Many factors can influence the health and well being of animals used in research and can in turn effect experimental results. The sections below focus on some of the most common complicating factors associated with animal research. The impact of many of these factors can be minimized by having a well designed animal facility and through good basic husbandry practices.
 

Source & Transportation

The research animal is a composite of a particular genetic make-up and all the environmental influences acting on it. You need to consider the source of your animal -- breeder or animal dealer or, in the case of wild caught animals, the geographic location, genetic purity of the strain or population.

Transportation is a stressful event -- elevates glucocorticoids -- may cause immunosuppression.

Animals need an acclimatization period once they arrive in the animal facility. UAF Veterinary Services imposes a 10-14 day acclimation/quarantine period for new animals. This gives animals time to adjust to their new environment and Veterinary Services time to assess the animal's health, thereby protecting other animals in the facility from potential infectious agents. No experimental procedures may be initiated during quarantine unless specifically authorized in an approved IACUC assurance.
 

Physical Factors

This cannot be controlled in a field setting but is of utmost importance in a captive setting. Physical factors in a captive environment must be tailored to the well-being of the animal under study and the research objectives. There are very specific requirements set in the various guidelines for conventional laboratory animals. However, guidelines and standards for the housing and care of captive wildlife are less precise or non-existent. Developing appropriate housing and animal care procedures for non-traditional species requires knowledge of the species' natural history, specific needs, and the research to be conducted.  

Temperature. Extremes may cause stress/distress, hyperthermia, or hypothermia.

Relative Humidity. Extremes may impact animal health, food quality, or negatively impact other aspects of the physical environment. High humidity may increase the prevalence of pneumonia, lead to excessive mold growth in the rooms, and spoiling of feed. Low humidity can cause ring tail in some strains of rats.

Ventilation. Poor ventilation may contribute to pneumonia and ocular problems. It may also contribute to the accumulation of harmful compounds such as ammonia.

Lighting. Excessive brightness may lead to retinal damage and blindness in albino animals. Consider photoperiod requirements for your animals. Exposure to constant light or darkness causes changes in the patterns of behavior, hormones, etc. in most animals and should typically be avoided unless part of the animal's natural conditions.

Noise. This should be obvious but is often ignored. Animals can be stressed when subjected to loud noises. Do not exceed 85 db. Be quiet during animal handling.
 

Chemical Factors

Bedding. Choose your bedding carefully. Some may contain chemicals that might influence your study. Wood shavings, if not kiln dried, contain aromatics that might induce the Hepatic Microsomal Enzyme systems. Understand that exposure to a wide variety of chemicals, including plant secondary compounds, may also influence the Hepatic Microsomal Enzyme system in free-ranging animals.

Diet. Consider natural vs. semi-purified. How much control is needed? Never presume that commercially manufactured diets contain exactly what they claim. Even large manufacturers have been known to make mistakes and introduce unwanted chemicals or create dietary deficiencies, excesses, or imbalances. Particular care should be taken with diet selection for wildlife species; although it may be a good starting point, it is important not to assume that a commercial diet for a related species will provide adequate nutrition for a wild species without empirical evidence. It is essential to monitor animal health closely when developing animal husbandry and care procedures for novel species.

Water. Know what is in your water! It is not always as pure as you might think. For example, past experience has shown that UAF water (tap or RO) is not suitable for housing frogs or fish.  The chemical composition of water is critical for housing aquatic species.

Miscellaneous chemicals in animal facilities. Many potent chemicals get used in animal facilities. Ensure that your animals are not affected by insecticides and/or disinfectants.
 

Microbiological Factors

Microbiological definitions. If you are working with conventional laboratory animals (mice, rats, dogs, pigs, etc) you can, if desired, purchase microbiologically defined animals. Please contact the UAF attending veterinarian if you wish to pursue this possibility.

Diseases caused by infectious agents. Be aware that all disease does not necessarily manifest with obvious clinical symptoms. Latency is an important issue in animal research. This is equally important in wildlife as well as conventional lab animals. You should know as much as possible about your research animal. For example, hepatitis B virus is endemic in all arctic ground squirrel populations that our captive animals are obtained from. You need to understand this and be confident that using hepatitis B infected animals won't affect your research. Additionally, in laboratory animals, we know about viruses that are essentially non-pathogenic but can cause problems with research. An example of this is "LDH elevating virus in mice". This virus does not affect mice other than cause spurious elevations in the serum enzyme LDH; if this is a variable under study in your work you should ensure that your colony is not infected! For those of you working with wildlife - you are disadvantaged because we know very little about clinical disease let alone subclinical infections.

Health Monitoring. Practice preventive medicine - not knee jerk responses to crises. Be proactive in identification of disease problems. Ensure that complete necropsies with diagnostics are done on animals that die. This is the only way to ensure that your animals have no subclinical problems. Sentinel animals are frequently utilized in laboratory animal facilities to monitor disease prevalence. Know your animals!

Containment or special facilities. If necessary, you can purchase containment and barrier cages. UAF has animal rooms capable of microbiological containment (up to ABSL 3).
 

Time Factors

So much work at UAF involves photoperiod and animal rhythms that addressing time and lighting factors should be second nature.

Age changes. Age is an important factor for basic physiology and behavior and, if not controlled in experimental design, can be a significant complicating factor when analyzing and interpreting research data.

Diurnal variations. Blood borne parasites have a diurnal rhythm within the host that needs to be considered when deciding what time of day you will collect samples. Likewise, there are significant diurnal changes in many internal processes and systems (e.g. hormone levels, neural activity, gross motor activity, and sleep).

Frequency of sampling. How frequently you handle our animal may influence the outcome of the experiment.

Time to sampling. Setting the time for sample collection is often a critical aspect of a research protocol. One variable that is important in deciding when to collect a sample is the latency period (time from stimulus to initial or maximal response) which varies between systems, animals and stimuli.
 

Husbandry and Manipulation Factors

Consistent routines are vital to high quality research. This means regular feeding schedules and minimizing unnecessary visitations from research staff, husbandry people, veterinary care staff and guests. In essence, keep to a schedule for all activities, whether it is research or husbandry, and keep the noise and traffic through the animal facilities to an absolute minimum.

Stress due to manipulations must be addressed whether you are doing field or laboratory based research. Within animal facilities investigators have the opportunity to ensure proper acclimation to manipulations thus minimizing the stress effect. However, keep in mind that in ALL activities, whether field or laboratory based, you must take every effort to minimize stress. This means using the most refined techniques possible, keeping the number of personnel to a minimum, eliminating loud noises (this includes loud talking and laughing), doing procedures in a procedure room and NOT in the animal room, etc.

Changes due to anesthetics and euthanasia agents employed. Everyone involved in a research project must understand the physiological effects of anesthetic agents and euthanasia methods employed and how they may impact the variables under observation. For example, use of alpha-2 agonists can alter blood glucose levels and may cause glucosuria. If you fail to recognize that this is a pharmacological effect of the sedative rather than an experimental effect you are liable to make a serious mistake!

Animal density factors. Careful with your housing. Animal density may affect behavior, induce stress, and increase the build-up of infectious agents in the animal's environment.
 

Pain and Distress

Pain & Pain Assessment. The following is taken directly from the Guide for the Care and Use of Laboratory Animals (p64, National Research Council, 1996) and serves as a good introduction to this topic:

An integral component of veterinary medical care is prevention or alleviation of pain associated with procedural and surgical protocols. Pain is a complex experience that typically results from stimuli that damage tissue or have the potential to damage tissue. The ability to experience and respond to pain is widespread in the animal kingdom. A painful stimulus prompts withdrawal and evasive action. Pain is a stressor and, if not relieved, can lead to unacceptable levels of stress and distress in animals. The proper use of anesthetics and analgesics in research animals is an ethical and scientific imperative. Recognition and Alleviation of Pain and Distress in Laboratory Animals (NRC 1992) is a source of information about the basis and control of pain (see also Appendix A).

Fundamental to the relief of pain in animals is the ability to recognize its clinical signs in specific species (Hughes and Lang 1983; Soma 1987). Species vary in their response to pain (Breazile 1987; Morton and Griffiths 1985; Wright and others 1985), 50 criteria for assessing pain in various species differ. Some species-specific behavioral manifestations of pain or distress are used as indicators, for example, vocalization, depression or other behavioral changes, abnormal appearance or posture, and immobility (NRC 1992). It is therefore essential that personnel caring for and using animals be very familiar with species-specific (and individual) behavioral, physiologic, and biochemical indicators of well being (Dresser 1988; Dubner 1987; Kitchen and others 1987). In general, unless the contrary is known or established it should be assumed that procedures that cause pain in humans also cause pain in animals (IRAC 1985).

The American Veterinary Medical Association (AVMA) has issued the following position statement regarding animal pain:

The AVMA believes that animal pain and suffering are clinically important conditions that adversely affect an animal's quality of life. Drugs, techniques, or husbandry methods used to prevent and control pain must be tailored to individual animals and should be based, in part, on the species, breed, age, procedure performed, degree of tissue trauma, individual behavioral characteristics, degree of pain, and health status. (current as of June 2005)

Another good discussion of animal pain, stress and distress is included in the introduction of the AVMA's 2007 Guidelines on Euthanasia.

All animal welfare laws, regulations and guidelines require the minimization of pain and distress.

Signs & Symptoms. Pain is typically subdivided into two main categories:

Acute - Pain having a rapid onset and short duration. Acute pain typically results from a specific tissue injury and resolves once the injury has healed.

Chronic - Pain having a gradual onset and long duration. It is typically less intense and the symptoms less obvious than seen for acute pain.

It is important that all animal care and research personnel be familiar with the following signs and symptoms of acute pain:

• Guarding - This term is used to refer to actions by the animal, including moving away from or biting the handler, to protect a painful area.
• Crying - Refers to vocalizations resulting from movement or palpation of the painful area.
• Self-mutilation - Is used to describe an animal's repeated licking, biting or scratching of the painful area. Repeated shaking of the limb or head is more typical when the pain is centered on a limb or ear.
• Restlessness - Pacing, constantly shifting weight or repeated changes in posture (lying or sitting down alternating with getting up) are signs of restlessness. Restlessness may be due to causes unrelated to pain/distress.
• Sweating - This is very species specific since may mammals have very few sweat glands. However, it is a good indicator in horses.
• Panting - This is the equivalent of sweating in animals that lack or have very few sweat glands (e.g. dogs, mice, rats, rabbits, and cats).
• Tooth grinding - Typically seen in larger species such as horses, sheep and rabbits. Not typically seen in rodents.
• Recumbency - Animal lies down from prolonged periods when it would normally be active. The posture assumed by the animal is not usually typical of its normal sleep or rest posture.
• Ambulation - Reluctance or difficulty moving or rising.
• Abnormal postures - Examples include, but are not limited to, head pressing, standing with a tucked abdomen, head hanging down, head tilting, stiff-leggedness.

In contrast to acute pain, chronic pain is usually less intense and tends to be intermittent. Therefore it is often more difficult to detect, especially for new animal caretakers or those not familiar with the particular species. This is one of the primary reasons why many animal facilities assign caretakers to specific animals or rooms of animals. This is especially important for long-term post-surgical care or in long term experiments where symptoms may appear gradually. Behavioral changes related to chronic pain include:

• Limping or carrying a limb.
• Licking or rubbing an area of the body.
• Reluctance to rise and move.
• Loss of appetite.
• Change in temperament or behavior toward handlers.
• Change in bowel and/or urinary activity.
• A lack of self-grooming (ruffled and soiled haircoat).

Animals showing symptoms of either acute or chronic pain MUST be reported IMMEDIATELY to Veterinary Services!

Distress. Distress is a generic term that can include a variety of responses to a given condition such as anxiety, fear, boredom, frustration, and so forth. The Institute for Laboratory Animal Research (ILAR) guidelines define distress as stress to which the animals cannot adequately adapt. Distress may be induced by psychologic, physiologic, or environmental factors.

There are potentially multiple expressions of distress. Like pain, level of distress is difficult to assess since an animal cannot directly report its state of well being. We have no way of knowing what relatively minor changes (for example in environment or experimental procedures) mean to the animal, and we have no way of knowing what impact different levels of well being have on a particular study. Environmental Conditions are discussed in more detail in the following section.

P.A. Flecknell's review article "Refinement of animal use - assessment and alleviation of pain and distress" (Laboratory Animal (1994) 28: 222-231) offers an excellent discussion of the need for objective measures to assess and the importance of refining experimental techniques to minimize pain and distress.

Environmental Considerations. Proper animal housing and animal facility management are essential to animal well-being. A good animal care program provides the conditions for animals to grow, mature, reproduce and maintain good health and provides for their well-being. There are many factors that must be considered in designing an animal housing situation that maximizes species-specific behaviors and minimizes stress-inducing behaviors. The environment in which animals are maintained should be appropriate to the species, its life history and its intended use.

Example 1: Fossorial mammals should be provided with dark retreats, preferably supplied with dirt or other material for burrowing, and ambient lighting should be dim.

Example 2: Normal room temperatures may be too warm for arctic animals in winter body condition. Likewise special accommodations may need to be made for desert adapted species.

Example 3: Species that have continuously growing teeth must be provided with appropriate gnawing material to prevent their teeth from overgrowing. The alternative is periodic trimming which may be stressful for the animal.

Example 4: Rats, chimpanzees and other highly social species (including herd animals) should be group housed whenever possible. Similarly, animals that are solitary by nature should be housed singly and if possible at low densities.

It is impossible to prevent stress, which may lead to distress, in research animals without a good understanding of their normal behavior, physiology and environmental conditions. Even with this information it is essentially impossible to create a research, teaching or testing environment that eliminates all potential stressors. At best we can minimize their impact on the animals (prevent distress) and therefore on the results of the study.

Whenever possible facilities and operational practices should be adapted as necessary to accommodate species specific differences.

Providing environmental enrichment can also reduce stress in many species. The following publications provide information and references related to enrichment for a variety of common research species:

Animal Welfare Institute website. This site provides a variety of links to sites and databases related to environmental enrichment. There are sections dedicated to animals in laboratories, agriculture, oceans, and the wild. Within the "animals in laboratories" section, there are numerous links to sites and databases dealing with refinement and environmental enrichment for both non-human primates and other research animals.

Environmental Enrichment Information Resources for Laboratory Animals: 1965 - 1995. AWIC Resource Series No.2. Published in 1995. Covers enrichment for Birds, Cats, Dogs, Farm Animals, Ferrets, Rabbits, and Rodents.

USDA Perspective on Environmental Enrichment for Animals. 2005 ILAR vol. 46 no. 2, pages 83-94.

There are a number of companies that provide environmental enrichment products for animals. AWIC maintains a list of suppliers complete with links and contact information.

It is important to recognize that different housing, enrichment, handling procedures may be indicated based on the sex, age, or reproductive state of an animal. Depending on the species and intended use you may also need to consider individual differences between animals.

Environmental conditions may need to be modified for animals following surgery or other painful or stressful procedures. For example it may be painful for an animal to lift its head to an overhead food bin if it has just had a neck or head surgery. In this case it may be appropriate to place food on the cage floor for the first few post operative days. Similarly, it may be necessary to eliminate or modify retreat openings or tunnels to accommodate an animal fitted with an external experimental apparatus.

Although enrichment products can reduce stress and stress related behaviors in research animals it may not be possible or advantageous to provide enrichment during an experiment. It is important to carefully consider how enrichment products may affect the animals' behavior and what effect that may have on their research, teaching, or testing results.  For example, in some situations it may be appropriate to provide enrichment to colony animals but not to experimental animals.  Although the UAF IACUC recommends the consideration of environmental enrichment for all animals, the ultimate decision about whether or not to use enrichment must take into account potential impacts on research, teaching or testing outcomes.

Pharmaceuticals. The use of pharmaceuticals is one way in which pain and distress can be minimized.  Veterinary Services can assist you in evaluating the potential for pain and distress due to capture, transport or housing conditions, experimental treatments, or surgical procedures, and help you develop a plan to minimize pain and distress that is consistent with your research goals.  Animal welfare regulations require minimization of pain and distress whenever possible, i.e. when it is consistent with scientific objectives, or documentation and justification for why it cannot be done. 

Use of pharmaceuticals for reducing pain or distress must be approved by Veterinary Services.  The IACUC Protocol must include the name of the drug(s), dosage, route of administration (including needle size and volume/quantity), and frequency of administration.  You must also describe what criteria will be used to determine when to start and stop administering the pharmaceuticals if a set schedule will not be used. 

Most of the drugs used to reduce pain or distress are prescription or controlled substances (see Pharmaceutical Basics and appropriate Drug Class information sheets) and can only be ordered by a licensed veterinarian.  Contact Veterinary Services well in advance to insure that adequate stock is on hand or ordered for your use.
 

Conditioning Animals Before Experiment

Once again, establishing a routine for your experimental animal will improve the data obtained. Be sure your animals are well acclimated to the housing and handling procedures. If animals are not properly acclimated your study will not be focused on the research questions under study, rather you are more likely to be faced with a stress response.

Normally a 14 day quarantine is imposed on wild-caught animals and a 10 day quarantine on purpose bred animals (i.e. those from a commercial supplier). This generally allows acclimation to the new housing and surroundings. Following quarantine it is recommended that animals be trained or worked in restraint devices prior to the start of an experiment.
 

Pre-experiment Instruction of Technicians/Students

Principal investigators are responsible for ensuring that all personnel working with live vertebrates are properly trained. All research personnel should know exactly what to do and expect during the project activities. Personnel with access to live vertebrates must, at a minimum, be familiar with the rules and regulations that govern the use of vertebrates and know who to contact with questions or concerns. Project personnel are typically appreciative of this training and your research will be better for the effort.

Your approved IACUC Protocol must be available to all individuals involved with your study. The best way to ensure this is to share the protocol with them via IRBNet; this will ensure that they always have access to the most current approved version of the protocol. 

It is extremely important that your IACUC Protocol accurately reflects current refined practices. Remember you may not make any changes to animal use procedures without the prior approval of the IACUC.  The one exception to that rule is that you may alter procedures to deal with an emergency situation.  Such situations must be immediately reported to the IACUC and Veterinary Services.  If after dealing with the emergency, you determine that changes to your approved procedures are necessary to prevent future occurences, you must request a modifcation to your protocol and receive IACUC approval before proceding with additional procedures or animals.