Determining the protocol requirements of in-home cat food digestibility testing.

In-home cat food digestibility testing has the potential to yield data that are highly representative of the pet population for which the food is intended. However, no standardized and validated in-home digestibility test protocols are currently available. Such protocols for in-home testing should address key factors that explain variation in cat food digestibility values and here we investigated the required period of adaptation, fecal collection and sample sizes. Thirty privately-owned indoor housed cats of various breeds (20♀ 10♂, 5.9 ± 3.9 yr, 4.5 ± 1.3 kg) received a relatively low and high digestible complete dry extruded food with the marker titanium (Ti) dioxide. Foods were given in a cross-over design of 2 periods of 8 consecutive days each. Owners collected feces daily for the determination of daily fecal Ti concentrations and digestibility of dry matter, crude protein, crude fat, and gross energy. Data originating from 26 cats were analyzed as mixed models and broken line regressions to investigate the required adaptation and fecal collection period. Bootstrap sampling was used to assess the impact of increasing the number of fecal collection days and sample size on the precision of the digestibility estimates. Feces were collected on 347 out of 416 study days (16 days/cat; 26 cats), implying the necessity for multiple collection days to account for cats not defecating every day. Cats showed stable fecal marker concentrations from day 2 onwards when fed the low digestible food and from 3 onwards when fed the high digestible food. Digestibility values were stable from day 1, 2 or 3 onwards, depending on the test food and nutrient. Increasing the number of fecal collection days from 1 to 6 days did not result in more precise digestibility estimates, whereas increasing the number of animals from 5 to 25 cats did. For future in-home digestibility tests of cat food, the findings support a minimum of 2 adaptation days and 3 fecal collection days. Appropriate sample sizes depend on the test food, the nutrient of interest, and the acceptable margin of error. The findings of this study support the protocol development for future in-home digestibility testing of cat foods.

Assessing the welfare impact of foot disorders in dairy cattle by a modeling approach.

Foot disorders are the main cause of dairy cow lameness and are considered to have a major impact on the welfare of dairy cattle. This study adopts a modeling approach, using a dynamic stochastic model, to provide more insight into the welfare impact of different types of foot disorders, both clinical and subclinical. The impact of specific foot disorders on welfare was assessed by simulating the incidence and duration of foot disorders and the pain associated with them. Pain assessment was based on locomotion scores, with underlying knowledge obtained from scientific literature and experts. The results demonstrated the seriousness of the welfare impact of foot disorders. The negative welfare impact was measured on a scale from 0 to 60, where the maximum outcome represents a cow having very severe pain during the whole year. On average, each cow achieves a welfare impact score of 12, which is 20% of the maximum welfare impact score. This welfare score equals having severe pain for a period of 3 months, indicating a serious impact on welfare. On average, digital dermatitis impacts most on welfare, which is caused by a high incidence of the painful clinical stage, followed by sole hemorrhages (SoH) and interdigital dermatitis and heel horn erosion (IDHE). The combination of a high incidence and long duration of SoH and IDHE causes this relatively high welfare impact of foot disorders that occur mostly subclinically. On average, over 1 year, 46% of the welfare impact due to foot disorders is caused by clinical foot disorders. The fact that subclinical foot disorders contribute more or less equally to the effects on welfare as clinical ones, indicates that farmers may readily underestimate the welfare impact by a factor two. Modeling welfare impact at cow level, individual cases of foot disorders, stresses the importance of pain intensity, indicating the importance of clinical foot disorders. This study demonstrated the serious welfare impact of foot disorders in dairy cattle and pointed out the considerable impact of subclinical foot disorders. Furthermore, the approach of welfare assessment, for example herd v. cow level, influenced the ranking of foot disorders for their impact on animal welfare. Potentially, this leads to different prioritization of specific solution strategies for dairy farmers, for example, focusing on cow comfort, hygiene or preventive medical treatments, foot trimming and/or health monitoring. The findings in this study support in raising awareness about this welfare issue.

Gene expression patterns in the ventral tegmental area relate to oestrus behaviour in high-producing dairy cows.

Reduced oestrus behaviour expression or its absence (silent oestrus) results in subfertility in high-producing dairy cows. Insight into the genomic regulation of oestrus behaviour is likely to help alleviate reproduction problems. Here, gene expression was recorded in the ventral tegmental area (VTA) of high milk production dairy cows differing in the degree of showing oestrus behaviour (H - highly expressing versus L - lowly expressing), which was then analysed. Genes regulating cell morphology and adhesion or coding for immunoglobulin G (IgG) chains were differentially expressed in VTA between cows around day 0 and 12 of the oestrus cycle, but only in cows that earlier in life tended to show high levels of oestrus behaviour (H0 versus H12). The comparisons between H and L groups of cows also revealed differential expression of several genes (e.g. those of the IgG family or encoding for pro-melanin-concentrating hormone). However, any significant changes in VTA genes expression were detected in the comparison of L0 versus L12 cows. Altogether, the genes expression profile in VTA of cows highly expressing oestrus behaviour changes together with phases of the oestrus cycle, while in case of cows expressing oestrus behaviour lowly it remains stable. This supports the existence of genomic regulation by centrally expressed genes on the expression of oestrus behaviour in dairy cows.

Estrous behavior in dairy cows: identification of underlying mechanisms and gene functions.

Selection in dairy cattle for a higher milk yield has coincided with declined fertility. One of the factors is reduced expression of estrous behavior. Changes in systems that regulate the estrous behavior could be manifested by altered gene expression. This literature review describes the current knowledge on mechanisms and genes involved in the regulation of estrous behavior. The endocrinological regulation of the estrous cycle in dairy cows is well described. Estradiol (E2) is assumed to be the key regulator that synchronizes endocrine and behavioral events. Other pivotal hormones are, for example, progesterone, gonadotropin releasing hormone and insulin-like growth factor-1. Interactions between the latter and E2 may play a role in the unfavorable effects of milk yield-related metabolic stress on fertility in high milk-producing dairy cows. However, a clear understanding of how endocrine mechanisms are tied to estrous behavior in cows is only starting to emerge. Recent studies on gene expression and signaling pathways in rodents and other animals contribute to our understanding of genes and mechanisms involved in estrous behavior. Studies in rodents, for example, show that estrogen-induced gene expression in specific brain areas such as the hypothalamus play an important role. Through these estrogen-induced gene expressions, E2 alters the functioning of neuronal networks that underlie estrous behavior, by affecting dendritic connections between cells, receptor populations and neurotransmitter releases. To improve the understanding of complex biological networks, like estrus regulation, and to deal with the increasing amount of genomic information that becomes available, mathematical models can be helpful. Systems biology combines physiological and genomic data with mathematical modeling. Possible applications of systems biology approaches in the field of female fertility and estrous behavior are discussed.

Gene expression patterns in anterior pituitary associated with quantitative measure of oestrous behaviour in dairy cows.

Intensive selection for high milk yield in dairy cows has raised production levels substantially but at the cost of reduced fertility, which manifests in different ways including reduced expression of oestrous behaviour. The genomic regulation of oestrous behaviour in bovines remains largely unknown. Here, we aimed to identify and study those genes that were associated with oestrous behaviour among genes expressed in the bovine anterior pituitary either at the start of oestrous cycle or at the mid-cycle (around day 12 of cycle), or regardless of the phase of cycle. Oestrous behaviour was recorded in each of 28 primiparous cows from 30 days in milk onwards till the day of their sacrifice (between 77 and 139 days in milk) and quantified as heat scores. An average heat score value was calculated for each cow from heat scores observed during consecutive oestrous cycles excluding the cycle on the day of sacrifice. A microarray experiment was designed to measure gene expression in the anterior pituitary of these cows, 14 of which were sacrificed at the start of oestrous cycle (day 0) and 14 around day 12 of cycle (day 12). Gene expression was modelled as a function of the orthogonally transformed average heat score values using a Bayesian hierarchical mixed model on data from day 0 cows alone (analysis 1), day 12 cows alone (analysis 2) and the combined data from day 0 and day 12 cows (analysis 3). Genes whose expression patterns showed significant linear or non-linear relationships with average heat scores were identified in all three analyses (177, 142 and 118 genes, respectively). Gene ontology terms enriched among genes identified in analysis 1 revealed processes associated with expression of oestrous behaviour whereas the terms enriched among genes identified in analysis 2 and 3 were general processes which may facilitate proper expression of oestrous behaviour at the subsequent oestrus. Studying these genes will help to improve our understanding of the genomic regulation of oestrous behaviour, ultimately leading to better management strategies and tools to improve or monitor reproductive performance in bovines.

Does enhancement of specific immune responses predispose laying hens for feather pecking?

To mimic airborne immune challenges, layer hens were intratracheally and concurrently challenged with various doses of the protein antigen human serum albumin (HuSA) and the pathogen-associated molecular pattern lipopolysaccharide (LPS) at 7 and 13 wk of age. All groups received 1 similar dose of HuSA plus LPS at 11 mo of age. Evaluation of plumage and body condition at 12 mo of age revealed that birds that had undergone intratracheal immunization with a high dosage of HuSA, irrespective of the concurrent dose of LPS, had significantly more feather damage but less wounds to the vent region, as opposed to birds not receiving HuSA. On the other hand, a high dosage of LPS was related to comb damage. These results suggest that stimulation of specific (humoral) immune responses (to HuSA) rather than innate responses (to LPS) at a young age may predispose layers for feather pecking (FP) behavior at later ages. Involvement of immune mechanisms in FP or vent damage may differ. Predisposal for FP behavior by specific immunity can have consequences for health and vaccine management.

Relationship between milk progesterone profiles and genetic merit for milk production, milking frequency, and feeding regimen in dairy cattle.

Milk progesterone profiles were determined from samples obtained twice weekly for 100 d postpartum in 100 Holstein primiparous cows at a Dutch experimental farm. Three treatments were applied in a 2 x 2 x 2 factorial arrangement with high-low genetic merit for overall production, high-low caloric density diet, and 2-3 times milking/day as factors. Milk progesterone profiles were characterized by start of first ovarian cyclical activity (commencement of luteal activity, C-LA), length and peak milk progesterone concentration of first ovarian cycle, and number of ovarian cycles in first 100 d postpartum, as well as classified into normal, delayed, prolonged, and interrupted ovarian cyclical activity. Cows with a greater milk production had lower peak progesterone concentrations, especially if the high milk production was caused by milking 3 times a day. A more negative energy and protein balance was associated with later C-LA and less ovarian cycles within 100 d postpartum. Relationships between protein balance and C-LA differed between cows with a high genetic merit and a low genetic merit. Cows with a high genetic merit for production showed delayed C-LA with more negative protein balances, whereas this association was not observed among cows with a low genetic merit. Cows in negative energy balance had greater risk for prolonged ovarian cycles when there was no delay in C-LA than when C-LA was delayed.

Expression profiles of genes regulating dairy cow fertility: recent findings, ongoing activities and future possibilities.

Subfertility has negative effects for dairy farm profitability, animal welfare and sustainability of animal production. Increasing herd sizes and economic pressures restrict the amount of time that farmers can spend on counteractive management. Genetic improvement will become increasingly important to restore reproductive performance. Complementary to traditional breeding value estimation procedures, genomic selection based on genome-wide information will become more widely applied. Functional genomics, including transcriptomics (gene expression profiling), produces the information to understand the consequences of selection as it helps to unravel physiological mechanisms underlying female fertility traits. Insight into the latter is needed to develop new effective management strategies to combat subfertility. Here, the importance of functional genomics for dairy cow reproduction so far and in the near future is evaluated. Recent gene profiling studies in the field of dairy cow fertility are reviewed and new data are presented on genes that are expressed in the brains of dairy cows and that are involved in dairy cow oestrus (behaviour). Fast-developing new research areas in the field of functional genomics, such as epigenetics, RNA interference, variable copy numbers and nutrigenomics, are discussed including their promising future value for dairy cow fertility.

Genetics and genomics to improve fertility in high producing dairy cows.

Improving dairy cow fertility by means of genetic selection is likely to become increasingly important, since it is now well established that declining fertility cannot only be arrested by improved management. Profit margins per kg milk produced are decreasing, therefore farmers need to reduce cost and increase herd size. This restricts the labor input per cow and the disposable cost of getting a cow pregnant, whilst at the same time hormone treatments have become less acceptable. This makes it unlikely that additional management interventions will maintain fertility at acceptable levels in the near future. Genetic improvement seems the obvious solution. Effective selection tools are available in most Western countries using traditional breeding value estimation procedures. Also, in addition to gene assisted selection using individual genes or QTL, high throughput Single Nucleotide Polymorphism (SNP) technology allows genetic improvement of fertility based on information from the whole genome (tens of thousands SNP per animal), i.e. genomic selection. Simulation studies have shown that genomic selection improves the accuracy of selecting juvenile animals compared with traditional breeding methods and compared with selection using information from a few genes or QTL only. Research in the areas genomics and proteomics promise to make genetic selection even more effective. The genomic and proteomics technologies combined with the bioinformatics tools that support the interpretation of gene functioning and protein expression facilitate an exciting starting point for the development of new management strategies and tools for the improvement of reproductive performance.

Effects of genotype by environment interactions on milk yield, energy balance, and protein balance.

Increases in genetic merit for milk yield are associated with increases in mobilization of body reserves. This study assessed the effects of genotype by environment (GxE) interactions on milk yield and energy and protein balances. Heifers (n = 100) with high or low genetic merit for milk yield were milked 2 or 3 times a day and received rations of low or high caloric density. The management factors were selected to induce substantial differences in milk production levels and model different management strategies. The 2 x 2 x 2 factorial arrangement enables the assessment of the effects of genotype, environment, and GxE interactions. Mean daily energy-corrected milk production in the first 100 d in milk varied between 21.8 and 35.2 kg among the groups. The experimental factors affected milk production in the presumed direction. Ration was the most determinant factor on milk production. Effects of milking frequency and genetic merit were significant only in the groups that were fed rations with high caloric density. Signs for severe negative energy balances, protein balances, and low body condition scores, all of which may be indicative of health risks, were not concentrated in the highest producing cows. Feed caloric density and milking frequency had stronger effects on energy balances and protein balances, with unfavorable effects of low caloric density feed and an extra milking. This emphasizes the possible effect of mismanagement on animal health risks. High genetic merit cows had significantly lower postpartum body condition scores. Genotype x environment interactions existed, but more information is needed to determine if cows of different genetic merit for milk yield are differently at risk for disease under specific conditions. High milk production levels per se will increase allostatic load, but need not compromise the health status of relatively young cows. Ongoing one-sided selection for high yield may be combined with good animal health, but because high genetic merit for milk yield seems intrinsically connected to the allocation of resources from maintenance toward milk, this puts increasing demands on farmers' time and management skills.

Effects of management and genetics on udder health and milk composition in dairy cows.

Milk production per cow has increased significantly as a result of breeding, feeding, and other management factors. This study aims to address concerns about udder health risks for low- and high-producing dairy cows. In a 2 x 2 x 2 factorial design, Holstein-Friesian heifers (n = 100) of low or high genetic merit for milk production, milked 2 or 3 times a day, and fed a mixed ration with low or high energy content, were compared during the first 14 wk of lactation. Milk composition and cell counts were determined weekly; quarter milk samples for bacteriology were taken in wk 2, 8, and 14; and teat condition was scored in wk 2, 6, 10, and 14 during the experiment. The experimental factors resulted in substantial differences in milk production between treatment groups (24.1 for low vs. 25.6 kg/d for high genetic merit; 23.3 for 2 times vs. 26.5 kg/d for 3 times daily milking; and 20.9 for low-energy ration vs. 29.0 kg/d for high-energy ration). Ration composition was the most important determining factor for milk production, but did not affect cell counts or intramammary bacterial infections, although cows that received low-energy rations had rougher teat ends than cows receiving high-energy rations. This indicates that high production itself is not a major risk factor for udder health in the first lactation. A higher milking frequency impaired teat condition and improved cell counts in general, but did not clearly influence bacteriological status. High genetic merit was related to higher cell count, more Staphylococcus, and less Bacillus and other environmental pathogens in cultures and did not affect teat condition. The effects of milking frequency and feeding on udder health were similar for cows with high and low genetic merit. Genetic selection on milk production, without taking udder health into account, reduces udder health. As a result, maintaining udder health will require increasing the skills and time of dairy farmers who have to divide their attention to more cows when farm sizes increase, or selection should put more emphasis on udder health traits.

Impact of nutrition on canine behaviour: current status and possible mechanisms.

Each year, millions of dogs worldwide are abandoned by their owners, relinquished to animal shelters, and euthanised because of behaviour problems. Nutrition is rarely considered as one of the possible contributing factors of problem behaviour. This contribution presents an overview of current knowledge on the influence of nutrition on canine behaviour and explores the underlying mechanisms by which diet may affect behaviour in animals. Behaviour is regulated by neurotransmitters and hormones, and changes in the availability of their precursors may influence behaviour. Tryptophan, the precursor of serotonin, may affect the incidence of aggression, self-mutilation and stress resistance. The latter may also be influenced by dietary tyrosine, a precursor to catecholamines. As diet composition, nutrient availability and nutrient interactions affect the availability of these precursors in the brain, behaviour or stress resistance may be affected. PUFA, especially DHA, have an important role as structural constituents in brain development, and dietary supply of n-3 and n-6 PUFA could modify aspects of the dopaminergic and serotonergic system and, consequently, cognitive performance and behaviour. Finally, persistent feeding motivation between meals can increase stereotyped behaviour and aggression and decrease resting time. This feeding motivation may be altered by dietary fibre content and source. At present, few studies have been conducted to evaluate the role of nutrition in canine (problem) behaviour through the above mentioned mechanisms. Studies that explore this relationship may help to improve the welfare of dogs and their owners.

Genetic correlations between milk production and health and fertility depending on herd environment.

High milk production in dairy cattle can have negative side effects on health and fertility traits. This paper explores the genetic relationship of milk yield with health and fertility depending on herd environment. A total of 71,720 lactations from heifers calving in 1997 to 1999 in the Netherlands were analyzed. Herd environment was described by 4 principal components: intensity, average fertility, farm size, and relative performance indicating whether herds had good (poor) health and fertility despite a high (low) production. Fertility was evaluated by days to first service and number of inseminations (NINS); somatic cell score was used as a measure of udder health. Data were analyzed with a multitrait reaction norm model. Genetic correlation within traits across environments ranged from 0.84 to unity. Genetic correlations of the 3 traits with milk yield were antagonistic but varied over environments. Genetic correlation of milk yield with days to first service varied from 0.30 in small herds to 0.48 in herds with low average fertility. Correlations with NINS varied from 0.18 in large herds to 0.64 in high fertility herds, and with somatic cell score from 0.25 in herds with a high fertility relative to production to 0.47 in herds with a relative low fertility. Selection in environments of average value resulted in different predicted responses over environments. For example, selection for a decrease of NINS of 0.1 in an average production environment decreased milk yield by 35 kg in low production herds, but by 178 kg in high production herds.

Effects of milk production capacity and metabolic status on HPA function in early postpartum dairy cows.

Increasing milk yields in modern dairy cows cause concern that high yield may impair the cows' health and welfare, for example, via negative effects on metabolic status and hypothalamo-pituitary-adrenocortical (HPA) function. This study aims to investigate whether a high level of milk production, and the associated metabolic status, affects HPA function in dairy cows and changes their adaptive capacity. Additionally, it aims to establish whether possible effects of milk production level only show under challenging conditions. Holstein-Friesian cows, which produced on average 11,443 and 7727 kg of fat and protein-corrected milk (FPCM)/305 d in their previous lactation, were compared. During the dry period, the cows were fed to requirements or overfed. High milk yield and the concomitant large energy deficit were associated with 1) increased pituitary (re)activity, i.e., increased ACTH baseline concentrations and higher ACTH concentrations after corticotropin-releasing hormone (CRH) administration, and 2) decreased adrenocortical reactivity, i.e., lower cortisol responses after ACTH administration. Although significant, the effects of milk production level on HPA function were relatively small. Animals showed seemingly normal hormonal responses to CRH and ACTH administration. Also, cortisol baseline concentrations were unaffected. It seems, therefore, unlikely that the adaptive capacity of the high-producing cows was significantly impaired compared with their low-producing herdmates.

Chronic stress in dogs subjected to social and spatial restriction. I. Behavioral responses.

Six weeks of social and spatial restriction were used as a model to induce chronic stress in Beagles. Behavioral and physiological measurements were performed during a period of enriched spacious outdoor housing in groups (GH) and during a subsequent period of solitary housing in small indoor kennels (IH). Behavioral parameters that may indicate chronic stress in dogs are reported. During IH, the dogs showed significantly (comparison-wise error rate <0.05) lower postures than during GH. IH induced enduring increments in frequencies of autogrooming, paw lifting, and vocalizing, and was associated with incidents of coprophagy and repetitive behavior. So far, we interpret the behavioral changes as signs of chronic stress. Relatively low levels of walking, digging, intentions to change from one state of locomotion to another, and increments in circling are conceived as obvious adaptations to the specific features of the IH system. By challenging the dogs outside their home kennel we tested whether the dogs' coping abilities were affected by IH. Dogs that were challenged were introduced into a novel environment, given the opportunity to escape from their home kennel, restrained, walked down an unfamiliar corridor, presented a novel object, exposed to loud noise, given food, or confronted with a conspecific. During IH, challenged dogs exhibited higher postures, showed more tail wagging, nosing, circling, urinating, and defecating, and changed more often from one state of locomotion (or posture) to another than during GH. These behavioral changes were observed across the different types of challenges, with the exception of the noise administration test. In the presence of conspecifics, the socially and spatially restricted male dogs behaved more dominantly and aggressive than during the time that they were kept in groups. Such behavior manifested as increased performances of raised hairs, growling, paw laying, and standing over. Both sexes showed increases in paw lifting, body shaking, ambivalent postures, intentions to change from one state of locomotion to another, and trembling in any of the challenges, excluding the walking down the corridor test. In short, during a variety of challenges, socially and spatially restricted dogs exhibited a heightened state of aggression, excitement, and uncertainty. Behavioral differences between dogs that had experienced pleasant and bad weather conditions during GH, suggested that "pleasant-weather individuals" had experienced early stress during the control period, and, as a result, responded to the subsequent period of IH differently. Regardless of the housing conditions, challenged bitches showed stronger indications of acute stress than male dogs. Gender did not affect the chronic stress responses to social and spatial restriction. A low posture and increased auto-grooming, paw lifting, vocalizing, repetitive behavior, and coprophagy may indicate chronic stress in dogs, and as such, can help to identify poor welfare. When challenged, chronically stressed dogs may show increased excitement, aggression, and uncertainty, but the nonspecificity of such emotional behavior will complicate its practical use with regard to the assessment of stress.

Chronic stress in dogs subjected to social and spatial restriction. II. Hormonal and immunological responses.

Two groups of beagles, accustomed to spacious group housing, were subjected to social and spatial restriction and studied for manifestations of chronic stress with a time interval of 7 weeks between the groups. The change from outside group housing (the control period) to individual housing in small indoor kennels resulted in sustained decreases in urinary adrenaline/creatinine and noradrenaline/creatinine ratios for the total group. Urinary dopamine/creatinine and noradrenaline/adrenaline ratios were statistically unaffected. Socially and spatially restricted dogs that had experienced pleasant weather during the control period showed (a) increased salivary and urinary cortisol concentrations, (b) a diminished responsiveness of the pituitary-adrenal axis to a sudden sound blast or exogenous CRH, (c) intact plasma ACTH and cortisol suppressions after dexamethasone administration, and (d) increased concanavalin A induced lymphocyte proliferations. When social and spatial restriction was preceded by a control period during which the weather was bad, these physiological responses were either augmented (lymphocyte proliferation), or offset (salivary and urinary cortisol), or directed oppositely (CRH-induced ACTH and cortisol responses). Together with the previously presented behavioral observations, these data suggest that bad weather conditions during spacious outdoor group housing induced early stress that attenuated the negative appraisal of the subsequent period of social and spatial restriction. In comparison to male dogs, bitches showed increased HPA responses to a sound blast or exogenous CRH. Their increased attenuations of the ACTH and cortisol responses to CRH after 5 weeks of restricted housing indicates that bitches are not only more susceptible to acute stress, but also to chronic housing stress. It is concluded that the quality of circumstances preceding a period of affected well-being determines the magnitude and even the direction of the behavioral and physiological stress responses. Basal salivary and urinary cortisol measurements are useful for the assessment of chronic stress, and of poor welfare in dogs. The use of urinary catecholamine, peripheral leucocyte, and lymphocyte proliferation measures requires further investigation.

The use of saliva cortisol, urinary cortisol, and catecholamine measurements for a noninvasive assessment of stress responses in dogs.

A problem in assessing animal welfare is that collecting data in itself may be stressful to the animals. Therefore, noninvasive methods for collecting data have to be devised and tested. A first step in investigating saliva cortisol, urinary cortisol, and urinary catecholamine as noninvasive indicators of canine well-being is the validation of these hormonal measures as alternatives for those in plasma. Using a model of insulin (0.2 U/kg)-induced hypoglycemia, we report on stress-induced responses in saliva cortisol, urinary cortisol, and urinary catacholamines relative to cortisol and catecholamine responses in plasma. Hypoglycemia in six dogs induced significant (P < 0.05) increases in plasma cortisol and adrenaline but not noradrenaline. Saliva cortisol responses expressed as net area under the response curve correlated significantly with plasma cortisol responses (r > 0.92). Saliva cortisol levels measured 7 to 12% of plasma cortisol concentrations. Cortisol/creatinine rations in urine were significantly higher when voided after insulin administeration, compared to when voided after saline treatment. Insulin-induced increments in cortisol/ creatinine ratios were nonsignificant when urine samples were assayed after dichloromethane extraction. Although urinary adrenaline/creatinine (A/C) ratios were significantly correlated with maximum plasma adrenaline values after insulin administration, A/C ratios did not differ significantly between insulin and saline treatment. The present experiment provides strong support for using saliva sampling and urine collection as noninvasive methods to establish stress-induced cortisol responses. For measuring acute plasma adrenaline responses, measuring A/C ratios may not be a valid alternative.