Laboratory Animals

The use of callitrichid primates in biomedical research was encouraged initially by their susceptibility to various diseases such as infectious hepatitis and rubella. However, there was no supporting biological knowledge which helped to keep them healthy and breed effectively in captivity. Further, various callitrichid biomedical models are endangered species; hence, animal welfare and primate conservation are interrelated. Recent information has extended our knowledge of the natural life styles of these species in various ways; but there is still little to predict persistent concerns of effective management in captivity. Hence, we need practical information such as those derived from laboratory records of housing, reproduction, diet and health, together with studies that compare specifically behaviour in different laboratory conditions.

More important in the long run, is that we study the biobehavioural propensities of different species. As yet callitrichids are largely unknown in these regards- situation that continues to present problems for the reliability, validity, standardization and generalization of experimental procedures.

Recommendations have been drawn up in different countries for the housing of non-human primates. Four of them are compared and it is clear that there is little consistency with regard to spatial allowances or suitable temperature and humidity ranges. Often the cage sizes quoted are incompatible with requirements to meet the behavioural needs of the animals. Only one provides guidance on primates' different social needs. To achieve greater consistency and credibility, guidelines should be regularly revised to include advances in our knowledge of the animals' needs.

Primates are used extensively in a variety of research settings. Federal regulations in the US mandate that caretakers provide for the 'psychological well-being of laboratory primates'. One of the difficulties in implementing this law has been both in the definition of psychological well-being and in the need to deal with each primate species and, in some cases, age or sex class, uniquely. Non-human primates exhibit distinct individual differences in their behavioural and physiological responses to experimental challenges and caretaking procedures. We have been investigating what factors can predict some of these individual differences, and have found that factors both intrinsic and extrinsic are significant. Extrinsic factors found to predict individual differences in response to stressors include the nature and prior experience with the challenge, the presence of familiar peers and availability of social support. Intrinsic factors include cognitive interpretations of the challenge and temperamental differences in reactivity. These studies highlight the importance of understanding the context and individual psychology of macaques in order to provide laboratory environments conducive to their welfare, and in order to understand the impact experimental and caretaking procedures are likely to have on the health and welfare of our subjects.

A system is described for the continuous measurement of blood pressure, heart rate and motor activity by telemetry in conscious marmosets freely moving in their home cages. Consistent diurnal variations in these parameters were observed under standard conditions. However these parameters were sensitive to changes in the environment. Blood pressure values were similar to those measured by non-telemetric methods in conscious restrained marmosets while heart rate values were significantly lower.

This paper records the effects of carbon dioxide when used for euthanasia, on behaviour, electrical brain activity and heart rate in rats. Four different methods were used. Animals were placed in a box (a) that was completely filled with carbon dioxide; (b) into which carbon dioxide was streamed at a high flow rate; (c) into which carbon dioxide was streamed at a low flow rate and (d) into which a mixture of carbon dioxide and oxygen was streamed at a fast rate. It was found that the cessation of behaviour was associated with an aberrant pattern of electrical brain activity together with an abnormally low heart rate. The time to reach this point was shortest in those animals placed in the box filled with pure carbon dioxide, longer when carbon dioxide was introduced at a high rate into the box, longer still when oxygen was added to the carbon dioxide gas, and longest when carbon dioxide was streamed slowly into the box. In the condition with pure carbon dioxide, signs of behavioural agitation and asphyxia were seen. This was also true for the two conditions in which carbon dioxide streamed into the box, but to a lesser degree. These signs occurred when some degree of consciousness may still have been present in the animals. Signs of agitation and asphyxia were almost completely absent in the condition where oxygen was added to the carbon dioxide. These results not only demonstrate the usefulness of behavioural criteria next to electrophysiological indices, but also demonstrate that the negative effects of carbon dioxide euthanasia can be prevented by an additional supply of oxygen.

Induction of anaesthesia in swine by thiopentone (27.1-35.7 mg/kg, mean 29.9 mg/kg) was followed by bolus doses and continuous infusion of midazolam and fentanyl (0.90 mg/kg followed by 0.90 mg/kg/h and 0.025 mg/kg followed by 0.025 mg/kg/h, respectively). This produced good anaesthesia and analgesia for up to 2 h in 6 Norwegian Landrace pigs (wt: 17-42 kg), based on responses to painful stimuli elicited by pinching the nasal septum, the mouth, the forefoot and the perineal skin area. The first responses occurred after 110 min of anaesthesia. No significant drop in rectal temperature due to the regimen was noted during monitoring periods (140-180 min). This combined intravenous anaesthetic regimen gave good anaesthesia and analgesia to pigs for up to 2 h as monitored by clinical signs. The regimen may not be sufficient for longer time periods. We cannot advocate the incorporation of neuromuscular blocking agents in this regimen.

Whole coronal slices from 6 levels of the brain of 16 cynomolgus monkeys (8 control and 8 treated by daily gavage with a novel pharmaceutical agent for one year) were examined histologically. Mineralized bodies were identified only in coronal sections passing through the optic chiasma and mammillary bodies.

Identical mineralized structures were present in the basal ganglia of both control and treated animals. The majority were seen in the globus pallidus, occasionally in the putamen and once in the nearby caudate nucleus. These structures were partially ferruginated and also partially calcified. They appeared to arise in relation to small vessels. They are part of the naturally occurring background pathology of several species of non-human primates and the incidence in this study (3/8 control and 5/8 treated) was approximately what might be expected from reports in the literature. Mineralized bodies of the basal ganglia of primates represent a spontaneous lesion with a characteristic distribution. They may cause confusion in interpretation of toxicological studies if their natural occurrence is not appreciated.

A transport and monitoring unit for fully anaesthetized piglets (weight 20-30 kg) was made. The unit provided easy transport for short or longer distances while at the same time making continuous monitoring of invasive blood pressure (IBP), temperature and HR possible. The animals stayed in the unit for the duration of the experiments which required that the anaesthetized animal be kept in exactly the same position for several hours. This, as well as the experimental animal's welfare, was ensured by the unit. Such a unit can be used for any lengthy transportation of experimental animals to research facilities located separately from animal laboratories.

Recognition and assessment of pain and distress is made by observing common clinical and behavioural signs. Observation usually occurs during a limited period of time and results can be biased by interpretation of an individual observer.

To improve objective assessment of distress we studied the locomotor activity pattern of mice during a 24-h interval. As a reference compound, Freund's complete adjuvant (FCA) was used. Mice were injected intraperitoneally with different doses FCA (0, 0.1, 0.2 and 0.5 ml) and observed for 5 to 7 days. Animals did not appear to be in pain and seemed to have a normal activity and behaviour pattern at first sight, however FCA induced a dose-dependent decrease of body weight. Open field activity (total distance run) measured during a limited period of time was not altered as a result of FCA. However, nocturnal activity was dose dependently decreased during the first 3 to 4 nights after treatment with FCA. The data presented indicate that using locomotor activity patterns over 24 h might be a useful adjunct and an objective approach to assess distress.

An enzyme-linked immunosorbent assay (ELISA) to measure Corynebacterium kutscheri antibodies in mice and rats was developed. Seven c. kutscheri isolates showed considerable serological relationship, but Japanese isolates differed from the British isolates. The ELISA appeared specific since c. kutscheri antigen did not react with antisera against 8 heterologous coryneform species. Antibodies to c. kutscheri were to a limited extent absorbed by autologous and homologous antigen, but not at all by the heterologous coryneform species.

In naturally infected wild Rattus norvegicus and laboratory NA rats, the ELISA demonstrated high ODs to c. kutscheri.

Interstitial nephritis was seen histologically in 19 (59%) out of 32 pure-breed beagle dogs (16 males and 16 females) subjected to standard safety tests. In these animals no clinical abnormalities were observed and all the tested parameters (haematology, biochemistry and urine analysis) were within the normal ranges. Leptospiral antibody titres ranging from 1:100 to 1:6400, against a serovar (hardio) belonging to the Sejroe serogroup, were detected by the microscopic agglutination test (MAT) in the serum of the 19 dogs with interstitial nephritis. All animals without renal lesions were seronegative. Leptospiral antigen was detected immunohistochemically in the kidneys of 4 dogs; leptospires were detected in Warthin-Starry stained sections of one dog. Leptospires were isolated from the kidneys of 3 of the 4 dogs examined by bacterial culture. The isolated strains were typed as serovar sejroe by restriction endonuclease digestion and Southern blot hybridization analysis of their DNA. It was concluded that Leptospira interrogans serovar sejroe, was responsible for an asymptomatic chronic renal infection which was widespread in this group of laboratory dogs.

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of Pasteurella pneumotropica antibodies in the sera of rats} mice, hamsters and Mastomys. P. pneumotropica from mice and rats showed cross-reactivity. The ELISA using P. pneumotropica NCTC 8284 detected more infected animals than selective culture in groups of rodents from which P. pneumotropica, Haemophilus sp and/or Actinobacillus sp were cultured. Cross reactivity between P. pneumotropica NCTC 8284 and haemophilus and actinobacillus isolates were not studied.

Thirty Pasteurellaceae strains isolated from gerbil, guineapig, hamster, mouse, muskrat and rat were reinvestigated and reclassified after comparison with reference strains. Strains originally described as Pasteurella pneumotropica were reclassified as [Pasteurella] pneumotropica Heyl biotype (7), [P.] pneumotropica Jawetz biotype (1), Pasteurella dagmatis (1) or Taxon 22 (2). Strains previously reported as Actinobacillus sp. were reclassified as [P.] pneumotropica biotype Jawetz (3), P. dagmatis (3) or Taxon 6 (7). Strains earlier described as Pasteurella gallinarum were renamed as SP group pasteurellas (4) or Taxon 25 (2). Some of these reclassified Pasteurellaceae have not been reported previously in rodents. The present findings underline the importance of extended characterization of isolates and comparison with reference strains to avoid misclassification within the family Pasteurellaceae Pohl 1981.

Approximately 18% of cryopreserved 2-cell mouse embryos of 26 different batches showed various degrees of morphological damage after the freeze-thaw process. Normal and damaged morphology were assessed by light microscopy and the ability of an embryo to develop in vitro to a blastocyst, or to develop to term, after transfer to foster mothers. Using vital stains such as Fluorescein-diacetate (FDA) and 4',6-Diamidino-2-Phenylindole (DAPI) it was found that in approximately 82% of the cases, both of the 2 blastomeres of the cryopreserved embryos survived the freeze-thaw process; in 10% only one cell survived the process; and in 8% none survived. Normally, only intact 2-cell embryos are considered for transfer. Here it was shown that over 60% of the partially damaged embryos developed in vitro to the blastocyst stage and, of those, 26% developed to term after transfer to suitable foster mothers. Although the inner cell mass (ICM) appeared to remain smaller during culture after the transfer of partially damaged 2-cell stage embryos, no difference during gestation period was found compared with intact embryos.

The influence of prostaglandins E₂ and F_2α on passage pressure across the uterotubal junction (UTJ) and isthmus were studied in rats that were either in the pro-oestrus, oestrus, metoestrus or dioestrus phases. Effects of these prostaglandins were also investigated in rats that had been either ovariectomized and treated with oestradiol or medroxiprogesterone acetate, or only ovariectomized. In each rat, the left UTJ was surgically resected and the isthmus anastomosed to the uterine horn, whereas the right UTJ was left untouched. The passage pressures across the left isthmus and the right UTJ were measured before and after prostaglandin treatment.

The pressures obtained in the UTJ in the oestrus phase and oestrogen-treated ovariectomized animals were lower than those registered in the remaining groups. Prostaglandin E₂ decreased the pressures when compared with pre-treatment measures in all groups. Significantly higher pressures were registered across the UTJ in prostaglandin F_2α than in E₂ treatment, with these higher pressures being similar to pre-treatment pressures. Both hormonal changes throughout the oestrous cycle and prostaglandin E₂ treatment had a similar influence on the passage pressure across the isthmus, as that described for UTT, but with lower values.

The results indicate that prostaglandin E₂ decreases the passage pressure across both UTJ and isthmus and can have an influence on the regulation of transport across these 2 areas.

This study was performed to investigate the potential effects of nocturnal, low-intensity light upon ovarian morphology of female Sprague-Dawley rats and to investigate the cause for ovarian changes which had been observed in an earlier study following transient exposure of control female Sprague-Dawley rats to indirect light of minimal intensity during the nocturnal 12-h dark cycle. Twenty female Sprague-Dawley CD rats (initial age: 5 weeks) were kept for 8 weeks under our standard laboratory conditions including a daily 12-h light cycle (light intensity: approximately 200 lux) followed by a 12-h dark cycle with exposure to an indirect light source of low intensity (approximately 30 lux). Ten female control rats of comparable age from a concurrent toxicology study housed in an adjacent animal room under our standard 12 h light/dark cycle served as controls. At the end of the study the rats were sacrificed, necropsied and the ovaries were evaluated histopathologically. In 5 of the 20 animals we found ovarian atrophy consisting of decreased number and size of corpora lutea and increased number of tertiary follicles and/or follicular cysts. Most corpora lutea present in these ovaries were old, indicating the absence of recent ovulations. In contrast, the incidence of ovarian changes in the control group was 0/10. In conclusion, nocturnal exposure of female Sprague-Dawley rats to light of minimal intensity produced a substantial incidence of ovarian changes and suggests that the incidence of ovarian atrophy observed in a previous study may have been due to transient exposure to indirect nocturnal light of minimal intensity.

Traditional methods for animal identification have a number of drawbacks. We evaluated a new system for individual identification using microchip implants in rabbits, guineapigs, woodchucks (Marmota monax) and amphibians (Xenopus laevis, Pleurodeles waltlii). Implantation procedure and long-term observations are described. Microchip implants proved to be a practicable and reliable system for animal identification without obvious adverse effects. The applicability of electronic animal identification in comparison with common methods and with regard to animal welfare and legal aspects is discussed.