Experimental Guidelines

Unanticipated Experimental Outcomes

Because scientific inquiry involves exploration of the unknown, the outcome of experimental procedures is often unpredictable. In the case of animal experimentation, this may result in situations in which animal health and/or welfare are adversely affected. Genetically manipulated animals (GMA) provide a good example. Initially, the overall effect(s) of a particular genetic manipulation may be unknown. However, once animals are generated the resulting novel phenotypes may include unexpected immune deficiency, metabolic disorders, behavioral abnormalities or other health issues that make those animals more susceptible to disease or distress. GMA breeding lines that exhibit abnormalities in health or behavior often require the establishment of revised husbandry and/or monitoring protocols. Humane endpoints specific for the GMA line may also need to be developed. More information on methods to reduce and refine the use of GMA can be found on the NC3R's site. In the case of a new GMA line it is recommended that the first generation be closely observed from birth to early adulthood for signs of disease, pain or distress. In addition, investigators should be aware that infertility or other causes of decreased breeding success may increase the number of animals used and require revisions of the IACUC protocol.

Animal Selection for Research

The goal of a scientific experiment is to determine the effects of defined variables in a controlled environment. Phenotypic variability between experimental animals can confound data analysis and lead to inaccurate or non-generalizable results. Investigators can reduce phenotypic variability by selecting appropriate animals for each experiment. This may also allow for the use of fewer animals per group/experiment. Factors that can increase variability in experimental results include:

• Use of animals with different or inaccurate genetic background
• Use of animals from different commercial vendors or facilities
• Stress associated with inadequate housing or husbandry
• Inflammation or infection associated with wounds or unrelieved pain and distress
• Infection with pathogenic bacteria, viruses or parasites
• Extravasation of irritating substances such as chemotherapeutics
• The presence of ulceration, necrosis or infection in or around a tumor
• Stereoptypic or other abnormal behaviors.

To reduce experimental variability:

• Use similar animals from the same source (e.g., inbred strain purchased from the same commercial vendor)
• Do not use animals with wounds or other signs of illness or injury
• Do not use animals that display abnormal behavior
• Ensure animals have appropriate food, shelter and environmental conditions
• Protect animals from infection with pathogenic microorganisms
• Select endpoints that will not introduce additional variables. Complete the study before animals become moribund or develop ulcerated, necrotic or infected tumors.
• Use aseptic technique when preparing for and performing surgery.

 

Rodent Genotyping

Genetic identification of research rodents is often accomplished through the use of PCR analysis of DNA extracted from tail or ear punch tissue. DNA may also be obtained from other samples, such as hair, blood and oral swabs. Deviations from PSU IACUC genotyping guidelines must be approved by the IACUC. Personnel must receive sufficient training to perform the procedures in a safe and humane manner.

Guidelines for tissue collection

Ear punch/ear snip: Tissue for genotyping may be collected by ear punch or snip at any age and does not require anesthesia. This is the preferred method of tissue collection for genotyping of rodents.

Ear punch - The amount of tissue collected from a 1-2 mm sterile ear punch is usually adequate for genotyping and may be performed on any age mouse without general anesthesia. Ear punches must be cleaned and disinfected between animals.

Ear snip - Using a sharp, sterile scissors, 2-3 mm may be snipped from the tip of the ear to collect tissue for genotyping. Scissor tips must be cleaned and disinfected between animals. This procedure may be performed on any age mouse without general anesthesia.

Tail Tip Amputation: Animals must be less than 3 1/2 weeks of age at the time of tail tip amputation for genotyping. DNA yield from tail tissue has been shown to be highest in 10-21 day old rodents. Tail tissue analysis and identification of desired mice prior to weaning may allow more efficient use of resources. In addition, cutting through the soft tissue in the tail of a young mouse is likely to be less painful than cutting through more extensively mineralized bone and mature tissue in older animals. Tail tip collection should be performed at as young an age as feasible.

Animals less than 3.5 weeks of age: Collection of tail tissue may be performed without general anesthesia. Local anesthetic methods are available. Please contact an ARP veterinarian for more information.

Animals greater than 3.5 weeks of age: Samples for genotyping must be collected by ear punch or ear snip.

In most cases, 2 mm removed from the tip of the tail is adequate for genotyping. No more than 5 mm of tissue may be removed from an animal. DNA yield does not proportionally increase as larger amounts of tail tissue are collected. Tail amputation should be done using a sharp, sterile scissors that is cleaned and disinfected between animals. Note: all tissue must be removed from the scissors after each animal for accurate DNA analysis via PCR.

Blood loss should be minimal from animals less than 3.5 weeks of age when proper procedures are used. If needed, bleeding may be controlled by applying direct pressure to the tip of the tail. Styptic powder/pencils and silver nitrate are chemical cauterizing agents that may be used if necessary. Use of heat to cauterize the tail end is not allowed.

If it is necessary to collect a second sample from an animal, the second sample must be collected via ear punch or ear snip.

How to Perform a 'Search for Alternatives'

Federal regulations require research investigators to search for alternatives to the use of animals when procedures causing more than momentary pain or distress will be used. In addition to non-animal alternatives, investigators must consider alternatives that can reduce the number of animals used, decrease animal pain or distress or replace the proposed animal species with a less sentient one.

The key to conducting an alternatives search is to focus on alternatives to the potentially painful or distressful procedures used in the study, not on alternatives to the topic or purpose of the research study.

Alternatives must be considered for each potentially painful or distressing procedure used in the research study. Investigators may use their own experience or consult with other professionals in the field as part of their search. However, many investigators rely on internet databases to conduct a comprehensive survey of their field of study. It takes thought and planning to conduct a good database search that yields beneficial information. Before beginning a literature search identify which experimental techniques or procedures used in the study have the potential to cause pain or distress in animals. Consider what the significance of these procedures is to the study and whether or not they can be refined or replaced. If painful or distressful procedures must be used, consider what endpoints will be chosen to determine when an animal will be removed from the study. When conducting a literature search use descriptive search keywords specific for the techniques and procedures noted above. Include the animal species you wish to use and select a database appropriate for the area of study. Do not include search terms like the name of the gene you are studying or scientific keywords relating to your study. The use of the term “alternatives” in the search is usually not very productive. 

Example 1: If a study proposes retro-orbital blood collection from mice search keywords could include: blood collection, method, and mouse. A search would provide alternative sites or methods for blood collection such as the submental veins, saphenous vein, and cardiac puncture. The investigator must then consider how much blood may be obtained with each method, whether or not repeated blood collection is needed and the potential for pain and distress with each method.

Example 2: For an experiment involving intra-cerebral injections in mice, alternative search keywords could include: drug, administration, method, and mouse. The search will provide other potential administration routes including intravenous and subcutaneous injections, oral gavage and subcutaneous implants. The investigator must determine if any of these alternate methods wil work for the specific needs of the experiment.

Please refer to the PSU Libraries Guide on Searching for Animal Alternatives or the UC Davis Center for Alternatives for more information and examples when planning and conducting alternatives searches. PSU investigators may also contact an ARP veterinarian for assistance.

Record Management in Animal Research

Research accuracy and integrity may be impacted by incomplete or careless record keeping. Good animal welfare is also dependent on timely and accurate care and treatment of laboratory animals to minimize pain and distress. Federal laws and regulations require that investigators maintain written documentation detailing the care and treatment of laboratory animals. Most medical and animal procedural records should be kept for the duration of the research activity and for an additional 3 years. Medical and surgical records must be maintained in or near the animal room readily available for inspection by veterinary staff and IACUC oversight. Individual medical and surgical records are required for non-rodent animals; group records may be used for rodents.

Cage cards or other cage-side identification for all animals must include, at a minimum, the following information:

• Investigator name
• IACUC protocol number
• Sex and birth date(s) of animals
• Individual identification information (if applicable) for each animal in cage

 

Types of Records

• Breeding colony
• Anesthesia/Analgesia
• Surgery/Post-procedural
• Controlled drug use
• Food/Water restriction protocols
• Neoplasia (tumor) monitoring

 

Breeding Colony Records

Rodent breeding colonies require accurate record keeping to maintain genetic integrity and reduce wasteful over- or under-breeding. The types of records needed for breeding colonies can include cage cards, computerized or paper record keeping systems and weaning records. Cage cards should include the following information:

• Dam and sire ID’s/ birthdates/genotype               
• Date paired/set up                         
• Cage ID number
• IACUC #
• Pup birth dates
• Disposition of litter (wean/died)

In addition to cage cards, a centralized record keeping system should be used to keep track of mating pair set-ups and breeding outcomes. Various types of colony management systems exist including commercial products, free databases and templates, or you can create your own. Information in centralized breeding records may include the following:

• Dam and sire ID’s/ birthdates/genotype(s)         
• Date paired/set up                         
• Date of birth/number of pups
• Disposition of litter (wean/died)                                                              
• Pedigree
• Tracking number
• Vendor or stock number
• Disposition
• Breeding/Performance history
• Noted Problems
• Breeding system used

 

Weaning Records

A record of the number of pups weaned in the colony should be maintained for both experimental and IACUC purposes. In addition, weaning records should indicate the following:

• Pup ID
• Date of birth
• Strain/Genotype information
• Dam and sire ID’s                            
• Designated use of pup (e.g., experimental, breeding, euthanasia)

Genotyping of mice typically uses ear punch or tail tip tissue samples although other tissue samples may be acceptable. DNA yield from tail tissue has been shown to be highest in 10-21 day old rodents. More information on rodent genotyping maybe found under Husbandry and Breeding on the ARP website.

Anesthesia/Analgesia/Surgical Records

 A sample surgical and post-operative record template may be found on the ARP website.

Investigators must maintain accurate records of anesthesia, surgery and postoperative care (including analgesic administration). Records serve as documentation that the procedures were conducted humanely and by appropriately trained individuals. Individual animal records are not necessary for groups of rodents treated similarly and at the same time. Records must be kept in the animal room or a nearby area accessible by ARP veterinary staff. Surgical records are examined by the IACUC every 6 months. Records of anesthesia/analgesia and peri-procedural care document adequate veterinary care and the alleviation of pain and distress during the conduct of these procedures.

• Physiological monitoring of an animal during general anesthesia is required and must continue until the animal is returned to its home cage.
• Anesthetic or analgesic drug treatments should be recorded so that a total dose is recorded or can be calculated (e.g., total mg or a volume plus a concentration).
• Anesthetic and analgesic administration records may be included within the surgical record.

 

Information to Include on Surgical Records

Surgical records must include the following information:

• Date of surgery
• Name of surgeon
• Animal identification e.g., number and group name
• A description of the surgical procedure*
• Name(s), dosage(s) and route(s) of anesthetic and analgesic drugs administered
• Names and dosages of experimental agents administered
• Post-operative monitoring that clearly documents daily observation and health status until the surgical wound is healed and complications resolved
• Medical treatments administered during or after surgery

*Surgical records should be kept in a notebook. It is strongly recommended that a detailed description of each surgical procedure be kept in the front of the notebook. Rather than rewriting the surgical description on each record, provide the name of the procedure that corresponds to the description provided in the front of the notebook. Also record any deviation from the detailed description.

Post-operative/procedural monitoring

Cages housing animals undergoing post-operative monitoring should be clearly marked so ARP staff can identify when surgery was done. Post-operative monitoring records must be kept accessible in the animal room and will be examined every 6 months by the IACUC. Animals recovering from general anesthesia must be monitored continuously until able to right themselves (i.e., sternal recumbency). In addition, animals must be observed and evaluated at least once a day until the surgical wound is healed and complications resolved. Written records detailing monitoring and treatment must be available to PSU veterinary staff.

Controlled Drug Records

Certain drugs used for anesthesia, analgesia, and euthanasia are regulated by the DEA (Drug Enforcement Administration) because of the potential for abuse.  Investigators using these drugs are responsible for complying with applicable regulations including storage in a substantial locked cabinet or safe and maintenance of written records accounting for quantities received and dispensed. Investigators who will be using controlled drugs must apply to the DEA for the appropriate license. Controlled drug records will be examined by the IACUC every 6 months.

Food and Water Restriction Protocols

Food and/or fluid restriction that deviates from normal husbandry care must be described in the IACUC proposal. Records for protocols in which an animal’s access to food and/or water is limited should include the following information:

• The approved restriction schedule
• The time/date and amount, if applicable, of latest feeding/watering
• Documentation that approved monitoring was conducted
• Contact information for responsible personnel (including phone)

In addition, cages should be clearly marked so animal caretakers and veterinary staff can identify which animals are included in the restriction protocol.

Experimental Neoplasia Protocols

Animals who are expected to develop internal or external tumors/neoplasia must be monitored as described in an approved IACUC protocol.  Humane endpoints used to prevent unnecessary animal suffering due to tumor growth must be described in the IACUC protocol. Monitoring records should be kept within the housing room until the death of the animal. Records must contain the following information:

• IACUC protocol number.
• Identification of the animals.
• Dates of monitoring.
• Tumor size, body weights, body condition scoring or other parameters used to evaluate the animal’s condition.
• Name of person(s) monitoring the animals.

 

Animal Use Guidelines and Regulations Governing the Use of Animals in Research, Teaching and Testing

The PHS Policy on Humane Care and Use of Laboratory Animals

The Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals is the most important law regulating the use of Laboratory Animals in NIH funded research. The PHS Policy on Humane Care and Use of Laboratory Animals

"sets forth the requirements that are applicable to all research, research training, biological testing, and related activities involving animals that are supported or conducted by agencies of the PHS. The Policy is mandated by the Health Research Extension Act of 1985 (Public law 99-158), and implements the U.S. Government Principles for the Utilization and Care of Vertebrate Animals used in Testing, Research, and Training. Included in the Policy are institutional responsibilities for Animal Welfare Assurances, Institutional Animal Care and Use Committees (IACUCs), review of projects, programmatic evaluations, facility inspections, record keeping and reporting. Specific criteria for IACUC review of projects, and frequency and methods of review are described. The information required by the PHS in applications or proposals when animals are to be involved, and PHS responsibilities for implementing the Policy, are also included."

The Office for Laboratory Animal Welfare at the National Institutes of Health has responsibility for the general administration and coordination of the Policy on behalf of the PHS.

Guide for the Care and Use of Laboratory Animals

The Guide is accepted by the scientific community as the main resource on laboratory animal care and use. The Guide is also recognized as the standard reference on laboratory animal care and use programs by the Association for the Assessment and Accreditation of Laboratory Animal Care International, the Public Health Service, and other private organizations and federal agencies. Its guidelines are based on established scientific principles, expert opinion, and experience with methods and practices consistent with high-quality, humane animal care. Copies of the Guide may be obtained from the National Academy Press.

Animal Welfare Act

The United States Animal Welfare Act contains provisions to ensure that animals used in research receive humane care. The law provides for regulating the transport, purchase, sale, housing, care, handling and treatment of covered animals. Currently mice of the genus Mus, rats of the genus Rattus and birds bred for research are not covered by the act.

Penn State Policy on the Care and Use of Vertebrate Animals

Penn State Policy RP04 is designed to provide the following assurances with respect to University projects involving the use of vertebrate animals at all University locations, excluding the College of Medicine at Hershey, which maintains a separate animal welfare assurance to OLAW.

• Vertebrate animals involved in any research, testing or teaching procedures receive humane care and treatment.
• Animal research is conducted in a well-controlled research environment.
• Concerns regarding the care and use of vertebrate animals at the University are addressed in a professional and responsible manner.
• Research involving the use of live animals is performed in an ethical manner designed to minimize pain and distress, and comply with applicable federal and state regulations.

 

Institutional Animal Care and Use Committee

Institutional Animal Care and Use Committees (IACUC) are required by federal law to oversee the humane care and treatment of animals used in research, education and testing. Penn State's IACUC is comprised of appointed members with specific backgrounds including research scientists, nonscientists, veterinarians and community members (who represent the public's interest). One of the primary responsibilities of the Penn State IACUC is to review and approve all proposed animal use activities by Penn State employees or students. In addition, the IACUC must evaluate all aspects of the University's animal care program semiannually. This is accomplished through inspections of animal husbandry, environment and facilities, and review of records, IACUC functions, veterinary care, training and safety programs. The IACUC is also responsible for reviewing and responding to any reported concerns involving the care and use of animals at the University.

The Penn State IACUC is responsible for the University Park campus and all outlying campuses except the Penn State Hershey campus, which has it's own IACUC. The IACUC reports directly to the Office of the Vice President for Research, who is the designated Institutional Official responsible for oversight of all programs involving the care and use of animals. The Office for Research Protections coordinates IACUC activities, provides online IACUC training and is the office from which appropriate animal use application forms may be obtained.