Fecal Cortisol Metabolite Concentrations Research Articles

Associations of peripartum markers of stress and inflammation with milk yield and reproductive performance in Holstein dairy cows

The objective was to evaluate the association of peripartum concentrations of fecal cortisol metabolites (11,17-dioxoandrostane; 11,17-DOA), plasma cortisol and haptoglobin (Hp), as well as two markers of negative energy balance, non-esterified fatty acid (NEFA) and postpartum β-hydroxybutyrate (BHBA), with milk yield and reproductive performance. Blood and fecal samples were collected weekly from 412 Holstein dairy cows from wk −3 through wk +1 relative to calving. Pregnancies by 150 days in milk (DIM) and projected 305-d mature equivalent (305ME) milk yield based on the 3rd Dairy Herd Improvement (DHI) test day (mean±SD; 102±17 DIM) were measured. Multivariable linear regression models were used to describe the associations of metabolites with 305ME milk yield. Semiparametric proportional hazards models were used to describe associations of the same metabolites with risk of conception by 150 DIM. Negative associations with milk yield were found for prepartum Hp in wk −2, −1, and +1 relative to calving (estimate±SE: 490±251, 564±259, and 464±136kg lower yield for every increase in Hp concentration by 1g/L, respectively) as well as with NEFA concentration in wk −2 (estimate±SE: 1465±541kg lower milk yield for an increase in NEFA concentration by 1mEq/L). Postpartum associations of NEFA with milk yield depended on parity; NEFA was associated with an increase in milk yield in primiparous animals only (estimate±SE: 1548±510kg increase for an increase in NEFA concentration by 1mEq/L). An increase in plasma cortisol concentration by 1μg/dL in wk +1 relative to calving was associated with an increase in milk yield (estimate±SE: 580±176kg). Prepartum 11,17-DOA was associated in all three prepartum sampling weeks with a reduced hazard ratio (HR) of conception (HR [95% CI]: 0.81 [0.67–0.97], 0.85 [0.72–0.99], and 0.85 [0.75–0.97] for every increase in concentration by 1mg/g fecal dry matter (DM) in wk −3, −2, and −1 relative to calving, respectively). Increased cortisol concentrations in wk −3 and −1 relative to calving were associated with decreased hazard of conception in primiparous animals only (HR [95% CI]: 0.54 [0.32–0.92] and 0.59 [0.35–0.99], respectively. Increases in postpartum metabolites Hp and BHBA had a negative association with hazard of conception (HR [95% CI]: 0.81 [0.70–0.97], 0.74 [0.56–0.98], respectively). Biomarkers of inflammation and stress around calving may be useful to assess opportunities for improved milk yield and reproduction.

Head partitions at the feed barrier affect behaviour of goats

Space allowance at the feeding places often forces goats to feed in close proximity, that is, less than their individual distances. In consequence, agonistic behaviour may increase as well as stress and injuries, while access to feed may decrease, especially in low-ranking goats. Partitions between single feeding places may reduce individual distances and may enhance the acceptance of goats feeding close by. The aim of this study was to investigate the influence of non-transparent head partitions at the feed barrier on agonistic behaviour, activity budget, feeding place occupancy, adrenocortical activity, nutritional status (body weight and body condition), and injuries in loose-housed dairy goats.The study involved 72 pregnant dairy goats of the German Improved Fawn breed. Two groups of 36 animals each were tested in a cross-over design. At the beginning, one group was provided with head partitions at a wooden palisade feed barrier, whereas the other group stayed without head partitions. After 11 experimental days, the head partitions were switched to the other group. Social interactions were recorded for 13h and 20min per treatment (2h and 40min on 5 days each group). Activity budget and feeding place occupancy were observed via scan sampling for 48h per treatment. Body weight, body condition score, and occurrence of injuries were assessed, and faeces were sampled for analysis of cortisol metabolites. Data were analysed by Wilcoxon-tests for dependent data, except for feeding place occupancy, where t-tests were used. Goats displayed less agonistic behaviour in the feeding area with head partitions at the feed barrier (p=0.003). In addition, with head partitions a lower number of displacements from feeding place by an actor standing inside the feed barrier was found (p=0.002). The impact was most pronounced in low-ranking animals (p=0.009), but effects were also found in middle-ranking goats (p=0.030). Low-ranking goats were observed less often feeding (p=0.009) and more often lying (p=0.026) during the first hour after feed supply with head partitions. With head partitions more goats were feeding directly next to each other, i.e. without an empty feeding place in between (p=0.017). Regarding the nutritional status of the goats, the lumbar body condition scores were higher in high-ranking animals with head partitions (p=0.007). Presence of head partitions had no effect on sternal body condition scores, body weight, concentrations of faecal cortisol metabolites, and occurrence of injuries. In summary, non-transparent head partitions seemed to reduce the accepted distance between goats and therefore showed beneficial effects in terms of lower levels of social disturbances during feeding. Thus, head partitions can be recommended for feed barriers in goat loose-housing systems.

Early transfer of mated females into the maternity unit reduces stress and increases maternal care in farm mink

Mated mammals on farms are typically transferred to another housing environment prior to delivery. We investigated whether the timing of this transfer – EARLY (Day −36), INTERMEDIATE (Day −18), or LATE (Day −3) relative to the expected day of birth (Day 0) – affects maternal stress, maternal care and the early kit vitality in farmed mink. We hypothesized that early transfer is beneficial for mink mothers and their offspring in comparison to intermediate or late movement closer to delivery, being the current practice in the commercial production. We used 180 double mated female yearlings in three equally sized groups (n=60): (i) ‘EARLY’, transfer to maternity unit immediately after the end of the mating period, March 23; (ii) ‘INTERMEDIATE’, transfer in the middle of the period, April 10; (iii) ‘LATE’, transfer late in the pregnancy period, April 25. Data collection included weekly determination of faecal cortisol metabolites (FCM) and evaluation of maternal care: nest building, in-nest temperature, plus kit-retrieval behaviour, kit mortality and growth day 0–7 postpartum. We document that mated mink females build and maintain a nest at least 1 month prior to delivery when transferred to an environment with free access to nest building material. During the weeks before delivery, INTERMEDIATE females had 50% higher FCM concentrations than the other two groups (P=0.002), indicative of stress. After delivery, late moved females had, in average, 2.7°C colder nests compared to early moved females (P=0.002). Additionally, the mortality in group LATE tended to be higher (P=0.085) in affected litters (N=92). Kits from early transferred females displayed less vocalization (17% vs. 40–41% in the two other groups, P=0.015), when tested away from the nest. This indicates enhanced offspring vitality from early moved females. In conclusion, transfer into the maternity unit early after mating, rather than later during the pregnancy period, reduces stress and increases maternal care in farm mink.

Maximizing the reliability of non-invasive endocrine sampling in the tiger (Panthera tigris): environmental decay and intra-sample variation in faecal glucocorticoid metabolites.

Evaluation of physiological stress in the tiger (Panthera tigris) using faecal cortisol metabolite (FCM) enzyme immunoassays (EIAs) provides a powerful conservation physiology tool for the species. However, it is important to validate non-invasive endocrine sampling techniques in field conditions to ensure that the method provides a reliable parameter of physiological stress in the species. This is because endocrine measurements are highly species specific and FCM concentrations can be influenced by environmental factors. Here, we studied the impact of the decay rate of FCMs and intra-sample variation of FCMs using a previously validated EIA. To determine the decay rate of FCMs, we measured FCMs in freshly deposited tiger faeces (n = 8 tigers and 48 scats) that were randomly exposed to the natural environment (dry conditions with no rainfall) for up to 192 h. To determine intra-sample variation in FCMs, we used 10 scats from 10 tigers, divided each sample into four sections and each section into four sub-sections and measured FCMs in each section and sub-section. The results of this decay-rate experiment showed that FCMs in tiger faeces began to decay after 48 h exposure to the environmental conditions available. Thus, FCMs within freshly deposited tiger faeces are influenced by available environmental conditions. Changes in weather conditions (e.g. increased rainfall and humidity) could influence the stability of FCMs. The results of the intra-sample variation study showed that inter-variation among scats accounted for 52% of the variations in FCMs, while intra-sample variation between sections (32%) was greater than the sub-sample variation (16%). Intra-sample variation can be reduced by homogenizing the entire lyophilized faecal sample prior to the EIA. In conclusion, careful evaluation of decay rate and complete homogenization of faeces prior to EIA analysis will increase the reliability of FCMs as a non-invasive index of physiological stress in the tiger.

Validation and use of hair cortisol as a measure of chronic stress in eastern chipmunks (Tamias striatus).

Stress levels of individuals are documented using glucocorticoid concentrations (including cortisol) in blood, saliva, urine or faeces, which provide information about stress hormones during a short period of time (minutes to days). In mammals, use of hair cortisol analysis allows for the assessment of prolonged stress over weeks and months and provides information on chronic stress levels without bias associated with handling. Here, we validate hair cortisol analysis in wild rodents using exogenous adrenocorticotrophic hormone (ACTH challenge) and apply the technique to evaluate stress in eastern chipmunks inhabiting logged and natural sites. Chipmunks were subjected to a mark-recapture study and injected weekly with ACTH (Synacthen Depot) or saline, with hair being collected at the conclusion of the challenge. Subsequently, faecal and hair samples were collected from chipmunks occupying logged and natural sites to assess the utility of hair cortisol in comparison with faecal cortisol metabolites. Following extraction, cortisol concentrations were quantified in hair and faecal extracts by enzyme immunoassay. Hair cortisol concentrations were significantly elevated in samples from ACTH-injected chipmunks compared with saline-injected control animals (five times higher). Chipmunks inhabiting logged sites had increased faecal cortisol metabolite concentrations compared with those in natural sites, but no differences were observed in hair cortisol concentrations. Faecal cortisol metabolite levels were positively correlated with hair cortisol levels in chipmunks. Hair cortisol levels reflect changes in circulating cortisol levels and can be used to evaluate the adrenal stress response, and thus stress, in natural populations. Nonetheless, because of the differences in the temporal scale of stress that hair and faeces represent, we caution the use of hair cortisol for detecting differences in physiological stress when comparing individuals within populations and suggest that it is best suited to examining population-level differences.

Events around weaning in semi-feral and stable-reared Konik polski foals: Evaluation of short-term physiological and behavioural responses

The annual weaning, stabling and subsequent sale of semi-feral Konik polski foals born in forest reserves ensure a stable population of free-roaming Konik polski horses. However, welfare concerns regarding the maternal and herd separation of young horses, manipulation by humans, transportation and stabling of forest-reared Koniks have arisen. The aim of the present study was to examine the physiological and behavioural responses of weaned, 7-to-9-month-old forest-reared Konik polski foals (FR, N=26) relative to those of stable-reared peers (SR, N=27). Fifty-three weanlings (24 colts and 29 fillies) were studied. FR foals from five distinct herds were captured and transported and stabled together in loose-housing stables (FR weaning scheme). Three weeks later, SR foals were weaned by separating them from their dams and joining them with FR foals (SR weaning scheme). Faecal cortisol metabolites (FCM), which reflect adrenocortical activity on the day preceding weaning (WD−1) and on the day of weaning (WD), were analysed to evaluate the effect of weaning on FCM concentrations. The time budget over the 6h following weaning on the day of weaning (WD) and one day post-weaning (WD+1) was recorded.Weaning caused a significant increase (P<0.0001) of adrenocortical activity in all horses. Sex (P=0.0064), but not rearing condition (P=0.1542) had an effect on FCM concentrations. Differences between FR and SR foals in terms of their behavioural responses to the two weaning schemes were observed, FR foals exhibiting less outward arousal (lower social and locomotor activity, less vocalisation) compared to SR foals. On WD+1, both groups tended to adapt to the post-weaning scheme. However, FR foals still stood for longer periods than their SR peers and presented less alert behaviour and lower locomotor and social activity. Increased FCM levels on weaning day were correlated with longer periods of standing (rs=0.32, P=0.0149) and shorter feeding bouts (rs=−0.42, P=0.0017).It seems that forest foals were more disturbed by all events around weaning. They presented higher apathy and more forest horses were among those showing higher FCM concentrations. However, as the FCM did not differed significantly between groups from different rearing conditions, the claim of a higher “stress” response to the weaning scheme in forest foals cannot be unequivocally supported based on behavioural and physiological measures in our study. For both groups, feeding behaviour appears to be a good indicator of post-weaning stress.

Climate-driven changes in diet composition and physiological stress in an arboreal folivore at the semi-arid edge of its distribution

Species, particularly foliovores, at the trailing edge of their geographical range are likely to be most vulnerable to climate change as they respond to physiological stress and the decline in the nutrient richness of their food source. We investigate the effect of environmental conditions on diet composition, resource use, and physiological stress of koalas (Phascolarctos cinereus) in the semi-arid landscapes of southwest Queensland, Australia, across three different biogeographic regions. Fresh faecal pellets were collected to measure cortisol metabolites and assess diet. Regression analyses were used to relate the diet composition and physiological stress of wild koalas to environmental variables. The impact of drought was apparent, with higher faecal cortisol metabolites (FCM) levels recorded during drought conditions compared with post-flood conditions. Diet composition also changed between drought and post-flood conditions, with diets during drought being mainly composed of species with high leaf-moisture content. Low minimum temperatures increased FCM concentrations, and these effects were greater during drought conditions. The results demonstrate the importance of integrating physiological assessments into ecological studies to identify stressors that have the potential to compromise the long-term survival of threatened species, as well as the need to identify the resources required for their continued survival.

Assessment of adrenocortical activity and behavior of the collared anteater (Tamandua tetradactyla) in response to food‐based environmental enrichment

One of the current standard approaches to the study of animal welfare is measuring hypothalamic-pituitary-adrenal activity, frequently in association with behavioral assessment. We studied the effects of food-based environmental enrichment on adrenocortical activity and behavior in zoo-housed collared anteaters (Tamandua tetradactyla; n = 5). We successfully validated measurements of fecal cortisol metabolites (FCMs) using an 11-oxoetiocholanolone enzyme immunoassay by stimulating (ACTH injection) and suppressing (dexamethasone administration) adrenocortical activity. Three months later, we subjected animals to an ABA-type experiment (three 6-week periods): pre-enrichment (routine diet: A), enrichment (modified diet: B), and post-enrichment (routine diet: A) periods. We assessed adrenocortical activity by collecting individual feces three times a week (total number of samples: 228), and evaluated behavior by performing 3 days of behavioral observations per period (with a total of 3,600 behavioral data points for the individuals studied). Statistical analysis revealed changes in FCM concentrations (µg/g) over the periods (3.04 ± 0.68, 2.98 ± 0.66, and 4.04 ± 0.90, respectively). Additionally, it showed that the number of FCM peaks was highly reduced during enrichment; meanwhile active natural behaviors were significantly increased. We consider that these changes in response to food-based environmental enrichment improved the welfare of individual zoo-housed collared anteaters. This research might contribute to in situ and ex situ studies on the physiology and behavior of this endemic South American species.

Additional foraging elements reduce abnormal behaviour – fur-chewing and stereotypic behaviour – in farmed mink (Neovison vison)

We investigated whether provision of additional appetitive and consummatory elements of foraging reduces baseline stress and abnormal behaviour – in terms of fur-chewing and stereotypic behaviour – in farmed mink. We studied 200 juveniles (n=100 females and 100 males) during the 5-month growth period with plenty of feed, and subsequently the females as adults during the 2-month feed restriction period before mating. The mink were distributed in four equally sized groups: (i) FARM, conventional finely ground feed (<8mm) without additional foraging elements; (ii) ROPE, access to biting ropes; (iii) CONS, chunky feed (parts up to 42mm), replacing conventional feed; (iv) BOTH, access to both biting ropes and chunky feed. In growing mink, biting ropes reduced fur-chewing (P=0.044) and chunky feed reduced stereotypic behaviour (P=0.038) and fur-chewing in female mink (P=0.019). During the season of feed restriction, the wear/tear of biting ropes increased. Females on the chunky diet had a higher concentration of faecal cortisol metabolites (P=0.033), probably due to a more severe slimming resulting in a 6.2% lower body weight (P=0.006) than the mink on the finely ground diet; still the chunky diet reduced time spent in pre-feeding stereotypies (P=0.001). In the restrictively fed females, fur-chewing was reduced both by access to biting ropes (P=0.005) and chunky feed (P=0.007). Consequently, 54% of group FARM mink displayed fur-chewing compared to 21% in group BOTH. In conclusion, stereotypic behaviour was reduced by provision of chunky feed, increasing the consummatory element in daily foraging. Fur-chewing was reduced upon access to either biting ropes or chunky feed in female mink throughout the study. Our findings support frustrated foraging, mainly consummatory, behind abnormal behaviour.

Age‐dependent baseline values of faecal cortisol metabolites in the American mink (Neovison vison) under semi‐natural housing conditions

The welfare of an animal is ensured if it is able to fully satisfy its essential species-typical needs in all functional aspects of behaviour. In mink, stereotypies and apathy, internal and/or external injuries as well as increased susceptibility to disease have been known to occur as a result of chronic stress. The non-invasive method of analysing faecal cortisol metabolites (FCM) allows conclusions to be drawn about the stress level in the respective housing system. The objective of this study is to find out how the cortisol metabolites content in the faecal changes with increasing age of the mink under semi-natural housing conditions. Thus, 40 American mink (Neovison vison) were housed in two outdoor enclosures imitating natural conditions. Throughout the entire study (13th to 32nd week of life), faecal samples were collected to measure cortisol metabolites. No differences in FCM concentrations between the two outdoor enclosures were found. In the young mink lower, less fluctuating FCM levels were found than in older animals. After the first faecal collection in the 13th/14th week of life, the level of metabolites decreased slightly (p = 0.032; 17th/18th week). From the 22nd/23rd week onwards until the 30th/31st week, shortly before the animals were pelted, continuously increasing concentrations were then measured. Increasing FCM levels with advancing age of the animals are probably attributable to the onset of sexual maturity and/or the respective season. This has to be taken into account in future studies using this method for assessing welfare and when comparing different mink housing systems.

The effect of ACTH upon faecal glucocorticoid excretion in the koala

Environmental changes result in physiological responses of organisms, which can adversely affect population dynamics and reduce resistance to disease. These changes are expressed in chronic levels of stress. The measurement of glucocorticoid (GC) concentrations in faeces is a non-invasive method for monitoring stress in wildlife. The metabolism and excretion of steroids differ significantly between species and, as a consequence, non-invasive methods must be physiologically validated for each species. Koalas (Phascolarctos cinereus) are declining in numbers through much of their range. The role of chronic stress in koala populations has not been identified. Prior to the assessment of faecal GC concentrations in wild koala populations, the excretion timing and concentrations of GCs need to be determined. In this study, we assessed a method for identifying and measuring the concentrations of GC metabolites in faecal pellets of captive koalas following ACTH treatment. The results show that an elevation of plasma cortisol concentrations, using sustained release of ACTH, results in elevated concentrations of faecal cortisol/cortisol metabolites. Taking into account the excretion time lag, an increase in faecal cortisol metabolite concentrations corresponds to the release of GCs from the adrenal cortex as early as 36 h before faecal pellet collection. The calculations of steroid partitioning of plasma cortisol showed that the ACTH-stimulated values were significantly different from the control values for the concentrations of free, corticosteroid-binding globulin-bound and albumin-bound cortisol. This study validates the use of faecal cortisol analysis to assess the activity of the hypothalamo-pituitary-adrenocortical axis in freshly collected koala faecal pellets and indicates that the method should be suitable to assess the adrenocortical status of koalas in wild populations.

Effect of the duration of road transport on the physiology and meat quality of lambs

To assess the effect of transport duration on animal welfare and meat quality of lambs, two trials were performed: Forty Ile-de-France × Merino lambs were used in Trial 1 and 40 Comisana lambs in Trial 2. In both trials, the lambs, aged between 14 and 16 weeks, were divided into two groups of 20 animals. One group was subjected to a 1-h period of transportation (T1) and the other to a 24-h period of transportation (T24), both in the same truck and arriving to the same slaughterhouse at the same time. The effect of transport on serum biochemistry variables (cortisol, aspartate transaminase, lactate dehydrogenase, blood urea nitrogen, creatine kinase, creatinine and total proteins), salivary cortisol, metabolites of cortisol in faeces, intra-ruminal temperature and meat quality (pH, conductivity, expressible juice, colour and shear force) was assessed. In both studies, the duration of transport did not affect serum and salivary cortisol concentration (P &gt; 0.05). However, in Trial 2, lambs exposed to 24-h transport had a higher concentration of faecal cortisol metabolites than did those transported for 1 h (P &lt; 0.05). Blood variables were not affected by transport in either trial (P &gt; 0.05), with the exception of blood urea nitrogen which was higher in Trial 1 for the T24 group (P &lt; 0.05) than it was to T1 group. Although no signs of dehydration were found, intra-ruminal loggers showed that animals did not drink during the transportation in the way they did before transport. In Trial 1, no effect of transport duration was found on meat-quality traits (P &gt; 0.05). Nevertheless, in Trial 2, lambs exposed to 24-h transport had higher values of colour attribute of a* (red trend) and less tenderness or higher values of shear force (P &lt; 0.05). The present study showed that although there is little effect on meat quality, signs of stress are detectable in lambs transported for 24 h. Therefore, in the case of lambs, the effect of long transportation periods must be considered more in terms of animal welfare than in terms of product quality.

Behavioural and physiological reactions of goats confronted with an unfamiliar group either when alone or with two peers

When introduced into a new herd, goats are confronted with unfamiliar animals. Their behavioural and physiological reactions during this confrontation are likely to differ depending on the presence or absence of familiar conspecifics (peers). To assess these reactions, we confronted 12 goats both alone and with two peers (confrontees) with established groups (n=4 groups) consisting of goats unfamiliar to the confrontee (unfamiliar goats) (12 goats×2 confrontations=24 confrontations in total). Each confrontation lasted for one hour. Agonistic interactions, sniffing behaviour and level of activity were recorded throughout the confrontations. In addition, concentrations of cortisol metabolites were measured in faecal samples taken in the evening before the confrontation and three successive samples after the confrontation. Before the start of the experiment, we evaluated the dominance relationships of the involved goats within their respective housing groups by direct observations made during the main feeding times. Data were analysed using generalised linear mixed-effects models with the fixed effects presence of peers (yes, no), rank category (high, medium, low) and repeated confrontation (numeric variable). For the analysis of activity level and concentrations of faecal cortisol metabolites, period (minutes 0–15, 16–30, 31–45, 46–60) and sample (control, 13, 14, 15h after the confrontation), respectively, were included as additional fixed effects. Unfamiliar goats directed fewer agonistic interactions towards confrontees when the latter were accompanied by peers compared to when they were alone (without peers: 57; with peers: 20 interactions per animal and confrontation). The same was true for the proportion of agonistic interactions involving physical contact (without peers: 69; with peers: 53%) and the number of sniffing behaviours (without peers: 16; with peers: 9 interactions per animal and confrontation). On the other hand, confrontees with peers were more likely to direct agonistic and sniffing behaviour towards unfamiliar goats than those on their own. Confrontees with peers had lower concentrations of faecal cortisol metabolites after confrontations (without peers: 273; with peers: 198ng/g). For confrontees (with and without peers), activity level was highest during the first 15min of the confrontation and decreased over its course. For the unfamiliar goats, the activity pattern was similar but was modulated by rank, with higher values for low-ranking goats than for medium- and high-ranking ones. In conclusion, our results indicate that the presence of peers is advantageous for goats being introduced into groups of unfamiliar goats.

Assessment of adrenocortical activity by non-invasive measurement of faecal cortisol metabolites in dromedary camels (Camelus dromedarius)

The aim of this study was to determine whether glucocorticoid production could be monitored non-invasively in dromedary camels by measuring faecal cortisol metabolites (FCMs). Five Sudanese dromedaries, two males and three females, were injected with a synthetic adrenocorticotropic hormone (ACTH) analogue. Blood samples were collected pre- and post-ACTH injection. Faeces were sampled after spontaneous defecation for five consecutive days (2 days before and 3 days after ACTH injection). Baseline plasma cortisol values ranged from 0.6 to 10.8 ng/ml in males and from 1.1 to 16.6 ng/ml in females, while peak values after ACTH injection were 10.9-41.9 in males and 10-42.2 ng/ml in females. Peak blood cortisol values were reached between 1.5 and 2.0 h after ACTH injection. The concentration of FCMs increased after ACTH injection in the faeces of both sexes, although steroid levels peaked earlier in males [24 h; (286.7-2,559.7 ng/g faeces)] than in females [36-48 h; (1,182.6-5,169.1 ng/g faeces)], reflecting increases of 3.1-8.3- and 4.3-8-fold above baseline levels. To detect chromatographic patterns of immunoreactive FCMs, faecal samples with high FCM concentrations from both sexes were pooled and subjected to reverse phase high performance liquid chromatography (RP-HPLC). RP-HPLC analysis revealed sex differences in the polarity of FCMs, with females showing more polar FCMs than males. We concluded that stimulation of adrenocortical activity by ACTH injection resulted in a measurable increase in blood cortisol that was reliably paralleled by increases in FCM levels. Thus, measurement of FCMs is a powerful tool for monitoring the adrenocortical responses of dromedaries to stressors in field conditions.

Measuring faecal glucocorticoid metabolites as a non-invasive tool for monitoring adrenocortical activity in South American camelids

AbstractThe welfare and productivity of South American camelids may be affected by stressful events. The purpose of this study was to validate a non-invasive method for stress monitoring using faecal samples and to apply it to evaluate a stressful event, such as confinement. For physiological validation, nine alpacas(Vicugna pacos)and six llamas(Lama glama)were subjected to pharmacological stimulation of their adrenal cortex. Serial faecal samples were collected during 48 h before and after stimulation. During confinement, faecal samples from six llamas were collected twice per day during six consecutive days. Faeces belonging to 18 vicuñas(Vicugna vicugna)were collected before and one day after their capture for confinement(Chacu). Faecal cortisol metabolites (FCM) were extracted from each sample and quantified by an 11-oxoaetiocholanolone enzyme immunoassay. Thirty-three and 28 h (median) after ACTH stimulation, FCM concentrations peaked with a ten- and eight-fold increase (median) above baseline in alpacas and llamas, respectively. There were no significant differences in FCM concentrations between sexes. In llamas, FCM concentrations peaked (4.7 times higher than baseline) after five days of confinement in females and after three days (2.7 times) in males. In vicuñas, three times higher FCM levels were observed the day after the start of confinement (in comparison to the starting values). Based on our findings, this non-invasive method is well suited to measure adrenocortical activity in alpacas, llamas and vicuñas. Thus, this method could help to improve management, handling and welfare in wild and domesticated South American camelids.

Factors influencing the welfare of goats in small established groups during the separation and reintegration of individuals

As a goat's separation from or reintegration into its group is likely to have an adverse effect on the welfare of both the separated goat and the remaining goats in the group, management procedures need to be carried out in a way that minimises their negative impact. In the present study, we tested the effects of two treatments of separation and reintegration by individually separating 12 goats from four experimental groups, each composed of seven horned, non-lactating female goats. In the ‘no-contact’ treatment the separation allowed for acoustic contact with the group only, whereas in the ‘contact’ treatment tactile and visual contact was also possible. The separation lasted two days, with each separated goat experiencing both treatments (i.e. there were 24 separations in total). Per group, one separated goat was of high, medium and low rank, respectively. The effects of separation and reintegration were assessed by evaluating social interactions, lying and feeding behaviour, and the concentration of cortisol metabolites in faecal samples of separated and resident goats. Data were collected during three phases: a reference period (days −7 to −1), a separation period (days 0 and 1) and a reintegration period (days 2, 3 and 4).Separated goats fed less and had higher concentrations of faecal cortisol metabolites during the separation than during the reference period. In addition, goats undergoing the ‘no-contact’ treatment spent less time lying during separation. On the first day of the reintegration period, newly reintroduced goats were more likely to display agonistic and sniffing behaviour towards resident goats and had higher concentrations of faecal cortisol metabolites than during the reference period. The faecal cortisol metabolite levels of the recently separated goats tended to be higher and the probability of recently separated goats displaying sniffing behaviour towards resident goats was higher in the ‘no-contact’ than in the ‘contact’ treatment. By contrast, resident goats were scarcely affected by the separation and reintegration of a group member. The rank of both separated and resident goats influenced behaviour per se, but did not interact with treatment or day.In conclusion, our results indicate that separation had a greater impact on the welfare of the individual goats than did reintegration. To mitigate negative effects on the goats’ welfare, it may therefore be advantageous to allow tactile, visual and acoustic contact during separation.

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

BackgroundStereotypic behaviours, i.e. repetitive behaviours induced by frustration, repeated attempts to cope and/or brain dysfunction, are intriguing as they occur in a variety of domestic and captive species without any clear adaptive function. Among the different hypotheses, the coping hypothesis predicts that stereotypic behaviours provide a way for animals in unfavourable environmental conditions to adjust. As such, they are expected to have a lower physiological stress level (glucocorticoids) than non-stereotypic animals. Attempts to link stereotypic behaviours with glucocorticoids however have yielded contradictory results. Here we investigated correlates of oral and motor stereotypic behaviours and glucocorticoid levels in two large samples of domestic horses (NStudy1 = 55, NStudy2 = 58), kept in sub-optimal conditions (e.g. confinement, social isolation), and already known to experience poor welfare states. Each horse was observed in its box using focal sampling (study 1) and instantaneous scan sampling (study 2). Plasma samples (collected in study 1) but also non-invasive faecal samples (collected in both studies) were retrieved in order to assess cortisol levels.ResultsResults showed that 1) plasma cortisol and faecal cortisol metabolites concentrations did not differ between horses displaying stereotypic behaviours and non-stereotypic horses and 2) both oral and motor stereotypic behaviour levels did not predict plasma cortisol or faecal cortisol metabolites concentrations.ConclusionsCortisol measures, collected in two large samples of horses using both plasma sampling as well as faecal sampling (the latter method minimizing bias due to a non-invasive sampling procedure), therefore do not indicate that stereotypic horses cope better, at least in terms of adrenocortical activity.

Behaviour and stress responses in horses with gastric ulceration

Only little is known about behaviour and stress responses in horses with gastric ulceration, despite the high prevalence of this condition. Our objectives in the present study was to (i) describe the severity of gastric ulceration in horses, housed under relatively standardised conditions, and (ii) to investigate whether horses with severe glandular gastric ulceration have increased baseline and response concentration of stress hormones and behave differently than control horses. We investigated stomachs of 96 horses at one stud, and compared an ulcer group (n=30; with severe lesions in the glandular mucosa) to paired controls (n=30; free from gastric ulcers). Baseline and response concentrations of faecal cortisol metabolites (FCM), heart rate and behaviour were measured in a novel object test (NOT, Day 1) and behaviour during postponed feeding (PF, Day 2). Glandular lesions occurred in 55.2% and non-glandular lesions in 40.6% of the horses. The amount of starch in the feed (P=0.006) and paternal stallion (P=0.031) influenced ulceration in the non-glandular region only; it should be noted that our study does not allow for separating hereditary from environmental influences, as offspring may be e.g. trained differently dependent on breeding line. Ulcer horses pawed more (P<0.001) and ate quicker (P=0.050) during PF. Although displayed by ulcers horses only during PF, we failed to demonstrate a significant association between glandular gastric ulceration and crib-biting/weaving; the total number of horses with these types of abnormal behaviour was low (n=5). Behaviour and heart rate did not differ between groups in the NOT. Baseline concentration of FCM was similar (P=0.79), however, ulcer horses responded stronger to novelty than controls (26% higher FCM; P=0.018). We conclude that the prevalence of gastric ulcers was high, and our results suggest different factors affecting ulceration in the glandular versus the non-glandular region of the horse stomach. Obvious external signs (e.g. poor body condition) identifying ulcer horses were absent. Horses with severe glandular ulcers had a higher stress hormone response to novelty, thus they were more stress sensitive. Consequently, management evoking stress in horses should be reduced to dampen the development of glandular ulceration, or to protect horses with this condition.

Non-invasive evaluation of physiological stress hormone responses in a captive population of the greater bilby Macrotis lagotis

Captive breeding programs are increasingly being used as a management option for threatened mammals. The greater bilby Macrotis lagotis, for example, is a vulnerable species which is maintained in captivity at several facilities in Australia. Non-invasive evaluation of stress hormones (cortisol in mammals) via excretory metabolites can be used to monitor physiological stress responses of captive individuals. In this study, we validated an enzyme-immunoassay (EIA) to measure cortisol metabolites in fresh faecal samples of adult male and female bilbies (n = 7) held in captivity at the Dreamworld Theme Park, Queensland, Australia. The faecal cortisol EIA was validated via parallelism and the recovery of exogenous cortisol added to pooled faecal extracts (>99% recovery). Female bilbies had higher average faecal cortisol metabolite concentrations and higher day-to-day variation than male bilbies; however, there was no relationship with bilby age. Cortisol metabolites for most individuals varied widely through time, with numerous peaks and troughs in response to long-term stressors (illnesses, injury and reproductive issues) and short-term stressors, such as use in shows at Dreamworld or public displays in local schools, manual restraint and short-term veterinary procedures (e.g. general anaesthesia). Overall, the higher mean cortisol metabolite concentrations of individuals suffering long-term stress was related to a greater response to short-term stressors. This suggests an interaction between responses to short-term and long-term stressors which is perhaps due to habituation and/or facilitation of long-term stressors. Non-invasive faecal monitoring of stress hormones could provide further information on the implications of captive breeding programs and the release of animals reared in captivity.

Integration into the dairy cow herd: Long-term effects of mother contact during the first twelve weeks of life

The objective of this study is to investigate the long-term effects of mother rearing on the ability to cope with the challenge of integration into the cow herd shortly before first parturition. Four groups of heifers with different levels of contact with their mothers during the first twelve weeks of life were compared. Two rearing groups were fed via an automatic milk feeder two (A2, n=5) or six times (A6, n=5) a day. The animals in these groups were compared to two treatment groups that were either kept separate from the cow herd and with limited contact with their mothers (M2, n=9) or permanent access to the herd and their mothers through selection gates (MP, n=7). At the age of 25±0.2months, heifers were integrated individually into the cow herd and observed for 33h. Social and other behaviour of each heifer was recorded by continuous behaviour sampling, and the identity of the nearest neighbour was determined by time sampling every 5min. Additionally, faecal samples were taken for measurement of adrenocortical activity. For statistical analysis ANOVA was performed with treatment and breed as fixed factors and herd size, except for social behaviour as covariate. For post hoc testing, heifers were allocated to groups according to contact with the mother (M2 and MP; mother) i.e. suckling by the mother compared to automatic feeding (A2 and A6; automat) and according to contact with the herd (MP; permanent suckling vs. M2, A2, A6; no herd). The treatment groups tended to differ in the frequency of self grooming (p=0.102), with mother-heifers tending to self-groom more often than automat-heifers (p=0.109). Permanent suckling-heifers performed self-grooming more often than no herd-heifers (p=0.048). Concerning social behaviour, the treatment groups tended to differ in submissive behaviour (p=0.062), with mother-heifers being submissive more often than automat-heifers (p=0.023) and permanent suckling-heifers more often than no herd-heifers (p=0.055). The increase in the concentration of faecal cortisol metabolites after the first two days of integration tended to be different between the treatment groups (p=0.088). In our nearest neighbour analysis, one MP-heifer and one M2-heifer could clearly identify their mothers in a herd of 50 cows after 2years of separation (p<0.05, over chance level). This suggests that rearing with contact with the mother in the first twelve weeks, even if very limited, may have an effect on later behaviour and may lead to possibly enhanced social skills in dairy heifers.