Cell Counts and Survival to Vitrification of Bovine In Vitro Produced Blastocysts Subjected to Sublethal High Hydrostatic Pressure

This work analyses the effects of a high hydrostatic pressure (HHP) treatment on in vitro survival of in vitro produced (IVP) bovine embryos vitrified with the Cryologic Vitrification Method (CVM). Consequences on embryo quality in terms of cell proliferation and differentiation, and levels of embryonic Heat Shock Protein 70 (Hsp‐70) were also examined. Day 7 and 8 bovine in vitro‐produced blastocysts were submitted to an HHP treatment (60 MPa, at 32°C for 1 h) and allowed to recover for 1 or 2 h in culture medium. The HHP treatment did not improve blastocyst survival rates after vitrification/warming. Survival (24 h post‐warming) and hatching (48 h post‐warming) rates were 79.3 ± 4.9 and 51.8 ± 4.2 vs 73.9 ± 4.2 and 44.7 ± 4.1 for untreated controls and HHP‐treated embryos, respectively. Total cell numbers measured in fresh embryos were reduced after 1 h at 32°C, with or without HHP treatment, indicating that cell proliferation was stopped as a result of stress. Vitrified HHP‐treated embryos that hatched at 48 h after warming showed increased cell numbers in their ICM compared with untreated controls (50.2 ± 3.1 vs 38.8 ± 2.7), indicating higher embryo quality. Treatment of blastocysts with HHP did not alter the level of the Hsp‐70 protein. In our conditions, HHP treatment did not affect the cryoresistance of these embryos. However, combination of HHP treatment and vitrification in fibreplugs resulted in an increase in the ICM cell number of hatched embryos 48 h post‐warming.     

Correlation between the Cell Number and Diameter in Bovine Embryos Produced In Vitro

This study was designed to examine the effects of age and developmental stage of in vitro‐produced bovine embryos on the cell number of the embryos and to investigate the correlation between the cell number and diameter in the embryos. The diameter and cell number in blastocysts and expanded blastocysts collected on days 7–9 after in vitro fertilization (IVF) were examined. Although the diameters of the blastocysts collected on days 7 and 8 after IVF were smaller than those of the expanded blastocysts collected on day 9, the cell number in both types of embryos was similar. The cell numbers of the blastocysts and expanded blastocysts decreased with increasing embryo age. There were positive correlations between the cell number and diameter in bovine embryos at each stage collected on each day after IVF. However, the value of the correlation coefficient in the day‐9 expanded blastocyst group tended to be higher than that in the other groups. These results indicate that the cell number of in vitro‐produced embryos is affected by the embryonic stage and age. The diameter of the embryo may be potentially used for the viability testing of the expanded blastocysts collected on day 9 after IVF.     

Effect of vitrification at different meiotic stages on epigenetic characteristics of bovine oocytes and subsequently developing embryos

Vitrification by the Cryotop method is frequently used for bovine oocyte cryopreservation. Nevertheless, vitrified oocytes still have reduced developmental competency compared with fresh counterparts. The objective of this study was to compare the effect of vitrification either at the germinal vesicle (GV) stage or at the metaphase II (MII) stage on epigenetic characteristics of bovine oocytes and subsequently developing embryos. Our results demonstrated that vitrification of oocytes at each meiotic stage significantly reduced blastocyst development after in vitro fertilization (IVF). However, vitrification at the GV stage resulted in higher blastocyst development than did vitrification at the MII stage. Irrespective of the meiotic stage, oocyte vitrification did not affect 5-methylcytosine (5mC) immunostaining intensity in oocyte DNA. However, at both stages, it caused a similar reduction of 5mC levels in DNA of subsequently developing blastocysts. Oocyte vitrification had no effect on the intensity of H3K9me3 and acH3K9 immunostaining in oocytes and subsequent blastocysts. The results suggest that irrespective of meiotic stage, oocyte vitrification alters global methylation in resultant embryos although such alteration in the oocytes was not detected. Oocyte vitrification might not influence histone acetylation and methylation in oocytes and resultant embryos. Vitrification at the immature stage was more advantageous for blastocyst development than at the mature stage.     

Total Cell Number and its Allocation to Trophectoderm and Inner Cell Mass in In Vitro Obtained Cats' Blastocysts

The aim of this study was to evaluate the developmental kinetics of cats' blastocysts in connection with their morphology and blastomeres allocation to trophoblast or embryoblast cells. We examined gross blastocyst morphology and the total number of blastomeres together with its allocation to inner cell mass (ICM) or trophectoderm (TE) cells in pre‐implantation feline embryos obtained from 6th to 9th day of in vitro culture. From all the investigated embryos, 61.8% developed to early blastocyst, 37.4% to full and 7.6% to hatching blastocyst stage. The total cell number (TCN) varied form 58 cells in early day 6 to 245 in hatching day 8 blastocyst, with the mean 84.9 cells in early, 156.7 in full, and 204.4 in hatching ones. Day 8 blastocyst had the highest number of total cells, together with the highest mean number of ICM regardless of its morphological assessment. Early blastocyst (apart from day 6) had the highest number of arrested cells, while dead cells were the highest in full day 9 blastocyst. More data about the relationship between blastocyst development and morphology would facilitate the selection of optimal blastocysts for further procedures.     

Development of large and small blastomeres from 2‐cell embryos produced in vitro in pigs

In the present study, we examined the development to blastocysts of large and small blastomeres from unevenly cleaved 2‐cell embryos (uneven 2‐cell embryos) in pigs. Proportion of blastocysts derived from large blastomeres (52.8 ± 6.4%) was significantly higher (P < 0.05) compared with small ones (32.1 ± 4.6%). However, there were no differences in total cell number, inner cell mass (ICM) cell number and ICM/total cells ratio between them. Of 53 sister blastomere pairs in the same embryos examined there were 12 pairs (22.6%) in which both blastomeres developed to blastocysts, 16 pairs (30.2%) in which only large blastomeres developed to blastocysts, and five pairs (9.4%) in which only small blastomeres developed to blastocysts. Relative total amount of active mitochondria in small blastomeres were lower (P < 0.05) than that of large blastomeres and blastomeres from evenly cleaved 2‐cell embryos. However, there was no difference in relative density of active mitochondria in these three types of blastomeres. In conclusion, blastocysts derived from small and large blastomeres in uneven 2‐cell embryos had comparable quality in terms of cell number, ICM number, ICM/total cell ratio and distribution of active mitochondria. The results suggest that these blastomeres may contribute multiple offspring production in pigs.     

Epidermal growth factor improves developmental competence and embryonic quality of singly cultured domestic cat embryos

This study examined the influence of EGF on the expression of EGF receptors (EGFR) and developmental competence of embryos cultured individually versus those cultured in groups. Cat oocytes were in vitro matured and fertilized (IVM/IVF), and cleaved embryos were randomly assigned to one of seven culture conditions: one group each in which embryos were subjected to group culture supplemented with or without 5 ng/ml EGF and five groups in which embryos were subjected to single-embryo culture supplemented with EGF (0, 5, 25, 50 or 100 ng/ml). Morulae, blastocysts and hatching blastocysts were assessed at days 5 and 7; post IVF, respectively, and total blastocyst cell numbers were assessed at day 7. Relative mRNA expressions of EGFR of 2–4-cell embryos, 8–16-cell embryos, morulae and blastocysts cultured in groups or singly with or without EGF supplementation were examined. OCT3/4 and Ki67 in blastocysts derived from the group or single-embryo culture systems with or without EGF supplementation were localized. A higher rate of embryos cultured in groups developed to blastocysts than individually incubated cohorts. Although EGF increased blastocyst formation in the single-embryo culture system, EGF did not affect embryo development in group culture. Expression levels of EGFR decreased in morulae and blastocysts cultured with EGF. An increased ratio of Ki67-positive cells to the total number of cells in the blastocyst was observed in singly cultured embryos in the presence of EGF. However, EGF did not affect the expression of OCT3/4. These findings indicate that EGF enhanced developmental competence of cat embryos cultured singly by stimulating cell proliferation and modulating the EGFR expression at various developmental stages.     

Mouse embryo co‐culture with autologous cumulus cells and fetal development post‐embryo transfer

This study was conducted to examine the potential for implantation and sustainable fetal development of mouse embryos cultured from the pronuclear to blastocyst stage. Pronuclear embryos from ICR mice (Harlan Sprague‐Dawley) were cultured in Sydney IVF sequential media (Cook) to the blastocyst stage in medium only or co‐cultured with autologous cumulus cells. We also experimented with co‐culture in 100 µL drops. Drop co‐culture produced blastocyst formation rates with a mean of 47.0%, which was significantly higher (P < 0.05) compared to embryos cultured in identical culture conditions except without cumulus cells at 27.3%. Blastocysts obtained in vitro in Cook medium only and co‐cultured in Cook medium with cumulus cells were transferred to pseudopregnant females of ICR strain. The day of blastocyst transfer into surrogate females was designated as post‐transfer of blastocyst day 1 (PT 1). The implantation and fetal development was compared to embryo transfer of in vivo derived blastocysts, which served as controls. There were no statistical differences for implantation and fetal development rates for blastocysts cultured in vitro in either Cook medium only or co‐culture in Cook medium with cumulus cells compared to in vivo‐derived blastocysts. The advantage of the co‐culture system is in generating more blastocysts available for transfer.     

Effect of different penetrating and non‐penetrating cryoprotectants and media temperature on the cryosurvival of vitrified in vitro produced porcine blastocysts

The aim of this study was to determine the most efficient vitrification protocol for the cryopreservation of day 7 in vitro produced (IVP) porcine blastocysts. The post‐warm survival rate of blastocysts vitrified in control (17% dimethyl sulfoxide + 17% ethylene glycol [EG] + 0.4 mol/L sucrose) and commercial media did not differ, nor did the post‐warm survival rate of blastocysts vitrified in medium containing 1,2‐propandiol in place of EG. However, vitrifying embryos in EG alone decreased the cryosurvival rate (55.6% and 33.6%, respectively, p < .05). Furthermore, the post‐warm survival rates of blastocysts vitrified with either trehalose or sucrose as the non‐penetrating cryoprotectant did not differ. There was also no significant difference in post‐warm survival of blastocysts vitrified in control (38°C) media and room temperature (22°C) media with extended equilibration times, although when blastocysts were vitrified using control media at room temperature, the post‐warm survival rate increased (56.8%, 57.3%, 72.5%, respectively, p < .05). The findings show that most cryoprotectant combinations examined proved equally effective at supporting the post‐warm survival of IVP porcine blastocysts. The improved post‐warm survival rate of blastocysts vitrified using media held at room temperature suggests that the cryoprotectant toxicity exerted in 22°C media was reduced.     

Effect of cryoprotectant composition on in vitro viability of in vitro fertilized and cloned bovine embryos following vitrification and in-straw dilution

In this study the efficacy of the combination of glycerol (GLY) and ethylene glycol (EG) as cryoprotectants in a vitrification method developed for direct embryo transfer was evaluated by in vitro development of in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos after vitrification. The IVF and SCNT blastocysts were vitrified in either 40% GLY, 30% GLY + 10% EG, or 20% GLY + 20% EG using French straws. After warming, the straws were held vertically for 1 min without shaking and were then placed horizontally for 5 min to dilute the cryoprotectants. After washing, the vitrified-warmed embryos were cultured in vitro for 72 h. There were no differences among the vitrification solutions with respect to the rates of vitrified-warmed IVF and SCNT embryos surviving and developing to the hatched blastocyst stage. However, the rates of development to the hatched blastocyst stage of the SCNT embryos vitrified with 40% GLY tended to be higher than those vitrified with 30% GLY + 10% EG or 20% GLY + 20% EG (26% vs. 7-8%, respectively). The development rates to the hatched blastocyst stage of the IVF and SCNT embryos vitrified with solution containing EG were significantly lower (P<0.05) than those of non-vitrified embryos. These results suggest that use of the combination of GLY and EG as cryoprotectants had no beneficial effect on the viability of embryos after in-straw dilution. However, this method is so simple that it can be used for practical direct transfer of vitrified embryos in the field.     

Cellular Composition and Viability of Cloned Bovine Embryos Using Exogene-Transfected Somatic Cells

The present study compared the efficiency of transgenic (TG) cloned embryo production by somatic cell nuclear transfer (SCNT) with fetal-derived fibroblast cells (FFCs) which were transfected with pEGFP-N1 to in vitro-fertilized (IVF), parthenogenetic and SCNT counterparts by evaluating the rates of cleavage and blastocyst formation, apoptosis rate at different developmental stages, cell number, ploidy and gene expression in blastocysts. In SCNT and TG embryos, the rates of cleavage and blastocyst formation were significantly lower (p < 0.05) than those of IVF controls, but it did not differ between SCNT and TG embryos. In IVF control, 86.7% embryos displayed diploid chromosomal complements and the rates were significantly (p < 0.05) higher than those of SCNT and TG embryos. Most TG embryos (79%) with FFCs expressed the gene by both PCR and under fluorescence microscopy. The expression of apoptosis by TUNEL was first detected at six to eight cell stages in all embryos of IVF, SCNT and TG groups, but the expression rate at each developmental stages was significantly higher (p < 0.05) in SCNT and TG embryos than in IVF counterparts. The expression rate in inner cell mass (ICM) of TG embryos was significantly higher (p < 0.05) than in SCNT and IVF embryos. These results indicate that the high occurrence of apoptosis observed in SCNT and TG embryos compared with IVF counterparts might influence the developmental competence. Moreover, the SCNT embryos derived using non-transfected donor cells exhibited a lower apoptosis expression in ICM cells than in TG embryos derived using pEGP-N1-transfected donor cells suggesting a possible role of negative gene effect in TG embryos.     

Vitrification of bovine matured oocytes and blastocysts in a paper container

In the present study, we aimed to determine the applicability of a paper container for the vitrification of in vitro matured (IVM) bovine oocytes. In experiment 1, IVM oocytes were exposed to vitrification solution (20% dimethylsulfoxide (DMSO), 20% ethylene glycol (EG), and 5 mol/L sucrose), using a two‐step method, for 30 s; loaded onto either a paper container or Cryotop; and stored in liquid nitrogen. No significant difference (P < 0.05) in the survival and blastocyst formation rates after in vitro vitrification was observed between the paper container and Cryotop. In experiment 2, IVM oocytes were exposed to either a two‐ or three‐step vitrification solution. The three‐step vitrification solution was not significantly different from the two‐step solution in terms of oocyte survival, cleavage and blastocyst rates. In experiment 3, in vitro produced blastocysts were graded according to the manual of the International Embryo Transfer Society (grades 1 and 2) and vitrified using the two‐ and three‐step methods. For grade 2 blastocysts, the three‐step method showed significantly higher (P < 0.05) survival and hatched blastocyst rates than the two‐step method, whereas for grade 1 blastocysts, no significant difference was observed. In conclusion, the paper device and three‐step technique are suitable for oocytes and embryo vitrification.     

Effect of co‐culture with intact embryos on development of bovine separated blastomeres

To improve embryo development in bovine separated blastomeres, we evaluated applicability of co‐culture with intact embryos. The morphological quality of blastocysts derived from separated blastomeres and rate of blastocyst formation were only slightly increased when the cells were co‐cultured with intact embryos, which did not provide significant differences when statistically analyzed. However, the cell count of inner cell mass (ICM), trophectoderm (TE) and total number of cells in Day 8 blastocysts were significantly higher when the cells were co‐cultured with the intact embryos than those with the cells cultured individually (P < 0.05). Transfer of four monozygotic pairs of blastocysts derived from the cells co‐cultured with intact embryos led to three pregnancies even when the blastomeres were produced by in vitro maturation and in vitro fertilization of oocytes collected by ovum pick‐up from elite cows. These results suggest that co‐culturing with intact embryos may enhance development of bovine separated blastomere.     

牛体外受精早期胚胎与小鼠胎仔成纤维细胞共培养的研究

探讨了人工合成培养液ＣＲ１ａａ和小鼠胎仔成纤维细胞对牛体外受精早期胚胎体外发育的影响。结果表明，牛体外受精卵在ＣＲ１ａａ液中的卵裂率达７６．２％，８细胞胚的比率达４４．８％。小鼠胎仔成纤维细胞能够显著促进牛体外受精的早期囊胚以上胚胎的发育。牛体外受精后第５、６天的早期胚胎分别与小鼠胎仔成纤维细胞共培养，在受精后第７天发育至囊胚以上的比率分别达１９．８％和２４．６％；受精后第８天，孵化的囊胚比例分别达５．２％和７．５％。实验表明，受精后第５、６天的牛体外受精早期胚胎与小鼠胎仔成纤维细胞共培养，可显著提高扩张囊胚和孵化囊胚数量。小鼠成纤维细胞对胚胎发育的支持作用取决于胚胎发育阶段     

Effects of ghrelin on in vitro development of porcine in vitro fertilized and parthenogenetic embryos

The objective of the present study was to investigate the effect of addition of ghrelin to in vitro culture medium on preimplantation development of porcine in vitro fertilized and parthenogenetic embryos. In Experiment 1, we sought to compare the in vitro developmental competence of IVF and parthenogenetic embryos. No significant (P<0.05) differences were detected for cleavage rate or blastocyst rate between the in vitro fertilization (IVF)- and parthenogenetic activation-derived embryos. In Experiment 2, parthenogenetic embryos were cultured in Porcine Zygote Medium-3 containing various concentrations of ghrelin. The blastocyst rate was remarkably (P<0.05) increased when 5 ng/ml (PA-5) and 500 ng/ml (PA-500) of ghrelin was added to in vitro culture medium compared with the other groups. Total cell number per blastocyst was slightly promoted in the ghrelin treatment groups compared with the controls. However, the ratio of inner cell mass (ICM) cell number/total cell number was significantly reduced in the PA-50 group compared with the controls (P<0.05). In Experiment 3, we cultured in vitro fertilized embryos in Porcine Zygote Medium-3 supplemented with ghrelin at different dosages. The rate of blastocyst formation was markedly (P<0.05) elevated when 500 ng/ml ghrelin was added to culture medium (IVF-500) compared with the controls. Increased total cell numbers (P<0.05) were observed when in vitro fertilized embryos were cultured in IVF-50 and IVF-500 compared with the controls. However, the ratio of ICM cell number/total cell number was decreased in the ghrelin treatment groups compared with the controls (P<0.05). Taken together, the results suggest that ghrelin can enhance blastocyst formation of porcine in vitro fertilized and parthenogenetic embryos while exerting a negative effect on the structural integrity of the blastocysts.     

Forced Collapse of the Blastocoel Cavity Improves Developmental Potential in Cryopreserved Bovine Blastocysts by Slow‐Rate Freezing and Vitrification

This study was conducted to evaluate the effectiveness of forced collapse of the blastocoel before slow‐rate freezing and vitrification of bovine blastocysts. Cryopreservation of bovine blastocysts has been proposed as a tool to improve the feasibility of cattle production using the embryo transfer technique. However, the low efficiency of frozen–thawed embryos survival and further development is a crucial problem. In this study, bovine in vitro and in vivo blastocysts were slow‐rate frozen and vitrified after forced blastocoele collapse (FBC) of the blastocyst cavity by puncturing the blastocoele with a pulled Pasteur pipet. Differences in the developmental potential of frozen–thawed blastocysts derived from FBC and non‐FBC groups were found in both slow‐rate freezing and vitrification. Furthermore, we found that the total cell number of blastocysts in FBC groups was increased and the index of apoptosis in FBC groups was decreased. Consistent with these results, real‐time RT‐PCR analysis data showed that expression of the anti‐apoptotic Bcl‐XL gene was significantly increased by FBC groups, whereas expression of the pro‐apoptotic Bax gene was significantly decreased by FBC groups. Our results also showed that pregnancy outcomes in both slow‐rate frozen and vitrified bovine in vivo blastocysts could be improved by reducing the fluid content after FBC of the blastocyst cavity. Therefore, we suggest that FBC of the blastocyst cavity with a pulled Pasteur pipet is an effective pre‐treatment technique for both slow‐rate freezing and vitrification of bovine blastocysts.     

Effects of Serum-Free In Vitro Maturation of Bovine Oocytes on Subsequent Embryo Development and Cell Allocation in Two Developmental Stages of Day 7 Blastocysts

Maturation of oocytes and the subsequent outcome of the in vitro production (IVP) are affected by the composition of in vitro maturation (IVM) medium. To determine the use of serum interfering with effects of single molecules, we aimed at developing simplified IVM medium. The experimental IVM media were: (1) M199-medium supplemented with hormones and serum (control), (2) as 1 but serum was substituted with fatty acid-free serum albumin (FAFBSA) and (3) M199-medium without hormonal and serum supplementation (M199). The quality of embryos was assessed on day 7 by morphology and cryotolerance, as well as by Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) and differential staining. Results showed that the nuclear maturation was suppressed in M199 group alone. Embryo cleavage and development rates, and the proportion of quality 1 blastocysts were lower in the FAFBSA and M199 groups compared to the control. Differences in the cell allocation of fresh embryos were observed at the blastocyst stage, but not at the expanded blastocyst stage. The control group blastocysts had larger number of cells allocated to the inner cell mass (ICM), and the FAFBSA group blastocysts larger apoptotic cell proportion compared to the blastocysts derived from other groups. After cryopreservation, the reduction of ICM proportion and increase of apoptotic cell proportion of embryos were equal between the experimental groups. In conclusion, exclusion of serum from the IVM media reduces embryo development and may cause perturbations in blastocyst development. Differences in the cell allocation of blastocysts between IVM media may appear only when the developmental stages are taken into account.     

Absence of Sperm Factors as in the Parthenogenesis Does Not Interfere on Bovine Embryo Sensitiveness to Heat Shock at Pre‐Implantation Stage

Oocyte has been considered the major contributor for embryo thermo‐tolerance. However, it was shown that sperm factors can be transferred to the oocyte during fertilization, raising the question of whether the absence of such factors could interfere on embryo thermo‐tolerance. In this study, we used parthenogenesis to generate bovine embryos without spermatozoa in order to test whether the absence of sperm factors could influence their thermo‐sensitiveness at early stages. In vitro fertilized (IVF) and parthenogenetic (PA) embryos at 44 h post‐insemination/chemical activation were exposed to 38.5°C (control) or 41°C (heat shock) for 12 h and then developed for 48 h and up to blastocyst stage. Apoptosis index and expression of PRDX1, GLUT1, GLUT5 and IGF1r genes in blastocysts derived from heat‐shocked embryos were also evaluated. The heat shock decreased the blastocyst rate at day seven (p < 0.05) for IVF embryos and at day eight (p < 0.01) for both IVF and PA embryos. Total cell number was not affected by heat shock in IVF and PA blastocysts, but there was an increased proportion (p < 0.05) of apoptotic cells in heat‐shocked embryos when compared to controls. There was no interaction (p > 0.05) between method of activation (IVF and PA) and temperature (38.5°C or 41.5°C) for all developmental parameters evaluated. Expression of GLUT1 gene was downregulated (p < 0.05) by heat shock in both IVF and PA blastocyst whereas expression of GLUT5 and IGF1r genes was downregulated (p < 0.05) by heat shock in PA blastocysts. Those data show that the heat shock affects negatively the embryo development towards blastocysts stage, increases the apoptotic index and disturbed the expression of some genes in both IVF and PA embryos, indicating that the presence or absence of sperm factors does not influence the sensitivity of the bovine embryo to heat shock.     

OPS玻璃化冷冻对小鼠四倍体胚胎体外发育的影响

为探究开放式拉长细管(OPS)玻璃化冷冻对四倍体胚胎发育的影响,本实验利用2-细胞胚胎电融合法制备四倍体胚胎,再对四倍体胚胎进行OPS玻璃化冷冻,分别观察记录二倍体胚胎、四倍体胚胎以及冷冻解冻后四倍体胚胎的发育情况。结果表明:2-细胞胚胎电融合效率为96.1%;二倍体胚胎组与电融合后四倍体胚胎组的囊胚率和孵化囊胚率差异不显著;冷冻解冻后四倍体胚胎的囊胚率(100%)与四倍体新鲜组(93.3%)差异不显著,其孵化囊胚率(72.3%)较新鲜组(64.9%)显著增高(P<0.05);四倍体冷冻解冻组的囊胚细胞数(31.96)与新鲜组(32.54)无显著差异;冷冻解冻后的四倍体早期囊胚进行体外培养时其发育速度比对照组更快。可见,冷冻对小鼠四倍体胚胎的囊胚率和囊胚细胞数均无显著影响,但孵化囊胚率显著提高,且OPS玻璃化冷冻后使四倍体胚胎的发育速度更快。     

Insulin‐like growth factor I enhances the developmental competence of yak embryos by modulating aquaporin 3

The objective of our present study was to determine the effects of insulin‐like growth factor I (IGF‐I) on the development of yak (Bos grunniens) embryos after cumulus–oocyte complex (COC) vitrification and warming followed by in vitro fertilization (IVF). In Experiment 1, the yak COCs underwent vitrification and then IVF. Embryos were incubated in synthetic oviductal fluid (SOF) supplemented with four concentrations (0, 50, 100 and 200 ng/ml) of IGF‐I, while the yak COCs without vitrification or IGF‐I supplementation acted as the control group; the BAX, BCL‐2, AQP3mRNA and aquaporin 3 (AQP3) protein expression levels in the five groups of blastocysts were evaluated using quantitative real‐time PCR and immunofluorescence analyses. In Experiment 2, the groups described above were fertilized and incubated. The cleavage rate, blastocyst rate, total cell count per blastocyst and the rate of growth of the inner cell mass (ICM) and trophectoderm (TE) were evaluated. The results were as follows: (1) the AQP3 gene expression and protein expression in the control and 100 ng/ml IGF‐I treatment groups were the highest. (2) The BAX gene expression was the lowest and the BCL‐2 gene expression was the highest in the control and 100 ng/ml IGF‐I treatment groups. (3) The rates of cleavage and blastocysts in the control and 100 ng/ml IGF‐I groups were higher than those in the other three groups. The total cell count per blastocyst in the vitrified and warmed 100 ng/ml IGF‐I group (106.7 ± 4.9) and the control group (107.3 ± 4.2) was higher than that in the vitrified and warmed 0 ng/ml IGF‐I (91.2 ± 3.1), 50 ng/ml IGF‐I (92.3 ± 3.7) and 200 ng/ml IGF‐I (92.4 ± 3.7) groups. Therefore, we conclude that IGF‐I can improve yak blastocyst developmental ability, cytomembrane permeability and formation of the blastocyst cavity after COC vitrification by improving the BAX, BCL‐2 and AQP3 expression levels.