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This research project had the aim to validate the possible alternative and less-painful sampling method of cutting feathers close to the skin instead of plucking them for subsequent feather corticosterone analysis, confirming recently-published results for other species in captivity. Analyzing CORTf is often used in animal welfare studies in combination with behavioral monitoring. The background of this idea was to act in the sense of animal welfare and reduce the burden of animal studies according to the 3-R-Principle (Replacement, Reduction, and Refinement) by refining procedures. To confirm the hypothesis that the sampling method itself has no influence on CORTf levels measured, plucked and cut samples of the respective bird were collected. Birds of two wild species were used: the Mallard (Anas platyrhynchos) and the Greater Flamingo (Phoenicopterus roseus). The CORTf was measured by using an enzyme-linked immunosorbent assay (ELISA). The determined values were inspected for their mean values, standard deviation (SD), and average differences. Afterwards, the CORTf levels of both species were compared, according to the sampling method, with the concordance correlation coefficient (CCC). In the Bland-Altman (BA) plot the differences of the methods were displayed against the mean values. Additionally, sex, as a possible factor influencing CORTf, was analyzed using the Mann-Whitney U test. The values of CCC showed poor agreement in the comparability of the two methods, whereas the concordance of the BA plot was decent. The average differences between the methods were marginal for both species (Mallards: −0.16 pg/mm, Flamingos −0.13 pg/mm). In summary, all anomalies or differences between the methods were negligible. Therefore, the alternative sampling method seems to be as suitable as the common standard method. No significant difference was found between females and males. Nevertheless, our results suggest that CORTf should not be interpreted in just considering the values themselves, but the results they should be analyzed in the context of a wider set of parameters. Hence, further studies are encouraged to create a larger data pool.

The pinioning of birds was previously one of the most-accepted forms of mutilation in zoos. Despite a lack of knowledge on the effects of deflighting procedures with regard to the well-being of deflighted birds, pelicans are often reversibly deflighted by feather-clipping to keep them in open enclosures, including those with ponds without netting. In the present study, we focused on the welfare implications of flight restraint on one of the most commonly kept types of birds in German zoos, the great white pelican. A combination of behavioral observations and feather corticosterone concentrations (CORTf) of pelicans with different deflighting statuses (i.e., irreversibly deflighted, reversibly deflighted, and airworthy) was used to evaluate the effects of deflighting status on pelican welfare. We observed 215 individuals in 21 different German zoos. The pelicans lived in differently designed exhibits. An ethogram for these species was developed and their behavior was evaluated by scan sampling. Feather samples from 182 individuals were collected to determine if different deflighting conditions influenced the CORTf and therefore stress levels. The hypothesis was that the CORTf values of airworthy pelicans differ from those of deflighted pelicans. Tendencies with regard to the flight status groups were found. Conversely, reversibly deflighted pelicans had higher CORTf levels than irreversible deflighted and airworthy pelicans. Tendencies with regard to CORTf values and the group size of the kept pelicans were observed. The CORTf values were lower in groups consisting of more than 10 animals. In addition, the frequency of fluttering behavior was positively associated with CORTf values. Pelicans that frequently showed fluttering had higher CORTf values. Therefore, fluttering behavior might be considered a sign of stress levels in pelicans. This study is one of the first important steps in assessing the impact of deflighting procedures on the welfare of great white pelicans kept in zoos.

The monitoring of stress physiology includes studying a wide range of endocrinological mechanisms, which can be assessed using multiple tissue samples. This study aimed to evaluate the seasonal variations of hair C, T and DHEA-S in horses for a whole year, as well as to assess the variations between seasons of C/DHEA-S and T/C ratios as a retrospective measure of the hypothalamic–pituitary–adrenal and hypothalamic–pituitary–gonadal axis activity. Ten pure-breed Menorca stallions were included in the study. The hair samples were collected approximately every two months following the shave-reshave method caudally to the sternum. After a methanol-based extraction, samples were analyzed by enzyme immunoassay for cortisol, testosterone, and dehydroepiandrosterone sulphate. Following our findings, we detected that cortisol, testosterone and dehydroepiandrosterone sulphate were significantly affected by seasonality, with the highest values of cortisol during summer and the lowest values of testosterone during spring. Dehydroepiandrosterone sulphate concentrations were increased in autumn compared to the other studied periods. Additionally, the studied hormone ratios showed variations between seasons. To conclude, season should, therefore, be considered when assessing sexual and stress hormones in stallion hair, since this variable can be a potential influencing factor and led to misinterpretations.

The most common feather sampling method for feather corticosterone measurement is by plucking the feathers from the bird’s skin. This procedure performed on living, restrained birds is qualified as an animal experiment according to German/European legislation, which has to be applied for from the competent authorities. The Directive 2010/63/EU requires the full implementation of the 3-R Principle of Russel and Burch in animal experiments, which means not only to replace the use of animals, but also to reduce the number of animals used and to refine procedures whenever possible. In response to this issue, the aim of this study was to validate an alternative, less invasive sampling method by cutting feathers close to the skin in comparison to the gold standard of plucking them. For this proof-of-principle study, a conventional poultry husbandry with trial groups of geese (Anser anser domesticus) and ducks (Anas sterilis) was selected. All birds were kept under the same living conditions to standardize the influencing factors regarding husbandry, and thus, their stress levels. Feather samples were collected between the shoulders from 46 geese and 51 ducks, both by cutting as well as by plucking, directly after slaughter for meat production. Feather corticosterone levels were measured with Enzyme-Linked Immunosorbent Assay (ELISA). Results were compared using Bland–Altman plots and concordance correlation coefficients (CCC). It could be seen that concordance between corticosterone levels in cut and plucked feathers was rather poor: 0.38 for Anser, and 0.57 for Anas. However, comparing the mean corticosterone values in pg/mm of each species with their respective standard deviations, the differences between the methods were negligible. As the results showed that the differences between the individuals were markedly greater than the differences between the methods, the determination of corticosterone levels in cut feathers is valid compared to using plucked feathers. The validation tests of ELISA showed only acceptable repeatability and reliability. Hence, the results should be verified in further studies. In conclusion, it is recommended for future research to use cut instead of plucked feathers for corticosterone measurement.

Deflighting zoo birds is a practice that receives increasing criticism due to its presumed incompatibility with animal welfare. To our knowledge, this is the first approach to address this problem in a scientific way. To do this, we compared feather corticosterone (CORTf) from Greater Flamingos (Phoenicopterus roseus, n = 151) of different flight status (i.e., pinioned, feather clipped, airworthy) from twelve different zoological institutions. To complement the hormone measurements, behavioral observations (scan samplings) were conducted prior to feather sampling within the presumed time frame of feather growth. We hypothesized that CORTf of the deflighted flamingos would differ from CORTf of their airworthy conspecifics. No significant difference in CORTf was found between the three groups, and our hypothesis was rejected. However, the impact of the institution itself (i.e., the housing conditions) proved to be the most dominant variable (variance between the institutions = 53.82%). Due to high variability, the behavioral observations were evaluated descriptively but did not give rise to doubt the findings in CORTf. Therefore, we assume that the method of flight restraint of Greater Flamingos does not have a measurable effect on CORTf. We consider this model for evaluating animal welfare of zoo birds a useful tool and provide ideas for further adjustments for consecutive studies.

Cortisol levels in fish skin mucus have shown to be good stress indicators in farm fish exposed to different stressors. Its applicability in free-ranging animals subject to long-term environmental stressors though remains to be explored. The present study was therefore designed to examine whether skin mucus cortisol levels from a wild freshwater fish (Catalan chub, Squalius laietanus) are affected by the habitat quality. Several well-established hematological parameters and cortisol concentrations were measured in blood and compared to variations in skin mucus cortisol values across three habitats with different pollution gradient. Fluctuations of cortisol in skin mucus varied across the streams of differing habitat quality, following a similar pattern of response to that detected by the assessment of cortisol levels in blood and the hematological parameters. Furthermore, there was a close relationship between cortisol concentrations in skin mucus and several of the erythrocytic alterations and the relative proportion of neutrophils to lymphocytes. Taken together, results of this study provide the first evidence that skin mucus cortisol levels could be influenced by habitat quality. Although results should be interpreted with caution, because a small sample size was collected in one studied habitat, the measurement of cortisol in skin mucus could be potentially used as a biomarker in freshwater fish.

The Mediterranean tortoise Testudo hermanni inhabits different regions bordering the northwestern Mediterranean. This species is vulnerable, protected by legislation, and involved in various breeding and reintroduction programs. Wild populations face numerous environmental and anthropogenic stressors that can potentially interfere with their conservation. While seasonal changes in stress-response biomarkers, such as glucocorticoids and thyroid hormones, have been widely studised in mammals and birds, there is a paucity of research in reptile species. Therefore, the present study aimed to evaluate the seasonal fluctuations in corticosterone and total triiodothyronine levels in adult and juvenile Hermann’s tortoises (Testudo hermanni) as a measure of the physiological stress response. Blood samples were collected seasonally (winter, spring, summer, and autumn) and posteriorly analyzed by using a specific and validated enzyme immunoassay for both hormones, respectively. The results showed that corticosterone levels varied seasonally and differed between sexes, whereas total triiodothyronine levels changed seasonally but did not differ between sexes. Notably, juveniles exhibited no seasonal changes in either corticosterone or total triiodothyronine levels. Additionally, no correlation between blood extraction duration and hormonal concentrations was observed. This study is pioneering in its comprehensive evaluation of corticosterone and total triiodothyronine changes across all four seasons, including winter, and its focus on juvenile Hermann’s tortoises.

The effectiveness of rabbit-sperm cryopreservation is still below average compared to other domestic species. After the sperm cryopreservation process, post-thawing parameters like motility and membrane integrity are significantly compromised. The use of new extender constituents is an approach that can be used to improve the effectiveness of cryopreservation. Accordingly, we used honey (1.25, 2.5, 5, and 10%), coenzyme Q10 (100 and 200 μM), and β-carotene/α-tocopherol (500 μM/620 μM and 250 μM/310 μM) as candidate components for rabbit-sperm extenders during cryopreservation. Ejaculates from commercial adult rabbit bucks (n = 5) were cryopreserved using conventional freezing. Several post-thawing sperm parameters were assessed, including total motility, membrane integrity, viability, nuclear membrane integrity, acrosome reaction, and mitochondrial membrane potential and activation. Additionally, we performed hormonal analyses of the seminal plasma. Moreover, we analyzed the post-thawing levels of a molecular marker of sperm quality, proAKAP4, which was used in rabbits for the first time. Our findings showed that the 2.5% honey supplementation increased the post-thawing sperm motility (13.75 ± 3.75%) compared to the greater concentrations employed. However, the post-thawing motility was negatively affected by the coenzyme Q10 (0%, in both groups) but was not affected by the β-carotene/α-tocopherol supplementation (22 ± 18.15%, and 11.67 ± 10.17%). In conclusion, the cryopreservation protocols of this study did not help to maintain the sperm parameters after thawing. Further studies are required to identify novel protocols to mitigate the damage caused to rabbit sperm during cryopreservation.

In many countries, horses remain involved in traditional equestrian events such as those celebrated in Menorca (Balearic Islands, Spain) every year since at least the 14th century. The present study aimed to evaluate the variations in salivary cortisol concentrations to estimate the physiological stress response in horses at the Menorca patronal festivals. Two different editions (years 2016 and 2018) of the festivals in honor of the Virgin of Grace in Maó (Menorca, Spain) were studied. Nineteen and seventeen Pure Breed Menorca stallions were included in the study, respectively. The stallions were aged between seven and twelve years. During celebrations, samples were collected before the start of the festivals between 8–9 a.m. and during the festivals at 8–9 p.m. On the second day of celebrations, the samples were collected at 8–9 a.m. and 3–4 p.m. Finally, on the day after the festivals, one sample was collected at 8–9 p.m. Additionally, a control group was sampled at 8–9 a.m., 3–4 p.m., and 8–9 p.m. Salivary cortisol concentrations were assessed by using a commercial enzyme immunoassay kit specially validated to quantify salivary cortisol in horses. Salivary cortisol concentrations did not show significant differences between sampling hours in the control group (p > 0.05). All the samples collected during festivals were significantly higher than samples of the control group (p < 0.05). Within the twenty-four hours after the end of the celebrations, cortisol concentrations returned to baseline levels and did not differ significantly from the control group (p > 0.05). Hence, the present study describes that the participation of the horses in these particular acts generate an acute and transitory stress response. Overall, the current work provides a reasonable basis for future research on the stress physiology and well-being of horses participating in traditional celebrations or similar events.

Monitoring the hypothalamic–pituitary–adrenal (HPA) axis through determination of fecal cortisol metabolite (FCM) levels is a non-invasive method useful for understanding how handling and social conditions may affect the physiological status of zoo animals. The present study used FCM analysis to evaluate whether the HPA axis activity of a lion pride was modified by a change in social and handling conditions after the death of the dominant male. Five African lions (Panthera leo bleyenberghi), two males and three females, were included in the study. Fecal samples were collected before and after the death of the dominant male. To avoid cohabitation conflicts between males before the dominant male died, subgroups were established and subjected to weekly changes between indoor and outdoor facilities. After the death of the dominant male, these management dynamics ceased, and the remaining four lions were kept together outdoors. Significant lower group FCM concentrations (p < 0.001) were detected after the decease of the dominant male, probably associated with a decrease in daily handling, together with a more stable social environment. Overall, the present study indicates the effect of different management scenarios on the HPA axis activity and differentiated physiological responses to the same situation between individuals.

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.

Rabbits are interesting as research animal models for reproduction, due to their condition of species of induced ovulation, with the release of endogenous gonadotropin-releasing hormone (GnRH) due to coitus. Glucocorticoid (GC) signaling, crucial for physiological homeostasis, is mediated through a yet unclear mechanism, by the GC receptor (NR3C1/GR). After mating, the female reproductive tract undergoes dynamic modifications, triggered by gene transcription, a pre-amble for fertilization and pregnancy. This study tested the hypothesis that when ovulation is induced, the expression of NR3C1 is influenced by sperm-free seminal plasma (SP), similarly to after mating (whole semen), along the different segments of the internal reproductive tract of female rabbits. Semen (mating) was compared to vaginal infusion of sperm-free SP (Experiment 1), and changes over time were also evaluated, i.e., 10, 24, 36, 68, and 72 h post-mating, corresponding to specific stages, i.e., ovulation, fertilization, and the interval of early embryo development up to the morula stage (Experiment 2). All does were treated with GnRH to induce ovulation. Samples were retrieved from seven segments of the reproductive tract (from the cervix to infundibulum), at 20 h post-mating or sperm-free SP infusion (Experiment 1) or at 10, 24, 36, 68, and 72 h post-mating (Experiment 2). Gene expression of NR3C1 was analyzed by qPCR. Results showed an increase in NR3C1 expression in the infundibulum compared to the other anatomical regions in the absence of spermatozoa when sperm-free SP infusion was performed (Experiment 1). Moreover, during the embryo transport through the oviduct, the distal isthmus was time-course upregulated, especially at 72 h, when morulae are retained in this anatomical region, while it was downregulated in the distal uterus at 68 h (Experiment 2). The overall results suggest that NR3C1, the GC receptor gene, assessed in the reproductive tract of does for the first time, shows differential expression changes during the interval of oviductal and uterine embryo transport that may imply a relevant role of the GC action, not only close to the site of ovulation and fertilization, but also in the endometrium.

The maternal environment modulates immune responses to facilitate embryo development and ensure pregnancy. Unraveling this modulation could improve the livestock breeding systems. Here it is hypothesized that the exposure of the female rabbit reproductive tract to semen, as well as to early embryos, modulates inflammation and angiogenesis among different tissue segments. qPCR analysis of the gene expression changes of the anti-inflammatory interleukin-10 (IL10) and transforming growth factor beta family (TGFβ1–3) and the angiogenesis mediator vascular endothelial growth factor (VEGF-A) were examined in response to mating or insemination with sperm-free seminal plasma (SP). Reproductive tract segment (cervix to infundibulum) samples were obtained in Experiment 1, 20 h after gonadotropin-releasing hormone (GnRH) stimulation (control), natural mating (NM) or vaginal infusion with sperm-free SP (SP-AI). Additionally, segmented samples were also obtained at 10, 24, 36, 68 or 72 h after GnRH-stimulation and natural mating (Experiment 2). The results of gene expression, analyzed by quantitative PCR, showed that NM effects were mainly localized in the uterine tissues, depicting clear temporal variation, while SP-AI effects were restricted to the oviduct. Changes in anti-inflammatory and angiogenesis mediators indicate an early response in the uterus and a late modulation in the oviduct either induced by semen or preimplantation embryos. This knowledge could be used in the implementation of physiological strategies in breeding systems to face the new challenges on rabbit productivity and sustainability.

Horse transportation for temporary relocation during rest periods is a common and widespread practice among horse owners, either from sport competition or working tasks. This study aimed to determine the effect of a relocation period and the multiple factors associated with a rest period on hair cortisol concentrations (HCCs) in horses. Additionally, this study reports the seasonal effect on HCCs and hair growth over a year. Thirteen police horses, Pure Spanish stallions of various ages (5–13 y), were selected to participate in this study. Hair sample collection was carried out approximately every 30 d for seven months (Study 1) and a year (Study 2). Cortisol determinations were performed by enzyme immunoassay. Interestingly, Study 1 revealed that relocated horses (n = 4) exhibited elevated HCCs compared with control horses (n = 4) after the relocation period (p < 0.05). Study 2 (n = 5) showed higher HCCs during summer compared with autumn and winter, and higher hair growth rates in winter compared with the other seasons (p < 0.05). Relocated horses had higher HCCs, suggesting a change in their welfare status, probably related to the sudden change in their surrounding conditions. However, these results should be interpreted cautiously due to the low sample size used. The nature of the relationship between HCCs and horse welfare needs to be further examined.