ANIMAL ACQUISITION 1. Procurement

All animals must be legally acquired. Animals received as donations for research should have been legally released (see Laboratory Animal Care, Identification and Records). It is almost always preferable to obtain standard laboratory species from an established breeder or licensed supplier. Several provinces have regulations governing the procurement of dogs (see also Responsibility for the Care and Use of Experimental Animals). In Ontario, laboratory animal supply facilities are licensed and inspected. All commercial producers of laboratory animals, regardless of whether or not they come under provincial legislation, are expected to provide housing facilities and to follow practices similar to those outlined in this Guide.

It is in the best interests of the user and the supplier to co-operate in the elimination of any undesirable condition affecting the health and quality of the animal. The institution (i.e., the receiver) should inform the supplier of any undesirable conditions observed in the stock received. The supplier should, if

requested, provide detailed information on health status monitoring, breeding, and husbandry practices followed.

The acquisition of animals should be dependent upon the prior approval of the project by the institutional Animal Care Committee (ACC). Acquisition procedures should be in place to ensure that the institution will have an up-to-date and ongoing inventory of all animal experiments for which it may be accountable, and to allow for the prior preparation of appropriate space and such other arrangements as may be necessary for the reception of the incoming animals.

2. Transportation a) Introduction

Depending upon the species and size of the animal, the modes of transportation can be by land, sea or air. For most laboratory species, the most common method is either by ground transportation, over relatively short distances, or by air for longer distances. The objective of any method of travel is to ensure the safety, security, and comfort of the animal in the container, and to take it to its destination as quickly and as safely as possible.

Although many agencies are concerned with animal transportation per se, those dealing with the transportation of experimental animals are few in number. The Animal Transportation Association (formerly known as the Animal Air Transportation Association) (AATA) is involved in making improvements to all modes of transportation. Publications are available on the subject of animal

transportation. The Canadian Federation of Humane Societies (CFHS) (1988), for example, conducted a survey of surface livestock transportation in Canada. A recent British Veterinary Association (BVA)

symposium has examined the welfare of animals in transit (Gibson, Paterson and Conville, 1986), as has a symposium of the World Association for Transport Animal Welfare and Studies (WATAWS) (Laing, 1991).

b) Regulations--Containers and Transportation

The International Air Transport Association (IATA) annually produces the IATA Live Animal Regulations, which includes information concerning the documentation, the containers and other requirements for humane transportation of live animals (IATA, 1992). Some 81 containers are described under the

headings of species, design and construction, preparations for dispatch, feeding guide, general care and loading. While the container information is specific for air transportation, the requirements for the containers are applicable to all modes of transport, for they ensure safety, comfort, and security for the animals (Rowsell, 1992).

In order to ensure accurate technical content, the IATA Regulations are prepared in consultation with representatives from the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) and the Office International des Epizooties (OIE). For over 10 years, the Canadian Council on Animal Care (CCAC), the International Council for Laboratory Animal Science (ICLAS), as well as the Eurogroup for Animal Welfare, have maintained close liaison with the IATA Live Animals Board,

addressing primarily the issues concerned with transportation of experimental animals.

Legislation in Canada addressing transportation includes the (federal) Health of Animals Act (C-66, June, 1990, rev. March, 1992; 38-39 Elizabeth II, Chapter 21), Ontario's Animals for Research Act (Revised Statutes of Ontario, 1980, Chapter 22 as amended by 1989, Chapter 72,s6 and Regulations

16,17,18,19), and, in Alberta, the Universities Act (Section 50, "Dog Control and Procurement"; Regs.

341-366). In 1972, Alberta Regulation 33-72 was expanded to cover the treatment of animals (Rowsell, 1974). As well, there is specific legislation governing the transportation of animals in provinces and specific bylaws in municipalities.

The transportation of animals from the United States into Canada is affected by the U.S. Department of Agriculture's Animal Welfare Act (1966) by specifying minimum and maximum temperatures to which animals in transit may be exposed. The U.S. Department of the Interior, U.S. Fish and Wildlife Service, is responsible for the enforcement of the importation of wild mammals and birds; as well, the U.S.

Department of the Interior is responsible for the enforcement of the Marine Mammal Protection Act.

c) Transportation Stress

One of the least controllable variables in animal experimentation is the effect on the research caused by moving animals from one area to another (Landi, Kreider, Lang et al. 1982, 1985; Aguila, Pakes, Lai et al. 1988; Bean-Knudsen and Wagner, 1987, Reinhardt, 1992). This might involve great distances between countries, or minor distances, such as within the animal facility itself. Even moving animals within the animal room affects the stress indices, which are more greatly influenced if the individual doing the transporting is a stranger (Gärtner, Büttner, Döhler et al. 1980).

The stressors in animal transportation include inexperienced handlers, the amount of time spent in preparation, in transit, and on arrival at the destination, and the state of the mode of transport, e.g., rough roads, rough rail beds, rough seas and air turbulence. Of importance are the comfort and suitability of the container, sufficient time spent for adaptation to the container prior to transport, and the

temperature and ventilation of both the container and the ambient temperature of the environment and the various temperature zones through which the animal may pass. It is essential to make appropriate pre-arrangements concerning the transport of the animal in order to minimize the length of time spent in transit.

Transportation stresses can be minimized by avoiding long distance shipping, slow modes of transport and, as mentioned, by acclimatizing the animals to the containers and, if possible, to the mode of transport. Kiley-Worthington (1990), in studies involving the transportation of animals in circuses and zoos, showed that unhandled, naive animals (i.e., those that are wild or have not been domesticated or are not used to confinement), will probably suffer the most. Although there is a great deal of concern about circus animals, show dogs, or competitive horses subject to regular transportation, this researcher reported that there was "no evidence suggesting that the transporting of circus animals is necessarily or even unusually distressing or traumatic for the animal, although it is for naive livestock."

The requirement for training those involved in the transportation of animals is often neglected. Although it is well established that inexperienced handlers can affect the animals significantly, little has been done

to ensure proper training of individuals engaged in animal transportation. In 1978, the CCAC produced an audio-slide presentation on "Humane Transportation of Live Animals" which was made available to all airlines and any of the other agencies engaged in the transportation of animals (Fletch, 1978; Rowsell, 1990).

The principles enunciated in the CCAC-produced training program continue to be applicable today. It notes, for example, that personnel engaged in the transportation of animals, including those employed by the animal facility, require knowledge of the various types of animals, the differences between species of birds and mammals, as well as consideration of the invertebrates. They must also be cognizant of the container requirements, and specification requirements for labelling and marking, and for completion of the proper documentation, which includes any licensing requirements, both for export and the country of designation. They should be knowledgeable of shippers' responsibilities as well as those of the individuals receiving the animals. They should realize the importance of making advance arrangements concerning the shipment and transport of animals.

The responsibility for knowing about the safe and humane transport of animals includes, as well, those who, in the conduct of their duties, are exposed to the animals or have responsibility for the animals they may be carrying, i.e., in the truck, on-board ship or in the aircraft. The institution receiving the animals should be prepared for accepting the animals by providing proper facilities and appropriate handling by trained, experienced personnel.

d) Animal Handling

The animal in a laboratory setting becomes conditioned to the environmental conditions such as temperature, humidity, air changes, noises, the habits of those working within the animal room, and animal or human pheremones (substances secreted and released by animals for detection and response by another of the same species). All of the foregoing may change when the animals are transported to the facility or moved within it (Slatnetz, Fratta, Crouse et al. 1957; Baker, Lindsey and Weisbroth, 1979;

Gibson, Paterson and Conville, 1986; Aguila, Pakes, Lai et al. 1988; Rowsell, 1988). Unfortunately, many of these changes have not been measured precisely (Yousef, 1988). As well, questions have been raised as to the suitability of certain tests used to measure the stress induced by environmental changes.

Behavioural changes such as increased agitation, difficulty in handling, reluctance to eat or to drink, bristling of the coat, hiding, and abnormalities in behaviour may go largely unnoticed, but may cause variations in experimental data. Therefore, it is essential that the animal be allowed to equilibrate to a new environment. Because the period for equilibration of each animal and between species varies, knowledge of the species and of the individual animals is essential.

When the animal arrives at its destination, the institution must ensure that it is then brought to the institution in a safe and humane manner. Air conditioned vehicles specifically designed for this purpose are essential in order to reduce stressors that may have increased during the transport period.

Acclimatization to the environment and a stabilization of the animal, physiologically and behaviourally, are essential prerequisites before the animal is used. Landi, Kreider, Lang et al. (1982) demonstrated that, following air transportation of rodents, a two-week period was required for blood and stressor parameters to return to normal. The adherence to the principles of humane transportation and handling throughout the transport period and on arrival at the institution should help ensure that, when animals are used in research, teaching, or testing, the results are meaningful and scientifically valid.

3. Breeding

A review of modern methods of breeding laboratory animals is beyond the scope of this Guide. However, it is axiomatic that all types of animals involved in a breeding program for animal production research purposes will require the best of care and that accurate breeding records must be kept (Box, 1976). It is essential that breeders and researchers who propose to breed animals for specific research purposes acquire detailed information on the anatomy, behaviour and physiology of reproduction relating to the stocks concerned (Altman and Dittmer, 1972; Greep, 1974; Hafez, 1970; Crawford, 1990). Blaffer-Hrdy and Whitten (1987) present comparative data for non-human primates (NHP) on cycle length, duration of

menstrual flow, visual signals, and the behaviour of males as well as females in estrus, for all species.

Breeders should refer to the sections of this Guide dealing with management, caging and special housing of the various species.

If an animal model of human disease is to be produced, it is mandatory that the scientist or breeder fully appreciates the basic physiological and pathological processes involved in the defect. The research facility is generally the best location for this type of breeding program. However, under some circumstances it may be preferable to contract for supply of a particular mutant strain, either with a reliable commercial enterprise or by arrangement with some other scientist who is already breeding and utilizing the same model (ILAR, 1979). The breeding of any laboratory animal must be done in accordance with the accepted genetic standards and genetic nomenclature (Festing, Kondo, Poiley et al. 1972; ILAR, 1979;

Lyon, 1981; Lyon and Searle, 1989), subjects which are beyond the scope of this Guide.

The decision to establish a breeding colony program in a research institution is one that the investigator and the institutional ACC should always study carefully in terms of the nature of the project. Unless breeding is an integral, even essential, part of the research or teaching exercise, thorough evaluation ought always to be undertaken of: a) the ultimate real cost (to the institution) of animals bred within the research facility; b) the occupancy of valuable and costly space which will no longer be available for other research; and c) the ultimate numbers of animals that will have to be produced versus the actual

numbers that will be utilized. Small in-house breeding programs almost always involve the need to dispose of animals superfluous to the needs of the project, and the maintenance of excess breeders in order to try to cope with fluctuating demands.

In commercial livestock production, selection may be based on ancestry, individual performance or progeny or combinations of these factors. However, in small laboratory species, emphasis has been placed on maintaining genetic purity (Festing, Kondo, Poiley et al. 1972). In recent years, questions raised about the genetic homogeneity of some strains has led to the genetic testing of animals. The larger suppliers of small laboratory animals provide genetic control for their stocks and strains, and may provide genetic testing services to investigators using other strains.

The genetic nature of an animal population can be changed in three ways: by selection, through

manipulation of the breeding system, or by altering the genome through the introduction of alien genes (DeTolla, 1991). Traditionally, the rapidity with which genetic change in a population is achieved will depend in part on the sex of the selected animals; the female has less effect on the maximal selection differential than the male, as the latter can produce many more progeny. The decision as to which animals are to be bred will depend on numerous criteria directly related to the purpose of the breeding exercise.

4. Breeding Transgenic Animals

The time required to produce large numbers of transgenic animals will depend on the reproductive capacity of the animal. Transgenic animals are now considered a standard biomedical research tool (Saffer, 1992) and are increasingly being used as animal models for human diseases (Merlino, 1991), gene therapy, the study of virus-induced disease, the physiology of foreign gene expression (Palmiter and Brinster, 1986; Geistfeld, 1991), probes into complex systems (Hanahan, 1989), and as models for genetic toxicology studies (Myhr and Brusick, 1991). Their use is increasing, and indeed, has been cited as a major cause in the first rise in animal use in the U.K. for many years (Anon., 1992).

Management of a transgenic mouse colony has recently been described by Geistfeld (1991), and a procedures manual has been published by Hogan, Constantini and Lacey (1986). As well, there are numerous research reports (Brinster, Chen, Trumbauer et al. 1985; Bishop and Smith, 1989;

Depamphilis, Herman, Martinez-salas et al. 1988; Gordon, Scangos, Plotkin et al. 1980; Jaenisch, 1976, 1988) describing transgenic animal use. Britain's Health and Safety Executive published Guidelines on Work with Transgenic Animals (1989) (Baynards House, 1 Chapstow Place, London, W2) which must be followed when creating, breeding or handling transgenic animals in the U.K. (Connor, 1989).

In describing management of a transgenic mouse colony Geistfeld (1991) suggests that Caesarian-derived pups be used in order to eliminate pathogens. Various breeding systems are feasible; usually a 2:1 to 3:1 harem mating system is effective, although there are no hard and fast rules.

Some of the problems associated with the breeding of transgenic mice have been outlined by Donnelly and Walsh-Mullen (1991). These include contamination of the media used in collecting eggs and blastocysts for microinjection, because of which the surrogate dam fails to deliver. They note that introducing foreign genes may cause deleterious insertions that prove lethal to the animal or that compromise reproduction.

The CCAC recently established a Committee on Animal Biotechnology (CABT), which has as its mandate:

"to develop ongoing guidelines for embryo manipulation, fetal research, and transgenic animals". The CABT considers it acceptable for transgenic/embryo manipulation research to produce animals which do not have a negative impact on the animal's well-being or on the environment, and which have a positive, scientifically justifiable endpoint. Should transgenic technology result in a new species or strain of animal, research techniques must be developed to adequately test the impact of such animals, in the view of the Committee. Some of Canada's institutions have prepared their own guidelines for research involving transgenics.

5. Animal Models with Special Needs

Animal models of human disease are used to study the causes and therapeutic and preventive methods for human disease, as well as to develop new drugs (Nomura, Katsuki, Yokoyama et al. 1987). Models are available for many diseases and conditions, e.g., hemophilia (Moake, 1988) atherosclerosis (Reddick, Read, Brinkhous et al. 1990; Farrell, Saunders, Freeman et al. 1986), Pasteurellosis (Morck, Costerton, Bolingbroke et al. 1990), intestinal disease (Pfeiffer, 1985), hepatic degeneration (Hultgren, Stevens and Hardy, 1986), enteric diseases such as Campylobacter jejuni (Fox, Ackerman, Taylor et al. 1987),

cardiomyopathy (Wagner, Reynolds, Weisman et al. 1986) and neurological disease (Barnes, 1986).

Animal models for advancing understanding of diseases such as hypertension, gastrointestinal tract and cardiovascular disease were discussed at a symposium of the British Laboratory Animals Veterinary Association (BLAVA) (Anon., 1986). Another meeting addressed the challenges facing researchers into the human immunodeficiency virus (HIV) and the need for animal models in this regard (Groopman, 1991).

Animal models of some conditions or diseases have special needs beyond those of normal, healthy laboratory animals. These special needs must be recognized and accommodated when such animal models are going to be used in research. It should be the responsibility of the principal investigator to take into consideration the special needs of the animals before embarking on the research project. These special needs will no doubt impact on the research budget in terms of additional animal care time,

materials, and equipment. ACC reviews of research proposals should include an assessment of these extra considerations for the animals.

The principle that encompasses this responsibility to attend to the special needs of animal models could be stated as follows: that any pain, suffering, distress, or deficits in function that negatively affect the animal's well-being, not scientifically "necessary" for the study, should be alleviated or minimized. Cost or convenience should not deter from this. Further, as soon as the study is done, the animal suffering should be terminated (Olfert, 1992).

6. Identification of the Sexes

Generally, sexes are kept separated subsequent to weaning, except for breeding purposes or because of experimental design requirements. Unwanted matings amongst stock animals should not occur, and under experimental conditions may compromise the experimental results.

The sexing of animals may be difficult in the newborn or in species with which one is unfamiliar. In addition to the genital organs themselves, secondary sex characteristics can be used in some cases (Valle, 1990). Detailed descriptions of the techniques/observations used to sex the various common laboratory animal species can be found in the species chapters of the CCAC Guide Volume 2, or in other general books on species used as laboratory animals (Poole, 1987). Descriptions of the techniques used to determine the sex of some of the less common species are also available (Goin and Goin, 1971; Frye, 1991; Marcus, 1981).

B. RESTRAINT AND MANIPULATIONS