Genome resource banking for wildlife conservation: promises and caveats.

The value of cryopreserved germplasm in agriculture, aquaculture and medicine was recognized in the mid-twentieth century following the discovery in the late 1940s of a method for recovering viable spermatozoa after freeze-thawing. Sir Alan Parkes (a founder of cryobiology as a discipline) remarked that "time and space has been abolished for cattle breeding", a phrase that continues to summarise the potential value of the Genetic Resource Bank (GRB) concept for all species. The underlying principle behind these remarks was based on the recognition that spermatozoa could remain viable for many years, and still achieve pregnancies even long after the semen donor had died. Nowadays, live mammalian embryos, amphibian spermatozoa and cultured somatic cells can also be stored for future use in conservation breeding programmes, where the overarching aim is to mitigate the deleterious impacts of inbreeding on the fitness and survival of populations. Revolutionary advances in the cryobiology of coral spermatozoa, embryos and larvae are also helping to counter the damaging effects of climate change and toxic chemicals on coral reefs. In this article we review the ways in which GRBs can contribute to global conservation activities, noting that species-specific biological differences can limit the success of standard animal breeding technologies such as artificial insemination and embryo transfer. These limitations mean that there is still a need for the development of novel, and possibly species-specific, GRB technologies.

Proteomic analysis of germinal vesicles in the domestic cat model reveals candidate nuclear proteins involved in oocyte competence acquisition.

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.

The mutual benefits of research in wild animal species and human-assisted reproduction.

Studying the reproductive biology of wild animal species produces knowledge beneficial to their management and conservation. However, wild species also share intriguing similarities in reproductive biology with humans, thereby offering alternative models for better understanding the etiology of infertility and developing innovative treatments. The purpose of this review is to raise awareness in different scientific communities about intriguing connections between wild animals and humans regarding infertility syndromes or improvement of fertility preservation. The objectives are to (1) highlight commonalities between wild species and human fertility, (2) demonstrate that research in wild species-assisted reproductive technologies can greatly enhance success in human reproductive medicine, and (3) recognize that human fertility preservation is highly inspiring and relevant to wild species conservation. In addition to having similar biological traits in some wild species and humans, the fact of sharing the same natural environment and the common needs for more options in fertility preservation are strong incentives to build more bridges that will eventually benefit both animal conservation and human reproductive medicine.

Presence of sucrose in the vitrification solution and exposure for longer periods of time improve post-warming follicle integrity in cat ovarian tissues.

Ovarian tissue cryopreservation followed by tissue culture is a promising approach to preserving the fertility of biomedical models and endangered species. The objective of this study was to investigate the impact of exposure time to vitrification solution and presence of sucrose using different exposure temperatures and base media on intra-ovarian follicle integrity. Peripubertal ovarian cortical pieces were obtained by isolating the cortex and dissecting it into 1 × 1 × 0.2 mm3 pieces. The cortical pieces were then exposed to equilibration solution and then vitrification solutions (VS) in one of the conditions mentioned above, plunged directly into liquid nitrogen and stored for ≥24 hr in liquid nitrogen. After thawing, the cortical pieces were cultured in vitro for 0, 1 or 7 days to determine the follicle integrity (through histological assessment) and the ability of the tissue to recover from cryoinjury. Fresh controls maintained a constant level of normal morphology (>60% of the total follicles) throughout the culture period. Cortical pieces exposed to VS with sucrose for 10 min had the highest percentage of normal follicles (approximately 20% after 7 days of culture) throughout the culture period. Other conditions using different base medium, lower exposure temperatures or different thawing methods did not improve the follicle integrity. This protocol provides a solid foundation on which to optimize ovarian tissue cryopreservation in the domestic cat and to investigate the molecular effects of vitrification.

Deciphering the mechanisms involving cenexin, ninein and centriolin in sperm maturation during epididymal transit in the domestic cat.

The sperm centrosome is an essential organelle with a key role in organizing the sperm aster for proper syngamy and formation of the first mitotic spindle. The sperm cell acquires the functional capability during epididymal transit by incorporation of key factors. The objective of the study was to identify these key maturation proteins, such as ninein and centriolin as well as cenexin-a scaffold protein that serves to bind ninein and centriolin. Epididymal samples were dissected from 17 adult cat testes (>1 year old) and spermatozoa were extracted from the different regions, including rete testis, caput, corpus, cauda and vas deferens. Tissue samples and sperm cells were fixed separately in 4% paraformaldehyde before immunostaining with anticenexin, ninein or centriolin antibodies. Results showed that the proportion of sperm cells with cenexin localized at the centrosome progressively increased along the tract with the lowest percentage of stained cells in the testis (mean = 45%) and highest in the cauda (mean = 81%). Although not significant, the intensity of cenexin immunofluorescence in positive cells increased twofold from the testis to vas deferens. There was no significant difference in the proportion of sperm labelled with centriolin or ninein (ranges of 21%-26% and 33%-48% between segments, respectively) or the intensity (±58% and ±63% change as compared to testis, respectively). Cenexin may serve as a scaffold protein for centriolin and ninein, as the vast majority of spermatozoa only displayed colocalization of these proteins when cenexin was also present (mean = 85% and 91% colocalization, respectively). In summary, these results could be applied to future efforts to create an in vitro culture system capable of rescuing the impaired centrosome of an infertile male, with particular potential for wild felid conservation.

The nuclear and developmental competence of cumulus-oocyte complexes is enhanced by three-dimensional coculture with conspecific denuded oocytes during in vitro maturation in the domestic cat model.

The objective of the study was to assess the efficacy of coculture with conspecific cumulus-denuded oocytes (CDOs) during in vitro maturation in a three-dimensional system of barium alginate microcapsules on the in vitro embryo development of domestic cat cumulus-oocyte complexes (COCs). In Experiment I, COCs were cocultured with conspecific CDOs or cultured separately in a 3D system for 24 hr of in vitro maturation, before assessing the meiotic progression. In Experiment II, the in vitro fertilization of COCs and CDOs was carried out with chilled epididymal spermatozoa and the presumptive zygotes were cultured in vitro separately for 7 days in 3D microcapsules before assesment of embryonic development. The results showed that the viability was maintained and that meiosis was resumed in the 3D culture system. The presence of CDOs during in vitro maturation improved the meiotic competence of the COCs, since the proportions of telophase I/metaphase II were higher than that in the groups cultured separately. The enrichment of the maturation system by companion oocytes also enhanced the ability of COCs to develop into embryos, and increased the percentages of morula and blastoycst stages. The COCs cocultured with CDOs developed at higher rates than the COCs cultured separately and the CDOs themselves. The beneficial effects of coculture with conspecific CDOs were presumably due to the paracrine action of some secreted factors that enhanced many molecular patterns related to the complex of cumulus oophorous cells. Further investigations to understand how the 3D microenvironment can influence the features of oocytes and embryos are required.

Mitigation of sperm tail abnormalities using demembranation approach in the clouded leopard (Neofelis nebulosa).

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.

Beneficial effect of extracellular adenosine 5'-triphosphate treatment on the Indochinese leopard (Panthera pardus delacouri) sperm quality after cryopreservation.

The Indochinese leopard (Panthera pardus delacouri) population, included in CITES Appendix I, has been declining for decades. Proper gamete preservation condition is critical for breeding programme management using artificial insemination or in vitro fertilization (IVF). The present study aimed at investigating the impact of post-thawing treatment of leopard semen with extracellular adenosine 5'-triphosphate (ATPe) on sperm quality (including morphological traits and ability to fertilize an oocyte). Semen from six adult male leopards was collected by electroejaculation (one ejaculation per cat). After the evaluation of the fresh sample quality, the semen was cryopreserved (10 × 106 cells per straw; two straws per cat). After thawing, the sperm sample from the first straw of each cat was divided into three aliquots: control (no ATPe), supplemented with 1.0 or 2.5 mM ATPe that were evaluated for sperm quality at 10, 30 min and 3 hr post-thawing. The sperm sample from the second straw, supplemented with 0, 1.0 or 2.5 mM ATPe for 30 min, was assessed for IVF with domestic cat oocytes. Sperm quality (all metrics) was negatively affected by the cryopreservation process (p ≤ .05). However, the percentage of sperm motility, level of progressive motility and percentage of plasma membrane integrity did not differ (p > .05) among post-thawing groups. The sperm mitochondrial membrane potential was enhanced (p ≤ .05) by ATPe treatment (1.0 and 2.5 mM; 10 min to 3 hr of incubation). Furthermore, incubation of ATPe (1.0 and 2.5 mM) for 30 min could promote sperm velocity patterns (curvilinear velocity; VCL and straight line velocity; VSL) (p ≤ .05). The percentage of pronuclear formation and cleaved embryos was increased (p ≤ .05) after 1.0 ATPe treatment (49.8 ± 2.8; 45.9 ± 1.5) compared to 0 mM (41.4 ± 3.3; 38.9 ± 0.5) whereas the number of sperm binding/oocyte did not significantly differ among groups. In summary, we suggest that ATPe activated the velocity of Indochinese leopard sperm motility that may lead to faster sperm/oocyte binding and sperm penetration (factors of successful embryo development).

Dynamic changes in mitochondrial DNA, distribution and activity within cat oocytes during folliculogenesis.

Mitochondria play fundamental roles during oocyte development. The accumulation and spatial redistribution of these energy-producing organelles have been linked to the developmental competence of mammalian oocytes. Here, we assessed the copy number, distribution and activity of mitochondria within cat oocytes during folliculogenesis. In Experiment 1, oocytes were recovered from primordial (n = 152), primary (112), secondary (95), early (131), small (118), antral (86) and advanced antral (5) stages follicles, and mitochondria DNA extracted and quantified using qPCR. In Experiment 2, oocytes from pre-antral (n = 44), early antral (n = 66), small antral (n = 59), antral (n = 41) and advanced antral (n = 21) follicles were isolated and stained with CMXRos MitoTracker dye to assess mitochondrial distribution pattern and activity levels. Oocyte's mitochondria DNA (mtDNA) copy numbers gradually increased as folliculogenesis progressed, with a significant shift at the small antral stage (0.5 to <1 mm in diameter). The location of mitochondria gradually shifted from a homogeneous distribution throughout the cytoplasm in pre-antral oocytes to a pericortical concentration in the advanced antral stage. Quantification of CMXRos fluorescent intensity revealed a progressive increase in mitochondrial activity in oocytes from the pre-antral to the large antral follicles. Taken together, these findings demonstrated that cat oocytes undergo dynamic changes in mitochondrial copy number, distribution and activity during folliculogenesis. These significant modifications to this crucial cytoplasmic organelle are likely associated with the acquisition of developmental competency by cat oocytes.

Influence of culture medium composition on relative mRNA abundances in domestic cat embryos.

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.

Special series on the role of the microbiome in reproduction and fertility.

Many parts of the animal and human body host groups of bacteria, viruses, and fungi that together are known as the microbiome. Microbiomes do not cause disease but are important for the healthy working of many systems in the body, including for reproduction and fertility. While the microbiome that lives in a reproductive tract play the most direct role, microbiomes from other areas of the body may also affect reproductive health. However, not much is known about how these groups of microorganisms regulate fertility as well as the health of parents and offspring and help animals to cope with environmental changes. Furthermore, compared to the large amount of research in laboratory species and humans, there is less information about domestic or wild animal species. This special series of Reproduction and Fertility on microbiomes is aimed at filling this gap with articles from experts highlighting important evidence in reproductive microbiomes, current research gaps, and new directions.

On the horizon for fertility preservation in domestic and wild carnivores.

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.

Cat and dog primordial follicles enclosed in ovarian cortex sustain viability after in vitro culture on agarose gel in a protein-free medium.

Our objective was to examine the influences of differing media, protein supplementation and the microenvironment on cat vs dog primordial follicle viability in vitro. Ovarian cortical slices were cultured for 3, 9 or 15 days in α-minimum essential medium (α-MEM) or MEM supplemented with 10% fetal bovine serum (FBS), 10% knock-out serum replacement (KSR) or 0.1% polyvinyl alcohol (protein free). In a separate study, cat and dog ovarian tissues were cultured in protein-free α-MEM and MEM, respectively, in cell culture inserts, on 1.5% agarose gel or in 24-well cell culture plates (control). Follicle viability was assessed in both studies using calcein AM/ethidium homodimer and histological evaluation with haematoxylin/eosin staining. No cat follicle sustained viability beyond 9 days of in vitro culture in α-MEM compared to 37.5% of those incubated for 15 days in MEM in protein-free condition (p < 0.05). In contrast, α-MEM was superior (p < 0.05) to MEM in maintaining dog follicle viability (32.7% vs 8.1%) in protein-free condition at 15 days. Serum was detrimental (p < 0.05) to follicle survival in both species. Knock-out serum replacement supplementation and a protein-free condition supported cat follicle viability, whereas the latter was superior (p < 0.05) to the former for sustaining dog follicle survival. Likewise, dog follicle viability was enhanced (p < 0.05) by the agarose gel compared to the cell culture insert and control groups after 3 and 9 days of culture. For the cat, the agarose gel better (p < 0.05) supported follicle viability compared to the control, but was equivalent to the cell culture insert. Therefore, sustaining primordial follicle survival from intracortical ovarian slices requires a different in vitro microenvironment for the cat vs the dog. A key factor to enhancing survival of these early stage follicles in culture appears to be the use of agarose gel, which enhances follicle viability, perhaps by promoting gas exchange.

Source of protein supplementation during in vitro culture does not affect the quality of resulting blastocysts in the domestic cat.

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.

Increase in histone methylation in the cat germinal vesicle related to acquisition of meiotic and developmental competence.

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 (H3K9me3), lysine 4 (H3K4me3), lysine 27 (H3K27me3) or H3 dimethylated at lysine 79 (H3K79me2). The vast majority of oocytes (range, 72.2-85.4%; p > 0.05) contained a GV with H3K9me3 or H3K27me3, regardless of follicular stage/size. However, the proportion of GVs with H3K4me3 or H3K79me2 was higher in early antral follicles (42.6%; p < 0.05) compared with other stages (range, 12.1-15.2%). Therefore, H3K4me3 and H3K79me2 (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, H3K9me3 and H3K27me3 (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.

The domestic dog and cat as models for understanding the regulation of ovarian follicle development in vitro.

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.

Birth of kittens after the transfer of frozen-thawed embryos produced by intracytoplasmic sperm injection with spermatozoa collected from cryopreserved testicular tissue.

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 µm 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/hCG)-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.

Connecting the spots: Understanding cheetah reproduction to improve assisted breeding and population management.

The North American cheetah (Acinonyx jubatus) population serves as both an insurance population for their rapidly decreasing wild cohorts as well as a research population to understand the unique biology of this species. This review focus on the complexity of the female cheetah reproductive system and the recent advances that have been made towards understanding basic biology and reproductive function, and application of assisted breeding technologies to enhance reproduction and maintain genetic diversity of this species in human care. Cheetah females are non-seasonal breeders that exhibit lengthy periods of anestrus that are not associated with age, environment, or reproductive potential. It is possible to collect good quality oocytes, that support fertilisation and successful early embryonic development, regardless of female age (from 2 to 12 yr old). However, the prevalence of uterine pathologies increases with age and prevents middle to advanced age females from establishing pregnancy. Pregnancy can be diagnosed in non-sedated cheetah females via ultrasonography (first month), steroid hormone analysis (second/third month) or radiography (third month). Fecal biomarkers, such as Immunoglobulin J, show great promise for diagnosing pregnancy at an early stage as well as other physiological states. Several decades of basic research have led to efficient management of natural breeding and recent successes in assisted reproduction.

The competence of germinal vesicle oocytes is unrelated to nuclear chromatin configuration and strictly depends on cytoplasmic quantity and quality in the cat model.

BACKGROUND: Chromatin configuration of the germinal vesicle (GV) and quality of the cytoplasm are critical factors in achieving oocyte meiotic and developmental capacity during folliculogenesis. Besides gaining new insights into the timing and cellular mechanisms associated with the acquisition and regulation of GV oocyte competence, the domestic cat model was used to examine (i) the relation between GV chromatin configuration and oocyte functionality during folliculogenesis and (ii) the role of the cytoplasmic environment on the GV competence and stability. METHODS: Structural and functional properties of GV oocytes were characterized after isolation from different follicle stages of non-stimulated cat ovaries. GV transfers, artificial chromatin compaction and oocyte vitrification were used to demonstrate the respective roles of GV and cytoplasm on the oocyte functionality. RESULTS: GVs acquired the intrinsic capability to resume meiosis during the pre-antral follicle stage, whereas the capacity to support embryo development occurred while the antrum started to form. Chromatin configuration of the GV did not undergo extensive modification during the acquisition of competence or during the arrest of transcriptional activity at the large antral follicle stage. However, the quality and quantity of the cytoplasm regulated and enhanced GV functionality. This finding also held for GVs transferred from incompetent or subpar oocytes into the cytoplasm of good quality oocytes or when chromatin was artificially modified or vitrified. CONCLUSIONS: The cat model provides a new insight into GV oocyte structure and function during folliculogenesis while challenging current concepts about oocyte quality criteria based on the GV morphology. This suggests alternative evaluative approaches for oocytes from other species too, including humans. Cat GVs also appear competent at an early follicle stage and are resilient to perturbations which designate this organelle as an attractive target for developing novel fertility preservation tactics.

In vitro compaction of germinal vesicle chromatin is beneficial to survival of vitrified cat oocytes.

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.