Current State of In Vitro Embryo Production in African Lion (Panthera leo).

In the last 30-40 years, in vitro maturation (IVM) and fertilization (IVF) of domestic cat oocytes have been established as part of the panel of assisted reproduction technologies. As a representative of wild felids, the African lion is not yet considered endangered. Nevertheless, the zoo population management of the African lion itself as well as other closely related felids would benefit from the establishment of an IVF system. Here, we aimed to investigate the transferability of domestic cat IVF technology to the African lion. From the ovaries of 42 lionesses aged between 0.75 and 15 years, a total of 933 IVF-suitable oocytes were retrieved and subjected to IVM and IVF. The overall maturation rate was 40.6% and 18.9% of these oocytes cleaved after fertilization, respectively. Embryos were generated by intracytoplasmic sperm cell injection as well as co-culture with epididymal sperm. Improvements in the model system also led to an improved outcome with in vitro produced embryos in the lion. Compared to domestic cats, the transportation of gonads to a specialized laboratory was time-consuming and influenced oocyte quality negatively. In conclusion, the domestic cat IVF system is adoptable for the African lion, although success rates are still lower.

Desiccated cat spermatozoa retain DNA integrity and developmental potential after prolonged storage and shipping at non-cryogenic temperatures.

PURPOSE: To evaluate the DNA integrity and developmental potential of microwave-dehydrated cat spermatozoa after storage at - 20 °C for different time periods and/or overnight shipping on dry ice. METHODS: Epididymal spermatozoa from domestic cats were microwave-dehydrated on coverslips after trehalose exposure. Dried samples were either assessed immediately, stored for various duration at - 20 °C, or shipped internationally on dry ice before continued storage. Dry-stored spermatozoa were rehydrated before assessing DNA integrity (TUNEL assays) or developmental potential (injection into in vitro matured oocytes followed by in vitro embryo culture for up to 7 days). RESULTS: Percentages of dried-rehydrated spermatozoa with intact DNA was not significantly affected (P > 0.05) by desiccation and short-term storage (range, 78.9 to 80.0%) but decreased (P < 0.05) with storage over 5 months (range, 71.0 to 75.2%) compared to fresh controls (92.6 ± 2.2%). After oocyte injection with fresh or dried-rehydrated spermatozoa (regardless of storage time), percentages of activation, pronuclear formation, and embryo development were similar (P > 0.05). Importantly, spermatozoa shipped internationally also retained the ability to support embryo development up to the morula stage. CONCLUSION: Results demonstrated the possibility to sustain DNA integrity and developmental potential of spermatozoa by dry-preservation, even after long-term storage and long-distance shipment at non-cryogenic temperatures. While further studies are warranted, present results demonstrate that dry preservation can be a reliable approach for simple and cost-effective sperm biobanking or shipment.

Amino acids activate mTORC1 to release roe deer embryos from decelerated proliferation during diapause.

Embryonic diapause in mammals leads to a reversible developmental arrest. While completely halted in many species, European roe deer (Capreolus capreolus) embryos display a continuous deceleration of proliferation. During a 4-mo period, the cell doubling time is 2 to 3 wk. During this period, the preimplantation blastocyst reaches a diameter of 4 mm, after which it resumes a fast developmental pace to subsequently implant. The mechanisms regulating this notable deceleration and reacceleration upon developmental resumption are unclear. We propose that amino acids of maternal origin drive the embryonic developmental pace. A pronounced change in the abundance of uterine fluid mTORC1-activating amino acids coincided with an increase in embryonic mTORC1 activity prior to the resumption of development. Concurrently, genes related to the glycolytic and phosphate pentose pathway, the TCA cycle, and one carbon metabolism were up-regulated. Furthermore, the uterine luminal epithelial transcriptome indicated increased estradiol-17ß signaling, which likely regulates the endometrial secretions adapting to the embryonic needs. While mTORC1 was predicted to be inactive during diapause, the residual embryonic mTORC2 activity may indicate its involvement in maintaining the low yet continuous proliferation rate during diapause. Collectively, we emphasize the role of nutrient signaling in preimplantation embryo development. We propose selective mTORC1 inhibition via uterine catecholestrogens and let-7 as a mechanism regulating slow stem cell cycle progression.

IGF-I Medium Supplementation Improves Singly Cultured Cat Oocyte Maturation and Embryo Development In Vitro.

Embryo production is a routine procedure in several species. However, in felids, the effectiveness of this approach is far behind that in the majority of laboratory species. The development of a suitable environment starts with the proper composition of culture media. Therefore, for the improvement of assisted reproduction techniques and their outcome in cats, this is an urgent task. As the addition of insulin-like growth factors (IGF-I, IGF-II) or granulocyte-macrophage colony-stimulating factor (GM-CSF) was beneficial in other mammalian species, this study aims to check whether these components, combined with other factors (such as type of fertilisation or type of culture) can provide a benefit in the felid culture system in current use. Thus, these supplements, in different concentrations and combinations, were merged with the use of two fertilisation techniques and randomly assigned to single or group culturing. The results showed that the addition of IGF-I and/or GM-CSF produced an increase in morula and blastocyst rate in a single culture system. In particular, the supplementation with 20 ng/mL of IGF-I incremented the maturation rate by 10% and significantly increased the morula and blastocyst rates in single culturing. This result is especially remarkable for wild felids, where only a few oocytes and/or embryos are available.

Decreasing glucocorticoid levels towards the expansion front suggest ongoing expansion in a terrestrial mammal.

Understanding the causes of range expansions in abundant species can help predict future species distributions. During range expansions, animals are exposed to novel environments and are required to cope with new and unpredictable stressors. Glucocorticoids (GCs) are mediators of the hormonal and behavioural mechanisms allowing animals to cope with unpredictable changes in the environment and are therefore expected to differ between populations at expansion edge and the historic range. However, to date, very few studies have evaluated the relationship between GCs and range expansion. The Egyptian mongoose has been rapidly expanding its range in Portugal over the past 30 years. In this study, we applied an information theoretic approach to determine the most important spatial and environmental predictors of hair GCs (hGCs) in the population, after controlling for normal patterns of hGC variation in the species. We observed a decrease in hGC as distance from the historic range increased (i.e. closer to the expansion front). This distance term was present in all of the top models and had a 95% confidence interval (95% CI) that did not overlap with zero, strongly supporting its influence on hGC. We estimated a 0.031 pg/mg (95% CI: -0.057, -0.004) decrease in hGCs for each kilometre distance to the Tagus River, which was once the limit of the species' distribution. Our results indicate that the species' expansion is unlikely to be limited by mechanisms related to or mediated by the physiological stress response. The decrease in hGC levels towards the expansion edge coupled with limited evidence of a negative effect of human population density suggests that the species' northward expansion in Portugal could continue.

Comparison of Different Materials for Self-Pressurized Vitrification of Feline Oocytes-First Results.

Cryobanking is a crucial part on species conservation. Nowadays, there is no suitable protocol for vitrification of feline oocytes. Self-pressurized rapid freezing of different cell types proved to mimic the advantages of high pressure freezing. As this method could also be applied for gamete rescue under field conditions, the aim here was to analyse the impact of self-pressurized vitrification on feline cumulus-oocyte-complexes (COCs) and to determine the appropriate material. Therefore, COCs of domestic cat were randomly vitrified (n = 189) in metal tubes of different materials: Aluminium, silver, and titanium. No significant differences were found on oocytes' competence after thawing. On average, 44% of the COCs presented normal morphology and 48.2% of them showed a polar body after in vitro maturation (IVM) and were subsequently fertilised. Aluminium tubes were positive on toxicity tests, producing the lowest cleavage rates. Silver tubes showed no toxic effect, but the cleavage rate was lower than with titanium tubes, and a previous association with embryotoxicity and biological alterations makes us aware of its indiscriminate use. Titanium seems to be the only inert material of them, presenting a slightly higher maturation (55.6%) and cleavage (20%) rates. Nevertheless, more studies should follow to increase embryo competence after warming.

Cloprostenol, a synthetic analog of prostaglandin F2α induces functional regression in cultured luteal cells of felids†.

In the present study, we investigated the effect of the synthetic analog of prostaglandin F2α (PGF2α)-cloprostenol-on cultured steroidogenic luteal cells of selected felid species over a 2-day culture period. The changes induced by cloprostenol were measured based on progesterone concentration and mRNA expression analysis of selected genes. Cloprostenol significantly reduced concentration of progesterone in cell culture medium of small luteal cells isolated from domestic cat corpora lutea (CL) at the development/maintenance stage (P < 0.05), but did not influence progesterone production in cultured cells from the regression stage. A decrease or complete silencing of progesterone production was also measured in cultured luteal cells of African lion (formation stage) and Javan leopard (development/maintenance stage). Gene-expression analysis by real-time PCR revealed that treatment with cloprostenol did not have an influence on expression of selected genes coding for enzymes of steroidogenesis (StAR, HSD3B, CYP11A1) or prostaglandin synthesis (PTGS2, PGES), nor did it effect hormone receptors (AR, ESR1, PGR, PTGER2), an anti-oxidative enzyme (SOD1) or factors of cell apoptosis (FAS, CASP3, TNFRSF1B, BCL2) over the studied period. Significant changes were measured only for expressions of luteinizing hormone (P < 0.05), prolactin (P < 0.05) and PGF2α receptors (P < 0.005) (LHCGR, PRLR, and PTGFR). The obtained results confirm that PGF2α/cloprostenol is a luteolytic agent in CL of felids and its impact on progesterone production depends on the developmental stage of the CL. Cloprostenol short-term treatment on luteal cells was associated only with functional but not structural changes related to luteal regression.

Luteinizing Hormone Effect on Luteal Cells Is Dependent on the Corpus Luteum Stage in Felids.

The objective of this study was to investigate the effect of luteinizing hormone (LH) on steroidogenic luteal cells obtained from corpora lutea (CL) of the domestic cat and selected wild felids. Luteal cells were isolated enzymatically from CL at different developmental stages and cultured for two days in the presence and absence of 100 ng/mL LH, respectively. Functionality was assessed by progesterone (P4) accumulation in cell culture media determined by ELISA. In addition, steroidogenic function was confirmed using immunohistochemistry for 3ß-hydroxysteroid dehydrogenase (HSD3B). The enzymatic method allowed for the isolation of mostly small luteal cells in all investigated felids. Treatment with LH resulted in an increase in P4 secretion of cultured luteal cells obtained from CL in the formation stage (African lion) and development/maintenance stage (domestic cat (p < 0.05), Javan leopard), whereas luteal cells from more advanced stages of luteal development (regression) responded moderately or not at all to LH stimulation (domestic cat, Asiatic golden cat, Asiatic lion). The protein signal for HSD3B on CL was visible until development/maintenance. In conclusion, this study shows that LH promotes P4 production in luteal cells only until the onset of regression, when morphological signs are visible on the CL of felids and HSD3B is no longer detectable.

Signalling pathways and mechanistic cues highlighted by transcriptomic analysis of primordial, primary, and secondary ovarian follicles in domestic cat.

In vitro growth (IVG) of dormant primordial ovarian follicles aims to produce mature competent oocytes for assisted reproduction. Success is dependent on optimal in vitro conditions complemented with an understanding of oocyte and ovarian follicle development in vivo. Complete IVG has not been achieved in any other mammalian species besides mice. Furthermore, ovarian folliculogenesis remains sparsely understood overall. Here, gene expression patterns were characterised by RNA-sequencing in primordial (PrF), primary (PF), and secondary (SF) ovarian follicles from Felis catus (domestic cat) ovaries. Two major transitions were investigated: PrF-PF and PF-SF. Transcriptional analysis revealed a higher proportion in gene expression changes during the PrF-PF transition. Key influencing factors during this transition included the interaction between the extracellular matrix (ECM) and matrix metalloproteinase (MMPs) along with nuclear components such as, histone HIST1H1T (H1.6). Conserved signalling factors and expression patterns previously described during mammalian ovarian folliculogenesis were observed. Species-specific features during domestic cat ovarian folliculogenesis were also found. The signalling pathway terms "PI3K-Akt", "transforming growth factor-ß receptor", "ErbB", and "HIF-1" from the functional annotation analysis were studied. Some results highlighted mechanistic cues potentially involved in PrF development in the domestic cat. Overall, this study provides an insight into regulatory factors and pathways during preantral ovarian folliculogenesis in domestic cat.

Ovary cold storage and shipment affect oocyte yield and cleavage rate of cat immature vitrified oocytes.

In feline species, cooled transport of ovaries can be employed without detrimental effects to retrieve immature oocytes intended for in vitro embryo production purposes. Indeed, this is the most common way to collect gametes from gonads of wild, valuable animals after they die or are castrated far from specialized laboratories. However, fresh retrieved gametes are generally used, and their cryosensitivity is not known. This study employed ovariectomy-derived domestic cat gonads as a model for wild felids, and aimed to compare the yield and developmental competence of Cryotop-vitrified oocytes (VOs) collected and cryopreserved right after ovary excision (In loco-VOs) or after 24 h cooled transport of ovaries (Shipped-VOs). The number of collected oocytes was higher in In loco-VOs than in Shipped-VOs (mean ± SD: 8 ± 3.36 vs 5.6 ± 3.1, p = 0.05). In vitro embryo production resulted in similar maturation (35% for both vitrified groups, p = 1) and fertilization rates (In loco-VOs: 29.1%; Shipped-VOs: 22.2%; p = 0.295), but showed a difference in cleavage (In loco-VOs: 25.6%; Shipped-VOs: 14.5%; p = 0.0495). No differences were found in further embryo development. Taken together, results suggested that delayed oocyte vitrification after cooled transport of organs was feasible and allowed embryo development. However, the number of collected oocytes and the cleavage rate of matured oocytes were higher when oocyte vitrification was performed without delay after ovary excision, and this should be considered in gamete conservation programs for endangered felids.

Maturation and fertilization of African lion (Panthera leo) oocytes after vitrification.

The African lion is an excellent model species for the highly endangered Asiatic lion. African lions reproduce well in zoos, leading to the fact that occasionally ovaries and testis are available for in-vitro experiments. We previously performed in-vitro maturation (IVM) and fertilization of lion oocytes and were able to produce advanced embryos after intracytoplasmic sperm injection (ICSI) with cryopreserved sperm. Here we examined whether our in-vitro method is also applicable after vitrification of immature oocytes. Oocytes of four lionesses (5-7 years old) were obtained after euthanasia and immediately processed on site. Half of the oocytes (n = 60) were subjected to IVM for a total of 32-34 h at 39 °C, 5% CO2 and humidified air atmosphere. The second group (59 oocytes) was vitrified instantly using the Cryotop method. Following 6 days of storage in liquid nitrogen, oocytes were warmed and subjected to IVM as well. Mature oocytes of both groups were fertilized with frozen-thawed African lion sperm using ICSI. Maturation rate was 55% and 49.2% for the control and vitrified group, respectively. In the control group, three oocytes cleaved and another three were arrested at the pronuclei stage. Due to the low fertilization result, a sperm sample of another male was used for the vitrified group. Of the vitrified oocytes 7 cleaved and 9 more oocytes stopped at pronuclei stage. All embryos of the vitrified group did not develop beyond 4 cell stage. This is the first time that African lion in-vitro-derived embryos have been produced following oocyte vitrification.

Inhibition of Apoptotic Pathways Improves DNA Integrity but Not Developmental Competence of Domestic Cat Immature Vitrified Oocytes.

Being a model for endangered wild felids, cryopreservation protocols for domestic cat oocytes are under continuous development. Immature vitrified oocytes (VOs) are a valuable resource for fertility preservation programs, but they often degenerate after warming and their in vitro development is poor. Since the exact mechanisms are not clear, this study assessed whether vitrification might trigger two apoptotic markers (DNA fragmentation and caspase activity, Experiment I) and the effects of a chemical inhibitor (i.e., the pan-caspase inhibitor Z-VAD-FMK) on the same markers (Experiment II) and on VOs in vitro development (Experiment III). The overarching aim was to check whether apoptosis inhibition might be a strategy to improve cat oocytes cryotolerance. In Experiment I, vitrification induced DNA fragmentation and increased caspase activity in VOs incubated for 24 h after warming (DNA fragmentation: 59.38%; caspase activity: 414.6 ± 326.8) compared to a fresh control (9.68%; 199.6 ± 178.3; p = 0.02). In Experiment II, the addition of Z-VAD-FMK to vitrification-warming and incubation media decreased DNA fragmentation and caspase activity (8.82%; 243.7 ± 106.9) compared to control (untreated) VOs (69.44%; 434.5 ± 248.3; p < 0.001). In Experiment III, Z-VAD-FMK brought maturation rates of treated VOs close to those of fresh oocytes (53.13 and 65.38%, respectively, p = 0.057), but there were no differences in VOs embryo development (cleavage rates; Z-VAD-FMK-treated VOs: 34.38%; control VOs: 31.78%; p = 0.69). In summary, vitrification increased apoptotic markers in cat VOs, and while Z-VAD-FMK was able to hinder DNA damage and caspase activity, its addition was not determinant for embryo development. To make the best use of VOs, other oocyte in vitro maturation and embryo culture strategies, such as the addition of other inhibitors or their prolonged use, should be investigated.

Meiotic Status Does Not Affect the Vitrification Effectiveness of Domestic Cat Oocytes.

Cryopreservation is important for animal fertility and biodiversity. Unfortunately, cryopreservation of feline oocytes is still an experimental technique. The aims of this study were to analyze the potential toxicity of the cryoprotectants in the vitrification solution (VS) on cat oocytes and to investigate whether the meiotic status of oocytes influences their developmental potential after vitrification. Two experiments were conducted with the VS composed of 20% ethylene glycol, 20% dimethyl sulfoxide, 20% fetal calf serum, 1.5 M trehalose, and 10% Ficoll PM-70: (1) toxicity assessment of the VS on immature cumulus oocyte complexes (COCs), and subsequently in vitro maturation (IVM) and in vitro fertilization; (2) assessment of the influence of the meiotic status on vitrification effectiveness, where immature and in vitro matured COCs were vitrified on the Cryotop. After rewarming, vitrified oocytes were subjected to IVM (immature) and intracytoplasmic sperm injection (ICSI) with fresh epididymal sperm. The toxicity test revealed no negative effect of oocyte exposure to the applied VS on their developmental potential (p > 0.05). Although the vitrification procedure itself significantly reduced the meiotic competence of oocytes, their meiotic status before vitrification (immature vs. in vitro matured) did not influence fertilization and morula rates. The only parameter affected by vitrification was the rate of oocytes suitable for ICSI, which was significantly lower for immature oocytes. Regardless of the meiotic status of vitrified oocytes, morphologically normal morulae were obtained. Moreover, the two meiotic stages examined are suitable for vitrification, with mature oocytes being a better choice when a well-equipped laboratory is available.

Sex steroids and glucocorticoid ratios in Iberian lynx hair.

Knowledge on species' reproductive biology is a fundamental pre-requisite of every conservation effort, but is often lacking. Sex steroids can provide valuable information for the assessment of reproductive success, whereas glucocorticoids are used to assess adrenocortical activity and stress-related bodily adaption. However, due to their perilous condition, access to animals is often difficult, which makes hormone measurement in non-invasively collected hair samples an attractive option. We determined cortisol, cortisone, corticosterone, testosterone and progesterone in Iberian lynx hair using enzyme immunoassay (EIA). Cross-validation was performed with high-performance liquid chromatography (HPLC) and high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). Finally, we statistically evaluated the variations of sex steroids and glucocorticoids according to age, sex, origin, behavior and management. All steroids except corticosterone were detectable in Iberian lynx hair. Hair progesterone measured by EIA was overestimated by cross-reaction with 5α-dihydroprogesterone, a biologically active gestagene, and was highly correlated with HPLC-MS/MS results. Progesterone was higher in adult females compared to all other age-sex groups. Cortisol measured by EIA was overestimated due to antibody cross-reactivity with cortisone and was correlated to the sum of HPLC-MS/MS measurements for cortisol and cortisone. Cortisol was higher in females than in males measured by HPLC-MS/MS, but the EIA results were confounded by the lack of specificity. When using cortisol-cortisone and cortisol-dihydroepiandrosterone ratios, differences were noted between wild-caught and captive-bred lynxes. Additionally, longitudinal EIA measurements of an Iberian lynx after a wildfire showed an inversion of the cortisol-cortisone ratio that later subsided. These results validate the use of hair progesterone measurement for Iberian lynx reproductive monitoring and add to the growing evidence supporting the need for a more comprehensive approach to hair steroid measurement that accounts for local interconversion and co-regulation mechanisms.

Preservation of female genetic resources in feline species.

The development of assisted reproduction techniques (ART) specifically for felids has been propagated for two main reasons: (i) most felids are threatened and faced with extinction in all or part of their native habitats (IUCN Red List of Threatened Species, www.catsg.org), and (ii) the domestic cat (Felis catus) can serve as a research model for the implementation of advanced assisted reproductive techniques (ART) to be applied in exotic cats. Domestic cat ovaries can be freshly obtained from veterinary clinics and are frequently used for research on preservation of genetic resources in feline species. The presented review will summarize recent advances and obstacles in biobanking of female genetic resources and discuss alternative approaches which are under investigation.

Hair cortisol analyses in different mammal species: choosing the wrong assay may lead to erroneous results.

Wild animals are faced with a broad range of environmental stressors and research is needed to better understand their effect on populations. Hormone analysis based on enzyme immunoassays (EIAs) can provide valuable information on adrenocortical activity (stress), and assessment of cortisol in hair may allow the quantification of cortisol production. To validate hair hormone analysis, we compared two EIAs based on antibodies against cortisol-3-CMO-BSA and cortisol-21-HS-BSA for hair glucocorticoid (hGC) measurements in Egyptian mongoose, Iberian lynx, Alpine marmot, Asiatic black bear, spotted hyena and cheetah, with results obtained by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) measurements. Both EIAs were also characterized by HPLC immunograms. Our results revealed that the cortisol-21-HS EIA measured 2.3- to 12-fold higher hGC concentrations than the cortisol-3-CMO assay. In dependence of the species, high-performance liquid chromatography (HPLC) immunograms showed that up to 70% of immunoreactivities determined by the cortisol-21-HS constituted of unknown unpolar compounds leading to an overestimation of hGC. The cortisol-3-CMO EIA expressed a better specificity, with 32.1-67.4% of immunoreactivity represented by cortisol and cortisone. The LC-MS/MS analyses (gold standard) revealed that the cortisol-3-CMO EIA also resulted in an (up to 3-fold) overestimation of hGC, but EIA results were correlated with LC-MS/MS in the mongoose, the lynx, the spotted hyena and the marmot. No correlation was obtained for Asiatic black bears. As a result of our study, we strongly recommend to test any cortisol EIA for its specificity towards extracted hair components. In all analyzed species, except the Asiatic black bear, cortisone and cortisol were simultaneously present in hair extracts; consequently, an appropriate EIA should cross-react to these two glucocorticoid hormones and express negligible affinity towards substances with less polarity than corticosterone. Choosing the wrong EIA for hGC analyses may lead to overestimations of hGC or-in the worst case-to results that do not mirror real adrenocortical activity.

Functional and Morphological Characterization of Small and Large Steroidogenic Luteal Cells From Domestic Cats Before and During Culture.

The current study aimed to isolate, culture and characterize small (SLC) and large (LLC) steroidogenic cells from the corpora lutea (CL) of non-pregnant domestic cats. Isolation of feline SLC was based on an enzymatic digestion of luteal tissue, whereas LLC were obtained by mechanical disruption of CL. To assess function of both cell types, progesterone secretion and mRNA expression of selected genes involved in steroid and prostaglandin synthesis were measured, as well as relative transcript abundance of hormone receptors and anti-oxidative enzymes, before and during culture. The cells were cultured for 3 or 5 days without gonadotropins. Isolated feline SLC and LLC had different sizes (12 ± 3 µm vs. 34 ± 5 µm, respectively), morphologies (amount of lipid droplets) and behaved differently in culture. SLC attached and proliferated or spread quickly, but lost their steroidogenic function during culture (significant decrease in progesterone secretion and expression of steroidogenic genes). The expression of receptors for gonadotropins and prolactin also decreased. Prostaglandin synthase (PTGS2) decreased steadily over time, whereas mRNA expression of PGE2 synthase (PGES) increased. The gene expression of anti-oxidative enzyme glutathione peroxidase 4 (GPX4), also increased during culture, but not of superoxide dismutase 1 (SOD1). In comparison to SLC, LLC did not attach to culture plates, secreted more progesterone per inoculated cells and maintained steroidogenic function during culture. Expression of prostaglandin synthases (PTGS2 and PGES) was almost non-detectable. The gene expression of hormone receptors for prostaglandin F2 alpha (PTGFR), gonadotropins (LHCHR and FSHR), and prolactin (PRLR), as well as of anti-oxidative enzymes (GPX4, SOD1), increased over time. To conclude, we successfully isolated and cultured different types of feline steroidogenic luteal cells and comprehensively characterized both isolated cell types. This knowledge can be used to better understand the CL lifecycle in felines more broadly, and the established cell cultures will provide a foundation for future studies on luteolytic and luteotrophic factors in the domestic cat, and for comparison with other feline species, particularly lynx.

Correction: Age, sex and storage time influence hair cortisol levels in a wild mammal population.

[This corrects the article DOI: 10.1371/journal.pone.0221124.].

Age, sex and storage time influence hair cortisol levels in a wild mammal population.

The measurement of hair cortisol is increasingly used to understand the effect of natural and anthropogenic stressors on wild animals, but it is potentially confounded by individual, seasonal and sex-dependant variations in baseline cortisol secretion. This study validated an enzyme-linked immunoassay for hair cortisol measurement and characterized its baseline variation in a wild population of Egyptian mongoose. The analysis encompassed individuals of both sexes and all ages, across a range of geographic, environmental and seasonal conditions that the species experiences in Portugal allowing us to account for spatial, temporal and biological factors that contribute to hair cortisol variation. Our results showed that age, sex and storage time had an effect on hair cortisol, but season did not. Hair cortisol was higher in early stage juveniles compared to other age cohorts, in males when compared to females, and decreased with longer storage time. By identifying the factors that influence baseline hair cortisol in this wild population, we establish the basis for its application as an indicator of the effect of natural and anthropogenic stressors.