Cryopreservation of mammalian embryos: Derivation of a method.

In 1972, a procedure was derived to cryopreserve mouse embryos. Over the past four decades, this procedure has been adapted to freeze embryos of more than twenty-five mammalian species. Cryopreservation of embryos has become a routine procedure in both veterinary and human medicine, having been used to freeze millions of embryos of mice and cattle, and many hundreds of thousands of human embryos.

Phenotypic and Molecular Characterization of Domestic Cat (Felis catus) Spermatogonial Stem Cells.

In many mammalian species, surface markers have been used to obtain enriched populations of spermatogonial stem cells (SSCs) for assisted reproduction and other applications; however, little is known about the expression patterns of feline SSCs. In this study, we assessed expression of the SSC surface markers commonly used in other species, KIT, ITGA6, CD9, GFRalpha1, ADGRA3, and THY1, in addition to the less frequently used pluripotent markers TRA-1-60, TRA-1-81, SSEA-1, and SSEA-4 in SSCs of both prepubertal and adult domestic cats (Felis catus). To further characterize cat SSCs, we sorted cells using SSC-specific markers and evaluated the expression of the pluripotent transcription factors NANOG, POU5F1, and SOX2 and the proto-oncogene MYC within these populations.

Birth of a domestic cat kitten produced by vitrification of lipid polarized in vitro matured oocytes.

The ability to cryopreserve oocytes is an effective method to retain valuable genetic material of mammals, including that of endangered animals. Embryos of domestic cats are amenable to cryopreservation, whereas their oocytes are much less cryo-tolerant. The capability of oocytes to survive cryopreservation is affected by several factors, one of which has been hypothesized to be the high concentration of intracellular lipids.

Different patterns of metabolic cryo-damage in domestic cat (Felis catus) and cheetah (Acinonyx jubatus) spermatozoa.

Felid spermatozoa are sensitive to cryopreservation-induced damage, but functional losses can be mitigated by post-thaw swim-up or density gradient processing methods that selectively recover motile or structurally-normal spermatozoa, respectively. Despite the importance of sperm energy production to achieving fertilization, there is little knowledge about the influence of cryopreservation or post-thaw processing on felid sperm metabolism. We conducted a comparative study of domestic cat and cheetah sperm metabolism after cryopreservation and post-thaw processing.

In vivo survival of domestic cat oocytes after vitrification, intracytoplasmic sperm injection and embryo transfer.

We evaluated: (1) cleavage rate after IVF or intracytoplasmic sperm injection (ICSI) of in vivo- and in vitro-matured oocytes after vitrification (experiment 1); and (2) fetal development after transfer of resultant ICSI-derived embryos into recipients (experiment 2). In vivo-matured cumulus-oocyte complexes (COCs) were recovered from gonadotropin-treated donors at 24 h after LH treatment. In vitro-matured oocytes were obtained by mincing ovaries (from local veterinary clinics) and placing COCs into maturation medium for 24 h.

Production of channel catfish with sperm cryopreserved by rapid non-equilibrium cooling.

This report describes the feasibility of using vitrification for fish sperm. Vitrification can be used to preserve samples in the field and offers an alternative to conventional cryopreservation, although it has not been systematically studied for sperm of aquatic species. The overall goal of the project was to develop streamlined protocols that could be integrated into a standardized approach for vitrification of aquatic species germplasm.

The principal variables of cryopreservation: solutions, temperatures, and rate changes.

Objective: To describe several fundamental variables that influence ultimate survival of oocytes and embryos when they are cryopreserved.

Design: The literature describing fundamental and applied aspects of cryobiological variables that determine the responses of oocytes and embryos has been reviewed.

Conclusion(s): When oocytes and embryos are to be cryopreserved, they are suspended in a solution of one of several low-molecular-weight solutes.

Integrating microsatellite and pedigree analyses to facilitate the captive management of the endangered Mississippi sandhill crane (Grus canadensis pulla).

The minimization of kinship in captive populations is usually achieved through the use of pedigree information. However, pedigree knowledge alone is not sufficient if pedigree information is missing, questionable, or when the founders of the captive population are related to one another. If this is the case, higher levels of inbreeding and lower levels of genetic diversity may be present in a captive population than those calculated by pedigree analyses alone.

Oxidative phosphorylation is essential for felid sperm function, but is substantially lower in cheetah (Acinonyx jubatus) compared to domestic cat (Felis catus) ejaculate.

Compared with the normospermic domestic cat, sperm metabolic function is compromised in the teratospermic cat and cheetah, but the pathway(s) involved in this deficiency are unknown. Glycolysis is essential for sperm motility, yet it appears to function normally in spermatozoa of either species regardless of structural morphology. We conducted a comparative study to further understand the mechanisms of energy production in felid spermatozoa, with the hypothesis that oxidative phosphorylation is required for normal sperm function and is impaired in teratospermic ejaculates.

Glycolytic enzyme activity is essential for domestic cat (Felis catus) and cheetah (Acinonyx jubatus) sperm motility and viability in a sugar-free medium.

We have previously reported a lack of glucose uptake in domestic cat and cheetah spermatozoa, despite observing that these cells produce lactate at rates that correlate positively with sperm function. To elucidate the role of glycolysis in felid sperm energy production, we conducted a comparative study in the domestic cat and cheetah, with the hypothesis that sperm motility and viability are maintained in both species in the absence of glycolytic metabolism and are fueled by endogenous substrates. Washed ejaculates were incubated in chemically defined medium in the presence/absence of glucose and pyruvate.

Evidence for compromised metabolic function and limited glucose uptake in spermatozoa from the teratospermic domestic cat (Felis catus) and cheetah (Acinonyx jubatus).

Cheetahs and certain other felids consistently ejaculate high proportions (≥ 60%) of malformed spermatozoa, a condition known as teratospermia, which is prevalent in humans. Even seemingly normal spermatozoa from domestic cat teratospermic ejaculates have reduced fertilizing capacity. To understand the role of sperm metabolism in this phenomenon, we conducted a comparative study in the normospermic domestic cat versus the teratospermic cat and cheetah with the general hypothesis that sperm metabolic function is impaired in males producing predominantly pleiomorphic spermatozoa.

Effect of cryopreservation and in vitro culture of bovine fibroblasts on histone acetylation levels and in vitro development of hand-made cloned embryos.

In this study, the relative acetylation levels of histone 3 in lysine 9 (H3K9ac) in cultured and cryopreserved bovine fibroblasts was measured and we determined the influence of the epigenetic status of three cultured (C1, C2 and C3) donor cell lines on the in vitro development of reconstructed bovine embryos. Results showed that cryopreservation did not alter the overall acetylation levels of H3K9 in bovine fibroblasts analysed immediately after thawing (frozen/thawed) compared with fibroblasts cultured for a period of time after thawing. However, reduced cleavage rates were noted in embryos reconstructed with fibroblasts used immediately after thawing.

Cryopreservation of canine ovarian and testicular fibroblasts.

To derive a practical procedure to store canine somatic cells, fibroblasts isolated from testicular or ovarian tissues were cryopreserved in 1.2 M ethylene glycol or in 1.2 M dimethylsulfoxide prepared in Dulbecco's Modified Eagle Medium as cryoprotectants, and were frozen either in plastic straws or vials.

Production of bovine cloned embryos with donor cells frozen at a slow cooling rate in a conventional freezer (-20 degrees C).

SummaryUsually, fibroblasts are frozen in dimethyl sulphoxide (DMSO, 10% v/v) at a cooling rate of 1 degrees C/min in a low-temperature (-80 degrees C) freezer (LTF) before storage in liquid nitrogen (LN2); however, a LTF is not always available. The purpose of the present study was to evaluate apoptosis and viability of bovine fibroblasts frozen in a LTF or conventional freezer (CF; -20 degrees C) and their subsequent ability for development to blastocyst stage after fusion with enucleated bovine oocytes. Percentages of live cells frozen in LTF (49.

Cryopreservation of oocytes and embryos: optimization by theoretical versus empirical analysis.

Embryos and oocytes were first successfully cryopreserved more than 30 years ago. This procedure has come to be an important, almost essential component in the practice of assisted reproduction in animals and humans. Literally millions of animals of more than 20 species and undoubtedly hundreds of thousands of children have been born from frozen embryos.

Effects of cryoprotectants and cryopreservation on germinal vesicle-stage cumulus-oocyte complexes of rhesus monkeys.

Objective: To determine the effect on rhesus germinal vesicle-stage oocytes enclosed within cumulus cells (COCs) of cryopreservation either by slow, equilibrium cooling or by rapid, non-equilibrium cooling.

Design: Experimental study.

Setting: University primate research center.

Cryopreservation of the germplasm of animals used in biological and medical research: importance, impact, status, and future directions.

Molecular genetics and developmental biology have created thousands of new strains of laboratory animals, including rodents, Drosophila, and zebrafish. This process will accelerate. A decreasing fraction can be maintained as breeding colonies; hence, the others will be lost irretrievably unless their germplasm can be cryopreserved.

Membrane transport properties of equine and macaque ovarian tissues frozen in mixtures of dimethylsulfoxide and ethylene glycol.

The rate at which equine and macaque ovarian tissue sections are first cooled from +25 degrees C to +4 degrees C has a significant effect on the measured water transport when the tissues are subsequently frozen in 0.85 M solutions of glycerol, dimethylsulfoxide (DMSO), or ethylene glycol (EG). To determine whether the response of ovarian tissues is altered if they are suspended in mixtures of cryoprotective agents (CPAs), rather than in solutions of a single CPA, we have now measured the subzero water transport from ovarian tissues that were suspended in mixtures of DMSO and EG.

Male-to-male differences in post-thaw motility of rhesus spermatozoa after cryopreservation of replicate ejaculates.

Background: The efficiency of controlled propagation to produce rhesus monkeys of particular genotypes can be maximized by use of cryopreserved spermatozoa collected from specific males to inseminate appropriate females. But this assumes that semen from males with different genotypes can be cryopreserved with equal effectiveness.

Methods: To investigate whether spermatozoa from different Macaca mulatta males can be effectively cryopreserved when frozen under identical conditions, we collected and froze semen specimens from 13 adult, fertile males maintained at three primate research centers.

Origins of techniques in human and animal embryology.

Increasingly innovative and imaginative techniques are being developed to investigate the development of animal and human embryos. Among the types of techniques that have been developed are ones that deal with oocyte maturation and culture, the isolation and utilization of stem cells, cryopreservation of reproductive cells and tissues, and various procedures to manipulate early embryos. To appreciate the derivation of these sophisticated techniques, it seems appropriate to consider the very early origins of these current techniques.

Subzero water transport characteristics and optimal rates of freezing rhesus monkey (Macaca mulatta) ovarian tissue.

The purpose of the present study was to examine the effect of two different suprazero (room temperature +25 degrees C to +4 degrees C) cooling conditions on the measured water transport response of primate (Macaca mulatta) ovarian tissue in the presence and absence of cryoprotective agents (CPAs). Freshly collected Macaca mulatta (rhesus monkey) ovarian tissue sections were cooled at either 0.5 degrees C/min or 40 degrees C/min from 25 to 4 degrees C.

Suprazero cooling conditions significantly influence subzero permeability parameters of mammalian ovarian tissue.

To model the cryobiological responses of cells and tissues, permeability characteristics are often measured at suprazero temperatures and the measured values are used to predict the responses at subzero temperatures. The purpose of the present study was to determine whether the rate of cooling from +25 to +4 degrees C influenced the measured water transport response of ovarian tissue at subzero temperatures in the presence or absence of cryoprotective agents (CPAs). Sections of freshly collected equine ovarian tissue were first cooled either at 40 degrees C/min or at 0.

Highly efficient vitrification method for cryopreservation of human oocytes.

Two experiments were performed to develop a method to cryopreserve MII human oocytes. In the first experiment, three vitrification methods were compared using bovine MII oocytes with regard to their developmental competence after cryopreservation: (i) vitrification within 0.25-ml plastic straws followed by in-straw dilution after warming (ISD method); (ii) vitrification in open-pulled straws (OPS method); and (iii) vitrification in <0.

Effect of cooling and exposure to ethylene glycol on in vitro maturation and embryo development of rhesus oocytes.

The meiotic spindle of metaphase II-stage oocytes is damaged when mature oocytes are cooled to temperatures close to 0 degrees C, as occurs during cryopreservation by equilibrium cooling. Since a spindle has not yet formed within a germinal vesicle-stage oocyte, it has been suggested that immature oocytes may be more resistant than metaphase II oocytes to cryopreservation by equilibrium cooling. To test this proposition, we examined the effects on rhesus macaque oocytes of chilling and exposure to ethylene glycol (EG) on their maturation and embryo development.