NIH Vertebrate Animals

Last updated: February 08, 2024

This attachment is required only for research involving vertebrate animals (e.g., mice, rats, non-human primates, frogs). It is usually one to two pages long, and needs to include:

  1. description of procedures
  2. justification for using animals
  3. description of how the animals' discomfort will be minimized
  4. how the animals will be euthanized (only required if your method of euthanasia is not consistent with the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals)  

If you are using animals, details of power analyses must be provided in the Research Strategy to facilitate review of rigor. 

Downloadable Template: Vertebrate Animals Section for mice  

NIH SF424 Instructions:

Examples of Vertebrate Animals Sections

Example: The laboratory mouse, Mus musculus, is/are commonly used and well described animal systems for [type]experiments: they are phylogenetically close to humans (there is a rat genome counterpart to almost all human genes known to be associated with diseases and 99% of mouse genes have a direct human counterpart); they have a relatively short life cycle and large little size (decreasing costs); as well as many molecular, genetics and other techniques and protocols are available for these species. Currently, there are no alternative models, such as non-vertebrate species or computer models, to conduct the studies that we are proposing. The mouse is the lowest species that fits this requirement and each species was selected since certain genetic experiments can only be performed with mice.  Even if we could mimic neurotransmission in a cell line or ES-derived cells, we would still have to confirm all results in primary neurons. Our ultimate aim is to understand Y and this cannot be achieved by non-animal-based studies.  To reach our goals, we will minimize the use of animals wherever possible and use techniques such as immunostaining and qPCR that require minimal amounts of material.

Example 1: The Institute of Comparative Medicine (ICM) at Columbia University is fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care, International (AAALAC) and complies with the regulations under the Animal Welfare Act (AWA) and the Health Research Extension Act of 1985, the National Research Council (NRC). There is a veterinarian on call 24 hours a day, including weekends and holidays. Members of Veterinary Services are present in the animal facilities 7 days per week. Veterinary Services does daily clinical rounds of all animal-housing rooms in each the animal facilities. Animals are examined for signs of unalleviated pain, discomfort, or distress, for the presence of clean and intact surgical incisions, and for signs of overall good health. Any changes in behavior or sign of the following humane endpoints below will lead to early termination of experiments and immediate euthanasia of animals: 1) Behavioral changes (aggression, guarding, hiding); 2) Neurological signs indicating the presence of brain tumors (seizures, ataxia, lethargy); 3) Licking, biting, scratching, or shaking of procedure site or any other self-mutilation; 4) Changes in hair coat (ruffled fur, lack of grooming, piloerection); 5) Changes in posture or ambulation (tense, stiff gait, ataxia); 6) Non-weight bearing for 24hrs (difficulty walking, inability to maintain upright posture); 7) Changes in activity level (restlessness, pacing, lethargy); 8) Infection unrelated to the protocol; or 9) Signs of moderate to severe pain or distress that was not anticipated by the study plan. Veterinary Services also confirm that appropriate post-operative analgesia has been administered and documented. Any concerns are immediately brought to the attention of the veterinarians and investigators. A clinical plan for these animal is developed in collaboration between the investigator and veterinarians at each institution, and according to humane endpoints described in the approved and pending IACUC protocols.

Example 2: To ensure limited discomfort, distress, and pain, mice are monitored for signs of pain or stress daily.  These signs include vocalization, increases in rate of respiration, weight loss, piloerection, withdrawal, hunched back, and crusted eyes.  In case any of these signs are observed, 0.1 cc of bupivacaine will be administered.  In the case that these signs persist even after Marcaine injections, the animals will be evaluated for treatment by the veterinary staff or euthanized by the researcher performing the study.  General anesthesia using inhalant anesthetic isoflurane (5% induction, 2% maintenance) in oxygen from a precision vaporizer is used to alleviate discomfort and distress during tail clipping and microchip implantation in young adult mice.  Hind limb toe pinch is used to assess depth of anesthesia.

Example 3: 

 Use of tranquilizers, analgesics and anesthetics (identify drugs by name/class)

  • Isoflurane:  This is 2-chloro-2-(difluoromethoxy)-1,1,1-trifluoro-ethane which is a halogenated ether used for inhalational anesthesia.  DEA Schedule:  None
  • Ketamine:  It is (RS)-2-(2-Chlorophenyl)-2-(methylamino)cyclohexanone.  Pharmacologically, ketamine is classified as an NMDA receptor antagonist, but it also acts at numerous other sites (including opioid receptors and monoamine transporters).  Like other drugs of the arylcyclohexylamine class, such as phencyclidine (PCP), ketamine is classified as a dissociative agent. DEA Schedule: III.
  • Xylazine:  It is N-(2,6-Dimethylphenyl)-5,6-dihydro-4H-1,3-thiazin-2-amine which is an analogue of clonidine and an agonist at the α2 class of adrenergic receptor which is used as a sedative, muscle relaxant, and analgesic.  DEA Schedule:  None
  • Bupivacaine:  It is (RS)-1-Butyl-N-(2,6-dimethylphenyl)piperidine-2-carboxamide which is a local anesthetic related chemically and pharmacologically to the aminoacyl local anesthetics.  DEA Schedule:  None.

Example: The method of euthanasia is consistent with the recommendations of the 2013 American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals and approved by Columbia University’s IACUC. Animals will be euthanized via CO2 and for a secondary physical method, thoracotomy will be used for rats and cervical dislocation for mice.


Register for K Award Program for On-time Proposal Preparation (K-POPP) Online Course

Through the course you can access detailed guidance for the Vertebrate Animals attachment. 

Register for the NIH Fellowship Online Course 

Through the course you can access detailed guidance and examples for the Vertebrate Animals attachment.