Chromosome-length genome assembly and karyotype of the endangered black-footed ferret (Mustela nigripes).

The black-footed ferret (Mustela nigripes) narrowly avoided extinction to become an oft-cited example of the benefits of intensive management, research, and collaboration to save a species through ex situ conservation breeding and reintroduction into its former range. However, the species remains at risk due to possible inbreeding, disease susceptibility, and multiple fertility challenges. Here, we report the de novo genome assembly of a male black-footed ferret generated through a combination of linked-read sequencing, optical mapping, and Hi-C proximity ligation. In addition, we report the karyotype for this species, which was used to anchor and assign chromosome numbers to the chromosome-length scaffolds. The draft assembly was ~2.5 Gb in length, with 95.6% of it anchored to 19 chromosome-length scaffolds, corresponding to the 2n = 38 chromosomes revealed by the karyotype. The assembly has contig and scaffold N50 values of 148.8 kbp and 145.4 Mbp, respectively, and is up to 96% complete based on BUSCO analyses. Annotation of the assembly, including evidence from RNA-seq data, identified 21,406 protein-coding genes and a repeat content of 37.35%. Phylogenomic analyses indicated that the black-footed ferret diverged from the European polecat/domestic ferret lineage 1.6 million yr ago. This assembly will enable research on the conservation genomics of black-footed ferrets and thereby aid in the further restoration of this endangered species.

Low estradiol production of non-laying whooping cranes (Grus americana) is associated with the failure of small follicles to enter follicular hierarchy.

For endangered species managed ex situ, production of offspring is a key factor to ensure healthy and self-sustaining populations. However, current breeding goals for the whooping crane (Grus americana) are impeded by poor reproduction. Our study sought to better understand mechanisms regulating ovarian function in ex situ managed whooping cranes and the regulatory function of the hypothalamic-pituitary-gonadal (HPG) axis in relation to follicle formation and egg laying. To characterize hormonal regulation of follicular development and ovulation, we collected weekly blood samples from six female whooping cranes during two breeding seasons, for a total of 11 reproductive cycles. The plasma samples were assessed for follicle stimulating hormone, luteinizing hormone, estradiol, and progesterone and the yolk precursors vitellogenin and very low-density lipoprotein. Ultrasonographic examination of the ovary was conducted at the time of blood collection. Preovulatory follicles (>12 mm) were present in laying cycles (n = 6) but absent in non-laying cycles (n = 5). The patterns of plasma hormone and yolk precursor concentrations corresponded to the stage of follicle development. Specifically, gonadotropin and yolk precursor concentrations increased as follicles transitioned from the non-yolky to yolky stage but did not increase further as the follicle advanced to preovulatory and ovulatory stages. Estrogen and progesterone concentrations increased as follicle size increased and reached peak concentrations (P < 0.05) when follicles developed to ovulatory and preovulatory stages, respectively. While overall mean circulating gonadotropin, progesterone, and yolk precursor concentrations did not differ for laying versus non-laying cycles, mean plasma estradiol in laying cycles was significantly higher than that in non-laying cycles. In summary, the findings suggested that disruption of mechanisms regulating follicle recruitment is likely responsible for the oviposition failure of the captive female whooping crane.

Comparison of genomic diversity and structure of sable antelope (Hippotragus niger) in zoos, conservation centers, and private ranches in North America.

As we enter the sixth mass extinction, many species that are no longer self-sustaining in their natural habitat will require ex situ management. Zoos have finite resources for ex situ management, and there is a need for holistic conservation programs between the public and private sector. Ex situ populations of sable antelope, Hippotragus niger, have existed in zoos and privately owned ranches in North America since the 1910s. Unknown founder representation and relatedness has made the genetic management of this species challenging within zoos, while populations on privately owned ranches are managed independently and retain minimal-to-no pedigree history. Consequences of such challenges include an increased risk of inbreeding and a loss of genetic diversity. Here, we developed and applied a customized targeted sequence capture panel based on 5,000 genomewide single-nucleotide polymorphisms to investigate the genomic diversity present in these uniquely managed populations. We genotyped 111 sable antelope: 23 from zoos, 43 from a single conservation center, and 45 from ranches. We found significantly higher genetic diversity and significantly lower inbreeding in herds housed in zoos and conservation centers, when compared to those in privately owned ranches, likely due to genetic-based breeding recommendations implemented in the former populations. Genetic clustering was strong among all three populations, possibly as a result of genetic drift. We propose that the North American ex situ population of sable antelope would benefit from a metapopulation management system, to halt genetic drift, reduce the occurrence of inbreeding, and enable sustainable population sizes to be managed ex situ.

Chromosomal-level genome assembly of the scimitar-horned oryx: Insights into diversity and demography of a species extinct in the wild.

Captive populations provide a valuable insurance against extinctions in the wild. However, they are also vulnerable to the negative impacts of inbreeding, selection and drift. Genetic information is therefore considered a critical aspect of conservation management. Recent developments in sequencing technologies have the potential to improve the outcomes of management programmes; however, the transfer of these approaches to applied conservation has been slow. The scimitar-horned oryx (Oryx dammah) is a North African antelope that has been extinct in the wild since the early 1980s and is the focus of a large-scale and long-term reintroduction project. To enable the selection of suitable founder individuals, facilitate post-release monitoring and improve captive breeding management, comprehensive genomic resources are required. Here, we used 10X Chromium sequencing together with Hi-C contact mapping to develop a chromosomal-level genome assembly for the species. The resulting assembly contained 29 chromosomes with a scaffold N50 of 100.4 Mb, and displayed strong chromosomal synteny with the cattle genome. Using resequencing data from six additional individuals, we demonstrated relatively high genetic diversity in the scimitar-horned oryx compared to other mammals, despite it having experienced a strong founding event in captivity. Additionally, the level of diversity across populations varied according to management strategy. Finally, we uncovered a dynamic demographic history that coincided with periods of climate variation during the Pleistocene. Overall, our study provides a clear example of how genomic data can uncover valuable insights into captive populations and contributes important resources to guide future management decisions of an endangered species.

Breeding Centers, Private Ranches, and Genomics for Creating Sustainable Wildlife Populations.

Human-induced changes to environments are causing species declines. Beyond preserving habitat (in situ), insurance (ex situ) populations are essential to prevent species extinctions. The Conservation Centers for Species Survival (C2S2) is leveraging space of breeding centers and private ranches to produce "source populations"-genetically diverse reservoirs that also support research and reintroductions. The initial focus is on four African antelopes. C2S2 has developed a program, the Source Population Alliance, that emphasizes animals living in spacious, naturalistic conditions in greater numbers than can be accommodated by urban zoos. Simulation modeling demonstrates how herds can rapidly increase population abundance and retain genetic diversity. Advances in genomics and resulting DNA data allow monitoring of genetic diversity and parentage as well as refined decision-making. This approach, neither pure in situ nor ex situ, but rather "sorta situ", is an innovative way of linking public and private sector resources to ensure that endangered species survive.

Whole Genome Sequencing and Re-sequencing of the Sable Antelope (Hippotragus niger): A Resource for Monitoring Diversity in ex Situ and in Situ Populations.

Genome-wide assessment of genetic diversity has the potential to increase the ability to understand admixture, inbreeding, kinship and erosion of genetic diversity affecting both captive (ex situ) and wild (in situ) populations of threatened species. The sable antelope (Hippotragus niger), native to the savannah woodlands of sub-Saharan Africa, is a species that is being managed ex situ in both public (zoo) and private (ranch) collections in the United States. Our objective was to develop whole genome sequence resources that will serve as a foundation for characterizing the genetic status of ex situ populations of sable antelope relative to populations in the wild. Here we report the draft genome assembly of a male sable antelope, a member of the subfamily Hippotraginae (Bovidae, Cetartiodactyla, Mammalia). The 2.596 Gb draft genome consists of 136,528 contigs with an N50 of 45.5 Kbp and 16,927 scaffolds with an N50 of 4.59 Mbp. De novo annotation identified 18,828 protein-coding genes and repetitive sequences encompassing 46.97% of the genome. The discovery of single nucleotide variants (SNVs) was assisted by the re-sequencing of seven additional captive and wild individuals, representing two different subspecies, leading to the identification of 1,987,710 bi-allelic SNVs. Assembly of the mitochondrial genomes revealed that each individual was defined by a unique haplotype and these data were used to infer the mitochondrial gene tree relative to other hippotragine species. The sable antelope genome constitutes a valuable resource for assessing genome-wide diversity and evolutionary potential, thereby facilitating long-term conservation of this charismatic species.

Activin promotes growth and antral cavity expansion in the dog ovarian follicle.

Understanding regulators of folliculogenesis remains limited in the domestic dog (Canis familiaris), which challenges our ability to develop in vitro follicle culture systems for canid genome rescue efforts. Here, we investigated the influence of activin on dog follicle development and survival, oocyte quality, and FSH receptor expression in culture. Preantral (150 - ≤230 µm diameter), early antral (231 - ≤330 µm), and antral (>330-550 µm) stage follicles were encapsulated in a fibrin-alginate hydrogel with 0, 100, or 200 ng/ml rhActivin plus 0, 0.1, 1, or 10 µg/ml FSH for 12 or 21 d of in vitro culture. All follicle groups increased in diameter (P < 0.05) with activin acting synergistically with FSH to improve (P < 0.05) growth and antral cavity expansion (to >630 µm) in early antral and antral cohorts. This complementary effect was not linked to changes in FSHR mRNA expression (P > 0.05). Although not influencing (P > 0.05) follicle survival or transzonal projection (TZP) density in shorter term 12 d culture, activin in the presence of 1 ng/ml FSH maintained TZP density from the 12-21 d interval. Activin also increased oocyte diameter and improved nuclear integrity compared to un-supplemented controls. These results indicate that activin acts synergistically with FSH to promote growth and antral cavity expansion of the dog follicle in vitro, information useful to formulating an effective culture microenvironment for this species.

Effect of sphingosine-1-phosphate on cryopreserved sheep testicular explants cultured in vitro.

Complete spermatogenesis has been achieved in vitro in mouse testicular explants with resulting sperm used to produce pups after Intra Cytoplasm Sperm Injection and Embryo Transfer. In the present study, we evaluated the influence of sphingosine-1-phosphate (S1P) on spermatogenesis of frozen-thawed lamb testis explants in vitro. Thawed testicular pieces were cultured for 12 d on agarose blocks in serum-free growth medium containing 0, 2, 5 or 10 µM S1P. At the end of D6 and D12, some pieces were fixed and processed for histology. Other pieces were processed for RNA isolation and quantitation of proliferation (PCNA, Ki67) and differentiation (PLZF) markers and genes involved in S1P signaling (S1PR1, SGPL1, SGPP1, AKT1 and NFKBIA) by qPCR. Histology revealed an increase (P < 0.05) in seminiferous cord (SC) diameter under all culture conditions, except 5 and 10 µM S1P by D6. In the presence of 5 µM S1P, percentage of gonocytes decreased (P < 0.05) by D6 (control, 24.9% vs. S1P, 10.3%) with a concomitant increase (P < 0.05) in spermatogonia formation (control, 74.4% vs. S1P, 88.1%). S1P induced PCNA or Ki67 expression by D6, whereas PLZF was up-regulated (P < 0.05) by D6 in 2 µM S1P and D12 in 5 & 10 µM S1P. Expression of SGPL1 and SGPP1 increased 4-12-fold in tissues cultured in 10 µM S1P by D12 compared to D12 control. AKT1 and NFKBIA mRNA expression was low (P < 0.05) in 5 and or 10 µM S1P treatments on D6. These results demonstrate that S1P promotes germ cell proliferation during first week of culture and may exert an anti-apoptotic influence on the seminiferous cord in sheep testicular explants in vitro.

Combinations of Growth Factors Regulating LIF/STAT3, WNT, and FGF2 Pathways Sustain Pluripotency-Related Proteins in Cat Embryonic Cells.

Propagation of pluripotent cells from early stage embryos in mouse and human highly depend on leukemia inhibitory factor (LIF)/signal transducer and activator of transcription 3 (STAT3) and FGF2/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways. However, mechanisms for maintaining pluripotency in embryonic stem cells using various combinations of growth factors (targeting LIF or FGF2 pathways) and inhibitors (targeting WNT/GSK3 or FGF2 pathways) still have to be deciphered in other models, including the domestic cat. Our objective was to understand how cytokines influence pluripotency in the cat inner cell mass (ICM) outgrowths. Cat ICM was isolated from in vitro-produced embryos and outgrowths were cultured for up to 6 days with single or combined cytokines. Cell proliferation was enhanced with almost all single growth factors and cytokine combinations. Based on gene expression and presence of NANOG, POU5F1, and Sex-determining region Y box 2 (SOX2) as cell state markers, single growth factors could not maintain similar levels in outgrowths as in the original ICMs, which is different from the response in mouse and human. In our conditions, cytokine combinations involving LIF, GSK3 inhibitor, and MEK inhibitor resulted in the most robust expression levels and allowed single-cell dissociation and propagation. However, further characterization of embryonic cells derived from ICM indicated that the pluripotent state was not fully preserved. The absence of detectable transcripts for BMP2-receptor and SMAD4, and very low levels of LIF-receptor and STAT3 in the cat ICM indicated that pluripotency regulatory machinery appear to be different in the cat from the predominant mouse and human models.

Retinoic acid promotes in vitro follicle activation in the cat ovary by regulating expression of matrix metalloproteinase 9.

Retinoic acid (RA) facilitates tissue morphogenesis by regulating matrix matalloproteinase (MMPs) expression. Our objective was to examine the influence of RA on in vitro development of follicles enclosed within domestic cat ovarian tissues. Ovarian cortices from 9 prepubertal and 13 adult cats were incubated for 7 d in medium containing 0 (control), 1 or 5 µM RA and then analyzed for viability. Cortices from additional three animals of each age group were cultured in the same condition and follicle morphology, stage and size were histologically evaluated. In a separate study, cortices from 14 donors (7 prepubertal; 7 adult cats) were incubated in 0 or 5 µM RA for 7 d and assessed for (1) MMP1, 2, 3, 7, 9 and TIMP1 expression by qPCR and (2) protein expression of MMP9 by immunohistochemistry. Donor age did not influence follicle response to RA. Collective data from both age groups revealed that percentages of primordial follicles in 5 µM RA treatment were lower (P < 0.05; 40.5 ± 4.5%) than in fresh cortices (66.7 ± 5.3%) or controls (60.1 ± 4.0%) with 1 µM-RA treatment producing intermediate (56.3 ± 4.0%) results. Proportion of primary follicles in 5 µM RA (21.7 ± 3.3%) was higher than in fresh cortices (4.9 ± 2.9%) and controls (9.0 ± 2.8%) with 1 µM-RA treatment producing an intermediate value (13.8 ± 2.0%). Furthermore, proportion of secondary follicles increased after 7 d in the presence of 5 µM RA (9.5 ± 2.7%) compared to other groups (fresh, 1.9 ± 0.8%; control, 2.6 ± 1.1%; 1 µM RA, 2.5 ± 0.2%). MMP9 transcript and protein were upregulated, whereas MMP7 mRNA was suppressed by 5 µM-RA treatment compared to fresh counterparts. RA did not impact MMP1, 2, 3, 13 or TIMP1 expression. In summary, RA activated cat primordial follicle growth likely via a mechanism related to upregulation of MMP9 and down-regulation of MMP7 transcripts.

Insulin promotes preantral follicle growth and antrum formation through temporal expression of genes regulating steroidogenesis and water transport in the cat.

The aims of the present study were to determine the effects of insulin, invitro, on: (1) the viability and growth of domestic cat ovarian follicles; (2) mRNA expression of genes regulating steroidogenesis (cytochrome P450 family 17 subfamily, A polypeptide 1 (Cyp17a1), cytochrome P450 family 19 subfamily, A polypeptide 1 (Cyp19a1) and steroidogenic acute regulatory protein (Star)) and water transport (aquaporins (AQPs) Aqp1, Aqp3, Aqp7, Aqp9); and (3) steroid production (17ß-oestradiol (E2), progesterone (P4), androstenedione (A4)). Cat secondary follicles were isolated from ovarian cortices and cultured in 0 (Control), 1 or 10µgmL-1 insulin for 14 days (Day 0=culture onset). Follicle and oocyte viability (based on neutral red staining), diameter and antrum formation were assessed every 72h and at the end of incubation (Day 14). Expression of steroidogenic and water transport genes was evaluated on Days 0, 6 and 12, and E2, P4 and A4 concentrations in the culture medium were determined on Day 12. By Day 14, 1 and 10µgmL-1 insulin had significantly promoted (P<0.05) both antrum formation in a mean (±s.e.m.) 26.9±9.0% and 78.0±10.0% of follicles respectively, and follicle growth (diameter 151.4±4.5 and 169.9±10.5µm respectively) compared with Control (antrum formation in 3.3±3.3% of follicles and follicle diameter 129.1±6.6µm). High insulin (10µgmL-1) treatment increased follicle viability compared with Control (86.0±9.8% vs 38.1±10.9% respectively; P<0.05). However, insulin had no beneficial effect (P>0.05) on oocyte diameter. Cyp17a1 expression on Days 6 and 12 was higher (P<0.05) in follicles cultured in the low (1µgmL-1) compared with high (10µgmL-1) insulin treatment, with no significant difference between low or high insulin vs Control groups. Star expression was higher (P<0.01) in the low insulin compared with Control group on Day 6, but Star was undetectable in the high insulin group by Day 12. Compared with high insulin, low insulin increased (P<0.05) Aqp1 expression on Day 6, but there were no significant differences between these two groups on Day 12. In contrast, high insulin decreased (P<0.05) Aqp9 transcript levels compared with Control. Only P4 production was affected by insulin, with P4 concentrations in the medium being higher (P<0.05) in the low compared with high insulin and Control groups. In summary, the findings indicate that insulin promotes cat ovarian follicle growth and survival invitro, including enhanced antrum formation, with the likely mechanism involving temporal expression of Cyp17a1, Star and Aqp9 genes.

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.

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.

Follicular size and stage and gonadotropin concentration affect alginate-encapsulated in vitro growth and survival of pre- and early antral dog follicles.

Understanding stage-specific requirements of mammalian folliculogenesis is limited in the domestic dog. The present study examined the effects of two potential regulators of dog follicle growth and survival in vitro, namely the original stage of the follicle (i.e. preantral (≤230µm diameter) vs early antral (diameter from >230 to ≤330µm) and FSH and/or LH concentrations. After isolation and alginate encapsulation, follicles were cultured in 0, 1, 10 or 100µgmL-1 FSH and 0, 1 or 10ngmL-1 LH for 20 days. Regardless of stage, FSH promoted growth, but LH did the same only in the absence of FSH. Production of 17ß-oestradiol and progesterone was detectable, indicating theca cell activity. The greatest growth occurred in preantral (mean (± s.d.) 61.4±25.9%) versus antral (42.6±20.3%) follicles, but neither developmental stage nor gonadotropin affected survival. Antrum detection was minimal due, in part, to antral collapse, and oocytes exhibited an increasingly pale appearance and chromatin degeneration over time. The results demonstrate that pre- and early antral stage dog follicles encapsulated in alginate grow significantly in vitro. However, because FSH and LH alone or in combination fail to promote antrum development, the next step is identifying factors that enhance antral expansion.

Stem cell factor promotes in vitro ovarian follicle development in the domestic cat by upregulating c-kit mRNA expression and stimulating the phosphatidylinositol 3-kinase/AKT pathway.

In the present study we examined the effects of stem cell factor (SCF; 50 vs 100ngmL-1) alone or in combination with epidermal growth factor (EGF; 100ngmL-1) on: (1) the in vitro viability and growth of cat follicles within ovarian cortices; (2) phosphatidylinositol 3-kinase (PI3K)/AKT and mitogen-activated protein kinase (MAPK) phosphorylation; and (3) c-kit and FSH receptor (FSHr) mRNA expression. At 100ngmL-1, SCF increased (P≤0.05) the percentage and size of secondary follicles after 14 days of in vitro culture and sustained AKT phosphorylation after 3 days incubation. EGF suppressed this beneficial effect and reduced (P≤0.05) the percentage of structurally normal follicles and FSHr expression when combined with 100ngmL-1 SCF. Expression of c-kit mRNA was higher (P≤0.05) in the presence of 100ngmL-1 SCF compared with fresh follicles and cohorts cultured under other conditions. A c-kit inhibitor suppressed follicle growth and reduced AKT phosphorylation. Collectively, the results demonstrate that SCF promotes cat follicle development by upregulating c-kit mRNA expression and AKT phosphorylation. EGF suppresses the stimulating effect of SCF, leading to downregulation of FSHr expression.

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.

Incidence of methylated histones H3K4 and H3K79 in cat germinal vesicles is regulated by specific nuclear factors at the acquisition of developmental competence during the folliculogenesis.

PURPOSE: This study aims to characterize the regulations of histone methylations, key epigenetic markers of oocyte competence, in germinal vesicle (GV) from different follicles (preantral, early, small, or large antral stage) using the domestic cat model. METHODS: In Experiment 1, the incidence of H3K4me3 or H3K79me2 was determined in GVs from the diverse follicle stages directly or after exposure to (1) a methyltransferase inhibitor, (2) sonication to fracture the cytoplasmic membranes and wash away the cytoplasmic content, or (3) methyltransferase inhibitor followed by sonication. In Experiment 2, the presence and maintenance of nuclear methyltransferases SMYD3 and DOT1L (regulating H3K4me3 and H3K79me2, respectively) was characterized in separate GV stages before and after sonication. Functionality of GVs from the various follicle stages (with or without transient isolation from the cytoplasm) then was assessed in Experiment 3 by transfer into recipient competent oocytes. RESULTS: The incidence of histones H3K4me3 and H3K79me2 within the GV were influenced by the cytoplasmic environment at all stages except at the transition to the early antral stage where nuclear regulating factors appeared to be mainly involved. The methyltransferase SMYD3 and DOT1L also appeared tightly bound to the nucleus at that transition. Interestingly, oocytes reconstructed with a GV isolated from the cytoplasm for a prolonged period had the capacity to form an embryo after fertilization which proved that communication between the donor GV and the host cytoplasm (likely including the regulation of epigenetic factors) could be restored. CONCLUSIONS: Histone methylation apparently becomes regulated by specific nuclear factors at the acquisition of competence during the folliculogenesis and does not seem to be disrupted by prolonged isolation from the surrounding cytoplasm.

Genomic legacy of the African cheetah, Acinonyx jubatus.

BACKGROUND: Patterns of genetic and genomic variance are informative in inferring population history for human, model species and endangered populations. RESULTS: Here the genome sequence of wild-born African cheetahs reveals extreme genomic depletion in SNV incidence, SNV density, SNVs of coding genes, MHC class I and II genes, and mitochondrial DNA SNVs. Cheetah genomes are on average 95 % homozygous compared to the genomes of the outbred domestic cat (24.08 % homozygous), Virunga Mountain Gorilla (78.12 %), inbred Abyssinian cat (62.63 %), Tasmanian devil, domestic dog and other mammalian species. Demographic estimators impute two ancestral population bottlenecks: one >100,000 years ago coincident with cheetah migrations out of the Americas and into Eurasia and Africa, and a second 11,084-12,589 years ago in Africa coincident with late Pleistocene large mammal extinctions. MHC class I gene loss and dramatic reduction in functional diversity of MHC genes would explain why cheetahs ablate skin graft rejection among unrelated individuals. Significant excess of non-synonymous mutations in AKAP4 (p<0.02), a gene mediating spermatozoon development, indicates cheetah fixation of five function-damaging amino acid variants distinct from AKAP4 homologues of other Felidae or mammals; AKAP4 dysfunction may cause the cheetah's extremely high (>80 %) pleiomorphic sperm. CONCLUSIONS: The study provides an unprecedented genomic perspective for the rare cheetah, with potential relevance to the species' natural history, physiological adaptations and unique reproductive disposition.

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.