Lameness assessment with automatic monitoring of activity in commercial broiler flocks.

The possibility of using automatic recordings of broiler chicken activity in commercial flocks to assess the birds΄ walking ability (lameness) was investigated. Data were collected from 5 commercial broiler farms in 4 European countries, using 16 flocks and 33 assessment occasions. Lameness was assessed using established gait scoring methods (Kestin et al., 1992; Welfare Quality®, 2009) and took place at 3, 4, and 5 wk of age. Gait score (GS) was used to assess the birds' walking ability, and automatic recordings of bird activity were collected using the eYeNamic™ camera system before, during, and after an assessor walked through the house. The variables used to predict the level of GS extracted from the camera system were: baseline activity, time from assessor leaving the house to resumption of baseline activity, average activity over that period, and Δ Amplitude (difference between highest activity peak after assessor left the house and baseline level). Age (<0.001) and Δ Amplitude (P = 0.0002) were significantly related to GS, with the gait getting poorer with increased age and Δ Amplitude decreasing with declining walking ability. Both measures are thus included in a predictive equation. The results demonstrate a potential method using image analysis techniques to realize an automated assessment of the level of lameness in commercial broiler flocks. This could be of use in future animal welfare assessment schemes.

Exploring the economic potential of reducing broiler lameness.

1. The present study was designed first to explore the potential economic benefits of adopting management practices to reduce lameness in broiler farms, and second to explore farmers' possible perceptions of this potential in the Swedish context. The likely financial effects were addressed using a normative economic model, whereas a questionnaire-based survey was used to obtain in-depth knowledge about the perceptions of a group of broiler farmers in Sweden. 2. The three alternative practices (out of 6 tested) which realised the greatest improvements in gross margin and net return to management compared to the conventional practice were feeding whole wheat, sequential feeding and meal feeding. 3. The model showed that the negative effect of feeding whole wheat on feed conversion rate was outweighed by the effect of a low feed price and the associated decrease in feed costs. The price of wheat played a major role in the improvement of economic performance, whereas the reduction of lameness itself made a relatively minor contribution. 4. Apparently, the surveyed farmers do not recognise the potential of the positive effects of changing feed or feeding practices on both broiler welfare and farm economics although their implementation can be of great importance in the broiler sector where profit margins are very tight.​.

Potential risk factors associated with contact dermatitis, lameness, negative emotional state, and fear of humans in broiler chicken flocks.

The objectives of this study were to 1) identify determinants of poor welfare in commercial broiler chicken flocks by studying the associations between selected resource-based measures (RBM, potential risk factors), such as litter quality and dark period, and animal-based welfare indicators (ABM), such as foot pad dermatitis and lameness, and 2) establish the breadth of effect of a risk factor by determining the range of animal welfare indicators associated with each of the risk factors (i.e., the number of ABM related to a specific RBM). Eighty-nine broiler flocks were inspected in 4 European countries (France, Italy, the United Kingdom, and the Netherlands) in a cross-sectional study. The ABM were contact dermatitis (measured using scores of foot-pad dermatitis and hock burn, respectively), lameness (measured as gait score), fear of humans (measured by the avoidance distance test and the touch test), and negative emotional state (measured using qualitative behavior assessment, QBA). In a first step, risk factors were identified by building a multiple linear regression model for each ABM. Litter quality was identified as a risk factor for contact dermatitis. Length of dark period at 3 wk old (DARK3) was a risk factor for the touch test result. DARK3 and flock age were risk factors for lameness, and the number of different stockmen and DARK3 were risk factors for QBA results. Next, the ABM were grouped according to risk factor and counted. Then, in a second step, associations between the ABM were investigated using common factor analysis. The breadth of a risk factor's effect was judged by combining the number (count) of ABM related to this factor and the strength of association between these ABM. Flock age and DARK3 appeared to affect several weakly correlated ABM, thus indicating a broad range of effects. Our findings suggest that manipulation of the predominant risk factors identified in this study (DARK3, litter quality, and slaughter age) could generate improvements in the related ABM and thereby enhance the birds' overall welfare status.

The benzodiazepine brotizolam reduces fear in calves exposed to a novel object test.

The present study examined the effects of the intravenous administration of the anxiolytic drug brotizolam on the behavioral and physiological responsiveness of calves to novelty in a dose response fashion. Holstein Friesian heifer calves (39-41 weeks of age; body weight 200-300 kg) received an intravenous injection of either a vehicle control (12 calves) or one of four doses of brotizolam (8 calves per dose): 0.0125, 0.05, 0.2 and 0.8 mg/100 kg body weight. They were then individually subjected to a 'combined' test involving exposure to a novel environment (open field, OF) for 5 min followed by the sudden introduction of a novel object (NO) that remained in place for a further 10 min. Behavioral, heart rate and plasma cortisol responses were recorded in all animals. Compared to vehicle treatment, the highest dose of brotizolam dose-dependently and significantly increased the time spent in locomotion and the distance travelled near the NO, as well as the time spent in contact with the NO. In addition, post-test plasma cortisol concentrations changed in a dose-dependent manner over time: they decreased between 0 and 10 min after the test in calves that had received the two highest doses of brotizolam, whereas they increased in vehicle-treated and low-dosage calves. There were no effects of brotizolam on vocalization or locomotion during the OF phase of the test or on vocalization following introduction of the NO. These findings strongly support the notion that interaction with a novel object in a novel arena represents a behavioral index of fear and fearfulness in calves, and that vocalization and locomotion in an OF reflect other independent characteristics.

Do low-density diets improve broiler breeder welfare during rearing and laying?

Low-density diets may improve welfare of restricted fed broiler breeders by increasing feed intake time with less frustration of feed intake behavior as a result. Moreover, low-density diets may promote satiety through a more filled gastrointestinal tract, and thus feelings of hunger may be reduced. Broiler breeders were fed 4 different diets during the rearing and laying periods. Behavioral and physiological parameters were measured at different ages as indicators of hunger and frustration of the feeding motivation. A diet of 8.4 MJ/kg as compared with a standard diet of 10.9 MJ/kg extended feeding time and reduced stereotypic object pecking at 6 and 10 wk of age. Furthermore, compensatory feed intake at 12 wk of age was reduced. During lay, differences in behavior were observed between the treatments that could be attributed to differences in feeding time. However, birds fed the diet with the lowest energy content (i.e., 9.2 MJ/kg) had higher heterophil to lymphocyte ratios (H/L) at 40 wk of age compared with the other treatments, indicating that they experienced more stress during the laying period than the other treatments. This result could have been due to the very long feeding time of this treatment group during lay, which may be stressful. In conclusion, a low-density diet of 8.4 MJ/kg may reduce hunger and frustration in the first half of the rearing period. However, for substantial improvement of broiler breeder welfare during rearing, more extreme diet modifications are required.

Responses of horses in behavioural tests correlate with temperament assessed by riders.

REASONS FOR PERFORMING STUDY: Behavioural tests as well as observers' ratings have been used to study horses' temperament. However, the relationship between the ratings and the responses in behavioural tests has not yet been studied in detail. OBJECTIVES: The aim of the present study was to examine this relationship between ratings and responses. METHODS: Eighteen mature Swedish Warmblood horses were subjected to 2 behavioural tests, one relating to novelty (novel object test) and one to handling (handling test). Subsequently, 16 of these horses were ridden by 16 equally experienced students, having no former experience with the horses. Immediately after each ride, the students scored the horse for 10 temperamental traits using a line rating method. RESULTS: It was shown that for each temperamental trait all 16 riders agreed on the ranking of the horses (0.212

Individual differences in behavioral and physiological responsiveness of primiparous dairy cows to machine milking.

An experiment was performed in primiparous dairy cows (n = 23) to examine consistency of individual differences in reactivity to milking, and correlations between measures of behavior, physiology, and milk ejection. Responsiveness to milking was monitored during the first machine milking, on d 2 of lactation, and during milkings on d 4 and 130 of lactation. Measurements included kicking and stepping behavior, plasma cortisol and plasma oxytocin, heart rate, milk yield, milking time, milk flow rate, and residual milk obtained after administration of exogenous oxytocin. With repeated early lactation milkings, residual milk and the incidence of abnormal milk flow curves decreased. On d 130 of lactation all heifers exhibited normal milk ejection. Except for higher plasma cortisol concentrations on d 2, all measures were consistent over time between d 2 and 4 of lactation as indicated by significant rank correlations. Individual differences in the behavioral response to udder preparation were consistent over time between early lactation milkings and d 130 of lactation. Residual milk, milk yield, maximum milk flow rate, plasma oxytocin and heart rate during udder preparation were similarly interrelated on d 2 and 4 of lactation. High heart rate responses on d 2 and 4 were associated with enhanced inhibition of milk ejection. In contrast, behavior recorded during the milking process was unrelated to ease of milk removal. Our results indicate that milking at the beginning of lactation may be stressful to some heifers, to the extent that milk ejection is inhibited, but less disturbing to others. The existence of consistent behavioral and physiological responses in the present study suggests that responsiveness of dairy heifers to milking is mediated by stable animal characteristics.

Effects of restricted feeding on physiological stress parameters in growing broiler breeders.

1. In previous studies, a lack of agreement in measurements of plasma corticosterone concentrations and heterophil:lymphocyte (H/L) ratio as physiological indices of stress, caused by hunger and frustration in restricted-fed broiler breeders, was observed. It could be suggested that the differences between previous studies were caused by differences in duration of restriction and time of the day of the measurements. Therefore, in the present study the plasma corticosterone concentration and the H/L ratio were again determined in restricted- and ad libitum-fed growing broiler breeders, taking possible causes of disagreement between previous studies into account. In addition, we measured the daily rhythm in body temperature and heart rate, and the corticosterone responses to an acute stressor as physiological indices of stress. 2. Female broiler breeders (64 per treatment, housed in groups of 4 birds) were used in the experiment. Behaviour, baseline plasma corticosterone concentrations and H/L ratio were determined at 21 d of age (immediately after the start of food restriction), and at 42 and 63 d of age. Body temperature, heart rate and activity were measured by radiotelemetry for 36 h at 49 and 70 d of age. In addition, the plasma corticosterone response to acute stress (5 min manual restraint) was measured at 77 or 78 d of age. 3. Restricted broiler breeders had higher plasma corticosterone concentrations at 42 and 63 d of age, but no differences in H/L. ratio were found between restricted birds and unrestricted control birds. Restricted broiler breeders had a higher corticosterone response to 5 min manual restraint than unrestricted birds. Restricted birds displayed a clear day-night rhythm in body temperature, heart rate and activity whereas such a rhythm was blunted in ad libitum-fed birds. 4. It is discussed that some physiological differences (plasma corticosterone concentrations, body temperature and heart rate) between ad libitum-fed and restricted broiler breeders may have been caused by differences in metabolic rate as well as by differences in the level of stress. It is concluded that a combination of behavioural measurements and a wide range of physiological parameters should be used for the assessment of stress in growing broiler breeders.

Heart rate and heart rate variability during a novel object test and a handling test in young horses.

Forty-one Dutch Warmblood immature horses were used in a study to quantify temperamental traits on the basis of heart rate (HR) and heart rate variability (HRV) measures. Half of the horses received additional training from the age of 5 months onwards; the other half did not. Horses were tested at 9, 10, 21 and 22 months of age in a novel object and a handling test. During the tests, mean HR and two heart variability indices, e.g. standard deviation of beat-to-beat intervals (SDRR) and root mean square of successive beat-to-beat differences (rMSSD), were calculated and expressed as response values to baseline measures. In both tests, horses showed at all ages a significant increase in mean HR and decrease in HRV measures, which suggests a marked shift of the balance of the autonomic nervous system towards a sympathetic dominance. In the novel object test, this shift was more pronounced in horses that had not been trained. Furthermore, statistical analysis showed that the increase in mean HR could not be entirely explained by the physical activity. The additional increase in HR, the nonmotor HR, was more pronounced in the untrained horses compared to the trained. Hence, it is suggested that this nonmotor HR might be due to the level of emotionality. HR variables showed consistency between years, as well as within the second year. These tests bring about a HR response in horses, part of which may indicate a higher level of emotionality; and horses show individual consistency of these HR variables over ages. Therefore, it is concluded that mean HR and HRV measures used with these tests quantify certain aspects of a horse's temperament.

Determination of the circadian rhythm in plasma corticosterone and catecholamine concentrations in growing broiler breeders using intravenous cannulation.

This paper describes a fast and simple technique for cannulation of the vena cutanea ulnaris (wing vein) in ad libitum fed growing broiler breeders of 5 weeks of age. Twenty-four hours after cannulation, blood was sampled every 4 h during 24 h. The circadian rhythm in plasma corticosterone and catecholamine concentrations was determined for the first time in growing broiler breeders. During the light period, baseline plasma corticosterone concentrations were higher than during the dark period, and reached the circadian peak at 11:00 h (lights on from 07:00 to 15:00 h). Plasma noradrenaline concentrations were higher during the light period than during the dark period, but there was no pronounced circadian rhythm in plasma adrenaline concentrations. Plasma corticosterone and catecholamine concentrations fell within the same range as previously reported in laying hens.

Adaptation to social isolation. Acute and long-term stress responses of growing gilts with different coping characteristics.

The present experiment studied the acute and long-term stress responses of reactive and proactive prepubertal gilts to social isolation. Gilts with either reactive or proactive features were identified according to behavioral resistance in a backtest at a young age (2-4 days), respectively being low (LR) and high resistant (HR) in this test. At 7 weeks of age, 12 gilts of each type were socially isolated. Initially, isolation was stressful for both types of gilts, as shown by increased cortisol concentrations and decreased body temperatures. Moreover, both types reacted with increases in exploration and vocalizations. Stress responses to isolation, however, differed in magnitude and/or duration between LR and HR gilts, which was in line with expected reaction patterns on the basis of preferred ways of coping. The cortisol response to isolation was higher in LR gilts, and they generally showed more explorative behavior. HR gilts seemed to be more engaged in walking/running behavior in the first hour after isolation, they generally vocalized more and their noradrenaline excretion in urine was higher at 3 weeks after the start of isolation. Several responses to isolation in the longer term pointed to a prolonged higher general state of stress of HR gilts. Body temperature in HR gilts, for instance, did not recover during 3 weeks of isolation, but values returned to "normal" within 1 day in LR gilts. At 1 week of isolation, relatively high parasympathetic responsivity to novelty was observed in HR gilts, probably due to stress-related high sympathetic reactivity. A shift in percentages of leucocyte subsets, typically occurring under conditions of stress, only developed in HR gilts during isolation. Finally, gastric ulceration was found in one HR gilt, but did not occur in LR gilts. To conclude, LR and HR gilts differed in their strategies to adapt to social isolation, and especially for HR gilts, this procedure seemed to become a chronic stressor.

Transgenesis may affect farm animal welfare: a case for systematic risk assessment.

This paper considers (potentially) harmful consequences of transgenesis for farm animal welfare and examines the strategy of studying health and welfare of transgenic farm animals. Evidence is discussed showing that treatments imposed in the context of farm animal transgenesis are by no means biologically neutral and may compromise animal health and welfare. Factors posing a risk for the welfare of transgenic farm animals include integration of a transgene within an endogenous gene with possible loss of host gene function (insertional mutations), inappropriate transgene expression and exposure of the host to biologically active transgene-derived proteins, and in vitro reproductive technologies employed in the process of generating transgenic farm animals that may result in an increased incidence of difficult parturition and fetal and neonatal losses and the development of unusually large or otherwise abnormal offspring (large offspring syndrome). Critical components of a scheme for evaluating welfare of transgenic farm animals are identified, related to specific characteristics of transgenic animals and to factors that may interact with the effects of transgenesis. The feasibility of an evaluation of welfare of transgenic farm animals in practice is addressed against the background of the objectives and conditions of three successive stages in a long-term transgenic program. Concrete steps with regard to breeding and testing of transgenic farm animals are presented, considering three technologies to generate transgenic founders: microinjection, electroporation and nuclear transfer, and gene targeting including gene knockout. The proposed steps allow for unbiased estimations of the essential treatment effects, including hemi- and homozygous transgene effects as well as effects of in vitro reproductive technologies. It is suggested that the implementation of appropriate breeding and testing procedures should be accompanied by the use of a comprehensive welfare protocol, specifying which parameters to monitor, at which stages of the life of a farm animal, and in how many animals. Some prerequisites and ideas for such a protocol are given. It is anticipated that systematic research into the welfare of farm animals involved in transgenesis will facilitate the use of the safest experimental protocols as well as the selection and propagation of the healthiest animals and, thereby, enable technological progress that could be ethically justified.

Farm animal welfare research in interaction with society.

Over the last 30 years concern about farm animal welfare has increased and has become a public issue in the Netherlands. Public discussion has stimulated research in this field, financed by both government and industry. Dutch society in general and consumers of animal products in particular, want to see high standards of welfare for production animals. Good animal welfare has gradually gained more impact in the total quality concept of the product. This will encourage scientists to continue to analyse the welfare status of animals and to come up with innovative solutions for the remaining problems. At ID-Lelystad much effort is put into farm animal welfare research. This research includes for example, the development of behavioural tests for quantifying and interpreting fear in cattle, investigations into the effects of dietary iron supply and a lack of roughage on behaviour, immunology, stress physiology, and pathology in veal calves, studies of the ontogeny of tail biting in finishing pigs and feather pecking in laying hens as well as evaluation of the welfare effects of automatic milking in dairy cows. The results of these projects contribute to concrete improvements in animal husbandry and expertise and support policy making and legislation. The animal industry as well as retailers should aim at the further implementation of this knowledge and to specify welfare standards to guarantee consumer acceptance of animal production.

Social isolation may influence responsiveness to infection with bovine herpesvirus 1 in veal calves.

An experiment was performed to develop a model to study the impact of stress on responsiveness to infection with bovine herpesvirus 1 (BHV1) in veal calves. Social isolation after previous group-housing was used as a putatively stressful treatment. Group-housed specific pathogen-free veal calves (n=8) were experimentally infected with BHV1 at the age of 12 weeks. Half of the calves were socially isolated at the time of infection. Clinical, virological and serological responses to BHV1, and adreno-cortical reactivity to exogenous ACTH were examined. In comparison with group-housed calves, calves socially isolated at the time of infection showed a diminished clinical and fever response, and delayed viral excretion after primary infection with BHV1. Four weeks after social isolation, basal cortisol levels before, and the integrated cortisol response after administration of a low dose of ACTH, were significantly depressed in socially isolated calves. The results suggest that social isolation in veal calves influences the response to an experimental BHV1 infection. A possible mechanism is discussed.

Effects of environmental enrichment on behavioral responses to novelty, learning, and memory, and the circadian rhythm in cortisol in growing pigs.

Previously we showed that pigs reared in an enriched environment had higher baseline salivary cortisol concentrations during the light period than pigs reared under barren conditions. In the present experiment, it was investigated whether these higher baseline salivary cortisol concentrations were a real difference in cortisol concentration or merely represented a phase difference in circadian rhythm. The effects of different cortisol concentrations on the behavioral responses to novelty and learning and long-term memory in a maze test were also studied in enriched and barren housed pigs. At 9 weeks of age enriched and barren housed pigs did not differ in baseline salivary cortisol concentrations nor in circadian rhythm, but at 22 weeks of age barren housed pigs had a blunted circadian rhythm in salivary cortisol as compared to enriched housed pigs. The differences in baseline salivary cortisol concentrations between enriched- and barren-housed pigs are age-dependent, and become visible after 15 weeks of age. Enriched- and barren-housed piglets did not differ in time spent on exploration in the novel environment test. Barren-housed pigs had an impaired long-term memory in the maze test compared to enriched-housed pigs; however, no differences in learning abilities between enriched- and barren-housed pigs were found. Because blunted circadian cortisol rhythms are often recorded during states of chronic stress in pigs and rats or during depression in humans, it is suggested that the blunted circadian rhythm in cortisol in barren-housed pigs similarily may reflect decreased welfare.

Short communication: effects of isolation stress on mammary tight junctions in lactating dairy cows.

Eighteen cows had been selected for their responsiveness to psychological stress during the first lactation and were classified as having low (n = 10) or high (n = 8) cortisol concentrations in response to isolation-induced stress. In the present study these cows, now in their second lactation, were used to determine the effect of social isolation stress on the permeability of mammary tight junctions. During the experiment, each cow was isolated from the rest of the herd for 55 h. After the 1st h of isolation, each cow received a bolus infusion of endotoxin in one hind quarter in order to challenge tight junctions. Blood samples were taken throughout to measure lactose, which was used as an indicator of tight-junction leakiness. After 1 h of isolation, stress caused an increase in tight junction permeability in both groups, which was further enhanced by the endotoxin treatment. Although the permeability did not differ significantly between the two groups, it was consistently higher in the high-cortisol group, which was also the most stress-responsive group. Thus, psychological stress may adversely affect milk quality by allowing serum components to leak into milk.

Coping styles in animals: current status in behavior and stress-physiology.

This paper summarizes the current views on coping styles as a useful concept in understanding individual adaptive capacity and vulnerability to stress-related disease. Studies in feral populations indicate the existence of a proactive and a reactive coping style. These coping styles seem to play a role in the population ecology of the species. Despite domestication, genetic selection and inbreeding, the same coping styles can, to some extent, also be observed in laboratory and farm animals. Coping styles are characterized by consistent behavioral and neuroendocrine characteristics, some of which seem to be causally linked to each other. Evidence is accumulating that the two coping styles might explain a differential vulnerability to stress mediated disease due to the differential adaptive value of the two coping styles and the accompanying neuroendocrine differentiation.

The effect of shot biopsy on behavior, salivary cortisol, and heart rate in slaughter pigs.

This paper describes behavioral and physiological responses of pigs to shot biopsy, an experimental method used to study muscle tissue processes or to predict meat quality. One biopsy sample from the longissimus muscle was obtained from 23-wk-old gilts (n = 10) using a cannula connected to a captive bolt. Ten other gilts were used as a control and received a sham shot. One week later, a second biopsy was taken from the same gilts. Behavioral and salivary cortisol responses to both biopsies were similar (P > .10). Pigs flinched in response to the biopsies. Salivary cortisol concentrations were increased (P < .05) 15 min after the biopsy as compared with pretreatment levels, but absolute levels were not different (P > .10) from the control group. In both biopsy and control groups, heart rate increased (P < .001) in response to the presence of the technician. In response to the first biopsy, heart rate increased (P < .01) as compared with the rate during the 5-s period before the biopsy, but heart rate did not increase in response to the second biopsy. The biopsy pigs showed a decrease (P < .05) in initiating contact with the technician in the second test. We conclude that shot biopsy had a significant acute effect on behavior and heart rate. Therefore, the usefulness of this technique in studies in which the behavioral and heart rate responses are measured is limited.

High carbon dioxide tension (PCO2) and the incidence of cardiac arrhythmias in rapidly growing broiler chickens.

The purpose of this investigation was to determine whether two-week-old rapidly growing broiler chickens with high metabolic activity have an increased risk of the development of heart failure three to five weeks later. The incidence of cardiac arrhythmias was assessed in broiler chickens with either a relatively high carbon dioxide tension (PCO2) or a low PCO2 in their venous blood. Their electrocardiograms (ECGS) were measured when the birds were between five and seven weeks old by means of a biotransplant which allowed them to move freely. Premature ventricular beats were observed in all the birds, but the largest numbers were observed in birds that had had a high PCO2 when they were two weeks old.

Effects of repeated jugular puncture on plasma cortisol concentrations in loose-housed dairy cows.

In three experiments, the effects of venipuncture on plasma cortisol concentrations were studied in loose-housed dairy cows. In Exp. 1, two blood samples were collected 18 min apart on three alternate days from 20 dairy cows for studying their adrenocortical response to a single venipuncture. To further evaluate the effect of cows anticipating venipuncture, in Exp. 2, 15 dairy cows were sequentially venipunctured once daily on 12 successive days in a randomized order in groups of five, starting 15 min apart. In Exp. 3, 10 primiparous cows were used on three alternate days to study habituation to serial sampling (i.e., collection of five blood samples by venipuncture, 15 min apart). In cows accustomed to handling, jugular puncture did not affect cortisol concentrations in plasma collected 18 min later. Average daily cortisol concentrations varied between 2.07 +/- .38 and 3.81 +/- .56 ng/mL in the first (t = 0) and between 1.43 +/- .15 and 2.61 +/- .72 ng/mL in the second (t = 18) blood samples. Likewise, when cows were sampled sequentially once a day, the order of sampling between and within groups did not influence (P > .05) plasma cortisol concentrations. In contrast, primiparous dairy cows that were less used to being handled showed an average increase in cortisol concentrations when five samples were collected by venipuncture 15 min apart. During successive sampling sessions, however, the cows did not decrease or increase plasma cortisol concentrations in response to repeated serial sampling at the group level (P > .05). Between individuals, the maximum effect of repeated venipuncture on cortisol concentrations (4.5 to 22.6 ng/mL), the time at which the effect reached its maximum (30 to 60 min), and the consistency of the response pattern over successive series varied largely. The results of this study show that in cows that were accustomed to handling and to being restrained, baseline cortisol concentrations can be measured in single blood samples that are collected by jugular puncture within 1 min after first approaching the cow. When successive blood samples need to be collected within 15 to 20 min, jugular puncture may induce an increase in cortisol concentration, which seems to depend on the handling experience of the animals and on individual differences.