The female effect-how female receptivity influences faecal testosterone metabolite levels, socio-positive behaviour and vocalization in male Southern white rhinoceroses.

Testosterone is known to be essential for sexual maturation as well as for the display of behavioural traits linked to reproduction. At the same time, external factors such as the presence of receptive females may affect testosterone levels, stressing the hormone's substantial role in reproductive success. It is therefore of major interest to investigate the links between androgens, behaviour and the social environment especially in species that rely on a resilient reproduction rate, such as the white rhinoceros (WR). We collected faecal samples of 16 male Southern WR (Ceratotherium simum simum) aged between 1 and 44 years from 11 European zoos. Audio and video recordings were simultaneously taken from five of the study males that were sexually mature and had direct contact with receptive females. Our results showed a positive correlation of faecal testosterone metabolite (fTM) concentrations and progressing age up until adulthood followed by a decline in older males. While previous reproductive success did not show any effect, the access to receptive females resulted in higher fTM levels. Thereby, fTM concentrations remained at the same level regardless of the receptivity phase, while social cohesion with respective females, affiliative behaviour as well as call rates of Pant and Hiss distinctly peaked during the receptive compared to the non-receptive periods. Conclusively, the immediate presence of receptive females poses a female effect that enhances the overall androgen levels in males and, thus, might facilitate their reproductive success. However, androgens do not seem to be the main driver of behavioural changes during courtship or mating. By linking endocrinological and socio-behavioural factors, we were able to provide an applicable basis for non-invasive monitoring of reproductive behaviour in male WR in captivity, thereby contributing to deeper understanding of potential reproduction impairments in a species whose population in captivity remains not fully self-sustaining.

Non-invasive monitoring of glucocorticoid metabolite concentrations in native Indian, as well as captive and re-wilded tigers in South Africa.

Over the last century, wild tiger (Panthera tigris) numbers have declined from over 100 000 individuals to fewer than 4 000, with animals now confined to less than 5% of their historic range due to habitat loss, persecution, inadequate management, and poaching. In contrast, 15 000-20 000 tigers are estimated to be housed in captivity, experiencing conditions vastly different than their wild counterparts. A total of 280 tigers are currently held at 44 different facilities within South Africa, including zoos, semi-captive 're-wilded' populations, and pets; these animals provide a unique opportunity to measure the impact of extrinsic factors, found in exotic habitats, on the adrenocortical activity of tigers. By monitoring and comparing stress-related faecal glucocorticoid metabolite (fGCM) concentrations of tigers housed at different locations, and free ranging tigers in natural tiger reserves, this project aimed to get a better understanding of the impact of extrinsic factors on adrenocortical function as a measure of stress. The results of this study showed no significant difference in fGCM concentrations between captive, re-wilded, and free-ranging tigers with the exception of one site. Furthermore, factors such as sex and season were not significant drivers of fGCM concentrations. One study group had elevated fGCM concentrations, showing population variation in the stress response. This indicates that populations are able to cope with exotic environments, however, as population-specific differences in the stress response exist, we suggest management protocols be created for each population. This study offered the unique opportunity to see how well tigers are faring outside of their native range and if having re-wilded tigers in exotic locations is a potential welfare-acceptable management option for tiger conservation globally.

Linking socio-sexual and vocal behaviour with faecal progesterone and oestrogen metabolite levels in Southern white rhinoceros females.

Progesterone and oestrogen are the main gonadal steroid hormones that regulate the ovarian activity and induce the fertile oestrus period in females. The monitoring of this receptive phase is particularly decisive for captive breeding and is commonly based on the observation of female behavioural patterns that coincide with their hormonal oestrus. However, in the white rhinoceros (WR), a species that is well known for its impaired reproductive rate in captivity, the female behavioural and vocal indicators of receptivity have not been systematically investigated or linked to their hormonal states so far. In order to close this gap, we combined behavioural and acoustic recordings, conducted over an average period of 32 days, with the analysis of faecal progesterone and oestrogen metabolite concentrations (fPM and fEM) in 27 adult Southern white rhinoceros (Ceratotherium simum simum; SWR) females from 10 European zoos. For eight of the study females, we were able to detect a receptive period indicated by their acceptance of sexual behaviour from the bulls. The comparison of behaviour and vocalization between receptive and non-receptive periods in these females demonstrated that particularly presenting and marking behaviour distinctly peaked during the receptive period, indicating the significance of olfactory signalling for female reproductive behaviour. Based on the analysis of fPM profiles, we were able to identify different reproductive states (cycling, non-cycling, pregnant) in 21 of 27 study females. In contrast, fEM profiles proved to be unsuitable for the detection of ovarian activity. For the majority (five of six females for which behavioural receptivity and endocrine cyclicity were determined), a coincidence of their receptive period and the hormonal oestrus, indicated by a nadir in fPM levels, could be detected. Conclusively, this study revealed a comprehensive behavioural repertoire that reflects the hormonal oestrus in SWR females and can therefore be reliably used for non-invasive ex situ reproduction monitoring.

A non-invasive method to assess the reproductive status of the European badger (Meles meles) from urinary sex-steroid metabolites.

Due to their unique reproductive physiology and behaviour, European badgers (Meles meles) are often used as a model to study mammalian reproduction. For reproductive endocrinology, circulating hormone levels are conventionally measured directly from blood samples. However, routine blood sampling is often not practical for wild animals and may induce stress affecting measurement accuracy. Non-invasive alternatives are thus of interest. Circulating hormones are metabolized through different routes, either by the kidneys, to be excreted through urine, or by the liver, to be excreted through faeces. These metabolites can thus be used as a proxy of hormone measurements, provided the species-specific metabolic characteristics are known. Here we tested the suitability of measuring urinary metabolites of circulating plasma sex-steroid hormones (testosterone in males and oestrogen in females) with enzyme immunoassays to assess the reproductive status of the European badger (Meles meles). Biological validation evidenced that urinary testosterone metabolite (UTM) and urinary total oestrogen metabolite (UEM) excretion patterns both corresponded with seasonal badger reproductive patterns on a population level, signaling correlation over a broad time frame. On an individual level, concurrent sampling of urine and plasma showed that male plasma testosterone and UTM levels correlated significantly across seasons, but no short term correlation was evident for total oestrogen and UEM in females. Thus, in badgers, urinary sex-steroid metabolites can be used reliably in the short term to assess male reproductive status at the individual level, but only at the broader population level for females.

Methodological Considerations for Using Fecal Glucocorticoid Metabolite Concentrations as an Indicator of Physiological Stress in the Brown Bear (Ursus arctos).

Reliable methods to measure stress-related glucocorticoid responses in free-ranging animals are important for wildlife management and conservation. Such methods are also paramount for our ability to improve our knowledge of the ecological consequences of physiological processes. The brown bear (Ursus arctos) is a large carnivore of ecological and cultural importance and is important for management. Here, we provide a physiological validation for an enzyme immunoassay (EIA) to quantify glucocorticoid metabolites in brown bear feces. We also provide an evaluation of the effects of sample exposure to ambient temperature on measured fecal glucocorticoid metabolite (fGCM) concentrations. We evaluated three EIA systems: a cortisol assay, an 11-oxoetiocholanolone assay, and an 11ß-hydroxyetiocholanolone assay. Of these, the cortisol assay provided the best discrimination between peak fGCM concentrations detected 1-4 d after injections of synthetic adrenocorticotrophic hormone and preinjection baseline concentrations in four individual brown bears. The time of exposure to ambient temperature had substantial but variable effects on measured fGCM concentrations, including variation both between samples from the same individual and among samples from different bears. We propose that the validated EIA system for measuring fGCM concentrations in the brown bear could be a useful noninvasive method to monitor stress in this species. However, we highlight that this method requires that fecal samples be frozen immediately after defecation, which could be a limitation in many field situations.

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.

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.

Monitoring corticoid metabolites in urine of white-tailed sea eagles: Negative effects of road proximity on breeding pairs.

The white-tailed sea eagle (Haliaeetus albicilla) is known to be sensitive to disturbance. To better understand potential stressors, we measured corticosterone metabolite levels in H. albicilla excreta and recorded the nest success of breeding pairs. We tested the ability of four enzyme immunoassays (EIA) to measure urinary glucocorticoid metabolites (uGM) in the excreta of one adult female eagle subjected to a controlled physiological stress treatment. We identified corticosterone-21-HS to be the most sensitive EIA to changes in uGM concentration. To exclude a sex bias, we confirmed the assay's applicability with samples collected from similar stress treatments in two juvenile males. We used the identified EIA to measure uGM in wild breeding pairs and tested effects of disturbance. Breeding pairs nesting closer to roads and paths had higher uGM concentrations (p = 0.02), which is likely an effect of human recreational activity and disturbance. There was no difference in uGM concentrations between failed and successful nests. Our results highlight the potential impact of road and path proximity on white-tailed sea eagles, with potential importance for species management and conservation, particularly with respect to nest protection zone legislation.

Environmental stressors may cause equine herpesvirus reactivation in captive Grévy's zebras (Equus grevyi).

Equine Herpesviruses (EHV) are common and often latent pathogens of equids which can cause fatalities when transmitted to non-equids. Stress and elevated glucocorticoids have been associated with EHV reactivation in domestic horses, but little is known about the correlation between stress and viral reactivation in wild equids. We investigated the effect of an environmental stressor (social group restructuring following a translocation event) on EHV reactivation in captive Grévy's zebras (Equus grevyi). A mare was translocated by road transport from Zoo Mulhouse, France, to join a resident group of three mares in Tierpark Berlin, Germany. We used an indirect sampling method to assess the frequency of EHV shedding for 14 days immediately after the translocation event (termed the 'experimental period'). The results were compared with those from two control periods, one preceding and one subsequent to the experimental period. In addition, we measured fecal glucocorticoid metabolite (fGCM) concentrations daily in all individuals from 6 days before, to 14 days after translocation. We found significantly higher EHV shedding frequencies during the experimental period, compared to each of the two control periods. All animals showed significantly elevated fGCM concentrations, compared to fGCM levels before translocation. Finally, we found that an increase in fGCM concentration was significantly associated with an increased likelihood of EHV shedding. Although the small number of animals in the study limits the conclusions that can be drawn from the study, taken together, our results support the hypothesis that environmental stressors induce viral reactivation in wild equids. Our results suggest that potentials stressors such as group restructuring and translocation should be considered in the management of zoological collections to reduce the risk of fatal EHV infections in novel hosts. Moreover, environmental stressors may play an important role in EHV reactivation and spread in wild equid populations.

Steroidogenic enzymes, their products and sex steroid receptors during testis development and spermatogenesis in the domestic cat (Felis catus).

In the present study we comprehensively characterize intratesticular sex steroid production, metabolism and receptors in the domestic cat to elucidate the role of testosterone, estradiol and progesterone in testis development, steroid synthesis and spermatogenesis. There is a great demand for new concepts of fertility control in domestic (feral) cats and wild felids. The acquired knowledge will help to understand the regulation of spermatogenesis in felids, and may reveal new target points for male contraception. Progesterone and androgens are produced throughout all stages of testicular development; their synthesizing enzymes are mainly expressed in Leydig cells, and to a much lesser extent also in tubular cells. Aromatase (CYP19A1), the estrogen synthesizing enzyme, is only present in the tubuli and is first detectable in spermatocytes and round spermatids at puberty. As shown by elevated expression of the enzymes steroid 5-α-reductase type 1 (SRD5A) and aldo-keto-reductase family 1 member C3 (AKR1C3), the capacity to metabolize particular steroids increases during testis development. Apparently, this refers to a decreasing intra-testicular testosterone concentration per mg tissue with increasing testis weight during postpuberty. The increasing potential of sulfation of E2 by estrogen sulfotransferase (SULT1E1) with ongoing development might be responsible for the low level of unconjugated intratesticular estradiol in all stages of development probably due to facilitated excretion of conjugated estrogens. For the first time, expression of the progesterone membrane receptor components 1 and 2 (PGRMC1, PGRMC2) was studied in mammalian testis tissue. Both of these and also the progesterone receptor (PGR) are expressed depending on the developmental stage and cell type, suggesting an important regulatory role of progesterone in the testis. Androgen receptor (AR) is present in almost all cell types except for some spermatogenic cells. The co-localization of aromatase with estrogen receptor alpha (ESR1) in spermatocytes and round spermatids of domestic cat testis indicates an auto-/paracrine function of estrogen in spermatogenesis. In summary, the testis of the domestic cat is an important source of sex steroids. All of them could act within the testis but additionally, at least androgens and estrogens are likely secreted by the testis, partly as conjugated steroids.

Cheetahs have a stronger constitutive innate immunity than leopards.

As a textbook case for the importance of genetics in conservation, absence of genetic variability at the major histocompatibility complex (MHC) is thought to endanger species viability, since it is considered crucial for pathogen resistance. An alternative view of the immune system inspired by life history theory posits that a strong response should evolve in other components of the immune system if there is little variation in the MHC. In contrast to the leopard (Panthera pardus), the cheetah (Acinonyx jubatus) has a relatively low genetic variability at the MHC, yet free-ranging cheetahs are healthy. By comparing the functional competence of the humoral immune system of both species in sympatric populations in Namibia, we demonstrate that cheetahs have a higher constitutive innate but lower induced innate and adaptive immunity than leopards. We conclude (1) immunocompetence of cheetahs is higher than previously thought; (2) studying both innate and adaptive components of immune systems will enrich conservation science.

Measuring fecal testosterone metabolites in spotted hyenas: Choosing the wrong assay may lead to erroneous results.

Enzyme-immunoassays (EIA) that detect fecal testosterone metabolites (fTM) are powerful tools to monitor gonadal activity non-invasively. However, a challenge with testosterone EIAs might be their potential for cross-reactivities with structurally similar glucocorticoid metabolites. Therefore, we aimed to verify the capability of four different testosterone EIAs to monitor fTM without reflecting changes in adrenocortical activity in spotted hyenas by analyzing fecal samples following testosterone and ACTH challenge tests. We demonstrated that none of the testosterone EIAs is appropriate to measure fTM as all of them showed substantial cross-reactivities to unknown metabolites. Our study underlines the importance of validating androgen EIAs.

Correction: Measuring Faecal Epi-Androsterone as an Indicator of Gonadal Activity in Spotted Hyenas (Crocuta crocuta).

[This corrects the article DOI: 10.1371/journal.pone.0128706.].

Synthesis and reception of prostaglandins in corpora lutea of domestic cat and lynx.

Felids show different reproductive strategies related to the luteal phase. Domestic cats exhibit a seasonal polyoestrus and ovulation is followed by formation of corpora lutea (CL). Pregnant and non-pregnant cycles are reflected by diverging plasma progesterone (P4) profiles. Eurasian and Iberian lynxes show a seasonal monooestrus, in which physiologically persistent CL (perCL) support constantly elevated plasma P4 levels. Prostaglandins (PGs) represent key regulators of reproduction, and we aimed to characterise PG synthesis in feline CL to identify their contribution to the luteal lifespan. We assessed mRNA and protein expression of PG synthases (PTGS2/COX2, PTGES, PGFS/AKR1C3) and PG receptors (PTGER2, PTGER4, PTGFR), and intra-luteal levels of PGE2 and PGF2α Therefore, CL of pregnant (pre-implantation, post-implantation, regression stages) and non-pregnant (formation, development/maintenance, early regression, late regression stages) domestic cats, and prooestrous Eurasian (perCL, pre-mating) and metoestrous Iberian (perCL, freshCL, post-mating) lynxes were investigated. Expression of PTGS2/COX2, PTGES and PTGER4 was independent of the luteal stage in the investigated species. High levels of luteotrophic PGE2 in perCL might be associated with persistence of luteal function in lynxes. Signals for PGFS/AKR1C3 expression were weak in mid and late luteal stages of cats but were absent in lynxes, concomitant with low PGF2α levels in these species. Thus, regulation of CL regression by luteal PGF2α seems negligible. In contrast, expression of PTGFR was evident in nearly all investigated CL of cat and lynxes, implying that luteal regression, e.g. at the end of pregnancy, is triggered by extra-luteal PGF2α.

Validation of an enzyme-immunoassay for the non-invasive monitoring of faecal testosterone metabolites in male cheetahs (Acinonyx jubatus).

In mammals, the sex hormone testosterone is the major endocrine variable to objectify testicular activity and thus reproductive function in males. Testosterone is involved in the development and function of male reproductive physiology and sex-related behaviour. The development of a reliable androgen enzyme-immunoassay (EIA) to monitor faecal testosterone metabolites (fTM) is a powerful tool to non-invasively assess the gonadal status of males. We validated an epiandrosterone EIA for male cheetahs by performing a testosterone radiometabolism study followed by high-performance liquid chromatography (HPLC) analyses and excluding possible cross-reactivities with androgenic metabolites not derived from testosterone metabolism. The physiological and biological relevance of the epiandrosterone EIA was validated by demonstrating (1) a significant increase in fTM concentrations within one day in response to a testosterone injection, (2) a significant increase in fTM concentrations within one day in response to a gonadotropin-releasing hormone (GnRH) injection, which failed following a placebo injection, and (3) significant differences in fTM concentrations between adult male and adult female cheetahs and between adult and juvenile male cheetahs of a free-ranging population. Finally, we demonstrated stability of fTM concentrations measured in faecal samples exposed to ambient temperatures up to 72h. Our results clearly demonstrate that the epiandrosterone EIA is a reliable non-invasive method to monitor testicular activity in male cheetahs.

Non-invasive monitoring of stress hormones in the bat Eptesicus isabellinus - Do fecal glucocorticoid metabolite concentrations correlate with survival?

Chronic stress may negatively impact fitness and survival in wildlife. Stress hormone analysis from feces is a non-invasive tool for identifying stressors and deducing about individual and population level fitness. Although many bat populations are endangered, fecal stress hormone analysis has not been established in bats as a method for focusing conservation efforts. The isabelline serotine bat, Eptesicus isabellinus, is exposed to human disturbance as its roosts are mostly found in anthropogenic structures. Moreover, this bat is host to various diseases and survival rates between colonies may vary significantly. To validate the analysis of fecal glucocorticoid metabolites, we applied an adrenocorticotropic hormone (ACTH) challenge and tested four different enzyme immunoassays (EIA) for measuring glucocorticoid concentrations. Cortisol and its metabolites showed the highest increase in blood and feces after the ACTH challenge, but corticosterone and its metabolites also increased significantly. Baseline fecal cortisol metabolite (FCM) concentrations did not increase until 1.5h after the animals were captured, which is a convenient time lag for sample collection from captured animals. We furthermore compared baseline FCM concentrations between five colonies of E. isabellinus in Andalusia, Spain, and tested for their correlation with survival rates. FCM concentrations did not vary between colonies, but FCM levels increased with the animals' age. FCM analysis may prove a useful tool for identifying bat colonies that experience uncommon environmental stress. However, inter-individual variation in hormone secretion, due to factors such as age, may require additional information to properly interpret differences in hormone concentrations.

Non-Invasive Pregnancy Diagnosis in Big Cats using the PGFM (13,14-dihydro-15-keto-PGF2α) Assay.

Non-invasive monitoring of hormones using feces has become a vital tool for reproductive management and reliable pregnancy diagnosis in big cats. Previous studies described the PGF2α metabolite (PGFM) as an indicator of pregnancy in various feline species. The present study aimed to standardize pregnancy detection in big cats like the tiger (Panthera tigris), jaguar (Panthera onca) and lion (Panthera leo) using fecal samples. High-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LCMS) were performed to identify PGFM in feces. An EIA developed against 9α,11α-dihydroxy-15-oxo-prost-5-en-1-oic acid-BSA was used to assay PGFM in fecal samples of the Bengal tiger, Asiatic lion and jaguar. The PGFM levels increased after 9 weeks of pregnancy and remained elevated until parturition. All animals showed elevated levels of PGFM in the last trimester of pregnancy, thus making PGFM a reliable tool for pregnancy diagnosis during this period that can be useful in captive breeding programs in these species.

Measuring Faecal Epi-Androsterone as an Indicator of Gonadal Activity in Spotted Hyenas (Crocuta crocuta).

Enzyme immunoassays (EIA) that measure faecal testosterone metabolites (fTM) are useful tools to monitor gonadal activity. The aim of this study was to validate an "in-house" epiandrosterone EIA to monitor fTM in spotted hyenas. FTM were characterised in a male and a female hyena that each received an injection of 3H-testosterone. High-performance liquid chromatography (HPLC) analyses revealed a cluster of highly polar enzyme-hydrolysable hormone metabolite conjugates. We performed hydrolysis using ß-glucuronidase to deconjugate metabolites and improve sensitivity of the assay. Because ß-glucuronidase from Helix pomatia has been reported to bias testosterone measurements in some species, we compared the enzymatic activity of the commonly used ß-glucuronidase extracted from H. pomatia with the same enzyme from Escherichia coli. Our results showed that ß-glucuronidases from both sources produced similar results from spotted hyena faeces. We therefore hydrolysed samples with H. pomatia enzymes. HPLC analyses also demonstrated that following hydrolysis the epiandrosterone EIA measured significant amounts of immunoreactive metabolites corresponding to radiolabelled metabolites in both sexes. Additionally, HPLC and GC-MS analyses confirmed the presence of epiandrosterone in faeces of spotted hyenas. The biological relevance of the epiandrosterone EIA was validated by demonstrating (1) a significant increase in fTM levels in response to a testosterone injection within 16 h, (2) no biological responsiveness to an adrenocorticotropic hormone (ACTH) injection and (3) significant differences in fTM levels between juvenile males and adult immigrant males in a free-ranging wild population. Our results clearly demonstrate that the epiandrosterone EIA is a reliable non-invasive method to monitor gonadal activity in spotted hyenas.

The corpus luteum of the domestic cat: histologic classification and intraluteal hormone profile.

In the present study, we aimed to histologically stage and characterize the structural life span of the CL in the domestic cat. Moreover, the intraluteal levels of progesterone and estrogens were determined throughout the pregnant and nonpregnant (pseudopregnant, PP) luteal phases. On the basis of observed histomorphology of CL, the following stages were identified: CL formation (preimplantation period, PP1), development/maintenance (Days 10-36 of pregnancy, PP2), early regression (Days 38 and 39 of pregnancy, PP3), late regression (Day 48 of pregnancy, PP4), and corpus albicans. The main cellular markers included luteal cell shape, the type and degree of vacuolation, nucleus condition, and the ratio of nonsteroidogenic to luteal cells. Intraluteal levels of progesterone and estrogens differed significantly throughout stages of pseudopregnancy (P < 0.01, progesterone; P < 0.0001, estrogens). The progesterone level in PP2 was higher compared with PP3 and PP4 (P < 0.05). The estrogen level in PP1 was higher compared with PP2 (P < 0.05), PP3 and PP4 (P < 0.005), as well as in PP2 compared with PP3 and PP4 (P < 0.005). The staging of the domestic cat luteal phase established here provides a basis for further research on feline luteotropic and luteolytic factors. These data will contribute to comparative studies in felids.