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[Ultrafast freezing of the embryos of outbred and inbred mouse strains].

Survival of 8-cell mouse embryos of inbred strains C57B1 and DBA and outbred strain NMRI was studied after ultrafast freezing. The embryos were placed in 10% glycerol for 10 min, transferred in plastic tubes filled with a mixture of glycerol and 1 M sucrose (3:7), and immersed in liquid nitrogen within 1.5 min. The tubes were thawed in air at the room temperature, the embryos were washed in 0.5 M sucrose and cultivated in a Dulbecco medium with 20% fetal calf serum at 37 degrees C for 48 h or transplanted to recipient females. The survival of embryos was estimated according to their capacity to develop in vitro to the blastocyst stage or form normal fetuses after transplantation. After thawing and cultivation, 80.4% of NMRI embryos retained viability (80.7% in the control). For C57B1 and DBA embryos, these indices were 90.2 and 88.7%, and 59.3 and 66.7%, respectively. The differences between the experiment and control was insignificant for all strains. After thawing and transplantation of NMRI embryos, 34.4% developed into normal live fetuses (in the control 33.3%). At the same time, after cryoconservation by the standard method using a programmed freezer, the viability of ICR and NMRI embryos decreased approximately by 20% even in in vitro experiments. Thus, no differences were found in survival of the embryos of the studied strains after ultrafast freezing. This method proved to be very efficient for the embryos of both inbred and outbred mouse strains, since their capacity for development practically did not decrease after cryoconservation. The method may be used for production of cryobanks of embryos of various mouse strains.

Animals↗
PubMed1992

The legal status of the embryo in comparative perspective.

Almost all decisions with regard to allowing or forbidding research with and on the embryo as well as any other diagnostic invasion into the embryo depend on what kind and range of protection human life in this early stage of its development is or should be entitled to. This question is commonly referred to as that of the 'moral status' of the embryo or-with special regard to legal provisions and sanctions-as its 'legal status'. The answer to this fundamental question, however, is much debated and highly controversial, both nationally and internationally. Therefore professional and legal regulations range from the rather permissive (as in the new English Human Fertilization and Embryology Act of 1990) on the one hand to the total prohibition of embryo research or certain reproductive procedures on the other (as has recently been enacted by the German Embryo Protection Act of 1990). Thus, trying to reach consensus with regard to an embryo's legal/ethical right to protection is made all the more difficult because such an opinion is often, consciously or unconsciously, prejudiced by the desire to give researchers either more or less freedom of action, depending on one's point of view: Those who wish to see diagnostic or other experimental procedures with embryos facilitated, are inclined to deny their human quality from the very start. They base their arguments on the lack of individual personality in the preinidation phase of development, or simply on the fact that in many countries abortion is not illegal at that stage. Those who, on the contrary, find abortion as well as embryo research indefensible, believe their position well-founded by assuming that the embryo, from the time of fertilization, has the individuality and personality of a human being and thus is entitled to its own basic legal rights. Since to me neither of these extreme positions seems to be particularly cogent, I will try in this article to show the reasons for and consequences of adopting a middle course which neither leaves the embryo at free disposition nor bars any kind of diagnostic or other scientific invasion.

Abortion, Legal↗
PubMed2000

Genetic reprogramming of lactate dehydrogenase, citrate synthase, and phosphofructokinase mRNA in bovine nuclear transfer embryos produced using bovine fibroblast cell nuclei.

Adult animal cloning has progressed to allow the production of offspring cloned from adult cells, however many cloned calves die prenatally or shortly after birth. This study examined the expression of three important metabolic enzymes, lactate dehydrogenase (LDH), citrate synthase, and phosphofructokinase (PFK), to determine if their detection in nuclear transfer (NT) embryos mimics that determined for in vitro produced embryos. A day 40 nuclear transfer produced fetus derived from an adult cell line was collected and fetal fibroblast cultures were established and maintained. Reconstructed NT embryos were then produced from this cell line, and RT-PCR was used to evaluate mRNA reprogramming. All three mRNAs encoding these enzymes were detected in the regenerated fetal fibroblast cell line. Detection patterns were first determined for IVF produced embryos (1-cell, 2-cell, 6-8 cell, morula, and blastocyst stages) to compare with their detection in NT embryos. PFK has three subunits: PFK-L, PFK-M, and PFK-P. PFK-L and PFK-P were not detected in bovine oocytes. PFK subunits were not detected in 6-8 cell embryos but were detected in blastocysts. Results from NT embryo RT-PCR demonstrated that PFK was not detected in 8-cell NT embryos but was detected in NT blastocysts indicating that proper nuclear reprogramming had occurred. Citrate synthase was detected in oocytes and throughout development to the blastocyst stage in both bovine IVF and NT embryos. LDH-A and LDH-B were detected in bovine oocytes and in all stages of IVF and NT embryos examined up to the blastocyst stage. A third subunit, LDH-C was not detected at the blastocyst stage in IVF or NT embryos but was detected in all earlier stages and in mature oocytes. In addition, LDH-C mRNA was detected in gonad isolated from the NT and an in vivo produced control fetus. These results indicate that the three metabolic enzymes maintain normal expression patterns and therefore must be properly reprogrammed following nuclear transfer.

Animals↗
PubMed2002

Brain expansion in the chick embryo initiated by experimentally produced occlusion of the spinal neurocoel.

The brain expands in the early chick embryo from pressure generated by accumulation of cerebrospinal fluid (CSF) in a closed neural tube. The sealing of the neural tube occurs as the result of occlusion of the spinal neurocoel rostral to and before closure of the posterior neuropore. We have previously demonstrated the dependence of normal brain expansion upon intraluminal pressure. We had yet to demonstrate, however, that brain expansion actually depends upon natural occlusion of the spinal neurocoel. To demonstrate such dependence, we experimentally occluded the spinal neurocoels of embryos 5 hr younger than stage 11 embryos (in which occlusion of the neurocoel occurs naturally). The stage 10 chick embryos were cultured ex ovo and critically staged, and their spinal neurocoels were occluded using microcautery. All embryos were photographed immediately and at 5, 12, and 24 hr after cautery. Serial sections were made of selected embryos, in which the areas of both the brain and the head were measured. Wilcoxon-Mann-Whitney rank-sum nonparametric tests, Hodges-Lehmann estimators, bootstrapping techniques, and resampling randomization tests were used to determine whether the increases in the brain and head areas for the experimental embryos were significantly different from those of the control embryos during three distinct intervals of expansion: 0-5, 5-12, and 0-12 hr. From 0 to 5 hr, the brains of the precociously occluded embryos expanded significantly more than the brains of the non-occluded controls. From 5 to 12 hr, the brains of the embryos with naturally occluded neurocoels grew significantly larger than the brains of the embryos with precociously occluded neurocoels. At 12 hr, there appeared to be no difference in brain size for these two groups. We conclude that the data support the hypothesis that brain expansion is directly dependent upon occlusion of the spinal neurocoel.

Animals↗
PubMed1982

Development of early somitic mouse embryos in static culture in vitro.

We have compared the in vitro development of 8.5-day early somitic mouse embryos cultured in static organ cultures dishes in human cord serum or rat serum with the development of embryos of the same age cultured in rotating tubes or developing in vivo. The growth of embryos cultured in rat serum in organ culture dishes was comparable during the first 24 hours, with the growth achieved in rotating tubes and paralleled the embryonic growth in vivo. The morphogenesis of embryos in static organ culture was delayed in comparison with the normal embryogenesis in vivo, but was not statistically different from embryos developing in rotating tube cultures. Embryos cultured for 36 hours continued to grow but were almost all lagging in development behind the embryos developing in utero or in the rotating tubes. Our data show that 24-hour culture of somitic mouse embryos in rat serum may be used to evaluate the growth of explanted embryos, and to study the in vitro development and function of certain embryonic structures, such as somite number, yolk sac circulation, heart function, and development of allantois. Static culture is inadequate for the study or evaluation of neural fold closure, branchial arch formation, anterior limb bud formation, and axial turning of the explanted embryos. Embryo cultures in rat serum yield better results than do cultures in human cord serum.

Animals↗
PubMed2003

Gene expression and in vitro development of inter-species nuclear transfer embryos.

This study examined the chromatin morphology, in vitro development, and expression of selected genes in cloned embryos produced by transfer of mouse embryonic fibroblasts (MEF) into the bovine ooplasm. After 6 hr of activation, inter-species nuclear transfer (NT) embryos (MEF-NT) had one (70%) or two pronuclei (20%), respectively. After 72 hr of culture in vitro, 62.6% of the MEF-NTs were arrested at the 8-cell stage, 31.2% reached the 2- to 4-cell stage, and only 6.2% had more than eight blastomeres, but none of these developed to the blastocyst stage. Whereas, 20% of NT embryos derived from bovine embryonic fibroblast fused with bovine ooplasm (BEF-NT) reached the blastocyst stage. Donor MEF nuclei expressing an Enhanced Green Fluorescent Protein (EGFP) transgene resulted in 1- to 8-cell stage MEF-NT that expressed EGFP. The expression of selected genes was examined in 8-cell MEF-NTs, 8-cell mouse embryos, enucleated bovine oocytes, and MEFs using RT-PCR. The mRNA for heat shock protein 70.1 (Hsp 70.1) gene was detected in MEF-NTs and MEF, but not in mouse embryos. The hydroxy-phosphoribosyl transferase (HPRT) mRNA was found in normal mouse embryos and MEF but not in MEF-NTs. Expression of Oct-4 and embryonic alkaline phospatase (eAP) genes was only detected in normal mouse embryos and not in the inter-species NT embryos. Abnormal gene expression profiles were associated with an arrest in the development at the 8-cell stage, but MEF-NT embryos appeared to have progressed through gross chromatin remodeling, typical of intra-species NT embryos. Therefore, molecular reprogramming rather than chromatin remodeling may be a better indicator of nuclear reprogramming in inter-species NT embryos.

Animals↗
PubMed1993

Nuclear transfer in cattle: effect of nuclear donor cells, cytoplast age, co-culture, and embryo transfer.

There are many factors affecting the efficiency of nuclear transfer technology. Some are evaluated here using our novel approach by enucleating oocytes at 20-22 hr after in vitro maturation (IVM), culturing the enucleated oocytes (cytoplasts) for 8-10 hr or 18-20 hr to gain activation competence and then conducting nuclear transfer. In the first experiment, we demonstrated that cumulus cell (CC) monolayer can support some cloned embryos to develop into morulae or blastocysts. Co-culture with CC and bovine oviduct epithelial cell (BOEC) monolayers resulted in no differences (P > 0.05) in supporting the development of cloned embryos (Experiment 2). When in vitro matured oocytes were enucleated at 22 hr after IVM followed by nuclear transfer 18-20 hr later, cleavage and morula or blastocyst development of the cloned embryos were similar to those resulting from the enucleated oocytes which had been matured in vivo (Experiment 3). Frozen embryos as nuclear donor cells worked equally well as fresh embryos for cloning in embryo development which was superior to IVF embryos (Experiment 4). However, fresh embryos resulted in a higher proportion (P < 0.05) of blastomere recovery than did frozen of IVF embryos. Finally, embryo transfer of cloned embryos from our procedure produced a viable calf, demonstrating the commercial value of this novel approach of the technology.

Animals↗
PubMed1988

Fine structure of early human embryos frozen with 1,2 propanediol.

Previous studies by a French group (Fertil Steril 44:645-651, 1985) have shown that two- to eight-cell human embryos can survive slow freeze-thawing with propanediol in a biological freezer. These embryos were assessed for morphological appearance by phase-contrast microscopy. We assessed the structure of 25 frozen-thawed one- to 12-cell embryos, obtained from our in vitro fertilization (IVF) and GIFT programmes, by phase-contrast and electron microscopy, using the same method of cryopreservation. One-fourth of the embryos examined had all cells intact, and more than one-half the embryos had over 50% of their cells well preserved. Some of these embryos had unequal blastomeres and cytoplasmic fragments. Ultrastructural assessment revealed good preservation of fine structure in the intact blastomeres of all embryos and maintenance of cell-to-cell contacts. Most cytoplasmic organelles, cell membranes, and nuclei were well preserved compared to nonfrozen controls. The cells that were cryoinjured showed varying degrees of disorganization of the cell membrane, cytosol, and cellular membranes, including swelling and disruption of the nuclear envelope. Disruption of the zona was somewhat rare. Small cytoplasmic fragments were less prone to cryoinjury than blastomeres. The use of propanediol for embryo cryopreservation seems to be feasible; frozen embryos with more than 50% cells intact have produced 10 pregnancies after embryo transfer (Fertil Steril 46:268-272, 1986). Replacement of 17 frozen embryos in seven patients has resulted in a twin pregnancy in Singapore. However, the effects of freezing on the mitotic spindles of embryonic cells need to be investigated further.

Blastomeres↗
PubMed2004

Ovine ooplasm directs initial nucleolar assembly in embryos cloned from ovine, bovine, and porcine cells.

Here we present ultrastructural and immunocytochemical evidence that ovine ooplasm is directing the initial assembly of the nucleolus independent of the species of the nuclear donor. Intergeneric porcine-ovine somatic cell nuclear transfer (SCNT) and intrageneric ovine-ovine SCNT embryos were constructed and the nucleolus ultrastructure and nucleolus associated rRNA synthesis examined in 1-, 2-, 4-, early 8-, late 8-, and 16-cell embryos using transmission electron microscopy (TEM) and light microscopical autoradiography. In addition, immunocytochemical localization by confocal microscopy of nucleolin, a key protein involved in processing rRNA transcripts, was performed on early 8-, late 8-, and 16-cell embryos for both groups of SCNT embryos. Intergeneric porcine-ovine SCNT embryos exhibited nucleolar precursor bodies (NPBs) of an ovine (ruminant) ultrastructure, but no active rRNA producing fibrillo-granular nucleoli at any of the stages. Unusually, cytoplasmic organelles were located inside the nucleus of two porcine-ovine SCNT embryos. The ovine-ovine SCNT embryos, on the other hand, revealed fibrillo-granular nucleoli in 16-cell embryos. In parallel, autoradiographic labeling over the nucleoplasm, and in particular, the nulcleoli was detected. Bovine-ovine SCNT embryos at the eight-cell stage were examined for nucleolar morphology and exhibited ruminant-type NPBs as well as structures that appeared as fibrillar material surrounded by a rim of electron dense granules, perhaps formerly of nucleolar origin. Nucleolin was localized throughout the nucleoplasm and with particular intensity around the presumptive nucleolar compartments for all developmental stages examined in porcine-ovine and ovine-ovine SCNT embryos. In conclusion, this study suggests that factors within the ovine ooplasm are playing a role in the initial assembly of the embryonic nucleolus in intrageneric SCNT embryos.

Animals↗
PubMed2005

Embryotropic effect of insulin-like growth factor (IGF)-I and its receptor on development of porcine preimplantation embryos produced by in vitro fertilization and somatic cell nuclear transfer.

Insulin-like growth factor (IGF)-I is a receptor-mediated autocrine/paracrine growth/survival factor for mammalian embryo development. The present study investigated the temporal expression and regulation of porcine IGF-I receptor (IGF-IR) mRNA and the role of IGF-I on development of porcine in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos. As assessed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), the level of IGF-IR mRNA expression was high in unfertilized oocytes, 2-cell and 4-cell embryos and gradually decreased in 8-cell embryos, morulae, and blastocysts in both IVF and SCNT series. The IVF or SCNT embryos were cultured with 0, 1, 10, 50, or 100 ng/ml IGF-I for 168 hr. Supplementing with 50 ng/ml IGF-I increased blastocyst formation and the number of cells in inner cell masses (ICMs) in both IVF and SCNT embryos. In a second experiment, more blastocysts were obtained when IVF or SCNT embryos were cultured for the first 48 hr or for the entire 168 hr with 50 ng/ml IGF-I compared to culturing without IGF-I for 48 hr or with IGF-I for the last 120 hr or without IGF-I for the entire 168 hr. Treating IVF or SCNT embryos with 50 ng/ml IGF-I significantly up-regulated IGF-IR mRNA compared to untreated control embryos. In conclusion, the present study demonstrated that IGF-IR mRNA is expressed in porcine IVF and SCNT embryos, and that IGF-I improved the developmental competence of IVF and SCNT embryos through its specific receptors.

Animals↗
PubMed2002

Homocysteine modifies development of neurulation and dorsal root ganglia in chick embryos.

BACKGROUND: The formation of the neural tube (neurulation) involves two mechanisms: primary and secondary neurulation. In chicks, there is also an overlap zone, where both mechanisms work together. Homocysteine (Hcy) may have an important teratogenic role in neural tube defects (NTD) when folic acid levels are considered normal. Recently, Hcy capability to generate NTD and modify neural crest cell migration has been demonstrated in chick embryos. This study was aimed to evaluate the effects of Hcy on neurulation and the development of the dorsal root ganglia (DRG). METHODS: Chick embryos were treated with L-Hcy thiolactone 20 micromol to produce the highest rate of survival with embryos carrying neural tube defect (NTD) in the spine. Embryos at stages (st) 3-10 were treated and harvested at st 18-23. Only externally normal embryos or those carrying spinal NTD embryos were considered. RESULTS: Histological sections of Hcy-treated embryos showed: open spina bifida (39% of embryos), more than one tube forming the spinal cord (26%), disorganized spinal cord (26%), always affecting lumbosacral regions, probably in the overlap zone. Additionally, 32% of embryos had small and continuous DRG, associated with a slimmed roof plate. Three-dimensional reconstruction showed unsegmented DRG until the C8 ganglion level. There was a 75% reduction of C3 DRG cells in treated embryos in comparison to untreated ganglia. CONCLUSION: Hcy teratogenicity in avian embryos affected the neural tube in the overlap zone, secondary neurulation and the cervical DRG.

Animals↗
PubMed1980

Differential embryonic cardiovascular responses to acute maternal uterine ischemia: an in vivo microscopic study of rabbit embryos with either intact or clamped umbilical cords.

The effects of acute episodes of maternal uterine ischemia on the cardiovascular system of New Zealand white rabbit embryos were studied by in vivo microscopic methods. On gestational day 12, gravid uteri were surgically externalized, and the walls of the implantation sites were carefully incised and reflected to reveal the embryos. Throughout all procedures, temperature, water, and electrolyte homeostasis were preserved. The embryos within each litter were divided into two groups: 1) two randomly selected embryos whose umbilical cords were clamped, and 2) the remaining embryos of the litter whose umbilical cords were uncompromised. Uterine ischemia was induced by clamping all uterine arterial and venous channels. Occlusion of the maternal uterine vessels produced severe uterine cyanosis within 1-2 min. The embryos whose umbilical cords were not clamped exhibited severe cardiovascular derangements beginning within 3-4 min. In contrast, the embryos whose umbilical cords were clamped (thereby interrupting both umbilical and vitelline circulations) had no observable cardiovascular alterations. The absence of effects in embryos with clamped umbilical cords suggests that acute episodes of hypoxia are well tolerated by young embryos. In contrast, the rapid appearance of cardiovascular derangements after interruption of maternal uterine circulation in embryos with unclamped umbilical cords emphasizes the idea that even brief intervals of uterine ischemia are deleterious to the rabbit embryo.

Animals↗
PubMed1984

The role of dysrhythmic heart function during cardiovascular teratogenesis in epinephrine-treated chick embryos.

Chick embryos were treated with a teratogenic dose (5 micrograms) of epinephrine hydrochloride at 4 days of development (stage 24). Heart rates were determined at 20, 40, and 60 minutes after treatment. The mean heart rate values for epinephrine-treated embryos were significantly less than values obtained for untreated and saline-treated control embryos. The decrements in mean heart rate could be explained by severe cardiac dysrhythmias that occurred in approximately 40% of epinephrine-treated embryos. The remaining 60% of embryos exhibited heartbeat patterns similar to controls. This finding enabled us to separate epinephrine-treated embryos into two physiologically distinct groups: dysrhythmia-positive and dysrhythmia-negative. Dysrhythmias were characterized by periods of bradycardia alternating with periods of asystole and were confirmed by electrocardiography. EKG data suggest that epinephrine induces cardiac conduction disturbances in some chick embryos. An additional experiment was conducted to identify the frequencies at which abnormally obliterated aortic arches, abnormally persistent aortic arches, ventricular septal defects, and embryonic death occurred in dysrhythmia-positive and negative embryos. Abnormally obliterated vessels and embryonic death occurred with significantly greater frequencies in dysrhythmia-positive embryos. Abnormally persistent vessels and ventricular septal defects occurred with significantly greater frequencies in dysrhythmia-negative embryos. We conclude that the production of specific malformations and decreased probability for survival in epinephrine-treated embryos are related to dysrhythmogenesis and bradycardia.

Abnormalities, Drug-Induced↗
PubMed1988

Strain differences in the teratogenicity induced by sodium valproate in cultured mouse embryos.

Strain differences in the teratogenicity of valproic acid (VPA) have been reported in mice. Finnell and Chernoff (Proc. Grnwd. Genet. Ctr. 5:162-163, 1985) showed that 300 mg/kg of VPA twice a day on days 6-8 of gestation induced exencephaly in 82% of SWV embryos but in 0% of C57BL/6J embryos. In the present experiment, we have collected similar results and investigated this strain difference using whole embryo culture in an attempt to determine whether maternal or embryonic factors are responsible for the difference. Mouse embryos were explanted on day 8.5 (plug day 0), and embryos at the 6-8-somite stage were cultured for 48 hours in rat serum containing various doses of sodium valproate (NaVP). All the embryos died within 24 hours with 4.5-mM and higher doses of NaVP in C57BL/6NCr1BR (C57) and with 3.0-mM and higher doses in SWV. Unfused brain folds were recognized in embryos treated with 3.0-mM and higher doses in C57, and with 1.0-mM and higher doses in SWV. Irregular somite formation was observed in many embryos treated with 1.6-mM and higher doses in C57 and with 1.0-mM and higher doses in SWV. These results indicate that SWV embryos have 1.5-3 times the sensitivity of C57 embryos to the embryolethal and teratogenic effects of NaVP. Furthermore, the results suggest that the basis of the strain difference resides within the embryo rather than the mother.

Animals↗
PubMed1991

Cadherin mRNAs during rat embryo development in vivo and in vitro.

Whole rat embryo cultures are being used in increasing numbers of laboratories to study the mechanisms by which teratogens disturb development. The development of early somite stage embryos in vitro is very similar morphologically to that in vivo, yet few biochemical comparisons have been made. The purpose of this study was to determine the steady-state mRNA concentrations of a family of Ca(2+)-dependent cell adhesion molecules, the cadherins, during rat embryonic development in vivo and in vitro. Embryos and yolk sacs were collected on days 10, 11, and 12 of gestation (in vivo); they were also obtained from day 10 embryos after growth in culture for 24 hr (day 11 in vitro) or 45 hr (day 12 in vitro). Total RNAs isolated from embryos and yolk sacs were studied by Northern blot analysis using specific cDNA probes for three cadherins, E-cadherin, N-cadherin, and P-cadherin. Although E-cadherin mRNA was detected in embryos, it was present at much higher concentrations in yolk sacs. In addition, multiple species of E-cadherin mRNA ranging from 3.0 to 13 kb were detected. Interestingly, the concentration of the major 4.5-kb E-cadherin mRNA species in yolk sac after 45 hr in culture was increased 2.8-fold over that on day 12 of gestation in vivo. Second, two species (4.3 and 3.5 kb) of N-cadherin mRNA were detected, almost exclusively in embryos. In yolk sac, N-cadherin mRNA was detected only after 45 hr in culture. Third, P-cadherin mRNA was detected as a single 3.5-kb species, mainly in embryos. P-cadherin mRNA concentrations in yolk sac after 45 hr in culture were 5.6-fold higher than in vivo. Thus, these results demonstrate that there is a differential distribution of cadherin mRNAs in rat embryos and yolk sacs. Further, there appear to be multiple species of mRNAs for E-cadherin and N-cadherin. Finally, while whole embryo culture in vitro did not significantly alter the steady-state concentrations of cadherin mRNAs in the embryo, these concentrations were dramatically increased in the yolk sac.

Animals↗
PubMed1995

In vitro and in vivo survival of cryopreserved sheep embryos.

Experiments were conducted to devise an efficient method to cryopreserve ovine embryos for field application. Embryos were surgically collected from superovulated ewes on Day 6 after natural breeding; oocytes were collected from ovaries obtained at the abattoir. Osmotic behavior of oocytes and embryos was determined by measuring their responses to hypertonic solutions of CsCl or sucrose. Embryos and oocytes contracted osmotically by decreasing their volumes proportionally to the reciprocal of the solution's osmolality. The respective nonosmotic volumes of embryos in CsCl and sucrose were 13.8 and 13.5% of their isotonic volume, and those of oocytes were 18.5 and 19.6%. Tests of the permeability of morulae to commonly used cryoprotectants, ethylene glycol (EG), propylene glycol (PG), dimethyl sulfoxide (DMSO), and glycerol (Glyc), showed that the order of permeability was EG > PG > DMSO approximately Glyc. Comparison of the efficacy of cryoprotective agents indicated that the respective survivals of embryos frozen in EG, PG, and DMSO were 76.9, 62.5, and 55.6%, based on their development into hatched blastocysts in vitro. Therefore, EG appeared to be superior to the other two cryoprotectants for freezing sheep embryos. To determine the functional survival of embryos in vivo, 67 embryos frozen in EG were thawed and directly diluted with phosphate-buffered saline; 47 of these (70%) appeared morphologically normal and were transferred into 14 recipients. Five of these recipients, which had received a total of 16 embryos, became pregnant. Ten lambs were born, showing that the method employed in this study for cryopreservation of sheep embryos followed by their direct dilution out of EG has potential application for practical field use.

Animals↗
PubMed1997

Chill sensitivity and cryoprotectant permeability of dechorionated zebrafish embryos, Brachydanio rerio.

The zebrafish (Brachydanio rerio) was used as a model for basic studies of the chilling sensitivity, permeability and toxicity of cryoprotectants. In both intact and dechorionated embryos, early-stage embryos (1.25, 1.5, 1.75, and 2 h) were more susceptible (P < 0.05) to chilling injury at 0 degrees C than late-stage embryos (50, 75, and 100% epiboly and three-somite stage). Moreover, enzymatic removal of the chorion did not alter (P > 0.05) this pattern of sensitivity to chilling. Eight-hour zebrafish embryos tolerated short-term exposures to temperatures ranging from 4 to 23 degrees C for 3.5 h with no detrimental developmental effects. The permeability of dechorionated embryos to cryoprotectants was examined by measuring the kinetics of volumetric change at various developmental stages (16 cells to six somites or ca. 1.25 to 14 h postfertilization) at 28.5 degrees C. The dechorionated zebrafish embryo is composed of two complex cellular compartments (i.e., a large yolk and the developing blastoderm). From 40 to 100% epiboly, the volumes of yolk and blastoderm remained constant, ca. 82 and 18%, respectively. However, these volumes changed rapidly after epiboly. For example, at the six-somite stage, the yolk composed 61% of the total volume, whereas the blastoderm composed 39%. When three- and six-somite embryos were placed in 1.5 and 2.0 M cryoprotectants (dimethyl sulfoxide and propylene glycol), osmometric measurement of volume changes indicated no permeation of the cryoprotectants. However, some permeation was observed for six-somite embryos immersed in a 2.0 M methanol solution, but not for 3-somite embryos. For up to 30 min at room temperature, these cryoprotectant solutions were toxic to zebrafish embryos; however, 1.5 M glycerol and ethylene glycol solutions were. We conclude that the complex nature of the zebrafish embryo reduces the effectiveness and predictive value of light microscopical measurements for cryoprotectant permeability studies.

Animals↗
PubMed1998

Production of monozygotic twins after freezing and thawing of bisected mouse embryos.

We examined the viability of mouse bisected embryos after freezing and thawing and produced monozygotic twin mice from these embryos. Two-cell embryos were collected from superovulated mature agouti, F1 hybrid (C57BL/6 x CBA) female mice. For bisection, one blastomere of the embryo was aspirated with a micropipette and injected into an empty zona pellucida. After culture for 24 to 28 h to the compacted 4- to 8-cell stage or 48 to 52 h to the late morula to blastocyst stage, the embryos were slowly frozen (-0.5 to -1.0 degrees C/min), thawed (30 degrees C/min), cultured for 24 h, and then transferred to recipient females. The bisected embryos without zonae pellucidae had developmental ability in vitro similar to those with zonae pellucidae (88% vs 89%). However, after freezing and thawing at the compacted 4- to 8-cell stage, bisected embryos with zonae pellucidae had higher viability than those without (60% vs 15%). Zona enclosed, bisected embryos frozen at the compacted 4- to 8-cell stage were more resistnat to freezing and thawing than those at the late morula to blastocyte stage (60% vs 23%). After transfer to recipients 26% of the zona enclosed bisected embryos frozen-thawed at the 4- to 8-cell stage developed to living fetuses a day 17.5 to 18.0 of pregnancy, which was slightly but not significantly lower than that of fresh bisected embryos (48%). On the other hand, only 5% of bisected embryos frozen-thawed at the late morula to blastocyst stage developed to young. The transfer of 15 sets of twin blastocysts as pairs that had been frozen and thawed at the compacted 4- to 8-cell stage yielded 2 (13%) sets of monozygotic twins.

Animals↗