ADMINISTRATIVE INFORMATION
A. Investigator Name:
Work Section/Group:
Phone:

B. Co-Investigator(s) Names(s):

C. Title of Proposal: Bleeding protocol for mice
Objective: To use the more humane and simple method of bleeding laboratory mice.

Significance:The present methods of bleeding laboratory mice are cumbersome and/or inhumane. The primary method used in the United State is retro-orbital bleeding. This is a rapid and efficient bleeding method but extremely inhumane for the mouse. In fact, the United Kingdom has banned this procedure. Alternatively, bleeding by clipping off the end of the tail is a method that is simple and slightly more humane, but yields a very limited amount of blood. Both of these methods are not useful when multiple samples are required from the same animal.

Bleeding from the Saphenous vein is much more humane and can be done multiple times to the same animal. However, this is a very cumbersome method that is not at all compatible with bleeding many animals. In short, this method involves shaving the area around the knee joint and poking the vein with a needle. Blood is collected in a tube from the puncture. Bleeding can be easily stopped by holding a sterile gauze pad on the puncture wound.

Use of the cheek pouch or Submandibular bleeding method has been done for many years. Briefly, mice have a large vein draining the head and the cheek skin area that passes down to the jaw at the back end of the cheek pouch (see enclosed illustration). We have used a scalpel to puncture this vein and blood is collected as in the saphenous method. A sterile gauze pad can be applied to stop the bleeding. The problem with this method is control of the scalpel. Poking with too much pressure leads to extensive damage to the mouse cheek. Poking with too little pressure yields little or no blood. This has been a very difficult method to teach and only a few investigators ever become proficient at bleeding mice with this method.

MEDIpoint, Inc., has designed and tested a mouse bleeding lancet, the Goldenrod animal bleeding lancet. Using the Submandibular site for bleeding, this lancet eliminates the problems experienced when using the scalpel. Instead, the lancet comes with specific point lengths of 4.0, 5.0, and 5.5 mm. The different point lengths are used for different size mice. The design of the lancet allows the investigator to stick the mouse with enough pressure to insure a good blood draw. As in human blood drawing lancets, the design makes it so the puncture is only as deep as the point of the lancet. Introducing this method to investigators presently using other mouse bleeding methods has shown that there is a rapid development of ease and comfort using this new method.

D. If procedures cause pain or distress, Federal regulations require that you provide a written narrative of the methods used to determine whether or not alternatives exist to procedures which may cause pain or distress.

The lancet procedure for bleeding causes only momentary pain and distress. All animals are in category I.

E. Pain and distress to animal subjects:

Species	Category I	Category II	Category III
	No pain/distress	Pain/distress avoided by the use of drugs	Pain/distress NOT avoided by the use of drugs
	# of Animals	# of Animals	# of Animals
Mice	all

F. Has a literature search been done to prevent duplication of previous experiments?
Yes

What databases were used?
PubMed (1974-2005), Google, Yahoo
Search performed: Jan 24, 2005.

List the key words used.
Mouse suppliers, IACUC, AAALAC, bleeding techniques, retro-orbital, Submandibular, saphenous.

Results all described bleeding methods reviewed in the Significance section above. Internet searches yielded many IACUC web sites with protocols approving retro-orbital bleeding of mice. Also, searches found ILAR and PHS guidelines and regulations also allowing retro-orbital bleeding of mice.