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Contents Innovations are emerging from the growing field of fertility preservation for humans and laboratory animals that are relevant to protecting and propagating valuable domestic and wild carnivores. These extend beyond the 'classical' approaches associated with sperm, oocyte and embryo freezing to include gonadal tissue preservation combined with in vitro culture or xenografting, all of which have potential for rescuing vast amounts of unused and wasted germplasm. Here, we review approaches under development and predicted to have applied value within the next decade, including the following: (i) direct use of early-stage gametes for in vitro fertilization; (ii) generation of more mature gametes from gonadal tissue or stem cells; (iii) simplification, enhanced safety and efficacy of cryopreservation methods; and (iv) biostabilization of living cells and tissues at ambient temperatures. We believe that all of these fertility preservation strategies will offer knowledge and tools to better manage carnivores that serve as human companions, valuable biomedical models or require assistance to reverse endangerment. [ABSTRACT FROM AUTHOR]

Contents The aim of this study is to produce live kittens from oocytes fertilized by intracytoplasmic sperm injection ( ICSI) with frozen/thawed testicular spermatozoa. Spermatozoa were collected from thawed testicular tissue and subsequently injected into in vitro matured cat oocytes. At 24 h post- ICSI, presumptive zygotes/cleaved embryos were treated with 10 μ m forskolin for 24 h to reduce intracellular lipid content of embryos (delipidation). At 48 h after oocyte injection, cleaved embryos (2- to 8-cell stage) were frozen in 10% (v/v) ethylene glycol-based medium by a slow controlled rate method and stored in liquid nitrogen. To evaluate in vitro and in vivo developmental competence, frozen embryos were thawed and then cultured for 6 days (n = 155) or cultured for 2 h before transferred (n = 209) to hormonal (equine chorionic gonadotropin/h CG)-treated cat recipients. Cleavage frequency at day 2 after ICSI with frozen/thawed testicular spermatozoa was ~30%. The percentages of frozen/thawed embryos that developed to morula and blastocyst stage (on day 3 and day 6 of in vitro culture, respectively) were significantly lower than that of fresh ICSI embryos (22.6 vs 45.2% and 21.3 vs 38.7%, respectively; p < 0.05). However, no difference was found in the number of blastomeres between frozen/thawed (242.5 ± 43.1) and fresh (320.2 ± 28.1) blastocysts. Three of seven cat recipients were pregnant and one pregnant cat delivered two healthy kittens. This is the first report of the birth of kittens after the transfer of frozen-thawed embryos produced by ICSI with frozen/thawed testicular sperm. [ABSTRACT FROM AUTHOR]

Ovarian tissue contains large pools of immature oocytes enclosed in primordial follicles, making it an attractive target for fertility preservation in female cancer patients, livestock and wild species. Compared to cryopreservation, desiccation and long-term storage of samples at supra-zero temperatures (using strategies inspired from small organisms to resist extreme environments) would be more cost-effective and convenient. The objective of the study was to characterize the influence of microwave-assisted dehydration on structural and functional properties of living ovarian tissues. While this method allows preservation of single cells (cat oocytes and sperm cells so far) using trehalose as the xeroprotectant, it has not been developed for multicellular tissues yet. Ovarian cortex biopsies were reversibly permeabilized, exposed to various concentrations of trehalose, and dried for different times using a commercial microwave under thermal control. Effective dehydration of samples along with proper trehalose retention were reached within 30 min of microwave drying. Importantly, the process did not affect morphology and DNA integrity of follicles or stromal cells. Moreover, transcriptional activity and survival of follicles were partially maintained following 10 min of drying, which already was compatible with storage at non-cryogenic temperatures. Present data provide critical foundation to develop dry-preservation techniques for long-term storage of living multicellular tissues., Competing Interests: The authors have declared that no competing interests exist.

The objective was to evaluate different permeating cryoprotectants to vitrify testicular tissue biopsies from adult collared peccaries. Five pairs of testicles were dissected into fragments (9 mm³) that were allocated to non-vitrified (control) and vitrified groups using a solid-surface method following exposure to different cryoprotectants (3.0 M dimethyl sulfoxide (DMSO), 3.0 M ethylene glycol (EG) or 1.5 M DMSO + 1.5 M EG). After warming, samples were evaluated for histomorphology, ultrastructure, viability, and proliferative capacity potential. The appropriate conservation of the ultrastructural organization of the seminiferous tubule in terms of lumen presence and cell junctions was only observed at the use of DMSO/EG combination. Regardless of the cryoprotectant, the vitrification effectively preserved cell nuclear visualization and condensation similarly as observed at the non-vitrified group. Moreover, DMSO/EG combination provided a better preservation of basal membranes of seminiferous tubules than DMSO (P < 0.05). The occurrence of cell swelling was more evident in the use of DMSO than EG (P < 0.05), but both isolate cryoprotectants were similar to the DMSO/EG combination. Only the DMSO/EG combination maintained the proliferative capacity potential for spermatogonia (3.69 NORs/cell) and Sertoli cell (3.19 NORs/cell) similar to controls (3.46 and 3.31 NORS/cell, respectively). Moreover, ~40% cell viability was found after vitrification independent of cryoprotectant. In conclusion, DMSO/EG in combination is better than DMSO or EG alone for SSV of testicular tissue biopsies from adult collared peccaries., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Understanding cellular and molecular damages in oocytes during exposure to extreme conditions is essential to optimize long-term fertility preservation approaches. Using the domestic cat (Felis catus) model, we are developing drying techniques for oocytes' germinal vesicles (GVs) as a more economical alternative to cryopreservation. The objective of the study was to characterize the influence of desiccation on nuclear envelope conformation, chromatin configuration, and the relative fluorescent intensities of histone H3 trimethylation at lysine 4 (H3K4me3) and at lysine 9 (H3K9me3) compared to vitrification. Results showed that higher proportions of dried/rehydrated GVs maintained normal nuclear envelope conformation and chromatin configuration than vitrified/warmed counterparts. Both preservation methods had a similar influence on epigenetic patterns, lowering H3K4me3 intensity to under 40% while maintaining H3K9me3 levels. Further analysis revealed that the decrease of H3K4me3 intensity mainly occurred during microwave dehydration and subsequent rehydration, whereas sample processing (permeabilization and trehalose exposure) or storage did not significantly affect the epigenetic marker. Moreover, rehydration either directly or stepwise with trehalose solutions did not influence the outcome. This is the first report demonstrating that the incidence of GV damages is lower after desiccation/rehydration than vitrification/warming., (© 2019 Wiley-Liss, Inc., A Wiley Company.)

Incomplete transgene-silencing remains a challenge in the generation of induced pluripotent stem cells (iPSC) in felids-a critical family in biomedical and biodiversity conservation science. In this study doxycycline-inducible transgenes (NANOG, POU5F1, SOX2, KLF4, and cMYC) were used to reprogram cat fetal fibroblasts with the objective of obtaining iPSC with fully silenced transgenes. Colony formation was slower (14 vs. 8 days) and at lower efficiency than mouse embryonic fibroblasts (0.002% vs. 0.02% of seeded cells). Alkaline-phosphatase positive colonies were grown on feeder cells plus LIF and GSK3, MEK, and ROCK inhibitors. Cells could be passaged singly and transgene expression was silenced at passage 3 (P3) after doxycycline removal at P2. NANOG, POU5F1, and SOX2 were expressed at P3, P6, and P10, although at lower immunostaining intensities than in cat inner cell masses (ICM). Transcripts related to pluripotency (NANOG, POU5F1, SOX2, KLF4, cMYC, and REX1) and differentiation (FGF5, TBXT, GATA6, SOX17, FOXF1, PAX6, and SOX1) were assessed by a reverse transcription-quantitative polymerase chain reaction in iPSC and embryoid bodies. The immunostaining patterns, relatively low levels of NANOG and REX1 in comparison to ICM along with the expression of TBXT (mesoderm) suggested that cells were a mix of reprogrammed pluripotent and differentiating cells., (© 2019 Wiley Periodicals, Inc.)

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., (Published by Elsevier Inc.)

The establishment of protocols for the control of the ovarian function of collared peccaries is recommended for the development of assisted reproductive techniques. The goals were to (1) compare a gonadotropin combination with prostaglandin analogue to synchronize timing of onset of estrus among animals, and (2) elucidate the effects of the most desirable protocol for performing an artificial insemination study and macroscopic evaluation of the ovaries. Three of five females treated with a double administration of 120 μg prostaglandin (cloprostenol) at a 9-day interval expressed symptoms of estrus 9 days after the second injection. One female presented estrus after 6 days, whereas other did not respond to the treatment. All females (5/5) treated with a single dose containing 400 IU eCG and 200 IU hCG manifested estrus 6 days after the hormone injection. In a second experiment, ten females that were estrous synchronized using eCG/hCG, were artificially inseminated with fresh semen and monitored for pregnancy every 30 days. Although there was no detection of fetuses by ultrasonic examination, seven females (7/10) had greater than basal progesterone values for 60 days after the treatments were imposed. Ovaries from two females treated with eCG/hCG were collected 6 days post-injection. There was confirmation of an ovarian stimulation as a result of the presence of 88 and 25 antral follicles, as well as three and eight hemorrhagic structures in ovaries of each female, respectively. It, therefore, is proposed that eCG/hCG can be used as an effective treatment for estrous synchronization in collared peccaries., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The hypothalamic neuropeptide RFRP3 can suppress hypothalamic GnRH neuron activation and inhibit gonadotropin release from the anterior pituitary. RFRP3 is also produced locally in the ovary and can inhibit steroidogenesis and follicle development in many vertebrates. However, almost nothing is known about the presence and regulatory action of RFRP3 in gonads of any carnivore species. Such knowledge is important for developing captive breeding programs for endangered carnivores and for inhibiting reproduction in feral species. Using the domestic cat as a model, our objectives were to (1) demonstrate the expression of feline RFRP3 (fRFRP3) and its receptor in the cat ovary and (2) assess the influence of fRFRP3 on ovarian follicle integrity, survival, and steroidogenesis in vitro . We first confirmed that fRFRP3 and its receptors (NPFFR1 and NPFFR2) were expressed in cat ovaries by sequencing PCR products from ovarian RNA. We then isolated and cultured preantral ovarian follicles in the presence of 10 or 1 µM fRFRP3 + FSH (1 µg/mL). We recorded the percentage of morphologically viable follicles (basal lamina integrity) over 8 days and calculated percentage survival of follicles on Day 8 (using fluorescent markers for cell survival and death). Last, we quantified progesterone accumulation in media. 10 µM fRFRP3 had no observable effect on viability, survival, or steroid production compared to follicles exposed to only FSH. However, 1 µM fRFRP3 decreased the percentage of morphologically viable follicles and the percentage of surviving follicles on Day 8. At the same time, 1 µM fRFRP3 increased the accumulation of progesterone in media. Our study shows, for the first time, direct action of RFRP3 on the follicle as a functional unit, and it is the first in a carnivore species. More broadly, our results support a conserved, inhibitory action of RFRP3 on ovarian follicle development and underscore the importance of comparative functional studies., Competing Interests: The authors declare there are no competing interests. George E. Bentley is an Academic Editor for PeerJ.

The objective of this study was to determine whether preantral follicles cultured in vitro for 7 days within ovine ovarian cortical strips could be isolated at the secondary follicles (SF) and grown until antral stage during an additional 6 days period of in vitro culture in the presence of aqueous extract of Justicia insularis. Fresh ovarian fragments from 16 adult sheep were fixed for histological analysis (Control 1) or in vitro cultured individually in α-MEM + supplemented with 0.3 mg/ml J. insularis (Step 1) for 7 days. Part of the fragments then were fixed for histological analysis (in vitro culture group). Remaining fragments were exposed stepwise to increasing trehalose concentrations before immediate isolation of SF and viability assessment (Control 2) or after 6 days of culture in α-MEM ++ supplemented with 0.3 mg/ml J. insularis (Step 2). In Step 1, percentage of follicular activation was 80%. In Step 2, a significant increase (p < 0.05) in follicular diameter and antrum formation within 6 days in vitro culture of isolated follicles was achieved. The total antioxidant capacity from both steps significantly increase (p < 0.05) from day 2 to day 6. Confocal analysis of oocytes showed 57.14% oocytes with homogeneous distribution and 42.86% with peri-cortical distribution. In conclusion, SF can be successfully isolated from sheep ovarian cortex after 7 days of culture and are capable of surviving and forming an antral cavity if cultured in vitro for an additional 6 days in the presence of 0.3 mg/ml J. insularis., (© 2019 Blackwell Verlag GmbH.)

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., (Copyright © 2019 Koepfli et al.)

Plastic polymers can be combined with additives that modify physical properties and stability of the material. However, the biocompatibility of those additives is not well known. The objective of the study was to characterize the impact of zinc stearate-a common additive-through the development of a novel three-dimensional (3-D) in vitro platform with endometrial cells from domestic cats. Epithelial and stromal cells from adult uteri were isolated and cultured in medium supplemented with 3% Matrigel for two weeks in plastic tissue culture dishes that had been identified as polystyrene with and without zinc stearate by Raman, FTIR, and X-ray fluorescence spectroscopies. Three-dimensional cell structures that were obtained were measured and categorized by shape. Cell viability, proliferation, differentiation, organization, and apoptosis then were assessed by immuno-staining. Results indicated that zinc stearate did not affect 3-D endometrial cell structure morphology, viability, or cellular composition. This first study of a new in vitro platform will be useful for studies testing the influence of other additives, drugs, or exogenous hormones., Competing Interests: The authors have declared that no competing interests exist.

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.

The previous edition of this book mainly provided a snapshot of the state of the art in terms of species-specific reproductive biology and emerging technologies. The influence of environmental changes on reproductive fitness was introduced but not fully explored. The objectives of this second edition were to (1) emphasize the need for holistic and global efforts to understand and sustain reproduction in a constantly changing environment and (2) provide more knowledge in the reproductive physiology of different taxa. The first section of the book is dedicated to survival and adaptation of species in a changing environment (including chapters on environmental impacts in different taxa, as well as the role of microbiomes). The second section focuses on progress in understanding, assisting or even suppressing reproduction in wild species, keeping in mind the influence of environmental factors as well. It contains chapters from the previous edition that were updated (reproduction in elephants, koalas, marsupials, amphibians, and corals), new chapters on species such as sharks and rays, and contributions about the increasing role of reproductive manipulations, such as assisted reproduction and contraception. While the present book emphasizes the overarching issue of environmental impacts on reproduction (resulting in infertility, subfecundity, or fitness), it also highlights the challenges of maintaining wild species in captivity, including those associated with ensuring good welfare. Captive environments can influence reproduction in a multitude of ways, some unexpected, such as the selection of unwanted genetic traits, an essential dimension to be considered to ensure the success of conservation breeding programs. Lastly, new approaches, such as the use of allostatic load indexes and reproductive microbiome analyses also will be closely examined for the first time in rare and endangered species to address conservation issues.

Communities of microbes have coevolved in animal organisms and are found in almost every part of the body. Compositions of those communities (microbiota) as well as their genomes and genes (microbiomes) are critical for functional regulations of the body organ systems-the digestive or 'gut' microbiome being the most described so far. Based on extensive research in humans, microbiomes in the reproductive tract may play a role in reproductive functions and pregnancy. However, in wild animal species, those microbiomes have been poorly studied, and as a result, little is known about their involvement in fertility or parental/offspring health. This emerging research area is highly relevant to conservation biology from captive breeding management to successful reintroduction or maintenance of wild populations. The objective of this chapter is to review current knowledge about reproductive microbiomes in healthy wild animal species. While recognizing the current technical limits of microbial identification in all animal species, we also explore the link between microbial communities (within female or male reproductive systems) and fertility, from conception to birth outcome. In addition, it is critical to understanding how reproductive microbiomes are affected by environmental factors (including captivity, contact with other individuals, or changes in the ecosystem) to optimize conservation efforts. Thus, reproductive microbiomes represent a novel dimension in conservation biology that will likely gain importance in the future.

While many of the traditional scientific disciplines have developed over centuries, animal conservation is a relative newcomer. It relies on multiple specialties with different levels of expertise that, eventually, generate vast amounts of data. More specifically, conservation physiology is an emerging area that can be defined as 'an integrative scientific discipline applying physiological concepts, tools, and knowledge to characterizing biological diversity and its ecological implications; understanding and predicting how organisms, populations, and ecosystems respond to environmental change and stressors; and solving conservation problems across the broad range of taxa, including microbes, plants, and animals' (Cooke et al. 2013). Reproductive biology is more focused, given that it mainly deals with the physiology underlying the production of gametes, embryos, and offspring, and the many associated processes that control these events. However, it is integrated into the different components of conservation physiology. In bringing together the various contributors for this book, the editors' purpose was to provide readers with a new perspective about the complexity behind reproduction and the role it plays in species conservation. Chapters highlight the diversity of reproductive mechanisms across taxa, and provide insight into how they may have evolved, and likely will continue to evolve in a changing environment. To conservation physiologists, the hope is that this information will be applied to sustain populations in both natural habitats and managed facilities. Ultimately, a major goal is to forecast and mitigate negative impacts of environmental change or anthropogenic pressures on animal fitness, which will only follow once we have acquired a solid understanding of reproductive processes.

Captive breeding of clouded leopards ( Neofelis nebulosa) is challenging because of mating incompatibility, high incidence of teratospermia in males, and inconsistent ovulation patterns in females. Assisted reproductive techniques, therefore, are necessary to overcome these issues and maintain the genetic diversity in the captive population. The objective was to use laparoscopic oviductal artificial insemination (AI) to breed genetically valuable females ( n = 4; aged 4.5-5 yr) that were unsuccessfully paired. Fecal hormone metabolites (estrogen and progesterone) were extracted and measured by enzyme immunoassay for monitoring of ovarian activity 45 days before and 65 days after laparoscopic AI. For timed insemination, females were injected with 200 IU equine chorionic gonadotropin and 1,000 IU porcine luteinizing hormone (pLH) at the 82-hr interval. Ovarian assessment was performed by laparoscopy 44 hr after pLH administration. One nulliparous female out of four presented two ovulation sites on each ovary. The single female that had ovulated was inseminated with chilled semen collected from two males (8 × 106 and 2.7 × 106 motile spermatozoa, respectively, in each oviduct). A significant increase in fecal progesterone concentrations was observed after AI with a concentration peak (500 μg/g dry feces) detected on day 24 after pLH injection, which was then sustained for more than 45 days after the pLH injection. The delivery of two cubs occurred on day 92 after pLH. Microsatellite marker analysis determined that both cubs were sired by the same male. This is the first report of a successful oviductal AI in the clouded leopard. [ABSTRACT FROM AUTHOR]

Contents Clouded leopards ( Neofelis nebulosa) produced high proportion of abnormal spermatozoa (mainly tail defects) that can limit sperm movement and conception. The study aimed to better identify the origin of those defects using a demembranation approach. Ejaculates (1-2 ejaculations/male; n = 9) were allocated to simple washing (control; resulting in 11.7% ± 1.9% coiled tails) and processed through colloid centrifugation to reduce the number of sperm with tail defects (treatment, resulting in 5.9% ± 0.9% coiled tails). Aliquots of semen were incubated in hypo-osmotic solution ( HOS, 60 mOsm fructose solution) containing 5 mM dithiothreitol ( DTT) (a reducing agent) to prevent oxidation of sperm membrane. Thereafter, 20% Triton X-100 ( TX) (a detergent) was added to the HOS/ DTT-treated samples. After HOS/ DTT incubation, the control samples and sperm-selected samples presented 73.4% ± 3.1% and 73.9% ± 2.5% swollen sperm (bent and coiled) indicating membrane intact, respectively. Most of the coiled tail in the raw ejaculates could not be opened by TX indicating that the cause of coiled sperm tails may be from testicular origin. The proportion of sperm with tightly coiled tail tended to be lower in the sperm-selected group than control group (18.8% ± 3.8% and 26.5% ± 3.4%; p = .1), whereas the sperm opened up by TX tended to be higher in the sperm-selected group (53.6% ± 10.4% and 21.1% ± 7.9%; p = .06). The results indicated TX was able to uncoil half of the tightly coiled sperm in the semen undergone preparation. In conclusion, the coiled sperm in the clouded leopard semen were likely not a defect of sperm volume regulation during post-ejaculate (osmotic swelling) but pre-ejaculate origin. Semen preparation demonstrated its ability to lessen the primary sperm defects and selected spermatozoa that were prone to be mitigated after demembranation. [ABSTRACT FROM AUTHOR]

The current gold standard for sperm preservation is storage at cryogenic temperatures. Dry preservation is an attractive alternative, eliminating the need for ultralow temperatures, reducing storage maintenance costs, and providing logistical flexibility for shipping. Many seeds and anhydrobiotic organisms are able to survive extended periods in a dry state through the accumulation of intracellular sugars and other osmolytes and are capable of returning to normal physiology postrehydration. Using techniques inspired by nature's adaptations, attempts have been made to dehydrate and dry preserve spermatozoa from a variety of species. Most of the anhydrous preservation research performed to date has focused on mouse spermatozoa, with only a small number of studies in nonrodent mammalian species. There is a significant difference between sperm function in rodent and nonrodent mammalian species with respect to centrosomal inheritance. Studies focused on reproductive technologies have demonstrated that in nonrodent species, the centrosome must be preserved to maintain sperm function as the spermatozoon centrosome contributes the dominant nucleating seed, consisting of the proximal centriole surrounded by pericentriolar components, onto which the oocyte's centrosomal material is assembled. Preservation techniques used for mouse sperm may therefore not necessarily be applicable to nonrodent spermatozoa. The range of technologies used to dehydrate sperm and the effect of processing and storage conditions on fertilization and embryogenesis using dried sperm are reviewed in the context of reproductive physiology and cellular morphology in different species. [ABSTRACT FROM AUTHOR]

Contents Different culture conditions have been used to produce domestic cat embryos. As part of the in vitro procedures, the medium composition significantly affects the quality of the embryo development also. Quality assessments based on cleavage kinetics and blastomere symmetry are useful, but embryos also can differ in their relative gene expression patterns despite similar morphological characteristics. The aim of this study was to compare cat embryos produced with two different in vitro culture systems routinely used in two different laboratories [Smithsonian Conservation Biology Institute, Washington D.C., USA (SCBI) and Leibniz Institute for Zoo and Wildlife Research, Berlin, Germany (IZW)]. Specifically, relative mRNA expression patterns of critical genes for pre-implantation embryo development were assessed in both conditions. Embryos were produced in parallel in both culture systems by IVF using frozen-thawed ejaculated semen in the United States and fresh epididymal sperm in Germany. Success of embryo development in vitro was recorded as well as relative mRNA abundances [DNA methyltransferases 1 and 3A (DNMT1, DNMT3A), gap junction protein alpha 1 (GJA1), octamer-binding transcription factor 4 [OCT4], insulin-like growth factors 1 and 2 receptors (IGF1R, IGF2R), beta-actin (ACTB)] in pools of days 4-5 morulae by semi-quantitative RT-PCR assay. Percentages of cleaved embryos were similar (p > 0.05) between both culture systems, regardless of the location. OCT4 mRNA abundance was higher (p < 0.05) in embryos derived in the SCBI culture system compared with those from the IZW system when epididymal sperm was used for IVF. No clear correlation between the expression pattern and the culture system could be found for all other genes. It is suggested that OCT4 expression might be affected by the media composition in some conditions and can be the indicator of a better embryo quality. [ABSTRACT FROM AUTHOR]

Contents The objective of this study was to assess and compare the quality of cat blastocysts produced in vitro using commercial blastocyst growth media supplemented with different sources of proteins (serum protein substitute from in vitro maturation through embryo development vs 4 mg/ml of bovine serum albumin for maturation and 5% foetal calf serum for fertilization and embryo development). Impact was specifically examined on the proportion of blastocyst formation, total number of blastomeres, proportion of inner cell mass and expression of pluripotency marker proteins NANOG and OCT-4. Blastocyst formation per total cleaved embryos was similar (p > 0.05) regardless of the protein supplementation. There were no differences (p > 0.05) between culture conditions regarding average number of blastomeres and proportion of inner cell mass in each embryo. Presence of OCT-4 protein was detected in nuclei of both trophectoderm and inner cell mass region, with a stronger signal in the latter regardless of the culture medium. NANOG protein also was present in the inner cell mass regardless of the in vitro culture condition. We therefore demonstrated that serum protein substitute was as good as semi-defined protein sources for the production of good-quality blastocysts and embryonic stem cells. In addition, a single defined medium could be successfully used for cat oocyte maturation, in vitro fertilization and embryo development. [ABSTRACT FROM AUTHOR]

Contents This study identified specific changes in histone lysine methylation patterns of the feline germinal vesicle ( GV) during pre-antral-to-antral follicle transition, the latter being a key interval for competence acquisition. Oocytes from adult cats were isolated from pre-antral, early (≤0.5 mm diameter), small (0.6-1 mm) or large (1-3.5 mm) antral follicles and immuno-stained with anti-histones H3 trimethylated at lysine 9 ( H3 K9me3), lysine 4 ( H3 K4me3), lysine 27 ( H3 K27me3) or H3 dimethylated at lysine 79 ( H3 K79me2). The vast majority of oocytes (range, 72.2-85.4%; p > 0.05) contained a GV with H3 K9me3 or H3 K27me3, regardless of follicular stage/size. However, the proportion of GVs with H3 K4me3 or H3 K79me2 was higher in early antral follicles (42.6%; p < 0.05) compared with other stages (range, 12.1-15.2%). Therefore, H3 K4me3 and H3 K79me2 (both known to be associated with selective gene activation) appear to be reliable markers of onset of GV competence during the pre-antral-to-antral transition phase. By contrast, H3 K9me3 and H3 K27me3 (both known to be related to selective gene repression) seem more linked to expression patterns during the GV stage and are less useful indicators during the entire folliculogenesis interval. [ABSTRACT FROM AUTHOR]

Contents The culture of ovarian follicles is an important tool for understanding the mechanisms controlling follicle development and differentiation of the oocyte. The benefit of recovering meiotically and developmentally competent oocytes from early stage follicles (primordial, primary, pre-antral and early antral) also would be significant, ranging from rescue of genomes from endangered species to preserving fertility in women facing cancer treatments. This research field is at an early stage of scientific discovery. To-date, live offspring from cultured primordial follicles that produced fertilizable oocytes has occurred only in the mouse. Progress in other more complex species has been limited because larger animals have longer durations of natural folliculogenesis, thereby requiring more culture time to generate fully grown follicles and oocytes. We believe the dog and cat are excellent models for understanding more about folliculogenesis in vitro. This review highlights what is known about this topic for these two species as well as future priorities. We have discovered that it is more challenging to maintain viability of primordial follicles within ovarian tissues in vitro in the dog than the cat. Nonetheless, it is possible to grow both isolated cat and dog pre-antral follicles in culture. Although the follicles of both species have the capacity to increase in size and produce steroids, only cat oocytes appear morphologically normal. The reason for this striking difference between these two species is an area of high research priority. While much more fundamental data are required, we envision advanced technology that will allow harvesting oocytes from the vast, unused follicle stores sequestered within carnivore ovaries. These gametes have utility for reproducing genetically valuable dogs and cats that are 'companions' or biomedical models for investigating human disorders as well as for salvaging the genomes of rare canid and felid species that die before contributing to genetic management programs. [ABSTRACT FROM AUTHOR]

The present study evaluated the effect of supplementing in vitro culture medium with J. insularis compared to FSH on isolated secondary follicles and in vitro maturation of oocytes from those follicles. Secondary follicles were isolated from sheep ovaries and individually cultured for 18 days in α-MEM+ (Control), α-MEM+ supplemented with 100 ng/mL recombinant bovine follicle stimulating hormone (FSH) or with 0.3, 1.25, or 2.5 mg/mL of J. insularis extract (JI0.3, JI1.25, and JI2.5, respectively). Culture medium collected every 2 days was used to measure ROS levels. At the end of the culture period, cumulus oocytes complex (COCs) were collected and matured in vitro. Follicular walls were used for mRNA quantitation. JI0.3 led to a higher (P < 0.05) percentages of intact follicles than other groups after 18 days of culture. While follicular diameter remained unchanged from Day 6 onwards with JI0.3 and FSH, percentages of antral cavity formation were higher (P < 0.05) with JI0.3 at Day 6 than in all other treatments. No differences were observed between controls and treatment groups regarding ROS levels and mRNA expression of genes. Viability of resulting oocytes was higher (P < 0.05) in JI0.3 compared to FSH. Interestingly, in control experiment, supplementation of maturation medium with JI0.3 led to higher (P < 0.05) percentages of metaphase II compared to controls. Although more validations will be needed, it seems that this natural extract could be used as a cheap and easily available alternative to commercial FSH., Competing Interests: The authors have declared that no competing interests exist.

Understanding critical roles of warming and reanimation is critical to improve the survival of vitrified testicular tissue in domestic cats. The objective was to study structural and functional properties of testicular tissues from prepubertal domestic cats after standard vitrification followed by two warming protocols (directly at 37°C or with a 5-second pre-exposure to 50°C) and three reanimation time points (immediately, 24 h and 5 days post-warming). In Experiment 1, tissues were evaluated for histo-morphology and mitochondrial activity immediately or 24 h after warming protocols. In Experiment 2, cell viability, DNA fragmentation, and germ cell composition were assessed immediately, 24 h, or 5 days after optimal warming. Preservation of seminiferous tubule structure was better using warming at 50°C for five seconds, and survival of somatic as well as germinal cells was higher compared to direct warming at 37°C for one minute. Short term in vitro culture (for reanimation) also proved that cellular composition and functionality were better preserved when warmed for a short time at 50°C. Collective data showed that short warming at 50°C led to better quality of seminiferous tubule structure and cell composition after vitrification and short-term culture. In addition, data suggest clear directions to further understand and optimize testicular tissue survival after fertility preservation procedures., Competing Interests: The authors have declared that no competing interests exist.

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 (17β-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.

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., Competing Interests: The authors have declared that no competing interests exist.

A better understanding of the factors influencing the biology of amphibian spermatozoa after release from the testis is a prerequisite to the development of sperm preservation methods. The objective of the study was to determine the effect of extracellular conditions (exposure to water and different temperatures) over time on the sperm motility and structural properties (including morphology and DNA integrity) collected from hormonally stimulated Atelopus zeteki. Following intraperitoneal injection of gonadotropin-releasing hormone agonist (des-Gly 10 , D-Ala 6 , Pro-NHEt 9 GnRH; 4 μg/g of body weight), human chorionic gonadotropin (hCG, 10 IU/gbw), or Amphiplex™ (0.4 μg/gbw GnRH-A + 10 μg/gbw metoclopramide hydrochloride), spermic urine samples from 27 males were collected and analyzed for sperm motility, morphology and DNA integrity while maintained at room temperature (23 °C), 4 °C, or diluted in water (hypo-osmotic environment) over a period of 46 min post-collection. Percentages of sperm motility and forward progressive motility remained high (>60%) when spermic urine was kept at room temperature or at 4 °C for 46 min regardless of the hormonal stimulation method. Dilution in water at room temperature greatly reduced the percentage of motile spermatozoa and forward progression (<50%) as well as DNA integrity (32.8% of intact cells) after 23 min while morphology did not differ (30.4% of normal cells), regardless of the hormone stimulation. This is the first systematic study on the effect of extracellular environment over time on A. zeteki sperm quality. This will contribute to the development of sperm handling protocols and reproductive technologies for this and other endangered Atelopus species., (Published by Elsevier Inc.)

Study Question: Do nuclear proteins in the germinal vesicle (GV) contribute to oocyte competence acquisition during folliculogenesis?, Summary Answer: Proteomic analysis of GVs identified candidate proteins for oocyte competence acquisition, including a key RNA processing protein-heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1)., What Is Known Already: The domestic cat GV, which is physiologically similar to the human GV, gains the intrinsic ability to resume meiosis and support early embryo development during the pre-antral-to-antral follicle transition. However, little is known about nuclear proteins that contribute to this developmental process., Study Design Size, Duration: GVs were enriched from pre-antral (incompetent) and antral (competent) follicles from 802 cat ovaries. Protein lysates were subjected to quantitative proteomic analysis to identify differentially expressed proteins in GVs from the two follicular categories., Participants/materials, Setting, Methods: Two biological replicates (from independent pools of ovaries) of pre-antral versus antral samples were labeled by tandem mass tags and then assessed by liquid chromatography-tandem mass spectrometry. Proteomic data were analyzed according to gene ontology and a protein-protein interaction network. Immunofluorescent staining and protein inhibition assays were used for validation., Main Results and the Role of Chance: A total of 174 nuclear proteins was identified, with 54 being up-regulated and 22 down-regulated (≥1.5-fold) after antrum formation. Functional protein analysis through gene ontology over-representation tests revealed that changes in molecular network within the GVs during this transitional phase were related to chromatin reorganization, gene transcription, and maternal RNA processing and storage. Protein inhibition assays verified that hnRNPA2B1, a key nuclear protein identified, was required for oocyte meiotic maturation and subsequent blastocyst formation., Large Scale Data: Data are available via ProteomeXchange with identifier PXD007211., Limitations Reasons for Caution: Proteins identified by proteomic comparison may (i) be involved in processes other than competence acquisition during the pre-antral-to-antral transition or (ii) be co-expressed in other macrostructures besides the GV. Expressional and functional validations should be performed for candidate proteins before downstream application., Wider Implications of the Findings: Collective results generated a blueprint to better understand the molecular mechanisms involved in GV competence acquisition and identified potential nuclear competence markers for human fertility preservation., Study Funding and Competing Interest(s): Funded by the National Center for Research Resources (R01 RR026064), a component of the National Institutes of Health (NIH) and currently by the Office of Research Infrastructure Programs/Office of the Director (R01 OD010948). The authors declare that there is no conflict of interest., (Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Contents The immature cat oocyte contains a large-sized germinal vesicle (GV) with decondensed chromatin that is highly susceptible to cryo-damage. The aim of the study was to explore an alternative to conventional cryopreservation by examining the influence of GV chromatin compaction using resveratrol (Res) exposure (a histone deacetylase enhancer) on oocyte survival during vitrification. In Experiment 1, denuded oocytes were exposed to 0, 0.5, 1.0 or 1.5 mmol/l Res for 1.5 h and then evaluated for chromatin structure or cultured to assess oocyte meiotic and developmental competence in vitro. Exposure to 1.0 or 1.5 mmol/l Res induced complete GV chromatin deacetylation and the most significant compaction. Compared to other treatments, the 1.5 mmol/l Res concentration compromised the oocyte ability to achieve metaphase II (MII) or to form a blastocyst. In Experiment 2, denuded oocytes were exposed to Res as in Experiment 1 and cultured in vitro either directly (fresh) or after vitrification. Both oocyte types then were assessed for meiotic competence, fertilizability and ability to form embryos. Vitrification exerted an overall negative influence on oocyte meiotic and developmental competence. However, ability to reach MII, achieve early first cleavage, and develop to an advanced embryo stage (8–16 cells) was improved in vitrified oocytes previously exposed to 1.0 mmol/l Res compared to all counterpart treatments. In summary, results reveal that transient epigenetic modifications associated with GV chromatin compaction induced by Res is fully reversible and beneficial to oocyte survival during vitrification. This approach has allowed the production of the first cat embryos from vitrified immature oocytes. [ABSTRACT FROM AUTHOR]

Contents Knowledge about reproduction is critical for predicting the viability of wildlife populations in nature and for managing breeding programmes in captivity. Intensive species-based studies are the priority, because reproductive mechanisms are extraordinarily diverse, even within the same taxonomic family. Carnivores deserve more attention as such species are highly vulnerable to environmental change and human persecution. The present review provides contemporary illustrations of how reproductive science is contributing to understand unique reproductive mechanisms that are both of fundamental and applied interest. In the case of the endangered African wild dog ( Lycaon pictus) free-living in South Africa, non-invasive faecal corticosteroid assessments have yielded new insights about the impact of animal relocation and reintroduction on adaptive responses, reproductive fitness and survival. For the maned wolf ( Chrysocyon brachyurus), advances have been made in characterizing and comparing reproductive traits in free-ranging vs captive individuals. For the cheetah ( Acinonyx jubatus), recent studies have focused on the cryosensitivity of sperm and the ability to develop a field-friendly sperm cryo-method. The by-product has been a large-scale frozen repository of sperm from wild-caught cheetahs useful for infusing new genes into ex situ populations. Finally, rigorous, multi-disciplinary and cross-institutional reproductive studies of the black-footed ferret ( Mustela nigripes), including the use of artificial insemination, have contributed to the remarkable recovery and restoration of this species, once on the brink of extinction. In summary, advances in reproductive science are not necessarily related to ‘assisted breeding’. However, understanding the unique ways of carnivore reproduction greatly contributes to species management and conservation. [ABSTRACT FROM AUTHOR]

Attempts to overcome the poor nuclear and cytoplasmic maturation in domestic cats, during in vitro maturation conducted during the annual non-breeding season. Collection of oocytes from July through November and cultured in a conventional IVM medium; Assessment of the nuclear status of oocytes after IVM or IVF; Blastocyte yield levels when oocytes were matured in medium containing higher FSH concentration and antioxidants; Demonstration that meiotic and developmental competences are inherent to the immature cat oocyte collected during non-breeding season.