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IACUC Protocol Literature Search

What is an alternatives literature search?

An alternatives literature search seeks to find non-animal models for the research described in the protocol OR find methods to improve animal welfare and reduce pain and distress. This follows a scientific method known as the 3Rs that incorporates some aspect of replacement, reduction, or refinement of animal use in pursuit of the minimization of animal pain and distress consistent with the goals of the research.

  • Replacement: Substituting sentient animals with insentient material
    • Full or partial replacement
      • Examples of full replacement: avoids the use of any research animals and instead use human tissues and cells, mathematical and computer models, established cell lines
      • Examples of partial replacement: includes the use of some animals that, based on current scientific thinking, are not considered capable of experiencing suffering such as invertebrates like drosophila, nematode worms, social amoebae, immature forms of vertebrates. Also include the use of primary cells and tissues taken from animals killed solely for this purpose.
  • Reduction: Reduction in the number of animals required per experiment or study design to achieve robust results.
    • Examples: Animals serving as their own control, pilot studies, sharing data and resources, imaging technologies, telemetry, appropriate experimental design and statistical evaluation of sample size, publishing results
  • Refinement: Methods that minimize animal suffering and improve animal welfare.
    • Examples: Use of anesthetics and analgesics, limiting clinical signs/humane endpoints, noninvasive methods of sampling, telemetry, modifications in capture/handling/restraint, imaging technology, knowledge of species physiology and recognition of normal/abnormal behavior, housing (social housing, cage design, etc), environmental enrichment
  • Other types of searches:
    • Duplication search: seeks to find published research like the protocol. This is to prevent possible duplication of animal studies that have already been performed.

The Virginia Tech IACUC requires that alternatives search be conducted for studies in pain categories D and E.

  • Searches should focus on procedures and disease processes that make the protocol a Pain Category D or E (what causes pain or distress).
  • Pain Category D: Use of procedures that would cause more than slight/momentary pain or distress, but are performed using appropriate anesthetics, analgesics, or tranquilizers to relieve pain (e.g., minor or major surgical procedures [survival or non-survival] performed under anesthesia; collection of cells or tissues prior to euthanasia; painful procedures performed under anesthesia [retro-orbital blood collection in rodents]).
  • Pain Category E: Use of procedures that cause more than slight/momentary pain or distress, but that cannot be performed using anesthetics, analgesics, or tranquilizers without adversely affecting the study (e.g., toxicity and lethal disease studies in which the animals are allowed to die without intervention and mortality is the endpoint). Mechanical restraint may, depending upon duration and type of restraint, be considered a category “E” procedure.
  • Additional information can be found in the IACUC Policy of Pain Category Assessment found on the IACUC Policies webpage under “Animal Care and Use”
  • Regulatory - To comply with the Animal Welfare Act and Public Health Safety (PHS) Policy, maintain accreditation with AAALAC
  • Scientific - Learn new approaches for less painful and invasive procedures, reduce animal stress, increase research efficiency
  • Economic - Reduce the expense of animal use in animal purchase, care costs, occupational health and safety costs, specialized facility infrastructure costs
  • Social - Respond to social pressure such as using non-animal alternatives and making research pain free
  • Humane - Ask ethical questions such as: Should animals be used in research, When and How should they be used?

Goal: To perform a refined search in order to obtain relevant results. This may require multiple search strings using syntax tools.

Characteristics of Incomplete versus Complete Search Strings

  • Incomplete or unrefined search
    • Not including search tools/syntax
    • Throwing keywords into the search with no specific order
    • Using only terms like replacement, reduction, refinement, or alternatives
  • Complete or refined search
    • Synonymous terms groups with parentheses
    • Use of search tools/syntax
    • Not only using the procedure/method you’re trying to replace, but rather using a combination of search terms

Syntax Tools


  • A symbol (usually an asterisk) added to the root of a word to represent extra characters
  • Example: behave* = behavior, behaves, behave, behaving, behaved, etc.

Boolean Logic

  • Allows you to link terms in specific ways, using AND, OR, and NOT
  • Example: (heart OR cardiac) AND (pig OR swine) NOT (guinea)


  • Used to combine synonymous terms in a search string
  • Example: (dog OR dogs OR canine) AND (pain OR discomfort OR distress)

Proximity Operators

  • Searching for words in the same sentence, or within a certain number of words of one another
  • Example: blood N3 collect* = blood collection, collection of arterial blood, collection blood

Quotation Marks

  • Used to search for exact phrases within literature and internet search engines
  • Example: “animal welfare” OR “animal health”

Coming Up with Search Terms

  • Construct a search string using concepts:
    • Disease keywords
    •  Animal keywords
    • 3Rs keywords
  • Come up with synonyms/equivalents of the search terms
  • Combine the disease, animal, and 3Rs terms in one search string using Boolean AND and OR.

Example One (Disease Model)

Step One

Concept/Keyword Type Concept/Keyword Type Search Terms
Disease Inflammatory bowel disease
Animal Mice
3Rs Humane endpoint

Step Two

Concept/Keyword Type Concept/Keyword Type Search Terms
Disease “inflammatory bowel disease” OR IBD OR colitis
Animal Murine OR mouse OR mice OR mus
3Rs “animal welfare” OR “humane endpoint” OR “non-invasive” OR imag* OR biomarker*

Step Three: Combined as a set in PubMed in Advanced Search

  • ((“animal welfare”(Title/Abstract) OR “humane endpoint*”(Title/Abstract) OR “non-invasive”(Title/Abstract) OR “noninvasive”(Title/Abstract) OR biomarker*(Title/Abstract) AND (mice(Title/Abstract) OR mouse(Title/Abstract) OR murine(Title/Abstract)) AND (“inflammatory bowel disease”(Title/Abstract) OR IBD(Title/Abstract) OR colitis(Title/Abstract))

Example Two (Organism Model):

Step 1

Concept/Keyword Type Search Terms
Organism “Cryptosporidium spp.”
Immune Response “Mucosal immune response”
Anatomy “Small intestine”
Non-animal model “Intestine-on-a-chip”

Step 2

Concept/Keyword Type Search Terms
Organism “Cryptosporidium spp.” OR helminth* OR parasite* OR hookworm* OR “crypto” OR “cryptosporidium” OR “microparasite*”
Immune Response “Mucosal immune response” OR “intestinal mucosa” OR “mucosal immune system” OR “mucosal immunity” OR “Th2 immunity” OR “intestinal tuft cells” OR “tuft cell*” OR “host-pathogen interaction*” OR “type 2 inflammatory response*” OR “intestinal inflammation”
Anatomy “Small intestine*” OR intestinal OR gastrointestinal OR “GI tract”
Non-animal model “Intestine-on-a-chip” OR organoid* OR enteroid* OR “microfluidic device*” OR “gut-on-a-chip” OR “cell culture” OR “cell assay” OR “non-animal” OR “animal use replacement”

Step 3: Combined as a set in PubMed in Advanced Search

  • (("small intestine*" OR intestinal OR gastrointestinal OR “GI tract”) AND ("intestinal mucosa" OR "mucosal immune system" OR "mucosal immunity" OR "Th2 immunity" OR "mucosal immune response" OR "Intestinal tuft cells" OR "tuft cell*" OR "Host-pathogen interaction*" OR "Type 2 inflammatory response*" OR "Intestinal inflammation") AND (organoid* OR enteroid* OR "3-D cell culture*" OR "3D cell culture*" OR "gut-on-a-chip" OR "intestine-on-a-chip" OR "microfluidic device*" OR "non-animal" OR "animal use replacement" OR “cell culture” OR “cell assay”) AND (helminth* OR parasit* OR hookworm* OR “crypto” OR “cryptosporidium” OR “cryptosporidium spp.” OR “microparasite*”))

Database Examples

How to perform an advanced search will vary by database. Below are examples of two common databases.


  • Click “Advanced”
  • Build search terms by selecting which field to search, entering the search term, and then selecting how to add. Once you select your add method, the query box will build your combination of search terms for you. See below for an example using Example 2 from above. Search terms for each concept are entered into the search term field together, and then added.
  • Once you have finalized your query box, click the search button.
  • Once searched, the search terms can be edited by clicking the “Advanced” link.

Google Scholar

  • To access the advanced search function, select the dropdown in the upper left.
  • Then select the Advance Search link.
  • Pop-up will appear. Less capability to build complex combinations, as there is a limit to the size of the text field. Type terms into appropriate text field..
  • Can filter searches on the left side of the screen, such as limiting to “Review articles”..

Evaluate and Refine the Search

  • ·Modify Search to Reduce Number of Results: focus on one particular aspect. For example, add additional keywords as a Title/Abstract field that are specifically about a pain category D or E procedure.
  • In most of the results are relevant, the search strategy doesn’t need another concept added
  • If the results are too broad, consider limiting the publication year or specific fields (ie title, keywords, abstract).
  • Other possible fixes:
    • Add a NOT group to exclude items
    • Fix any “unfortunate” truncations

Red Flags to Avoid in Search Evaluations

  • Only one database searched
  • Terms only for painful aspects without searching for other 3R terms
  • The term “alternative” used alone with no other 3Rs terms
  • Keywords listed no relevant to protocol
  • Keywords and concepts linked in an incorrect manner (ie wrong Boolean operators)
  • Search doesn’t cover adequate time period (5-10 years)

Terminology for Refinement Alternatives

  • Analgesic or analgesia or pain reduction
  • Anesthetic or anasthetic or anaesthetic
  • Animal welfare or well-being or wellness or colony management
  • Assay or technique or method or procedure
  • Bedding or substrate
  • Behavior or behaviour or ethology
  • Blood draw or blood sampling or sample sites (intravenous, tail vein, saphenous vein, etc.)
  • Challenge method or infection
  • Environmental enrichment
  • Euthanasia or humane endpoint
  • Grimace scales (for pain monitoring)
  • Handling or humane handling or humane restraint or humane treatment
  • Husbandry or housing or caging
  • Imaging or scanning (MRI, magnetic resonance imagery, PET scans, positron emission tomography, CAT scans, bioluminescent scanning)
  • Injection or injection site
  • Non-invasive or less-invasive
  • Pain or stress or distress
  • Positive reinforcement training or animal training
  • Refinement
  • Social housing or group housing
  • Telemetry device, monitoring device, biotelemetry

Most search terms are obtained from the protocol and area of study.

Terminology for Reduction Alternatives

  • Animal model
  • Animal study registries
  • Auto control (using animal as its own control)
  • Biomarker or biological marker
  • Computational methods, computational models, in silico
  • Data repositories or reuse data
  • Experimental or statistical design (pilot study, variation, sample size, etc.)
  • Imaging or scanning (MRI, magnetic resonance imagery, PET scans, positron emission tomography, CAT scans, bioluminescent scanning)
  • Microsampling
  • Monitoring device or telemetry device, biotelemetry
  • Pilot study
  • Reduce or reduction or minimize or “use fewer animals”
  • Reusing or sharing or repurposing animals (between studies)
  • Sample size or “number of animals”
  • Tissue banks
  • Variation (i.e. minimizing variation among animals in study)

Terminology for Replacement Alternatives

  • Animal testing alternative or animal use alternative or alternative (useful primarily in toxicology and education)
  • Cadaver or carcass
  • Cell culture, cell line
  • Computer aided instruction, computer assisted instruction
  • Computational methods or models (in silico)
  • Computer (simulation or application or model)
  • Digital imaging
  • Interactive
  • In vitro
  • Isolated (cell, tissue, organ)
  • Mannequin or manikin or model (non-animal)
  • Mathematical (computational biology or model or simulation)
  • Model (animal, cadaveric, interactive, mathematical, statistical, theoretical), modeling (US spelling or modelling (UK spelling)
  • Non-animal model
  • Organ-on-a-Chip or microfluidic device
  • Organoids or 3-D organoids
  • Plastinate or plastination
  • Software
  • Replacement alternatives
  • Simulation or simulator or trainer
  • Structure evaluated system
  • Tissue culture or organ culture
  • Tissue engineering
  • Video (disc, display)
  • Virtual (reality)