Differences in hepatic cytochrome P450 activity correlate with the strain-specific biotransformation of medetomidine in AX/JU and IIIVO/JU inbred rabbits.

Medetomidine is an alpha(2)-adrenoceptor agonist with sedative and analgesic properties. Previously we demonstrated significant differences in the response to medetomidine between two inbred rabbit strains, denoted IIIVO/JU and AX/JU. The aim of the present study was twofold: first, to compare the hepatic CYP450 enzyme activities between these rabbit strains [n = 13(male male,7 female female)/strain]. To this end, liver microsomes were incubated with known fluorescent substrates for the major drug-metabolizing CYP450 isoforms. A comparison of the obtained results indicated significant gender differences as well as differences between the two rabbit inbred strains. Secondly, the biotransformation rate of medetomidine in liver microsomes of both rabbit strains was determined using liquid chromatography coupled to tandem mass spectrometry. The rate of hydroxymedetomidine and medetomidine carboxylic acid formation was found to be significantly higher in the AX/JU strain. Specific CYP2D and CYP2E inhibitors could decrease the formation of both metabolites. Significant correlations were found between the rate of biotransformation of medetomidine and the activities of CYP2D and CYP2E, as well as between CYP450 enzyme activities and the anaesthetic response to medetomidine.

The effects of buprenorphine on behaviour in the ACI and BN rat inbred strains.

Buprenorphine is a partial mu, kappa agonist that has been shown to influence spontaneous behaviour in animals. Previously, we have demonstrated significant differences in the analgesic response to buprenorphine between the August Copenhagen Irish (ACI)/SegHsd and the Brown Norway (BN)/RijHsd inbred rat strains. The purpose of this study was to determine whether these strains also differed in their behavioural response to buprenorphine in order to provide an additional parameter for the genetic analysis and localization of genes involved in this response. Male and female rats of both strains were used (n = 6/strain/sex) for this study. Each rat was subjected, respectively, to three treatment regimens at 15:00 h: (A) unchallenged; (B) intravenous saline; (C) intravenous buprenorphine (0.05 mg/kg) according to a crossover design. The relative duration (s/h) of locomotion, grooming, drinking and eating behaviour was subsequently determined from 15:30 to 07:00 h using the automatic registration system, Laboratory Animal Behaviour Registration and Analysis System(trade mark). Significant strain differences were observed in unchallenged behaviour between the ACI and the BN rats. ACI rats, but not BN rats, responded to buprenorphine treatment with decreased levels of locomotion, drinking and eating behaviour. The same treatment resulted in an increased grooming behaviour in both strains. Slight but significant sex differences were observed for locomotion and eating in the analysis of variance procedure, but did not reach the level of statistical significance in the multiple comparison procedure. The results of this study emphasize the possibility that strain-specific effects must be taken into account when using behavioural parameters for the assessment of the analgesic effects of buprenorphine in rats.

Differences in response to anaesthetics and analgesics between inbred rat strains.

Differences in response to analgesic and anaesthetic drugs can partly be attributed to variations in the genetic background of experimental animals. This study was carried out to determine differences in the response of inbred rat strains to a selection of analgesics and drugs used in anaesthetic protocols. A cross between the most contrasting strains can then be phenotyped in future studies in order to localize quantitative trait loci (QTLs) involved in analgesic/anaesthetic drug sensitivity. Eight inbred strains (n = 6 rats/strain) were selected for the study: the pigmented ACI, BN and COP strains and the albino F344, LEW, SHR, WAG and WKY strains. Each rat was injected intravenously with two analgesics (buprenorphine 0.05 mg/kg and nalbuphine 1 mg/kg) and three drugs used in anaesthetic protocols (propofol 25 mg/kg, medetomidine 50 microg/kg and ketamine 10 mg/kg), respectively, using a crossover design. Analgesic responses were assessed using an analgesiometric procedure. The sleep time of the rat and, where applicable, the interval between injection and loss of righting reflex were used to determine the anaesthetic response. Six out of eight strains responded significantly different from each other to the analgesic effect of buprenorphine with the ACI strain as hyper-responder. The tail withdrawal latency at 55 degrees C of the F344 and WKY rats using buprenorphine was not significantly different from baseline tail withdrawal latencies. In this study, all strains were non-responsive to the analgesic effects of nalbuphine. The response to all three drugs used in anaesthetic protocols differed significantly among the strains. The F344 and BN strains were relatively resistant to the sedative effects of medetomidine. Use of ketamine was abandoned in the ACI and BN strains when the first two animals of both strains died soon after induction. With all three drugs the sleep time of albino rats was significantly longer compared with that of the pigmented ones. We conclude that the results from this study can be used in future studies where QTLs for the sensitivity to anaesthetic/analgesic drugs are localized.

Influence of environmental enrichment and handling on the acute stress response in individually housed mice.

In this study we investigated the effect of environmental enrichment and handling on the acute physiological stress response caused by short periods of restraint in individually housed female mice. Heart rate (HR) and body temperature (BT) were measured by radiotelemetry and compared with plasma corticosterone (pCORT) levels. Also, postmortem thymus weight and tyrosine hydroxylase (TH) activity were assessed. The acute stress response was seen in both HR and BT. Enrichment and handling were found to increase rather than decrease this stress response, but pCORT values, measured 90 min after restraint, suggested a lower stress response in the enriched groups. No effect was found with thymus weight or TH as parameters.

Effect of restraint and injection methods on heart rate and body temperature in mice.

Routine procedures in the laboratory, inducing acute stress, will have an impact on the animals and might thereby influence scientific results. In an attempt to gain more insight into quantifying this acute stress by means of the parameters heart rate (HR) and body temperature (BT), we subjected mice to different restraint and injection methods. We first compared the treatment response of HR and BT, measured by means of radiotelemetry, with the treatment response of plasma corticosterone (pCORT), a common and well-validated parameter for measuring acute stress responses. It was found that HR, and to a lesser extent also BT, parallels pCORT values after subjecting the animals to different methods of restraint. Secondly, the acute stress response caused by different injection methods was evaluated. Again, HR was found to be a more sensitive parameter than BT. We found that, in case of sham injections, the acute stress response after an intraperitoneal (i.p.) injection was more pronounced than after intramuscular (i.m.) or subcutaneous (s.c.) injections, but this difference was found to be inconsistent when saline was used as injection fluid. In a third experiment we investigated if the level of experience of the animal technician influenced the stress response after s.c. injections, but no differences were found. Overall, the results have indicated that HR might be considered as a useful parameter for measuring acute stress responses to routine procedures, but the value of BT seems to be of limited value in this respect.

Long-term effects of husbandry procedures on stress-related parameters in male mice of two strains.

In socially unstable groups of male laboratory mice, individuals may experience a chronic stress situation. Previous experiments have shown that the transfer of specific olfactory cues during cage cleaning, and the provision of nesting material decrease aggression and stress in group-housed male mice. In this study, the combined effect of these husbandry procedures were tested for their long-term effect on stress in groups of moderately aggressive (BALB/c) and severely aggressive (CD-1) male mice. The physiological and behavioural stress-related parameters used were body weight, food and water intake, spleen and thymus weight, adrenal tyrosine hydroxylase activity, urine corticosterone levels and behaviour in a cage emergence test. Long-term provision of nesting material and its transfer during cage cleaning was found to influence several stress-related physiological parameters. Mice housed in cages enriched with nesting material had lower urine corticosterone levels and heavier thymuses, and they consumed less food and water than standard-housed mice. Furthermore, marked differences were found between strains. CD-1 mice were less anxious in the cage emergence test, weighed more, ate and drank more, and had heavier thymuses but lighter spleens and lower corticosterone levels than BALB/c mice. We conclude that the long-term provision of nesting material, including the transfer of nesting material during cage cleaning, reduces stress and thereby enhances the welfare of laboratory mice.

Preference for social contact versus environmental enrichment in male laboratory mice.

Due to their aggressive nature, male mice are less frequently used than female mice in biomedical research. When aggressive males are being used, individual housing is common practice. The question arises whether this is an acceptable housing for a social species. The present study was designed to gain more insight into the nature of inter-male social contact and into the potential of a form of environmental enrichment (nesting material) to compensate for the lack of social contact. In a series of tests, we analysed whether male mice of different ages preferred to spend time (1) near a familiar cage mate versus an empty cage, or (2) near to a familiar cage mate versus direct contact with nesting material (tissues). Dwelling time in each of the test cages and sleeping sites was recorded, as was the behaviour of the test mice. Results indicated that when other conditions were similar, male mice preferred to sleep in close proximity to their familiar cage mate. Furthermore, the need to engage in active social behaviour increased with age. Tissues were used to a large extent for sleeping and sleep-related behaviour. It is concluded that single housing in order to avoid aggression between male mice is a solution with evident negative consequences for the animals. When individual housing is inevitable due to excessive aggressive behaviour, the presence of nesting material could partly compensate for the deprivation of social contact.

Male management: Coping with aggression problems in male laboratory mice.

In a laboratory environment, aggressive interactions between male mice may exceed normal levels leading to negative effects both on the well-being of the animals and on the validity of experimental results. In this paper we review results from the literature and our own research with regard to coping with excessive aggressive behaviour in male laboratory mice. Based on this review practical recommendations concerning the housing and care of male laboratory mice are formulated. In short, it is recommended to avoid individual housing, to transfer odour cues from the nesting area during cage cleaning and to apply nesting material as environmental enrichment. Furthermore, group size should be optimized to three animals per cage. Further research, in particular into the effects of frequency, duration, type and severity of disturbances during an experiment on the degree of aggression, is recommended.

Mapping of rabbit microsatellite markers using chromosome-specific libraries.

Recently, rabbit microsatellite markers were developed from a chromosome 1-specific library, and seven new markers were incorporated into the genetic map of the rabbit. We have now developed microsatellite markers from chromosomes 3-, 5-, 6-, 7-, 12-, and 19-specific libraries. Linkage analysis was performed with use of these new markers, five recently physically mapped markers (PMP2, TCRB, ALOX15, MT1, and Sol33), microsatellite markers located in the HBA gene cluster, the MHC region and FABP6 gene, and seven biochemical markers (Es-1, Es-3, Est-2, Est-4, Est-6, Est-X, and HP). This analysis enabled us to verify the specificity of the libraries and to determine the position and orientation of the linkage groups on the chromosomes.

Detection of universal variable fragments as markers for genetic studies. A novel technology for DNA fingerprinting.

A novel DNA technology enables the detection of universal variable fragments (UVF), thus revealing genetic variation without a priori sequence information. The detection of UVF markers is based on two amplifications of genomic DNA with the polymerase chain reaction. In the first amplification, two short oligonucleotide primers produce a large number of fragments. One primer is based on a microsatellite sequence, whereas the second primer can have any sequence. In the second amplification, the length of the primers is increased in order to decrease the number of amplicons. This enables the selection of polymorphic fragments. Restriction digestion can be used to further increase the number of polymorphisms. Until now, we have demonstrated UVF in several different species. In addition, with the present study we have contributed to the linkage map of the rabbit by localizing 11 UVF markers on different linkage groups. Mendelian inheritance was shown in this linkage study through a backcross of two inbred rabbit strains. The power of the UVF technique is based on the selection for microsatellite variation in combination with the detection of single-nucleotide polymorphisms. UVF thus offers the possibility of increasing the clustering of markers and localizing genes in species for which sequence information is either not present or only scarcely present.

Chromosomal localization of genes involved in biosynthesis, metabolism or transport of cholesterol in the rat.

Several genes involved in biosynthesis, transport or metabolism of cholesterol have been localized on rat chromosomes by using a radiation hybrid (RH) panel. The genes, coding for squalene epoxidase (Sqle), mevalonate kinase (Mvk), and farnesyl diphosphate farnesyl transferase 1 (Fdft1) which are involved in cholesterol biosynthesis, have been mapped on chromosome 7, 12, and 15, respectively. The genes coding for phospholipid transfer protein (Pltp), sterol carrier protein-2 (Scp2), ATP binding cassette reporter A7 (Abca7), scavenger receptor class B, type 1 (Cd36l1), steroidogenic acute regulatory protein (Star), and lecithin:cholesterol acyl transferase (Lcat), which are involved in the transfer and/or metabolism of cholesterol, have been mapped on chromosome 3, 5, 7, 12, 16, and 19, respectively. Each of the genes Scp2, Sqle and Fdft1 maps close to a QTL for serum total cholesterol in rat, suggesting that these three genes might represent candidate genes for the previously mapped QTLs.

Fourteen chromosomal localizations and an update of the cytogenetic map of the rabbit.

In order to improve the informativeness of the cytogenetic map of the rabbit genome, fourteen markers were regionally mapped to individual chromosomes. The localizations comprise eleven gene loci (PRLR, GHR, HK1, ACE, TF, 18S+28S rDNA, CYP2C4, PMP2, TCRB, ALOX15 and MT1) and three microsatellite loci (Sat13, Sol33 and D1Utr6). Five of the genes contain known microsatellite sequences. To achieve these localizations, homologous and heterologous small insert clones, and clones from a rabbit Bacterial Artificial Chromosome (BAC) library were used as probes for fluorescence in situ hybridization experiments. Results indicate that especially BAC clones are a valuable tool for cytogenetic mapping. Some of the genes were selected for mapping on the basis of human- rabbit comparative painting data, to achieve localizations on gene-poor rabbit chromosomes. Our data are, in general, in agreement with the human-rabbit comparative painting data. By mapping microsatellite sequences that have also been used in linkage studies, links are provided between the genetic and physical maps of the rabbit genome. Linkage groups I, VI and XI could be assigned to chromosomes 1, 5 and 3 respectively. Moreover, in this paper we give an overview of the current status of the rabbit cytogenetic map. This map now comprises 62 physically mapped genes, which are scattered over all autosomes, except chromosome 2, and the X chromosome.

Application of AFLP markers for QTL mapping in the rabbit.

Two rabbit (Oryctolagus cuniculus) inbred strains (AX/JU and IIIVO/JU) have been used for genetic analysis of quantitative traits related to dietary cholesterol susceptibility. Application of the AFLP (amplified fragment length polymorphism) technique with 15 primer combinations revealed 226 polymorphisms between the 2 inbred strains. A total of 57 animals from a backcross progeny (IIIVO/JU x [IIIVO/JU x AX/JU]F1) were available for the genetic analysis. These backcross animals were fed a commercial pelleted diet fortified with 0.3% w/w cholesterol during a test period that lasted five weeks. A male genetic map could be constructed, consisting of 12 linkage groups and 103 AFLP markers. Linkage analysis between the cholesterol-related traits and marker loci revealed a significant LOD score for the relative weight of adrenal glands in males (LOD score = 3.83), whereas suggestive linkages were found for basal serum total cholesterol levels in females (LOD score = 2.69), for serum total cholesterol response (area under the curve) in males (LOD score = 2.21), and for hematocrit in males (LOD score = 3.24).

Use of animals in research: a science - society controversy? The European perspective.

The use of animals for research and testing purposes has decreased substantially during the last two decades but is now increasing again, mainly because of the increased use of transgenic animal models. Presently, 10-12 millions of vertebrate animals are used per year within EU Member States. Scientists who are using animals in research are frequently criticised by animal protection groups and blamed that they do not respect the integrity of an animal's life. The European Science Foundation (ESF) is recognising that legislation cannot be the only answer to those worries in society and that it is essential to take a stand and to clearly explain what the Foundation's position with regard to the use of animals is. In a recently published position paper, ESF has discussed its views and has adopted guidelines for the use of animals in research. The document explicitly states that laboratory animals have an intrinsic value, which must be respected. The consequences of recognising the intrinsic value have been elaborated in the position paper and include, among others, strong endorsement of the replacement, reduction, refinement principles. It is stated that, prior to the performance of an animal experiment, the protocol should be subjected to independent expert review including the weighing of the likely benefit versus the likely animal suffering. Also, the development and organisation of accredited courses on laboratory animal science, including information on animal alternatives, welfare and ethics are encouraged. The guidelines, as formulated in the position paper, can be seen as reinforcement of developments that have already been started in several countries, but have not always received full support from the scientific community. With this document, the association of 70 leading national science organisations in Europe has taken a position that, in several aspects, exceeds current European legislative regulations. For bridging the science - society controversy, it is essential to continue this initiative by promoting the implementation of the guidelines at all levels of the scientific community. This requires commitment and scientific leadership.

A rat linkage map based on BC x LEW intercross.

A genetic linkage map consisting of 258 polymorphic loci has been constructed on the basis of an F2 intercross between the BC/CpbU and LEW/OlaHsd inbred rat strains. When compared to previously published maps a discrepancy was found for rat chromosome 7. The map spans a sex-averaged genetic length of 1790 cM and has an average marker spacing of 7.7 cM. It was estimated that this genetic map is linked to about 90% of the DNA in the rat genome. Because LEW/OlaHsd and BC/CpbU strains differ for dietary cholesterol susceptibility and hepatic copper content, the map is considered to be a valuable tool for studying the genetic background of these complex traits.

Quantitative trait loci influencing hepatic copper in rats.

Significant differences in liver copper content have been observed between rat inbred strains. To define loci controlling this trait, the offspring (n = 190) from an (LEW/OlaHsd x BC/CpbU) F(2)-intercross was genetically analyzed. From each F(2) animal, liver copper content was determined and genomic DNA was screened with polymorphic DNA markers. We found a major quantitative trait locus (QTL) for liver copper content in females on chromosome 2 and in males on chromosome 10. Both QTLs accounted for approximately 20% of the genetic variance. In addition, suggestive linkage for liver copper content was found on rat chromosomes 1, 8, 10, 12, 14, and 19. The regions on these chromosomes contain genes that are responsible for 9.0-15.5% of the genetic variance of liver copper content.

Validation of a new system for the automatic registration of behaviour in mice and rats.

A newly developed behaviour registration system, Laboratory Animal Behaviour Observation, Registration and Analysis System (LABORAS) for the automatic registration of different behavioural elements of mice and rats was validated. The LABORAS sensor platform records vibrations evoked by animal movements and the LABORAS software translates these into the corresponding behaviours. Data obtained by using LABORAS were compared with data from conventional observation methods (observations of videotapes by human observers). The results indicate that LABORAS is a reliable system for the automated registration of eating, drinking, grooming, climbing, resting and locomotion of mice during a prolonged period of time. In rats, grooming, locomotion and resting also met the pre-defined validation criteria. The system can reduce observation labour and time considerably.