Management of dairy heifers: Can operant conditioning be an effective and feasible tool to decrease stress and ease animals' close contact and handling?

Besides health monitoring, a regular check of dairy heifers' growth rate is desirable, but it is rarely done because procedures that require restraint and handling can be associated with substantial stress for both animals and farmers. Inexperienced heifers, especially if they are highly responsive to humans, may find restraint and handling potentially aversive. This study investigated whether training heifers of different age and responsiveness toward humans (RTH), through operant conditioning, could reduce stress in animals, ease close contact and handling, and be feasible in terms of farmers' effort. We assessed 60 Holstein heifers of 2 age classes (young, n = 29, 291 ± 39 d; old, n = 31, 346 ± 62 d) according to the avoidance distance test and classified them as confident (n = 20), neutral (n = 21), or nonconfident (n = 19). Half of the heifers of each age and RTH class were trained (n = 29), whereas the other half was not (n = 31). The trained heifers were subjected to target training for 8 sessions and positively reinforced with feed to allow being touched on the muzzle, rump, and perineum. If a heifer refused positive reinforcement, the trainer stepped back as negative reinforcement. In the last week of the experiment, the effect of training on the reaction to handling was assessed in all heifers. We measured heart rate, root mean square of successive interbeat interval differences (RMSSD), and fecal cortisol metabolites (FCMet). The presence of behavioral distress signs was recorded as well. The avoidance distance test was performed a second time 24 h after the measuring session. All of the trained heifers, regardless of RTH class, successfully accomplished the target training task in 6 sessions, each spending on average 25.3 s per session. All of the trained heifers allowed touches on the rump and perineum at the end of the fourth session. Training nonconfident heifers required more time compared with the others. Trained heifers showed higher RMSSD than nontrained heifers (14.2 vs. 16.9 ms, respectively), indicating a lower vagal tone, and thus, a slightly lower stress level than nontrained heifers. Training did not lead to differences in HR, FCMet, or presence of stress behavioral signs. Nonconfident heifers had the highest mean baseline FCMet values compared with neutral and confident heifers (38.4 vs. 30.3 vs. 29.1 ng/g, respectively). Nonconfident heifers also showed the lowest value of FCMet 12 h after the measuring session (36.7 vs. 44.6 vs. 49.7 ng/g), likely due to a decreased responsiveness of the adrenal gland to a stressor. The average avoidance distance decreased between the beginning and the end of the experiment, especially for neutral and nonconfident heifers, regardless of whether they were trained or not. These results show how using operant conditioning on some heifers not only decreased their vagal tone, but also reduced the responsiveness to humans of all the animals, trained and not trained; in the latter case, this reduction was through nonassociative learning, such as habituation.

Effects of scent enrichment on behavioral and physiological indicators of stress in zoo primates.

Captive breeding is vital for primate conservation, with modern zoos serving a crucial role in breeding populations of threatened species and educating the general public. However, captive populations can experience welfare issues that may also undermine their reproductive success. To enhance the wellbeing of endangered zoo primates, we conducted a study to assess the effects of a new scent enrichment program on captive red-ruffed lemurs (Varecia rubra), black howler monkeys (Alouatta caraya), siamangs (Symphalangus syndactylus), lar gibbons (Hylobates lar) and orangutans (Pongo pygmaeus pygmaeus). We combined behavioral observations and fecal endocrinology analyses to evaluate the effects of a series of essential oils (benzoin, lavender, lemongrass) on five captive troops (N = 19) housed at Dudley Zoo & Castle and Twycross Zoo (UK). We recorded observations of natural species-specific and abnormal stress-related behaviors for 480 h using instantaneous scan sampling. We collected 189 fecal samples and measured the fecal cortisol concentrations using radioimmunoassay. We found a significant effect of the scent enrichment on behaviors, with red-ruffed lemurs and black howler monkeys reducing their social interactions, as well as red-ruffed lemurs and lar gibbons decreasing their stress-related behaviors after they were exposed to the series of essential oils. We also found that red-ruffed lemurs displayed a significant increase in fecal glucocorticoids following exposure to essential oils. Our contradictory findings suggest that the effects of this series of essential oils may change depending on the species-specific social lives and olfactory repertoires of primates. In conclusion, we cannot recommend using these essential oils widely with zoo primates without additional evaluation.

The stress of being alone: Removal from the colony, but not social subordination, increases fecal cortisol metabolite levels in eusocial naked mole-rats.

In many social species, hierarchical status within the group is associated with differences in basal adrenocortical activity. We examined this relationship in naked mole-rats (Heterocephalus glaber), eusocial rodents with arguably the most extreme social hierarchies of all mammals. This species lives in colonies where breeding is restricted to one socially dominant 'queen' and her male consorts, and all other individuals are reproductively suppressed 'subordinates'. The relationship between cortisol and social status in naked mole-rats has not fully been elucidated, as prior results on this topic have been contradictory. We used non-invasive feces sampling to measure baseline cortisol levels in eight laboratory colonies of naked mole-rats, to either replicate or reject rank differences. First, we successfully validated an assay to measure fecal cortisol metabolites (FCMs). Removal from the colony for the validation experiment, either alone or with an opposite sex conspecific, induced prolonged elevation of FCM levels on a scale of days to weeks. This increase in cortisol did not prevent the removed animals from sexually maturing. In colony-housed animals, we found no relationship between rank in the social hierarchy and FCM levels. Further, queens, breeding males, and reproductively suppressed subordinates all had equivalent FCM levels. We conclude that this species shows little evidence of the 'stress of dominance' or 'stress of subordination' and that reproductive suppression in naked mole-rats is not driven by elevated cortisol levels.

Not all urban landscapes are the same: interactions between urban land use and stress in a large herbivorous mammal.

Urbanization significantly impacts the health and viability of wildlife populations yet it is not well understood how urban landscapes differ from non-urban landscapes with regard to their effects on wildlife. This study investigated the physiological response of eastern grey kangaroos (Macropus giganteus) to land use at a landscape scale. Using fecal glucocorticoid metabolites (FGM) we compared stress levels of kangaroo populations in urban and non-urban environments. We modeled FGM concentrations from 24 kangaroo populations against land use (urban or non-urban) and other anthropogenic and environmental factors, using a linear modeling approach. We found that land use was a significant predictor of FGM concentrations in eastern grey kangaroos with significant differences in concentrations between urban and non-urban populations. However, the direction of the relationship differed between northern and southern regions of Australia. In the northern study sites, kangaroos in urban areas had significantly higher FGM levels than their non-urban counterparts. In contrast, in southern sites, where kangaroos occur in high densities in many urban areas, urban kangaroos had lower FGM concentrations than non-urban kangaroos. Rainfall and temperature were also significant predictors of FGM and the direction of the relationship was consistent across both regions. These results are consistent with the contrasting abundance and persistence of kangaroo populations within the urban matrix between the two study regions. In the northern region many populations have declined over the last two decades and are fragmented, also occurring at lower densities than in southern sites. Our study indicates that it is the characteristics of urban environments, rather than the urban environment per se, which determines the extent of impacts of urbanization on kangaroos. This research provides insights into how the design of urban landscapes can influence large mammal populations.

Characterization and correlations of behavioral and adrenocortical activities of zoo-housed lesser anteaters (Tamandua tetradactyla).

We characterized behavioral and adrenocortical activities of Tamandua tetradactyla under human care driven by the hypothesis that they vary between males and females. We also assessed the potential association between natural or abnormal behaviors and adrenocortical activity. We kept females and males T. tetradactyla in individual, contiguous enclosures at Córdoba Zoo (Argentina), under natural photoperiod and temperature. During 29 consecutive days we monitored the animals' behavior by recording their activity pattern every 5 min using infrared cameras (8352 records/individual). We collected all feces and measured fecal glucocorticoid metabolites (FGM) with an 11-oxoaetiocholanolone enzyme immunoassay. We found individual differences in all behavioral variables. We detected that females exhibited lower total activity than males (23.8 ± 0.2% and 32.3 ± 0.3%, respectively; p = .005). Females were more active at night and males during the day (p < .05) and exhibited less abnormal behaviors than males (p = .05). Although we did not find sex-related differences for average FGM, we detected individual differences (p < .0001). We found that daily FGM showed negative (-0.39) and positive (0.38) correlations with natural and abnormal behaviors, respectively (p < .0001). Thus, we consider that individual input and sex are factors to be considered in stress responses of the species in captivity. Natural and abnormal behaviors may demand different levels of adrenocortical activity. Our findings may prove useful as normative data for ex situ management of conservation programs.

Linking social environment and stress physiology in feral mares (Equus caballus): group transfers elevate fecal cortisol levels.

Feral horses (Equus caballus) have a complex social structure, the stability of which is important to their overall health. Behavioral and demographic research has shown that decreases in group (or band) stability reduce female fitness, but the potential effects on the physiological stress response have not been demonstrated. To fully understand how band stability affects group-member fitness, we need to understand not only behavioral and demographic, but also physiological consequences of decreases to that stability. We studied group changes in feral mares (an activity that induces instability, including both male and female aggression) on Shackleford Banks, NC. We found that mares in the midst of changing groups exhibit increased fecal cortisol levels. In addition, mares making more group transfers show higher levels of cortisol two weeks post-behavior. These results offer insights into how social instability is integrated into an animal's physiological phenotype. In addition, our results have important implications for feral horse management. On Shackleford Banks, mares contracepted with porcine zona pellucida (PZP) make approximately 10 times as many group changes as do untreated mares. Such animals may therefore be at higher risk of chronic stress. These results support the growing consensus that links between behavior and physiological stress must be taken into account when managing for healthy, functional populations.

Instability of fecal glucocorticoid metabolites at 4°C: Time to freeze matters.

The use of fecal corticosteroid metabolites (FCMs) has proven to be well suited to evaluate adrenocortical activity, a major component of the stress response, particularly in wildlife. As with any tools, confounding factors and drawbacks must be carefully considered. Among them, sample preservation and storage are of particular importance, as they can affect stability of FCMs and lead to biased results and interpretations. Arguably, immediate freezing of fecal samples upon collection is the best practice to preserve FCM integrity, however, for logistical reasons, this condition is rarely feasible in the field. It is generally argued that temporary storage of samples at low above-zero temperature is an acceptable way of preserving samples in the field before freezing them for long-term storage. However, to our knowledge, there is no empirical study that demonstrates the stability of fecal metabolites in samples stored at +4°C. In this study, we collected a fresh fecal sample from 20 captive roe deer, each of which was homogenized and split into three subsamples (60 subsamples in total) to investigate the effects on FCMs levels of temporary storage at +4°C for 24 h and 48 h before freezing versus immediate freezing at -20°C after feces collection. Compared to immediate freezing, mean FCMs levels decreased by 25% every 24 h when feces were stored at +4°C before freezing. The variance of FCMs levels followed the same pattern, leading to a clear reduction in the ability to detect biological effects. Minimizing the storage time at +4°C before freezing should therefore be seriously considered when establishing sampling and storage protocols for feces in the field for adequate hormonal profiling.

Effect of intensity and duration of anthropic noises on European mink locomotor activity and fecal cortisol metabolite levels.

Human activities involving noise emission can affect wild animals. European mink was exposed to road noise and human voice playbacks to analyze how sound intensity level and duration of both noises altered the time that individuals were active and if their fecal cortisol metabolite (FCM) levels varied. A Hierarchical Analysis Cluster was performed to establish 2 mink groups with respect to both noise source type: short duration/low intensity (SL) and long duration/high intensity (LH). We performed general linear mixed models to evaluate the variation in locomotor activity duration (s) and FCM (nanogram per gram) levels, respectively. The results showed both road noise and human voices decreased locomotor activity duration in SL more sharply compared with LH, and human voices were the triggers that induced the most pronounced response to both exposure conditions. FCM (ng/g) levels increased in SL compared with LH during road noise while the opposite happened during human voices. Differences based on sex and age of individuals were observed. In conclusion, noise characteristics given by the sound type determined the variations in locomotor activity duration while noise exposure level determined the variations in FCM (ng/g) levels. Attention should be paid to noisy activities (e.g., recreational activities for visitors in protected natural areas) and loud groups of people to conserve wildlife, especially noise sensitive species.

Intestinal Parasites and Fecal Cortisol Metabolites in Multi-Unowned-Cat Environments: The Impact of Housing Conditions.

Housing conditions were assessed in different unowned multi-cat management models in order to evaluate their impact on the occurrence of intestinal parasites and fecal cortisol metabolite (FCM) levels. Fresh stool fecal samples were collected from rescue shelters, catteries and feline colonies for coprological analyses in order to detect intestinal parasite patency and fecal cortisol metabolites. A questionnaire provided information about the facilities, management and housing conditions of cats, including information about dog exposure, enclosure size, environment enrichment and changes to group composition. Overall, intestinal parasite infection was detected in 58.2% of fecal samples collected. The occurrence of intestinal parasites detected in free-roaming cats was 82.2%, mainly due to helminth infection. The parasite infection rate was 57.3% in rescue shelters and 34.6% in catteries. In confined cats, protozoa infection was more likely detected in rescue shelters than in catteries (RR = 2.02 (1.30-3.14), p = 0.0012). Although the FCM values were very variable between cats, the enclosure size and parasite infection were correlated with the average FCM. A small enclosure size was correlated with high fecal cortisol metabolites (p = 0.016). Protozoa-positive samples showed higher FCM levels than negative samples (p = 0.0150). High dog exposure was statistically associated with protozoa infection (p = 0.0006). The results indicated that improving housing, especially in terms of floor space and avoiding dog exposure, reduces stress and can thus be applied to make control strategies in multi-unowned-cat environments more efficient, especially when cats are confined.

Reflections of ecological differences? Stress responses of sympatric Alpine chamois and red deer to weather, forage quality, and human disturbance.

Depending on the habitats they live in, temperate ungulates have adapted to different degrees to seasonally changing forage and weather conditions, and to specific escape strategies from predators. Alpine chamois, a mountain ungulate, and red deer, originally adapted to open plains, would therefore be expected to differ in their physiological responses to potential stressors. Based on 742 chamois and 1557 red deer fecal samples collected year-round every 2 weeks for 4 years at the same locations within a strictly protected area in the Swiss Alps, we analyzed glucocorticoid metabolite (FGM) concentrations for both species. Results from linear mixed effects models revealed no physiological stress response to changing visitor numbers, but instead to drought conditions for both species during summer. In winter, FGM concentrations increased with increasing snow height in both species, but this response was modulated by temperature in red deer. Chamois showed a stronger stress response to increasing snow height during November and December than between January and March, while FGM concentrations increased with decreasing temperature throughout winter. An increase in FGM concentrations with decreasing forage digestibility during winter was found only for red deer. The results are thus partly in contradiction to expectations based on feeding type and adaptations to different habitats between the two species. The lack of a response to forage digestibility in chamois may reflect either better adaptation to difficult feeding conditions in subalpine forests, or, by contrast, strong constraints imposed by forage quality. The similar responses of both species to weather conditions in winter suggest that climatic factors at the elevations examined here are sufficiently harsh to be limiting to temperate ungulates regardless of their specific adaptations to this environment.

The cost of associating with males for Bornean and Sumatran female orangutans: a hidden form of sexual conflict?

ABSTRACT: Sexual coercion, in the form of forced copulations, is relatively frequently observed in orangutans and generally attributed to their semi-solitary lifestyle. High ecological costs of association for females may be responsible for this lifestyle and may have prevented the evolution of morphological fertility indicators (e.g., sexual swellings), which would attract (male) associates. Therefore, sexual conflict may arise not only about mating per se but also about associations, because males may benefit from associations with females to monitor their reproductive state and attempt to monopolize their sexual activities. Here, we evaluate association patterns and costs for females when associating with both males and females of two different orangutan species at two study sites: Suaq, Sumatra (Pongo abelii), and Tuanan, Borneo (Pongo pygmaeus wurmbii). Female association frequency with both males and females was higher in the Sumatran population, living in more productive habitat. Accordingly, we found that the cost of association, in terms of reduced feeding to moving ratio and increased time being active, is higher in the less sociable Bornean population. Males generally initiated and maintained such costly associations with females, and prolonged associations with males led to increased female fecal cortisol metabolite (FCM) levels at Tuanan, the Bornean population. We conclude that male-maintained associations are an expression of sexual conflict in orangutans, at least at Tuanan. For females, this cost of association may be responsible for the lack of sexual signaling, while needing to confuse paternity. SIGNIFICANCE STATEMENT: Socioecological theory predicts a trade-off between the benefits of sociality and the ecological costs of increased feeding competition. Orangutans' semi-solitary lifestyle has been attributed to the combination of high association costs and low predation risk. Previous work revealed a positive correlation between association frequencies and habitat productivity, but did not measure the costs of association. In this comparative study, we show that females likely incur costs from involuntary, male-maintained associations, especially when they last for several days and particularly in the population characterized by lower association frequencies. Association maintenance therefore qualifies as another expression of sexual conflict in orangutans, and especially prolonged, male-maintained associations may qualify as an indirect form of sexual coercion. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00265-020-02948-4.

Timed environmental exposure indicates sample stability for reliable noninvasive measurement of fecal cortisol metabolite concentrations in sheep.

The use of noninvasive techniques to evaluate stress responses in animals has become an increasingly popular method of animal welfare assessment in both production animals and wildlife. In particular, using fecal samples to measure fecal cortisol metabolites (FCMs) as a quantitative measure has proven ideal as samples can be collected remote to the animal after defecation without the need for invasive procedures. Colorimetric enzyme immunoassays (EIAs) have been shown to have a high level of selectivity and sensitivity for FCM concentration analysis, equivalent to the traditionally used radioimmunoassay. Regardless of the assay system used, species- and sample-specific validation must be undertaken to ensure the reliability of results, particularly where sampling is undertaken in a novel species or where environmental conditions might impact FCM stability in the fecal sample. To determine the limit of environmental exposure acceptable for analysis of FCM concentrations in ovine scat samples collected from a paddock under conditions of stable heat and humidity, this study quantified FCMs in ovine feces shortly after defecation (2-7 h) and after timed environmental exposure (1-9 d). Samples were determined to show stable FCM concentrations for up to 5 d by this analysis. Understanding the impacts of environmental exposure, and therefore the viability of remote fecal collection methods for quantitative analysis of FCM by EIA, is important to assess the utility of noninvasive measures of endocrine status in animals where the exact timing of defecation may not be known.

Response of Bolivian gray titi monkeys (Plecturocebus donacophilus) to an anthropogenic noise gradient: behavioral and hormonal correlates.

Worldwide urban expansion and deforestation have caused a rapid decline of non-human primates in recent decades. Yet, little is known to what extent these animals can tolerate anthropogenic noise arising from roadway traffic and human presence in their habitat. We studied six family groups of titis residing at increasing distances from a busy highway, in a park promoting ecotourism near Santa Cruz de la Sierra, Bolivia. We mapped group movements, sampled the titis' behavior, collected fecal samples from each study group and conducted experiments in which we used a mannequin simulating a human intrusion in their home range. We hypothesized that groups of titi monkeys exposed to higher levels of anthropogenic noise and human presence would react weakly to the mannequin and show higher concentrations of fecal cortisol compared with groups in least perturbed areas. Sound pressure measurements and systematic monitoring of soundscape inside the titis' home ranges confirmed the presence of a noise gradient, best characterized by the root-mean-square (RMS) and median amplitude (M) acoustic indices; importantly, both anthropogenic noise and human presence co-varied. Study groups resided in small, overlapping home ranges and they spent most of their time resting and preferentially used the lower forest stratum for traveling and the higher levels for foraging. Focal sampling analysis revealed that the time spent moving by adult pairs was inversely correlated with noise, the behavioral change occurring within a gradient of minimum sound pressures ranging from 44 dB(A) to 52 dB(A). Validated enzyme-immunoassays of fecal samples however detected surprisingly low cortisol concentrations, unrelated to the changes observed in the RMS and M indices. Finally, titis' response to the mannequin varied according to our expectation, with alarm calling being greater in distant groups relative to highway. Our study thus indicates reduced alarm calling through habituation to human presence and suggests a titis' resilience to anthropogenic noise with little evidence of physiological stress.

Welfare Assessment in Shelter Dogs by Using Physiological and Immunological Parameters.

This study aimed to evaluate the state of welfare of a group of dogs during the first month after entering the shelter by using different stress parameters. Blood and fecal samples were collected from a group of 71 dogs at the time of admission to the shelter. In 46 of these dogs, sampling was repeated after four weeks. Well-recognized welfare biomarkers, such as fecal cortisol and leukocytes, as well as some innovative parameters (ß-endorphin and lysozyme) were determined. Uni- and multivariate statistical analyses were used to evaluate their interactions and changes over time. Neutrophils (p < 0.01), lysozyme (p < 0.05), and fecal cortisol (p < 0.05) decreased, while lymphocytes (p < 0.05) increased after four weeks compared to the first days of being in the shelter, suggesting an improvement in the dogs' welfare over time. A principal component analysis extracted three bipolar components (PCs), explaining 75% of the variance and indicating negative associations between neutrophil and lymphocyte (PC1), lysozyme and ß-endorphin (PC2), cortisol and lysozyme (PC3). The associations between these variables within each PC also confirmed the intricate relationships between the hypothalamic-pituitary-adrenal (HPA) axis and the immune system as well as the importance of a multiparametric approach in evaluating welfare.

HPLC-QTOF method for quantifying 11-ketoetiocholanolone, a cortisol metabolite, in ruminants' feces: Optimization and validation.

Studies of animal ecology can benefit from a quantified understanding of eco-physiological processes and, in particular, of the physiological responses in free-ranging animals to potential stressors. The determination of fecal cortisol metabolites as a noninvasive method for monitoring stress has proved to be a powerful tool. High-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) has emerged as the most accurate method for avoiding problems related to the nonspecificity of immunoassays. In this study, we optimize and validate a reliable method using HPLC-MS/MS for quantifying 11-ketoetiocholanolone (11-k), a representative fecal cortisol metabolite in ruminants. An appropriate extraction and purification procedure was developed taking into account the complex nature of feces. The final extract obtained was then analyzed with HPLC-MS/MS using a quadrupole-time-of-fly (QTOF) tandem mass spectrometer with an electrospray ionization interface operating in positive mode, which allowed an unequivocal determination of the metabolite due to its accurate mass capabilities. After rigorous optimization of both sample extraction and the HPLC-QTOF parameters, making use of feces from free-ranging Iberian ibex, ideal conditions were established. Matrix-matched standards were used to calibrate the method. The limit of detection and quantification was 13- and 40- ng/g, respectively. The validation of the method was performed with recoveries in the range of 85-110%, a figure much higher than the 60% obtained with the previous extraction methods used in our laboratory, and with relative standard deviations (RSDs) no higher than 15% for the complete analytical procedure, including extraction and analysis. The time required for the fecal 11-k analysis was greatly reduced in comparison with the previous work carried out in our laboratory. This is the first time that QTOF mass detection coupled with HPLC has been validated for 11-k quantification in feces from free-ranging ruminants such as Iberian ibex. Given the high selectivity and sensitivity attained, our method could become a useful tool for noninvasive stress quantification in ruminants.

Physiological and behavioral responses to routine procedures in captive common marmosets (Callithrix jacchus).

The effect of routine captive procedures on the welfare of species used as experimental models in biomedical research is of great interest, since stress may alter the generalization and interpretation of results. This study investigated behavioral and endocrine (fecal cortisol) reactivity patterns in common marmoset (Callithrix jacchus) adult males (N = 10) and females (N = 9) subjected to three types of routine procedures in captivity: (1) moving to a same-sized cage (P1), to a smaller cage (P2), and (2) first-time pair formation (P3). Sexually dimorphic cortisol responses were detected in animals submitted to a physical environmental stressor (cage change). Females showed an increased response throughout P1, in relation to baseline (BP) cortisol, and a trend during P2. Males increased cortisol only during P2. On the other hand, males and females showed a similar endocrine response when management involved social challenge (pair formation), with both sexes increasing cortisol levels, but females exhibited a more intense and longer-lasting cortisol increase. Males and females exhibited similar behavioral responses to cage change, except for autogrooming, with males decreasing this behavior in P1. Only females demonstrated a significantly higher increase in piloerection frequency than that of males during the pair formation phase. These endocrine and behavioral changes must be taken into account when interpreting research data that involve these types of procedures. Further studies on the impacts of routine colony management are required to devise and include protocols in official husbandry guidelines.

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 ß-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.

Stress assessment in small ruminants kept on city farms in southern Germany.

Sheep and goats are frequently used in nonhuman animal-assisted activities on city farms. There are few data available on this type of usage of small ruminants. Health evaluations, behavioral observations (feeding, resting, comfort, explorative and social behaviors), behavioral tests (human approach tests and touch test), and measurements of fecal cortisol metabolites and heart rate were performed to assess stress levels in 25 sheep and 32 goats on 7 city farms and 2 activity playgrounds in Germany. No evidence was found that the animals suffered from major distress. Health evaluations, behavioral observations, and behavioral tests proved to be the methods of stress assessment most suitable for routine on-farm checks in these settings.

Effects of crowding and sex on fecal cortisol levels of captive forest musk deer

BACKGROUND: Restricted space and close contact with conspecifics in captivity may be stressful for musk deer, as they are highly territorial and solitary in the wild. So we tested the effects of crowding on stress of forest musk deer (Moschus berezovskii) in heterosexual groups, using fecal cortisol analysis as a non-invasive method. 32 healthy adults during non-breeding seasons were chose as our experimental objects. Group 1 was defined as higher crowding condition, with 10-15 m²/deer (6 enclosures, 10♀ and 6♂); group 2 was defined as lower crowding condition, with 23-33 m²/deer (6 enclosures, 1010♀ and 6♂). Every enclosure contained 1 male and 3 female. These patterns had been existed for years. RESULTS: The results showed that females in lower crowding condition (217.1 ± 9.5 ug/g) had significantly higher fecal cortisol levels than those in higher crowding condition (177.2 ± 12.1 ug/g). Interestingly, crowding seemed have no effect on male fecal cortisol levels (148.1 ± 9.1 ug/g and 140.5 ± 13.3 ug/g, respectively). At both groups, cortisol was significantly lower in males than in females. CONCLUSIONS: These results showed that chronic crowding may affect stress status of captive forest musk deer. The captive environment should consider the space need for musk deer.