Microwave-assisted dehydration, long-term storage at non-freezing temperatures, and rehydration of cat germinal vesicles.

Germinal vesicles (GVs) are alternative targets for female fertility preservation due to their availability and high resilience against non-physiological conditions. Preserved GVs can then be transferred to fresh cytoplasts to reconstitute viable oocytes. Here, we describe a GV preservation method that employs non-ionizing microwave radiations imparting energy to water molecules, which results in rapid and homogeneous drying of the sample. Trehalose is added as a xero-protectant before the radiations, enabling isothermal vitrification of the disaccharide sugar during drying. While the technique is still considered experimental, studies have shown that DNA and structural integrity can be effectively maintained in dried/rehydrated GVs. Importantly, the dry-preservation approach allows supra-zero temperature storage of the samples, offering a cost-effective and energy-saving alternative to traditional methods relying on ultra-low freezing temperatures. The protocol outlines a comprehensive procedure involving GV oocyte collection, trehalose loading, microwave drying, storage, and rehydration. The simplicity of the protocol facilitates ease of manipulation, making it an accessible method for researchers. While initially developed for domestic cats, the protocol can be adapted for other species with necessary modifications, considering potential species-specific responses to dehydration stress.

Semen proteome and transcriptome of the endangered black-footed ferret (Mustela nigripes) show association with the environment and fertility outcome.

The ex situ population of the endangered black-footed ferret (Mustela nigripes) has been experiencing declines in reproductive success over the past 30 years of human-managed care. A potential cause may be environmental-dependent inbreeding depression with diet being one of the contributing factors since ferrets are not fed their natural diet of prairie dogs. Here, we generated and analyzed semen proteome and transcriptome data from both wild and ex situ ferrets maintained on various diets. We identified 1757 proteins across all samples, with 149 proteins unique to the semen of wild ferrets and forming a ribosomal predicted protein-protein interaction cluster. Wild ferrets also differed from ex situ ferrets in their transcriptomic profile, showing enrichment in ribosomal RNA processing and potassium ion transport. Successful fertility outcomes documented for ex situ ferrets showed the strongest association with the semen transcriptome, with enrichment in genes involved in translation initiation and focal adhesion. Fertility also synergized with the effect of diet on differentially expressed transcriptomes, mainly affecting genes enriched in mitochondrial function. Our data and functional networks are important for understanding the causes and mechanisms of declining fertility in the ex situ ferret population and can be used as a resource for future conservation efforts.

Markers of fertility in reproductive microbiomes of male and female endangered black-footed ferrets (Mustela nigripes).

Reproductive microbiomes contribute to reproductive health and success in humans. Yet data on reproductive microbiomes, and links to fertility, are absent for most animal species. Characterizing these links is pertinent to endangered species, such as black-footed ferrets (Mustela nigripes), whose populations show reproductive dysfunction and rely on ex-situ conservation husbandry. To understand microbial contributions to animal reproductive success, we used 16S rRNA amplicon sequencing to characterize male (prepuce) and female (vaginal) microbiomes of 59 black-footed ferrets at two ex-situ facilities and in the wild. We analyzed variation in microbiome structure according to markers of fertility such as numbers of viable and non-viable offspring (females) and sperm concentration (males). Ferret vaginal microbiomes showed lower inter-individual variation compared to prepuce microbiomes. In both sexes, wild ferrets harbored potential soil bacteria, perhaps reflecting their fossorial behavior and exposure to natural soil microbiomes. Vaginal microbiomes of ex-situ females that produced non-viable litters had greater phylogenetic diversity and distinct composition compared to other females. In males, sperm concentration correlated with varying abundances of bacterial taxa (e.g., Lactobacillus), mirroring results in humans and highlighting intriguing dynamics. Characterizing reproductive microbiomes across host species is foundational for understanding microbial biomarkers of reproductive success and for augmenting conservation husbandry.

Epigenetic changes to gene pathways linked to male fertility in ex situ black-footed ferrets.

Environmental variation can influence the reproductive success of species managed under human care and in the wild, yet the mechanisms underlying this phenomenon remain largely mysterious. Molecular mechanisms such as epigenetic modifiers are important in mediating the timing and progression of reproduction in humans and model organisms, but few studies have linked epigenetic variation to reproductive fitness in wildlife. Here, we investigated epigenetic variation in black-footed ferrets (Mustela nigripes), an endangered North American mammal reliant on ex situ management for survival and persistence in the wild. Despite similar levels of genetic diversity in human-managed and wild-born populations, individuals in ex situ facilities exhibit reproductive problems, such as poor sperm quality. Differences across these settings suggest that an environmentally driven decline in reproductive capacity may be occurring in this species. We examined the role of DNA methylation, one well-studied epigenetic modifier, in this emergent condition. We leveraged blood, testes, and semen samples from male black-footed ferrets bred in ex situ facilities and found tissue-type specificity in DNA methylation across the genome, although 1360 Gene Ontology terms associated with male average litter size shared functions across tissues. We then constructed gene networks of differentially methylated genomic sites associated with three different reproductive phenotypes to explore the putative biological impact of variation in DNA methylation. Sperm gene networks associated with average litter size and sperm count were functionally enriched for candidate genes involved in reproduction, development, and its regulation through transcriptional repression. We propose that DNA methylation plays an important role in regulating these reproductive phenotypes, thereby impacting the fertility of male ex situ individuals. Our results provide information into how DNA methylation may function in the alteration of reproductive pathways and phenotypes in artificial environments. These findings provide early insights to conservation hurdles faced in the protection of this rare species.

Cloning for the Twenty-First Century and Its Place in Endangered Species Conservation.

Cloning as it relates to the animal kingdom generally refers to the production of genetically identical individuals. Because cloning is increasingly the subject of renewed attention as a tool for rescuing endangered or extinct species, it seems timely to dissect the role of the numerous reproductive techniques encompassed by this term in animal species conservation. Although cloning is typically associated with somatic cell nuclear transfer, the recent advent of additional techniques that allow genome replication without genetic recombination demands that the use of induced pluripotent stem cells to generate gametes or embryos, as well as older methods such as embryo splitting, all be included in this discussion. Additionally, the phenomenon of natural cloning (e.g., a subset of fish, birds, invertebrates, and reptilian species that reproduce via parthenogenesis) must also be pointed out. Beyond the biology of these techniques are practical considerations and the ethics of using cloning and associated procedures in endangered or extinct species. All of these must be examined in concert to determine whether cloning has a place in species conservation. Therefore, we synthesize progress in cloning and associated techniques and dissect the practical and ethical aspects of these methods as they pertain to endangered species conservation.

Gut bacterial composition shows sex-specific shifts during breeding season in ex situ managed black-footed ferrets.

The gut microbiome of mammals engages in a dynamic relationship with the body and contributes to numerous physiological processes integral to overall health. Understanding the factors shaping animal-associated bacterial communities is therefore paramount to the maintenance and management in ex situ wildlife populations. Here, we characterized the gut microbiome of 48 endangered black-footed ferrets (Mustela nigripes) housed at Smithsonian's National Zoo and Conservation Biology Institute (Front Royal, Virginia, USA). We collected longitudinal fecal samples from males and females across two distinct reproductive seasons to consider the role of host sex and reproductive physiology in shaping bacterial communities, as measured using 16S rRNA amplicon sequencing. Within each sex, gut microbial composition differed between breeding and non-breeding seasons, with five bacterial taxa emerging as differentially abundant. Between sexes, female and male microbiomes were similar during non-breeding season but significantly different during breeding season, which may result from sex-specific physiological changes associated with breeding. Finally, we found low overall diversity consistent with other mammalian carnivores alongside high relative abundances of potentially pathogenic microbes such as Clostridium, Escherichia, Paeniclostridium, and (to a lesser degree) Enterococcus - all of which have been associated with gastrointestinal or reproductive distress in mammalian hosts, including black-footed ferrets. We recommend further study of these microbes and possible therapeutic interventions to promote more balanced microbial communities. These results have important implications for ex situ management practices that can improve the gut microbial health and long-term viability of black-footed ferrets.

The Effect of Choline Salt Addition to Trehalose Solution for Long-Term Storage of Dried and Viable Nuclei from Fully Grown Oocytes.

Although drying techniques are exciting alternatives to cryopreservation, it remains challenging to maintain tightly controlled temperatures and humidity levels during storage of dried products. The objective of this study was to determine if the addition of choline acetate to trehalose solution could enable a wider range of storage conditions for preservation of nuclei from fully grown oocytes, by allowing temporary humidity excursions (>44% relative humidity) that may lead to crystallization of trehalose and loss of DNA integrity. Using domestic cat germinal vesicle oocytes as a model, we characterized the recovery as well as the integrity of samples after microwave-assisted dehydration. Exposure to choline acetate alone did not impair the germinal vesicle's DNA integrity and only had a negative impact on the chromatin configuration. Choline acetate addition enabled us to reach lower moisture contents after 25 min of microwave-assisted drying. Sample recovery after rehydration was also better in the presence of choline acetate. The integrity of the germinal vesicle's DNA was not affected, while the chromatin configuration was impaired by the presence of choline acetate during dehydration. Importantly, choline acetate addition helped to maintain an amorphous state (absence of detrimental crystallization) during excursion from ideal humidity conditions.

Segmental differentiation of the murine epididymis: identification of segment-specific, GM1-enriched vesicles and regulation by luminal fluid factors†.

The murine epididymis has 10 distinct segments that provide the opportunity to identify compartmentalized cell physiological mechanisms underlying sperm maturation. However, despite the essential role of the epididymis in reproduction, remarkably little is known about segment-specific functions of this organ. Here, we investigate the dramatic segmental localization of the ganglioside GM1, a glycosphingolipid already known to play key roles in sperm capacitation and acrosome exocytosis. Frozen tissue sections of epididymides from adult mice were treated with the binding subunit of cholera toxin conjugated to AlexaFluor 488 to label GM1. We report that GM1-enriched vesicles were found exclusively in principal and clear cells of segment 2. These vesicles were also restricted to the lumen of segment 2 and did not appear to flow with the sperm into segment 3, within the limits of detection by confocal microscopy. Interestingly, this segment-specific presence was altered in several azoospermic mouse models and in wild-type mice after efferent duct ligation. These findings indicate that a lumicrine factor, itself dependent on spermatogenesis, controls this segmental differentiation. The RNA sequencing results confirmed global de-differentiation of the proximal epididymal segments in response to efferent duct ligation. Additionally, GM1 localization on the surface of the sperm head increased as sperm transit through segment 2 and have contact with the GM1-enriched vesicles. This is the first report of segment-specific vesicles and their role in enriching sperm with GM1, a glycosphingolipid known to be critical for sperm function, providing key insights into the segment-specific physiology and function of the epididymis.

Collection, Handling, and Preservation of Wild Bird Semen: Current Status, Challenges, and Perspectives.

Semen preservation is a significant biotechnology used to safeguard the genetic material of birds, especially those with declining populations, through biobanking. However, there are limited reports on the successful chilling or cryopreservation of wild bird semen. In general, these techniques are not yet well-established for several species of wild birds and pose several challenges such as the need for bird handling and training, contamination of semen samples, low volume of semen collected, and inefficient preservation protocols. To address these challenges and improve post-thawing outcomes, new possibilities are being investigated, including alternative collection methods to traditional digital massage, the use of antioxidants and enzymes in the medium for chilling or freezing, storage methods using different straws from the usual pellet, and slower freezing rates. This review aims to discuss the various aspects of applying semen preservation in wild birds to create germplasm banks, highlighting the primary results obtained and the challenges that need to be addressed.

Trehalose delivered by cold-responsive nanoparticles improves tolerance of cumulus-oocyte complexes to microwave drying.

PURPOSE: Trehalose is a non-permeable protectant that is the key to preserve live cells in a dry state for potential storage at ambient temperatures. After intracellular trehalose delivery via cold-responsive nanoparticles (CRNPs), the objective was to characterize the tolerance of cat cumulus-oocyte complexes (COCs) to different levels of microwave-assisted dehydration. METHODS: Trehalose was first encapsulated in CRNPs. After exposure to trehalose-laden CRNPs, different water amounts were removed from cat COCs by microwave drying. After each dehydration level, meiotic and developmental competences were evaluated via in vitro maturation, fertilization, and embryo culture. In addition, expressions of critical genes were assessed by quantitative RT-PCR. RESULTS: CRNPs effectively transported trehalose into COCs within 4 h of co-incubation at 38.5 °C followed by a cold-triggered release at 4 °C for 15 min. Intracellular presence of trehalose enabled the maintenance of developmental competence (formation of blastocysts) as well as normal gene expression levels of HSP70 and DNMT1 at dehydration levels reaching up to 63% of water loss. CONCLUSION: Intracellular trehalose delivery through CRNPs improves dehydration tolerance of COCs, which opens new options for oocyte storage and fertility preservation at ambient temperatures.

Changes in Sperm Morphology, Morphometry, and Motility from the Epididymis to the Vas Deferens in Rheas (Rhea americana, Linnaeus, 1758).

The objective was to characterize morphological, morphometric, and ultrastructural changes in rhea spermatozoa between the epididymis and the vas deferens. Sperm samples were collected from the reproductive tracts of seven adult individuals and evaluated for sperm characteristics using brightfield microscopy as well as ultrastructural features using scanning electron microscopy (SM). Mean sperm count tended to increase in the vas deferens (378.0 ± 135.0 × 106) compared to the epididymis (201.0 ± 77.4 × 106). Percentages of motile sperm grew from 37.0 ± 4.9% in the epididymis to 58.5 ± 7.7% in the vas deferens. The proportion of normal spermatozoa was 75.6 ± 1.8% and most common defects were bent tails (9.7 ± 0.9%). However, these proportions were not different between epididymis and vas deferens. SM analysis revealed further features of rhea spermatozoa. Normal rhea spermatozoa were threadlike with an acrosome (0.95 ± 0.0 µm), head (7.53 ± 0.01 µm), midpiece (2.08 ± 0.01 µm), and tail (30.7 ± 0.06 µm). Lengths of sperm acrosome, head, midpiece, and tail were longer in the vas deferens compared to the epididymis. Our findings suggest that rhea spermatozoa undergo a maturation process during the passage from the epididymis to the vas deferens.

Immune priming prior to pathogen exposure sheds light on the relationship between host, microbiome and pathogen in disease.

Dynamic interactions between host, pathogen and host-associated microbiome dictate infection outcomes. Pathogens including Batrachochytrium dendrobatidis (Bd) threaten global biodiversity, but conservation efforts are hindered by limited understanding of amphibian host, Bd and microbiome interactions. We conducted a vaccination and infection experiment using Eastern hellbender salamanders (Cryptobranchus alleganiensis alleganiensis) challenged with Bd to observe infection, skin microbial communities and gene expression of host skin, pathogen and microbiome throughout the experiment. Most animals survived high Bd loads regardless of their vaccination status and vaccination did not affect pathogen load, but host gene expression differed based on vaccination. Oral vaccination (exposure to killed Bd) stimulated immune gene upregulation while topically and sham-vaccinated animals did not significantly upregulate immune genes. In early infection, topically vaccinated animals upregulated immune genes but orally and sham-vaccinated animals downregulated immune genes. Bd increased pathogenicity-associated gene expression in late infection when Bd loads were highest. The microbiome was altered by Bd, but there was no correlation between anti-Bd microbe abundance or richness and pathogen burden. Our observations suggest that hellbenders initially generate a vigorous immune response to Bd, which is ineffective at controlling disease and is subsequently modulated. Interactions with antifungal skin microbiota did not influence disease progression.

Transfer of Galectin-3-Binding Protein via Epididymal Extracellular Vesicles Promotes Sperm Fertilizing Ability and Developmental Potential in the Domestic Cat Model.

Key proteins transferred by epididymal extracellular vesicles (EVs) to the transiting sperm cells contribute to their centrosomal maturation and developmental potential. Although not reported in sperm cells yet, galectin-3-binding protein (LGALS3BP) is known to regulate centrosomal functions in somatic cells. Using the domestic cat model, the objectives of this study were to (1) detect the presence and characterize the transfer of LGALS3BP via EVs between the epididymis and the maturing sperm cells and (2) demonstrate the impact of LGALS3BP transfer on sperm fertilizing ability and developmental potential. Testicular tissues, epididymides, EVs, and spermatozoa were isolated from adult individuals. For the first time, this protein was detected in EVs secreted by the epididymal epithelium. The percentage of spermatozoa with LGALS3BP in the centrosome region increased as cells progressively incorporated EVs during the epididymal transit. When LGALS3BP was inhibited during in vitro fertilization with mature sperm cells, less fertilized oocytes and slower first cell cycles were observed. When the protein was inhibited in epididymal EVs prior to incubation with sperm cells, poor fertilization success further demonstrated the role of EVs in the transfer of LGALS3BP to the spermatozoa. The key roles of this protein could lead to new approaches to enhance or control fertility in clinical settings.

Current knowledge in the biology of gametes and embryos from Carnivora.

In addition to companion animals and laboratory species, about 270 carnivore species play fundamental ecological roles in different ecosystems. However, almost 40% of carnivore species are now threatened or endangered in the wild because of human activities. While protection of natural habitats is critical, it is equally important to better understand carnivore reproduction, including a solid knowledge in sperm, oocyte, and embryo biology, to maintain sustainable populations in the wild and in conservation breeding centers. Characterizing gamete and embryo biology is also needed to develop cryopreservation and assisted reproductive technologies to enhance conservation efforts. The objective of this review is to provide the most recent knowledge in the biology of sperm cells, oocytes, and early embryos across all carnivore families. Overall, most data originate from populations maintained in breeding centers or zoos. Characterizations of sperm biology and cryopreservation are far more advanced than for oocytes and embryos. Currently, sperm biology is mainly studied in Canids, Felids, Ursids, and Mustelids, with more emphasis on structural than functional properties. Importantly, fundamental studies of gamete and embryo biology in domestic dogs, cats, and ferrets have paved the way for more precise characterizations in wild counterparts as well as the development of cryopreservation and assisted reproductive technologies. A striking feature of spermatozoa across a wide range of Canids and Felids is the presence of teratospermia (>60% of abnormal sperm cells), which is related to the loss of genetic diversity in some populations. Although sperm structures differ across carnivore families, sperm biology remains difficult to compare because of the small amount of data in many species. Regarding oocyte biology and embryology, data are much scarcer than in sperm cells, with too few studies going beyond structural descriptions. More carnivore species and more individuals (especially from wild populations in addition to captive ones) must be studied to improve our understanding about comparative germplasm biology and develop adequate conservation breeding strategies including the use of cryobanking and assisted reproductive technologies.

Modeling genetic benefits and financial costs of integrating biobanking into the conservation breeding of managed marsupials.

Managed breeding programs are an important tool in marsupial conservation efforts but may be costly and have adverse genetic effects in unavoidably small captive colonies. Biobanking and assisted reproductive technologies (ARTs) could help overcome these challenges, but further demonstration of their potential is required to improve uptake. We used genetic and economic models to examine whether supplementing hypothetical captive populations of dibblers (Parantechinus apicalis) and numbats (Myrmecobius fasciatus) with biobanked founder sperm through ARTs could reduce inbreeding, lower required colony sizes, and reduce program costs. We also asked practitioners of the black-footed ferret (Mustela nigripes) captive recovery program to complete a questionnaire to examine the resources and model species research pathways required to develop an optimized biobanking protocol in the black-footed ferret. We used data from this questionnaire to devise similar costed research pathways for Australian marsupials. With biobanking and assisted reproduction, inbreeding was reduced on average by between 80% and 98%, colony sizes were on average 99% smaller, and program costs were 69- to 83-fold lower. Integrating biobanking made long-standing captive genetic retention targets possible in marsupials (90% source population heterozygosity for a minimum of 100 years) within realistic cost frameworks. Lessons from the use of biobanking technology that contributed to the recovery of the black-footed ferret include the importance of adequate research funding (US$4.2 million), extensive partnerships that provide access to facilities and equipment, colony animals, appropriate research model species, and professional and technical staff required to address knowledge gaps to deliver an optimized biobanking protocol. Applied research investment of A$133 million across marsupial research pathways could deliver biobanking protocols for 15 of Australia's most at-risk marsupial species and 7 model species. The technical expertise and ex situ facilities exist to emulate the success of the black-footed ferret recovery program in threatened marsupials using these research pathways. All that is needed now for significant and cost-effective conservation gains is greater investment by policy makers in marsupial ARTs.

Los programas de reproducción controlada son una herramienta importante para los esfuerzos de conservación de marsupiales, aunque pueden resultar costosos y tener efectos genéticos adversos en las colonias cautivas incapaces de aumentar en tamaño. Los biobancos y las tecnologías de reproducción asistida (TRA) podrían ayudar a superar estos problemas, pero es necesario seguir demostrando su potencial para mejorar su adopción. Utilizamos modelos genéticos y económicos para analizar si la introducción de esperma fundador proveniente de biobancos mediante tecnologías de reproducción asistida a poblaciones cautivas hipotéticas de los marsupiales Parantechinus apicalis y Myrmecobius fasciatus podría reducir la endogamia, disminuir el tamaño efectivo de las colonias y reducir el costo de los programas. También pedimos a los profesionales del programa de recuperación en cautiverio del hurón de patas negras (Mustella nigripes) que respondieran un cuestionario para analizar los recursos y los métodos de investigación de las especies modelo necesarias para desarrollar un protocolo de biobanco optimizado para el hurón de patas negras. Utilizamos los datos de este cuestionario para diseñar métodos de investigación con costos similares para los marsupiales australianos. Con el biobanco y la reproducción asistida, la endogamia se redujo en promedio entre un 80 y un 98%, el tamaño de las colonias fue en promedio un 99% más pequeño y los costos del programa entre 69 y 83 veces menores. La integración del biobanco posibilitó los objetivos de retención genética en cautiverio a largo plazo en marsupiales (90% de heterocigosidad de la población de origen durante un mínimo de 100 años) dentro de un marco realista de costos. Entre el aprendizaje extraído del uso de la tecnología de biobancos que contribuyó a la recuperación del hurón de patas negras figuran la importancia de una financiación adecuada de la investigación (4.2 millones de dólares), colaboraciones profundas que faciliten el acceso a instalaciones y equipos, colonias de animales, especies modelo adecuadas para la investigación y el personal profesional y técnico necesario para abordar las lagunas de conocimiento y ofrecer un protocolo optimizado para los biobancos. Una inversión en investigación aplicada de 133 millones de dólares australianos para la investigación de los marsupiales podría proporcionar protocolos de biobancos para 15 de las especies de marsupiales australianos en mayor riesgo y 7 especies modelo. Existen los conocimientos técnicos y las instalaciones ex situ para emular el éxito del programa de recuperación del hurón de patas negras en marsupiales amenazados utilizando estas vías de investigación. Ahora sólo se necesita una mayor inversión por parte de los responsables políticos de las TRA para marsupiales para obtener beneficios de conservación significativos y rentables.

Development of assays for the characterization of sperm motility parameters, viability, and membrane integrity in the epididymis and vas deferens of the greater rhea (Rhea americana).

The objectives of the study were to (1) describe the kinematic parameters of spermatozoa (2) compare methods of evaluating sperm viability (3) validate assays of functionality and integrity of the sperm membrane and (4) evaluate possible changes between spermatozoa from the epididymis and the vas deferens of the greater rhea. Semen samples were recovered from 7 adult individuals. Sperm motility was characterized by adjusting the set-up for Computer-assisted semen analysis (CASA) to that new species. For sperm viability evaluation, smears of bromophenol blue and eosin-nigrosine dyes were used. Five solutions of different osmolarities were then tested for the hypoosmotic swelling test (HOST). The combination of fluorescent probes (propidium iodide - IP and Hoechst 33342) was also used to assess plasma membrane integrity. Data were presented as mean ± SEM. Rhea spermatozoa from the vas deferens had an overall motility of 14.6 ± 2.5%. The bromophenol blue staining technique revealed that 64.6 ± 5.2% sperm were viable, while that proportion was 72.1 ± 2.5% using eosin-nigrosine. An average of 77.6 ± 4.8% of spermatozoa reacted to the HOST with distilled water at 0 mOsm/l. Fluorescent probes indicated that 65.3 ± 2.6% of spermatozoa had intact membranes. Interestingly, no statistical differences were observed between the parameters analyzed in the epididymal spermatozoa and the vas deferens. These new assays set reference values that can now be used to further exploration of sperm handling conditions and freezing protocols in rheas.

Damages and stress responses in sperm cells and other germplasms during dehydration and storage at nonfreezing temperatures for fertility preservation.

Long-term preservation of sperm, oocytes, and gonadal tissues at ambient temperatures has the potential to lower the costs and simplify biobanking in human reproductive medicine, as well as for the management of animal populations. Over the past decades, different dehydration protocols and long-term storage solutions at nonfreezing temperatures have been explored, mainly for mammalian sperm cells. Oocytes and gonadal tissues are more challenging to dehydrate so little to no progress have been made. Currently, the detrimental effects of the drying process itself are better characterized than the impact of long-term storage at nonfreezing temperatures. While structural and functional properties of germ cells can be preserved after dehydration, a long list of damages and stresses in nuclei, organelles, and cytoplasmic membranes have been reported and sometimes mitigated. Characterizing those damages and better understanding the response of germ cells and tissues to the stress of dehydration is fundamental. It will contribute to the development of optimal protocols while proving the safety of alternative storage options for fertility preservation. The objective of this review is to (1) document the types of damages and stress responses, as well as their mitigation in cells dried with different techniques, and (2) propose new research directions.

Feline microRNAome in ovary and testis: Exploration of in-silico miRNA-mRNA networks involved in gonadal function and cellular stress response.

The aim of the study was to perform the first in-depth analysis of miRNAs in ovarian and testicular tissues of the domestic cat, a critical biomedical model. Specifically, potential miRNA involvement was explored in gonadal function, testis development, and cellular stress response to preservation protocols. We performed miRNA-sequencing on 20 ovarian and 20 testicular samples from 15 cats, including different ages and tissue treatments. Using fresh tissues (n = 15), we confirmed gonadal expression of 183 miRNA precursors and discovered additional 52 novel feline candidate precursors. We integrated the mRNA data from our previous study on the same age and treatment groups to create in-silico miRNA-mRNA networks and their functional enrichment, which allows comprehensive exploration into possible miRNA functions in cat gonads. Clusters of miRNAs united by shared differentially expressed mRNA targets are potentially involved in testicular development and spermatogenesis. MicroRNAs could play a significant role in ovarian tissue response to stress from microwave-assisted dehydration, with smaller roles in cellular response to vitrification in both ovary and testis. This new list of miRNAs with potential function in cat gonads is a major step towards understanding the gonadal biology, as well as optimizing fertility preservation protocols.