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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). info:eu-repo/semantics/publishedVersion

The purpose of the present study was to evaluate the effects of season on the in vitro fertilizing ability of bovine spermatozoa and subsequent embryo development. Bovine oocytes were matured and fertilized in vitro with Holstein dairy bull sperm cells collected and frozen in different seasons (winter, spring, and summer). On d 2 and 8 postinsemination, cleavage and blastocyst rates, respectively, were recorded; the blastocysts were graded for morphology. The number of sperm cells binding to the zona pellucida of oocytes, together with the number of nuclei in the developing blastocysts, were assessed after staining with Hoechst. No significant differences were observed among seasons in cleavage and embryo development rate. However, the proportion of "advanced blastocysts" was significantly higher in spring compared with winter and summer, with a corresponding decrease in the proportion of early blastocysts in spring compared with winter and summer. The number of sperm cells binding per oocyte was significantly lower in the oocytes inseminated with sperm samples collected in summer compared with winter or spring. Moreover, a significant interaction was observed in the number of sperm cells binding per oocyte between bull and season. Although no significant differences were observed among seasons in the number of nuclei per blastocyst, a significant interaction was observed between bull and season for this variable. Embryo development rate in in vitro fertilization appeared to be affected by season of semen collection, with sperm samples collected in spring being associated with a higher proportion of advanced blastocysts and better morphology than those collected at other times of the year.

Altres ajuts: acords transformatius de la UAB 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 (R = 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.

Measuring corticosterone concentrations in feathers (FCC) has been increasingly used as an integrated index of bird’s response to stressors, ofering valuable insights into subsequent carry-over efects. However, it is still unclear to what extent corticosterone levels deposited in feathers during the moulting period relate to individual physiology in high-energetic demanding situations. In addition, the stability and repeatability of FCC over the same feather generation and across successive generations are still unresolved questions. In this study, we assessed FCC in 24 captive Northern Bald Ibis, Geronticus eremita. We sampled body feathers on three occasions during two consecutive years (2015–2016) to explore within-individual stability of FCC in feathers grown during the same generation and test whether FCC was repeatable or varied between two consecutive generations. Then, we explored whether FCC (a) was associated with individual health status, (b) correlated with previous reproductive success and (c) was related to future reproductive success. Results showed high stability of FCC among feathers of the same generation, while FCC difered between successive generations. We also observed that FCC, age and health status were negatively associated with subsequent reproductive success the following season, while no association was found with previous reproductive success. Overall, our study supports the use of feathers as a stable measure of corticosterone over the same feather replacement generation and emphasizes the usefulness of FCC as a biomarker of subsequent reproductive success. © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

The aim of the present study was to make a retrospective analysis of the relationship between climatic factors and sperm quality of frozen-thawed semen from bulls kept in temperate climates. Semen samples from 21 European dairy bulls from 2 countries were collected and cryopreserved in winter, spring, and summer. Sperm quality parameters such as kinematics, morphology, plasma membrane integrity, mitochondrial membrane potential, sperm chromatin structure assay, and reactive oxygen species were analyzed and correlated retrospectively with climate factors recorded by the local meteorological office. This study demonstrated that sperm quality parameters are more likely to be correlated with climate factors 1 or 2 mo before semen collection than in the month of semen collection. During the month of sperm collection, sperm kinematics, DNA fragmentation, and hydrogen peroxide production were the only sperm quality parameters related to climate factors, whereas 1 and 2 mo before sperm collection, normal morphology and additional sperm kinematics, in addition to DNA fragmentation and hydrogen peroxide production, were correlated with climate factors. In conclusion, dairy bull sperm quality is affected by climatic conditions, even in so-called temperate zones. The timing of heat stress during spermatogenesis determines which aspects of sperm quality are likely to be affected. Husbandry conditions for bulls used for semen collection should be adapted to allow the animals' physiological responses for temperature regulation within the scrotum to operate fully, to mitigate the effects of increased temperature and humidity. Extremes of temperature should be avoided.

Cortisol, the end product of the hypothalamus-pituitary-interrenal (HPI) axis, has been traditionally measured in blood as indicator of stress in fish, however, the degree of invasiveness inherent to blood collection is not always possible or desirable. Instead, cortisol measurement in skin mucus is far less invasive, but as blood, this method potentially provides only a brief window of information of the HPI axis activity. The newly described method of cortisol measurement from scales may serve as a long-term, integrated measure of the HPI axis activity in fish. While skin mucus and scales cortisol measurement present practical and conceptual advantages, there are still several unclear issues related to their biological relevance that need deeper study. Accordingly, we aimed to evaluate whether skin mucus and scales cortisol levels can be reliably used as stress indicators by subjecting fish to prolonged, continuous stressful conditions. The present study demonstrates that the measurement of cortisol in skin mucus reflect circulating cortisol concentrations when fish are responding to stress with an intense activation of the HPI axis. Results also revealed that cortisol content in scales strongly correlates to circulating cortisol levels in chronically stressed fish. Besides, we provide further support that scales cortisol assessment offer a retrospective measure of the past stress experience in fish. While this study provides a good basis for future research applying the methods presented, our results open the question of whether these matrices have additional sources of cortisol other than blood, and the route of incorporation or diffusion. Further knowledge about the general robustness and stability of scales cortisol in fish subjected to prolonged stress would largely help strengthen the interpretation of hormone fluctuations in this matrix.

Simple Summary The monitoring of a horse’s endocrine status can provide valuable information of its welfare and sexual and health conditions. The use of non-invasive matrixes for hormonal monitoring, as alternatives to blood samples, are being increasingly used. Among them, the measurement of hormones deposited in the hair shaft has been widely used as a retrospective hormonal biomarker. In the present study, we aimed to evaluate the seasonal variations in 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. The results showed how cortisol, testosterone and dehydroepiandrosterone sulphate and their ratios were significantly affected by season. Therefore, as shown in this study, season should be considered when analysing sexual and stress hormones in stallion hair. Abstract 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.

This study was designed to the optimize vitrification and in-straw warming protocol of in vitro-produced bovine embryos by comparing two different equilibration periods, short equilibrium (SE: 3 min) and long equilibrium (LE: 12 min). Outcomes recorded in vitrified day seven (D7) and day eight (D8) expanded blastocysts were survival and hatching rates, cell counts, apoptosis rate, and gene expression. While survival rates at 3 and 24 h post-warming were reduced (p &lt, 0.05) after vitrification, the hatching rates of D7 embryos vitrified after SE were similar to the rates recorded in fresh non-vitrified blastocysts. The hatching rates of vitrified D8 blastocysts were lower (p &lt, 0.05) than of fresh controls regardless of treatment. Total cell count, and inner cell mass and trophectoderm cell counts were similar in hatched D7 blastocysts vitrified after SE and fresh blastocysts, while vitrified D8 blastocysts yielded lower values regardless of treatment. The apoptosis rate was significantly higher in both treatment groups compared to fresh controls, although rates were lower for SE than LE. No differences emerged in BAX, AQP3, CX43, and IFNτ gene expression between the treatments, whereas a significantly greater abundance of BCL2L1 and SOD1 transcripts was observed in blastocysts vitrified after SE. A shorter equilibration vitrification protocol was found to improve post-warming outcomes and time efficiency after in-straw warming/dilution.

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 &lt, 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.

Simple Summary Multi-unowned-cat environments can be highly stressful for cats, and infectious and parasite diseases spread quickly and are difficult to prevent. This study aimed to determine the occurrence of intestinal parasites and fecal cortisol metabolites (FCM) in cat feces collected from different multi-cat environments and assess the effect of housing conditions on intestinal parasites and FCM levels in order to develop more efficient control strategies. Cat fecal samples from rescue shelters, catteries and feline colonies were analyzed with coprological methods to detect intestinal parasite patency and determine FCM. Helminth infection was mainly detected in free-roaming cats. In confined cats, protozoa infections were more likely detected in shelter cats than in cattery cats. Exposure to dogs was associated with parasite infection and cats highly exposed to dogs with visual contact and audible barking showed higher intestinal protozoa prevalence than cats that were not exposed to dogs. FCM levels were correlated with enclosure size and protozoa infection. Reducing stress by improving housing in terms of enclosure size and avoiding exposure to dogs may have an impact on the occurrence of intestinal parasites, especially protozoa. Abstract 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.

Background VitTrans is a device that enables the vitrification and warming/dilution of in vitro produced bovine embryos followed by their direct transfer to recipient females in field conditions. This study sought to improve the VitTrans method by comparing two equilibration times: short (SE: 3 min) and long (LE: 12 min). Outcome measures recorded in vitrified D7 and D8 expanded blastocysts were survival and hatching rates, differential cell counts, apoptosis rate and gene expression. Results While survival rates at 3 h and 24 h post-warming were reduced (P

This study aimed to examine whether the addition of glutathione ethyl ester (GSH-OEt) to the in vitro maturation (IVM) medium would improve the resilience of bovine oocytes to withstand vitrification. The effects of GSH-OEt on spindle morphology, levels of reactive oxygen species (ROS), mitochondrial activity and distribution, and embryo developmental potential were assessed together with the expression of genes with a role in apoptosis (BAX, BCL2), oxidative-stress pathways (GPX1, SOD1), water channels (AQP3), implantation (IFN-&tau, ) and gap junctions (CX43) in oocytes and their derived blastocysts. Vitrification gave rise to abnormal spindle microtubule configurations and elevated ROS levels. Supplementation of IVM medium with GSH-OEt before vitrification preserved mitochondrial distribution pattern and diminished both cytoplasmic and mitochondrial ROS contents and percentages of embryos developing beyond the 8-cell stage were similar to those recorded in fresh non-vitrified oocytes. Although not significantly different from control vitrified oocytes, vitrified oocytes after GSH-OEt treatment gave rise to similar day 8-blastocyst and hatching rates to fresh non-vitrified oocytes. No effects of GSH-OEt supplementation were noted on the targeted gene expression of oocytes and derived blastocysts, with the exception of GPX1, AQP3 and CX43 in derived blastocysts. The addition of GSH-OEt to the IVM medium before vitrification may be beneficial for embryo development presumably as the consequence of additional anti-oxidant protection during IVM.

Presence of the hyaluronan (hyaluronic acid, HA) receptor CD44 on spermatozoa has been difficult to pursue, mostly obeying to the use of different commercial mono- and/or polyclonal antibodies, often lacking proper controls. Here, we describe how the presence (Western blotting) and specific location (immunocytochemistry) of the CD44 receptor differs in ejaculated pig spermatozoa depending on the type of antibody and protocol used. While we were able to detect binding to spermatozoa and mark its presence in the sperm membrane, the use of blocking peptides clearly indicated that only the monoclonal antibody could confirm the specific presence and location of the CD44 receptor, whereas the polyclonal antibody was detecting multiple presumed CD44 isoforms or degraded proteins thus proving unspecific. These results call for strict protocols when attempting immunological determination of sperm membrane receptors. Funding Agencies|Lions Forskningsfond 2016-postdoc, Linkoping, Sweden [DNR LIU-2016-00641]; Forskningsradet i Sydostra Sverige [473121, 745971]; Svenska Forskningsradet Formas [2017-00946]

letrozole is an aromatase inhibitor that stops the production of estrogen through interrupting the entrance of hormone androgen into a small amount of estrogen. Therefore, the current study was developed to estimate orally administrated Letrozole on the reproductive performance and relative abundance of Foxj1, PVRL3, and LPR2 mRNA in aged roosters. Fifty-five-week old ROSS 308 breeder roosters (n = 18) were orally treated using letrozole. Primarily, the body weight of the animals was recorded, and they were randomly classified into three groups (n = 6 birds/group) receiving different doses of Letrozole, including 0, 0.015, and 0.03 mg/kg body weight/day for three weeks. At the end of the trial, seminal traits, plasma, testicular hormone levels (testosterone, estradiol, and FSH), histopathological studies, in vitro fertility, and relative abundance of testis PVRL3, epidydimal Foxj1, and LPR2 mRNA were evaluated. Based on the results, the sperm quality variables were statistically higher in the 0.03 group compared to the controls. Greater histologic parameters, such as diameter of seminiferous tubules, thickness of seminiferous epithelium, categorized epididymal region, and in vitro fertility rates were estimated for the treated groups(p 0.001). Plasma and testicular testosterone, estradiol concentrations, and plasma FSH levels were significantly influenced by letrozll treatment (p 0.001). Relative mRNA transcript abundance increased for PVRL3 and decreased for Foxj1 and LPR2 in treated groups. Overall, aromatase inhibitors can enhance the reproductive performance of aged commercial broiler breeder roosters. However, it can impact endocytosis and ciliogenesis events via reducing estradiol.

Persistent organic pollutants (POPs) have the potential to impair the endocrine regulation of organisms and alter their ability to respond to environmental changes. We studied whether polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) affected the endocrine regulation of free-living and captive red kites (Milvus milvus) through studying the dynamics of corticosterone (CORT) and dehydroepiandrosterone (DHEA). We sampled migratory free-living kites coming from northern Europe and captive kites born in a rehabilitation center in Spain. We used body feathers from the interscapular region as a minimally-invasive and integrative matrix. The most abundant compound detected in free-living kites was 4,4′-dichlorodiphenyldichloroethylene (4,4′-DDE; 6.10 ± 1.56 ng g−1 dw feather) followed by CB-153 (3.10 ± 0.63 ng g−1 dw feather) and CB-180 (2.43 ± 1.08 ng g−1 dw feather). In captive kites, the most abundant compounds were 4,4′-dichlorodyphenyltrichloroethane (4,4′-DDT; 2.38 ± 1.30 ng g−1 dw feather), CB-153 (2.15 ± 0.47 ng g−1 dw feather) and hexachlorobenzene (HCB; 2.03 ± 0.45 ng g−1 dw feather) at similar concentrations. Free-living kites showed higher levels of 4,4′-DDE and CB-180 in comparison to captive kites. Age influenced HCB and CB-101 levels, whereas body mass was inversely related to CB-180 and 4,4′-DDT. Interestingly, captive kites showed a ratio DDT/DDE higher than 1 suggesting a relatively recent exposure of DDT, in contrast to free-living kites. Regarding hormonal levels, free-living kites showed higher levels of CORT (3.30 ± 0.22 pg mm−1 feather) than captive (2.40 ± 0.16 pg mm−1 feather), reflecting higher allostatic load. In addition, a positive association between PCBs and DDTs and adrenal hormones was found in free-living kites, suggesting an increase of CORT as a response of the endocrine system to cope with stressors and a subsequent elevation of DHEA to ameliorate the potential negative effects that high CORT levels could cause to the organism.

Porcine circovirus 3 (PCV‐3) prevalence has been minimally investigated in wild boar; dynamics of infection and viral tissue distribution are currently unknown. In this study, serum samples from 518 wild boar (from years 2004 to 2018) were used to study frequency of infection. Also, serum samples from 19 boar captured and recaptured at least two times for a period of time from 1 month to 1 year were collected to determine PCV‐3 infection dynamics. Finally, to elucidate PCV‐3 DNA organic distribution, sera, different tissues and faeces were obtained from 35 additional wild boar. PCV‐3 DNA was extracted and amplified with a conventional PCR. For the PCV‐3 PCR‐positive sera from the longitudinally sampled and different tissue types, a quantitative PCR was performed. Genome sequence was obtained from a number of PCV‐3 PCR‐positive samples from different years, different time‐points of infection and tissues. Obtained results confirmed the susceptibility of wild boar to the virus, showing high frequency of PCV‐3 detection (221 out of 518, 42.66%) and demonstrating circulation at least since 2004. Compiled data indicate the possibility of long‐term infections, since 5 out of 10 PCV‐3 PCR‐positive boars longitudinally sampled showed positivity in samplings separated for more than 5 months. All tested tissue types' harboured PCV‐3 genome, with the highest percentage of PCR positivity in submandibular lymph node, tonsil, lung, liver, spleen and kidney. The amount of DNA in all tested PCV‐3 PCR‐positive samples was moderate to low. All partial and complete PCV‐3 sequences obtained from wild boar displayed high nucleotide identity, higher than 98%. In conclusion, this study further confirms that wild boar is susceptible to PCV‐3 infection, showing high frequency of detection in this animal species. Furthermore, PCV‐3 can be found in different tissues of wild boar and is apparently able to cause persistent infection. Instituto Nacional de Investigación y Tecnologia Agraria y Alimentaria. Grant Number: E‐RTA2017‐00007‐00‐00

Contents Heat-shock protein 70 (HSP70) plays a crucial role as intracellular cytoprotectant and molecular chaperone. A phenomenon of heat stress (HS) leads to production of these proteins that could be beneficial to cells during cryopreservation, which is also a stressful process for the cell. This study aimed to evaluate the protective effect of exposure of bovine oocytes to moderate HS during in vitro maturation (IVM) prior vitrification. First, oocytes were subjected to HS (41.5°C for 1 hr) at 0, 4, 8, 12 or 16 of IVM. Oocytes in vitro matured for 20 hr served as control group. Presence of HSP70 was detected at 20 hr by immunofluorescence. HSP70 expression was significantly higher when oocytes were subjected to HS at 8 hr of IVM. Next, oocytes were distributed into four groups: Control: IVM oocytes; VIT: oocytes vitrified/warmed at 20 hr of IVM; HS: oocytes subjected to HS at 8 hr of IVM; HS-VIT: oocytes subjected to HS at 8 hr of IVM and vitrified/warmed at 20 hr of IVM. Oocytes were fertilized at 24 hr of IVM, and cleavage and blastocyst yield were assessed. No significant differences were observed among treatments when cleavage rate was evaluated. However, fresh control and HS oocytes resulted in a significantly higher (p

Concentrations of different steroid hormones have been used in cows as a measure of adrenal or gonadal activity and, thus, as indicators of stress or reproductive state. Detecting cortisol and progesterone in cow hair provides a long-term integrative value of retrospective adrenal or gonadal/placental activity, respectively. Current techniques for steroid detection require a hormone-extraction procedure that involves time, several types of equipment, management of reagents, and some assay procedures (which can also be time-consuming and can destroy the samples). In contrast, near-infrared reflectance spectroscopy (NIRS) is a multi-component predictor technique, characterized as rapid, nondestructive for the sample, and reagent-free. However, as a predictor technique, NIRS needs to be calibrated and validated for each matrix, hormone, and species. The main objective of this study was to evaluate the predictive value of the NIRS technique for hair cortisol and progesterone quantification in cows by using specific enzyme immunoassay as a reference method. Hair samples from 52 adult Friesian lactating cows from a commercial dairy farm were used. Reflectance spectra of hair samples were determined with a NIR reflectance spectrophotometer before and after trimming them. Although similar results were obtained, a slightly better relationship between the reference data and NIRS predicted values was found using trimmed samples. Near infrared reflectance spectroscopy demonstrated its ability to predict cortisol and progesterone concentrations with certain accuracy (R2 = 0.90 for cortisol and R2 = 0.87 for progesterone). Although NIRS is far from being a complete alternative to current methodologies, the proposed equations can offer screening capability. Considering the advantages of both fields, our results open the possibility for future work on the combination of hair steroid measurement and NIRS methodology.

Members of the interleukin-6 (IL-6) family of cytokines are important for reproductive function that are mediated through changes in gene and miRNA expression. Herein, we characterized the expression of miR-21, miR-155, miR-34c and miR-146a in bovine oocytes and cumulus cells during in vitro maturation (IVM) with leukemia inhibitory factor (LIF), IL-6 and IL-11 or unsupplemented controls. LIF-exposed COCs showed higher expression of miR-21 and miR-155 in oocytes, whereas miR-146a expression was increased in oocytes matured with IL-6 and IL-11. In cumulus cells, miR-155 expression was elevated by all treatments while only LIF increased miR-21 expression. Based on these results, we next examined how LIF exposure during IVM affected oocyte competence, through IVF and the expression of specific genes in GV- and MII-oocytes, in 2- and 8-cell embryos, and in Day 8-blastocysts. LIF supplementation did not affect cleavage rate, blastocyst yield or several other developmental parameters, but did increase hatching rate. LIF suppressed DPPA3, ZAR1 and NPM2 expression in 2 cell- and/or 8-cell embryos. LIF increased the expression of KAT2A and HSPA1A in MII-oocytes, and that of HDAC1, KAT2A and HSP90AA1 and the BAX:BCL2L1 ratio in 2-cell embryos. In contrast, HDAC1, KAT2A and HSP90AA1 expression and BAX:BCL2L1 ratio was lower in 8-cell embryos derived from LIF oocytes. IVM with LIF also increased the expression of DNMT3A, HSPA1A and HSP90AA1 in blastocysts. In conclusion, supplementation with LIF during IVM was consistently associated with changes in the relative abundance of transcripts in mature bovine oocytes and in specific embryo developmental stages Tis study was supported by the Spanish Ministry of Science and Innovation (Project AGL2016-79802-P and grant CTQ2014-59632-R) and the Generalitat de Catalunya (Project No. 2017 SGR 1229). Dr. Mertixell VendrellFlotats was supported by an FPI scholarship (BES-2014-071075). Collaboration with Dr. LaMarre was supported by a Fellowship under the OECD Co-operative Research Programme: Biological Resource Management for Sustainable Agricultural Systems and funded by NSERC (Canada)

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&ndash, 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 &lt, 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 &lt, 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.

The avian seminal fluid (SF) is a protein-rich fluid, derived from the testis, the rudimentary epididymis and, finally, from the cloacal gland. The SF interacts with spermatozoa and the inner cell lining of the female genital tract, to modulate sperm functions and female immune responsiveness. Its complex proteome might either be free or linked to extracellular vesicles (EVs) as it is the case in mammals, where EVs depict the tetraspanin CD9; and where those EVs derived from the epididymis (epididymosomes) also present the receptor CD44. In the present study, sperm-free SF from Red Jungle Fowl, White Leghorn and an advanced intercross (AIL, 12th generation) were studied using flow cytometry of the membrane marker tetraspanin CD9, Western blotting of the membrane receptor CD44 and electron microscopy in non-enriched (whole SF) or enriched fractions obtained by precipitation using a commercial kit (Total Exosome Precipitation Solution). Neither CD9- nor CD44 could be detected, and the ultrastructure confirmed the relative absence of EVs, raising the possibility that avian SF interacts differently with the female genitalia as compared to the seminal plasma of mammals.

Simple Summary The analysis of circulating cortisol has been by far the most common method used as a means to assess fish stress responses and, thus, animal welfare. To avoid many of the drawbacks inherent to blood sampling, cortisol can be less-invasively detected in fish skin mucus. The measurement of cortisol in skin mucus however, has, to date, only been demonstrated as suitable for farm fish, although its application to free-ranging animals would offer many advantages. The present study was therefore designed to evaluate the applicability of skin mucus cortisol analysis as a potential tool to assess habitat quality. To that end, wild fish residing in environments of different habitat quality were sampled for blood and skin mucus. First, several physiological endpoints typically used as indicators of exposure to pollutants were accurately related to the habitat quality in the Catalan chub (Squalius laietanus). Second, cortisol levels in blood were also compared between habitats, and they were successfully correlated to skin mucus cortisol concentrations. Finally, we contrasted the patterns of response of all the endpoints assessed to skin mucus cortisol levels across the sites. The strong linkages detected in this study provide new evidence that the measurement of cortisol in skin mucus could be potentially used as a biomarker of habitat quality in freshwater fish. Abstract 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 aim of this study was to induce the cold-inducible RNA-binding protein (CIRBP) expression on cumulus-oocyte complexes (COCs) through exposure to a sub-lethal cold shock and determine the effects of hypothermic temperatures during the in vitro maturation of bovine oocytes. Nuclear maturation, cortical granule redistribution and identification of cold-inducible RNA-binding protein (CIRBP) were assessed after 24 hr of in vitro maturation of control (38.5 degrees C) and cold-stressed oocytes (33.5 degrees C). The presence of CIRBP was assessed by Western blot in COCs or denuded oocytes and their respective cumulus cells. Based on the odds ratio, cold-stressed oocytes presented higher abnormal cytoplasmic distribution of cortical granules and nuclear maturation than the control group. Although CIRBP was detected in both control and cold-stressed groups, cold-stressed COCs had 2.17 times more expression of CIRBP than control COCs. However, when denuded oocytes and cumulus cells were assessed separately, CIRBP only was detected in cumulus cells in both groups. In conclusion, cold shock induced CIRBP expression, but it negatively affected nuclear maturation and cortical granule distribution of bovine oocytes. Moreover, the expression of CIRBP was only identified in cumulus cells but not in oocytes. Funding Agencies|FI grant [2018 FI_B 00236]; [AGL2016-79802-P]; [AGL2016-81890-REDT]; [FPU2015/06029]; [IJCI-2015-24380]

In birds, integrated levels of corticosterone (CORT) measured in feathers (CORTf) allow us to make inferences on past levels of stress demands. It has been suggested that levels of CORTf track carry-over effects across seasons. Nevertheless, our understanding of how this measure can be used to assess future health status is far from complete. In this study, we aimed to investigate whether CORT deposited in feathers over the moulting period was related to subsequent mortality and health status in wild raptors admitted to rehabilitation centres. Thirty-four Eurasian Sparrowhawks (Accipiter nisus) admitted during the non-moulting period were sampled. Body condition (BC) was used as an indicator of health status to classify individuals’ health as good, poor or cachexia depending on their pectoral muscle score. Mortality was recorded over the non-moulting period. Other potential sources of CORTf variation were assessed, such as sex, age and feather type, primary or body covert feathers. While CORTf did not vary with age or sex, significant differences were found between primary and body feathers, highlighting the importance of sampling the same feather type. Our results also revealed that birds in poor BC showed higher CORTf levels than individuals in good condition; however, CORTf levels in cachectic birds did not differ from those in good condition. This finding suggests caution when assuming that only high CORTf levels represent individuals in poor condition, and limits the utility of CORTf for the prediction of BC. The present study also showed that individuals which died following admission had higher CORTf levels than individuals which survived, suggesting the potential utility of CORTf as a metric for the study of subsequent mortality.

In recent times, the detection of cortisol in hair is intended to be used as an animal-based indicator for the assessment of chronic stress. However, the relationship between the ability to report average values of long-term circulating cortisol concentrations and the sensitivity to acute peaks of cortisol is still unclear. To gain insight into this relationship, 24 Holstein-Friesian bull calves under the same management conditions were used in this study. Two injections of ACTH (at D0 and D7) were administrated to twelve animals with the aim to create two acute increases of serum cortisol concentrations. Blood samples were taken in order to determine the duration of serum cortisol peaks and to confirm a mediated response by the administration of ACTH. Cortisol concentrations from 14-day-old white hair samples collected from forehead and hip were analysed separately by EIA and compared with those from the control group. Serum cortisol analyses revealed an acute increase of cortisol concentrations for approximately three hours after each ACTH administration. Concentrations of hair cortisol from forehead and hip locations showed no differences between ACTH-administrated and control animals. Hair cortisol concentrations from 14-day old samples were not altered by two acute elevations of serum cortisol suggesting that hair cortisol is not masked by short and non-recurrent moments of stress. These results are a step forward in the validation of hair cortisol detection as a robust integrative measure of serum cortisol concentrations from an extended period of time.

In the warm months the function of the spermatozoa can be affected by the temperature of the reproductive tract of the female exposed to hyperthermic conditions. The aim of this study was to evaluate the impact of heat stress on sperm parameters in an in vitro model and to determine if there were seasonal effects on sperm heat tolerance. Sperm samples from 32 New Zealand White rabbits were collected in two seasons and incubated at scrotal (32.5°C), body (37°C) or hyperthermic (42°C) temperatures for 3h. Sperm viability and morphology were evaluated using nigrosin-eosin staining. Motility and metabolic activity parameters were determined using computer-assisted sperm analysis and the QBlue cell viability test, respectively. The incubation of spermatozoa at 42°C decreased (P0.05) the mean values of total motility, curvilinear (VCL) and mean velocity (VAP) as well as the metabolic activity with respect to the incubation at 32.5°C and 37°C. No seasonal effects were observed except for the highest percentages of bent and coiled tails in the cold season, and the highest mean values of VCL, linear velocity and VAP in the warm season (P0.01). The interaction between in vitro heat stress and season was significant for metabolic activity (P=0.02). Our results suggest that rabbit spermatozoa parameters are largely modified by a short exposure to hyperthermic conditions, in terms of metabolic activity and motility parameters. Thus, a short exposure of spermatozoa to an environment of 42°C in temperature for only 3h may compromise sperm functionality. Additionally, sperm metabolic activity is influenced by season.

Ensuring welfare in captive wild animal populations is important not only for ethical and legal reasons, but also to maintain healthy individuals and populations. An increased level of social behaviors such as aggression can reduce welfare by causing physical damage and chronic stress to animals. Recently, cortisol in hair has been advanced as a non-invasive indicator to quantify long-lasting stress in many species. The sensitivity of social behavior and hair cortisol concentration was evaluated in several groups of dorcas gazelles (Gazella dorcas). Four different groups of gazelles from three different zoos were observed and the expression of intra-specific affiliative and negative social behaviors was assessed across the different groups. Hair samples were taken from sub-groups of animals and analyzed for cortisol concentrations. Significant differences between groups of dorcas gazelles were found in frequency of negative social behavior and hair cortisol concentration. Despite the low sample size, these two parameters had a positive Spearman correlation coefficient (rs = +0.80, P = 0.20). These results suggest that hair cortisol levels are sensitive to differences in the social structure of dorcas gazelles. Zoo Biol. 35:467–473, 2016. © 2016 Wiley Periodicals, Inc.

The Dorcas gazelle (Gazella dorcas), in the family Bovidae, is one of the smallest species of the genus Gazella. Its population is decreasing and it is classified as vulnerable by the International Union of Conservation of Nature Red List of Threatened Species. The subspecies Saharan Dorcas gazelle (G. dorcas osiris), originating in the central-western Saharan and Sahel regions, is almost extinct in the wild because of over-hunting. Thereon, assisted reproductive techniques (ARTs) could help in its population management. Here, we performed invitro maturation (IVM) of oocytes recovered from an adult female of Saharan Dorcas gazelle (10 years old) to attempt IVF with frozen-thawed epididymal sperm cells from an adult male (9 years old) of the same subspecies, following protocols for ARTs in cattle. Epididymal sperm was obtained after postmortem examination by cannulation of vas deferens with commercial semen extender Gent A (Minitub). Motility features were assessed using a computer-assisted sperm analysis system (Proiser SL), and viability was evaluated using eosin/nigrosin staining. After collection, viability (88.3%), total (17.8%) and progressive (8.7%) motility were assessed. A total of 820.5×106 sperm cells was obtained and diluted (1:1) in a semen extender with permeable cryoprotectants (Gent B, Minitub). Then, the sample was refrigerated in straws (200×106 mL−1; 0.5mL) at 4°C for 1h and exposed to liquid nitrogen vapors (LN2) for 10min before being directly plunged into and kept on LN2. A total of 35 oocytes were recovered by slicing, 27 of which were selected for IVM (24h, 38.5°C, 5% CO2 in humidified air). Motile sperm were obtained by centrifugation of thawed semen (10min, 1000×g) by performing a density gradient (BoviPure, Nidacon). Post-thawing viability (72.9%) and total (14%) and progressive (4%) motility were evaluated just before IVF. After IVM, 17 oocytes were incubated for IVF for 20h with 1×106 thawed sperm cells mL−1 (although the first polar body extrusion was observed only in 5 of the 17 oocytes before IVF; 29.4%). Also, 7 oocytes selected for IVM were vitrified and kept for the genetic bank, and 3 were fixed. Thereafter, all oocytes that underwent IVF were incubated for invitro embryo culture (38.5°C, 5% CO2, 5% O2 in humidified air) to avoid discarding any potential zygote. Cleavage was assessed at 48h post-IVF. From those, only 2 oocytes successfully achieved IVF out of the 5 that showed first polar body extrusion (40%), although cleavage was asymmetric and embryos did not progress. The rest of oocytes (12) did not achieved IVM, IVF, or cleavage. Results suggest that optimal protocols and appropriate individualized conditions for vulnerable nondomestic ungulates are required for successful ARTs. Future efforts are needed to improve preservation strategies for the gametes/embryo conservation of endangered species. JG and MRC contributed equally to this work. JG is supported by AGAUR (2018 FI_B 00236) and MRC by FPU (Training for Academic Staff) (FPU15/06029).

Rooster fertility peaks between 30 and 40 weeks of age and declines rapidly from 50 weeks of age. This is linked to a reduction in the number of spermatozoa in the ejaculate due to high density of spermatids in the seminiferous tubules of low-fertility roosters. In this study, we assessed the effects over spermatogenesis of both letrozole, an aromatase inhibitor, and a commercial herbal extract designed for improving fertility composed by Tribulus, Cinnamomum, Zingiber and Sativus. Forty-two-week-old Ross 308 roosters (n = 24) were distributed into four groups: control, letrozole (0.03 mg/kg), herbal extract (0.04 ml/kg) and letrozole+herbal extract. After 14 weeks of daily oral supplementation, their testes and epididymides were weighed, fixed and sectioned for assessment of spermatogenesis and quantification of sperm cells inside the lumen. Differences in seminiferous tubules measurements and density of sperm cells were tested using R software (version 3.0.1). Although body weight was not affected by the treatment, testes from animals treated with the combination of letrozole and herbal extracts were heavier than those from control animals. Animals treated with either letrozole or herbal extract, or their combination, showed a significant higher number of sperm cells inside the seminiferous tubules and epididymis than control animals (p

Contents Oocyte vitrification causes less cell stress than slow cooling, but cytoskeletal and spindle alterations may occur affecting the oocyte competence. In vitro maturation (IVM) supplementation with different antioxidant molecules has been performed to attenuate this harmful stress. Coenzyme Q10 (CoQ10) supplementation has previously shown to have positive effects in bovine and mouse in vitro embryo development. The aim of this study was to evaluate the effects of CoQ10 during bovine oocyte IVM and vitrification. Cumulus–oocyte complexes (COCs) (n = 311) were cultured under standard maturation conditions with 0 μM (control), 25 μM and 50 μM CoQ10 supplementation. After 22 hr, a cohort of 170 oocytes both from the control and from CoQ10-supplemented groups were vitrified, warmed and returned to incubation until 24 hr of maturation, while the rest of the oocytes (n = 141) remained fresh. Then, oocyte survival was assessed morphologically by stereomicroscopy. Oocytes from all groups were then fixed and stained for assessing cortical granules (CG) migration and nuclear stage. High rates of oocyte MII progression and appropriate CG migration as a continuous layer beneath the plasma membrane were obtained both in control and in CoQ10 groups. Results showed that although vitrification has great impact in survival of IVM bovine oocytes, 50 μM CoQ10 supplementation significantly improved oocyte survival (p = .045) and reduced the premature CG exocytosis, helping to preserve the CG migration pattern (31.3% control vs. 54.5% in 50 μM CoQ10; p = .039), attenuating the negative effects of vitrification.

Persistent organic pollutants (POPs) are still globally distributed and some have been shown to interact with the endocrine system of birds. However, the relationship between POPs and the stress response mediated by the hypothalamic-pituitary-adrenal (HPA) axis is still poorly understood. Raising concerns are now focused on the toxic properties of emergent organophosphate ester flame retardants (OPEs), but whether OPEs interact with the HPA axis response has not yet been investigated. We measured corticosterone concentrations in feathers (CORTf) as a long-term biomarker of the bird HPA axis response and we investigated their relationship with POP and OPE concentrations in down feathers of nestling cinereous vultures (Aegypius monachus). We also examined whether high contaminant burden and high CORTf concentrations impacted the duration of chick development. The most predominant compounds were the following: p,p′-DDE (3.28 ± 0.26 ng g−1 dw) > γ-HCH (0.78 ± 0.09 ng g−1 dw) > BDE-99 (0.73 ± 0.09 ng g−1 dw) > CB-153 (0.67 ± 0.04 ng g−1 dw). The most persistent POP compounds (CB-170, -177, -180, -183, -187, -194 and p,p′-DDE) were associated (P = 0.02) with high concentrations of CORTf (range: 0.556.09 pg mm−1), while no relationship was found when OPEs were tested (P > 0.05). Later egg-laying was positively associated to high levels of CORTf (P = 0.02) and reduced duration of chick development (P

7 pages, 2 figures, 3 tables, supplementary data https://doi.org/10.1016/j.ygcen.2019.01.015, A significant body of literature suggests that aquatic pollutants can interfere with the physiological function of the fish hypothalamic-pituitary-interrenal (HPI) axis, and eventually impair the ability to cope with subsequent stressors. For this reason, development of accurate techniques to assess fish stress responses have become of growing interest. Fish scales have been recently recognized as a biomaterial that accumulates cortisol, hence it can be potentially used to assess chronic stress in laboratory conditions. We, therefore, aimed to evaluate the applicability of this novel method for cortisol assessment in fish within their natural environment. Catalan chub (Squalius laietanus) were sampled from two sites; a highly polluted and a less polluted (reference) site, in order to examine if habitat quality could potentially influence the cortisol deposition in scales. We also evaluated the seasonal variation in scale cortisol levels by sampling fish at three different time points during spring-summer 2014. In each sampling, blood was collected to complement the information provided by the scales. Our results demonstrated that blood and scale cortisol levels from individuals inhabiting the reference site were significantly correlated, therefore increasing the applicability of the method as a sensitive-individual measure of fish HPI axis activity, at least in non-polluted habitats. Since different environmental conditions could potentially alter the usefulness of the technique, results highlight that further validation is required to better interpret hormone fluctuations in fish scales. Scale cortisol concentrations were unaffected by habitat quality although fish from the polluted environment presented lower circulating cortisol levels. We detected a seasonal increase in scale cortisol values concurring with an energetically costly period for the species, supporting the idea that the analysis of cortisol in scales reveals changes in the HPI axis activity. Taken together, the present study suggests that cortisol levels in scales are more likely to be influenced by mid-term, intense energetically demanding periods rather than by long-term stressors. Measurement of cortisol in fish scales can open the possibility to study novel spatio-temporal contexts of interest, yet further research is required to better understand its biological relevance

Biological molecules isolated from organisms that live under subzero conditions could be used to protect oocytes from cryoinjuries suffered during cryopreservation. This study examined the cryoprotectant role of exopolysaccharides of Pseudomonas sp. ID1 (EPS ID1) in the vitrification of prepubertal and adult cow oocytes. IVM oocytes were vitrified and warmed in media supplemented with 0, 1, 10, 100 or 1000µgmL−1 EPS ID1. After warming, oocytes were fertilised and embryo development, spindle morphology and the expression of several genes in Day 8 blastocysts were assessed. Vitrification led to significantly lower proportion of prepubertal oocytes exhibiting a normal spindle configuration. In fresh control oocytes and most groups of vitrified adult oocytes, similar percentages of oocytes with a normal spindle configuration were observed. Percentages of Day 8 blastocysts were similar for prepubertal oocytes vitrified in the absence or presence of 1 or 10µgmL−1 EPS ID1 and for adult oocytes vitrified in the presence of 10µgmL−1 EPS ID1 compared with non-vitrified oocytes. EPS ID1 supplementation had no effect on solute carrier family 2 member 3 (SLC2A3), ubiquitin-conjugating enzyme E2A (UBE2A) and histone deacetylase 1 (HDAC1) expression in Day 8 blastocysts form adult oocytes. However, supplementation with 10 and 100µgmL−1 EPS ID1 led to increased expression of genes involved in epigenetic modifications (DNA methyltransferase 3 alpha (DNMT3A) and K (lysine) acetyltransferase 2A (KAT2A)) and apoptosis (BCL2 associated X apoptosis regulator (BAX) and BCL2-like 1 (BCL2L1)). The lowest BAX:BCL2L1 ratio was found in the 10µgmL−1 EPS ID1-supplemented group. The results suggest that 10µgmL−1 EPS ID1 added to vitrification and warming media may help protect bovine oocytes against cryodamage.

The measurement of fecal progesterone metabolites (fPM) by enzyme immunoassay analysis is a non-invasive technique that permits gathering reproductive information from wildlife without the stress associated with restraint. In the collared peccary (Pecari tajacu), a high correlation between serum progesterone and fPM levels (r2 = 0.783) suggests that fPM can be used to monitor their reproductive function. We monitored fPM during the estrous cycle of 15 collared peccary females. Estrous cycles averaged 27.9 ± 4.5 days (n = 28), ranging from 21 to 36 days. The luteal phase was 22.2 ± 4.8 days and the inter-luteal phase was 4.3 ± 1.4 days. Mean concentration of fPM across pregnancy were not different from those found during the luteal phase (1230 ± 718 and 1265 ± 584 ng/100 mg dried feces, respectively), however, significant differences were found when luteal phase concentrations were compared only against fPM concentrations during late pregnancy. In addition, late pregnancy fPM concentrations (1893 ± 551 ng/100 mg) were also significantly higher than those in the early (639 ± 339 ng/100 mg) and mid (1134 ± 449 ng/100 mg) pregnancy. For females during the early post-partum period, fPM concentrations were significantly increased (243 ± 118 ng/100 mg) than those of non-cycling females (103 ± 89 ng/100 mg). The analysis of fPM is a simple, non-invasive methodology to detect the ovarian activity in the collared peccary; moreover, it provides a husbandry tool, which may be used to help understand how social structure may impact reproduction.

The zona pellucida (ZP) is an extracellular matrix that surrounds mammalian oocytes. In eutherians it is formed from three or four proteins (ZP1, ZP2, ZP3, ZP4). In the few marsupials that have been studied, however, only three of these have been characterised (ZP2, ZP3, ZP4). Nevertheless, the composition in marsupials may be more complex, since a duplication of the ZP3 gene was recently described in one species. The aim of this work was to elucidate the ZP composition in marsupials and relate it to the evolution of the ZP gene family. For that, an in silico and molecular analysis was undertaken, focusing on two South American species (gray short-tailed opossum and common opossum) and five Australian species (brushtail possum, koala, Bennett’s wallaby, Tammar wallaby and Tasmanian devil). This analysis identified the presence of ZP1 mRNA and mRNA from two or three paralogues of ZP3 in marsupials. Furthermore, evidence for ZP1 and ZP4 pseudogenes in the South American subfamily Didelphinae and for ZP3 pseudogenes in two marsupials is provided. In conclusion, two different composition models are proposed for marsupials: a model with four proteins (ZP1, ZP2 and ZP3 (two copies)) for the South American species and a model with six proteins (ZP1, ZP2, ZP3 (three copies) and ZP4) for the Australasian species.

Raptor feathers have been increasingly used to assess pollutants in ecotoxicological monitoring studies. However, the suitability of down feathers to detect pollutants has not yet been investigated. In this study, concentrations of persistent organic pollutants (POPs) and organophosphate ester flame retardants (OPEs) were assessed in down and juvenile contour feathers of Spanish cinereous vulture (Aegypius monachus) nestlings (circa 73 days old) and contaminant concentrations were compared between both types of feathers from the same individuals. Concentrations of polychlorinated biphenyls (PCBs: 1.30 -6.16 ng g(-1) dw feather), polybrominated diphenyl ethers (PBDEs: 0.23-1.35 ng g(-1) dw feather), p,p'-dichlorodiphenyldichloroethylene (pp-DDE: 0.09-6.10 ng g(-1) dw feather) and tris (1-chloro-2-propyl) phosphate (TCiPP: 0.86-48.96 ng g(-1) dw feather) were significantly higher in down than in contour feathers. In contrast, contour feathers showed higher levels of the more volatile POP, lindane (0.25 -3.12 ng g(-1) dw feather). Concentrations of hexachlorobenzene (HCB) and OPEs (except TCiPP) were similar between the two types of feathers. By showing high accumulation of the most persistent POPs investigated, down feathers presented a contamination profile similar to that previously described in raptor eggs. As these feathers grow during the first days of a vulture chicks life, they probably reflect the contaminant burden of the chick due to maternal transfer to the egg. Overall, the present study provides the first indication that down feathers may be useful for biomonitoring studies. Further research is needed to confirm whether nestling down feathers reflect the concentrations in the egg. (C) 2018 Elsevier Ltd. All rights reserved.

The study of fish stress is usually assessed by measuring blood cortisol. Nevertheless, blood provides only a snapshot of the hormonal profile at one point in time. An alternative source of cortisol may be found in scales, providing a new approach for assessing long-term hormonal levels. The present study aimed to develop and validate a methodology for detecting cortisol in scales of goldfish (Carassius auratus). The study highlights the importance of an initial isopropanol washing procedure to completely eliminate external contaminations of cortisol. Additionally, the biochemical validation of the enzyme immunoassay verifies the ability to detect cortisol with repeatability and reliability in goldfish scales. In conclusion, this study provides validated information about a new methodology to measure cortisol in scales. The incorporation of this biomarker could provide retrospective hormonal measurements from species and time periods that are usually difficult or impossible to obtain, thus offering key data of an animal's physiology

Hair may be a useful matrix to detect cumulative cortisol concentrations in studies of animal welfare and chronic stress. The aim of this study was to validate a protocol for cortisol detection in hair from dairy cattle by enzyme immunoassay (EIA). Seventeen adult Holstein-Friesian dairy cows were used during the milking period. Hair cortisol concentration was assessed in 25-day-old hair samples taken from the frontal region of the head, analysing black and white coloured hair separately. Concentrations of cortisol metabolites were determined in faeces collected twice a week during the same period of time. There was a high correlation between cortisol values in faeces and cortisol in white colour hair samples but such correlation was not significant with the black colour hair samples. The intra- and inter-assay coefficients of variation were 4.9% and 10.6%, respectively. The linearity showed R 2=0.98 and mean percentage error of -10.8 ± 1.55%. The extraction efficiency was 89.0 ± 23.52% and the parallelism test showed similar slopes. Cortisol detection in hair by using EIA seems to be a valid method to represent long-term circulating cortisol levels in dairy cattle.

The objective is to study the local perceptions on the conservation of wild chimpanzees (Pan troglodytes verus) in the Réserve Naturelle Communautaire de Dindéfélo (RNCD), southeast of Senegal, to design specific actions to improve conservation management. We conducted 338 semi-structured interviews in three main villages of RNCD. Three-fourths of the population were farmers. Of those interviewed, 29% received elementary education. Two of the three villages participated in a project to plant trees as fences. On average, 66% of the respondents were animists. Of the respondents who were afraid to see a chimpanzee, 68% answered because they attack. Seventy-seven percent washed their clothes in the forest river because there was more water than in the village wells. Of the interviewees who threw the old clothes into the forest, 50% did it due to tradition. Ninety-six percent of respondents stated that chimpanzees do not feed from their crops. The main problems of the locals with the Reserve were lack of water and basic resources and not been allow to cut trees in protected areas. There were significant relationships between education (1 relationship), environmental project (4 relationships) and animism (11 relationships) with local perceptions. The 93% of the respondents who had the traditional belief that “if the old clothes were burnt, children would become sick” feel fear of chimpanzees, while those who did not have this animistic belief the 6% are afraid (χ2 = 1.57, P < 0.02). These local perceptions allow us to design specific course of action to improve chimpanzee conservation and sustainable coexistence in this complex period of the Anthropocene.

Assisted reproductive technology (ART) can benefit from the features of microfluidic technologies, such as the automation of time-consuming labor-intensive procedures, the possibility to mimic in vivo environments, and the miniaturization of the required equipment. To date, most of the proposed approaches are based on polydimethylsiloxane (PDMS) as platform substrate material due to its widespread use in academia, despite certain disadvantages, such as the elevated cost of mass production. Herein, we present a rapid fabrication process for a cyclic olefin copolymer (COC) monolithic microfluidic device combining hot embossing—using a low-temperature cofired ceramic (LTCC) master—and micromilling. The microfluidic device was suitable for trapping and maturation of bovine oocytes, which were further studied to determine their ability to be fertilized. Furthermore, another COC microfluidic device was fabricated to store sperm and assess its quality parameters over time. The study herein presented demonstrates a good biocompatibility of the COC when working with gametes, and it exhibits certain advantages, such as the nonabsorption of small molecules, gas impermeability, and low fabrication costs, all at the prototyping and mass production scale, thus taking a step further toward fully automated microfluidic devices in ART.

Hair cortisol concentrations (HCCs) and hair progesterone concentrations (HPCs) allow monitoring long-term retrospective steroid levels. However, there are still gaps in the knowledge of the mechanisms of steroid deposition in hair and its potential application in dairy cattle research. This study aimed to evaluate the potential uses of hair steroid determinations by studying the interrelations between HCC, HPC, physiological data from cows, and their milk production and quality. Cortisol and progesterone concentrations were analyzed in hair from 101 milking Holstein Friesian cows in a commercial farm. Physiological data were obtained from the 60 d prior to hair collection. Moreover, productive data from the month when hair was collected and the previous one were also obtained as well as at 124 d after hair sampling. Significant but weak correlations were found between HCC and HPC (r = 0.25, P0.0001) and between HPC and age (r = 0.06, P = 0.0133). High HCC were associated with low milk yields from the 2 previous months to hair sampling (P = 0.0396) and during the whole lactation (P0.0001). High HCC were also related to high somatic cell count (P = 0.0241). No effect of HCC on fat or protein content was detected. No significant correlations were detected between hair steroid concentrations and pregnancy status, days of gestation, parturition category (primiparous vs multiparous), number of lactations or days in milk. The relationship between physiological variables and HCC or HPC could depend on the duration of the time period over which hair accumulates hormones. Steroid concentrations in hair present high variability between individuals but are a potential tool for dairy cattle welfare and production research by providing a useful and practical tool for long-term steroid monitoring.