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Systematic cryo-banking of reproductive tissues could enhance reproductive management and ensure sustainability of rare mammalian genotypes. Testicular tissues contain a vast number of germ cells, including at early stages (spermatogonia and spermatocytes), that can potentially develop into viable spermatozoa after grafting or culture in vitro, and the resulting sperm cells then can be used for assisted reproductive techniques. The objective of this review was to describe current advances, limitations, and perspectives related to the use of testicular tissue preservation as a strategy for the conservation of male fertility. Testes can be obtained from mature or prepubertal individuals, immediately postmortem or by orchiectomy, but testicular biopsies could also be an alternative to collect samples from living individuals. Testicular fragments can be then cryopreserved by using slow or ultra-rapid freezing, or even vitrification methods. The composition of cryopreservation media can vary according to species-specific characteristics, especially regarding the cryoprotectant type and concentration. Finally, spermatozoa have been usually obtained after xenografting of testicular fragments into severely immunodeficient mice, while this method still has to be optimized after in vitro culture conditions. [ABSTRACT FROM AUTHOR]

The extinction rate of mammalian species has been accelerated in recent decades. It therefore is important to preserve and store genetic materials in cryobanks for research purposes and subsequent production of offspring through assisted reproductive techniques. Along with the systematic collection and storage of germplasm, research efforts focusing on in vitro culture to produce mature gametes are critical. Specifically, obtaining mature oocytes from in vitro culture of preantral follicles is a great option to enhance female fertility preservation since these early follicles represent 90%–95% of the follicular population on ovarian cortex biopsy. This review presents current advances and discusses limitations and prospects about isolation, cryopreservation/banking, and in vitro culture of preantral follicles from wild species. [ABSTRACT FROM AUTHOR]

Neotropical carnivores include a large number of threatened and endangered species. It is critical to develop conservation efforts to ensure the sustainability of populations in situ and ex situ. The highest priorities are to protect natural habitats and better understand the biology of rare species. Conservation efforts also are directed toward the implementation of breeding programs and the development of reproductive biotechnologies in which the cryopreservation of male gametes plays a major role. It also is fundamental to create semen banks that contribute to maintaining genetic diversity in small and endangered populations. The present article aims at reviewing the state of the art in cryopreservation of semen from neotropical carnivores and discuss the development of systematic banking for the conservation of these understudied species. [ABSTRACT FROM AUTHOR]

Lipids are among the most abundant and essential cell components. Specifically, cytoplasmic lipid droplets (LDs) play crucial roles in cellular energy homeostasis. The foci of this review are (1) the composition and roles of lipids during oocyte maturation and early embryonic development, (2) possible causes of cryoinjuries in lipid-rich oocytes/embryos, and (3) ways to overcome these detrimental effects. Recent reports show that LDs in oocytes and embryos are not only energy depots but also are active organelles, possessing many other functions. In addition, analysis of the current literature confirms that lipid phase transition followed by phase separation during cryopreservation is one of the major causes of cryodamage in lipid-rich oocytes and embryos. While LDs and cell membranes are sensitive to chilling and freezing conditions, recent advances in vitrification and delipidation of lipid-rich oocytes and embryos partly mitigate cryodamage. The multidisciplinary approach is critical to reveal mechanisms underlying cryodamage and provides a theoretical basis for optimal cryopreservation of lipid-rich oocytes/embryos. [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]

The ability to compact and inject the cat germinal vesicle (GV) into a recipient cytoplast allows exploration of a new fertility preservation strategy that avoids whole oocyte freezing. The objective of the present study was to understand the impact of water loss and storage time on GV DNA integrity. Immature cat oocytes were exposed to 1.5 M trehalose for 10 min before microwave-assisted dehydration for 0, 5, 10, 15, 20, 25, 30, or 40 min. Oocytes then were rehydrated to assess chromatin configuration and the incidence of DNA fragmentation (TUNEL assay). The moisture content progressively decreased ( p<0.05) from 1.7 to 0.1 gH2O/gDW over the first 30 min, but did not decrease further ( p>0.05) after 40 min. Chromatin configuration was unaffected ( p>0.05) over time. The percentage of GVs with DNA fragmentation was unaltered ( p>0.05) from 0 to 30 min of treatment (range, 6.1%-12%), but increased ( p<0.05) to 32.5% after 40 min. Next, the influence of storage at two different supra-zero temperatures after 30 min of drying was investigated. Oocyte-loaded, microwave-treated filters were individually sealed in Dri-Shield moisture barrier bags and stored at 4°C or ambient temperature for 0 to 8 weeks. Moisture contents gradually decreased ( p<0.05) from 0.12 to 0.10 gH2O/gDW after 8 weeks of storage at 4°C or ambient temperature. The percentage of GVs with DNA fragmentation more than doubled ( p<0.05) from 0 (14.3%) to 2 days (30.0%-33.0%), but remained stable ( p>0.05) thereafter (1 through 4 weeks, 25.0%-35.0%). Collective results demonstrate the feasibility of using microwave processing to dehydrate the mammalian GV to a moisture content that is nonlethal and enables nonfrozen storage, an alternative approach for preserving the maternal genome at cool or ambient temperature. [ABSTRACT FROM AUTHOR]

The author discusses the collecting units of the Smithsonian Institution in the U.S. that comprised of management activities which directly support and enhance exhibition, research and educational programs of the institution. He cites the fundamental factors to the Smithsonian's mission and strategic plan which include acquisition, preservation, and study of collections. He notes the significant role of Smithsonian collections in addressing scientific and societal issues of the 21st century.

Somatic cell biobanking and related technologies, somatic cell nuclear transfer (SCNT), and induction of pluripotent stem cells offer significant promise for wildlife conservation, but have yet to achieve optimal success. Inefficiency and variability in outcome have been linked to incomplete nuclear reprogramming, highlighting the importance of donor cell contribution. Studies show significant differences in SCNT outcome in donor cell lines within and between individuals, highlighting the necessity for a standardized characterization method to evaluate cell line reprogramming potential. Stringently standardized bovine fibroblast cell lines were generated and assessed for inter- and intraindividual variability on cellular (morphology, chromosome number, apoptotic incidence; Experiment 1) and molecular (pluripotency and epigenetic-related gene expression; Experiment 2) levels encompassing putative biomarkers of reprogramming potential. Cellular parameters were similar across cell lines. While some statistically significant differences were observed in DNMT1, DNMT3B, and HAT1, but not HDAC1, their biological relevance could not be determined with the information at hand. This study lays the foundation for understanding cellular characteristics in cultured cell lines; however, further studies are required to determine any correlation with reprogramming potential. [ABSTRACT FROM AUTHOR]

Somatic cell nuclear transfer (SCNT) is an important technique for biological science research. Cytoplasm injection cloning technology (CICT) was developed to improve the reprogramming efficiency as well as to overcome the limitations of SCNT. CICT uses an additional cytoplasm fused with an enucleated oocyte to restore the cytoplasmic volume of the cloned embryo, and this method could improve the reprogramming efficiency of the cloned embryo. In this study, we show that CICT can be adapted to mouse species to overcome the inefficiency of the SCNT method. In this study, results indicate that the two-cell embryo and blastocyst rates of cloned embryos with the use of the CICT method were significantly higher (p < 0.05) than that of the SCNT method (96.6% ± 1.1% vs. 86.7% ± 6.0%, 29.5% ± 2.6% vs. 22.1% ± 3.0%, respectively). Furthermore, the apoptotic cell number per blastocyst was significantly lower in the CICT group than that in the SCNT group (1.7 ± 0.2 vs. 2.9 ± 0.3, p < 0.05). Moreover, the acH3K9/K14 expression level in the CICT group was greater than that of the SCNT group (p < 0.05), and the relative acH3K56 level in the CICT group was significantly (p < 0.05) higher than that in the SCNT group. These results indicate that CICT helps improve the in vitro developmental competence and quality of cloned embryos. [ABSTRACT FROM AUTHOR]

The genetic diversity of Neotropical deer is increasingly jeopardized, owing to declining population size. Thus, the formation of cryobanking of somatic cells is important for the preservation of these species using cloning. The transformation of these cells into viable embryos has been hampered by a lack of endangered species oocytes. Accordingly, the aim of this study was to produce brown brocket deer embryos by interspecific somatic cell nuclear transfer (iSCNT), using goat or cattle oocytes as cytoplasts, and to elucidate embryo mitochondrial activity by measuring the expression levels of ATP6, COX3, and ND5. Cattle embryos produced by in vitro fertilization (IVF) were used as a control. There were no differences in the development of embryos produced by traditional SCNT and iSCNT when using either the goat cytoplasts (38.4% vs. 25.0% cleaved and 40.0% vs. 50.0% morula rates, respectively) or cattle cytoplast (72.8% vs. 65.5% cleaved and 11.3% vs. 5.9% blastocyst rates, respectively). Concerning the gene expression, no significant difference was observed when goat oocytes were used as cytoplasts. However, when using cattle oocytes and 16S as a reference gene, the iSCNT upregulated COX3, when compared with SCNT group. In contrast, when GAPDH was used as a reference gene, all the evaluated genes were upregulated in the iSCNT group, when compared with the IVF group. When compared with the SCNT group, only the expression of ATP6 was statistically different. In conclusion, it was demonstrated that interspecific nuclear transfer is a potentially useful tool for conservation programs of endangered similar deer species. [ABSTRACT FROM AUTHOR]

Reproductive cloning by somatic cell nuclear transfer (SCNT) is a valuable method to propagate service dogs with desirable traits because of higher selection rates in cloned dogs. However, incomplete reprogramming is a major barrier to SCNT, and the assessment of reprogramming is limited to preimplantation embryos and tissues from dead and/or adult tissue. Thus, lifelong monitoring in SCNT dogs can be useful to evaluate the SCNT service dogs for propagation. We applied microarray and qRT-PCR to profile of mRNA and miRNA in whole blood samples collected from four cloned dogs (S), three age-matched control dogs (A), and a donor dog (D). In the analysis of differentially expressed genes in S-A, A-D, and S-D pairs, most genomes were completely reprogrammed and rejuvenated in the cloned offspring. However, several RNAs were differentially expressed. Interestingly, the altered genes are associated with aging and senescence. Furthermore, we identified potential biomarkers such as mirR-223 (NFIB; CLIC4), miRN-494 (ARHGEF12), miR-106b (PPP1R3B; CC2D1A), miR-20a (CC2D1A; PPP1R3B), miR-30e (IGJ; HIRA), and miR-19a (TNRC6A) by miRNA-target mRNA pairing for monitoring rejuvenation, aging/senescence, and reprogramming in cloned dogs. The novel comparative transcriptomic information about SCNT and age-matched dogs can be used to assess the lifelong health of cloned dogs and to facilitate the selection of training animals with minimal invasive procedures. [ABSTRACT FROM AUTHOR]

Mammals are important for balancing the natural ecosystem, but in the past few decades, several species have rapidly been entered under threatened category worldwide. The environmental changes, loss of natural habitats, human activities, and thereby stress are responsible for a gradual decline in reproductive outcome. Stress induces generation of reactive oxygen species (ROS). High physiological level of ROS drives abortive spontaneous egg activation (SEA), while beyond the physiological level causes oxidative stress (OS). The OS induces apoptosis and deteriorates egg quality that limits reproductive outcome. The reduced reproductive outcome is one of the major causes for gradual decline in population size of several mammalian species. Despite having several conservation programs, a gradual decline in species reproductive outcome and their population size is the serious concern for the existence of threatened mammalian species. Thus, it is important to identify and prevent the underlying causes responsible for abortive SEA, which could be an emerging problem for several mammalian species that are threatened or at the verge of extinction. [ABSTRACT FROM AUTHOR]