Rapid replacement of somatic linker histones with the oocyte-specific linker histone H1foo in nuclear transfer

The most distinctive feature of oocyte-specific linker histones is the specific timing of their expression during embryonic development. In Xenopus nuclear transfer, somatic linker histones in the donor nucleus are replaced with oocyte-specific linker histone B4, leading to the involvement of oocyte-specific linker histones in nuclear reprogramming. We recently have discovered a mouse oocyte-specific linker histone, named H1foo, and demonstrated its expression pattern in normal preimplantation embryos. The present study was undertaken to determine whether the replacement of somatic linker histones with H1foo occurs during the process of mouse nuclear transfer. H1foo was detected in the donor nucleus soon after transplantation. Thereafter, H1foo was restricted to the chromatin in up to two-cell stage embryos. After fusion of an oocyte with a cell expressing GFP (green fluorescent protein)-tagged somatic linker histone H1c, immediate release of H1c in the donor nucleus was observed. In addition, we used fluorescence recovery after photobleaching (FRAP), and found that H1foo is more mobile than H1c in living cells. The greater mobility of H1foo may contribute to its rapid replacement and decreased stability of the embryonic chromatin structure. These results suggest that rapid replacement of H1c with H1foo may play an important role in nuclear remodeling.     

In vitro development of reconstructed porcine oocytes after somatic cell nuclear transfer

This study was designed to examine the developmental ability of porcine embryos after somatic cell nuclear transfer. Porcine fibroblasts were isolated from fetuses at Day 40 of gestation. In vitro-matured porcine oocytes were enucleated and electrically fused with somatic cells. The reconstructed eggs were activated using electrical stimulus and cultured in vitro for 6 days. Nuclear-transferred (NT) embryos activated at a field strength of 120 V/mm (11.6 +/- 1.6%) showed a higher developmental rate as compared to the 150-V/mm group (6.5 +/- 2.3%) (P: < 0.05), but the mean cell numbers of blastocysts were similar between the two groups. Rates of blastocyst development from NT embryos electrically pulsed at different times (2, 4, and 6 h) after electrofusion were 11.6 +/- 2.9, 6.6 +/- 2.3, and 8.1 +/- 3.3%, respectively. The mean cell numbers of blastocysts developed from NT embryos were gradually decreased (30.4 +/- 10.4 > 24.6 +/- 10.1 > 16.5 +/- 7.4 per blastocyst) as exposure time (2, 4, and 6 h) of nuclei to oocyte cytoplast before activation was prolonged. There was a significant difference in the cell number between the 2- and 6-h groups (P: < 0. 05). Nuclear-transferred embryos (9.4 +/- 0.9%) had a lower developmental rate than in vitro fertilization (IVF)-derived (21.4 +/- 1.9%) or parthenogenetic embryos (22.4 +/- 7.2%) (P: < 0.01). The mean cell number (28.9 +/- 11.4) of NT-derived blastocysts was smaller than that (38.6 +/- 10.4) of IVF-derived blastocysts (P: < 0. 05) and was similar to that (29.9 +/- 12.1) of parthenogenetic embryos. Our results suggest that porcine NT eggs using somatic cells after electrical activation have developmental potential to the blastocyst stage, although with smaller cell numbers compared to IVF embryos.     

Development of vitrified-thawed bovine oocytes after in vitro fertilization and somatic cell nuclear transfer

Cryopreservation could be a useful technique for providing a steady source of oocytes for nuclear transfer and in vitro embryo production. The purpose of this study was to develop a method for cryopreservation of bovine oocytes while maintaining the developmental potential following subsequent in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT). Following vitrification-thawing, the surviving oocytes were (a) used for parthenogenetic activation, (b) examined for pronuclear formation after IVF, (c) examined for embryo development after IVF, and (d) used for SCNT employing fetal fibroblasts transfected with green fluorescent protein (GFP) gene. While most of the oocytes survived vitrification when the microdrop method was used (92.50%), the cleavage and blastocyst formation rates after parthenogenetic activation were lower (46.5% and 11.1%) than that in the non-vitrified control (86.6% and 13.5%). After IVF, the pronuclear formation (2PN) of fertilized embryos was lower in the vitrified group than in the control (21.7% and 59.9%). After SCNT, fusion rates were similar in control (58.33%) and vitrified-thawed oocytes (53.19%). However, the cleavage (73.1% and 46.3%) and blastocyst formation rates (22.2%, 7.4%; p<0.05) differed between control and vitrified-thawed oocytes. In vitrified-thawed or control oocytes, all embryos reconstructed using fetal fibroblasts transfected with GFP gene showed GFP expression. To evaluate the complete developmental potential, embryos derived from vitrified-thawed and fresh control oocytes were non-surgically transferred to 27 recipients (16 for control and 11 for vitrified-thawed). In the vitrified-thawed group, two pregnancies were detected at day 60, and one of them lasted until day 222. While in the fresh group, one pregnancy maintained to term. In conclusion, vitrified-thawed bovine oocytes could support development into the subsequent stages after IVF and SCNT. In addition, this study showed the possibility of the vitrified-thawed bovine oocytes in the production of transgenic cloned animals. In addition, further studies are required to increase the efficiency of oocyte vitrification for the practical uses and production of live offspring.     

Differential cytoplast requirement for embryonic and somatic cell nuclear transfer in cattle

Effective activation of a recipient oocyte and its compatibility with the nuclear donor are critical to the successful nuclear reprogramming during nuclear transfer. We designed a series of experiments using various activation methods to determine the optimum activation efficiency of bovine oocytes. We then performed nuclear transfer (NT) of embryonic and somatic cells into cytoplasts presumably at G1/S phase (with prior activation) or at metaphase II (MII, without prior activation). Oocytes at 24 hr of maturation in vitro were activated with various combinations of calcium ionophore A23187 (A187) (5 microM, 5 min), electric pulse (EP), ethanol (7%, 7 min), cycloheximide (CHX) (10 micro g/ml, 6 hr), and then cultured in cytochalasin D (CD) for a total of 18 hr. Through a series of experiments (Exp. 1-4), an improved activation protocol (A187/EP/CHX/CD) was identified and used for comparison of NT efficiency of embryonic versus somatic donor cells (Exp. 5). When embryonic cells from morula and blastocysts (BL) were used as nuclear donors, a significantly higher rate of blastocyst development from cloned embryos was obtained with G1/S phase cytoplasts than with MII-phase cytoplasts (36 vs. 11%, P < 0.05). In contrast, when skin fibroblasts were used as donor cells, the use of an MII cytoplast (vs. G1/S phase) was imperative for blastocyst development (30 vs. 6%, P < 0.05). Differential staining showed that parthenogenetic, embryonic, and somatic cloned BL contained 26, 29, and 33% presumptive inner cell mass (ICM) cells, respectively, which is similar to that of frozen-thawed in vivo embryos at a comparable developmental stage (23%). These data indicate that embryonic and somatic nuclei require different recipient cytoplast environment for remodeling/ reprogramming, and this is likely due to the different cell cycle stage and profiles of molecular differentiation of the transferred donor nuclei.     

Use of strontium in the activation of bovine oocytes reconstructed by somatic cell nuclear transfer

As an important step in the nuclear transfer (NT) procedure, we evaluated the effect of three different treatments for oocyte activation on the in vitro and in vivo developmental capacity of bovine reconstructed embryos: (1) strontium, which has been successfully used in mice but not yet tested in cattle; (2) ionomycin and 6-dimethylaminopurine (6-DMAP), a standard treatment used in cattle; (3) ionomycin and strontium, in place of 6-DMAP. As regards NT blastocyst development, no difference was observed when strontium (20.1%) or ionomycin/6-DMAP (14.4%) were used. However, when 6-DMAP was substituted by strontium (3), the blastocyst rate (34.8%) was superior to that in the other activation groups (p < 0.05). Results of in vivo development showed the possibility of pregnancies when NT embryos activated in strontium were transferred to recipient cows (16.6%). A live female calf was obtained when ionomycin/strontium were used, but it died 30 days after birth. Our findings show that strontium can be used as an activation agent in bovine cloning procedures and that activation with a combination of strontium and ionomycin increased the in vitro developmental capacity of reconstructed embryos. This is the first report of a calf produced by adult somatic cell NT in Latin America.     

DNA甲基化修饰与体细胞核移植的关系

成功的体细胞核移植（somatic cell nuclear transfer,SCNT）有赖于供体细胞的基因组通过重编程恢复到支持胚胎发育的全能性状态。但是,相比起自然受精后发生的重编程来说,要诱导一个已经分化的供体细胞重编程为全能性状态,往往在时间上和程度上都是迟滞的和不完全的。同时,DNA甲基化状况又是影响克隆胚胎发育和基因表达的关键因素之一。因此,深入研究主导DNA甲基化修饰的分子机理,探讨DNA去甲基化在供体细胞重编程过程中扮演的角色,从而进一步提高供体细胞重编程效率,提高克隆胚的发育潜能,这对于体细胞核移植效率的提高具有重要的意义。     

Bovine SNRPN methylation imprint in oocytes and day 17 in vitro-produced and somatic cell nuclear transfer embryos

Findings from recent studies have suggested that the low survival rate of animals derived via somatic cell nuclear transfer (SCNT) may be in part due to epigenetic abnormalities brought about by this procedure. DNA methylation is an epigenetic modification of DNA that is implicated in the regulation of imprinted genes. Genes subject to genomic imprinting are expressed monoallelically in a parent of origin-dependent manner and are important for embryo growth, placental function, and neurobehavioral processes. The vast majority of imprinted genes have been studied in mice and humans. Herein, our objectives were to characterize the bovine SNRPN gene in gametes and to compare its methylation profile in in vivo-produced, in vitro-produced, and SCNT-derived Day 17 elongating embryos. A CpG island within the 5' region of SNRPN was identified and examined using bisulfite sequencing. SNRPN alleles were unmethylated in sperm, methylated in oocytes, and approximately 50% methylated in somatic samples. The examined SNRPN region appeared for the most part to be normally methylated in three in vivo-produced Day 17 embryos and in eight in vitro-produced Day 17 embryos examined, while alleles from Day 17 SCNT embryos were severely hypomethylated in seven of eight embryos. In this study, we showed that the SNRPN methylation profiles previously observed in mouse and human studies are also conserved in cattle. Moreover, SCNT-derived Day 17 elongating embryos were abnormally hypomethylated compared with in vivo-produced and in vitro-produced embryos, which in turn suggests that SCNT may lead to faulty reprogramming or maintenance of methylation imprints at this locus.     

Vitamin C enhances in vitro and in vivo development of porcine somatic cell nuclear transfer embryos

The reprogramming of differentiated cells into a totipotent embryonic state through somatic cell nuclear transfer (SCNT) is still an inefficient process. Previous studies revealed that the generation of induced pluripotent stem (iPS) cells from mouse and human fibroblasts could be significantly enhanced with vitamin C treatment. Here, we investigated the effects of vitamin C, to our knowledge for the first time, on the in vitro and in vivo development of porcine SCNT embryos. The rate of blastocyst development in SCNT embryos treated with 50 μg/mL vitamin C 15 h after activation (36.0%) was significantly higher than that of untreated SCNT embryos (11.5%). The enhanced in vitro development rate of vitamin C-treated embryos was associated with an increased acetylation level of histone H4 lysine 5 and higher Oct4, Sox2 and Klf4 expression levels in blastocysts, as determined by real-time PCR. In addition, treatment with vitamin C resulted in an increased pregnancy rate in pigs. These findings suggest that treatment with vitamin C is beneficial for enhancement of the in vitro and in vivo development of porcine SCNT embryos.     

Caffeine treatment prevents age-related changes in ovine oocytes and increases cell numbers in blastocysts produced by somatic cell nuclear transfer

Maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) are key regulators of both meiotic and mitotic cycles. Oocytes arrested at metaphase of the second meiotic division (MII) contain high levels of both kinases; however, these activities decline with age. Caffeine (an inhibitor of Myt1/Wee1 activity) can increase MPF and MAPK activities in ovine oocytes; however, the effects of caffeine treatment on the activation, nuclear configuration and developmental potential of ovine SC nuclear transfer (SCNT) embryos were unknown. We examined the effects of aging and caffeine treatment on MPF and MAPK activities, activation, development, and nuclear remodeling of SCNT embryos. Both kinases reached maximum activities at 24-h postonset of maturation (hpm) and then decreased with time. The decline in MPF activity occurred rapidly, whereas MAPK activity declined more slowly. Caffeine treatment (10.0 mM) of aging oocytes prevented the decline in activities associated with both kinases and prevented the acquisition of activation competence by a single activation stimulus. However, treatment of aged oocytes with caffeine could not increase kinase activities or reverse the acquisition of activation competence. Enucleation did not affect kinase activities, but caffeine treatment significantly increased both. Caffeine treatment did not affect the decline in MPF or MAPK activities following activation or significantly affect development of parthenogenetically activated oocytes. When SCNT reconstructed embryos were treated with caffeine following fusion, no increase in the frequency of development to blastocyst was observed; however, a significant increase in the occurrence of nuclear envelope break-down (NEBD) and an increase in total cell numbers occurred.     

体细胞核移植后核重编程的影响因素

近年来,人类核移植胚胎干细胞建系成为一项炙手可热的研究,用再生医学的理念治疗退行性疾病及器官移植为这一研究带来无穷的魅力和生命力;但是核重编程仍是核移植技术的瓶颈,制约了重构胚胎干细胞的研究。核重编程是指供体细胞核移入卵母细胞后必须停止本身的基因表达程序并恢复为胚胎发育所必需的特定的胚胎表达程序。只有供核发生完全重编程,重构胚胎才能正常发育。核重编程与供核者的年龄,供核细胞的组织来源、分化状态、细胞周期、传代次数,供核的表遗传标记以及供卵者的年龄、卵子的成熟度等因素有关。一般来说,颗粒细胞作为核供体最易被核重编程。供核者为胎体或新生体,供核细胞处于低分化状态或已传数代,供核细胞经过去表遗传标记处理,供卵者性成熟且年龄轻、卵子核与胞浆都成熟等均为有利于核重编程的因素。重构胚胎的培养方法对核重编程也至关重要,目前主张使用序贯培养及体细胞化培养。创造各种适于核重编程的条件有利于从更高的起点开展核移植胚胎干细胞研究,提高重构胚胎干细胞建系效率。     

Lowering storage temperature during ovary transport is beneficial to the developmental competence of bovine oocytes used for somatic cell nuclear transfer

The objective of this study was to determine the effect of storage temperature during ovary transport on the developmental competence of bovine oocytes for use in somatic cell nuclear transfer (SCNT). Ovaries obtained from a slaughterhouse were stored in physiological saline for 3-4h at one of the three temperatures: 15 °C, 25 °C, or 35 °C. The developmental competence of oocytes used for SCNT was ascertained by cleavage and blastocyst formation rate, total cell number, apoptosis index, and the relative abundance of Bax and Hsp70.1 in day 7 blastocysts. Ovaries stored at 35 °C for 3-4h reduced the recovery rate of grade I and II oocytes compared with those stored at 25 °C or 15 °C (45.1±0.7% vs. 76.7±1.2% or 74.8±2.0%, P<0.05). The proportion of oocytes matured to the MII stage (maturation rate) for oocytes stored at 35 °C was significantly lower than those stored at 25 °C or 15 °C (51.3±0.9% vs. 75.1±1.4% or 71.7±1.3%, P<0.05). Cleavage rate (77.7±2.1%, 77.9±1.1% and 72.1±0.7% for 15 °C, 25 °C and 35 °C groups, respectively) and blastocyst formation rate (39.1±0.5%, 36.8±1.4% and 32.2±0.9% for 15 °C, 25 °C and 35 °C groups, respectively) following SCNT were not significantly different between treatments. Oocytes from ovaries stored at 15 °C, however, produced blastocysts with higher cell numbers (97.3±8.6 vs. 80.2±10.8 or 77.4±11.7; P<0.05) and lower apoptotic index (5.1±1.3 vs. 13.5±1.6 or 18.6±1.1, P<0.05) than those stored at 25 °C or 35 °C. The relative abundance of Bax and Hsp70.1 in day 7 blastocysts produced from oocytes derived from ovaries stored at 15 °C was lower than those stored at 25 °C or 35 °C (P<0.05). It was concluded that a storage temperature of 15 °C for a 3-4h period had a significant beneficial effect on the quality and developmental competence of oocytes used for SCNT due to the alleviation of stresses on the oocytes compared with those subjected to storage temperatures of 25 °C or 35 °C.     

Maintenance of meiotic arrest and developmental potential of porcine oocytes after parthenogenetic activation and somatic cell nuclear transfer

Several studies report that meiotic maturation of porcine oocytes can be reversibly preserved. The present study examined how long meiotic maturation can be suppressed. The first experiment determined the preservation medium suitable for reversibly suppressing meiotic maturation of porcine oocytes. The second experiment examined the in vitro developmental potential of oocytes maintained in meiotic arrest after parthenogenetic activation and nuclear transfer of somatic cells. Preservation of cumulus-oocyte complexes with NCSU-37 medium containing 10% follicular fluid, 1 mM dibutyryl cyclic AMP, and follicular shell pieces for 24-96 h at 39 degrees C did not affect oocyte maturation compared with controls (94-98% vs. 98%). The potential of parthenogenetically activated and nuclear-transferred oocytes maintained in meiotic arrest for 24-48 h to develop into blastocysts was not significantly different from that of controls (20-25% vs. 18% and 8-11% vs. 9%, respectively). The present study demonstrated that meiotic maturation of porcine oocytes can be suppressed after preservation for 48 h at 39 degrees C without decreasing oocyte maturation competence or the ability of oocytes to develop to at least the blastocyst stage.     

Improvement of canine somatic cell nuclear transfer procedure

The purpose of the present study on canine somatic cell nuclear transfer (SCNT) was to evaluate the effects of fusion strength, type of activation, culture media and site of transfer on developmental potential of SCNT embryos. We also examined the potential of enucleated bovine oocytes to serve as cytoplast recipients of canine somatic cells. Firstly, we evaluated the morphological characteristics of in vivo-matured canine oocytes collected by retrograde flushing of the oviducts 72 h after ovulation. Secondly, the effectiveness of three electrical strengths (1.8, 2.3 and 3.3 kV/cm), used twice for 20 micros, on fusion of canine cytoplasts with somatic cells were compared. Then, we compared: (1) chemical versus electrical activation (a) after parthenogenetic activation or (b) after reconstruction of canine oocytes with somatic cells; (2) culture of resulting intergeneric (IG) embryos in either (a) mSOF or (b) TCM-199. The exposure time to 6-DMAP was standardized by using bovine oocytes reconstructed with canine somatic cells. Bovine oocytes were used for SCNT after a 22 h in vitro maturation interval. The fusion rate was significantly higher in the 3.3 kV/cm group than in the 1.8 and 2.3 kV/cm treatment groups. After parthenogenesis or SCNT with chemical activation, 3.4 and 5.8%, respectively, of the embryos developed to the morula stage, as compared to none of the embryos produced using electrical activation. Later developmental stages (8-16 cells) were transferred to the uterine horn of eight recipients, but no pregnancy was detected. However, IG cloned embryos (bovine cytoplast/canine somatic cell) were capable of in vitro blastocyst development. In vitro developmental competence of IG cloned embryos was improved after exposure to 6-DMAP for 4 h as compared to 0, 2 or 6h exposure, although the increase was not significantly different among culture media. In summary, for production of canine SCNT embryos, we recommend fusion at 3.3 kV/cm, chemical activation, culture in mSOF medium and transfer of presumptive zygotes to the oviduct of recipient animals. The feasibility of IG production of cloned canine embryos using bovine cytoplasts as recipient of canine somatic cells was demonstrated.     

Recent advancements in cloning by somatic cell nuclear transfer

Somatic cell nuclear transfer (SCNT) cloning is the sole reproductive engineering technology that endows the somatic cell genome with totipotency. Since the first report on the birth of a cloned sheep from adult somatic cells in 1997, many technical improvements in SCNT have been made by using different epigenetic approaches, including enhancement of the levels of histone acetylation in the chromatin of the reconstructed embryos. Although it will take a considerable time before we fully understand the nature of genomic programming and totipotency, we may expect that somatic cell cloning technology will soon become broadly applicable to practical purposes, including medicine, pharmaceutical manufacturing and agriculture. Here we review recent progress in somatic cell cloning, with a special emphasis on epigenetic studies using the laboratory mouse as a model.     

Current status and applications of somatic cell nuclear transfer in dogs

Although somatic cell nuclear transfer (SCNT) technology and applications are well developed in most domesticated and laboratory animals, their use in dogs has advanced only slowly. Many technical difficulties had to be overcome before preliminary experiments could be conducted. First, due to the very low efficiency of dog oocyte maturation in vitro, in vivo matured oocytes were generally used. The nucleus of an in vivo matured oocyte was removed and a donor cell (from fetal or adult fibroblasts) was injected into the oocyte. Secondly, fusion of the reconstructed oocytes was problematic, and it was found that a higher electrical voltage was necessary, in comparison to other mammalian species. By transferring the resulting fused oocytes into surrogate females, several cloned offspring were born. SCNT was also used for producing cloned wolves, validating reproductive technologies for aiding conservation of endangered or extinct breeds. Although examples of transgenesis in canine species are very sparse, SCNT studies are increasing, and together with the new field of gene targeting technology, they have been applied in many fields of veterinary or bio-medical science. This review summarizes the current status of SCNT in dogs and evaluates its potential future applications.     

Birth of Beagle dogs by somatic cell nuclear transfer

The present study was undertaken to evaluate two enucleation methods for somatic cell nuclear transfer (SCNT), and to standardize the optimum number of embryos for transfer to each recipient for canines. Oocytes retrieved from outbreed dogs were reconstructed with adult somatic cells from a male Beagle dog. A total of 134 or 267 oocytes were enucleated either by aspiration or squeezing method, fused with two DC pulses of 1.75 kV/cm for 15 μs electrical stimulation, chemically activated after 1 h of fusion using 10 μM calcium ionophore for 4 min and cultured 4 h in 1.9 mM 6-dimethylaminopurine. Finally, 103 or 214 embryos for aspiration or squeezing method were transferred to 6 or 11 naturally synchronized recipients, respectively. A total of 53, 317 and 342 embryos were transferred to 7, 17 and 12 recipients for the group of 4–10, 11–25 and 26–40 embryos, respectively. There was no difference between fusion rate (76.87% vs. 80.15%), full term pregnancy rate (16.66% vs. 27.27%) and percent of live puppies born (0.97% vs. 1.87%) for aspiration and squeezing method (P > 0.05). Production efficiency of cloned dogs was significantly affected by the number of embryos transferred to each recipient. No pregnancy was established for the group of 4–10 embryos (n = 7) and 26–40 embryos (n = 12) while pregnancy was detected in 23.53% recipients received a group of 11–25 embryos (n = 17). Among them, five (1.76%) live puppies were born (P < 0.05). These data show an increase in the overall efficiency of SCNT in canine species.     

Production of goats by somatic cell nuclear transfer.

In this study, we demonstrate the production of transgenic goats by nuclear transfer of fetal somatic cells. Donor karyoplasts were obtained from a primary fetal somatic cell line derived from a 40-day transgenic female fetus produced by artificial insemination of a nontransgenic adult female with semen from a transgenic male. Live offspring were produced with two nuclear transfer procedures. In one protocol, oocytes at the arrested metaphase II stage were enucleated, electrofused with donor somatic cells, and simultaneously activated. In the second protocol, activated in vivo oocytes were enucleated at the telophase II stage, electrofused with donor somatic cells, and simultaneously activated a second time to induce genome reactivation. Three healthy identical female offspring were born. Genotypic analyses confirmed that all cloned offspring were derived from the donor cell line. Analysis of the milk of one of the transgenic cloned animals showed high-level production of human antithrombin III, similar to the parental transgenic line.     

High developmental rates of vitrified bovine oocytes following parthenogenetic activation, in vitro fertilization, and somatic cell nuclear transfer

Successful cryopreservation of mammalian oocytes would provide a steady source of materials for nuclear transfer and in vitro embryo production. Our goal was to develop an effective vitrification protocol to cryopreserve bovine oocytes for research and practice of parthenogenetic activation, in vitro fertilization, and nuclear transfer. Bovine oocytes matured in vitro were placed in 4% ethylene glycol (EG) in TCM 199 plus 20% fetal bovine serum (FBS) at 39 degrees C for 12-15 min, and then transferred to a vitrification solution (35% EG, 5% polyvinyl-pyrrolidone, 0.4 M trehalose in TCM 199 and 20% FBS). Oocytes were vitrified in microdrops on a precooled (-150 degrees C) metal surface (solid-surface vitrification). The vitrified microdrops were stored in liquid nitrogen and were either immediately thawed or were thawed after storage for 2-3 wk. Surviving oocytes were subjected to 1) parthenogenetic activation, 2) in vitro fertilization, or 3) nuclear transfer with cultured adult fibroblast cells. Treated oocytes were cultured in KSOM containing BSA or FBS for 9 to 10 days. Embryo development rates were recorded daily and morphologically high-quality blastocysts were cryopreserved for nuclear transfer-derived embryos at Day 7 or Day 8 of culture. Immediate survival of vitrified/thawed oocytes varied between 77% and 86%. Cleavage and blastocyst development rates of vitrified oocytes following in vitro fertilization or activation were lower than those of the controls. For nuclear transfer, however, vitrified oocytes supported embryonic development as equally well as fresh oocytes.