Puppy whines mediate maternal behavior in domestic dogs.

In mammals, offspring vocalizations typically encode information about identity and body condition, allowing parents to limit alloparenting and adjust care. But how do these vocalizations mediate parental behavior in species faced with the problem of rearing not one, but multiple offspring, such as domestic dogs? Comprehensive acoustic analyses of 4,400 whines recorded from 220 Beagle puppies in 40 litters revealed litter and individual (within litter) differences in call acoustic structure. By then playing resynthesized whines to mothers, we showed that they provided more care to their litters, and were more likely to carry the emitting loudspeaker to the nest, in response to whine variants derived from their own puppies than from strangers. Importantly, care provisioning was attenuated by experimentally moving the fundamental frequency (fo, perceived as pitch) of their own puppies' whines outside their litter-specific range. Within most litters, we found a negative relationship between puppies' whine fo and body weight. Consistent with this, playbacks showed that maternal care was stronger in response to high-pitched whine variants simulating relatively small offspring within their own litter's range compared to lower-pitched variants simulating larger offspring. We thus show that maternal care in a litter-rearing species relies on a dual assessment of offspring identity and condition, largely based on level-specific inter- and intra-litter variation in offspring call fo. This dual encoding system highlights how, even in a long-domesticated species, vocalizations reflect selective pressures to meet species-specific needs. Comparative work should now investigate whether similar communication systems have convergently evolved in other litter-rearing species.

Synchronous citizen science with dogs.

Citizen science approaches have grown in popularity over the years, partly due to their ability to reach a wider audience and produce more generalizable samples. In dogs, these studies, though, have been limited in their controls over materials or experimental protocols, with guardians typically reporting results without researcher supervision. Over two studies, we explored and validated a synchronous citizen science approach. We had dog guardians act as experimenters while being supervised by a researcher over Zoom. In study 1, we demonstrated that synchronous citizen science produced equivalent levels of performance to in-lab designs in a choice task. Consistent with past in-lab research, dogs selected a treat (vs. an empty plate) in a two-alternative forced-choice task. In study 2, we showed that Zoom methods are also appropriate for studies utilizing looking time measures. We explored dogs' looking behaviors when a bag of treats was placed in an unreachable location, and dogs' guardians were either attentive or inattentive while dogs attempted to retrieve the treats. Consistent with past work, dogs in the attentive condition looked at their guardian for longer periods and had a shorter latency to first look than dogs in the inattentive condition. Overall, we have demonstrated that synchronous citizen science studies with dogs are feasible and produce valid results consistent with those found in a typical lab setting.

Comparative Proteome and Cis-Regulatory Element Analysis Reveals Specific Molecular Pathways Conserved in Dog and Human Brains.

Brain development and function are governed by precisely regulated protein expressions in different regions. To date, multiregional brain proteomes have been systematically analyzed only for adult human and mouse brains. To understand the underpinnings of brain development and function, we generated proteomes from six regions of the postnatal brain at three developmental stages of domestic dogs (Canis familiaris), which are special among animals in terms of their remarkable human-like social cognitive abilities. Quantitative analysis of the spatiotemporal proteomes identified region-enriched synapse types at different developmental stages and differential myelination progression in different brain regions. Through integrative analysis of inter-regional expression patterns of orthologous proteins and genome-wide cis-regulatory element frequencies, we found that proteins related with myelination and hippocampus were highly correlated between dog and human but not between mouse and human, although mouse is phylogenetically closer to human. Moreover, the global expression patterns of neurodegenerative disease and autism spectrum disorder-associated proteins in dog brain more resemble human brain than in mouse brain. The high similarity of myelination and hippocampus-related pathways in dog and human at both proteomic and genetic levels may contribute to their shared social cognitive abilities. The inter-regional expression patterns of disease-associated proteins in the brain of different species provide important information to guide mechanistic and translational study using appropriate animal models.

CanISO: a database of genomic and transcriptomic variations in domestic dog (Canis lupus familiaris).

BACKGROUND: The domestic dog, Canis lupus familiaris, is a companion animal for humans as well as an animal model in cancer research due to similar spontaneous occurrence of cancers as humans. Despite the social and biological importance of dogs, the catalogue of genomic variations and transcripts for dogs is relatively incomplete. RESULTS: We developed CanISO, a new database to hold a large collection of transcriptome profiles and genomic variations for domestic dogs. CanISO provides 87,692 novel transcript isoforms and 60,992 known isoforms from whole transcriptome sequencing of canine tumors (N = 157) and their matched normal tissues (N = 64). CanISO also provides genomic variation information for 210,444 unique germline single nucleotide polymorphisms (SNPs) from the whole exome sequencing of 183 dogs, with a query system that searches gene- and transcript-level information as well as covered SNPs. Transcriptome profiles can be compared with corresponding human transcript isoforms at a tissue level, or between sample groups to identify tumor-specific gene expression and alternative splicing patterns. CONCLUSIONS: CanISO is expected to increase understanding of the dog genome and transcriptome, as well as its functional associations with humans, such as shared/distinct mechanisms of cancer. CanISO is publicly available at https://www.kobic.re.kr/caniso/ .

High Investment in Reproduction Is Associated with Reduced Life Span in Dogs.

AbstractProminent differences in aging among and within species present an evolutionary puzzle. The theories proposed to explain evolutionary differences in aging are based on the axiom that selection maximizes fitness, not necessarily life span. This implies trade-offs between investment in self-maintenance and investment in reproduction, where high investments in growth and current reproduction are associated with short life spans. Fast growth and large adult size are related to shorter life spans in the domestic dog, a bourgeoning model in aging research; however, whether reproduction influences life span in this system remains unknown. Here we test the relationship between reproduction and differences in life span among dog breeds, simultaneously controlling for shared ancestry and recent gene flow. We found that shared ancestry explains a higher proportion of the among-breed variation in life history traits, in comparison with recent gene flow. Our results also show that reproductive investment negatively impacts life span, and more strongly so in large breeds, an effect that is not merely a correlated response of adult size. These results suggest that basic life history trade-offs are apparent in a domestic animal whose diversity is the result of artificial selection and that among-breed differences in life span are due to a combination of size and reproduction.

Dog Life Spans and the Evolution of Aging.

AbstractThe basic tenets of the evolutionary theories of senescence are well supported. However, there has been little progress in determining the relative influences of mutation accumulation and life history optimization. The causes of the well-established inverse relationship between life span and body size across dog breeds are used here to test these two classes of theories. The life span-body size relationship is confirmed for the first time after controlling for breed phylogeny. The life span-body size relationship cannot be explained by evolutionary responses to differences in extrinsic mortality either of contemporary breeds or of breeds at their establishment. The development of breeds larger and smaller than ancestral gray wolves has occurred through changes in early growth rate. This may explain the increase in the minimum age-dependent mortality rate with breed body size and thus higher age-dependent mortality throughout adult life. The main cause of this mortality is cancer. These patterns are consistent with the optimization of life history as described by the disposable soma theory of the evolution of aging. The dog breed life span-body size relationship may be the result of the evolution of greater defense against cancer lagging behind the rapid increase in body size during recent breed establishment.

Evolutionary history of the extinct wolf population from France in the context of global phylogeographic changes throughout the Holocene.

Phylogeographic patterns in large mammals result from natural environmental factors and anthropogenic effects, which in some cases include domestication. The grey wolf was once widely distributed across the Holarctic, but experienced phylogeographic shifts and demographic declines during the Holocene. In the 19th-20th centuries, the species became extirpated from large parts of Europe due to direct extermination and habitat loss. We reconstructed the evolutionary history of the extinct Western European wolves based on the mitogenomic composition of 78 samples from France (Neolithic-20th century) in the context of other populations of wolves and dogs worldwide. We found a close genetic similarity of French wolves from ancient, medieval and recent populations, which suggests the long-term continuity of maternal lineages. MtDNA haplotypes of the French wolves showed large diversity and fell into two main haplogroups of modern Holarctic wolves. Our worldwide phylogeographic analysis indicated that haplogroup W1, which includes wolves from Eurasia and North America, originated in Northern Siberia. Haplogroup W2, which includes only European wolves, originated in Europe ~35 kya and its frequency was reduced during the Holocene due to an expansion of haplogroup W1 from the east. Moreover, we found that dog haplogroup D, currently restricted to Europe and the Middle East, was nested within the wolf haplogroup W2. This suggests European origin of haplogroup D, probably as a result of an ancient introgression from European wolves. Our results highlight the dynamic evolutionary history of European wolves during the Holocene, with a partial lineage replacement and introgressive hybridization with local dog populations.

Appeasement function of displacement behaviours? Dogs' behavioural displays exhibited towards threatening and neutral humans.

Appeasement signals are behavioural patterns displaying an animal's non-aggressive attitude and are hypothesized to reduce the aggressive behaviours in the receiver. In domestic dogs, specific displacement behaviours (i.e., behavioural patterns exhibited without an apparent function related to the ongoing situation), have been suggested to function as appeasement signals. To test this possibility, we assessed whether the occurrence of these behaviours was dependent on a social conflict context, predicting that, if displacement behaviours also function as appeasement signals, they should be more prevalent in a conflict vs. non-conflict context. Fifty-three dogs were exposed to two unfamiliar humans approaching them in either a mildly threatening or neutral way. We categorized the attitude of the dogs towards the strangers as "reactive", i.e., barking and lunging towards the stimulus, and "non-reactive", i.e., remaining passive in front of the stimuli. We coded dogs' displacement activities and modelled their duration or frequency as a function of the interaction between the test condition and the attitude of the dog. Displacement behaviours of "blinking", "nose licking" and "lip wiping" were associated with a "non-reactive" attitude, independently from the test condition, confirming an association with a non-aggressive intention. "Head turning" was associated with a "non-reactive" attitude in the threatening condition. In conclusion, dogs with a non-aggressive attitude exhibited more putative appeasement signals; however, these were not strictly associated with a conflict-ridden situation, calling for further investigation of their function.

Inflammaging in domestic dogs: basal level concentrations of IL-6, IL-1ß, and TNF-α in serum of healthy dogs of different body sizes and ages.

Inflammaging, a "hallmark of aging," refers to a chronic, progressive increase in the proinflammatory status of mammals as they age, and this phenotype has been associated with many age-related diseases, including cardiovascular disease, arthritis and cancer. Though, inflammaging research is common in humans, there is a lack of data for this process for the domestic dog. Here, serum concentrations of IL-6, IL-1ß and TNF-α in healthy dogs of different body sizes and ages were measured to determine whether inflammaging may play a mechanistic role in aging rates in dogs, similar to those found in humans. Using a four-way ANOVA, a significant decrease in IL-6 concentrations in young dogs with the rest of the age categories showing increased IL-6 concentrations was found, similar to humans. However, only young dogs have decreased IL-6 concentrations, with adult dogs having similar IL-6 concentrations to senior and geriatric dogs, implying differences in aging rates between humans and dogs. And, there was a marginally significant interaction between sex*spayed or neutered status and IL-1ß concentrations with intact females having the lowest IL-1ß concentrations compared with intact males, and spayed and neutered dogs. The presence of estrogen in intact females may, overall, decrease inflammatory pathways. This implies that age at spaying or neutering may be an important aspect to consider for inflammaging pathways in dogs. Furthermore, sterilized dogs often die of immune-related diseases, which could be linked to the increases in IL-1ß in sterilized dogs found in this study.

Head-mounted mobile eye-tracking in the domestic dog: A new method.

Humans rely on dogs for countless tasks, ranging from companionship to highly specialized detection work. In their daily lives, dogs must navigate a human-built visual world, yet comparatively little is known about what dogs visually attend to as they move through their environment. Real-world eye-tracking, or head-mounted eye-tracking, allows participants to freely move through their environment, providing more naturalistic results about visual attention while interacting with objects and agents. In dogs, real-world eye-tracking has the potential to inform our understanding of cross-species cognitive abilities as well as working dog training; however, a robust and easily deployed head-mounted eye-tracking method for dogs has not previously been developed and tested. We present a novel method for real-world eye-tracking in dogs, using a simple head-mounted mobile apparatus mounted onto goggles designed for dogs. This new method, adapted from systems that are widely used in humans, allows for eye-tracking during more naturalistic behaviors, namely walking around and interacting with real-world stimuli, as well as reduced training time as compared to traditional stationary eye-tracking methods. We found that while completing a simple forced-choice treat finding task, dogs look primarily to the treat, and we demonstrated the accuracy of this method using alternative gaze-tracking methods. Additionally, eye-tracking revealed more fine-grained time course information and individual differences in looking patterns.

Searching high and low: domestic dogs' understanding of solidity.

Physical reasoning appears central to understanding how the world works, suggesting adaptive function across the animal kingdom. However, conclusive evidence for inferential reasoning about physical objects is limited to primates. We systematically tested a central feature-understanding of solidity-in domestic dogs, by adapting a validated procedure (the shelf task) previously used to test children and non-human primates. Dogs watched a treat dropped into an apparatus with a shelf either present (treat landing on the shelf) or absent (treat landing on the bottom surface) and chose where to search for it (above or below the shelf). Across four studies (n = 64), we manipulated visual access to the treat trajectory and apparatus interior. Dogs correctly inferred the location of treats using physical cues when the shelf was present (Study 1), and learned rapidly when visual cues of continuity were limited (Study 2), and when the shelf was absent (Study 3). Dogs were at chance when the apparatus was fully occluded, and the presence and absence of the shelf varied across trials within subjects, and showed no evidence of learning (Study 4). The findings of these four studies suggest that dogs may be able to make some inferences using solidity and continuity and do not exhibit proximity or gravity biases. However, dogs did not always search correctly from Trial 1, and failed to search correctly when the rewarded location varied within-subjects, suggesting a role for learning, and possible limits to their ability to make inferences about physical objects.

Canine Endotheliochorial Placenta: Morpho-Functional Aspects.

In the domestic dog, placentation arises from central implantation, passing through a transitional, yet important stage of choriovitelline placenta (yolk sac placenta), on the way to the formation of the definite, deciduate, zonary (girdle) allantochorionic endotheliochorial placenta.Sharing some similarities with other invasive types of placentation, e.g., by revealing decidualization, it is characterized by restricted (shallow) invasion of trophoblast not affecting maternal capillaries and maternal decidual cells. Thus, being structurally and functionally placed between noninvasive epitheliochorial placentation and the more invasive hemochorial type, it presents an interesting and important model for understanding the evolutionarily determined aspects of mammalian placentation. More profound insights into the biological mechanisms underlying the restricted invasion of the fetal trophoblast into maternal uterine structures and the role of decidual cells in that process could provide better understanding of some adverse conditions occurring in humans, like preeclampsia or placenta accreta. As an important endocrine organ actively responding to ovarian steroids and producing its own hormones, e.g., serving as the source of gestational relaxin or prepartum prostaglandins, the canine placenta has become an attractive research target, both in basic and clinical research. In particular, the placental feto-maternal communication between maternal stroma-derived decidual cells and fetal trophoblast cells (i.e., an interplay between placenta materna and placenta fetalis) during the maintenance and termination of canine pregnancy serves as an interesting model for induction of parturition in mammals and is an attractive subject for translational and comparative research. Here, an updated view on morpho-functional aspects associated with canine placentation is presented.

Partial rewarding during clicker training does not improve naïve dogs' learning speed and induces a pessimistic-like affective state.

Clicker training is considered a welfare-friendly way of teaching novel behaviors to animals because it is mostly based on the positive reinforcement. However, trainers largely vary in their way of applying this training technique. According to the most, a reward (e.g., food) should follow every click, while others claim that dogs learn faster when the reward is sometimes omitted. One argument against the use of partial rewarding is that it induces frustration in the animal, raising concerns over its welfare consequences. Here, we investigated the effect of partial rewarding not only on training efficacy (learning speed), but also on dogs' affective state. We clicker-trained two groups of dogs: one group received food after every click while the other group received food only 60% of the time. Considering previous evidence of the influencing role of personality on reactions to frustrated expectations, we included measurements of dogs' emotional reactivity. We compared the number of trials needed to reach a learning criterion and their pessimistic bias in a cognitive bias test. No difference between the two groups emerged in terms of learning speed; however, dogs that were partially rewarded during clicker training showed a more pessimistic bias than dogs that were continuously rewarded. Generally, emotional reactivity was positively associated with a more pessimistic bias. Partial rewarding does not improve training efficacy, but it is associated with a negatively valenced affective state, bringing support to the hypothesis that partial rewarding might negatively affect dogs' welfare.

Spatial and temporal dynamics of space use by free-ranging domestic dogs Canis familiaris in rural Africa.

Variation in the spatial ecology of animals influences the transmission of infections and so understanding host behavior can improve the control of diseases. Despite the global distribution of free-ranging domestic dogs Canis familiaris and their role as reservoirs for zoonotic diseases, little is known about the dynamics of their space use. We deployed GPS loggers on owned but free-ranging dogs from six villages in rural Chad, and tracked the movements of 174 individuals in the dry season and 151 in the wet season. We calculated 95% and core home ranges using auto-correlated kernel density estimates (AKDE95 and AKDEcore ), determined the degree to which their movements were predictable, and identified correlates of movement patterns. The median AKDE95 range in the dry season was 0.54 km2 and in the wet season was 0.31 km2 , while the median AKDEcore range in the dry season was 0.08 km2 and in the wet season was 0.04 km2 . Seasonal variation was, in part, related to owner activities; dogs from hunting households had ranges that were five times larger in the dry season. At least 70% of individuals were more predictably "at home" (<50 m from the household) throughout the day in the dry season, 80% of dogs demonstrated periodicity in activity levels (speed), and just over half the dogs exhibited periodicity in location (repeated space use). In the wet season, dogs mostly exhibited 24-h cycles in activity and location, with peaks at midday. In the dry season, dogs exhibited both 12- and 24-h cycles, with either a single peak at midday, or one peak between 06:00 and 12:00 and a second between 18:00 and 22:00. Strategies to control canine-mediated zoonoses can be improved by tailoring operations to the local spatial ecology of free-ranging dogs. Interventions using a door-to-door strategy in rural Chad would best conduct operations during the dry season, when access to dogs around their household more reliably exceeds 70% throughout the day. Given the importance of use in hunting for explaining variation in dog space-use, targeting approaches to disease control at the household level on the basis of owner activities offers potential to improve access to dogs.

Untangling life span and body mass discrepancies in canids: phylogenetic comparison of oxidative stress in blood from domestic dogs and wild canids.

Canids are a morphological and physiological diverse group of animals, with the most diversity found within one species, the domestic dog. Underlying observed morphological differences, there must also be differences at other levels of organization that could lead to elucidating aging rates and life span disparities between wild and domestic canids. Furthermore, small-breed dogs live significantly longer lives than large-breed dogs, while having higher mass-specific metabolic rates and faster growth rates. At the cellular level, a clear mechanism underlying whole animal traits has not been fully elucidated, although oxidative stress has been implicated as a potential culprit of the disparate life spans of domestic dogs. We used plasma and red blood cells from known aged domestic dogs and wild canids, and measured several oxidative stress variables: total antioxidant capacity (TAC), lipid damage, and enzymatic activities of catalase, superoxide dismutase, and glutathione peroxidase (GPx). We used phylogenetically informed general linear mixed models and nonphylogenetically corrected linear regression analysis. We found that lipid damage increases with age in domestic dogs, whereas TAC increases with age and TAC and GPx activity increases as a function of age/maximum life span in wild canids, which may partly explain longer potential life spans in wolves. As body mass increases, TAC and GPx activity increase in wild canids, but not domestic dogs, highlighting that artificial selection may have decreased antioxidant capacity in domestic dogs. We found that small-breed dogs have significantly higher circulating lipid damage compared with large-breed dogs, concomitant to their high mass-specific metabolism and higher growth rates, but in opposition to their long life spans.

What smells? Gauging attention to olfaction in canine cognition research.

One of the challenges of animal cognition research is overcoming anthropocentric sensory biases-in particular, favoring visual information and cues despite the dominance of other sensory cues in many nonhuman research subjects. As such, it is particularly important for animal cognition researchers to explicitly mention steps taken to control for and attend to the sensory world of their study species. Dogs are well known for their reliance on olfaction, but the extent to which dog cognition and behavior research accounts for olfactory cues or incorporates olfactory controls is unknown. With this bibliographic study, we reviewed canine research published in the past 10 years (2008-2018) in 13 scientific journals and coded the 481 resulting papers for mentions of olfactory or odor cues or controls. Our findings indicate that despite widespread acceptance of the significance of olfaction to dogs, scientific methodology rarely takes olfactory information processing into account. Finally, we propose a simple rubric of recommended reporting of olfactory information in research contexts, with the aims to help attune researchers to the umwelt of their study subjects, and to enhance the methodological reproducibility of canine cognition research.

Comparing pet and detection dogs (Canis familiaris) on two aspects of social cognition.

Interspecific communication between dogs and humans enables dogs to occupy significant roles in human society, both in companion and working roles. Dogs excel at using human communicative signals in problem-solving tasks, and solicit human contact when unable to solve a problem. Dogs' sociocognitive behavior likely results from a selection for attention to humans during domestication, but is highly susceptible to environmental factors. Training for particular tasks appears to enhance dog-human communication, but effects may depend on the nature of the relationship determined by their role. Our aim was to examine two types of social cognition (responsiveness to human gestures, and human-directed communicative behavior in an unsolvable task) in pet dogs (n = 29) and detection dogs (n = 35). The groups did not differ in their ability to follow human signals, but pets were less responsive to signals given by a stranger than by their owner. Pets also exhibited more human-directed gazing in the unsolvable task, showing a bias for gazing at their owner compared with the stranger, whereas detection dogs showed greater persistence in attempting to solve the task compared with pets. Thus, different aspects of dogs' sociocognitive behavior may differentially vary as a function of selection or training for particular roles.

Body size, inbreeding, and lifespan in domestic dogs.

Inbreeding poses a real or potential threat to nearly every species of conservation concern. Inbreeding leads to loss of diversity at the individual level, which can cause inbreeding depression, and at the population level, which can hinder ability to respond to a changing environment. In closed populations such as endangered species and ex situ breeding programs, some degree of inbreeding is inevitable. It is therefore vital to understand how different patterns of breeding and inbreeding can affect fitness in real animals. Domestic dogs provide an excellent model, showing dramatic variation in degree of inbreeding and in lifespan, an important aspect of fitness that is known to be impacted by inbreeding in other species. There is a strong negative correlation between body size and lifespan in dogs, but it is unknown whether the higher rate of aging in large dogs is due to body size per se or some other factor associated with large size. We used dense genome-wide SNP array data to calculate average inbreeding for over 100 dog breeds based on autozygous segment length and found that large breeds tend to have higher coefficients of inbreeding than small breeds. We then used data from the Veterinary medical Database and other published sources to estimate life expectancies for pure and mixed breed dogs. When controlling for size, variation in inbreeding was not associated with life expectancy across breeds. When comparing mixed versus purebred dogs, however, mixed breed dogs lived about 1.2 years longer on average than size-matched purebred dogs. Furthermore, individual pedigree coefficients of inbreeding and lifespans for over 9000 golden retrievers showed that inbreeding does negatively impact lifespan at the individual level. Registration data from the American Kennel Club suggest that the molecular inbreeding patterns observed in purebred dogs result from specific breeding practices and/or founder effects and not the current population size. Our results suggest that recent inbreeding, as reflected in variation within a breed, is more likely to affect fitness than historic inbreeding, as reflected in variation among breeds. Our results also indicate that occasional outcrosses, as in mixed breed dogs, can have a substantial positive effect on fitness.

Right nostril biases to experimental scents in Canis familiaris.

Although olfactory laterality in canids has been demonstrated experimentally, the extent to which nostril bias occurs in "nature" is not well known. We tested whether there was olfactory laterality of untrained dogs in various off-leash dog parks within Victoria, British Columbia to manipulated scents placed at the tail base of full-size dog replica. Using video-playback, we found that of 192 separate approaches (N = 119 different subjects), dogs used the right nostril first greater than 66% of the time and for longer periods when investigating estrous dog secretions, deer urine and coyote urine. Similar trends were observed when using scents on a similar-sized box rather than the dog model. There was no side preference for the scent of commercial pet food. These results support right hemisphere control of the sympathetic-hypothalamic-pituitary-adrenal axis and encourage more detailed evaluations of olfactory laterality in wild canids and other carnivores where olfaction is the major sensory modality.

Training pet dogs for eye-tracking and awake fMRI.

In recent years, two well-developed methods of studying mental processes in humans have been successively applied to dogs. First, eye-tracking has been used to study visual cognition without distraction in unrestrained dogs. Second, noninvasive functional magnetic resonance imaging (fMRI) has been used for assessing the brain functions of dogs in vivo. Both methods, however, require dogs to sit, stand, or lie motionless while yet remaining attentive for several minutes, during which time their brain activity and eye movements are measured. Whereas eye-tracking in dogs is performed in a quiet and, apart from the experimental stimuli, nonstimulating and highly controlled environment, MRI scanning can only be performed in a very noisy and spatially restraining MRI scanner, in which dogs need to feel relaxed and stay motionless in order to study their brain and cognition with high precision. Here we describe in detail a training regime that is perfectly suited to train dogs in the required skills, with a high success probability and while keeping to the highest ethical standards of animal welfare-that is, without using aversive training methods or any other compromises to the dog's well-being for both methods. By reporting data from 41 dogs that successfully participated in eye-tracking training and 24 dogs IN fMRI training, we provide robust qualitative and quantitative evidence for the quality and efficiency of our training methods. By documenting and validating our training approach here, we aim to inspire others to use our methods to apply eye-tracking or fMRI for their investigations of canine behavior and cognition.