Animal Welfare Worldwide, the Opinion of Practicing Veterinarians.

The objective of this study was to investigate the animal welfare issues considered the most important by companion animal veterinarians worldwide. For this purpose, a global survey of several potential animal welfare issues was distributed via SurveyMonkey® in multiple languages. The distribution of survey responses differed by region. The main animal welfare concern reported worldwide was obesity, although there were differences across regions, possibly due to cultural and socioeconomic factors. Anthropomorphism (attributing human qualities or characteristics to an animal) was an issue in western countries but less so in Asia, Africa, and Oceania. There were significant differences between Asia and Europe, Africa, and Oceania in the importance and prevalence of convenience euthanasia. There were also age and sex differences in participant responses, with older veterinarians reporting fewer welfare problems than younger veterinarians, and female veterinarians reporting more welfare issues than their male counterparts.

An improved procedure for integrated behavioral z-scoring illustrated with modified Hole Board behavior of male inbred laboratory mice.

BACKGROUND: Guilloux et al. introduced: integrated behavioral z-scoring, a method for behavioral phenotyping of mice. Using this method multiple ethological variables can be combined to show an overall description of a certain behavioral dimension or motivational system. However, a problem may occur when the control group used for the calculation has a standard deviation of zero or when no control group is present to act as a reference group. NEW METHOD: In order to solve these problems, an improved procedure is suggested: taking the pooled data as reference. For this purpose a behavioral study with male mice from three inbred strains was carried out. The integrated behavioral z-scoring methodology was applied, thereby taking five different reference group options. The outcome regarding statistical significance and practical importance was compared. RESULTS: Significant effects and effect sizes were influenced by the choice of the reference group. In some cases it was impossible to use a certain population and condition, because one or more behavioral variables in question had a standard deviation of zero. Based on the improved method, male mice from the three inbred strains differed regarding activity and anxiety. COMPARISON WITH EXISTING METHOD: Taking the method described by Guilloux et al. as basis, the present procedure improved the generalizability to all types of experimental designs in animal behavioral research. CONCLUSIONS: To solve the aforementioned problems and to avoid getting the diagnosis of data manipulation, the pooled data (combining the data from all experimental groups in a study) as reference option is recommended.

Consomic mouse strain selection based on effect size measurement, statistical significance testing and integrated behavioral z-scoring: focus on anxiety-related behavior and locomotion.

BACKGROUND: Selecting chromosome substitution strains (CSSs, also called consomic strains/lines) used in the search for quantitative trait loci (QTLs) consistently requires the identification of the respective phenotypic trait of interest and is simply based on a significant difference between a consomic and host strain. However, statistical significance as represented by P values does not necessarily predicate practical importance. We therefore propose a method that pays attention to both the statistical significance and the actual size of the observed effect. The present paper extends on this approach and describes in more detail the use of effect size measures (Cohen's d, partial eta squared - η p (2) ) together with the P value as statistical selection parameters for the chromosomal assignment of QTLs influencing anxiety-related behavior and locomotion in laboratory mice. RESULTS: The effect size measures were based on integrated behavioral z-scoring and were calculated in three experiments: (A) a complete consomic male mouse panel with A/J as the donor strain and C57BL/6J as the host strain. This panel, including host and donor strains, was analyzed in the modified Hole Board (mHB). The consomic line with chromosome 19 from A/J (CSS-19A) was selected since it showed increased anxiety-related behavior, but similar locomotion compared to its host. (B) Following experiment A, female CSS-19A mice were compared with their C57BL/6J counterparts; however no significant differences and effect sizes close to zero were found. (C) A different consomic mouse strain (CSS-19PWD), with chromosome 19 from PWD/PhJ transferred on the genetic background of C57BL/6J, was compared with its host strain. Here, in contrast with CSS-19A, there was a decreased overall anxiety in CSS-19PWD compared to C57BL/6J males, but not locomotion. CONCLUSIONS: This new method shows an improved way to identify CSSs for QTL analysis for anxiety-related behavior using a combination of statistical significance testing and effect sizes. In addition, an intercross between CSS-19A and CSS-19PWD may be of interest for future studies on the genetic background of anxiety-related behavior.

Substrain and light regime effects on integrated anxiety-related behavioral z-scores in male C57BL/6 mice-Hypomagnesaemia has only a small effect on avoidance behavior.

Magnesium (Mg) has been described to possess an anxiolytic function, but a number of studies present inconsistent results on this matter. In this study the effect of Mg deficiency on anxiety-related behavior, brain and blood plasma Mg in young adult male C57BL/6JOlaHsd and C57BL/6NCrl mice was studied. The animals were put on a control or Mg deficient diet from day 0 and significant hypomagnesaemia was evident from day 12 onwards in the test animals. Housing and test conditions were under either conventional light regime (white light behavioral test conditions) or reverse light regime (red light behavioral test conditions). The animals were tested in three tests for unconditioned anxiety: the modified Hole Board (day 14), the light-dark test (day 21) and the elevated plus maze (day 28). Overall integrated behavioral z-scores were calculated over these three behavioral tests. Mg showed a structure dependent distribution at the level of the brain, that differed between C57BL/6 substrain and light regime (conventional versus reverse), respectively. Likewise, total brain Mg did differ between substrain and light regime, but was not affected by the diet. Animals on the Mg deficient diet housed under conventional light regime had a higher final (day 28) blood plasma corticosterone level as compared to controls. Animals housed under reverse light regime exhibited no diet effect of plasma corticosterone levels. The significant hypomagnesaemia at blood plasma level resulted in an effect of Mg deficiency on avoidance, but not overall anxiety-related behavior. Significant differences regarding avoidance behavior were found between the two substrains and light regimes, respectively.

A candidate syntenic genetic locus is associated with voluntary exercise levels in mice and humans.

Individual levels of physical activity, and especially of voluntary physical exercise, highly contribute to the susceptibility for developing metabolic, cardiovascular diseases, and potentially to psychiatric disorders. Here, we applied a cross-species approach to explore a candidate genetic region for voluntary exercise levels. First, a panel of mouse chromosome substitution strains was used to map a genomic region on mouse chromosome 2 that contributes to voluntary wheel running levels - a behavioral readout considered a model of voluntary exercise in humans. Subsequently, we tested the syntenic region (HSA20: 51,212,545-55,212,986) in a human sample (Saint Thomas Twin Register; n=3038) and found a significant association between voluntary exercise levels (categorized into excessive and non-excessive exercise) and an intergenic SNP rs459465 (adjusted P-value of 0.001). Taking under consideration the methodological challenges embedded in this translational approach in the research of complex phenotypes, we wanted to further test the validity of this finding. Therefore, we repeated the analysis in an independent human population (ALSPAC data set; n=2557). We found a significant association of excessive exercise with two SNPs in the same genomic region (rs6022999, adjusted P-value of P=0.011 and rs6092090, adjusted P-value of 0.012). We explored the locus for possible candidate genes by means of literature search and bioinformatics analysis of gene function and of trans-regulatory elements. We propose three potential human candidate genes for voluntary physical exercise levels (MC3R, CYP24A1, and GRM8). To conclude, the identified genetic variance in the human locus 20q13.2 may affect voluntary exercise levels.

Cross-species genetics converge to TLL2 for mouse avoidance behavior and human bipolar disorder.

Interspecies genetic analysis of neurobehavioral traits is critical for identifying neurobiological mechanisms underlying psychiatric disorders, and for developing models for translational research. Recently, after screening a chromosome substitution strain panel in an automated home cage environment, chromosomes 15 and 19 were identified in female mice for carrying genetic loci that contribute to increased avoidance behavior (sheltering preference). Furthermore, we showed that the quantitative trait locus (QTL) for baseline avoidance behavior on chromosome 15 is homologous with a human linkage region for bipolar disorder (8q24). Similarly, we now performed comparative analysis on the QTL for avoidance behavior found on chromosome 19 and correspondingly revealed an overlap of the mouse interval and human homologous region 10q23-24, which has been previously linked to bipolar disorders. By means of a comparative genetic strategy within the human homologous region, we describe an association for TLL2 with bipolar disorder using the genome-wide association study (GWAS) data set generated by the Wellcome Trust Case Control Consortium (WTCCC). On the basis of genetic homology and mood stabilizer sensitivity, our data indicate the intriguing possibility that mouse home cage avoidance behavior may translate to a common biochemical mechanisms underlying bipolar disorder susceptibility. These findings pave new roads for the identification of the molecular mechanisms and novel treatment possibilities for this psychiatric disorder, as well as for the validity of translational research of associated psychiatric endophenotypes.

Genetic dissection of granulomatous enterocolitis and arthritis in the intramural peptidoglycan-polysaccharide-treated rat model of IBD.

BACKGROUND: Inflammatory arthropathies are common extraintestinal manifestations of inflammatory bowel diseases (IBD). As genetic susceptibility plays an important role in the etiology of IBD, we questioned how granulomatous enterocolitis and arthritis are genetically controlled in an experimental animal model displaying both conditions. METHODS: Chronic intestinal and systemic inflammation was induced by intramural injection of peptidoglycan-polysaccharide (PG-PS) polymers in the ileocecal region of female F2 progeny derived from susceptible LEW and resistant F344 rats. Animals were followed for 24 days after injection and phenotyped by evaluating gross gut lesions, liver weight and granulomas, hematocrit, white blood cell count, and change in rear ankle joint diameters. Coinheritance of the phenotypic parameters with polymorphic DNA markers was analyzed by genome-wide quantitative trait locus (QTL) analysis. RESULTS: Linkage analysis revealed significant QTLs for enterocolitis and/or related phenotypes (liver granulomas, white blood cell count) on chromosomes 8 and 17. The QTL on chromosome 8 also showed suggestive linkage to arthritis. Significant QTLs for arthritis were detected on chromosomes 10, 13, 15, and 17. Analyses of the modes of inheritance showed arthritogenic contributions by both parental genomes. In addition, several other loci with suggestive evidence for linkage to 1 or several phenotypes were found. CONCLUSIONS: Susceptibility to PG-PS-induced chronic intestinal and systemic inflammation in rats is under complex multigenic control in which the genetic loci regulating arthritis are largely different from those controlling enterocolitis. Possible candidate genes within these QTL (including Tnfrsf11a/RANK, Gpc5, Il2ra, and Nfrkb) are also implicated in the respective human diseases.

High-resolution genetic mapping of mammalian motor activity levels in mice.

The generation of motor activity levels is under tight neural control to execute essential behaviors, such as movement toward food or for social interaction. To identify novel neurobiological mechanisms underlying motor activity levels, we studied a panel of chromosome substitution (CS) strains derived from mice with high (C57BL/6J strain) or low motor activity levels (A/J strain) using automated home cage behavioral registration. In this study, we genetically mapped the expression of baseline motor activity levels (horizontal distance moved) to mouse chromosome 1. Further genetic mapping of this trait revealed an 8.3-Mb quantitative trait locus (QTL) interval. This locus is distinct from the QTL interval for open-field anxiety-related motor behavior on this chromosome. By data mining, an existing phenotypic and genotypic data set of 2445 genetically heterogeneous mice (http://gscan.well.ox.ac.uk/), we confirmed linkage to the peak marker at 79 970 253 bp and refined the QTL to a 312-kb interval containing a single gene (A830043J08Rik). Sequence analysis showed a nucleotide deletion in the 3' untranslated region of the Riken gene. Genome-wide microarray gene expression profiling in brains of discordant F(2) individuals from CS strain 1 showed a significant upregulation of Epha4 in low-active F(2) individuals. Inclusion of a genetic marker for Epha4 confirmed that this gene is located outside of the QTL interval. Both Epha4 and A830043J08Rik are expressed in brain motor circuits, and similar to Epha4 mutants, we found linkage between reduced motor neurons number and A/J chromosome 1. Our findings provide a novel QTL and a potential downstream target underlying motor circuitry development and the expression of physical activity levels.

Evidence for epigenetic interactions for loci on mouse chromosome 1 regulating open field activity.

The expression of motor activity levels in response to novel situations is under complex genetic and environmental control. Several genetic loci have been implicated in the regulation of this behavioral phenotype, but their relationship to epigenetic and epistatic interactions is relatively unknown. Here, we report on a quantitative trait locus (QTL) on mouse chromosome 1 for novelty-induced motor activity in the open field, using chromosome substitution strains derived from a high active host strain (C57BL/6J) and a low active donor strain (A/J). The QTL for open field (horizontal distance moved) peaked at the location of Kcnj9, however, QTL detection was initially masked by an interplay of both grandparent genetic origin and genetic co-factors influencing behavior on chromosome 1. Our findings indicate that epigenetic interactions can play an important role in the identification of behavioral QTLs and must be taken into consideration when applying behavioral genetic strategies.

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.

Rat strains differ in antibody response to natural Haemophilus species infection.

Antibody response to Haemophilus species in rat strains was monitored by enzyme-linked immunosorbent assay (ELISA) using antigens of two Haemophilus strains and a Pasteurella pneumotropica strain. Five rat strains from a breeding colony naturally infected by Haemophilus were significantly different in ELISA antibody activity and in the number of seropositive animals. BN and RP rats were (relatively) high and low responders, respectively and BUF, LEW and WAG rats were intermediate. In a second study, five rat strains were exposed to Haemophilus-infected rats, and, after six weeks, were also significantly different in ELISA antibody activity and in numbers of seropositive animals. Here, BN and LEW rats were (relatively) high and low responders, respectively, and BD IX, F344 and WKY rats were intermediate.

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.

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).

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.

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.