Skip to content Skip to main navigation Report an accessibility issue

Guidelines for the use of Complete Freund’s Adjuvant in Rabbits

These guidelines are based, in part, on the National Institutes of Health intramural “Guidelines for the Use of Freund’s Adjuvant in Laboratory Animals.” The Committee recognizes that the scientist is the best qualified person to select the appropriate adjuvant to be used. It will consider justified applications for use of adjuvants outside these guidelines. Uses of Complete Freund’s Adjuvant (CFA) in species or by routes not covered in these guidelines must be addressed and justified in the protocol.

The IACUC, in its deliberations over protocols, most closely examines those likely to inflict pain or distress on the animals. As the level of “ethical cost” increases, the scientist’s burden of justification increases. This is especially true in the case of adjuvants. The use of CFA does increase the “ethical cost.” Consequently, the IACUC will expect the use of less pathogenic alternatives whenever possible.

Alternatives which reduce the number of animals required (tissue culture, chicken eggs, etc.), or utilize less traumatic adjuvants must be considered.

Complete Freund’s Adjuvant is a water in oil emulsion containing killed, dried Mycobacterium butyricum which has been used to enhance antigenicity and stimulate an immune response greater than antigen alone. Incomplete Freund’s Adjuvant (IFA), water in oil emulsion only, is used for similar reasons. The improper or unnecessary use of CFA may cause inflammation, induration, and/or necrosis in laboratory animals. The most severe inflammatory responses in animals are seen following multiple injections of CFA. The intention of the following guidelines is to minimize potential animal discomfort associated with the use of adjuvants in research.

  1. CFA must be used only when absolutely necessary. (i.e. small amounts of antigen available, weak antigens, etc.)
  2. If CFA is used, it must be limited to the initial immunizing dose. If more than one dose is proposed, it must be strongly justified with objective data.
  3. The normal routes of injection of CFA are intramuscular (IM), subcutaneous (SC), or intradermal (ID). It must never be given intravenously. Even when given by the approved routes, CFA may cause severe local and systemic pathology if not properly used. ID injection, in particular, may result in skin necrosis and sloughing. Intramuscular injection, especially in the leg, can cause large areas of inflammation and necrosis. The use of footpad injections is prohibited unless justified with objective data. Regardless of route of injection; site, dose and volume guidelines given below must be followed:
    • All intradermal injection sites must be clipped, cleaned and disinfected.
    • For intradermal inoculation, no more than .05 ml is injected per site. The FCA: antigen emulsified mixture of 1:1 is commonly used. If the emulsion is complete, a 23 gauge needle on a 1/2 ml syringe will allow for accurate dosing. If the emulsion is not complete, dosing will be difficult. Injection sites must be separated from each other widely enough to ensure continued blood supply to adjacent areas of skin and subcutis. Number of injection sites should not exceed 10.
    • For subcutaneous inoculation, no more than 0.25 ml is injected per site. Injection sites must be separated from each other widely enough to ensure continued blood supply to adjacent area of skin and subcutis. A 20 gauge needle or smaller is recommended. Number of injection sites should not exceed 10.
    • For intramuscular inoculation in the rabbit, no more than 0.3 ml is given in one hind leg of the rabbit. Only one leg may be used in each animal. Reportedly, much less pathology results from the intramuscular injection in the lumbar muscles. If the lumbar muscles are used, up to 0.3 ml may be injected on each side.
  4. An inoculum must be free of extraneous microbial or other particulate contamination when possible.
  5. Animals exhibiting signs of pain, discomfort, or distress must receive appropriate relief unless written scientific justification is provided in the animal use protocol and approved by the IACUC.
  6. The USDA has declared that the use of Complete Freund’s Adjuvant (CFA) may cause more than momentary pain or distress and an alternative must be considered. A written narrative description of the methods and sources used to search for alternatives to painful procedures must be provided in the animal use protocol. The minimal written narrative should include:
    • the databases searched or other sources consulted
    • the date of the search and the years covered by the search
    • the key words and/or search strategy used by the Principal Investigator when considering alternatives or descriptions of other methods and sources used to determine that no alternatives were available to the painful or distressful procedure
    • The narrative should be such that the IACUC can readily assess whether the search topics were appropriate and whether the search was sufficiently thorough.


  1. Anon. Guidelines for use of Freund’s Adjuvant in Laboratory Animals. NIH Intramural Guidelines.
  2. Broderson J. A Retrospective Review of Lesions Associated with the use of Freund’s Adjuvant. Lab An Science, 1989; 39:400-405.
  3. Harlow E, Lane D. Antibodies, A Laboratory Manual. Cold Spring Harbor Laboratory, 1988; 96-97.
  4. Carroll SB, Stoller BD. Antibodies to Calf Thymus RNA Polymerase from Egg Yolks of Immunized Hens. J. Biol. Chem., 1983; 258:24-26.
  5. Polson A. et al. Isolation of Viral IgY Antibodies from Yolks of Immunized Hens. Immunological Communications, 1980; 9:475-493.
  6. Smith HC. et al. Macromolecular Domains Containing Nuclear Protein p107 and U-snRNP Protein p28: Further Evidence for an in situ Nuclear Matrix. Molec. and Cell. Biochem, 1986; 70:151-168.
  7. Smith HC. et al. Alterations in Chromatin Conformation are Accompanied by Reorganization of Nonchromatin Domains that Contain U-sn RNP Protein and Nuclear Protein p107. J. Cell Biology, 1985; 101:560-567.
  8. Niemi SM, Fox JG, Brown LR, Langer R. Evaluation of Ethylene-vinyl Acetate Copolmer as Non-inflammatory Alternative to Freund’s Complete Adjuvant in Rabbits. Lab An Science 1985; 35:609-612.
  9. Bomford R., Adjuvants for Anti-parasite Vaccines. Parasitology Today 1989; 5:41-46.
  10. McGuill MW, Rowan AN. Refinement of Monoclonal Antibody Production and Animal Well-Being. ILAR News 1989; 31:7-11.
  11. Rudbach JA, Cantrell JL, Ulrich JT. Immunological Adjuvants for Bioengineered Antigens. Proc. Biotech San Francisco International Conf 1986.
  12. Anon. Ribi Adjuvant System (RAS). RIBI Immunochem Research, Inc. Helena, Montana 1985.
  13. USDA Policy #11, “Painful/Distressful Procedures, April 14, 1997
  14. USDA Policy #12, “Written Narrative for Alternatives to Painful Procedures, April 14, 1997.

Please see: Institutional Policies and Guidelines on Adjuvants and Antibody Production, Lynn R. Jackson and James G. Fox, ILAR Journal, Volume 37, Number 3 1995 Pages 141-152.