Changes in salivary cortisol concentrations during a 24-hour period in dogs.

The purpose of this investigation was to clarify whether salivary cortisol secretion in dogs had a circadian rhythm. Saliva sampling during a 24-hour period was performed in 4 non-consecutive days. Eight adult beagle dogs (4 males and 4 females) were divided into 4 groups, and 2 dogs (1 male and 1 female) were used for each repetition. Saliva samples were taken at 1 h intervals from 9:00 a. m. to 9:00 a. m. of the following day. Salivary cortisol concentrations were determined using enzyme-linked immunosorbent assay. No circadian rhythm was detected for salivary cortisol, and the differences among salivary cortisol concentrations measured every hour were not demonstrated during a 24-hour period in dogs.     

Differences in Behavior and Activity Associated with a Poly(A) Expansion in the Dopamine Transporter in Belgian Malinois

In Belgian Malinois dogs, a 38-base pair variable number tandem repeat in the dopamine transporter gene (SLC6A3) is associated with behavior changes in Malinois. By additional sequencing in SLC6A3, we identified an intronic 12-nucleotide poly(A) insertion (“PolyA(22)”) before the terminal exon that was associated with seizure, “glazing over” behaviors, and episodic biting behaviors in a sample of 138 Malinois. We next investigated whether PolyA(22) was associated with 1) increased locomotor activity and 2) response to novelty. Using a sample of 22 Malinois and 25 dogs of other breeds, dogs’ activity was monitored in a novel and non-novel environment while wearing activity monitoring collars. All dogs were more active in novel compared with non-novel environments, and Malinois were more active overall than other breeds. There was an effect of PolyA(22) genotype on activity levels, and this effect appeared to underlie the difference detected between Malinois and other breeds. There was no effect of PolyA(22) genotype on the relative decrease in activity between novel and non-novel environments for either group or all dogs considered together. In addition to an association between PolyA(22) and owner reports of seizure, “glazing over” behaviors, and episodic biting behaviors, these findings support an effect of PolyA(22) on dopamine transporter function related to activity. Further investigation is required to confirm mechanistic effects of PolyA(22) on SLC6A3. The complex polygenic nature of behavior and the range of behaviors associated with this insertion predict that effects are likely also modified by additional genetic and environmental factors.     

The Shepherds’ Tale: A Genome-Wide Study across 9 Dog Breeds Implicates Two Loci in the Regulation of Fructosamine Serum Concentration in Belgian Shepherds

Diabetes mellitus is a serious health problem in both dogs and humans. Certain dog breeds show high prevalence of the disease, whereas other breeds are at low risk. Fructosamine and glycated haemoglobin (HbA1c) are two major biomarkers of glycaemia, where serum concentrations reflect glucose turnover over the past few weeks to months. In this study, we searched for genetic factors influencing variation in serum fructosamine concentration in healthy dogs using data from nine dog breeds. Considering all breeds together, we did not find any genome-wide significant associations to fructosamine serum concentration. However, by performing breed-specific analyses we revealed an association on chromosome 3 (p_corrected ≈ 1:68 × 10^-6) in Belgian shepherd dogs of the Malinois subtype. The associated region and its close neighbourhood harbours interesting candidate genes such as LETM1 and GAPDH that are important in glucose metabolism and have previously been implicated in the aetiology of diabetes mellitus. To further explore the genetics of this breed specificity, we screened the genome for reduced heterozygosity stretches private to the Belgian shepherd breed. This revealed a region with reduced heterozygosity that shows a statistically significant interaction (p = 0.025) with the association region on chromosome 3. This region also harbours some interesting candidate genes and regulatory regions but the exact mechanisms underlying the interaction are still unknown. Nevertheless, this finding provides a plausible explanation for breed-specific genetic effects for complex traits in dogs. Shepherd breeds are at low risk of developing diabetes mellitus. The findings in Belgian shepherds could be connected to a protective mechanism against the disease. Further insight into the regulation of glucose metabolism could improve diagnostic and therapeutic methods for diabetes mellitus.     

An overview of types of aggressive behaviour in dogs and methods of treatment

In 223 cases of dogs presented to a specialist behavioural clinic in Brisbane, Australia, 87 (39%) were for severe aggression. The classes of aggression included dominance (31.6%), territorial (29%), predatory (12.3%), intermale (12.3%), sibling rivalry (7.9%), fear biting (6%) and idiopathic rage (0.9%). The breeds most represented which attacked humans were the Bull Terrier (16%), German Shepherd and crosses (15%), Cattle dog breeds (Blue Heeler and crosses, 9.2%), Terrier breeds (9.2%), Labrador (8%), Poodle and Cocker Spaniel (both 5.7%) and Rottweiler (4.6%). The dangerous dog list put out by the local Brisbane City Council includes the first three breeds mentioned and the Rottweiler as the top four breeds causing aggression problems.

Hospital records in Victoria and Queensland confirm that most damage is caused to humans by Bull Terriers and German Shepherds. Many breeds similar to those in our study are also represented in American data on aggressive breeds.

Treatments included obedience training only, restraint only, obedience and restraint, synthetic progestins and obedience, castration, progestins and obedience, castration and obedience, use of chlorpromazine and as a last resort, euthanasia (12.6%). Entire males formed the largest group (44%), followed by castrated males and females (both 21%) and spayed females (15%).

Several breeds (Boxer, Briand, Samoyed and St. Bernard) only attacked other animals and birds.

This study reinforces evidence that social disruption is caused by aggressive dogs, but it also indicates that many responsible clients seek advice on how to deal with this behavioural problem.     

Deletion of the SELENOP gene leads to CNS atrophy with cerebellar ataxia in dogs

We investigated a hereditary cerebellar ataxia in Belgian Shepherd dogs. Affected dogs developed uncoordinated movements and intention tremor at two weeks of age. The severity of clinical signs was highly variable. Histopathology demonstrated atrophy of the CNS, particularly in the cerebellum. Combined linkage and homozygosity mapping in a family with four affected puppies delineated a 52 Mb critical interval. The comparison of whole genome sequence data of one affected dog to 735 control genomes revealed a private homozygous structural variant in the critical interval, Chr4:66,946,539_66,963,863del17,325. This deletion includes the entire protein coding sequence of SELENOP and is predicted to result in complete absence of the encoded selenoprotein P required for selenium transport into the CNS. Genotypes at the deletion showed the expected co-segregation with the phenotype in the investigated family. Total selenium levels in the blood of homozygous mutant puppies of the investigated litter were reduced to about 30% of the value of a homozygous wildtype littermate. Genotyping >600 Belgian Shepherd dogs revealed an additional homozygous mutant dog. This dog also suffered from pronounced ataxia, but reached an age of 10 years. Selenop^-/- knock-out mice were reported to develop ataxia, but their histopathological changes were less severe than in the investigated dogs. Our results demonstrate that deletion of the SELENOP gene in dogs cause a defect in selenium transport associated with CNS atrophy and cerebellar ataxia (CACA). The affected dogs represent a valuable spontaneous animal model to gain further insights into the pathophysiological consequences of CNS selenium deficiency.     

Novel repeat polymorphisms of the dopaminergic neurotransmitter genes among dogs and wolves

Genetic polymorphisms of the neurotransmission systems are intensively studied in the human because of a possible influence on personality traits and the risk of psychiatric disorders. The investigation of genetic variations of the dog genome has recently been a promising approach, as a considerable similarity can be observed between dogs and humans, in both genetic and social aspects, suggesting that the dog could become an appropriate animal model of human behavioral genetic studies. The aim of our study was the identification and analysis of variable number of tandem repeats polymorphisms (VNTRs) in the genes of the dopaminergic neurotransmitter system of dogs. The in silico search was followed by the development of PCR-based techniques for the analysis of the putative VNTRs. Highly variable repetitive sequence regions were found in the tyrosine hydroxylase (TH), dopamine transporter (DAT), and dopamine β-hydroxylase (DBH) genes. Allele frequency and genotype distribution of these novel polymorphisms together with the exon 3 and exon 1 VNTR of the dopamine D4 receptor gene were determined in a large sample involving four dog breeds (German Shepherd, Belgian Tervueren, Groenandael, and Malinois) and European Grey Wolves. A significant difference of allele and genotype frequencies was demonstrated among the analyzed breeds; therefore, an association analysis was also carried out between the activity–impulsivity phenotype and the described VNTRs. Preliminary findings are presented that polymorphisms of the DRD4, DBH, and DAT genes can be associated with attention deficit among Belgian Tervuerens.     

Use of Accelerometers to Measure Stress Levels in Shelter Dogs

Stress can compromise welfare in any confined group of nonhuman animals, including those in shelters. However, an objective and practical method for assessing the stress levels of individual dogs housed in a shelter does not exist. Such a method would be useful for monitoring animal welfare and would allow shelters to measure the effectiveness of specific interventions for stress reduction. In this pilot study, activity levels were studied in 13 dogs using accelerometers attached to their collars. Behavioral stress scores as well as urinary and salivary cortisol levels were measured to determine if the dogs' activity levels while confined in the kennel correlated with behavioral and physiological indicators of stress in this population. The results indicated that the accelerometer could be a useful tool to study stress-related activity levels in dogs. Specific findings included a correlation between the salivary cortisol and maximum activity level (r = .62, p = .025) and a correlation between the urine cortisol-to-creatinine ratio and average activity level (r = .61, p = .028) among the study dogs. Further research is needed to better understand the complex relationship between stress and activity level among dogs in a kennel environment.     

Effects of weight, temperature and behaviour on the circadian rhythm of salivary cortisol in growing pigs

In farm animals, salivary cortisol has become a widely used parameter for measuring stress responses. However, only few studies have dealt with basal levels of concentration of cortisol in pigs and its circadian rhythm. The aim of this study was to examine the effects of ambient temperature and thermoregulatory behaviour on the circadian rhythm of salivary cortisol levels in fattening pigs. Subjects were 30 fattening pigs of different weight (60 to 100 kg), kept in six groups in an uninsulated building in pens with partly slatted floors. Saliva samples were taken every 2 h over periods of 24 h at different ambient temperatures at two times in winter and four times in summer. Thermoregulatory behaviour was recorded in the same 24-h time periods. The effect of time of day, body weight, ambient temperature and behaviour on the cortisol level was analysed using a mixed-effects model. Two peaks of cortisol levels per day were found. This circadian pattern became more pronounced with increasing weight and on days where thermoregulatory behaviour was shown. Mean cortisol levels per day were affected by weight but not by thermoregulatory behaviour. From our data, we conclude that long-term variations in cortisol concentration may be influenced by increasing age and weight more than by the respective experimental situation. In assessing animal welfare, it seems more reliable to consider the circadian pattern of cortisol concentration instead of only one value per day.     

Reduction of saliva immunoglobulin levels by swim training

Saliva immunoglobulin A (IgA) and cortisol levels were measured in 21 male members of a major midwestern swim team. Saliva samples were collected before and after training sessions four times during the fall season; the training intensity was light, moderate, heavy and during the taper period before a major competitive meet. Saliva IgA levels were decreased after each training session, reaching statistical significance with the moderate training intensity. Over the 3-month training period the pre-session and post-session IgA levels both decreased significantly during the heavy and taper training intensities later in the fall season. Cortisol levels were significantly elevated only after the heavy-intensity training session. The Profile of Mood States (POMS) was used to assess the swimmers' overall mood on each test day. No significant correlations were found between the global POMS score and IgA or cortisol. Also, cortisol and IgA were not significantly correlated except after the light training session. Results from this study indicate that acute bouts of exercise can reduce salivary IgA levels and that chronic exercise of high intensity can reduce the resting levels of IgA. These changes may render the athletes more vulnerable to respiratory infections after exercise and even at rest during the later stages of the competitive season.     

Characterization of the dog <Emphasis Type="Italic">Agouti</Emphasis> gene and a <Emphasis Type="Italic">nonagouti</Emphasis>mutation in German Shepherd Dogs

The interaction between two genes, Agouti and Melanocortin-1 receptor (Mc1r), produces diverse pigment patterns in mammals by regulating the type, amount, and distribution pattern of the two pigment types found in mammalian hair: eumelanin (brown/black) and pheomelanin (yellow/red). In domestic dogs (Canis familiaris), there is a tremendous variation in coat color patterns between and within breeds; however, previous studies suggest that the molecular genetics of pigment-type switching in dogs may differ from that of other mammals. Here we report the identification and characterization of the Agouti gene from domestic dogs, predicted to encode a 131-amino-acid secreted protein 98% identical to the fox homolog, and which maps to chromosome CFA24 in a region of conserved linkage. Comparative analysis of the Doberman Pinscher Agouti cDNA, the fox cDNA, and 180 kb of Doberman Pinscher genomic DNA suggests that, as with laboratory mice, different pigment-type-switching patterns in the canine family are controlled by alternative usage of different promoters and untranslated first exons. A small survey of Labrador Retrievers, Greyhounds, Australian Shepherds, and German Shepherd Dogs did not uncover any polymorphisms, but we identified a single nucleotide variant in black German Shepherd Dogs predicted to cause an Arg-to-Cys substitution at codon 96, which is likely to account for recessive inheritance of a uniform black coat.Genbank accession numbers are AC092250 (bacterial artificial chromosome clone RP81-20712) and AY714374 (Doberman Pinscher Agouti cDNA).     

Training methods of military dog handlers and their effects on the team's performances

While only a few studies have analysed training methods used on working dogs, a recent survey in 303 Belgian military handlers revealed the use of harsh training methods on military working dogs (MWD). The present work aims at analysing the training methods used on Belgian MWD and the behaviour of handlers to objectify the performances of the dog handlers teams (DH teams) and the welfare of the animals.A standardized evaluation, including obedience and protection work exercises, was conducted on DH teams (n = 33). Every evaluation was done twice to assess the reliability of the observation methods. The behaviours of MWD and handlers were recorded on videotape and subsequently analysed. Results showed that handlers rewarded or punished their dogs intermittently. Stroking and patting the dogs were the most frequently used rewards. Pulling on the leash and hanging dogs by their collars were the most commonly used aversive stimuli.The team's performance was influenced by the training method and by the dog's concentration: (1) low-performance dogs received more aversive stimuli than high-performance dogs; (2) dog's distraction influenced the performance: distracted dogs performed less well.Handlers punished more and rewarded less at the second evaluation than at the first one. This suggests that handlers modified their usual behaviour at the first evaluation in view to present themselves in a positive light. During the second evaluation the dogs reacted to this higher frequency of aversive stimuli as they exhibited a lower posture after aversive stimuli. The authors cannot prove that the welfare of these dogs had been hampered, but there is an indication that it was under threat.Low team performances suggest that DH teams should train more regularly and undertake the usefulness of setting a new training system that would rely on: the use of more positive training methods, an increased training frequency, the elaboration of a course on training principles, and an improvement of dog handler relationship.     

Mapping quantitative trait loci for canine hip dysplasia in German Shepherd dogs

Canine hip dysplasia (CHD) is a common hereditary developmental disease of the coxofemoral joints. CHD is characterized by subluxation of the femoral head and deformation of the acetabulum leading to a painful osteoarthrosis. Analyses of mode of inheritance have shown the involvement of a major gene in expression of CHD in German Shepherd dogs. Thus, a whole genome scan for quantitative trait loci (QTL) was performed in German Shepherd dogs. For this purpose 11 paternal half-sib families, including a total of 459 purebred German Shepherd dogs with sires, dams, and offspring, were genotyped for 261 microsatellites. These markers were equidistantly distributed over all 38 autosomes and the X chromosome with an average marker distance of 11.7 cM. The mean observed heterozygosity of the marker set was 50%. The CHD status for the dogs was scored according to the official rules of the Fédération Cynologique Internationale. At the genome-wide level of significance at p < 0.05, QTL for CHD were located on nine different canine chromosomes: 1, 3, 4, 8, 9, 16, 19, 26, and 33. The minimal QTL regions containing the CHD genes spanned on average 5 Mb with a range between 1 and 8.2 Mb. Chromosome-wide level of significance at p < 0.05 was found for QTL on 19 chromosomes. Further analyses can now be performed to refine these map positions of QTL already identified in German Shepherd dogs. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Salivary cortisol assessment for stress detection in the Asian elephant (Elephas maximus): A pilot study

Effects of introducing an unfamiliar female into an Asian elephant herd at Tierpark Berlin were monitored by means of salivary cortisol assessment. Saliva samples were obtained from a second female for comparative purposes. The period of familiarization was characterized by an enhanced cortisol level in both animals, with a maximum on the second day after joining. Cortisol returned to normal on the following day. Manipulations of the keepers caused a transitory increase on two other days. Possibilities for the use of this noninvasive method of stress monitoring in various management situations are indicated. © 1992 Wiley-Liss Inc.     

Diurnal variation of salivary cortisol in captive African elephants (Loxodonta africana) under routine management conditions and in relation to a translocation event

The present study assessed the diurnal variation in salivary cortisol in captive African elephants during routine management (baseline) and in relation to a potential stressor (translocation) to evaluate to what extent acute stress may affect diurnal cortisol patterns. Under baseline conditions, we collected morning and afternoon saliva samples of 10 animals (three zoos) on different days in two study periods (n = 3–10 per animal, daytime and period). Under stress conditions, we sampled the transported cow (newcomer) and the two cows of the destination zoo before and after the transport in the morning and afternoon (n = 3–9 per animal, daytime and transport phase), as well as after the first introduction of the newcomer to the bull (n = 1 per animal). Cortisol was measured in unextracted samples by enzyme immunoassay. Under baseline conditions, we observed the expected diurnal variation with higher cortisol levels in the morning than in the afternoon. Under stress conditions, neither a significant difference between pre- and posttransport, nor between morning and afternoon levels was found. The percentage difference between morning and afternoon cortisol after the transport, however, was remarkably lower than before the transport in the newcomer potentially indicating a stress response to familiarization. Saliva samples taken immediately after the introduction of the newcomer to the bull revealed a marked cortisol increase. Our findings indicate that stressors may disturb the diurnal cortisol rhythm. Furthermore, provided that samples can be collected promptly, salivary cortisol is a useful minimally invasive measure of physiological stress in the African elephant.     

Quantifying blood leakage into the oral mucosa and its effects on the measurement of cortisol, dehydroepiandrosterone, and testosterone in saliva

The impact of blood leakage due to microinjury to the oral cavity on the measurement of salivary hormones was examined. Saliva samples were collected before, immediately after, and then every 15 min for 1 h following vigorous tooth brushing. Blood in saliva was quantified by visual inspection of discoloration, Hemastix reagent strips to detect hemoglobin, and an immunoassay for transferrin. The presence of blood in saliva immediately after microinjury was confirmed by all methods. Hemoglobin and transferrin levels remained elevated over baseline for at least 30 min. Levels of salivary testosterone increased over baseline and remained elevated for 30 min in response to microinjury. Microinjury induced change in salivary testosterone was more closely associated with the change in transferrin than hemoglobin levels or discoloration ratings. On average, levels of salivary dehydroepiandrosterone (DHEA) did not increase in response to microinjury. However, individual differences in microinjury induced change in DHEA were associated with discoloration ratings. Salivary cortisol levels, on average, were neither responsive to microinjury, nor were individual differences in cortisol change associated with blood contamination measures. Neither diurnal nor gender-related differences in baseline hormone levels predicted the impact of blood leakage on quantitative salivary measurements. The findings suggest ecologically valid minor-to-moderate level microinjuries to the oral cavity have negligible effects on the measurement of salivary cortisol, but may be important to quantify and control when assessing other hormones especially testosterone.     

Saliva sampling to assess cortisol levels in unrestrained common marmosets and the effect of behavioral stress

We report a method for taking saliva samples from unrestrained, captive marmosets (Callithrix jacchus) to assess levels of free cortisol. Saliva samples can be obtained reliably, without any habituation, by encouraging the marmosets to lick and chew a cotton-wool bud coated in banana. Saliva is thus left on the bud. We also tested sweetened fruit-drink crystals and a number of other substances, but none of these attracted all of the marmosets, and even flavors that were effective once soon lost their attraction. The presence of banana in the samples collected was found to lower the measured concentration of cortisol; however, as shown in samples taken with and without the banana coating on the bud, it did so in a linear and consistent way, and did not vary significantly among subjects. Therefore, a simple conversion factor could be applied to correct for the presence of banana. A first experiment showed that the marmosets exhibited a rise in salivary cortisol levels in response to social isolation. A second experiment showed elevation of cortisol during a period when the marmosets were disturbed by increased human activity and noise levels in the building in which they were housed. Hence, this method of saliva sampling is a convenient, noninvasive means of assessing cortisol levels in marmosets.     

Cortisol and behavioral responses to enrichment in military working dogs

Our objective was to compare the effects of two schedules of enrichment exposure, a distributed (high frequency) and regular regime versus a mass (low frequency) and irregular regime, on the cortisol and behavioral responses of 14 kennelled military working dogs. The enrichment experiences were exercise coupled with human contact. Both groups of dogs were exposed to approximately the same total amount of enrichment per week for a total of 7 weeks. The distributed and regular regime (DRR) furnished enrichment for 20-min intervals three to four times a day every weekday; the mass and irregular regime (MIR) randomly furnished enrichment for 16 or 24 continuous hours 1 day out of 5. Our data show that: (1) cortisol concentrations of DRR dogs significantly decreased between the first and the last week of observations; this was not the case in MIR dogs; (2) at the 7th week DRR dogs had lower cortisol concentrations than did MIR dogs; (3) all dogs in each group exhibited stereotypies. Our results suggest that MIR dogs underwent a decline in welfare throughout the experiment, whereas DRR dogs appeared to undergo a period of acute stress at the beginning of their training that resolved over 7 weeks. This study suggests that the welfare of kennelled dogs, particularly military working dogs, is affected among other factors by schedules of exercise and contact with people.     

Circadian Rhythm of Salivary Cortisol in Asian Elephants (Elephas maximus): A Factor to Consider During Welfare Assessment

Elevated glucocorticoid levels during an extended time period might be a stress indicator in nonhuman animals. Therefore, knowledge of the circadian pattern of cortisol secretion is very important to correctly interpret data obtained for welfare assessment of animals in captivity through salivary cortisol. In order to define the circadian rhythm of salivary cortisol secretion in the Asian elephant (Elephas maximus), morning and evening saliva samples of 3 Asian elephants were collected and analyzed by radioimmunoassay. Significantly higher salivary cortisol concentrations were found in the morning than in the evening in all individuals. These results show that salivary cortisol of Asian elephants follows a diurnal pattern of secretion, which could be taken into account when using this methodology to assess welfare in captive Asian elephants.     

Plasma cortisol and faecal cortisol metabolites concentrations in stereotypic and non-stereotypic horses: do stereotypic horses cope better with poor environmental conditions?

Background

Stereotypic behaviours, i.e. repetitive behaviours induced by frustration, repeated attempts to cope and/or brain dysfunction, are intriguing as they occur in a variety of domestic and captive species without any clear adaptive function. Among the different hypotheses, the coping hypothesis predicts that stereotypic behaviours provide a way for animals in unfavourable environmental conditions to adjust. As such, they are expected to have a lower physiological stress level (glucocorticoids) than non-stereotypic animals. Attempts to link stereotypic behaviours with glucocorticoids however have yielded contradictory results. Here we investigated correlates of oral and motor stereotypic behaviours and glucocorticoid levels in two large samples of domestic horses (N _Study1 = 55, N _Study2 = 58), kept in sub-optimal conditions (e.g. confinement, social isolation), and already known to experience poor welfare states. Each horse was observed in its box using focal sampling (study 1) and instantaneous scan sampling (study 2). Plasma samples (collected in study 1) but also non-invasive faecal samples (collected in both studies) were retrieved in order to assess cortisol levels.

Results

Results showed that 1) plasma cortisol and faecal cortisol metabolites concentrations did not differ between horses displaying stereotypic behaviours and non-stereotypic horses and 2) both oral and motor stereotypic behaviour levels did not predict plasma cortisol or faecal cortisol metabolites concentrations.

Conclusions

Cortisol measures, collected in two large samples of horses using both plasma sampling as well as faecal sampling (the latter method minimizing bias due to a non-invasive sampling procedure), therefore do not indicate that stereotypic horses cope better, at least in terms of adrenocortical activity.