Guideline #29
Categorizing Pain or Distress
1. References:
1.1. Animal Welfare Act CFR 9, Chapter 1, Subchapter A – Animal Welfare, Parts 1-4.
1.2. Animal and Plant Health Inspection Service, USDA, Animal Care Tech Note, APHIS-23-027, July 2023.
1.3. Sikes R, et al. 2016 Guidelines of the American Society of Mammalogists for the use of wild animals in research and education. Journal of Mammalogy, 97(3):663-688, 2016.
1.4. Guide for the Care and Use of Agricultural Animals in Research and Teaching, 4th edition.
2. Purpose/Scope: The Animal Welfare Act defines a painful procedure as “any procedure that would reasonably be expected to cause more than slight pain or distress in a human being to which that procedure is applied, that is, pain in excess of that caused by injections or other minor procedures.” The Institutional Animal Care and Use Committee (IACUC) is responsible to ensure that investigators avoid or minimize discomfort, pain and distress to animals; consider alternatives to any procedure that may cause more that slight or momentary pain or distress; and consulted the Attending Veterinarian when planning these procedures. Vertebrate animals used for teaching, research and demonstration will be assigned to USDA pain and distress categories and approved by the IACUC committee. This guideline is to provide guidance to Kansas State University IACUC and investigators.
3. Recognition of Pain and/or Distress: It is the responsibility of the animal care staff, the research staff, clinical veterinarians, and the IACUC to continue to monitor animals for pain, distress, illness, morbidity or mortality throughout the research study. If unexpected pain and/or distress occurs, and is more than an isolated incident, it is the PI’s responsibility to consult with the Attending Veterinarian (AV) and submit a modification delineating the unexpected outcome and the proposed resolution (e.g., administration of analgesics, wound treatment, etc.). The AV and/or IACUC has the authority to suspend activities associated with the protocol until intervention strategies have been approved. Alternatively, the Principal Investigator (PI) could justify the need for unrelieved pain and/or distress in the amendment, and in the case of USDA covered species, in a Category E justification (see appendix 2).
Appendix 1: Clinical Signs of Pain/Distress by Species
| Species | Behavior | Appearance |
| Rodents | Decreased activity; excessive licking/scratching; self-mutilation; avoidance or aggression; abnormal locomotion (stumbling); no nest building | Piloerection; rough or stained haircoat; abnormal stance or hunched back; porphyrin staining (rats); rapid, shallow respirations |
| Dog | Excessive licking; increased aggression or avoidance; increased vocalizations (whimpering, howling and growling); self-mutilation; reluctance to ambulate | Stiff body movements; rough haircoat, lack of grooming; trembling; guarding; rapid, shallow respirations; hypersalivation |
| Cat | Hiding; increased vocalizations (growling, hissing); increased aggression | Stiff body movements; rough haircoat, lack of grooming; trembling; guarding; rapid, shallow respirations; hunched posture; tail twitching; flattened ears |
| Swine | Increased vocalization and aggression; reluctance to move; altered gait; decreased activity; will stand but lays down, lack of interest in eating or drinking | Immobile; piloerection; rapid, shallow respirations; increased muscle tension around the eyes; hunched posture |
| Ruminants | Increased vocalization; isolation; bruxism; decreased activity or reluctance to move; restlessness; altered gait | Stiff body movements; rapid, shallow respirations; tucked abdomen |
| Horses | Aggression or avoidance to handling; restlessness; kicking at abdomen; altered gait; reluctance to move; anxious while restrained; decreased interest in food/water | Anxious appearance; profuse sweating; increased respiratory rate |
| Rabbits | Head pressing; bruxism; aggressive or avoidance; increased vocalizations; reluctant to ambulate; self-mutilation | Hypersalivation; hunched posture; rapid, shallow respirations |
| Fish | Improper buoyancy; lethargy; surface breathing | Opercular flaring; sloughed mucus; clamped fins; petechiation or hemorrhage; change in body color; scale loss; whirling |
| Birds | Inappetence; altered gait or posture | Wasting (decreased pectoral muscles); ruffled feathers; rapid |
Other behavioral signs or abnormalities in appearance may be noted and determined to be associated with pain and/or distress.
4. USDA Pain and Distress Categories: Regulatory agencies require all vertebrate animals used in research, teaching and demonstration be assigned to an appropriate pain category. Appendix 2 shows the USDA pain categories and examples that may apply to each category. Due to the variety of protocols submitted to IACUC, this list cannot be all inclusive. Using USDA guidance, the IACUC will review all protocols to determine the proper categorization. When anesthetics, analgesics, or sedatives are used for the purpose of improved animal welfare, restraint and safety for procedures that do not involve pain or distress, these animals should be placed in Category C. However, an AV consult will be required to ensure that the safety and efficacy of the anesthetic, analgesic or sedative has been fully considered. All protocols with animals reported in Category D and E will require an AV Consult. If multiple procedures are performed that involve different pain and distress categories for the same animal, the animal should be placed in the category consistent with the greatest amount of pain or distress experienced.
Appendix 2: USDA Categories of Pain and Distress and Examples
| Category | Explanation | Examples |
| B | Animals being bred, conditioned, or held for use in teaching, testing, experiments, research, or surgery but not yet used for such purposes | • Animals on a holding protocol • Animals on a breeding protocol with no research or experimental component • Animals held in quarantine or acclimation period prior to use • Observing animal behavior in their home enclosure/environment without manipulation |
| C | Animals upon which teaching, research, experiments, or tests were conducted involving no or only momentary pain or distress, and no use of pain-relieving drugs |
• Observing animal behavior in the lab • Administering an anesthetic, analgesic, or tranquilizing drug to an animal for restraint purposes to perform a procedure that involves no or only momentary pain or distress such as radiography, ultrasound, blood collection, abdominocentesis, intraperitoneal injection, cystocentesis, urinary catheterization, nasogastric intubation rectal palpation, etc.* |
| D | Animals upon which teaching, research, experiments, or tests were conducted involving accompanying pain or distress to the animals and for which appropriate anesthetic, analgesic, or tranquilizing drugs were used | • General anesthesia • Surgical manipulations (survival or non-survival) or laparoscopy in which the animals received appropriate pre, intra-, and post-operative anesthetics and analgesics • Routine agricultural husbandry procedures such as castration, disbudding, dehorning, etc. if alleviation of pain/distress occurs • Surgical exposure of blood vessels for catheter placement • Tail snipsʶ or tattooing in rodents >21 days old that requires general anesthesia • Using Freund’s Complete Adjuvantˀ, Incomplete Freund’s Complete Adjuvant, Resiniferatoxin (RTX), capsaicin or other inflammatory agents if alleviation of pain/distress occurs • Tumor induction or implantation if alleviation of pain/distress occurs# • Induced infections or antibody production • Retro-orbital blood collection in mice and rats • Forced exercise or behavioral testing protocols with approved interventions and end points • Exsanguination under anesthesia followed by euthanasia |
| E | Animals upon which teaching, research, experiments, or tests were conducted involving accompanying pain or distress to the animals and for which the use of appropriate anesthetic, analgesic, or tranquilizing drugs would have adversely affected the procedures, results, or interpretation of the teaching research, experiments, surgery, or tests | • Any Category D procedure for which needed analgesics, tranquilizers, or anesthetics are withheld for justifiable study purposes • Paralysis or immobilization of a conscious animal • LD50 studies • Exposure to extreme environmental conditions • Using Freund’s Complete Adjuvantˀ, Incomplete Freund’s Complete Adjuvant, Resiniferatoxin (RTX), capsaicin or other inflammatory agents that cause unalleviated pain or distress or may cause tissue sloughing • Toxicological, microbiological, or infectious disease research that requires continuation after clinical signs are evident without medical care or that requires death as an endpoint • Forced exercise or behavioral testing protocols that could reasonably be expected to cause distress or exhaustion • Applying noxious stimuli that the animal cannot avoid/escape (i.e. electric shock) • Long-term restraint (days to weeks) |
*Category C procedures that require an AV consult; ʶFollow KSU IACUC Guideline #5; #Follow KSU IACUC Guideline #18; ±Within Blood Collection: Maximum Volumes and Fluid Replacement limits of total volume and frequency; ˀFollow KSU IACUC Guideline #12
5. For additional information please contact:
• The University Research Compliance Office (URCO) – 785-532-3224
• Comparative Medicine Group (CMG) - 785-532-5640
LAST REVIEWED AND ADOPTED BY THE IACUC: May 1, 2024