The effects of cell size and ploidy on cell allocation in mouse chimaeric blastocysts

In a previous study of mouse tetraploid<-->diploid chimaeric blastocysts, tetraploid cells were found to be more abundant in the trophectoderm than the inner cell mass (ICM) and more abundant in the mural trophectoderm than the polar trophectoderm. This non-random allocation of tetraploid cells to different regions of the chimaeric blastocyst may contribute to the restricted tissue distribution seen in post-implantation stage tetraploid<-->diploid chimaeras. However, the tetraploid and diploid embryos that were aggregated together differed in several respects: the tetraploid embryos had fewer cells and these cells were bigger and differed in ploidy. Each of these factors might underlie a non-random allocation of tetraploid cells to the chimaeric blastocyst. A combination of micromanipulation and electrofusion was used to produce two series of chimaeras that distinguished between the effects of cell size and ploidy on the allocation of cells to different tissues in chimaeric blastocysts. When aggregated cells differed in cell size but not ploidy, the derivatives of the larger cell contributed significantly more to the mural trophectoderm and polar trophectoderm than the ICM. When aggregated cells differed in ploidy but not cell size, the tetraploid cells contributed significantly more to the mural trophectoderm than the ICM. In both experiments the contributions to the polar trophectoderm tended to be intermediate between those of the mural trophectoderm and ICM. These experiments show that both the larger size and increased ploidy of tetraploid cells could have contributed to the non-random cell distribution that was observed in a previous study of tetraploid<-->diploid chimaeric blastocysts.     

Identical triplets and twins developed from isolated blastomeres of 8- and 16-cell mouse embryos supported with tetraploid blastomeres

We studied the developmental potential of single blastomeres from early cleavage mouse embryos. Eight- and sixteen-cell diploid mouse embryos were disaggregated and single blastomeres from eight-cell embryos or pairs of sister blastomeres from sixteen-cell embryos were aggregated with 4, 5 or 6 tetraploid blastomeres from 4-cell embryos. Each diploid donor embryo gave eight sister aggregates, which later were manipulated together as one group (set). The aggregates were cultured in vitro until the blastocyst stage, when they were transferred (in sets) to the oviducts of pseudopregnant recipients. Eighteen live foetuses or pups were obtained from the transfer (11.0% of transferred blastocysts) and out of those, eleven developed into fertile adults (one triplet, one pair of twins and four singletons). In all surviving adults, pups and living foetuses, only diploid cells were detected in their organs and tissues as shown by analysis of coat pigmentation and distribution of glucose phosphate isomerase isoforms. In order to explain the observed high rate of mortality of transferred blastocysts, in an accompanying experiment, the diploid and tetraploid blastomeres were labelled with different fluorochromes and then aggregated. These experiments showed the diploid cells to be present not only in the inner cell mass (ICM) but also in the trophectoderm. The low number of diploid cells and the predominance of tetraploid cells in the ICM of chimaeric blastocysts might have been responsible for high postimplantation mortality of our experimental embryos.     

Characterization of DNA synthesis during the 2-cell stage and the production of tetraploid chimeric pig embryos

The DNA content of nuclei during the 2-cell stage as well as in presumptive tetraploid embryos was investigated. In vivo produced pig zygotes were cultured to the 2-cell stage and either monitored for cleavage to the 4-cell stage or mounted at various times postcleavage and DNA content determined. The length of the 2-cell stage was 14.8 ± 3.0 hr. There was a significant increase in the length of the 2-cell stage due to the time in vitro as a zygote (P < 0.001: R² = 0.866). The DNA content increased (P < 0.05) each 2 hr postcleavage until 10 hr postcleavage. This suggested that there is a short G₁ and G₂ phase and a relatively long phase of DNA synthesis. Next, 2-cell stage embryos were pulsed with electricity to induce cell-to-cell fusion. Whereas only about half fused within 30 min (55%), most (96%) developed to the blastocyst stage. The DNA content of the nuclei of the embryos was consistent with them being tetraploid. A final experiment was designed to evaluate the ability of the tetraploid embryo to form a chimera with isolated inner cell mass (ICM) cells. Inner cell masses were isolated from d 6 embryos, cut into thirds, labeled with DiO (a membrane die) and injected into the perivitelline space of 4-cell-stage tetraploid embryos. Twelve of 17 formed blastocysts. In most 8/12), the ICM of the resulting blastocyst was labeled, whereas in one the only fluorescence was in the trophectoderm, and in two fluorescence was evenly distributed between the ICM and trophectoderm. These results suggest that it may be possible to create a fetus derived from ICM cells, or potentially stem cells, that has a tetraploid trophoblast. © 1996 Wiley-Liss, Inc.     

Generation and Developmental Characteristics of Porcine Tetraploid Embryos and Tetraploid]diploid Chimeric Embryos

The aim of this study was to optimize electrofusion conditions for generating porcine tetraploid(4n)embryos and produce tetraploid/diploid(4n/2n)chimeric embryos.Different electric feld intensities were tested and 2 direct current(DC)pulses of 0.9 kV/cm for 30 ls was selected as the optimum condition for electrofusion of 2-cell embryos to produce 4n embryos.The fusion rate of 2-cell embryos and the development rate to blastocyst of presumably 4n embryos,reached85.4%and 28.5%,respectively.68.18%of the fused embryos were found to be 4n as demonstrated by fluorescent in situ hybridization(FISH).Although the number of blastomeres in 4n blastocysts was signifcantly lower than in 2n blastocysts(P<0.05),there was no signifcant difference in developmental rates of blastocysts between 2n and 4n embryos(P>0.05),suggesting that the blastocyst forming capacity in 4n embryos is similar to those in 2n embryos.Moreover,4n/2n chimeric embryos were obtained by aggregation of 4n and 2n embryos.We found that the developmental rate and cell number of blastocysts of 4-cell(4n)/4-cell(2n)chimeric embryos were signifcantly higher than those of 2-cell(4n)/4-cell(2n),4-cell(4n)/8-cell(2n),4-cell(4n)/2-cell(2n)chimeric embryos(P<0.05).Consistent with mouse chimeras,the majority of 4n cells contribute to the trophectoderm(TE),while the 2n cells are mainly present in the inner cell mass(ICM)of porcine4n/2n chimeric embryos.Our study established a feasible and effcient approach to produce porcine4n embryos and 4n/2n chimeric embryos.     

Allocation of cells to inner cell mass and trophectoderm lineages in preimplantation mouse embryos

Inner cell mass (ICM) and trophectoderm cell lineages in preimplantation mouse embryos were studied by means of iontophoretic injection of horseradish peroxidase (HRP) as a marker. HRP was injected into single blastomeres at the 2- and 8-cell stages and into single outer blastomeres at the 16-cell and late morula (about 22- to 32-cell) stages. After injection, embryos were either examined immediately for localization of HRP (controls) or they were allowed to develop until the blastocyst stage (1 to 3.5 days of culture) and examined for the distribution of labeled cells. In control embryos, HRP was confined to one or two outer blastomeres. In embryos allowed to develop into blastocysts, HRP-labeled progeny were distributed into patches of cells, showing that there is limited intermingling of cells during preimplantation development. A substantial fraction of injected blastomeres contributed descendants to both ICM and trophectoderm (95, 58, 44, and 35% for injected 2-cell, 8-cell, 16-cell, and late morula stages, respectively). Although more than half of the outer cells injected at 16-cell and late morula stages contributed descendants only to trophectoderm (53 and 63%, respectively), some outer cells contributed also to the ICM lineage even at the late morula stage. Although the mechanism for allocation of outer cells to the inner cell lineage is unknown, our observation of adjacent labeled mural trophectoderm and presumptive endoderm cells implicated polarized cell division. This observation also suggests that mural trophectoderm and presumptive endoderm are derived from common immediate progenitors. These cells appear to separate into inner and outer layers during the fifth cleavage division. Our results demonstrate the usefulness of HRP as a cell lineage marker in mouse embryos and show that the allocation of cells to ICM or trophectoderm begins after the 2-cell stage and continues into late cleavage.     

Cell specific loss of polarity-inducing ability by later stage mouse preimplantation embryos

The individual blastomeres of the preimplantation mouse embryo become polarized during the 8-cell stage. Microvilli become restricted to the free surface of the embryo and this region of the membrane shows increased labeling with FITC-Con A and trinitrobenzenesulfonate (TNBS). Previous studies have shown that this polarity develops in response to asymmetric cell-cell contact with stage specific induction competent blastomeres. In the present study, the ability of later stage embryos to induce 8-cell polarization has been investigated. Newly-formed, nonpolar 8-cell stage blastomeres (1/8 cells) were isolated, then aggregated with morulae, inner cell clusters (from morulae), blastocysts, or inner cell masses (ICM) and cultured for 8 hr. Aggregates were then assayed for polarity. The results show a hierarchy of inducing ability, with the ICM and IC cluster possessing greater activity than the morula and polar trophectoderm of the early blastocyst, while the mural trophectoderm shows very little inducing activity. Furthermore, the inducing ability of the polar trophectoderm decreases with complete expansion and hatching of the blastocyst. These results indicate that the ability to induce 8-cell blastomere polarization is retained by the embryo beyond the 8-cell stage and that this ability is lost with further differentiation.     

小鼠遗传背景对4n胚胎及2n-4n聚合胚制作效率的影响

不同品系小鼠的 2 细胞期胚胎 (二倍体 ,2n) ,经电融合后 ,获得发育的 4 细胞期四倍体胚胎 (4n)的能力上存在着差异。将不同品系小鼠的 2n、 4n胚胎分别配对作聚合 ,所获 2n 4n聚合胚的发育结果表明 :在着床前 ,2n 4n聚合胚的获得率因胚胎品系组合的不同而异 ;胚胎移植后 ,聚合胚在与 4n胚胎相同或相近品系的移植受体中 ,其着床率较高 ;在着床后胎儿及出生仔鼠的获得率上 ,采用遗传杂合性的 2n胚胎所组成的 2n 4n聚合组合较高。上述结果提示 :小鼠的遗传背景可影响到 4n胚胎及相应 2n 4n聚合胚的制作效率。以GFP标记跟踪2n 4n聚合胚 4n细胞着床后的发育命运 ,发现 :妊娠中后期的孕体中 ,4n细胞限制性地分布至胚外组织     

Development of rat tetraploid and chimeric embryos aggregated with diploid cells

In the present study, we examined the preimplantation and postimplantation development of rat tetraploid embryos produced by electrofusion of 2-cell-stage embryos. Developmental rate of tetraploid embryos to morula or blastocyst stage was 93% (56/60) and similar to that found in diploid embryos (95%, 55/58). After embryo transfer, rat tetraploid embryos showed implantation and survived until day 8 of pregnancy, however the conceptuses were aberrant on day 9. In mouse, tetraploid embryos have the ability to support the development of blastomeres that cannot develop independently. As shown in the present study, a pair of diploid blastomeres from the rat 8-cell-stage embryo degenerated immediately after implantation. Therefore, we examined whether rat tetraploid embryos have the ability to support the development of 2/8 blastomeres. We produced chimeric rat embryos in which a pair of diploid blastomeres from an 8-cell-stage green fluorescent protein negative (GFP-) embryo was aggregated with three tetraploid blastomeres from 4-cell GFP-positive (GFP+) embryos. The developmental rate of rat 2n(GFP-) <--> 4n(GFP+) embryos to the morula or blastocyst stages was 93% (109/117) and was similar to that found for 2n(GFP-) <--> 2n(GFP+) embryos (100%, 51/51). After embryo transfer, 2n(GFP-) <--> 4n(GFP+) conceptuses were examined on day 14 of pregnancy, the developmental rate to fetus was quite low (4%, 4/109) and they were all aberrant and smaller than 2n(GFP-) <--> 2n(GFP+) conceptuses, whereas immunohistochemical analysis showed no staining for GFP in fetuses. Our results suggest that rat tetraploid embryos are able to prolong the development of diploid blastomeres that cannot develop independently, although postimplantation development was incomplete.     

Cytokeratin filament assembly in the preimplantation mouse embryo

The timing, spatial distribution and control of cytokeratin assembly during mouse early development has been studied using a monoclonal antibody, TROMA-1, which recognizes a 55,000 Mr trophectodermal cytokeratin (ENDO A). This protein was first detected in immunoblots at the 4-cell stage, and became more abundant at the 16-cell stage and later. Immunofluorescence analysis revealed assembled cytokeratin filaments in some 8-cell blastomeres, but not at earlier stages. At the 16-cell stage, filaments were found in both polarized (presumptive trophectoderm; TE) and apolar (presumptive inner cell mass; ICM) cells in similar proportions, although polarized cells possessed more filaments than apolar cells. By the late 32-cell, early blastocyst, stage, all polarized (TE) cells contained extensive filament networks whereas cells positioned inside the embryo tended to have lost their filaments. The presence of filaments in inside cells at the 16-cell stage and in ICM cells was confirmed by immunoelectron microscopy. Lineage tracing techniques demonstrated that those cells in the ICM of early blastocysts which did possess filaments were almost exclusively the progeny of polar 16-cell blastomeres, suggesting that these filaments were directly inherited from outside cells at the 16- to 32-cell transition. Inhibitor studies revealed that proximate protein synthesis but not mRNA synthesis is required for filament assembly at the 8-cell stage. These results demonstrate that there are quantitative rather than qualitative differences in the expression of cytokeratin filaments in the inner cell mass and trophectoderm cells of the mouse embryo.     

Characterization of embryos derived from calf oocytes: kinetics of cleavage, cell allocation to inner cell mass, and trophectoderm and lipid metabolism

Embryos derived from calf oocytes were compared with adult cow oocyte-derived embryos (1) by studying the kinetics of embryo development using time-lapse cinematography (2) by evaluating the ratio between inner cell mass (ICM) and trophectoderm (TE) cells in blastocysts (3) by measuring the triglyceride content of the blastocysts. The rate of calf oocyte-derived embryos reaching the blastocyst stage was reduced (26 vs. 46% for adult derived embryos). Calf oocyte-derived embryos preferably arrested their development before the 9-cell stage. Those that developed into blastocysts had cleaved earlier to reach the 2-cell or 3-cell stages than embryos that arrested before the 9-cell stage. The 9-cell stage tended to appear later in calf oocyte-derived embryo that reached the blastocyst stage than in adult-derived embryos. This difference became significant at the morula stage. Accordingly, the fourth cell cycle duration was longer for calf oocyte-derived embryos. Day 8 blastocysts from both sources had similar total cell numbers (calf: 89 +/- 20; cow: 100 +/- 30) and cell distribution between TE and ICM. The triglyceride content of day 7 blastocysts was similar for both sources (64 +/- 15 vs. 65 +/- 6 ng/embryo, respectively). In conclusion, calf oocyte-derived embryos are characterized by a higher rate of developmental arrest before the 9-cell stage and by a longer lag phase preceding the major onset of embryonic genome expression. These changes might be related to insufficient "capacitation" of the calf oocyte during follicular growth. Despite these differences, modifications in the quality of the resulting blastocysts were not detected.     

Evidence for selection against tetraploid cells in tetraploid<-->diploid mouse chimaeras before the late blastocyst stage

Tetraploid (4n) cells do not contribute equally to all tissues of midgestation mouse chimaeras and mosaics. Our previous studies of early blastocysts showed that 4n cells are preferentially allocated to the mural trophectoderm of the early blastocyst and this may contribute to the restricted distribution pattern seen at later stages. In this study of later-stage blastocysts we found evidence for selection against 4n cells. The contribution of 4n cells to 4n<-->2n chimaeric blastocysts decreased between E3.5 and E4.5 days, whereas the composition of 2n<-->2n controls changed little over this period. These results suggest that, prior to implantation, blastocysts have already lost some tetraploid cells from their embryonic and extra-embryonic lineages due to a combination of preferential allocation of 4n cells to the mural trophectoderm and selection against 4n cells throughout the embryo.     

Individual blastomeres of 16- and 32-cell mouse embryos are able to develop into foetuses and mice

Cell and developmental studies have clarified how, by the time of implantation, the mouse embryo forms three primary cell lineages: epiblast (EPI), primitive endoderm (PE), and trophectoderm (TE). However, it still remains unknown when cells allocated to these three lineages become determined in their developmental fate. To address this question, we studied the developmental potential of single blastomeres derived from 16- and 32-cell stage embryos and supported by carrier, tetraploid blastomeres. We were able to generate singletons, identical twins, triplets, and quadruplets from individual inner and outer cells of 16-cell embryos and, sporadically, foetuses from single cells of 32-cell embryos. The use of embryos constitutively expressing GFP as the donors of single diploid blastomeres enabled us to identify their cell progeny in the constructed 2n↔4n blastocysts. We showed that the descendants of donor blastomeres were able to locate themselves in all three first cell lineages, i.e., epiblast, primitive endoderm, and trophectoderm. In addition, the application of Cdx2 and Gata4 markers for trophectoderm and primitive endoderm, respectively, showed that the expression of these two genes in the descendants of donor blastomeres was either down- or up-regulated, depending on the cell lineage they happened to occupy. Thus, our results demonstrate that up to the early blastocysts stage, the destiny of at least some blastomeres, although they have begun to express markers of different lineage, is still labile.     

A quantitative analysis of cell allocation to trophectoderm and inner cell mass in the mouse blastocyst

The allocation of cells to the trophectoderm and inner cell mass (ICM) in the mouse blastocyst has been examined by labelling early morulae (16-cell stage) with the short-term cell lineage marker yellow-green fluorescent latex (FL) microparticles. FL is endocytosed exclusively into the outside polar cell population and remains autonomous to the progeny of these blastomeres. Rhodamine-concanavalin A was used as a contemporary marker for outside cells in FL-labelled control (16-cell stage) and cultured (approximately 32- to 64-cell stage) embryos, immediately prior to the disaggregation and analysis of cell labelling patterns. By this technique, the ratio of outside to inside cell numbers in 16-cell embryos was shown to vary considerably between embryos (mean 10.8:5.2; range 9:7 to 14:2). In cultured embryos, the trophectoderm was derived almost exclusively (over 99% cells) from outside polar 16-cell blastomeres. The origin of the ICM varied between embryos; on average, most cells (75%) were descended from inside nonpolar blastomeres with the remainder derived from the outside polar lineage, presumably by differentiative cleavage. In blastocysts examined by serial sectioning, polar-derived ICM cells were localised mainly in association with trophectoderm and were absent from the ICM core. In nascent blastocysts with exactly 32 cells an inverse relationship was found between the proportion of the ICM descended from the polar lineage and the deduced size of the inside 16-cell population. From these results, it is concluded that interembryonic variation in the outside to inside cell number ratio in 16-cell morulae is compensated by the extent of polar 16-cell allocation to the ICM at the next division, thereby regulating the trophectoderm to ICM cell number ratio in early blastocysts.     

Influence of chromosomal determinants on development of androgenetic and parthenogenetic cells

We have examined the role of germline-specific chromosomal determinants of development in the mouse. Studies were carried out using aggregation chimaeras between androgenetic----fertilized embryos and compared with similar parthenogenetic----fertilized chimaeras. Several adult chimaeras were found with parthenogenetic cells but none were found with androgenetic cells. Analysis of chimaeras at mid-gestation showed that parthenogenetic cells were detected in the embryo and yolk sac but that androgenetic cells were found only in the trophoblast and yolk sac and not in the embryo. The contribution of parthenogenetic cells to the embryo and yolk sac was increased by aggregating 2-cell parthenogenetic and 4-cell fertilized embryos but the contribution of parthenogenetic cells in extraembryonic tissues remained negligible even after aggregation of 4-cell parthenogenetic and 2-cell fertilized embryos. Furthermore, parthenogenetic cells were primarily found in the yolk sac mesoderm and not in the yolk sac endoderm. These results suggest that maternal chromosomes in parthenogenetic cells permit their participation in the primitive ectoderm lineage but these cells are presumably eliminated by selective pressure or autonomous cell lethality from the primitive endoderm and trophectoderm lineages. Conversely paternal chromosomes in androgenetic cells confer opposite properties since the embryonic cells can be detected in the trophoblast and the yolk sac but not in the embryos, presumably because they are eliminated from the primitive ectoderm lineage. The spatial distribution of cells with different parental chromosomes may occur partly because of differential expression of some genes, such as proto-oncogenes, and partly due to their ability to respond to a variety of diffusible growth factors.     

Size regulation does not cause the composition of mouse chimaeras to become unbalanced.

Mouse chimaeras made by aggregating two 8-cell stage embryos undergo size regulation shortly after implantation. Thus chimaeric pups are approximately normal size at birth despite their origin from two complete embryos. Chimaeras of some strain combinations are genotypically unbalanced such that cells of one strain almost always predominate. For example, the BALB/c inbred strain often makes a low contribution to chimaeras. This genotypic imbalance in the composition could arise by selection against BALB/c cells. Selection may be particularly acute at the time of size regulation. To investigate if the mechanism(s) responsible for size regulation could cause the low contribution of BALB/c cells, we compared the composition of an unbalanced series of chimaeras, produced by aggregating two complete 8-cell stage embryos, with a similar series of chimaeras made by aggregating two half 8-cell stage embryos. In each case the unbalanced strain combination was BALB/c<-->[(C57BL x CBA/Ca)F1 x TGB] and parallel studies were undertaken with a genotypically balanced strain combination. For each chimaera, the composition of the fetus, placenta and extraembryonic membranes were determined at E12.5. When two half embryos were aggregated the BALB/c strain still made a poor contribution to all the tissues of the mid-gestation conceptus. This implies that this strain combination remained unbalanced even when size regulation was absent or minimal. Therefore, size regulation did not play a major role in reducing the contribution of BALB/c cells and producing the phenotypic imbalance in the chimaeras.     

Developmental changes in the incidence of chromosome anomalies of bovine embryos fertilized in vitro.

In total, 196 two- to 32-cell bovine embryo and 104 blastocysts were obtained by the in vitro fertilization of follicular oocytes matured in vitro, and 15 blastocysts fertilized in vivo were used. Chromosomal anomalies in these embryos and the inner cell mass (ICM) separated immunologically were investigated. Chromosomal anomalies were observed in 12.1% (5/41) of 2-cell embryos, 20.0-36.4% of 4- to 16-cell embryos, 7.1% (1/14) of 17- to 32-cell embryos, 44.2% (15/34) of blastocysts, and 18.6% (13/70) of ICM cells derived from in vitro fertilization. These anomalies were mainly 3N and 4N at 2-cell stage, 1N and 1N/2N at 4- to 32-cell stages, and 2N/4N in blastocysts and in their ICM cells. Chromosomal anomalies of blastocysts from in vivo fertilization and their ICM were observed in 20.0% (1/5) of blastocysts and 33.3% (3/9) of ICM cells and these compositions were mainly 2N/4N. These results indicate that the abnormalities at early and blastocyst stages of embryos derived from in vitro fertilization were caused by abnormal fertilization in vitro and abnormal cleavage, respectively. Furthermore, a definite location of the chromosomal anomalies was observed in the trophectoderm of blastocysts derived from in vitro fertilization.     

小鼠四倍体早期胚胎基因组甲基化模式

利用小鼠抗5-甲基胞嘧啶(5MeC)单克隆抗体检测了体外培养小鼠四倍体早期胚胎的基因组甲基化模式。结果表明: 利用电融合方法制备的小鼠四倍体胚胎在体外培养体系中经历细胞质融合、细胞核融合及细胞继续分裂发育直到囊胚期的过程, 在细胞质融合的时候胚胎卵裂球同体内体外培养二倍体胚胎一样, 呈现高度甲基化状态; 在细胞核开始融合的时候, 甲基化水平急速下降, 在细胞核完全融合的时候甲基化水平达到最低点; 随着胚胎继续分裂, 胚胎甲基化水平逐渐增加, 在桑葚胚期甲基化水平最高; 但是囊胚期四倍体胚胎内细胞团同滋养层细胞甲基化荧光信号没有差别, 这与体内体外培养二倍体囊胚内细胞团细胞甲基化荧光强度高于滋养层细胞甲基化荧光强度不同。因此, 小鼠体外培养四倍体胚胎的甲基化模式是不正常的, 这可能是四倍体小鼠难以发育到妊娠足月的原因之一。这是对小鼠四倍体早期胚胎基因组甲基化模式的首次报道。     

Vitrification of in vitro produced ovine embryos at various developmental stages using two methods

This study was conducted to evaluate the effects of developmental stage of in vitro produced (IVP) ovine embryos and the type of vitrification procedure used on embryo cryotolerance.The IVP embryos were vitrified at five different developmental stages: 4-, 8- and 16-cell, morula, and blastocyst. For each stage, half of the embryos were vitrified in either 30 μl 3.4 M glycerol + 4.6 M ethylene glycol in straw (method 1) or in <0.1 μl 2.7 M ethylene glycol + 2.1 M Me₂SO + 0.5 M sucrose placed on the inner surface of a straw (method 2) of vitrification solution, based on two different procedures. After warming embryo viability was determined by assessing the rates of re-expansion, survival, and blastocyst formation. The quality of surviving embryos was evaluated by their hatching rate and blastocyst cell numbers. In both vitrification methods, embryo survival progressively increased as the developmental stage progressed. In method 1 few of the early cleavage stage embryos (4-, 8- and 16-cell) could reach to the blastocyst stage following warming. There was no significant difference in blastocyst cell numbers (total, ICM, and trophectoderm cells) or hatching rate of blastocysts derived from vitrified embryos at different developmental stages. The number of dead cells in vitrified blastocysts in method 1 was higher than for non-vitrified blastocysts (P < 0.05). The number of apoptotic cells in vitrified blastocysts was higher than for non-vitrified counterparts (P < 0.05). In conclusion, both the developmental stage of IVP ovine embryos and the method of vitrification have a significant effect on the viability and developmental competence of sheep embryos.