An Investigation of Ovarian and Adrenal Hormone Activity in Post-Ovulatory Cheetahs (Acinonyx jubatus).

Cheetahs have been the subject of reproductive study for over 35 years, yet steroid hormone activity remains poorly described after ovulation. Our objective was to examine and compare fecal progestagen (fPM), estrogen (fEM), and glucocorticoid (fGM) metabolite concentrations post-ovulation in pregnant and non-pregnant animals to better understand female physiology (1) during successful pregnancy, (2) surrounding frequent non-pregnant luteal phases, and (3) after artificial insemination (AI) to improve the low success rate. Secondarily, the authors also validated a urinary progestagen metabolite assay, allowing pregnancy detection with minimal sample collection. Fecal samples were collected from 12 females for ≥2 weeks prior to breeding/hormone injection (the PRE period) through 92 days post-breeding/injection. Samples were assessed for hormone concentrations using established enzyme immunoassays. Urine samples were collected for 13 weeks from 6 females after natural breeding or AI. There were no differences among groups in fGM, but in pregnant females, concentrations were higher (p < 0.01) in the last trimester than any other time. For pregnant females that gave birth to singletons, fGM was higher (p = 0.0205), but fEM tended to be lower (p = 0.0626) than those with multi-cub litters. Our results provide insight into the physiological events surrounding natural and artificially stimulated luteal activity in the cheetah.

Assessing puberty in female cheetahs (Acinonyx jubatus) via faecal hormone metabolites and body weight.

With fewer than 7500 cheetahs remaining in the wild, ex situ cheetah populations serve as an insurance policy against extinction and a resource to study species' biology. This study aimed to identify the age of pubertal onset in ex situ female cheetahs using non-invasive faecal steroid hormone monitoring and body weights. Faecal samples from nine female cheetahs were collected two to three times weekly from 2 to 36months of age and body weights were recorded every 3months. Faecal oestrogen metabolites (FOM) and faecal glucocorticoid metabolites (FGM) were analysed using enzyme immunoassays and samples were categorised into 6-month intervals to compare endocrine characteristics. Faecal hormone and body weight data were analysed using generalised linear mixed models. Age was a significant predictor of mean and baseline FOM concentrations, number of FOM peaks, mean and maximum FOM peak concentrations and the number of cycles. Female cheetahs aged 24-30months exhibited a marked rise in mean FOM concentration and the number of FOM peaks and cycles increased with age until 24-30months. Females attained adult body weight by 21months of age. Mean and baseline FGM concentrations were highest at the 0-6 and 12-18months of age groups and did not follow the same FOM patterns. Based on body weight data, the FOM concentrations and peak patterning, females were considered pubertal from 24 to 30months of age. Characterisation of cheetah puberty has direct and significant implications for the improvement of management and reproductive success of cheetahs under human care. This information is particularly informative for identifying important windows of development, littermate dispersal and breeding introductions.

First Birth of Cheetah Cubs from In Vitro Fertilization and Embryo Transfer.

Approximately 30% of the Association of Zoos and Aquariums cheetah population (~350 total animals) is unlikely to breed naturally due to advanced age, health, or behavioral issues. Aging cheetah females (≥9 y old) are unlikely to become pregnant via natural breeding if they are nulliparous. We previously demonstrated that oocytes recovered from aged females were of similar quality compared with those recovered from younger females (2-8 y old). We hypothesize that transfer of 4-8 cell embryos produced by in vitro fertilization with oocytes from old donors could result in pregnancy after transfer into younger recipients. Female cheetahs (n = 3 aging donors and n = 3 young recipients) received 300 IU equine Chorionic Gonadotropin (eCG) and 3000 IU Luteinizing Hormone (LH) while fecal metabolites of estrogens and progestogens were closely monitored. At 28 h post-LH injection, oocytes were aspirated laparoscopically from donors and inseminated in vitro with cryopreserved sperm. After 48 h of in vitro culture, resulting embryos (4-8 cells) were transferred into the oviducts of recipient females. Pregnancy was confirmed in one recipient via ultrasound 32 days after transfer and by radiograph 62 days after transfer. Two cubs were born naturally after 90 days of gestation, representing the first cheetah births resulting from transfer of embryos produced in vitro.

Assessing puberty in ex situ male cheetahs (Acinonyx jubatus) via fecal hormone metabolites and body weights.

Cheetahs are one of the most heavily studied felid species, with numerous publications on health, disease, and reproductive physiology produced over the last 30 years. Despite this relatively long history of research, there is a paucity of crucial biological data, such as pubertal onset, which has direct and significant applications to improved management of ex situ cheetah populations. This study aimed to determine age of pubertal onset in ex situ male cheetahs using non-invasive fecal steroid hormone monitoring and body weights. Fecal samples from 12 male cheetahs from four institutions were collected 2-3 times weekly from 1 to 42 months of age. Fecal androgen and glucocorticoid metabolites were analyzed using enzyme immunoassays previously validated for use with cheetah feces. Animal body weights were recorded monthly. Fecal hormone and body weight data were analyzed using generalized linear mixed models. Androgen concentrations exhibited an increase to levels similar to those observed in adult males by 18-24 months of age, and males attained adult body weights by 21 months of age. Based on these weight data and the initial increase in androgens toward adult concentrations, males were considered pubertal from 18 to 24 months of age. Glucocorticoid concentrations and amplitude of concentration over baseline were also increased during this period. Knowledge about the physiological changes associated with puberty is useful for management and improving reproductive success of cheetah populations under human care, particularly for determining timing of litter separation from dam, littermate dispersal and when to introduce potential breeding pairs.

Non-invasive identification of protein biomarkers for early pregnancy diagnosis in the cheetah (Acinonyx jubatus).

Approximately 80% of cheetahs living in typical zoological collections never reproduce. In more than 60% of breedings, the female is confirmed to ovulate, but parturition fails to occur. It is unknown if these non-pregnant intervals of elevated progesterone (deemed luteal phases) are conception failures or a pregnancy terminating in embryonic/fetal loss. There have been recent advances in metabolic profiling and proteome analyses in many species with mass spectrometry used to identify 'biomarkers' and mechanisms indicative of specific physiological states (including pregnancy). Here, we hypothesized that protein expression in voided cheetah feces varied depending on pregnancy status. We: 1) identified the expansive protein profile present in fecal material of females; and 2) isolated proteins that may be candidates playing a role in early pregnancy establishment and diagnosis. Five hundred and seventy unique proteins were discovered among samples from pregnant (n = 8), non-pregnant, luteal phase (n = 5), and non-ovulatory control (n = 5) cheetahs. Four protein candidates were isolated that were significantly up-regulated and two were down-regulated in samples from pregnant compared to non-pregnant or control counterparts. One up-regulated candidate, immunoglobulin J chain (IGJ; an important component of the secretory immune system) was detected using a commercially available antibody via immunoblotting. Findings revealed that increased IGJ abundance could be used to detect pregnancy successfully in >80% of 23 assessed females within 4 weeks after mating. The discovery of a novel fecal pregnancy marker improves the ability to determine reproductive, especially gestational, status in cheetahs managed in an ex situ insurance and source population.

Fecal estrogen, progestagen and glucocorticoid metabolites during the estrous cycle and pregnancy in the giant anteater (Myrmecophaga tridactyla): evidence for delayed implantation.

BACKGROUND: Declining numbers of wild giant anteaters highlight the importance of sustainable captive populations. Unfortunately, captive reproductive management is limited by the lack of external physical indicators of female reproductive status and the aggressive behavior of males. We examined the endocrinology of the estrous cycle and pregnancy, and whether delayed implantation is a gestational strategy for giant anteaters as described for other xenarthrans. METHODS: Feces were collected from seven captive females 3-5 times weekly and mating was recorded. Concentrations of estrogen (estrone-glucuronide, E1, and estradiol-17ß, E2), progestagen (20-oxo-progestagens, P4), and glucocorticoid (GC) metabolites were examined in fecal extracts by enzyme immunoassay. RESULTS: Estrous cycles for nulliparous females (6 cycles, n = 2) compared to the multiparous female (6 cycles, n = 1) were shorter (47.3 +/- 4.3 days versus 62.5 +/- 2.6 days) with relatively lower luteal phase concentrations of P4 (49.4 +/- 2.9 ng/g versus 136.8 +/- 1.8 ng/g). The four remaining females had unclear ovarian activity: two females exhibited apparent luteal activity but unclear fluctuations in estrogens, while the other two females had parallel fecal P4 and estrogens concentrations. Pregnancy ranged 171-183 days with females returning to estrus post-partum as early as 60 days (n = 3, 1.8-4 years of age at mating). Delayed implantation was indicated by a biphasic elevation in fecal P4 metabolites: the initial 4-fold increase occurred for 81-105 days and was followed by a 26-fold secondary rise in P4 metabolites lasting 66-94 days prior to parturition. Fecal GC was correlated with fecal estrogens and greatest during estrus, late pregnancy, and six days prior to parturition (estrous cycle GC, 14.4-62.8 ng/g; pregnancy GC, 13.6-232.7 ng/g). CONCLUSIONS: Estrous cycles of giant anteaters occurred year-round, but were shorter and more intermittent in younger nulliparous animals compared to a multiparous female. A pronounced elevation in fecal P4, estrogen, and GC occurred during late gestation after an initial post-mating delay providing evidence for delayed implantation. Adrenocorticoid activity indicated impending parturition. Differences in estrous cycle characteristics with age and the protracted but variable gestation length must be considered to improve reproductive success and neonatal survival in giant anteaters.