The forest fly (Hippobosca equina) is an obligate haematophagous dipteran insect (order Diptera) that primarily infests horses and may contribute to the circulation of vector-borne pathogens. This study aimed to investigate the presence of Anaplasma phagocytophilum, Borrelia burgdorferi s.l., Babesia caballi, and Theileria equi, important vector-borne pathogens of equids, in forest flies collected from horses in endemic areas of Spain. A total of 170 forest flies were collected from 39 equids across four geographical regions in Spain (Segovia, Madrid, Toledo, and Menorca) and blood samples were collected from 27 of these horses. All flies were morphologically and molecularly identified as H. equina, and DNA extracted from flies and equine blood was screened using multiplex real-time and nested PCR, followed by sequencing and phylogenetic analysis. Neither flies nor horses tested positive for A. phagocytophilum, whereas one fly was positive for B. burgdorferi s.l. (0.6%). In contrast, T. equi and B. caballi DNA were detected in 11.2% and 1.2% of flies, respectively, and all positive flies were collected from horses positive for equine piroplasmosis (T. equi/B. caballi infection), with identical 18S rRNA sequences between hosts and flies. Nested PCR showed a higher detection rate than real-time PCR for the detection of these piroplasms in flies and blood samples. These findings provide the first molecular evidence of EP pathogens in H. equina and support further investigation into the epidemiological importance of forest flies in equine pathogen surveillance.

Fecal glucocorticoid metabolites (FGM) are widely used to assess hypothalamic-pituitary-adrenal (HPA) activity in vertebrates, but their applicability to reptiles, and more specifically to snakes, remains poorly explored. The objective of the present study was to determine how changes in housing conditions, from a standard racking system to enriched terraria, affect fecal glucocorticoid metabolite concentrations as a proxy potentially related to endocrine activity and stress response. A total of 58 fecal samples were collected across three different phases from 12 snakes representing four species: Boa constrictor (BC), Lampropeltis polyzona (LP), Pantherophis guttatus (PG), and Python regius (PR). FGM concentrations ranged from 54.9 to 832.2 ng/g, with a mean value of 298.4 ± 171.6 ng/g (mean ± SD). Data showed marked inter-individual variability, while within-individual concentrations remained relatively stable across experimental phases. PG exhibited the highest FGM concentrations during the study, with LP showing intermediate levels and PR and BC the lowest. The results showed that housing conditions did not have a significant effect on FGM concentrations in the studied population. In conclusion, the present study showed that fecal hormone analysis provides an integrated measure of glucocorticoid metabolites concentrations over time, potentially reflecting physiological differences among snake species.

Background Hippoboscid flies are obligate hematophagous Diptera that infest mammals and birds worldwide. Hippobosca equina, commonly known as the forest fly, primarily affects horses and is considered both a mechanical and/or biological vector of various bacteria, viruses, and protozoa. Equine piroplasmosis (EP) is a tick-borne disease caused by three intra-erythrocytic hemoprotozoans: Theileria equi, Babesia caballi, and the recently identified species Theileria haneyi. To date, no studies have reported the detection of any Piroplasmida in H. equina. This study provides the first evidence of the presence of T. equi and B. caballi in forest flies infesting horses in endemic areas, identified using molecular techniques. Methods A total of 170 forest flies were collected from 35 Equidae across five locations in Spain (Segovia, Madrid, Toledo, and Menorca Island). Blood samples for equine piroplasmosis (EP) testing were drawn from 27 examined horses. All insects were identified both morphologically and molecularly, and genomic DNA from each fly and their equine host blood samples was screened for EP using real-time and nested PCR. Phylogenetic analyses were performed on all obtained T. equi and B. caballi sequences. Results All collected hippoboscids were identified as H. equina using a stereomicroscope and taxonomic keys, as well as by sequencing the mitochondrial cytochrome oxidase subunit 1 (COI) gene. Molecular screening detected T. equi DNA in 19 of 170 (11.2%) collected forest flies and B. caballi DNA in 2 of 170 (1.2%). All EP-positive flies were collected from EP-positive horses, with sequences from the flies and their equine hosts being identical. Two distinct T. equi and two B. caballi sequences were identified. Conclusions This study is the first to report the detection of T. equi and B. caballi in H. equina collected from horses. Given that approximately one-quarter of the flies sampled from EP-positive horses tested positive for the disease, sampling forest flies could be a practical method for detecting equine piroplasmosis in feral horse populations. Experimental transmission studies are required to assess the vector competence of H. equina for EP and other equine diseases.

ABSTRACT Blood serum (BS) and seminal plasma (SP) share a plethora of compounds that might present an individual and/or temporal concentration variation. We aimed to determine whether BS and SP concentrations of albumin, calcium, citrate, creatinine, fructose, glucose, lactate, total protein, urea, zinc, cortisol, anti‐Müllerian hormone (AMH) and testosterone are related to weekly collections in New Zealand White (NZW) adult rabbit bucks. During a 12‐week study, blood samples were obtained at the beginning and the end of the study period, and semen samples were taken twice a week from four NZW adult rabbit bucks, starting at 6–7 months of age. After semen collection, the sperm motility was subjectively assessed, and SP was obtained by centrifugation. BS and SP were evaluated for the above‐mentioned metabolites using a Biosystems BA400 automated analyser with commercial‐specific kits or enzyme immunoassay (EIA) kits, assessing the effects of the male and time of collection. In addition, a correlation analysis aimed at disclosing associations between parameters in BS and SP was performed. Male effect was not significant for BS, but it was significant for SP albumin, citrate, fructose, glucose, lactate and total protein. In addition, all the correlations in BS were positive, whereas they were more balanced in SP, being close to half of the correlations. In conclusion, variations of some metabolites (albumin, citrate, fructose, glucose, lactate and total protein) appear to be potential biomarkers for rabbit SP, although further studies should test their usefulness for sperm fertility assessment.

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 brain regulates multiple metabolic processes, such as food intake, energy expenditure, insulin secretion, hepatic glucose production, and glucose and fatty acid metabolism in adipose tissue, which are fundamental for the maintenance of energy and glucose homeostasis during lactation and pregnancy. In addition, brain expression has a fundamental impact on the development of maternal behavior. Although brain functions are partly regulated by long noncoding RNAs (lncRNAs), their expression profiles have not been characterized in depth in any ruminant species. We have sequenced the transcriptome of 12 brain tissues from 3 goats that were 1 mo pregnant and 4 nonpregnant goats to investigate their lncRNA expression patterns. Between 4,363 (adenohypophysis) and 4,604 (olfactory bulb) lncRNAs were expressed in brain tissues, leading us to establish a set of 794 already annotated lncRNAs and 5,098 novel lncRNA candidates. The detected lncRNAs shared features with those of other mammals, and tissue-specific lncRNAs were enriched in brain development-related terms. Differential expression analyses between goats that were 1 mo pregnant and nonpregnant goats showed that the lncRNA expression profiles of certain brain regions experience substantial changes associated with early pregnancy (238 lncRNAs are differentially expressed in the olfactory bulb), but others do not. Enrichment analysis showed that differentially expressed lncRNAs from the olfactory bulb are co-expressed with genes previously linked to behavioral changes related to pregnancy. These findings provide a first characterization of the landscape of lncRNA expression in the goat brain and provides valuable clues to understand the molecular events triggered by early pregnancy in the central nervous system.

Immunoglobulin A (IgA) has been investigated as a stress biomarker with the potential to complement glucocorticoid measurements in welfare assessments. This study aimed to develop the methodology and validate an enzyme immunoassay (EIA) for quantifying IgA in feces (FIgA) of lions (Panthera leo), investigate excretion patterns of FIgA under baseline conditions in captive lions, and explore its relationship with fecal glucocorticoid metabolites (FGM). Feces were collected from 11 lions housed in stable social groups at four Spanish zoos over a period of two to six weeks. FIgA was reliably quantified using a commercial EIA, with concentrations ranging from 0.28 to 794.17 μg IgA/g feces, showing substantial intra- and inter-individual variability. Females had significantly higher FIgA concentrations than males (113.10 vs 54.96 μg IgA/g feces; p < 0.01). Additionally, FIgA concentrations varied across zoos (p < 0.001). Positive correlations were found between FIgA and FGM for all samples combined (rho = 0.43, p < 0.001) and across individual means (rho = 0.70, p < 0.05), but not consistently when examining each lion separately. This study demonstrates for the first time that IgA can be reliably quantified in lion feces, paving the way for its application in welfare studies.

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.

Background: The brain is an extraordinarily complex organ with multiple anatomical structures involved in highly specialized functions related with behavior and physiological homeostasis. Our goal was to build an atlas of protein-coding gene expression in the goat brain by sequencing the transcriptomes of 12 brain regions in seven female Murciano-Granadina goats, from which three of them were 1-month pregnant. Results: Between 14,889 (cerebellar hemisphere) and 15,592 (pineal gland) protein-coding genes were expressed in goat brain regions, and most of them displayed ubiquitous or broad patterns of expression across tissues. Principal component analysis and hierarchical clustering based on the patterns of mRNA expression revealed that samples from certain brain regions tend to group according to their position in the anterior-posterior axis of the neural tube, i.e., hindbrain (pons and medulla oblongata), midbrain (rostral colliculus) and forebrain (frontal neocortex, olfactory bulb, hypothalamus, and hippocampus). Exceptions to this observation were cerebellum and glandular tissues (pineal gland and hypophysis), which showed highly divergent mRNA expression profiles. Differential expression analysis between pregnant and non-pregnant goats revealed moderate changes of mRNA expression in the frontal neocortex, hippocampus, adenohypophysis and pons, and very dramatic changes in the olfactory bulb. Many genes showing differential expression in this organ are related to olfactory function and behavior in humans. Conclusion: With the exception of cerebellum and glandular tissues, there is a relationship between the cellular origin of sampled regions along the anterior-posterior axis of the neural tube and their mRNA expression patterns in the goat adult brain. Gestation induces substantial changes in the mRNA expression of the olfactory bulb, a finding consistent with the key role of this anatomical structure on the development of maternal behavior.

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.

The measurement of glucocorticoid hormones, such as cortisol, is a technique being increasingly used as a welfare or stress indicator (Mormède et al., 2007; Palme, 2019). In response to perceived stressors, these hormones are released into the bloodstream through activation of the hypothalamic–pituitary–adrenal (HPA) axis. The release of cortisol, a principal glucocorticoid in many mammals, enables the body to cope with threatening or demanding situations. Although this adaptive response is usually beneficial for the animal, chronic activation of the HPA axis and long-term cortisol elevations can reduce growth and reproduction, compromise the immune system, and impair the individual's ability to respond to subsequent stressors (Moberg & Mench, 2000). Blubber, a specialized hypodermic adipose tissue found in most marine mammals, has emerged as a practical alternative tissue wherein steroid hormones can be measured. The blubber tissue is thought to accumulate cortisol over hours to days that passively diffuse from blood with a delay of one to several hours after the onset of the stressor (Champagne et al., 2017; Kellar et al., 2015). These attributes of hormone integration offer a potentially new method of studying baseline cortisol levels that otherwise might be difficult to obtain. The growing number of endocrine studies using blubber suggests that this tissue may become the preferred method of hormone analysis in wild cetaceans (Beaulieu-McCoy et al., 2017; Kellar et al., 2015; Trana et al., 2015). Despite its popularity, few studies have addressed whether collection protocols can influence steroid hormone levels present in blubber. For instance, the state of sample decomposition could be a source of variability of these levels in the blubber of humpback (Megaptera novaeangliae) and beluga (Delphinapterus leucas) whales (Mello et al., 2017; Trana et al., 2015). As demonstrated in belugas and harbor porpoises (Phocoena phocoena), cortisol is stratified through the blubber, thus caution should also be taken when different sample depths are collected (Kershaw et al., 2017; Trana et al., 2015). Similarly, the body location of blubber collection has been shown to influence sex hormone levels in some species (Kellar et al., 2006; Mello et al., 2017). Regarding blubber cortisol, Kershaw et al. (2017) did not detect differences in blubber cortisol among three body regions in samples collected from stranded harbor porpoise. As observed in terrestrial mammals, where hair hormone levels can be influenced by body location in some species (Ashley et al., 2011; Macbeth et al., 2010; Terwissen et al., 2013) but not in others (Carlsson et al., 2016; Macbeth et al., 2012; Tallo-Parra et al., 2017), species-specific patterns could also be possible in marine mammals (Trana et al., 2015). Revealing whether and how cortisol levels vary with blubber location will help interpret physiological metrics and improve future collection protocols. To improve accuracy and interpretation of hormone measurements in this sample matrix, the present study was designed to evaluate whether sample location within the body influences blubber cortisol concentration in striped dolphins (Stenella coeruleoalba). As reviewed elsewhere, two different reference denominators have been used to estimate blubber cortisol levels (Champagne et al., 2018); blubber tissue mass and amount of lipid extracted. Therefore, the present study also aims to highlight the influence that the reference denominator can have on blubber cortisol concentrations. A total of 10 striped dolphins (Table 1), stranded along the Catalan coast (northwestern Mediterranean Sea) and in a well-preserved state according to classification of Jauniaux et al. (2002), were transported to Veterinary School of the Autonomous University of Barcelona for necropsy. Blubber samples were collected from five different locations: cranial and caudal areas in relation to the dorsal fin, and dorsal, medial, and ventral areas in relation to the pectoral flipper (Figure 1). Samples were surgically excised before necropsy using a scalpel. To avoid potential effects of cortisol stratification (Kershaw et al., 2017; Trana et al., 2015), full-depth blubber samples were uniformly collected. The epidermis and the outer layer of the dermis, mostly composed of connective tissue, were discarded leaving only fat-filled panniculus adiposus, referred to as blubber. Then, a full-depth subsample of 100 mg from the original block was taken for hormone extraction. Cortisol was extracted from blubber as described by Kellar et al. (2015) with some modifications. In brief, 100 mg of each blubber sample were placed in a homogenization microtube with five 0.7 mm balls and ground using a ball mill for 15 min at 25 Hz (MM200; Retsch, Haan, Germany). The homogenized tissue was first separated, rinsed with ethanol, and the washed contents transferred to a new 15 ml polypropylene tube. Ethanol was also added to the homogenization microtube and all contents transferred to the polypropylene tube. The rinse step was repeated once more obtaining a tube with the sample soaked with a total of 1.5 ml of ethanol. Afterwards, we added 2 ml of a 4:1 ethanol:acetone solution, vortexed for 5 min and centrifuged at 3,000 g for 15 min. The supernatant was transferred into a new weighed 15 ml tube and was incubated in an oven at 37°C. After evaporation the entire polypropylene tube with residue was weighted to obtain the amount of lipid extracted by subtraction of the first weight (before evaporation) from the second weight obtained after evaporation of the contents (Mello et al., 2017). We measured the amount of lipid extracted in each individual sample and calculated the proportion of extracted lipid by: proportion extracted lipid = blubber lipid weight (grams)/total blubber weight (0.1 g). Then, 2 ml of diethyl ether were added to the tube, vortexed for 5 min, and centrifuged at 3,000 × g for 15 min. The supernatant collected was transferred into a 10 ml-glass vial and introduced into the oven (Heraeus model T6; Kendro Laboratory Products, Langenselbold, Germany) with continuous venting at 30°C. The boiling point of diethyl ether is at 34.6°C, therefore, this step should always be performed with caution. Once completely evaporated, the residue was resuspended in 1.5 ml of acetonitrile and lightly vortexed for 5 min. A total of 1.5 ml of hexane was then added to the acetonitrile-lipid mixture, vortexed for 5 min and centrifuged (1,500 × g, 15 min). After centrifugation, 1.5 ml of the acetonitrile present in the lower layer was transferred into a new 10 ml glass vial and the process was repeated once more. The final volume of acetonitrile (3 ml) was transferred into a 15 ml polypropylene tube and introduced into the oven at 37°C. The evaporated contents were resuspended in 0.25 ml of enzyme immunoassay (EIA) buffer provided by the EIA kit and stored at −20°C until analysis. Cortisol concentrations from blubber extracts were measured by EIA (Cortisol EIA KIT; Neogen Corporation, Ayr, UK). The assay was validated for the species and sample of interest (Buchanan & Goldsmith, 2004; Reimers & Lamb, 1991) following the criteria for an immunological validation (Midgley et al., 1969; Reimers & Lamb, 1991). Blubber extracts from 20 different samples were first pooled for the assay validation. Intra- and interassay coefficients of variation (CV) from duplicated samples analyzed (intra-assay CV, n = 11 samples; interassay CV, n = 3 samples) were calculated for precision assessment. The specificity was tested with the linearity of dilution, determined by using 1:1, 1:2, 1:5, and 1:10 dilutions of the pool with EIA buffer. Through the spike-and-recovery test we assessed the accuracy of the EIA; different volumes of the pool were spiked with different volumes of hormone standard of known concentrations provided by the EIA kit (0.19, 0.45, and 1.05 ng cortisol/ml). Finally, we measured the sensitivity of the test, which was given by the smallest amount of cortisol that the assay can distinguish and measure from the blubber extracts previously diluted. Mean intra- and interassay coefficients of variation were 4.15% and 4.92%, respectively, indicating high repeatability of the test. The dilution test showed an R2 = 99.82% and a mean percentage error (± SD) of 0.92% ± 12.07%, demonstrating a high correlation between obtained and theoretical cortisol values (Figure 2). The spike-and-recovery values from the spike recovery test ranged from 0.61 ng/ml to 1.17 ng/ml (M = 0.9 ng/ml) and the average recovery percentage was 107.5% ± 7.65%, suggesting that other potential compounds present in the tissue may not interfere with the analytical assay. Finally, the sensitivity of the test was 0.124 ng cortisol/g blubber. These results indicate that cortisol detection in striped dolphin blubber by the EIA kit used and with the methodology presented is precise, specific, accurate, and sensitive. R software (Version 3.5.3; R Core Team, 2016) was used to analyze the data, with a p-value below .05 as a criterion for significance. First, normality of the data distribution was tested by applying the Shapiro–Wilk test. Generalized linear mixed models (GLMMs) were used to investigate the effect of body location on cortisol concentrations. The GLMMs was applied since this model allows considering for potential variation among individuals. Individual was accordingly treated as a random effect. To account for the skewed and continuous positive data of hormone concentrations, the GLMMs followed a gamma distribution with a log link function. First, one GLMM (glmer function in the R package lme4) was applied with concentration measured as ng of cortisol per gram of blubber tissue (ng cortisol/g blubber) and a second GLMM was applied with concentration measured as ng cortisol per gram of lipid extracted (ng cortisol/g lipid). Because body condition can influence cortisol concentrations (Kershaw et al., 2017), this variable was introduced as a covariate in both models. Body condition was calculated following the Quetelet's index (mass/length2) as this formula has been demonstrated to be the most appropriate for small cetaceans (Kershaw et al., 2017). The interaction between body condition and body location was also added in the models. In both cases, variables were eliminated in a backward stepwise procedure, and selection of the best fit model was performed based upon Akaike's information criterion corrected for small sample size (AICc), ΔAICc (difference between each model's AICc and that of the lowest model) and Akaike weight (Wi). Candidate set models were chosen for which ΔAIC ≤2 (Table 2). When significant, a multiple comparison post hoc test (glht function from the R package multcomp) was applied to determine which body location differed. Finally, the correlation between blubber cortisol concentration and proportion of lipid extracted was studied with a GLMM in which individual was also treated as a random effect. Hormone levels obtained from all five body locations and estimated using blubber tissue mass were used to study the correlation. While the measurement of steroid hormones in blubber samples is becoming increasingly popular, only one study to date has focused on elucidating whether the body region from which samples are collected influences blubber cortisol levels (Kershaw et al., 2017). In the present study, we compared blubber cortisol levels across different body sites in order to assess the influence of sample location on hormone concentrations in striped dolphins. To our knowledge, this is the first time that levels of blubber cortisol are reported in this species (Tables 3 and S1). Importantly, results demonstrated that the chosen denominator used to estimate hormone levels could potentially influence the interpretation of hormone concentration. The best fit model based on AIC selection for the blubber cortisol concentrations measured as a function of blubber wet tissue (ng cortisol/g blubber) failed to find differences across the five body regions evaluated in striped dolphins (p > .05; Figure 3a), similarly to results reported previously in harbor porpoises (Kershaw et al., 2017). However, differences between regions were detected when cortisol assessments were made using the total amount of lipid as the denominator (ng cortisol/g lipid; Figure 3b). Under this scenario, the dorsal area to the pectoral flipper presented higher cortisol levels when compared to the caudal (p = .002), cranial (p < .001) and medial (p < .001) areas, and concentrations of the ventral area differed significantly to levels of medial samples (p < .05). In both cases, the AIC selection indicated that the best fit model included body condition as a covariate. The GLMM in turn revealed that this covariate was negatively related to both estimates of blubber cortisol concentrations (p < .01). The layer from which samples are collected and the sample mass itself can both be a source of variation in the extraction efficiency and consequently, in the levels of cortisol measured (Mello et al., 2017; Trana et al., 2015). To avoid the potential influence of these intrinsic factors, full-depth blubber was used in the present study and the mass was kept constant across all samples processed. Differences between hormone estimates could therefore be driven by blubber lipid differences. The blubber tissue has different functions across the body (i.e., insulator, buoyancy, and energy storage), and as such, the lipid content can vary depending on the anatomical region (Gómez-Campos et al., 2015; Tornero et al., 2004). The ventral region in striped dolphins, similar to harbor porpoises and common dolphins (Koopman et al., 2002; Tornero et al., 2004), presents higher lipid concentration and as such, this location has been suggested to have the function of energy storage (Gómez-Campos et al., 2015). Higher number of adipocytes and lipid concentration could explain the higher blubber cortisol concentrations detected in the ventral region. The reason why the dorsal area also presented higher hormone levels measured as ng cortisol/g lipid is unclear. In striped dolphins, the dorsal area presents the lower adipocyte number and lipid content compared to the ventral locations (Gómez-Campos et al., 2015), further hindering interpretation. Blubber has been recognized as a site of active steroid metabolism (Galligan et al., 2018) and evidence in other mammals suggests that the hormonal activity of the adipose tissue differs across body regions (Lindsay et al., 2003). Therefore, results could reflect a different diffusion and local transformation of the hormone across the body blubber. Nevertheless, although we collected fresh tissue whenever possible, we must recall that samples were taken from stranded animals and cortisol values may not represent the healthy population. The units used to reflect blubber cortisol concentrations vary among reports, making interpretations and direct comparisons difficult. If blubber cortisol is predominantly dependent on fat content, then "ng cortisol/g lipid extracted" is the appropriate unit to use. Accordingly, our results here could reflect a different rate of cortisol deposition across the body and anatomical variation should be considered to ensure consistent sampling. Conversely, if cortisol incorporation into blubber is not only dependent on fat content, using the amount of lipid extracted as the reference denominator would be acting as a confounding factor. In this context, our results would suggest a homogeneous distribution of cortisol across the blubber. Given that the use of one denominator over the other remains unclear, and results can vary depending on this factor (Mello et al., 2017), defining the anatomical sampling location on the body should be a relevant prerequisite for the appropriate physiological interpretation of blubber analyses. To date, the dorsal area has been the preferred body location sampled as it is easily accessible, especially in wild living animals biopsied by remote methods. We therefore suggest caution when making straightforward equivalences between the dorsal samples and the whole-body regions and recommend studying different sites simultaneously until more validation work is available. Finally, body condition, a measure that reflects the energy stores of an individual (Aguilar & Borrell, 1990), was negatively related to both estimates of blubber cortisol concentrations. In general, individuals sampled with a chronic disease (SC1 and SC6) presented worst body condition indices compared to those accidentally captured, and accordingly, higher cortisol levels. Certain pathogens and diseases can increase cortisol concentrations (Cook, 2012; Hunt et al., 2018). This hormone in turn may increase lipolysis to provide energy resources (Galligan et al., 2019; Khudyakov et al., 2017; Reynaert et al., 1976), as supported by the significant relationship detected between blubber cortisol levels and proportion of lipid extracted (Figure 4; p < .001). Overall, although the present study was not specifically designed to evaluate which reference denominator best fits cortisol blubber assessments, these findings are indeed biologically relevant because they highlight that results depend on which of the two measurements is assigned to the denominator. Given that standardization of data is a key element for proper comparison between studies, future effort should integrate results using both wet-tissue mass and lipid content to estimate hormone concentrations in order to gain further consensus into this important issue. We encourage forthcoming studies to use biopsy samples from live free-ranging striped dolphins and to evaluate whether the active steroid metabolism takes place homogeneously within the whole-body blubber tissue to better understand how cortisol varies across anatomical sites. We thank all those people and organizations that were involved in the fieldwork and helped with the collection of stranded dolphin samples, and Itziar Martínez for all her assistance in the laboratory tasks. Appendix S1: Supplementary Information Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

The impacts of environmental changes and anthropogenic threats in marine mammals are a growing concern for their conservation. In recent years, efforts have been directed to understand how marine mammals cope with stressors and to assess and validate stress biomarkers, mainly levels of glucocorticoid hormones (e.g. cortisol) in certain body tissues. The aims of this study were to assess the impact of different causes of stranding (chronically affected and bycaught striped dolphins) on cortisol concentrations in serum and in blubber; and to evaluate the association between cortisol levels in these tissues. Blubber and blood samples were collected from striped dolphins (n = 42) stranded on the Mediterranean coast between 2012 and 2018. Cortisol concentrations were measured by using enzyme immunoassay. A high correlation was found between circulating and blubber cortisol concentrations (R2 = 0.85, p < 0.01). Necropsies and pathological studies concluded that a third of the dolphins were bycaught in fishing nets and released by fishermen (Bycaught animals group), while the other two thirds were euthanized, or died, due to a disease or chronic condition (e.g. calves separated from the mother or animals infected with dolphin morbillivirus or Brucella ceti) that impeded survival (Chronically affected animals group). Cortisol concentrations (mean ± SD) were six times higher in chronically affected animals (35.3 ± 23 ng cortisol/g blubber and 6.63 ± 3.22 μg cortisol/dl serum) compared to those bycaught in fishing nets (6.2 ± 4.3 ng cortisol/g blubber and 1.15 ± 1.51 μg cortisol/dl serum). Results suggests that serum and blubber cortisol concentrations can contribute in inferring the overall health and welfare of free-ranging cetaceans. However, further research is required to understand better the kinetics of blubber cortisol incorporation and removal, the factors involved in these processes, and the local conversion of cortisol in the blubber.

The cold-inducible proteins (CIPs) are essential for post-transcriptional gene regulation playing diverse tissue-specific roles in maintaining normal cellular function and morphogenesis. The potential implications of CIPs in reproductive events raise questions about their role in the physiology of the bovine reproductive tract. However, the expression changes of CIPs during the bovine estrous cycle have not been studied so far. Here, we hypothesized that the bovine estrous cycle could affect the mRNA expression of the CIPs and other candidate transcripts in the reproductive tract. This study aimed to examine estrous cycle-dependent mRNA expression patterns in the bovine endometrium and ampulla of three of the major described CIPs (CIRBP, RBM3, SRSF5), a set of inflammatory cytokines (IL-10, IL-18, IL-1β), and other candidate genes (IL-10RA, IL-10RB, BCL2, NLRP3, STAT1, STAT3, STAT5A, STAT6). Endometrial and ampullar tissues were assessed by RT-qPCR. Additionally, the mRNA expression levels were correlated among them and with follicular progesterone and estradiol concentrations. The transcript levels of CIPs increased in the endometrium during stage III (Days 11-17) compared to stage I (Days 1-4) and IV (Days 18-20). In the ampulla, the mRNA expression of CIRBP increased during the late luteal phase (stage III), but no differences in the expression of other CIPs were observed. This study expands the current knowledge regarding mRNA expression in the endometrium and oviductal ampulla of cycling heifers, focusing mainly on the CIPs. A better understanding of the mechanisms within the uterus and oviduct during the estrous cycle is crucial to improving the fertility rate.

The bovine reproductive tract exhibits changes during the estrous cycle modulated by the interplay of steroid hormones. Glucocorticoids can be detrimental when stress-induced but are relevant at baseline levels for appropriate reproductive function. Here, an analysis of quantitative real-time PCR was performed to study the bovine glucocorticoid-related baseline gene transcription in endometrial and ampullar tissue samples derived from three time points of the estrous cycle, stage I (Days 1-4), stage III (Days 11-17) and stage IV (Days 18-20). Our results revealed expression differences during stages, as expression observed in the ampulla was higher during the post-ovulatory phase (stage I), including the glucocorticoid receptor NR3C1, and some of its regulators, involved in glucocorticoid availability (HSD11B1 and HSD11B2) and transcriptional actions (FKBP4 and FKBP5). In contrast, in the endometrium, higher expression of the steroid receptors was observed during the late luteal phase (stage III), including ESR1, ESR2, PGRMC1 and PGRMC2, and HSD11B1 expression decreased, while HSD11B2 increased. Moreover, at protein level, FKBP4 was higher expressed during the late luteal phase, and NR3C1 during the pre-ovulatory phase (stage IV). These results suggest that tight regulation of the glucocorticoid activity is promoted in the ampulla, when reproductive events are taking place, including oocyte maturation. Moreover, most expression changes in the endometrium were observed during the late luteal phase, and may be related to the embryonic maternal recognition. In conclusion, the glucocorticoid regulation changes across the estrous cycle and may be playing a role on the reproductive events occurring in the bovine ampulla and endometrium.

Kimberly M. Davenport1, Alisha T. Massa2, Michelle R. Mousel3,4, Maria K. Herndon2, Stephen N. White2,3,5, Mazdak Salavati6, Emily Clark6, Alan Archibald6, Suraj Bhattarai7, Stephanie D. McKay7, Kim C. Worley8, Brian Dalrymple9, James Kijas10, Alex Caulton11, Shannon Clarke11, Rudiger Brauning11, Tracy Hadfield12, Noelle E. Cockett12, Timothy P.L. Smith13, and Brenda M. Murdoch1,5 on behalf of The Ovine FAANG Project Consortium

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.

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.