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Guidelines for Immunization of Research Animals



While a variety of adjuvants are available for use in research animals, Freund's adjuvant is perhaps the most commonly used. Complete Freund's adjuvant (CFA) may cause inflammation, induration, pain and necrosis at the injection site in mammals. Despite the common perception that CFA is a superior adjuvant, numerous studies have demonstrated comparable results with less irritating adjuvants.  It is important to note that complications are still possible with other adjuvants, as well.  

In order to eliminate or reduce discomfort from Complete Freund's adjuvant, the following guidelines have been established by the IACUC. Departures from these guidelines require adequate scientific justification on your Animal Care and Use Protocol form for approval by the IACUC. 

  • The use of other adjuvants must be considered.
  • CFA may be used only for the first (priming) dose. Subsequent immunizations should be with incomplete Freund's or another adjuvant unless justified. This is rarely warranted and if allowed, an interval of at least three weeks should be given between doses.

Route of Administration

Because of the potential for complications from certain routes of administration of immunizations, the following guidelines have been established. 

  • The inoculum should be free of extraneous microbial contamination. Millipore filtration of the antigen before mixing with the adjuvant is recommended when possible.
  • Injection sites should be cleaned to remove debris that may result in contamination and infection.
  • Injections containing Freund's complete adjuvant should be given subcutaneous (SQ), rather than intradermal (ID), intramuscular (IM), intravenous (IV) or intraperitoneal (IP). ID injections frequently result in skin necrosis and sloughing. IM injections can result in temporary or permanent lameness. IV injections can cause pulmonary lipid embolism.
  • For subcutaneous injections, the inoculum containing the adjuvant should be divided into fractions so that no more than 0.1 ml is injected per site for rabbits and 0.05 ml per site for mice.  If skin necrosis results while following these guidelines, future  injections should be spaced farther apart.
  • The IACUC discourages injection of foot pads because animals may develop chronic pain and secondary infections in the inflamed areas. If this procedure is to be used, it must be described and its use scientifically justified, including documentation that injections in other sites do not produce adequate antibody titers for the specific antigen being used. If used, only one hind foot may be injected with a maximum volume of 0.05 ml per site, and injections must be spaced at 10 day to 2 week intervals. Animals that have received foot pad injections must be housed on contact bedding rather than a metal mesh floor.

The IACUC has not established guidelines for immunization of birds.  Complete Freund's Adjuvant does not seem to result in the same types of lesions as in mammals.  However, investigators still must specify the details of the immunization procedure in the Animal Usage Form.  In addition, investigators should be aware that the IACUC may request changes in immunization protocols if they result in abscess formation or other painful lesions in birds.


The information contained in this site is intended as a reference for University of Minnesota investigators, and animal husbandry and veterinary staff. Drug information and dosages are derived from a variety of sources and do not necessarily guarantee safety or efficacy. Information obtained through this site should not be relied upon as professional veterinary advice. Any medications administered or procedures performed on animals should only be performed by or under order of a qualified, licensed veterinarian.