Laboratory Animals

A Working Party of the UK group of European Primate Resources Network (EUPREN) considered primate supply for scientific work in the UK. Through a questionnaire, which achieved a very good response, it obtained details of primate use, sources and breeding in the UK and it put forward options to ensure that animal welfare is the best possible whilst ensuring continued supply.

The questionnaire showed that contract research laboratories and pharmaceutical companies use about 80% of the 4233 primates used annually at the moment, with the rest accounted for by academic establishments and public sector laboratories. Fifty-four per cent are cynomolgus macaques (Macaca fascicularis), of which nearly 90% are captive-bred outside the European Union (EU), the remainder being bred in the UK. Nearly 90% of cynomolgus macaques are used by only five institutions. Thirty-seven per cent of primates used are marmosets (Callithrix jacchus jacchus), all of which are bred in the UK. Most of the rest are rhesus macaques (Macaca mulatta), about half of which are captive-bred outside the EU, the other half being bred in the UK. Overall primate use has increased from about 3000 per year in 1990 and users predict that requirements for all species except baboons (Papio sp.) will be maintained or increase.

Marmoset breeding in the UK is already closely matched to use, and it could be increased reasonably easily if necessary. Some of the existing breeding centres of macaques in the UK would be prepared to consider expanding to supply others, although investment and imported breeding stock would be needed and it is likely that a large investment would be needed to breed a significant fraction of the macaque use in the UK. A further problem is that the users of only about 10% of the cynomolgus macaques said that they could replace this species by rhesus macaques, which are easier to breed in the UK.

The questionnaire showed that much of the use of macaques would be transferred to other countries equally remote from the natural source countries of the animals, if constraints on primate use became more severe in the UK. Users felt that it is unlikely that much of the work could be transferred to the natural source countries themselves.

A review of the literature revealed a paucity of information on the effects of transport on primate welfare. The importance of obtaining this information before making decisions about alternative means of supply is stressed. Current schemes for the accreditation of primate breeders were reviewed.

A list of options is presented for discussion. Users vary so much in their requirements that it is unlikely that one means of supply will be applicable to all. Animal welfare will benefit and supply will be more certain if cooperation between those concerned (preferably through the UK group of EUPREN) is maintained.

The transportation of primates has become an important welfare issue and the outcome of the debate over its cost to the animal will have effects on the future of medical research using these species. There is a paucity of scientific studies on transport relating to primates and the need for gathering of further scientific evidence is highlighted.

Salivary cortisol is a non-invasive and easy-to-assess measure of the activity of the hypothalamo-pituitary-adrenocortical (HPA) system. Here we report that salivary cortisol determination can be used in squirrel monkeys (Saimiri sciureus) to monitor variations in HPA system activity induced by both housing and experimental conditions. Saliva cortisol assessment has several advantages over blood cortisol analysis such as stress-free frequent sampling, laboratory independence and lower costs. Therefore, this non-invasive measure can be the method of choice in primatological research projects and routine programmes related to the well-being of these laboratory animals.

This paper reports the results of an examination of the 'methods' sections of a range of experimental research papers in biomedical science, focusing on the descriptions of animal use and housing. Detailed descriptions in the methods should enable replication, and also enable readers to judge scientific quality. Relatively few papers sampled gave adequate descriptions of housing conditions and many failed to give details of physiologically relevant variables such as weight of animals. Thirty per cent of papers omitted the number of animals used, and the deaths of animals (whether as part of the protocol, or accidental death) were not always recorded.

Adequate reporting of the conditions of animal maintenance and use are important, both in relation to the quality of the science produced, and also because of public concerns about the ethics of animal experiments.

During the last decades, an increase is apparent in the use of analgesics for laboratory animals in situations where this was previously considered unnecessary. Mice with advanced tumours often show clear signs of discomfort which may be a result of chronic pain or a result of general ill-being. The syngeneic murine tumour model most frequently used in our experiments was used to investigate whether this discomfort can be reduced with an analgesic. Twenty DBA/2 mice bearing SL2 lymphoma were given 0.5 mg/kg buprenorphine (Temgesic^®) in food gel twice daily, 20 tumour-bearing mice were given control food gel at the same times. Indicators of well-being were monitored daily. These included behavioural parameters such as exploration, grooming, and posture; food and water consumption and fur quality. All mice showed a clear increase of discomfort with time: explorative behaviours and grooming decreased, while sitting in hunched posture increased. Food and water consumption and fur quality also decreased. Major significant differences between the buprenorphine treated group and the control group were not apparent. In conclusion, we could not document a positive effect or buprenorphine on discomfort in mice as evaluated by our scoring system. It remains possible that pain itself was not the primary cause of the discomfort in mice bearing these tumours, or that the analgesic effect of buprenorphine was insufficient under these circumstances.

Coelomocytes were extruded from three earthworm species: Lumbricus terrestris, Eisenia fetida and Octolasion tyrtaeum. Featuring a simple low-vacuum holding device, the proposed methodology allows the recovery of cells with minimum risk of contamination by faecal material. The viability of O. tyrtaeum coelomocytes was highly reproducible (average 93%), with an average yield of 0.92 × 10⁶ viable cells per earthworm. Cell viability for L. terrestris and E. fetida averaged ~68% but the cell yields were higher (respectively 1.67 × 10⁶ and 1.28 × 10⁶). Large inter-individual differences in cell yields were observed with L. terrestris. Flow cytometric analyses indicated species to species differences in cell populations. Coelomocytes from E. fetida were the smallest with ~57% of the total viable cells recovered being monitored between 2 and 10 µm. Large granulated cells (≥20 µm) were detected in fairly large proportions in L. terrestris and O. tyrtaeum [~52 and ~96%, respectively) while they were less abundant in E.fetida (~9%). Using the vital dye neutral red to assess functional integrity, average cellular uptakes were significantly higher for L. terrestris and O. tyrtaeum than for E. fetida (2.94, 2.66 and 0.64 µg/2 × 10⁵ cells, respectively). In summary, the extrusion methodology herein described is applicable for the recovery of coelomocytes from a wide range of earthworm sizes and species. Moreover, this study strengthens the fact that extruded coelomocytes could be used for the evaluation of cell dysfunction and/or cell death following an in vitro and/or in vivo treatment.

Our results showed more rapid falls in pulse rate and blood pressure in rats euthanized in a chamber precharged with carbon dioxide (CO₂), when compared with rats euthanized more slowly, but death still took over 5 min in the former group. There was no behavioural evidence of pain or distress in either group during euthanasia. Initial ataxia and dyspnoea was punctuated by a lag before death, thus separating euthanasia into three clearly defined phases. All visual signs of death preceded complete vascular collapse by about 1 min in both groups, so we recommend that gas flow be maintained for at least 1 min after apparent death.

Prolonged exposure to hypoxia elicits a variety of time-related morphologic and physiologic changes in the pulmonary vasculature of mammals, including humans. The study of hypoxia-induced changes in rodents generally requires a prolonged exposure to 9% oxygen for a minimum of 10 days in an airtight chamber, which has only been generally described in the literature as large (200-400 l), sealed acrylic chambers. To assist in the search for better therapies for diseases associated with chronic hypoxia using animal models, we have custom-built an airtight chamber for hypoxic exposure of rodents, and characterized the effect of chronic hypoxia on functional and morphologic changes in the pulmonary vasculature of the guineapig using this system. This chamber has been designed to alleviate any unnecessary stress related to food or water intake, cleanliness and excess illumination to the animals during the hypoxic-exposure period. Chronic exposure of the guineapig to hypoxia (0-21 days) produced time-related physiologic, morphologic, and haematologic changes. For example, after 10 days in hypoxia (9% oxygen), pulmonary artery pressure was significantly increased from 13±1 mmHg in normoxic controls (day 0, n=6) to 26±0 mmHg (day 10, n=4, P<0.01). Right ventricular hypertrophy in hypoxic animals, presented as a ratio of right ventricle free wall weight to body weight, showed a significant increase from 0.054±0.004 (day 0) to 0.069±0.004 on day 10 (P<0.05), while age-matched normoxic animals showed no changes in right ventricular weight (day 0=0.059; day 10=0.058; P>0.05). Red blood cell count significantly increased over the same time period, from 5.9±0.1 (day 0) to 6.4±0.1 (day 10, P<0.05) as did haematocrit, 48±0.7 (day 0) to 61±0.9 (day 10, P<0.05) and haemoglobin, l6±0.2 (day 0) to 20±0.1 (day 10, P<0.05). It is concluded that considerations for the well-being of the test animals (i.e. continuous water, ample food supplies, burrow-like hiding places, sanitation and protection from excess illumination) can easily be incorporated into a hypoxic chamber. The purpose of the present study was to explore modifications that may provide the animal with an optimized environment which will reduce anxiety and stress, as seen in their behaviour when inside the chambers, and to thoroughly characterize the morphologic and physiologic changes associated with chronic hypoxia which develop in a consistent time-related manner.

Individual housing has been reported to modify animal behaviour. The present study compares the plasma levels of glucose, triglycerides and total cholesterol, weight, and food and water intake in two groups of female rats. Group A: 10 rats who remained grouped in two cages for 21 days; and Group B: 10 rats housed in two cages for 7 days, then isolated in individual cages from day 8 to day 15, and finally grouped together again for the last 7 days of the study.

The results showed that the plasma values of glucose declined (P<0.05) in the Group B rats when they had been returned to group condition (4.79±0.72 mM) than when they had been isolated (5.45±0.94 mM). Plasma triglyceride levels were lower (P<0.05) in isolated rats (0.70±0.26 mM) than in any determination of the grouped rats. Group B: 1st week 1.21±0.21 mM, 3rd week 0.88±0.20 mM; and Group A: 1.22±0.20, 0.96±0.16, and 0.96±0.36 mM, in the first, second, and third week, respectively. There were no statistically significant differences in total cholesterol values as a function of the individual housing of animals. While there was no weight difference between the two groups of rats that could be ascribed to individual housing, there was a statistically significant increase (P<0.05) in the food intake of isolated rats (17.5±3.2 g) with respect to values in the same Group B animals when they were housed together (1st week, 16.6±2.8 g; 3rd week, 16.8±3.1 g).

These results therefore confirm that individual housing of female rats provoke variations in certain biochemical parameters, and that if this is not taken into account in performing different scientific studies, it could give rise to unreliable or even dubious results.

Laboratory rabbits kept in barren 'traditional' cages tend to develop stereo typic behaviours and bone deformities. We have used an alternative regime, housing adult does as groups of four or five in floor pens (2.5-3 m²) supplied with hiding places and bedding. High- and low-ranking members of each group were identified, and their immunological status compared in terms of blood leucocyte function (chemiluminescence and mitogen tests), complement activity, and antibody production to soluble and cellular antigens. We found no evidence of immunosuppression, either in groups of a 'docile' breed (New Zealand White) or Dutch crosses. These results, together with the animals' general health and ease of handling, lead us to conclude that group-housed does are suitable for raising antisera and other purposes, provided that they are adequately monitored.

A case of spontaneous occurrence of systemic mast cell disease in a non-treated laboratory-reared gerbil is reported. This case corresponds to the form of systemic mastocytosis with bone marrow and visceral involvement associated with skin disease and displays clinically aggressive behaviour. The histopathological, histochemical and ultrastructural features are described.