Vitrification of murine mature metaphase II oocytes perturbs DNA methylation reprogramming during preimplantation embryo development

Accurate reprogramming of DNA methylation occurring in preimplantation embryos is critical for normal development of both fetus and placenta. Environmental stresses imposed on oocytes usually cause the abnormal DNA methylation reprogramming of early embryos. However, whether oocyte vitrification alters the reprogramming of DNA methylation (5 mC) and its derivatives in mouse preimplantation embryo development remains largely unknown. Here, we found that the rate of cleavage and blastocyst formation of embryos produced by IVF of vitrified matured oocytes was significantly lower than that in control counterparts, but the quality of blastocysts was not impaired by oocyte vitrification. Additionally, although vitrification neither altered the dynamic changes of 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5 fC) before 4-cell stage nor affected the levels of 5 mC and 5-carboxylcytosine (5caC) throughout the preimplantation development, vitrification significantly reduced the levels of 5hmC and 5 fC from 8-cell stage onwards. Correspondingly, vitrification did not alter the expression patterns of Tet3 in preimplantation embryos but apparently reduced the expression levels of Tet1 in 4-cell and 8-cell embryos and increased the expression levels of Tet2 at morula stage. Taken together, these results demonstrate that oocyte vitrification perturbs DNA methylation reprogramming in mouse preimplantation embryo development.     

Effect of oocyte vitrification on DNA damage in metaphase II oocytes and the resulting preimplantation embryos

As an assisted reproduction technology, vitrification has been widely used for oocyte and embryo cryopreservation. Many studies have indicated that vitrification affects ultrastructure, gene expression, and epigenetic status. However, it is still controversial whether oocyte vitrification could induce DNA damage in metaphase II (MII) oocytes and the resulting early embryos. This study determined whether mouse oocytes vitrification induce DNA damage in MII oocytes and the resulting preimplantation embryos, and causes for vitrification‐induced DNA damage. The effects of oocyte vitrification on reactive oxygen species (ROS) levels, γ‐H2AX accumulation, apoptosis, early embryonic development, and the expression of DNA damage‐related genes in early embryos derived by in vitro fertilization were examined. The results indicated that vitrification significantly increased the number of γ‐H2AX foci in zygotes and two‐cell embryos. Trp53bp1 was upregulated in zygotes, two‐cell embryos and four‐cell embryos in the vitrified group, and Brca1 was increased in two‐cell embryos after vitrification. Vitrification also increased the ROS levels in MII oocytes, zygotes, and two‐cell embryos and the apoptotic rate in blastocysts. Resveratrol (3,5,4′‐trihydroxystilbene) treatment decreased the ROS levels and the accumulation of γ‐H2AX foci in zygotes and two‐cell embryos and the apoptotic rate in blastocysts after vitrification. Overall, vitrification‐induced abnormal ROS generation, γ‐H2AX accumulation, an increase in the apoptotic rate and the disruption of early embryonic development. Resveratrol treatment could decrease ROS levels, γ‐H2AX accumulation, and the apoptotic rate and improve early embryonic development. Vitrification‐associated γ‐H2AX accumulation is at least partially due to abnormal ROS generation.     

小鼠不同阶段胚胎玻璃化冷冻保存的比较研究

The cryopreservation of different embryo stages collected from ICR, C57BL/6 and F1 of DBA*C57BL/6 was carried out by using vitrification method. The morphology, in vitro development and birth rates of these embryos were compared after frozen-thawed. The results showed that more than 75% of the morphology from 2-cell embryos to morula stages from different strains was normal, the normal morphology rates of 8-cell embryos being the highest, while those of blastulas being the lowest. The in vitro development rates became higher as the embryos developed. The morphology of in vivo and in vitro fertilized frozen 2-cell embryos showed no difference, but the development rate of in vivo fertilized frozen 2-cell embryos was significantly higher than that of in vitro ones. Embryos that underwent 3 times frozen-thawing remained normal morphology. The pregnant rate and birth rate of frozen 2-cell embryos after embryo transfer were 64% and 40% respectively, but lower than those of fresh 2-cell embryo transfer.     

小鼠不同阶段胚胎玻璃化冷冻保存的比较研究

采用玻璃化冷冻法对ICR、C57BL/6、DBA~*C57BL/6杂交F1代三种品系小鼠的不同阶段胚胎进行冷冻保存,比较胚胎解冻后形态良好率、体外发育率和移植后的出生率,结果表明解冻后各品系小鼠胚胎从2细胞到桑椹胚形态良好率在75%以上,其中8细胞胚胎形态良好率在83%以上,而囊胚的形态良好率仅在40%左右。解冻后胚胎体外培养的发育率随胚胎发育阶段的提高而提高,桑椹胚的发育达93%以上。体外受精2细胞冷冻胚与体内受精2细胞冷冻胚比较,二者形态良好率差异无显著意义(74%∶75%),但体内受精冷冻胚的发育率明显高于体外受精冷冻胚(76%:40%,p<0.01);胚胎经过三次反复冻融后形态良好率无显著差别;冷冻2细胞胚移植后的受孕率与仔鼠出生率分别达64%和40%,但均低于新鲜2细胞胚。     

Resveratrol promotes the embryonic development of vitrified mouse oocytes after <Emphasis Type="Italic">in vitro</Emphasis> fertilization

Vitrification of oocytes is closely associated with the lower embryonic developmental potential, which involves the cryopreservation injury occurred during vitrification. It indicates that vitrification may need to be further optimized. Therefore, we studied the effects of resveratrol, an antioxidant, on the developmental potential of vitrified mouse oocytes after in vitro fertilization. After adding a series of concentrations of resveratrol (0, 1, 10, 25, and 50 μM) into vitrification, warming, and post-warming mediums, we found that 25 and 50 μM resveratrol increased the blastocyst formation rate of vitrified oocytes. We further showed that 25 μM resveratrol increased the mean cell numbers of blastocyst from vitrified oocytes. 25 μM resveratrol reduced oxidative stress of vitrified oocytes through decreasing the levels of reactive oxygen species (ROS) and increasing the levels of glutathione (GSH), and 25 μM resveratrol alleviated the abnormal mitochondrial distribution pattern of oocytes after vitrification. In conclusion, our study implied that resveratrol could diminish the cryopreservation injuries during the vitrification of mouse oocytes and further confirmed that resveratrol may be an effective antioxidant to optimize vitrification.     

Studies on replacing most of the penetrating cryoprotectant by polymers for embryo cryopreservation.

Solutions used for vitrification or rapid cooling of embryos usually contain high concentrations of penetrating cryoprotectants. At these concentrations embryos can tolerate the penetrating cryoprotectants for only short periods of time without damage. This study designed and tested cryoprotectant solutions that combined high polymer concentrations with low penetrating cryoprotectant concentrations. Mouse 2-cell embryo development was not compromised by up to 15-min exposure to 30 wt% solutions of the polymers Ficoll 70,000 MW or dextran 69,000 MW at room temperature. However, our batches of polyvinylpyrrolidone (PVP) 10,000 and PVP 40,000 were embryo-toxic even after extensive dialysis against Milli-Q water. As both Ficoll and dextran contribute to a solution's physical vitrification properties, we formulated vitrifying solutions containing only 11 to 27 wt% ethylene glycol (EG) by including 34 to 49 wt% polymers (27 wt% EG + 34 wt% Ficoll, 27 wt% EG + 34 wt% dextran, 16 wt% EG + 39 wt% Ficoll, or 11 wt% EG + 49 wt% Ficoll, in phosphate-buffered saline (PBS)). Novel solutions were designed for 0.25 ml straw as a viscous matrix for encapsulation of embryos. These yielded high rates of development of 2-cell mouse embryos after rapid cooling and warming (> or = 96% expanded blastocysts in vitro and > or = 62% viable fetuses as assessed on day 15 of gestation in vivo) in all tested solutions. All control 2-cell embryos formed expanded blastocysts in vitro and 78% formed fetuses in vivo. Comparable results were obtained with both 4-cell and 8- to 16-cell mouse embryos. The lower toxicity of Ficoll and dextran may explain why these new solutions gave better results than had previously been reported for solutions containing 7.5% PVP and low concentrations of EG (2 M).     

Vitrification of rat embryos at various developmental stages

The effect of developmental stage on the survival of cryopreserved rat embryos was examined. Wistar rat embryos at various developmental stages were vitrified by a 1-step method with EFS40, an ethylene glycol-based solution, or by a 2-step method with EFS20 and EFS40. After warming, the survival of the embryos was assessed by their morphology, their ability to develop to blastocysts (or expanded blastocysts for blastocysts) in culture, or their ability to develop to term after transfer. Most (91-100%) of the embryos recovered after vitrification were morphologically normal in all developmental stages. However, the developmental ability of 1-cell embryos was quite low; exposing them to EFS40 for just 0.5 min decreased the in vitro survival rate from 76 to 9%. The survival rates of 2-cell embryos and blastocysts, both in vitro and in vivo, were significantly higher with a 2-step vitrification process than with a 1-step vitrification process. Very high in vitro survival rates (94-100%) were obtained in 4- to 8-cell embryos and morulae in the 1-step method. Although survival rates in vivo of 4-cell (40%) and 8-cell (4%) embryos vitrified by the 1-step method were comparatively low, the values were similar to those obtained in non-vitrified fresh embryos. When morulae vitrified by the 1-step method were transferred to recipients, the in vivo survival rate (61%) was high, and not significantly different from that of fresh embryos (70%). These results show that rat embryos at the 2-cell to blastocyst stages can be vitrified with EFS40, and that the morula stage is the most feasible stage for embryo cryopreservation in this species.     

Effects of fetal calf serum, phenazine ethosulfate and either glucose or fructose during in vitro culture of bovine embryos on embryonic development after cryopreservation

This study investigated effects of hexoses, fetal calf serum (FCS), and phenazine ethosulfate (PES) during the culture of bovine embryos on blastocyst development and survival after cryopreservation by slow freezing or vitrification. The basal, control medium was chemically defined (CDM) plus 0.5% fatty acid-free BSA. In vitro-produced bovine zygotes were cultured in CDM-1 with 0.5 mM glucose; after 60 hr, 8-cell embryos were cultured 4.5 days in CDM-2. The 8-cell embryos were randomly allocated to a 2 x 3 x 2 x 3 factorial experimental design with two energy substrates (2 mM glucose or fructose); three additives (0.3 microM PES, 10% FCS, and control); two cryopreservation methods using no animal products (conventional slow freezing or vitrification); and semen from three bulls with two replicates for each bull. A total of 1,107 blastocysts were produced. Fructose resulted in 13% more blastocysts per oocyte than glucose (37.2% vs. 32.9%), and per 8-cell embryo (51.3% vs. 45.3%; P < 0.01). No differences were found for additives (P > 0.1) control, FCS, or PES for blastocysts per oocyte or per 8-cell embryo. There was a significant interaction (P < 0.05) between additives and hexoses for blastocyst production; although trends were similar, the benefit of fructose compared to glucose was greater for controls than for FCS or PES. Culture of embryos with PES, which reduces cytoplasmic lipid content, improved cryotolerance of bovine embryos; post-cryopreservation survival of blastocysts averaged over vitrification and slow freezing (between which there was no difference) was 91.9%, 84.9%, and 60.2% of unfrozen controls (P < 0.01) for PES, control, and FCS groups, respectively.     

Morphologic evaluation and actin filament distribution in porcine embryos produced in vitro and in vivo

Porcine embryos produced in vitro have a small number of cells and low viability. The present study was conducted to examine the morphological characteristics and the relationship between actin filament organization and morphology of porcine embryos produced in vitro and in vivo. In vitro-derived embryos were produced by in vitro maturation, in vitro fertilization (IVF), and in vitro development. In vivo-derived embryos were collected from inseminated gilts on Days 2-6 after estrus. In experiment 1, in vitro-derived embryos (相似文献   

Carboxylated ε-poly-L-lysine,a cryoprotective agent,is an effective partner of ethylene glycol for the vitrification of embryos at various preimplantation stages

It has been known that different protocols are used for embryo preservation at different stages due to different sensitivity to the physical and physiological stress caused by vitrification. In this study, we developed a common vitrification protocol using carboxlated ε-poly-l-lysine (COOH-PLL), a new cryoprotective agent for the vitrification of mouse embryos at different stages. The IVF-derived Crl:CD1(ICR) x B6D2F1/Crl pronuclear, 2-cell, 4-cell, and 8-cell, morula and blastocyst stage embryos were vitrified with 15% (v/v) ethylene glycol (EG) and 10% (w/v) COOH-PLL (E15P15) or 15% (v/v) EG and 15% (v/v) dimethyl sulfoxide (E15D15) using the minimal volume cooling method. The survival of vitrified embryos from pronuclear to blastocyst stages was equivalent between E15P15 and E15D15 groups. However, the rate of development to blastocysts was significantly lower in E15P15 than E15D15. The rates of survival and development to blastocysts were dramatically improved by a slight modification of EG and COOH-PLL concentrations (E20P10). After transferring 17 (E20P10) and 15 (E15D15) vitrified/warmed blastocysts, 8 and 7 pups were obtained (47.1% and 46.7%, respectively). Taken together, these results indicate that our vitrification protocol is appropriate for the vitrification of mouse embryos at different stages.     

Development of 4-cell mouse embryos after re-vitrification

This paper reports studies on the effects of re-vitrification by the CPS (Closed Pulled Straw) method on the development of 4-cell stage mouse embryos. The procedure involved culturing 2-cell mouse embryos in G-1 medium until the 4-cell stage followed by the division of the normal 4-cell stage embryos into a control group (non-vitrified) and two experimental subgroups (vitrified and re-vitrified). Embryos in the vitrified subgroup were cryopreserved by the CPS vitrification method. In the second experimental subgroup (re-vitrified), embryos that were already vitrified were warmed and cryopreserved again by the same method. There was no significant reduction in the rate of blastocyst formation after vitrification and re-vitrification. However, re-vitrification reduced the total cell number, ICM (inner cell mass) percent and blastocyst diameter (P<0.05). These results showed that vitrification and re-vitrification by the CPS method did not negatively affect the development of vitrified-warmed 4-cell mouse embryos, whereas re-vitrification significantly reduced both the cell number and diameter of blastocysts.     

Open-pulled straw vitrification differentiates cryotolerance of in vitro cultured rabbit embryos at the eight-cell stage

The objective was to determine cryotolerance of in vitro cultured rabbit embryos to the open-pulled straw (OPS) method. Overall, 844 rabbit embryos at pronuclear, 2- to 4-cell, 8-cell, and morula/blastocyst stages were vitrified, and ≥ 1 mo later, were sequentially warmed, rehydrated, and subjected to continuous culture (n = 691) or embryo transfer (ET, n = 153). Embryos vitrified at the 8-cell stage or beyond had greater survival, expanded blastocyst and hatched blastocyst rates in vitro, and better term development than those vitrified at earlier stages. The 8-cell group had 70.1% expanded blastocysts, 63.7% hatched blastocysts, and 25.7% term development, as compared to 1.5-17.7%, 1.5-4.3% and 2.8-3.7% in the pronuclear, 2-cell and 4-cell embryos, respectively (P < 0.05). The expanded and hatched blastocyst rates in vitrified morula/blastocyst post-warming were higher than that in the 8-cell group; however, their term development after ET was similar (8-cell vs morula/blastocyst: 25.7 vs 19.4%, P > 0.05). Development after ET was comparable between vitrified-warmed embryos and fresh controls at 8-cell and morula/blastocyst stages (19.4-25.7 vs 13.7-26.6%, P > 0.05). For embryos at pronuclear or 2- to 4-cell stages, however, term rates were lower in the vitrified-warmed (2.8-3.7%) than in fresh controls (28.6-35.6%, P < 0.05). Therefore, cultured rabbit embryos at various developmental stages had differential crytolerance. Under the present experimental conditions, the 8-cell stage appeared to be the critical point for acquiring cryotolerance. We inferred that for this OPS cryopreservation protocol, rabbit embryos should be vitrified no earlier than the 8-cell stage, and stage-specific protocols may be needed to maximize embryo survival after vitrification and re-warming.     

Cryopreservation of porcine embryos derived from in vitro-matured oocytes

This study describes a cryopreservation method for porcine in vitro-produced (IVP) embryos using as a model parthenogenetic embryos derived from in vitro-matured (IVM) oocytes. IVP embryos at the expanded blastocyst stage were cryopreserved by vitrification using the minimum volume cooling (MVC) method and exhibited an embryo survival rate of 41.2%. Survival was then significantly improved (83.3%, P < 0.05) by decreasing the amount of cytoplasmic lipid droplets (delipation) prior to vitrification. IVP embryos at the 4-cell stage also survived cryopreservation when vitrified after delipation (survival rate, 36.0%), whereas post-thaw survival of nondelipated embryos was quite low (9.7%). Furthermore, it was demonstrated that porcine IVP morulae can be cryopreserved by vitrification following delipation by a noninvasive method (survival rate, 82.5%). These results clearly confirm that porcine embryos derived from IVM oocytes can be effectively cryopreserved with high embryo survival using the MVC method in conjunction with delipation.     

Obesity does not aggravate vitrification injury in mouse embryos: a prospective study

ABSTRACT: BACKGROUND: Obesity is associated with poor reproductive outcomes, but few reports have examined thawed embryo transfer in obese women. Many studies have shown that increased lipid accumulation aggravates vitrification injury in porcine and bovine embryos, but oocytes of these species have high lipid contents (63 ng and 161 ng, respectively). Almost nothing is known about lipids in human oocytes except that these cells are anecdotally known to be relatively lipid poor. In this regard, human oocytes are considered to be similar to those of the mouse, which contain approximately 4 ng total lipids/oocyte. To date, no available data show the impact of obesity on vitrification in mouse embryos. The aim of this study was to establish a murine model of maternal diet-induced obesity and to characterize the effect of obesity on vitrification by investigating the survival rate and embryo developmental competence after thawing. METHODS: Prospective comparisons were performed between six--eight-cell embryos from obese and normal-weight mice and between fresh and vitrified embryos. Female C57BL/6 mice were fed standard rodent chow (normal-weight group) or a high-fat diet (obese group) for 6 weeks. The mice were mated, zygotes were collected from oviducts and cultured for 3 days, and six--eight-cell embryos were then selected to assess lipid content in fresh embryos and to evaluate differences in apoptosis, survival, and development rates in response to vitrification. RESULTS: In fresh embryos from obese mice, the lipid content (0.044 vs 0.030, P<0.01) and apoptosis rate (15.1% vs.9.3%, P<0.05)were significantly higher, the survival rate (83.1% vs. 93.1%, P<0.01) on day 5 was significantly lower, and embryo development was notably delayed on days 3--5 compared with the normal-weight group. After vitrification, no significant difference was found between thawed embryos from obese and normal-weight mice in apoptosis, survival, and development rates on days 4 and 5. In both groups, pre- and post-vitrification embryo apoptosis, survival, and development rates were similar. CONCLUSIONS: This study demonstrated that differences in survival and developmental rates between embryos from obese and normal-weight mice were eliminated after vitrification. Thus, maternal obesity does not aggravate vitrification injury, but obesity alone greatly impairs pre-implantation embryo survival and development.     

Characteristic of structural and functional organization of two-cell mouse embryos exposed to inhibitors of cell proliferation

Within the framework of studying the "2-cell block in vitro" phenomenon, two variants of inhibitory-induced stoppage of development at a two-cell stage were produced and analysed. Mimosine arrested the cleavage on the G1/S interface, and genistein at G2 stage of the second cell cycle. In the experimentally blocked embryos a detailed study was made of the ultrastructural organization of blastomeres and intracellular localization of mitochondria vitally stained with rhodamine 123. The light and electron microscope observations testify to the viability of the embryos within a 22-24 hour exposure to inhibitors. Adhesive contacts between blastomeres were seen to slack after the treatment with both the inhibitors, resp., but in particular after genistein treatment. At the ultrastructural level no significant destructive modifications in blastomere organization were noticed. The cytoplasm of the control and treated embryo cells displayed diffusely distributed sheets of intermediate filaments, morphologically looking immature mitochondria and numerous aggregated lipid inclusions. The nuclear morphology was similar in both cases. Mitochondria of the treated embryo cells kept their ability to accumulate rhodamine 123, which testifies to their functional activity. However, the character of mitochondrial intracellular distribution was seen to change from diffuse to clustered. Numerous mitochondria clusters were concentrated mainly in the perinuclear area of blastomeres. As in the control ones, in the treated embryos the position of the nuclei was visualized by ring-like concentrations of mitochondria in the central part of blastomeres; in mimosine-treated cells the "rings" were thickened and contained mitochondria clusters. In genistein-treated embryos, mitochondria form numerous tiny clusters uniformly distributed in the cytoplasm; the perinuclear "rings" are still present, though less distinct than in the control embryos. Thus, it may be concluded that although the inhibitory treatment of two-cell embryos truly modified the mitochondrial distribution in these, the eventual pattern of such changes differed considerably from that characteristic of embryos in the state of "2-cell block in vitro". These results support the view on the unique character of morphofunctional modifications that occur in the latter embryos.     

自制载体冷冻小鼠原核期胚胎效果分析

目的探讨自制冷冻载体冷冻保存昆明小鼠体内原核期胚胎的可行性。方法首先,比较了两种流行的商业化载体:开放式拉长麦管(open pulled straw,OPS)和冷冻帽(cryotop)开展小鼠原核胚玻璃化冷冻保存效果。其次,以cryotop为对照,利用自制简易载体(cryotip)开展小鼠原核期胚胎的玻璃化冷冻保存。之后,利用ANOVA对各组胚胎在复苏后的体外培养卵裂率、囊胚率进行统计分析。结果 OPS和cryotop两组之间,胚胎在玻璃化冷冻/复苏后发育的2-细胞率、4-细胞率和囊胚率差异均无显著性(P0.05),但cryotop冷冻效果更接近对照组;cryotip玻璃化冷冻载体与cryotop相比,胚胎复苏后各组差异均无显著性(P0.05),数值上除了2-细胞发育率外,cryotip其他几项结果都稍微高于cryotop组。结论 OPS,cryotop,cryotip冷冻保存昆明小鼠体内原核期胚胎均是可行的;cryotop在冷冻效果上要优于OPS,笔者自制的cryotip因其成本低,制作简单,操作安全可靠,在实验中替代昂贵的商业化载体OPS和cryotop是可行的。     

Tolerance to vitrification of rat embryos at various developmental stages

Numerous genetically engineered rat strains have been produced via genome editing. Although freezing of embryos is helpful for the production and storage of these valuable strains, the tolerance to freezing of embryos varies at each developmental stage of the embryo. This study examined the tolerance to freezing of rat embryos at various developmental stages, particularly at the pronuclear stage. Embryos that had developed to the pronuclear, 2-cell, and morula stages were frozen via vitrification using ethylene glycol- and propylene glycol-based solutions. More than 90% of the embryos at all developmental stages survived after warming. The developmental rates to offspring of thawed embryos at the pronuclear, 2-cell, and morula stages were 19%, 41%, and 52%, respectively. Pronuclear stage embryos between the early and late developmental stages were then vitrified. The developmental rates to offspring of the thawed pronuclear stage embryos collected at 24, 28, and 31 h after the induction of ovulation were 17%, 21%, and 23%, respectively. These results indicated that the tolerance to vitrification of rat embryos increased with the development of embryos. The establishment of vitrification method of rat embryos at various developmental stages is helpful for improving the production and storage of valuable rat strains used for biomedical science.     

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.