Safeguarding global health is a moving target, with changes in climate and human activity raising new threats to public health, animal health and animal welfare. Decision support in the context of health management requires tools capable of making sense of large volumes of complex and multidisciplinary evidence. A One Health living risk assessment tool, L'ORA, was developed to assess the incursion risk for zoonotic and animal diseases using a One Health approach and allowing for automatic updates of the risk assessment. L'ORA estimates the incursion risk as the resultant of four steps: 1) Disease occurrence in source areas based on the distribution of diseases worldwide; 2) Rate of incursion based on the individuals or products moved from source areas to target areas and their probability of infection or contamination and contact with susceptible hosts in target areas; 3) Extent of disease spread in target areas, considering both domestic and wild host populations, vectors if the disease is vector-borne, and humans if the disease is zoonotic; and 4) Impact of the disease outbreak and concurrent control measures on public health, animal health and animal welfare, accounting for economic, societal, and environmental impacts. L'ORA is a decision support tool that was built as a generic tool comparing the relative risks of multiple diseases across multiple target areas. L'ORA monthly evaluates the incursion risk for each NUTS2 region in the European Union. Results can be used to identify areas of greater risk for each of the diseases to inform risk managers on where to focus attention and resources, and to inform, e.g., risk-based surveillance.

An unhealthy prepubertal diet can have long-lasting effects throughout life. This study investigated hair concentrations of adrenal and sex steroids, in an in vivo mouse model of juvenile obesity subjected to control (CTRL), obesogenic (HFHC) diet, or nutraceutical supplementation (silymarin or coconut oil) diets. 87 3-week-old C57BL/6 mice (42 females, 45 males) were fed CTRL or HFHC diets for 8 weeks. Afterward, the CTRL group continued on CTRL diet while the HFHC diet group was divided into five groups: HFHC, HFHC→CTRL, HFHC→CTRL + silymarin (SIL), HFHC→HFHC + SIL and HFHC→HFHC + Coconut oil. At 4 weeks, the HFHC group showed increased cortisol/dehydroepiandrosterone (DHEA) ratio compared to CTRL group. At 20 weeks, the HFHC→HFHC group showed higher levels of progesterone (P4) and dehydroepiandrosterone sulfate (DHEA-S) and lower levels of estradiol (E2) compared to the CTRL→CTRL group. The switch from HFHC→CTRL was the optimal therapy because the body weight and almost all the hormones were close to those observed for the CTRL diet group. Supplement with SIL or Coconut oil reduced DHEA-S and increased in E2 compared with the endocrine setting seen with the HFHC diet. These interventions should be considered as supportive measures rather than substitutes for dietary correction.

Background: Unlike psychological distress, which has been extensively studied during the COVID-19 pandemic, the impact of the pandemic on stress hormones has been overlooked. The aim of this study is to examine the hair cortisol/dehydroepiandrosterone sulfate (DHEA-S) ratios as markers of HPA axis dysregulation in healthcare workers and their patients. Methods: A total of 200 healthcare workers and 161 “patients” patients with special healthcare needs due to chronic illness or motor disabilities were included in this study. The hormone concentrations were measured using a radioimmunoassay. Results: Our results show that the patients had significantly higher cortisol/DHEA-S ratios than the workers. A high cortisol/DHEA-S ratio in the patients reflects higher cortisol concentrations (p < 0.001) and lower DHEA-S (p < 0.05) concentrations compared to those of the healthcare workers, suggesting that they may be exposed to a greater degree of stress and a decrease in their ability to cope with their disease. The cut-off value of the hair cortisol/DHEA-S ratio in our study for detecting people with needs that require special consideration and attention was 1.46 (p ≤ 0.01). Conclusions: Assessing the hair cortisol/DHEA-S ratios in both healthcare workers and the patients allowed us to identify a non-homeostatic condition that could lead to disease and to understand psychophysical well-being during the COVID-19 pandemic. They also play a crucial role in preventive and personalized medicine.

Feces are a noninvasive and easily collectible matrix that may help determine cumulative hormone metabolite concentrations over medium-to-long times. To date, 11-oxoetiocholanolone, an important metabolite of cortisol, has been measured in equine and bovine feces solely by an in-house enzyme immunoassay (EIA). Therefore, we validated the use of a commercial ELISA kit (11-oxoetiocholanolone ELISA kit; Cayman Chemical), which had been validated on sheep feces and human urine, to measure 11-oxoetiocholanolone in feces from 42 horses and 32 bulls. The ELISA kit had good precision (intra- and inter-assay CVs: 5.8% and 11.2% for equine feces; 9.9% and 11.2% for bovine feces, respectively), analytical sensitivity (0.186 ng/mL for both equine and bovine feces), and accuracy (parallelism and recovery tests) in determining 11-oxoetiocholanolone concentrations in feces from both species. We found ranges of 11-oxoetiocholanolone concentrations of 1-109 ng/g in equine feces and 40-302 ng/g in feces of bulls. The Cayman Chemical ELISA kit offers a simple and accessible means of analysis of 11-oxoetiocholanolone in equine and bovine fecal samples.

Several matrices are available to detect the concentrations of cortisol and dehydroepiandrosterone (DHEA) as markers of allostatic load and resilience but most of them provide a measurement of the concentration at a single -time point or within a 12- 24 h period and are subjected to daily fluctuations; conversely, keratinized de-rivatives, as hair is, provide a measure of the hormonal concentrations over medium-and long-term periods, appearing unaffected by circadian changes or by factors inducing short-term variations. Aim of this study was to evaluate hair cortisol (HC), hair DHEA (HDHEA) and their ratio (HC/HDHEA ratio) as biomarkers of allostatic load and resilience in sows around farrowing, in relation to four different models of farrowing crates, and in relation to different batches by a non-invasive sampling. The study has been conducted on 296 pregnant sows managed in a batch farrowing system from February to June. Sows were kept in multiple pens of 10-15 sows until 5 days before farrowing, then were randomly divided in 4 different models of farrowing crates. The hair samples were taken by shaving for the first time (ST1) 2.6 +/- 1.6 days before the expected delivery date and for the second time (ST2) 88.9 +/- 3.3 days after parturition. The environmental temperature and the relative hu-midity data were recorded and used to calculate the Temperature Humidity Index (THI). HC and HDHEA were determined using a solid-phase microtiter radioimmunoassay (RIA) while HC/HDHEA ratio has been calculated. No differences in terms of hair steroids concentration were found between the four models of farrowing crates and the sampling times (P > 0.05). The interaction between batch and sampling time has been significant for all the biomarkers considered (HC (P < 0.01), HDHEA (P < 0.05) and their ratio (P < 0.01)). During the pregnancy period in collective pens (ST1) HC/HDHEA ratios were never lower (with an exclusion of February) than those in the following characterized by the early postpartum and lactation in individual crates (ST2). It seems that an aggregate effect of different stressors acts on the sows exhausting their ability to cope with adverse events; it should be further investigated the influence of the social interaction in collective pens that cyclically re-occurs in the standard swine production system.

Cortisol is the main glucocorticoid hormone commonly measured to assess adrenocortical activity in vertebrates in response to potential stressors. Competition and habitat quality may act as chronic stressors leading to an increase in cortisol accumulation. Moreover, chronic cortisol concentrations may seriously hamper the reproductive potential, health status and body condition of a species. Using hair collected from the rump of 88 adult roadkill roe deer Capreolus capreolus in the north-eastern Italy, we aimed to assess the effect of both ungulate competitor population densities and habitat suitability on the long-term physiological stress response of roe deer, using hair cortisol as a reference parameter. Our findings revealed that roe deer showed higher cortisol concentrations in response to higher red deer Cervus elaphus population density, most likely as a consequence of direct interspecific competition. No significant cortisol changes were however recorded in response to both wild boar Sus scrofa and roe deer population densities, probably as a result of reduced competition because of abundant feeding resources, different species population densities and spatial/temporal avoidance among species. Moreover, the absence of a significant effect as far as habitat suitability is concerned, suggest that roe deer are well adapted to live even in poor quality habitats most likely as long as they are close to better quality habitats which provide access to food resources and refuge sites. Our results highlight the importance of hair cortisol as a suitable parameter to assess physiological chronic stress response towards different environmental/ecological conditions.

In humans, allopregnanolone plays important roles in a number of different neurodegenerative disorders, and it has been proposed for use in some therapies. Horses are commonly used as animal models for human neurodegenerative diseases, mental and behavioral disorders, and neuropsychiatric disorders, and there is interest in using hair as a biological sample to study hormones in these conditions. We validated the use of a commercial ELISA kit (DetectX allopregnanolone kit; Arbor Assays), which was designed for serum, plasma, feces, urine, and tissue samples, to assess allopregnanolone in hair samples from 30 humans and 63 horses. The ELISA kit had good precision (intra- and inter-assay CVs: 6.4% and 11.0% for equine hair; 7.3% and 11.0% for human hair, respectively), sensitivity (50.4 pg/mL for both equine and human hair), and accuracy (parallelism and recovery tests) in determining allopregnanolone concentrations in hair from both species. The allopregnanolone concentrations in human hair were 7.3–79.1 pg/mg; the concentrations were 286 ± 141 pg/mg (x̄ ± SD) in mares on the day of parturition and 16.9 ± 5.5 pg/mg in nonpregnant mares. The DetectX ELISA kit offered a simple and accessible analysis of allopregnanolone in human and equine hair samples.

Measurements of cortisol levels in hair are a non-invasive method to study potential chronic stress that may affect carnivores' welfare. Using hair from 15 frozen and 18 taxidermied road-kill individuals, we aimed to provide information on the long-term physiological response of European wildcats (Felis silvestris silvestris) to habitat fragmentation and potential interspecific competition with golden jackals and red foxes. Our findings revealed that wildcats seemed to be unaffected by habitat fragmentation, suggesting that the facultative specialist behaviour of the species may lead to better toleration of human-altered environments. Red fox presence did not affect cortisol levels. However, significantly higher cortisol levels were measured in hairs of wildcats exposed to golden jackals, suggesting that the potential competition between the two species may lead to an increase in allostatic load in wildcats.

In a recent study, we observed that starch-rich diets used in mid lactation induced lower milk production persistency and higher body fat accumulation in dairy ewes compared with dairy goats. Because these species differences could be linked to hormonal mechanisms that drive energy partitioning, in the same experiment, we explored the evolution of metabolic and hormonal status during lactation to test this hypothesis. Twenty mature Sarda dairy ewes and 20 mature Saanen goats [15-134 ± 11 d in milk (DIM), mean ± SD] were compared simultaneously. In early lactation, each species was allocated to one dietary treatment: high-starch diet [HS: 20.4% starch, on dry matter (DM) basis], whereas from 92 ± 11 DIM, each species was allocated to 1 of 2 dietary treatments: HS (20.0% starch, on DM basis) and low-starch (LS: 7.8% starch, on DM basis) diets. Blood samples were collected in the morning to analyze glucose, nonesterified fatty acids (NEFA), growth hormone (GH), insulin, and insulin-like growth factor I (IGF-I). Data were analyzed using the PROC MIXED procedure of SAS with repeated measurements (SAS Version 9.0). The HS and LS diets applied in mid lactation did not affect metabolic status of the animal within species; thus, only a comparison between species was carried out. From early to mid lactation, plasma glucose concentration was higher in ewes than in goats (54.57 vs. 48.35 ± 1.18 mg/dL), whereas plasma NEFA concentration was greater in goats than in ewes (0.31 vs. 0.25 ± 0.03 mmol/L). Goats had higher plasma GH concentration and lower plasma insulin content than ewes (4.78 vs. 1.31 ng/mL ± 0.47; 0.11 vs. 0.26 μg/L ± 0.02). Plasma IGF-I concentration did not vary between species. The comparison of metabolic and hormonal status of lactating Sarda dairy ewes and Saanen goats, carried out by studying simultaneously the 2 species in the same stage of lactation and experimental conditions, suggests that the higher insulin and glucose concentration observed in Sarda ewes explains why they partitioned more energy toward body reserves than to the mammary gland, especially in mid lactation. This can justify the negative effect of high-starch diets in mid-lactating Sarda ewes. Conversely, the highest GH and NEFA concentration observed in Saanen goats explain why they partitioned more energy of starch diets toward the mammary gland than to body reserves and justify the positive effect of high-starch diet in mid lactation. Together, these different responses contribute to explain why specialized dairy goats, such as the Saanen breed, have a higher milk production persistency than specialized dairy sheep breeds, such as the Sarda.

Radioimmunoassay (RIA) methods have always represented a technique of choice for the determination of steroids in biological samples. The Amplified Luminescent Proximity Homogenous Assay-Linked Immunosorbent Assay (AlphaLISA) is now emerging as the new-generation immunoassay technology that does not require washing/separation steps. The aim of this study was to adapt the Perkin-Elmer's AlphaLISA kit for wool cortisol and compare it with a RIA wool cortisol assay. Wool from lambs, 35 at birth (A0) and 54 at two months old (A2), was collected and each extract was evaluated for wool cortisol concentrations (HCC) both by RIA and AlphaLISA immunoassay. The two methods showed good precision, sensitivity and specificity for determining HCC. Both methods were able to detect significant differences between the high and the low HCC assessed in lambs at A0 and A2 (P < 0.01). The HCC assessed with RIA were significantly higher than those assessed with AlphaLISA (P < 0.01). Moreover, the correlation between HCC measured using the AlphaLISA and RIA methods was strong (r = 0.878). The regression analyses show a constant and not proportional error. This could be due to the diversity in the dosage steps and to the diversity of the molecules used in the two methods. Results support the validity of using AlphaLISA as an alternative method to RIA for the quantification of cortisol in sheep wool and considering the performances showed it has a great potential to be further applied as an excellent tool to evaluate HCC in samples derived from animal species.

This study was conducted to assess concentrations of 17 beta-estradiol (E2) and testosterone (T) in toe claws of puppies collected at birth, at 30 and at 60 days of age, evaluating changes relating to age and effect of puppy sex, Apgar score, bodyweight at birth, "litter effect", litter size, and maternal age. Puppies (n = 89), 46 males and 43 females, with normal weight and without malformations, were assigned for the study. Within 12 h of birth tips of toe claws were clipped, and the re-growth tissue of the claws was collected at 30 and 60 days of age. Steroid quantifications occurred using a radioimmunoassay. The results indicated there were lesser concentrations (P < 0.001) of both hormones at 30 and 60 days of age than at birth and that concentrations were similar at 30-60 days of age. There were greater (P < 0.001) T concentrations in males than females, with there being an interaction between sex and sampling time (P < 0.01). The Apgar score was positively (P < 0.001) related to T concentrations in toe claws at birth. The bodyweight was positively correlated (P < 0.05) with T concentrations, with an interaction among puppy sex, bodyweight and sampling time (P < 0.05). Results of the present study confirmed the usefulness of toe claws as a matrix for study of hormonal changes in perinatology of dogs. Results of the study also indicate there are greater E2 and T concentrations at birth compared with 30 and 60 days of age that could be the result of these prenatal steroids affecting fetal development.

The aim of the present pilot study was to determine the allostatic load by analysing the hair cortisol and dehydroepiandrosterone‑sulphate (DHEA‑S) of commercial pigs belonging to different farms having good overall animal welfare according to the CREnBA (Centro di Referenza Nazionale per il Benessere Animale - Brescia, Italy) assessment protocol. The study was conducted on 86 pigs belonging to three farms with a grade higher than 8 on the CReNBA welfare evaluation system. Hair samples were taken by shaving on sows 1‑10 days after weaning (Farms 1 and 2) and at the age of 8‑10 months (Farm 3). The hormone concentrations were measured by radioimmunoassay. From the box plots, it is evident that the hair cortisol concentrations of animals were different between farms. Conversely, the variability of the hair DHEA‑S concentration was similar between the F1 and F2 farms but much lower at F3. For all the variables considered (cortisol, DHEA‑S and cortisol/DHEA‑S ratio), F2 showed a higher hair concentration level than F1 (P < 0.05). The study's results indicate that the measurement of cortisol and DHEA‑S in pig hair shows a different allostatic load between them, although the official welfare evaluation method assessed the farms as having good overall animal welfare.

Because of the need to improve the knowledge about canine perinatology, and given the major role of fetal fluids in sustaining the course of pregnancy and fetal development, an in-depth analysis to better understand the role of some hormones in these compartments is essential. Among all, leptin is recognized to play a key role not only on the energetic homeostasis, but also at multiple levels, influencing the control of reproduction, food assumption and metabolism. Even if in humans and other species it is reported the presence of leptin receptors during fetal development, very little is known about the canine species, in which the role of leptin still needs to be fully understood. The present study aimed to assess the amniotic fluid leptin (AFL) concentrations at term pregnancy in healthy dogs, and to evaluate the possible influence played by breed body-size (after assessment of correlation with maternal bodyweight and placental weight), or other maternal (age, parity, and the so-called "litter effect") and neonatal (gender, birth weight, litter size) parameters on AFL concentrations, analyzed by ELISA test. The study was performed on 90 healthy, viable and normal weighted puppies, 39 small-sized (adult body weight < 10 kg) and 51 large-sized (adult body weight > 25 kg), born by 29 purebred, healthy bitches, submitted to elective Caesarean section because of breed-related or individual high risk for dystocia. The results showed that the mean AFL concentration in the small-sized puppies was significantly (p < 0.05) higher in comparison to large-sized puppies (867.48 vs 698.42 pg/ml), while all the other studied parameters did not show to influence AFL concentrations. In conclusions, the present study showed significant higher at term AFL concentrations in small-sized as compared to large-sized breeds, suggesting an influence of breed body-size on fetal metabolism, as previously reported for NEFA and IGF-I.

The aim of this study was to analyze the feasibility and reliability of using hair as a matrix to determine the dehydroepiandrosterone (DHEA) and sexual steroid concentrations and the cortisol/DHEA ratio in fattening pigs. The results could be also used to plan future research to identify threshold values in order to set up strategies to control the allostatic load and increase the resilience of fattening pigs before slaughter. The study was conducted on 107 commercial crossbred rearing pigs. The hair samples were taken by shaving at the age of 36 weeks, and concentrations of the hormones were measured using a solid-phase microtiter radioimmunoassay. Females had significantly higher cortisol levels (p < 0.01), significantly lower DHEA concentrations (p < 0.05) and significantly higher cortisol/DHEA ratios (p < 0.01) than barrows. Progesterone was significantly higher in gilts than in barrows (p < 0.01). Testosterone and 17β-estradiol were significantly higher in barrows than in gilts (p < 0.05). If future research can produce threshold values for the different markers examined, the evaluation of animals under subclinical stress conditions will be possible.

The quantification of glucocorticoid metabolites in hair is a non-invasive tool that provides important information regarding the endocrine status and represents a valuable method for studying potential stressors that may affect carnivores under both natural and non-natural conditions. Cortisol is the main glucocorticoid hormone of the hypothalamic-pituitary-adrenal gland axis and is considered a standard stress indicator for animal welfare. The current study aimed to compare cortisol levels extracted from hair of both dead, frozen European wildcats (Felis silvestris silvestris) and living feral individuals (Felis silvestris catus) living in different environmental conditions. The results obtained revealed that wild individuals exhibited a significantly (p < 0.001) higher cortisol concentration (n = 15, mean ± SD = 8.91 ± 4.48 pg/mg) than feral ones (n = 10, mean ± SD = 3.57 ± 1.25 pg/mg), probably as a result of both the physiological and/or environmental factors to which each subspecies was subject. This is the first study in which cortisol concentrations have been compared within the Felis silvestris subspecies, thus enriching the scarce information available for the Felidae. Nevertheless, further research is needed to better understand the various physiological and ecological factors affecting the adrenocortical activity of species or populations living in different environmental contexts.

The objective of the G-RAID project was the mutual exchange of knowledge between the consortium members on the development of generic risk assessment (RA) tools for animal disease incursion. Seven generic RA tools were compared considering objectives, inputs, algorithms and outputs. All tools were designed for rapid risk assessment and could assess the incursion risk for multiple diseases and pathways. Specific objectives of the tools, however, varied from immediate response to new disease events to prioritization of diseases and horizon scanning, resulting in different approaches to evaluate the incursion risk of infectious animal diseases. Cross-validation was explored as a method to validate the generic RA tools. All tools were applied to a case study for African swine fever (ASF) in which the incursion risk for the Netherlands and Finland was assessed for the 2017 situation and two hypothetical scenarios with ASF cases reported in Germany. The generic RA tools were parameterized using the same global databases for disease occurrence and trade in live animals and animal products. Disease-related parameters, however, could not be standardized because of the different levels of detail included in the model calculations. A comparison of absolute results of the tools was not possible, because output parameters represented different endpoints, varied from qualitative probability levels to quantitative numbers, and were expressed in different units. Therefore, relative risks across countries and scenarios were calculated for each tool and compared. The risk assessment tools largely agreed upon the ranking of countries and scenarios based on relative risks and would thus indicate similar priorities for risk management As such, the cross-validation increased the credibility of results obtained with the generic RA tools. The cross-validation also contributed to the internal validation and further development of the tools. Results from the G-RAID project were disseminated to risk assessors and risk managers at a one-day symposium.

Abstract The use of a non-invasive approach to collect biological samples from natural populations represents a great means of gathering information while avoiding handling animals. Even if corticosterone is the main glucocorticoid investigated in birds, there has been observed a proportional direct link between corticosterone and cortisol concentrations. Dehydroepiandrosterone (DHEA) can be produced by the adrenal cortex and should have prominent antiglucocorticoid properties also in birds. The aim of this study was to verify if there is any difference in the cortisol and DHEA feather concentrations between clinically normal and physiologically compromised Griffon vultures (Gyps fulvus) through the non-invasive approach of collecting moulted feathers without having to pluck them from the bird. The study was carried out using 8 physiologically compromised (PC) Griffons and 9 clinically normal Griffons considered as the control (CTRL) group that were necropsied or from the wildlife rehabilitation centre. Primary and secondary covert feathers were either collected directly from the birds’ cage floors, or, in the case of dead Griffons, they were plucked off the animals. The results, obtained by RIA, revealed that both cortisol (P<0.01) and DHEA (P<0.05) feather concentrations were higher in the PC than in the CTRL group. No difference was observed by comparing the cortisol/DHEA ratio between the two evaluated groups (P=0.15). Pearson’s correlation coefficients showed no correlation between feather hormone concentrations in the PC group (r=0.01, P=0.96) while a positive correlation in the CTRL group (r=0.65, P=0.006) was observed. In conclusion, our study reveals that moulted feathers can be a non-invasive and an interesting tool to evaluate the allostatic load of wild birds and they allowed better understanding the relationship between hormones of the hypothalamic–pituitary–adrenal axis and the physiological status of the birds.

The aim of this study was to assess hair cortisol concentrations in New Zealand white rabbits (Oryctolagus cuniculus) that were subjected to relocation and surgery to evaluate HPA-axis activity; in addition, we used this marker of cortisol secretion to evaluate the allostatic load of animals undergoing surgery. After a period of acclimatisation, which lasted 40 days from their arrival at the enclosure, 19 rabbits were subjected to T1-T12 dorsal arthrodesis (RS), 19 were sham-operated (SS), and 19 were non-operated (CON). Hair samples were collected at the time of arrival (ST1) at the animal facility, and seven other sets of hair samples were collected at 40-day intervals from the same area of skin for a period of 240 days as re-shaved hair (anagen phase): immediately before surgery (ST2) and after the surgery (ST3, ST4, ST5, ST6, ST7, and ST8). The transition from the rabbitry to the animal breeding facility led to a significant increase in cortisol concentration (ST2) in all of the groups. At ST3, the RS group presented higher cortisol concentrations than those of the SS group and the CON group. At ST4, the experimental groups showed similar values that remained constant until ST8. The results show that the management of rabbits undergoing surgery should be evaluated very carefully, and hair cortisol concentrations may provide a means of avoiding the dangerous cumulative effects of additional stressors close to surgery.

Despite the high rates of perinatal mortality in dogs, the study of canine perinatology did not receive strong scientific interests until recently, probably due to the difficulties in studying long term changes evaluations without incur with ethical issues. In the recent years, however, the usefulness of new matrices, such as the claws, collectable without invasiveness and providing information about a long-term, retrospective window of time were reported to be a suitable tool for the study of canine perinatology in spontaneously dead puppies. The present study was therefore aimed to assess the usefulness of claws as matrices collectable without invasiveness for the study of immunoreactive cortisol and dehydroepiandrosterone (sulfate) in alive puppies at birth, at 30 and at 60 days of age. Beside the effect played by the sampling time, the possible effect of type of birth, vaginal delivery (VD) or elective Cesarean section (CS), as well as of some other maternal or neonatal factors was assessed. The results showed a significant decrease of both hormones from birth to 30 and to 60 days of age (p < 0.001) and from 30 to 60 days of age (p < 0.05), and highlighted, for both hormones, the influence of the type of birth, with newborns born by VD having higher concentrations of immunoreactive cortisol (p < 0.01) and dehydroepiandrosterone (sulfate) (p < 0.001) than the newborns born by elective CS in collections made at birth. No other significant effect was detected. The study confirmed the usefulness of claws as matrix collectable without invasiveness for the retrospective, long-term assessment of hormonal changes in alive newborn puppies and that both hormones declined from birth to 60 days of age. The differences between puppies born by vaginal delivery or elective Caesarean section suggest a possible different HPA activation in puppies born by the two types of birth.