study. This protocol aims to offer simple and reliable blood sampling alternatives to researchers.
Both methods can be done by a single operator and require no extensive prior training, which allows for standardized procedure. When an animal is under anesthesia, the effect of isoflurane can lead to vessel spasm and make the tail vein puncture unsuitable for blood collection. Our protocol offers an alternative in this situation, the penile vein puncture, which presents a better success rate and yields significantly more amounts of blood in less time.
To begin, secure the rat in a plastic restraining cone and close the large end of the cone around the base of the tail. Ensure that the animal is comfortable and breathing is unrestricted throughout the procedure. Dip the tail in warm water for about one minute to dilate the vein, and dry the tail with a paper towel.
In the restrainer, position the animal facing down with the tail lying on a heating pad. To select the appropriate tail vein for sampling, rotate the entire animal to the right or left side. Use the terminal third of the tail for blood vessel puncturing, since the vessels become more superficial in this zone.
Wipe the tail with 70%ethanol wipes at the puncture site, and place it on the edge of the heating pad, creating an angle in the terminal third of the tail. This brings the vein to the surface and creates more space for taking the sample. For blood sampling, 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.
Secure the tail flat on the heating pad using the non-dominant index and middle finger. Place the middle finger proximally and the index finger distally with the puncture site between these two fingers. Slide the insulin syringe against the index finger with the eye of the needle pointing upward until it is inserted into the vein.
This creates an angle of 35 degrees between the needle and the tail. Once the needle has entered the vein, blood will flow into the syringe. Slowly withdraw the plunger of the syringe at a steady rate to collect the desired volume.
Remove the syringe from the tail, and allow a blood drop to form on the puncture side of the tail. Aspirate this blood to collect a few more microliters of blood in case of a non-sterile procedure. Apply pressure to the puncture site to stop the bleeding, and wipe the area with a new alcohol wipe.
After removing the rat from the cone, return it to its cage. The success rates with the tail vein puncture methods were compared with the success rate of the penile vein puncture method under anesthesia. Interestingly, under anesthesia, the tail vein became very unreliable and had a success rate of only 25%The success rates showed almost similar results for the tail vein puncture in conscious rats, and the penile vein puncture under anesthesia.
The blood sampling was performed in less than six minutes in the tail vein puncture without anesthesia, and the penile vein puncture method under anesthesia, whereas the tail vein puncture method under anesthesia took more than eight minutes due to multiple failures. 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.
