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.

Oviductal Extracellular Vesicles Improve Post-Thaw Sperm Function in Red Wolves and Cheetahs.

Artificial insemination (AI) is a valuable tool for ex situ wildlife conservation, allowing the re-infusion and dissemination of genetic material, even after death of the donor. However, the application of AI to species conservation is still limited, due mainly to the poor survival of cryopreserved sperm. Recent work demonstrated that oviductal extracellular vesicles (oEVs) improved cat sperm motility and reduced premature acrosomal exocytosis. Here, we build on these findings by describing the protein content of dog and cat oEVs and investigating whether the incubation of cryopreserved red wolf and cheetah sperm with oEVs during thawing improves sperm function. Both red wolf and cheetah sperm thawed with dog and cat oEVs, respectively, had more intact acrosomes than the non-EV controls. Moreover, red wolf sperm thawed in the presence of dog oEVs better maintained sperm motility over time (>15%) though such an improvement was not observed in cheetah sperm. Our work demonstrates that dog and cat oEVs carry proteins important for sperm function and improve post-thaw motility and/or acrosome integrity of red wolf and cheetah sperm in vitro. The findings show how oEVs can be a valuable tool for improving the success of AI with cryopreserved sperm in threatened species.

Immunoglobulin J chain as a non-invasive indicator of pregnancy in the cheetah (Acinonyx jubatus).

The North American cheetah population serves as a reservoir for the species, and acts as a research population to help understand the unique biology of the species. Little is known about the intrauterine physiology of the cheetah, including embryo differentiation, implantation, and the development of the placenta. After mating, cheetah females frequently experience (30-65% of matings) a non-pregnant luteal phase where progestogen metabolite levels match those found in pregnant females for the first ~55 days of gestation, but parturition does not occur. Immunoglobulin J chain (IgJ) is a molecule that is involved in the activation of the secretory immune response and has been found to be indicative of pregnancy in the cheetah using fecal monitoring. In this study, western blotting was employed to track IgJ abundance in pooled weekly fecal samples following natural breeding or exogenous stimulation to ovulate, and IgJ levels were compared between individuals undergoing a pregnant (n = 12) and non-pregnant (n = 19) luteal phase. It was revealed that IgJ abundance was increased in pregnant females compared to non-pregnant females at week 4 and week 8 post-breeding, indicating the potential modulation of maternal immunity in response to sensitive events such as implantation and the increased secretory activity of the placenta. IgJ levels also tended to be higher early after breeding in females that were bred naturally with intact males compared to exogenously stimulated females with no exposure to seminal plasma, potentially indicating a response to the act of intromission or the stress of breeding, or possibly demonstrating an immune response resulting in the promotion of maternal tolerance to seminal antigens present upon embryonic implantation. Monitoring fecal IgJ may be a potential method to determine gestational status in the cheetah and will aid future conservation efforts of the species.

Responsiveness of the cheetah (Acinonyx jubatus) ovary to exogenous gonadotropins after preemptive oral progestin treatment.

Control of ovarian function in cheetahs is sub-optimal, which currently limits the integration of assisted reproductive techniques into the genetic management of that endangered species. The objective of this study was to determine the effect of preemptive progestin treatment on the quality of ovarian responses after exogenous gonadotropin stimulation in cheetahs. Adult females received either 1) 200 IU equine chorionic gonadotropin (eCG) followed with 3,000 IU porcine luteinizing hormone (pLH) (intramuscular route) (n = 5; control group) or 2) similar eCG/pLH administration preceded by a 7-day treatment with oral progestin (0.1 mg/kg altrenogest; ALT group; n = 7). At 42 h post-pLH administration, a series of metrics was assessed via laparoscopy (number of follicles ≥ 2 mm, number of corpora lutea, oviduct and uterine cornua diameter and overall vascularization). Concentrations of fecal estradiol, progesterone and glucocorticoid metabolites (FEM, FPM, and FGM, respectively) were measured by enzyme immunoassay for 3 wk before ALT treatment (Period 1), 7 d during ovarian suppression period (Period 2), throughout eCG/LH treatment and laparoscopy (Period 3), and 6 wk following laparoscopy (Period 4). Overall, nine out of 12 cheetahs (4/5 in control and 5/7 ALT group) had freshly-formed corpora lutea at the time of laparoscopy. Mean follicle and corpora lutea numbers in the control versus ALT group were not different (P > 0.05). Overall measurements and vascularization scores also did not differ (P > 0.05) among groups. FEM average concentrations increased (P ≤ 0.05) in response to eCG for the ALT-treated females between Periods 2 and 3 and were sustained during Period 4. However, FEM average concentrations did not vary (P > 0.05) for control females throughout Periods 1-4. Post-ovulatory FPM average concentrations (Period 4) did not differ (P > 0.05) between the ALT-treated females and controls. FPM average concentration from both groups increased in Period 4 compared to Periods 1-3 (P ≤ 0.05). Females receiving the ALT treatment also had lower (P ≤ 0.05) FGM metabolite average concentrations than control females during ovarian suppression (suggesting adrenal suppression). Collective results suggest that ovarian response to gonadotropin treatment in the cheetah was improved following oral progestin administration due to the normative increase in estradiol following stimulation for these females compared with control. This treatment should lead to more effective timed assisted reproduction procedures for this species.

Analysis of metabolic flux in felid spermatozoa using metabolomics and 13C-based fluxomics†.

Spermatozoa from three feline species-the domestic cat (Felis catus), the cheetah (Acinonyx jubatus), and the clouded leopard (Neofelis nebulosa)-were analyzed using metabolomic profiling and 13C-based fluxomics to address questions raised regarding their energy metabolism. Metabolic profiles and utilization of 13C-labeled energy substrates were detected and quantified using gas chromatography-mass spectrometry (GC-MS). Spermatozoa were collected by electroejaculation and incubated in media supplemented with 1.0 mM [U13C]-glucose, [U13C]-fructose, or [U13C]-pyruvate. Evaluation of intracellular metabolites following GC-MS analysis revealed the uptake and utilization of labeled glucose and fructose in sperm, as indicated by the presence of heavy ions in glycolytic products lactate and pyruvate. Despite evidence of substrate utilization, neither glucose nor fructose had an effect on the sperm motility index of ejaculated spermatozoa from any of the three felid species, and limited entry of pyruvate derived from these hexose substrates into mitochondria and the tricarboxylic acid cycle was detected. However, pathway utilization was species-specific for the limited number of individuals (four to seven males per species) assessed in these studies. An inhibitor of fatty acid beta-oxidation (FAO), etomoxir, altered metabolic profiles of all three felid species but decreased motility only in the cheetah. While fluxomic analysis provided direct evidence that glucose and fructose undergo catabolic metabolism, other endogenous substrates such as endogenous lipids may provide energy to fuel motility.

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.

Impact of mimicking natural dispersion on breeding success of captive North American Cheetahs (Acinonyx jubatus).

This paper examines the effects of transfer away from natal facility and littermate presence on cheetah breeding success in the AZA Species Survival Plan (SSP) population. Transfer and breeding history data for captive males and females were gathered from seven and four AZA SSP breeding facilities, respectively, to identify factors influencing breeding success. The results indicate that transfer history (p = 0.032), age at transfer (p = 0.013), and female littermate presence/absence (p = 0.04) was associated with breeding success, with females transferred away from their natal facility before sexual maturity and without littermates present accounting for the highest breeding success. Keeping males at their natal facility and/or removing them from their coalitions did not negatively affect their breeding success. Males appeared to demonstrate the same fecundity regardless of transfer history or coalition status, indicating that dispersal away from natal environment was not as critical for the breeding success of males compared with female cheetahs. These results highlight the significance of moving females away from their natal environment, as would occur in the wild, and separating them from their female littermates for optimization of breeding success in the ex situ population.

Age-associated and deslorelin-induced declines in serum anti-Müllerian hormone concentrations in female cheetahs, Acinonyx jubatus.

Anti-Müllerian hormone (AMH) is widely used in human medicine to non-invasively estimate the size of the ovarian follicle reserve and to predict the ovarian response to gonadotropin stimulation in the context of assisted reproductive technologies (e.g., IVF). These applications of AMH testing have recently expanded to non-human mammals, with production animals, such as cows, goats and sheep being the primary focus of AMH research. However, few investigations have involved exotic species, and in particular carnivores. In this study, we measured AMH concentrations (0.078-3.078ng/mL) in archived serum samples that had been collected from 36 adult female cheetahs across their reproductive lifespan (2-15years of age). Similar to other mammals, AMH concentration in cheetahs declined with age, and its variability among females of the same age was considerable. The rates at which AMH declined over time in individual cheetahs were also highly variable. Five cheetahs had been contracepted with the long-acting GnRH agonist deslorelin for 6-18months prior to sample collection, and their AMH concentrations were relatively low compared to untreated females. In this first study of AMH in an exotic carnivore, the findings demonstrate that the age-associated decline in AMH is highly variable and that deslorelin appears to suppress AMH concentration in serum. Owing to the increased use of assisted reproductive technologies in ex situ populations of threatened and endangered species, such as cheetahs, the present study's findings will need to be taken into consideration if AMH is to be used successfully to optimize breeding management decisions in exotic species.

Group management influences reproductive function of the male cheetah (Acinonyx jubatus).

Although the free-ranging cheetah is generally socially solitary, as many as 60% of males live in same-sex (usually sibling) coalitions. Under ex situ conditions, the cheetah experiences low reproductive success with only ~18% of males having ever produced young. Most male cheetahs (85%) are managed in captivity in coalitions, but with no data on the influence of social grouping on reproductive parameters. We examined the influence of singleton versus coalition management on various male cheetah physiological traits, including ejaculate quality and gonadal and adrenal hormone metabolite concentrations. We also assessed behaviour within coalitions for evidence of social hierarchy through initiation of interactions with group mates and relatedness to physiological traits. Ejaculate quality (including total motile and structurally normal spermatozoa per ejaculate) and androgen concentration profiles were higher (P<0.05) in coalition compared with singleton males. These results support the conclusion that testis function in the cheetah, specifically related to the development of normal, motile spermatozoa and androgen production, is influenced by management with same-sex conspecifics. The findings have implications for ex situ conservation breeding programs by suggesting that reproductive quality can be enhanced through group maintenance of cheetah males.

Circumventing the natural, frequent oestrogen waves of the female cheetah (Acinonyx jubatus) using oral progestin (Altrenogest).

Cheetah are induced ovulators, experiencing short, variable oestrogen waves year-round. Exogenous gonadotrophin administration induces ovulation, but success is variable and often improves if ovaries are quiescent. After affirming the presence of short-term oestrogenic waves, we examined the effect of the timing of administration of exogenous equine and human chorionic gonadotrophins (eCG-hCG) within the oestrogen concentration pattern on subsequent follicle development and oocyte and corpus luteum quality. We also investigated ovarian suppression using an oral progestin (Altrenogest, 7 days) and assessed whether Altrenogest moderated adrenal activity by reducing glucocorticoid metabolites. All cheetahs exhibited short (every ~7-10 days), sporadic, year-round increases in faecal oestradiol punctuated by unpredictable periods (4-10 weeks) of baseline oestradiol (anoestrous). Gonadotrophin (eCG-hCG) efficacy was not affected by oestradiol 'wave' pattern if administered ≥3 days after an oestrogen peak. Such cheetahs produced normative faecal progestagen patterns and higher numbers (P<0.06) of mature oocytes than females given gonadotrophins ≤2 days after an oestradiol peak. Altrenogest supplementation expanded the interval between oestradiol peaks to 12.9 days compared with 7.3 days without progestin pretreatment. Altrenogest-fed females excreted less (P<0.05) glucocorticoid metabolites than non-supplemented counterparts. Results show that Altrenogest is effective for suppressing follicular activity, may contribute to reduced glucocorticoid production and may result in more effective ovulation induction via gonadotrophin therapy.

Public exposure and number of conspecifics have no influence on ovarian and adrenal activity in the cheetah (Acinonyx jubatus).

Cheetahs in managed zoological collections do not reproduce efficiently, a problem that may be related to environmental/management stressors. In this study, we examined 17 adult female cheetahs to determine the influence of two environmental factors, (1) being housed on- or off-exhibit and (2) number of adult conspecifics (males and/or females) in nearby enclosures, on profiles and concentrations of ovarian and adrenal hormones. Secondarily, we assessed a subset of group-housed siblings (n=5 females in groups of 2 or 3) for effects of long-term cohabitation. All of the females demonstrated waves of estrogen excretion (indicative of ovarian activity) as well as occasional periods of no estrogen production (anestrus). Glucocorticoid and estrogen concentrations were correlated within an individual (rs=0.53; P<0.05), and overall there was a higher frequency of days with elevated glucocorticoid concentrations in association with elevated estrogen excretion. However, none of the management factors had an impact (P>0.05) on estrogen or glucocorticoid metabolite excretory patterns. Although we recently reported that public exposure can negatively affect sperm production, ovarian steroidogenesis in females was unaffected. There also was no evidence of hyper-adrenal activity. Thus, different methods of ex situ management appear to have minimal influence on ovarian function or stress susceptibility of female cheetahs.

Serum Amyloid A Protein Concentration in Blood is Influenced by Genetic Differences in the Cheetah (Acinonyx jubatus).

Systemic amyloid A (AA) amyloidosis is a major cause of morbidity and mortality among captive cheetahs. The self-aggregating AA protein responsible for this disease is a byproduct of serum amyloid A (SAA) protein degradation. Transcriptional induction of the SAA1 gene is dependent on both C/EBPß and NF-κB cis-acting elements within the promoter region. In cheetahs, 2 alleles exist for a single guanine nucleotide deletion in the putative NF-κB binding site. In this study, a novel genotyping assay was developed to screen for the alleles. The results show that the SAA1A (-97delG) allele is associated with decreased SAA protein concentrations in the serum of captive cheetahs (n = 58), suggesting genetic differences at this locus may be affecting AA amyloidosis prevalence. However, there was no significant difference in the frequency of the SAA1A (-97delG) allele between individuals confirmed AA amyloidosis positive versus AA amyloidosis negative at the time of necropsy (n = 48). Thus, even though there is evidence that having more copies of the SAA1A (-97delG) allele results in a potentially protective decrease in serum concentrations of SAA protein in captive cheetahs, genotype is not associated with this disease within the North American population. These results suggest that other factors are playing a more significant role in the pathogenesis of AA amyloidosis among captive cheetahs.

Motile Sperm Output by Male Cheetahs (Acinonyx jubatus) Managed Ex Situ Is Influenced by Public Exposure and Number of Care-Givers.

The collective cheetah (Acinonyx jubatus) population in zoological institutions has never been self-sustaining because of challenges in natural reproduction. A retrospective analysis of North American zoo-breeding records has revealed that >90% of litters produced since 2003 occurred in facilities 'off-display' from the public. We examined seminal, endocrine, and behavioral traits of 29 adult male cheetahs that were: 1) managed in public exhibit or off-display facilities; 2) maintained by different numbers of cheetah-specific care-givers; and 3) living adjacent to varying numbers of adult conspecifics. Cheetahs housed off-display produced more total motile sperm/ejaculate (P = 0.04) than on-exhibit males. This finding was mirrored in our laboratory's historical records where two-fold more total motile sperm (P < 0.01) were measured in ejaculates from individuals with no public exposure (n = 43) compared to on-exhibit (n = 116) counterparts. Males at institutions with ≤3 care-givers also produced more total motile sperm/ejaculate (P < 0.03) and spent more time behaviorally active (P < 0.01) than at facilities using >3 care-givers. Exposure to high numbers of conspecifics within the same institution did not impact (P > 0.05) seminal traits, and presence of the public, care-giver number, or animals/facility had no influence (P > 0.05) on androgen or glucocorticoid excretion or other behavioral metrics. Findings indicate that male cheetahs are sensitive to general public exposure and too many care-givers, resulting in compromised motile sperm output/ejaculate with mechanism of action unrelated to altered androgen or glucocorticoid excretion.

INDUCTION OF CYTOKINE PRODUCTION IN CHEETAH (ACINONYX JUBATUS) PERIPHERAL BLOOD MONONUCLEAR CELLS AND VALIDATION OF FELINE-SPECIFIC CYTOKINE ASSAYS FOR ANALYSIS OF CHEETAH SERUM.

Peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood of cheetahs (Acinonyx jubatus ; n=3) and stimulated with lipopolysaccharides (LPS) to induce the production of proinflammatory cytokines TNF-α, IL-1ß, and IL-6 for establishment of cross-reactivity between these cheetah cytokines and feline-specific cytokine antibodies provided in commercially available Feline DuoSet® ELISA kits (R&D Systems, Inc., Minneapolis, Minnesota 55413, USA). This study found that feline-specific cytokine antibodies bind specifically to cheetah proinflammatory cytokines TNF-α, IL-1ß, and IL-6 from cell culture supernatants. The assays also revealed that cheetah PBMCs produce a measurable, cell concentration-dependent increase in proinflammatory cytokine production after LPS stimulation. To enable the use of these kits, which are designed for cell culture supernatants for analyzing cytokine concentrations in cheetah serum, percent recovery and parallelism of feline cytokine standards in cheetah serum were also evaluated. Cytokine concentrations in cheetah serum were approximated based on the use of domestic cat standards in the absence of cheetah standard material. In all cases (for cytokines TNF-α, IL-1ß, and IL-6), percent recovery increased as the serum sample dilution increased, though percent recovery varied between cytokines at a given dilution factor. A 1:2 dilution of serum resulted in approximately 45, 82, and 7% recovery of TNF-α, IL-1ß, and IL-6 standards, respectively. Adequate parallelism was observed across a large range of cytokine concentrations for TNF-α and IL-1ß; however, a significant departure from parallelism was observed between the IL-6 standard and the serum samples (P=0.004). Therefore, based on our results, the Feline DuoSet ELISA (R&D Systems, Inc.) kits are valid assays for the measurement of TNF-α and IL-1ß in cheetah serum but should not be used for accurate measurement of IL-6.

Progestin priming before gonadotrophin stimulation and AI improves embryo development and normalises luteal function in the cat.

Exogenous gonadotrophins administered before AI can adversely alter endocrine dynamics and inhibit embryo development in felids. In the present study, we tested the hypothesis that priming the domestic cat ovary with progestin mitigates the negative influence of gonadotrophin therapy by normalising early embryogenesis and luteal function. Queens were given either: (1) progestin pretreatment plus chorionic gonadotrophins (n=8; primed); or (2) gonadotrophins only (n=8; unprimed). Ovulatory response was assessed laparoscopically, and cats with fresh corpora lutea (CL) were inseminated in utero. Ovariohysterectomy was performed 3 days later to recover intra-oviductal embryos for in vitro culture; one ovary was prepared for histology, and CL from the remaining ovary were excised and assessed for progesterone content and targeted gene expression. Of the six primed and seven unprimed queens inseminated, embryo(s) were recovered from five individuals per group. Embryos from progestin-primed donors more closely simulated normal stage in vivo development (P<0.05). No 2- or 4-cell embryos from either group developed beyond 16-cells in vitro; however, 50% of unprimed and 66.7% of primed (P>0.05) 5-16-cell embryos progressed to morulae or blastocysts by Day 4 of culture. Although histological characteristics were unaffected by progestin priming (P>0.05), luteal progesterone was unusually high (P<0.05) in unprimed compared with primed cats (72.4±5.8 vs. 52.2±5.5 ng mg(-1), respectively). Two genes associated with progesterone biosynthesis (luteinising hormone receptor and 3ß-hydroxysteroid dehydrogenase) were upregulated in unprimed versus primed individuals (P=0.05 and P<0.05, respectively), indicating potential mechanistic pathways for the protective influence of pre-emptive progestin treatment. Building on earlier findings that progestin priming prevents spontaneous ovulation, increases ovarian sensitivity to gonadotrophins and ensures a normative endocrine environment, the present study demonstrates that pretreatment with this steroid also benefits embryo development and normalisation of early luteal function.

Oral progestin priming increases ovarian sensitivity to gonadotropin stimulation and improves luteal function in the cat.

As the only domesticated species known to exhibit both induced and spontaneous ovulation, the cat is a model for understanding the nuances of ovarian control. To explore ovarian sensitivity to exogenous gonadotropins and the influence of progestin priming, we conducted a study of queens that were down-regulated with oral progestin or allowed to cycle normally, followed by low or high doses of equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG). Our metrics included 1) fecal steroid metabolite profiles before and after ovulation induction, 2) laparoscopic examination of ovarian follicles and corpora lutea (CL) on Days 2 and 17 (Day 0 = hCG administration), and 3) ovariohysterectomy (Day 17) to assess CL progesterone concentrations, morphometrics, and histology. Reproductive tracts from time-matched, naturally mated queens (n = 6) served as controls. Every progestin-primed cat (n = 12) produced the desired response of morphologically similar, fresh CL (regardless of eCG/hCG dose) by Day 2, whereas 41.7% of unprimed counterparts (n = 12) failed to ovulate or had variable-aged CL suggestive of prior spontaneous ovulation (P < 0.05). The ovarian response to low, but not high, eCG/hCG was improved (P < 0.05) in primed compared to unprimed cats, indicating increased sensitivity to gonadotropin in the progestin-primed ovary. Progestin priming prevented hyperelevated fecal steroid metabolites and normalized CL progesterone capacity, but only when combined with low eCG/hCG. However, priming failed to prevent ancillary CL formation, smaller CL mass, or abnormal luteal cell density, which were common to all eCG/hCG-treated cats. Thus, the domestic cat exposed to eCG/hCG produces CL with structural and functional aberrations. These anomalies can be partially mitigated by progestin priming, possibly due to a protective effect of progestin associated with enhanced ovarian sensitivity to gonadotropins.

Different patterns of metabolic cryo-damage in domestic cat (Felis catus) and cheetah (Acinonyx jubatus) spermatozoa.

Felid spermatozoa are sensitive to cryopreservation-induced damage, but functional losses can be mitigated by post-thaw swim-up or density gradient processing methods that selectively recover motile or structurally-normal spermatozoa, respectively. Despite the importance of sperm energy production to achieving fertilization, there is little knowledge about the influence of cryopreservation or post-thaw processing on felid sperm metabolism. We conducted a comparative study of domestic cat and cheetah sperm metabolism after cryopreservation and post-thaw processing. We hypothesized that freezing/thawing impairs sperm metabolism and that swim-up, but not density gradient centrifugation, recovers metabolically-normal spermatozoa. Ejaculates were cryopreserved, thawed, and processed by swim-up, Accudenz gradient centrifugation, or conventional washing (representing the 'control'). Sperm glucose and pyruvate uptake, lactate production, motility, and acrosomal integrity were assessed. Mitochondrial membrane potential (MMP) was measured in cat spermatozoa. In both species, lactate production, motility, and acrosomal integrity were reduced in post-thaw, washed samples compared to freshly-collected ejaculates. Glucose uptake was minimal pre- and post-cryopreservation, whereas pyruvate uptake was similar between treatments due to high coefficients of variation. In the cat, swim-up, but not Accudenz processing, recovered spermatozoa with increased lactate production, pyruvate uptake, and motility compared to controls. Although confounded by differences in non-specific fluorescence among processing methods, MMP values within treatments were positively correlated to sperm motility and acrosomal integrity. Cheetah spermatozoa isolated by either selection method exhibited improved motility and/or acrosomal integrity, but remained metabolically compromised. Collectively, findings revealed a metabolically-robust subpopulation of cryopreserved cat, but not cheetah, spermatozoa, recovered by selecting for motility rather than morphology.