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The first record of captive-bred red foxes (Vulpes vulpes) dates to 1896 when a breeding enterprise emerged in the provinces of Atlantic Canada. Because its domestication happened during recent history, the red fox offers a unique opportunity to examine the genetic diversity of an emerging domesticated species in the context of documented historical and economic influences. In particular, the historical record suggests that North American and Eurasian farm-bred populations likely experienced different demographic trajectories. Here, we focus on the likely impacts of founder effects and genetic drift given historical trends in fox farming on North American and Eurasian farms. A total of 15 mitochondrial haplotypes were identified in 369 foxes from 10 farm populations that we genotyped (n = 161) or that were previously published. All haplotypes are endemic to North America. Although most haplotypes were consistent with eastern Canadian ancestry, a small number of foxes carried haplotypes typically found in Alaska and other regions of western North America. The presence of these haplotypes supports historical reports of wild foxes outside of Atlantic Canada being introduced into the breeding stock. These putative Alaskan and Western haplotypes were more frequently identified in Eurasian farms compared to North American farms, consistent with historical documentation suggesting that Eurasian economic and breeding practices were likely to maintain low-frequency haplotypes more effectively than in North America. Contextualizing inter- vs. intra-farm genetic diversity alongside the historical record is critical to understanding the origins of this emerging domesticate and the relationships between wild and farm-bred fox populations., (© The Author(s) 2024. Published by Oxford University Press on behalf of The American Genetic Association. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Although the silver fox (Vulpes vulpes) has been largely overlooked by neuroscientists, it has the potential to serve as a powerful model for the investigation of brain-behavior relationships. The silver fox is a melanistic variant of the red fox. Within this species, the long-running Russian farm-fox experiment has resulted in different strains bred to show divergent behavior. Strains bred for tameness, aggression, or without selection on behavior present an excellent opportunity to investigate neuroanatomical changes underlying behavioral characteristics. Here, we present a histological and MRI neuroanatomical reference of a fox from the conventional strain, which is bred without behavioral selection. This can provide an anatomical basis for future studies of the brains of foxes from this particular experiment, as well as contribute to an understanding of fox brains in general. In addition, this can serve as a resource for comparative neuroscience and investigations into neuroanatomical variation among the family Canidae, the order Carnivora, and mammals more broadly., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Since 1959, the Russian Farm-Fox study has bred foxes to be either tame or, more recently, aggressive, and scientists have used them to gain insight into the brain structures associated with these behavioral features. In mice, hippocampal area CA2 has emerged as one of the essential regulators of social aggression, and so to eventually determine whether we could identify differences in CA2 between tame and aggressive foxes, we first sought to identify CA2 in foxes (Vulpes vulpes). As no clearly defined area of CA2 has been described in species such as cats, dogs, or pigs, it was not at all clear whether CA2 could be identified in foxes. In this study, we cut sections of temporal lobes from male and female red foxes, perpendicular to the long axis of the hippocampus, and stained them with markers of CA2 pyramidal cells commonly used in tissue from rats and mice. We observed that antibodies against Purkinje cell protein 4 best stained the pyramidal cells in the area spanning the end of the mossy fibers and the beginning of the pyramidal cells lacking mossy fibers, resembling the pattern seen in rats and mice. Our findings indicate that foxes do have a "molecularly defined" CA2, and further, they suggest that other carnivores like dogs and cats might as well. With this being the case, these foxes could be useful in future studies looking at CA2 as it relates to aggression., (© 2023 The Authors. Hippocampus published by Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

The Russian farm-fox experiment is an unusually long-running and well-controlled study designed to replicate wolf-to-dog domestication. As such, it offers an unprecedented window onto the neural mechanisms governing the evolution of behavior. Here we report evolved changes to gray matter morphology resulting from selection for tameness versus aggressive responses toward humans in a sample of 30 male fox brains. Contrasting with standing ideas on the effects of domestication on brain size, tame foxes did not show reduced brain volume. Rather, gray matter volume in both the tame and aggressive strains was increased relative to conventional farm foxes bred without deliberate selection on behavior. Furthermore, tame- and aggressive-enlarged regions overlapped substantially, including portions of motor, somatosensory, and prefrontal cortex, amygdala, hippocampus, and cerebellum. We also observed differential morphologic covariation across distributed gray matter networks. In one prefrontal-cerebellum network, this covariation differentiated the three populations along the tame-aggressive behavioral axis. Surprisingly, a prefrontal-hypothalamic network differentiated the tame and aggressive foxes together from the conventional strain. These findings indicate that selection for opposite behaviors can influence brain morphology in a similar way. SIGNIFICANCE STATEMENT Domestication represents one of the largest and most rapid evolutionary shifts of life on earth. However, its neural correlates are largely unknown. Here we report the neuroanatomical consequences of selective breeding for tameness or aggression in the seminal Russian farm-fox experiment. Compared with a population of conventional farm-bred control foxes, tame foxes show neuroanatomical changes in the PFC and hypothalamus, paralleling wolf-to-dog shifts. Surprisingly, though, aggressive foxes also show similar changes. Moreover, both strains show increased gray matter volume relative to controls. These results indicate that similar brain adaptations can result from selection for opposite behavior, that existing ideas of brain changes in domestication may need revision, and that significant neuroanatomical change can evolve very quickly, within the span of <100 generations., (Copyright © 2021 the authors.)

The underlying neurological events accompanying dog domestication remain elusive. To reconstruct the domestication process in an experimental setting, silver foxes (Vulpes vulpes) have been deliberately bred for tame vs aggressive behaviors for more than 50 generations at the Institute for Cytology and Genetics in Novosibirsk, Russia. The hypothalamus is an essential part of the hypothalamic-pituitary-adrenal axis and regulates the fight-or-flight response, and thus, we hypothesized that selective breeding for tameness/aggressiveness has shaped the hypothalamic transcriptomic profile. RNA-seq analysis identified 70 differentially expressed genes (DEGs). Seven of these genes, DKKL1, FBLN7, NPL, PRIMPOL, PTGRN, SHCBP1L and SKIV2L, showed the same direction expression differences in the hypothalamus, basal forebrain and prefrontal cortex. The genes differentially expressed across the three tissues are involved in cell division, differentiation, adhesion and carbohydrate processing, suggesting an association of these processes with selective breeding. Additionally, 159 transcripts from the hypothalamus demonstrated differences in the abundance of alternative spliced forms between the tame and aggressive foxes. Weighted gene coexpression network analyses also suggested that gene modules in hypothalamus were significantly associated with tame vs aggressive behavior. Pathways associated with these modules include signal transduction, interleukin signaling, cytokine-cytokine receptor interaction and peptide ligand-binding receptors (eg, G-protein coupled receptor [GPCR] ligand binding). Current studies show the selection for tameness vs aggressiveness in foxes is associated with unique hypothalamic gene profiles partly shared with other brain regions and highlight DEGs involved in biological processes such as development, differentiation and immunological responses. The role of these processes in fox and dog domestication remains to be determined., (© 2019 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.)

While the number of mammalian genome assemblies has proliferated, Y-chromosome assemblies have lagged behind. This discrepancy is caused by biological features of the Y-chromosome, such as its high repeat content, that present challenges to assembly with short-read, next-generation sequencing technologies. Partial Y-chromosome assemblies have been developed for the cat ( Felis catus ), dog ( Canis lupus familiaris ), and grey wolf ( Canis lupus lupus ), providing the opportunity to examine the red fox ( Vulpes vulpes ) Y-chromosome in the context of closely related species. Here we present a data-driven approach to identifying Y-chromosome sequence among the scaffolds that comprise the short-read assembled red fox genome. First, scaffolds containing genes found on the Y-chromosomes of cats, dogs, and wolves were identified. Next, analysis of the resequenced genomes of 15 male and 15 female foxes revealed scaffolds containing male-specific k -mers and patterns of inter-sex copy number variation consistent with the heterogametic chromosome. Analyzing variation across these two metrics revealed 171 scaffolds containing 3.37 Mbp of putative Y-chromosome sequence. The gene content of these scaffolds is consistent overall with that of the Y-chromosome in other carnivore species, though the red fox Y-chromosome carries more copies of BCORY2 and UBE1Y than has been reported in related species and fewer copies of SRY than in other canids. The assignment of these scaffolds to the Y-chromosome serves to further characterize the content of the red fox draft genome while providing resources for future analyses of canid Y-chromosome evolution., Competing Interests: The authors declare no conflict of interest.

Animal domestication efforts have led to a shared spectrum of striking behavioral and morphological changes. To recapitulate this process, silver foxes have been selectively bred for tame and aggressive behaviors for more than 50 generations at the Institute for Cytology and Genetics in Novosibirsk, Russia. To understand the genetic basis and molecular mechanisms underlying the phenotypic changes, we profiled gene expression levels and coding SNP allele frequencies in two brain tissue specimens from 12 aggressive foxes and 12 tame foxes. Expression analysis revealed 146 genes in the prefrontal cortex and 33 genes in the basal forebrain that were differentially expressed, with a 5% false discovery rate (FDR). These candidates include genes in key pathways known to be critical to neurologic processing, including the serotonin and glutamate receptor pathways. In addition, 295 of the 31,000 exonic SNPs show significant allele frequency differences between the tame and aggressive populations (1% FDR), including genes with a role in neural crest cell fate determination., Competing Interests: The authors declare no conflict of interest.

Strains of red fox (Vulpes vulpes) with markedly different behavioural phenotypes have been developed in the famous long-term selective breeding programme known as the Russian farm-fox experiment. Here we sequenced and assembled the red fox genome and re-sequenced a subset of foxes from the tame, aggressive and conventional farm-bred populations to identify genomic regions associated with the response to selection for behaviour. Analysis of the re-sequenced genomes identified 103 regions with either significantly decreased heterozygosity in one of the three populations or increased divergence between the populations. A strong positional candidate gene for tame behaviour was highlighted: SorCS1, which encodes the main trafficking protein for AMPA glutamate receptors and neurexins and suggests a role for synaptic plasticity in fox domestication. Other regions identified as likely to have been under selection in foxes include genes implicated in human neurological disorders, mouse behaviour and dog domestication. The fox represents a powerful model for the genetic analysis of affiliative and aggressive behaviours that can benefit genetic studies of behaviour in dogs and other mammals, including humans.

In the version of this Article originally published, there were some errors in the affiliations: Stephen J. O'Brien's affiliations were incorrectly listed as 8,9; they should have been 7,9. Affiliation 3 was incorrectly named the Institute of Cytology and Genetics of the Russian Academy of Sciences; it should have read Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences. Affiliation 4 was incorrectly named the Institute of Molecular and Cell Biology of the Russian Academy of Sciences; it should have read Institute of Molecular and Cellular Biology of the Siberian Branch of the Russian Academy of Sciences. These have now been corrected.

The genome of a red fox ( Vulpes vulpes ) was recently sequenced and assembled using next-generation sequencing (NGS). The assembly is of high quality, with 94X coverage and a scaffold N50 of 11.8 Mbp, but is split into 676,878 scaffolds, some of which are likely to contain assembly errors. Fragmentation and misassembly hinder accurate gene prediction and downstream analysis such as the identification of loci under selection. Therefore, assembly of the genome into chromosome-scale fragments was an important step towards developing this genomic model. Scaffolds from the assembly were aligned to the dog reference genome and compared to the alignment of an outgroup genome (cat) against the dog to identify syntenic sequences among species. The program Reference-Assisted Chromosome Assembly (RACA) then integrated the comparative alignment with the mapping of the raw sequencing reads generated during assembly against the fox scaffolds. The 128 sequence fragments RACA assembled were compared to the fox meiotic linkage map to guide the construction of 40 chromosomal fragments. This computational approach to assembly was facilitated by prior research in comparative mammalian genomics, and the continued improvement of the red fox genome can in turn offer insight into canid and carnivore chromosome evolution. This assembly is also necessary for advancing genetic research in foxes and other canids.

Domesticated species exhibit a suite of behavioral, endocrinological, and morphological changes referred to as "domestication syndrome." These changes may include a reduction in reactivity of the hypothalamic-pituitary-adrenal (HPA) axis and specifically reduced adrenocorticotropic hormone release from the anterior pituitary. To investigate the biological mechanisms targeted during domestication, we investigated gene expression in the pituitaries of experimentally domesticated foxes ( Vulpes vulpes ). RNA was sequenced from the anterior pituitary of six foxes selectively bred for tameness ("tame foxes") and six foxes selectively bred for aggression ("aggressive foxes"). Expression, splicing, and network differences identified between the two lines indicated the importance of genes related to regulation of exocytosis, specifically mediated by cAMP, organization of pseudopodia, and cell motility. These findings provide new insights into biological mechanisms that may have been targeted when these lines of foxes were selected for behavior and suggest new directions for research into HPA axis regulation and the biological underpinnings of domestication., (Copyright © 2018 Hekman et al.)

The de novo assembly of the red fox (Vulpes vulpes) genome has facilitated the development of genomic tools for the species. Efforts to identify the population history of red foxes in North America have previously been limited by a lack of information about the red fox Y-chromosome sequence. However, a megabase of red fox Y-chromosome sequence was recently identified over 2 scaffolds in the reference genome. Here, these scaffolds were scanned for repeated motifs, revealing 194 likely microsatellites. Twenty-three of these loci were selected for primer development and, after testing, produced a panel of 11 novel markers that were analyzed alongside 2 markers previously developed for the red fox from dog Y-chromosome sequence. The markers were genotyped in 76 male red foxes from 4 populations: 7 foxes from Newfoundland (eastern Canada), 12 from Maryland (eastern United States), and 9 from the island of Great Britain, as well as 48 foxes of known North American origin maintained on an experimental farm in Novosibirsk, Russia. The full marker panel revealed 22 haplotypes among these red foxes, whereas the 2 previously known markers alone would have identified only 10 haplotypes. The haplotypes from the 4 populations clustered primarily by continent, but unidirectional gene flow from Great Britain and farm populations may influence haplotype diversity in the Maryland population. The development of new markers has increased the resolution at which red fox Y-chromosome diversity can be analyzed and provides insight into the contribution of males to red fox population diversity and patterns of phylogeography., (© The American Genetic Association 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Individuals involved in a social interaction exhibit different behavioral traits that, in combination, form the individual's behavioral responses. Selectively bred strains of silver foxes (Vulpes vulpes) demonstrate markedly different behaviors in their response to humans. To identify the genetic basis of these behavioral differences we constructed a large F 2 population including 537 individuals by cross-breeding tame and aggressive fox strains. 98 fox behavioral traits were recorded during social interaction with a human experimenter in a standard four-step test. Patterns of fox behaviors during the test were evaluated using principal component (PC) analysis. Genetic mapping identified eight unique significant and suggestive QTL. Mapping results for the PC phenotypes from different test steps showed little overlap suggesting that different QTL are involved in regulation of behaviors exhibited in different behavioral contexts. Many individual behavioral traits mapped to the same genomic regions as PC phenotypes. This provides additional information about specific behaviors regulated by these loci. Further, three pairs of epistatic loci were also identified for PC phenotypes suggesting more complex genetic architecture of the behavioral differences between the two strains than what has previously been observed.

Work on laboratory and wild rodents suggests that domestication may impact on the extent of adult hippocampal neurogenesis and its responsiveness to regulatory factors. There is, however, no model of laboratory rodents and their nondomesticated conspecifics that would allow a controlled comparison of the effect of domestication. Here, we present a controlled within-species comparison of adult hippocampal neurogenesis in farm-bred foxes (Vulpes vulpes) that differ in their genetically determined degree of tameness. Quantitative comparisons of cell proliferation (Ki67) and differentiating cells of neuronal lineage (doublecortin, DCX) in the hippocampus of foxes were performed as a proxy for neurogenesis. Higher neurogenesis was observed in tameness-selected foxes, notably in an extended subgranular zone of the middle and temporal compartments of the hippocampus. Increased neurogenesis is negatively associated with aggressive behavior. Across all animals, strong septotemporal gradients were found, with higher numbers of proliferating cells and young neurons relative to resident granule cells in the temporal than in the septal hippocampus. The opposite gradient was found for the ratio of DCX/Ki67- positive cells. When tameness-selected and unselected foxes are compared with rodents and primates, proliferation is similar, while the number of young neurons is higher. The difference may be mediated by an extended period of differentiation or higher rate of survival. On the background of this species-specific neurogenic pattern, selection of foxes for a single behavioral trait key to domestication, i.e., genetic tameness, is accompanied by global and region-specific increases in neurogenesis., (© 2015 Wiley Periodicals, Inc.)

The silver fox (Vulpes vulpes) offers a novel model for studying the genetics of social behavior and animal domestication. Selection of foxes, separately, for tame and for aggressive behavior has yielded two strains with markedly different, genetically determined, behavioral phenotypes. Tame strain foxes are eager to establish human contact while foxes from the aggressive strain are aggressive and difficult to handle. These strains have been maintained as separate outbred lines for over 40 generations but their genetic structure has not been previously investigated. We applied a genotyping-by-sequencing (GBS) approach to provide insights into the genetic composition of these fox populations. Sequence analysis of EcoT22I genomic libraries of tame and aggressive foxes identified 48,294 high quality SNPs. Population structure analysis revealed genetic divergence between the two strains and more diversity in the aggressive strain than in the tame one. Significant differences in allele frequency between the strains were identified for 68 SNPs. Three of these SNPs were located on fox chromosome 14 within an interval of a previously identified behavioral QTL, further supporting the importance of this region for behavior. The GBS SNP data confirmed that significant genetic diversity has been preserved in both fox populations despite many years of selective breeding. Analysis of SNP allele frequencies in the two populations identified several regions of genetic divergence between the tame and aggressive foxes, some of which may represent targets of selection for behavior. The GBS protocol used in this study significantly expanded genomic resources for the fox, and can be adapted for SNP discovery and genotyping in other canid species.

The red fox (Vulpes vulpes) demonstrates a variety of coat colors including platinum, a common phenotype maintained in farm-bred fox populations. Foxes heterozygous for the platinum allele have a light silver coat and extensive white spotting, whereas homozygosity is embryonic lethal. Two KIT transcripts were identified in skin cDNA from platinum foxes. The long transcript was identical to the KIT transcript of silver foxes, whereas the short transcript, which lacks exon 17, was specific to platinum. The KIT gene has several copies in the fox genome: an autosomal copy on chromosome 2 and additional copies on the B chromosomes. To identify the platinum-specific KIT sequence, the genomes of one platinum and one silver fox were sequenced. A single nucleotide polymorphism (SNP) was identified at the first nucleotide of KIT intron 17 in the platinum fox. In platinum foxes, the A allele of the SNP disrupts the donor splice site and causes exon 17, which is part of a segment that encodes a conserved tyrosine kinase domain, to be skipped. Complete cosegregation of the A allele with the platinum phenotype was confirmed by linkage mapping (LOD 25.59). All genotyped farm-bred platinum foxes from Russia and the US were heterozygous for the SNP (A/G), whereas foxes with different coat colors were homozygous for the G allele. Identification of the platinum mutation suggests that other fox white-spotting phenotypes, which are allelic to platinum, would also be caused by mutations in the KIT gene., (© 2015 Stichting International Foundation for Animal Genetics.)

Foxes long time selected for tame and aggressive behavior were compared on ability to focus attention on the object of food reinforcement. Attenuation of this behavior and rate of training for motor skills also has been examined. Maximal duration of eye focusing was significantly higher in aggressive foxes, in comparison with tame ones. Our experiments allowed divide the group of tame foxes into two subgroups "calm" and "emotional", on the base of emotionality and motor activity during tests. Features of behavior of these two subgroups steadily differed in all tests. "Calm" tame foxes at the extinction test continued the trained skill longer than "emotional" and aggressive ones. Tame foxes were more successful than aggressive in the training for motor skills. The possible reasons of the data obtained are discussed.

This study presents a first direct comparison of vocal type, call rate and time spent vocalizing among Unselected, Tame and Aggressive strains of silver fox ( Vulpes vulpes ) in three modes of human approach (Provoking, Approach-Retreat, and Static). Also, it provides a first comparison of male and female vocal output in the Provoking test. Vocal types were found strain-specific irrespective of the fox sex or the test. Males had higher call rates and spent shorter times vocalizing than females. These results support the evidence of genetic-based emotional states, triggering vocal behavior in silver fox strains, and suggest sex dimorphism in vocal activity toward humans.

The silver fox provides a rich resource for investigating the genetics of behavior, with strains developed by intensely selective breeding that display markedly different behavioral phenotypes. Until recently, however, the tools for conducting molecular genetic investigations in this species were very limited. In this review, the history of development of this resource and the tools to exploit it are described. Although the focus is on the genetics of domestication in the silver fox, there is a broader context. In particular, one expectation of the silver fox research is that it will be synergistic with studies in other species, including humans, to yield a more comprehensive understanding of the molecular mechanisms and evolution of a wider range of social cognitive behaviors.

Background: Two strains of the silver fox (Vulpes vulpes), with markedly different behavioral phenotypes, have been developed by long-term selection for behavior. Foxes from the tame strain exhibit friendly behavior towards humans, paralleling the sociability of canine puppies, whereas foxes from the aggressive strain are defensive and exhibit aggression to humans. To understand the genetic differences underlying these behavioral phenotypes fox-specific genomic resources are needed., Results: cDNA from mRNA from pre-frontal cortex of a tame and an aggressive fox was sequenced using the Roche 454 FLX Titanium platform (> 2.5 million reads & 0.9 Gbase of tame fox sequence; >3.3 million reads & 1.2 Gbase of aggressive fox sequence). Over 80% of the fox reads were assembled into contigs. Mapping fox reads against the fox transcriptome assembly and the dog genome identified over 30,000 high confidence fox-specific SNPs. Fox transcripts for approximately 14,000 genes were identified using SwissProt and the dog RefSeq databases. An at least 2-fold expression difference between the two samples (p < 0.05) was observed for 335 genes, fewer than 3% of the total number of genes identified in the fox transcriptome., Conclusions: Transcriptome sequencing significantly expanded genomic resources available for the fox, a species without a sequenced genome. In a very cost efficient manner this yielded a large number of fox-specific SNP markers for genetic studies and provided significant insights into the gene expression profile of the fox pre-frontal cortex; expression differences between the two fox samples; and a catalogue of potentially important gene-specific sequence variants. This result demonstrates the utility of this approach for developing genomic resources in species with limited genomic information.

During the second part of the twentieth century, Belyaev selected tame and aggressive foxes (Vulpes vulpes), in an effort known as the "farm-fox experiment", to recapitulate the process of animal domestication. Using these tame and aggressive foxes as founders of segregant backcross and intercross populations we have employed interval mapping to identify a locus for tame behavior on fox chromosome VVU12. This locus is orthologous to, and therefore validates, a genomic region recently implicated in canine domestication. The tame versus aggressive behavioral phenotype was characterized as the first principal component (PC) of a PC matrix made up of many distinct behavioral traits (e.g. wags tail; comes to the front of the cage; allows head to be touched; holds observer's hand with its mouth; etc.). Mean values of this PC for F1, backcross and intercross populations defined a linear gradient of heritable behavior ranging from tame to aggressive. The second PC did not follow such a gradient, but also mapped to VVU12, and distinguished between active and passive behaviors. These data suggest that (1) there are at least two VVU12 loci associated with behavior; (2) expression of these loci is dependent on interactions with other parts of the genome (the genome context) and therefore varies from one crossbred population to another depending on the individual parents that participated in the cross.

The foxes at Novosibirsk, Russia, are the only population of domesticated foxes in the world. These domesticated foxes originated from farm-bred silver foxes (Vulpes vulpes), whose genetic source is unknown. In this study we examined the origin of the domesticated strain of foxes and two other farm-bred fox populations (aggressive and unselected) maintained in Novosibirsk. To identify the phylogenetic origin of these populations we sequenced two regions of mtDNA, cytochrome b and D-loop, from 24 Novosibirsk foxes (8 foxes from each population) and compared them with corresponding sequences of native red foxes from Europe, Asia, Alaska and Western Canada, Eastern Canada, and the Western Mountains of the USA. We identified seven cytochrome b - D-loop haplotypes in Novosibirsk populations, four of which were previously observed in Eastern North America. The three remaining haplotypes differed by one or two base change from the most common haplotype in Eastern Canada. Φ(ST) analysis showed significant differentiation between Novosibirsk populations and red fox populations from all geographic regions except Eastern Canada. No haplotypes of Eurasian origin were identified in the Novosibirsk populations. These results are consistent with historical records indicating that the original breeding stock of farm-bred foxes originated from Prince Edward Island, Canada. Mitochondrial DNA data together with historical records indicate two stages in the selection of domesticated foxes: the first includes captive breeding for ~50 years with unconscious selection for behaviour; the second corresponds to over 50 further years of intensive selection for tame behaviour.

Domestication affects behavioral and vocal responses, involved in communication with humans; in particular, those that attract human attention. In this study, we found that silver foxes of Tame strain, experimentally domesticated for a few tenses of generation, displayed bursts of vocal activity during the first minute after appearance of an unfamiliar human, that faded quickly during the remaining time of the test, when the experimenter stayed passively before the cage. Distinctively, foxes of Aggressive strain, artificially selected for tenses of generation for aggressive behavior toward humans, and the control group of Unselected for behavior silver foxes kept steady levels of vocal activity for the duration of the tests. We found also that Aggressive foxes vocalized for a larger proportion of time than Unselected foxes for all 5 min of the test. We discuss the obtained data in relation to proposal effects of domestication on mechanisms directed to involving people into human-animal interactions and structural similarity between human laughter and vocalization of Tame foxes., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Directional asymmetry (DA) is a characteristic of most vertebrates, most strikingly exhibited by the placement of various organs (heart, lungs, liver, etc.) but also noted in small differences in the metrics of skeletal structures such as the pelvis of certain fish or sauropsids. We have analyzed DA in the skeleton of the fox (V. vulpes), using ∼1,000 radiographs of foxes from populations used in the genetic analysis of behavior and morphology. Careful measurements from this robust data base demonstrate that: 1) DA occurs in the limb bones, the ileum, and ischium and in the mandible; 2) regardless of the direction of the length asymmetry vector of a particular skeletal unit, the vectorial direction of length is always opposite to that of width; 3) with the exception of the humerus and radius, there is no correlation or inverse correlation between vectorial amplitudes or magnitudes of bone asymmetries. 4) Postnatal measurements on foxes demonstrate that the asymmetry increases after birth and continues to change (increasing or decreasing) during postnatal growth. 5) A behavior test for preferential use of a specific forelimb exhibited fluctuating asymmetry but not DA. None of the skeletal asymmetries were significantly correlated with a preferential use of a specific forelimb. We suggest that for the majority of fox skeletal parameters, growth on the right and left side of the fox are differentially biased resulting in fixed differences between the two sides in either the rate of growth or the length of the period during which growth occurs. Random effects around these fixed differences perturb the magnitude of the effects such that the magnitudes of length and width asymmetries are not inversely correlated at the level of individual animals., (© 2010 Wiley-Liss, Inc.)

Vocal indicators of welfare have proven their use for many farmed and zoo animals and may be applied to farmed silver foxes as these animals display high vocal activity toward humans. Farmed silver foxes were selected mainly for fur, size, and litter sizes, but not for attitudes to people, so they are fearful of humans and have short-term welfare problems in their proximity. With a human approach test, we designed here the steady increase and decrease of fox-human distance and registered vocal responses of 25 farmed silver foxes. We analyzed the features of vocalizations produced by the foxes at different fox-human distances, assuming that changes in vocal responses reflect the degrees of human-related discomfort. For revealing the discomfort-related vocal traits in farmed silver foxes, we proposed and tested the algorithm of "joint calls," equally applicable for analysis of all calls independently on their structure, either tonal or noisy. We discuss that the increase in proportion of time spent vocalizing and the shift of call energy toward higher frequencies may be integral vocal characteristics of short-term welfare problems in farmed silver foxes and probably in other captive mammals.

The involvement of glucocorticoid hormones in the appearance of white spottings during embryogenesis in domesticated gray rats was studied. It was shown that prenatal stress and exposure to dexamethasone on the 12-14 days of pregnancy of fully pigmented gray rats elicited the slowing of melanoblast migration and its development in embryos. It was associated with a 4-fold increase of the offspring percentage with the depigmentation on the ventral side of body in adults. It was also demonstrated that response of H PA axis to emotional stress was lower in adult offsprings from prenatal-stressed and dexamethason-treated mothers than in adult offspring from control mothers. The role of glucocorticoids in the appearance of coat depigmentation under animal domestication is discussed.

We examined the production of different vocalizations in three strains of silver fox (unselected, aggressive, and tame) attending three kinds of behavior (aggressive, affiliative, and neutral) in response to their same-strain conspecifics. This is a follow-up to previous experiments which demonstrated that in the presence of humans, tame foxes produced cackles and pants but never coughed or snorted, whilst aggressive foxes produced coughs and snorts but never cackled or panted. Thus, cackle/pant and cough/snort were indicative of the tame and aggressive fox strains respectively toward humans. Wild-type unselected foxes produced cough and snort toward humans similarly to aggressive foxes. Here, we found that vocal responses to conspecifics were similar in tame, aggressive and unselected fox strains. Both cackle/pant and cough/snort occurred in foxes of all strains. The difference in the use of cackle/pant and cough/snort among these strains toward humans and toward conspecifics suggest that silver foxes do not perceive humans as their conspecifics. We speculate that these vocalizations are produced in response to a triggering internal state, affiliative or aggressive, that is suppressed by default in these fox strains toward humans as a result of their strict selection for tame or aggressive behavior, whilst still remaining flexible toward conspecifics., (Copyright 2010 Elsevier B.V. All rights reserved.)

The genetic basis of the effects of domestication has previously been examined in relation to morphological, physiological and behavioural traits, but not for vocalizations. According to Belyaev [Belyaev, D.K., 1979. Destabilizing selection as a factor in domestication. J. Hered. 70, 301-308], directional selection for tame behaviour toward humans resulted in domestication. This hypothesis has been confirmed experimentally on the farm-bred silver fox Vulpes vulpes population that has undergone 45 years of artificial selection for tameness and 35 years of selection for aggressiveness. These foxes, with their precisely known attitudes toward people, provide a means of examining vocal indicators of tameness and aggressiveness to establish the genetic basis for vocal production in canids. We examined vocalizations toward people in foxes selected for tameness and aggressiveness compared to those of three kinds of crosses: Hybrids (Tame x Aggressive), A-Backcrosses (Aggressive x Hybrid) and T-Backcrosses (Tame x Hybrid). We report the effects of selection for tameness on usage and structure of different vocalizations and suggest that vocal indicators for tameness and aggressiveness toward people are discrete phenotypic traits in silver foxes.

High-quality sequencing of the dog (Canis lupus familiaris) genome has enabled enormous progress in genetic mapping of canine phenotypic variation. The red fox (Vulpes vulpes), another canid species, also exhibits a wide range of variation in coat color, morphology, and behavior. Although the fox genome has not yet been sequenced, canine genomic resources have been used to construct a meiotic linkage map of the red fox genome and begin genetic mapping in foxes. However, a more detailed gene-specific comparative map between the dog and fox genomes is required to establish gene order within homologous regions of dog and fox chromosomes and to refine breakpoints between homologous chromosomes of the 2 species. In the current study, we tested whether canine-derived gene-containing bacterial artificial chromosome (BAC) clones can be routinely used to build a gene-specific map of the red fox genome. Forty canine BAC clones were mapped to the red fox genome by fluorescence in situ hybridization (FISH). Each clone was uniquely assigned to a single fox chromosome, and the locations of 38 clones agreed with cytogenetic predictions. These results clearly demonstrate the utility of FISH mapping for construction of a whole-genome gene-specific map of the red fox. The further possibility of using canine BAC clones to map genes in the American mink (Mustela vison) genome was also explored. Much lower success was obtained for this more distantly related farm-bred species, although a few BAC clones were mapped to the predicted chromosomal locations.

The effects of selection of agouti rats (with genotype AAHH) on the tame and aggressive behavior and dietary methyl given to females from the eighth day of pregnancy to the fifth day after the birth of the offspring on the intensity of the agouti coat color in the offspring have been studied. The morphometric parameters of hair determining the darkness of the agouti color (the total length of guard hairs, the lengths of their eumelanin end and pheomelanin band, the ratio between the lengths of the eumelanin and pheomelanin portions of the hair, the total length of the awn hairs, and the relative length of their widened "lanceolate" upper end) have been compared. It has been found that selection of agouti rats for aggressive behavior is accompanied by darkening of the coat color compared to tame rats due to an increase in the ratio of the length of the black eumelanin end of the guard hairs to the length of the yellow pheomelanin band. Methyl-containing additives to the diet of females affect the intensity of the agouti coat color in the offsprings with both types of behavior, but to different extents. Aggressive offspring is more sensitive to the mother's methyl-containing diet: the percentage of animals that are darker than control rats is higher among aggressive animals than among tame ones due to a greater increase in the ratio between dark and light portions of hairs. The possible mechanisms of differences in the phenotypic modifications of coat color in control and experimental agouti rats with different types of behavior are discussed.

The characteristics of learning in the Morris water test were studied in gray rats subjected to prolonged selection for elimination (the tame strain) and enhanced (the aggressive strain) aggressivity towards humans. Blood corticosterone levels at different stages of learning were also estimated. Tame rats learned to locate the invisible platform better than aggressive rats. The time spent seeking the platform by aggressive rats increased because they spent more time at the periphery of the basin. The duration of vertical investigative activity while on the platform was greater in tame rats than in aggressive rats. Fixation of the memory trace was demonstrated by the observation that rats of both strains spent more time in the sector in which the platform had been located during the training period. Rats of the two strains showed essentially no difference in terms of the time spent seeking the platform when it was placed in the opposite sector. After one day of training, blood corticosterone was significantly lower in tame than in aggressive rats. On subsequent training days, hormone levels in tame animals increased and were no different from those in aggressive rats. It is suggested that decreased emotionality and stress reactivity facilitated the learning process in tame rats in the Morris water test.

To better understand the biology of tameness, i.e. tolerance of human presence and handling, we analyzed two lines of wild-derived rats (Rattus norvegicus) artificially selected for tameness and defensive aggression towards humans. In response to a gloved human hand, tame rats tolerated handling, whereas aggressive rats attacked. Cross-fostering showed that these behavioral differences are not caused by postnatal maternal effects. Tame rats were more active and explorative and exhibited fewer anxiety-related behaviors. They also had smaller adrenal glands, larger spleens and lower levels of serum corticosterone. Blood glucose levels were lower in tame rats, whereas the concentrations of nine amino acids were higher. In the brain, tame rats had lower serotonin and higher taurine levels than aggressive rats. Our findings reinforce the notion that tameness is correlated with differences in stress response and will facilitate future efforts to uncover the genetic basis for animal tameness.

Strains of silver foxes, selectively bred at the Institute of Cytology and Genetics of the Russian Academy of Sciences, are a well established, novel model for studying the genetic basis of behavior, and the processes involved in canine domestication. Here we describe a method to measure fox behavior as quantitative phenotypes which distinguish populations and resegregate in experimental pedigrees. We defined 50 binary observations that nonredundantly and accurately distinguished behaviors in reference populations and cross-bred pedigrees. Principal-component analysis dissected out the independent elements underlying these behaviors. PC1 accounted for >44% of the total variance in measured traits. This system clearly distinguished tame foxes from aggressive and wildtype foxes. F1 foxes yield intermediate values that extend into the ranges of both the tame and aggressive foxes, while the scores of the backcross generation resegregate. These measures can thus be used for QTL mapping to explore the genetic basis of tame and aggressive behavior in foxes, which should provide new insights into the mechanisms of mammalian behavior and canine domestication.

Genetic variation in functionally integrated skeletal traits can be maintained over 10 million years despite bottlenecks and stringent selection. Here, we describe an analysis of the genetic architecture of the canid axial skeleton using populations of the Portuguese Water Dog Canis familiaris) and silver fox (Vulpes vulpes). Twenty-one skeletal metrics taken from radiographs of the forelimbs and hind limbs of the fox and dog were used to construct separate anatomical principal component (PC) matrices of the two species. In both species, 15 of the 21 PCs exhibited significant heritability, ranging from 25% to 70%. The second PC, in both species, represents a trade-off in which limb-bone width is inversely correlated with limb-bone length. PC2 accounts for approximately 15% of the observed skeletal variation, approximately 30% of the variation in shape. Many of the other significant PCs affect very small amounts of variation (e.g., 0.2-2%) along trade-off axes that partition function between the forelimbs and hind limbs. These PCs represent shape axes in which an increase in size of an element of the forelimb is associated with a decrease in size of an element of the hind limb and vice versa. In most cases, these trade-offs are heritable in both species and genetic loci have been identified in the Portuguese Water Dog for many of these. These PCs, present in both the dog and the fox, include ones that affect lengths of the forelimb versus the hind limb, length of the forefoot versus that of the hind foot, muscle moment (i.e., lever) arms of the forelimb versus hind limb, and cortical thickness of the bones of the forelimb versus hind limb. These inverse relationships suggest that genetic regulation of the axial skeleton results, in part, from the action of genes that influence suites of functionally integrated traits. Their presence in both dogs and foxes suggests that the genes controlling the regulation of these PCs of the forelimb versus hind limb may be found in other tetrapod taxa.

Maternal choline diet is known to affect the processes of spatial learning. We report here our studies of learning ability in the Morris swimming test in the adult offspring of maternal rats given a methyl-containing supplement enriched with choline and betaine during pregnancy and lactation. Increases in the time taken to find the invisible platform and the duration of swimming close to the vessel walls were seen, these demonstrating worsening of learning ability in response to the maternal diet. Changes in the platform search strategy were not associated with increases in anxiety in male rats. The possible role of a maternal methyl-containing diet in altering the expression of genes controlling the development of the nervous system is discussed.

Some features of learning in Morris water test were studied in gray rats after a long-term selection for elimination (tame strain) and enhancement (aggressive strain) of aggressiveness towards human. The content of plasma corticosteroids was estimated at different stages of learning. It was shown that tame rats were better in performance of a special Morris task than aggressive ones. The time of search for invisible platform was increased in aggressive rats owing to the fact that they spent more time near the pool walls. Also, time of rearing at the platform was higher in tame rats compared to aggressive animals. In the retention test, rats of both strains spent significantly more time in the target quadrant than they did in other quadrants. Rats of both strains did not differ in time of search for invisible platform when it was replaced to the opposite quadrant. After the first day of learning, the corticosteroid plasma level was lower in tame rats than aggressive animals. During the following days of training, the content of the hormone increased in tame rats and did not differ from aggressive rats. It is supposed that, in tame rats, low emotionality and stress response facilitated learning in Morris water test.

A meiotic linkage map is essential for mapping traits of interest and is often the first step toward understanding a cryptic genome. Specific strains of silver fox (a variant of the red fox, Vulpes vulpes), which segregate behavioral and morphological phenotypes, create a need for such a map. One such strain, selected for docility, exhibits friendly dog-like responses to humans, in contrast to another strain selected for aggression. Development of a fox map is facilitated by the known cytogenetic homologies between the dog and fox, and by the availability of high resolution canine genome maps and sequence data. Furthermore, the high genomic sequence identity between dog and fox allows adaptation of canine microsatellites for genotyping and meiotic mapping in foxes. Using 320 such markers, we have constructed the first meiotic linkage map of the fox genome. The resulting sex-averaged map covers 16 fox autosomes and the X chromosome with an average inter-marker distance of 7.5 cM. The total map length corresponds to 1480.2 cM. From comparison of sex-averaged meiotic linkage maps of the fox and dog genomes, suppression of recombination in pericentromeric regions of the metacentric fox chromosomes was apparent, relative to the corresponding segments of acrocentric dog chromosomes. Alignment of the fox meiotic map against the 7.6x canine genome sequence revealed high conservation of marker order between homologous regions of the two species. The fox meiotic map provides a critical tool for genetic studies in foxes and identification of genetic loci and genes implicated in fox domestication.

Maternal choline diet influences the spatial learning processes. In this work, the learning ability of adult progeny of mothers who had received methyl diet enriched with choline and betain during pregnancy and lactation was studied in Morris test. The introduction of the diet to pregnant rats resulted in an increase in the time of search for invisible platform and time of swimming near the pool walls in offsprings, which meant a worsening of their learning ability. It was also found that change in platform searching strategy was not associated with an increase in anxiety of male rats. Possible involvement of maternal methyl diet in the change of expression of genes which control development of the nervous system is discussed.

The effect of methyl supplements to the diet of pregnant homozygous (AAHH) female rats with agouti coat color mated with homozygous (aahh) males on the phenotypic modification of the coat color of their heterozygous offspring (AaHh) has been studied. Comparative morphological analysis of the main parameters of hair that determine coat color, including the total length of hairs of different types and the length of the upper black (eumelanin) and light (pheomelanin) parts of awn hairs has been performed. The pattern of pigment granule distribution among hair layers has been analyzed. The melanin content of the hair has been determined using electron spin resonance (ESR). Although all offspring have a typical agouti coat color (alternating black and light portions of hair), 39% of them have a darker coat color than control and other experimental rats have. The main differences between the offspring with darkened and standard coat colors are accounted for by the ratio between the eumelanin and pheomelanin portions of awn hairs. In darkened offspring, this ratio is significantly higher than in control rats. The possible mechanisms of the phenotypic modification of agouti coat color in experimental animals are discussed.

Plant and animal karyotypes sometimes contain variable elements, that are referred to as additional or B-chromosomes. It is generally believed that B-chromosomes lack major genes and represent parasitic and selfish elements of a genome. Here we report, for the first time, the localization of a gene to B-chromosomes of mammals: red fox (Vulpes vulpes) and two subspecies of raccoon dog (Nyctereutes procyonoides). Identification of the proto-oncogene C-KIT on B-chromosomes of two Canidae species that diverged from a common ancestor more than 12.5 million years ago argues against the current view of B-chromosomes. Analyses of fox B-chromosomal C-KIT gene from a flow-sorted fox B-chromosome-specific library revealed the presence of intron-exon boundaries and high identity between sequenced regions of canine and fox B-chromosomal C-KIT copies. Identification of C-KIT gene on all B-chromosomes of two canid species provides new insight into the origin and evolution of supernumeraries and their potential role in the genome.

Silver foxes from a commercial population (farm bred or unselected for behavior control) and from populations selected for tame behavior and enhanced aggressiveness towards man have been investigated. Plasma cortisol and adrenocorticotropic hormone (ACTH) levels, pituitary ACTH levels, POMC gene expression in the anterior pituitary, and corticotropin-releasing factor (CRF) gene expression in the hypothalamus were assessed. The results indicate that the males from the tame-behavior group have lower plasma cortisol and ACTH levels and POMC gene expression in the anterior pituitary in response to capture and handling in comparison with unselected ones. Foxes from the aggressive behavior group also have lower POMC expression, although plasma cortisol and ACTH levels remain the same as in unselected ones. The three groups of animals show no significant changes in the ACTH level in the pituitary and CRF expression in the hypothalamus.

This paper is a review of the results of the authors obtained in a long-term experiment on fox domestication. Debatable issues of dog evolution are discussed in light of these results. It is demonstrated that genetic physiological mechanisms of the behavior transformation during selection and the nature of the arising phenotypic changes are associated with retarded development of corresponding ontogenetic processes. As a result of this retardation, the adult animals retain juvenile traits of behavior and morphology (the phenomenon of neoteny). The role of hormonal changes caused by domestication in the evolutionary origin of neoteny is discussed.

The silver fox, a variant of the red fox (Vulpes vulpes), is a close relative of the dog (Canis familiaris). Cytogenetic differences and similarities between these species are well understood, but their genomic organizations have not been compared at higher resolution. Differences in their behavior also remain unexplained. Two silver fox strains demonstrating markedly different behavior have been generated at the Institute of Cytology and Genetics of the Russian Academy of Sciences. Foxes selected for tameness are friendly, like domestic dogs, while foxes selected for aggression resist human contact. To refine our understanding of the comparative genomic organization of dogs and foxes, and enable a study of the genetic basis of behavior in these fox strains, we need a meiotic linkage map of the fox. Towards this goal we generated a primary set of fox microsatellite markers. Four hundred canine microsatellites, evenly distributed throughout the canine genome, have been identified that amplify robustly from fox DNA. Polymorphism information content (PIC) values were calculated for a representative subset of these markers and population inbreeding coefficients were determined for tame and aggressive foxes. To begin to identify fox-specific single nucleotide polymorphisms (SNPs) in genes involved in the neurobiology of behavior, fox and dog orthologs of serotonin 5-HT1A and 5-HT1B receptor genes have been cloned. Sequence comparison of these genes from tame and aggressive foxes reveal several SNPs. The close relationship of the fox and dog enables canine genomic tools to be utilized in developing a fox meiotic map and mapping behavioral traits in the fox., (Copyright 2004 The American Genetic Association)

The goal of this work was to find out to which degree the black coat color mutation nonagouti that appears in wild Norway rat in the course of breeding for the maintenance and enhancement of aggressiveness towards a human affects selected and other behavioral traits as well as stress reactivity and brain neurochemistry. Nonagouti rats displayed increased emotionality in the open-field and forced-swimming tests as compared to agouti animals, whereas there were no differences in anxiety estimated in the elevated plus-maze. Neither were there any differences in glucocorticoid reactions to a combined stressful procedure such as forced swimming. The dopamine content in the frontal cortex and striatum and noradrenaline content in the frontal cortex were increased in black rats as compared to gray rats. The result suggest that the nonagouti mutation in gray aggressive rats enhances the effects of selection for aggressiveness on some behavioral traits and brain catecholamine level.

Several reproductive parameters were studied in males homozygous (hh) or heterozygous (Hh) for the hooded mutation as compared with completely pigmented wild-type males (HH). Histological analysis of the testes was carried out in males of the three genotypes. The proportion of sterile males in homogeneous matings of homozygotes hh was twice as high as in matings of heterozygotes. The proportion of sterile males in matings yielding no progeny was also twice higher in homozygotes hh as compared with heterozygotes. No sterile males were detected in matings of completely pigmented wild-type animals. Unilateral cryptorchidism, a hypoplastic testis combined with a hyperplastic one, or hypoplasia of both testes were observed in some males homozygous for the hooded mutation. Morphologically, these defects were associated with underdevelopment or the complete absence of spermatogenic epithelium or with the presence of gaps and cells with large nondivided nuclei in the epithelium. The results showed that the hooded coat-color mutation exerts a pleiotropic effect on male fertility in rat.

Directional left-sided asymmetry of the adrenals was typical of black and silver foxes, American minks, and gray rats selected by their behavior. In domesticated, but to a greater extent, in aggressive animals, the weight of the left adrenal and the width of its medulla and cortex markedly surpassed the corresponding parameters of the right adrenal. In aggressive animals enlargement of the left adrenal cortex was associated with widening of the zona reticularis, while in domesticated animals with enlargement of the zona fasciculata.