Blastocysts derived from in vitro-fertilized cat oocytes after vitrification and dilution with sucrose

Experiments were conducted to find an optimal incubation period in a sucrose solution during dilution of cryoprotectants for obtaining a higher level of survival and development of cat oocytes cryopreserved by vitrification method. In the first experiment, in vitro-matured fresh oocytes were exposed to 0.5M sucrose solution for 1 or 5 min before in vitro fertilization (IVF). The percentage of development to the blastocyst stage significantly decreased in oocytes exposed for 5 min, compared with oocytes exposed for 1 min and control oocytes without exposure to sucrose (P<0.05). In the second experiment, oocytes that had been vitrified in 40% ethylene glycol and 0.3M sucrose were liquefied and then incubated in 0.5M sucrose for 0.5, 1 or 5 min to dilute the cryoprotectant. The percentage of cleavage (>or=2-cell stage) of vitrified-liquefied oocytes incubated for 0.5 min was significantly higher (P<0.05) than that of other groups. Development of vitrified-liquefied oocytes to the morula and blastocyst stages after IVF was observed only in oocytes incubated in sucrose for 0.5 min. The present study indicates that the oocytes have sensitivity to the toxic effect of sucrose and that the incubation period during dilution of the cryoprotectant is of critical importance for developmental competence of vitrified-liquefied cat oocytes.     

Nuclear maturation and embryo development of porcine oocytes vitrified by cryotop: Effect of different stages of in vitro maturation

The present study was designed to evaluate the viability, meiotic competence and subsequent development of porcine oocytes vitrified using the cryotop method at different stages of in vitro maturation (IVM). Cumulus–oocyte complexes (COCs) were cultured in IVM medium supplemented with 1 mM dibutyryl cAMP (dbcAMP) for 22 h and then for an additional 22 h without dbcAMP in the medium. Germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), anaphase I/telophase I (AI/TI) and metaphase II (MII) were found to occur predominantly at 0–22, 26, 32, 38 and 44 h of IVM, respectively. Oocytes were exposed to cryoprotectant (CPA) or vitrified after different durations of IVM (0, 22, 26, 32, 38 and 44 h). After CPA exposure and vitrification, surviving oocytes that were treated before completion of the 44 h maturation period were placed back into IVM medium for the remaining maturation period, and matured oocytes were incubated for 2 h. CPA treatment did not affect the viability of oocytes matured for 26, 32, 38 or 44 h, but significantly decreased survival rate of oocytes matured for 0 or 22 h. CPA treatment had no effect on the ability of surviving oocytes to develop to the MII stage regardless of the stage during IVM; however, blastocyst formation following PA was severely lower (P < 0.05) than that in the control. At 2 h post-warming, the survival rates of oocytes vitrified at 26, 32, 38 and 44 h of IVM were similar but were higher (P < 0.05) than those of oocytes vitrified at 0 or 22 h of IVM. The MII rates of surviving oocytes vitrified at 0 and 38 h of IVM did not differ from the control and were higher (P < 0.05) than those of oocytes vitrified at 22, 26 or 32 h of IVM. After parthenogenetic activation (PA), both cleavage and blastocyst rates of vitrified oocytes matured for 22, 26, 32, 38 and 44 h did not differ, but all were lower (P < 0.05) than those matured 0 h. In conclusion, our data indicate that survival, nuclear maturation and subsequent development of porcine oocytes may be affected by their stage of maturation at the time of vitrification; a higher percentage of blastocyst formation can be obtained from GV oocytes vitrified before the onset of maturation.     

Effects of oxygen tension and follicle cells on maturation and fertilization of porcine oocytes during in vitro culture in follicular fluid

The objective was to investigate the effects of oxygen tension and follicle cells (FCs) during in vitro maturation of porcine oocytes in only porcine (Sus scrofa domesticus) follicular fluid (pFF), using static and non-static (rotating) culture systems, on the nuclear maturation and subsequent in vitro fertilization of the oocytes. In the first experiment, cumulus-oocyte complexes (COCs) were matured for 48 h in pFF supplemented with (+) or without (−) FCs (5.2 × 10⁶ cells/mL), using the static (S) and rotating (R) culture systems (+FC/S, −FC/S, +FC/R, and −FC/R) under 5% or 20% O₂. Co-culture with FCs in the static culture system (+FC/S) had a detrimental effect on the meiotic competence of oocytes, whereas co-culture with FCs in the rotating culture system (+FC/R) increased maturation rates. In both culture systems, oxygen tension had no apparent effects on meiotic competence of oocytes, irrespective of culture system and FC addition. In the second experiment, COCs were matured under 5% or 20% O₂ using the −FC/S or +FC/R culture systems and then fertilized. Oxygen tension had no significant effects on fertilization parameters, irrespective of the culture system. The rotating culture system increased rates of sperm penetration and male pronuclear formation and decreased polyspermic fertilization compared with the static culture system (P < 0.05). In conclusion, both −FC/S and +FC/R culture systems supported meiotic competence, irrespective of oxygen tension. However, the +FC/R culture system may be superior to the −FC/S culture system for promoting fertilization.     

Effect of vitrification on meiotic maturation and expression of genes in immature goat cumulus oocyte complexes

The aim of the study was to evaluate meiotic maturation, and expression of genes coding for oocyte secreted factors (GDF9, BMP15, TGFBR1, and BPR2) and apoptosis (BCL2, BAX and P53) after vitrification of immature goat cumulus oocyte complexes (COCs) and in vitro maturation. COCs were vitrified in a solution containing ethylene glycol, dimethyl sulfoxide and sucrose using either a conventional straw (CS), open pulled straw (OPS), cryoloop (CL), hemistraw (HS) or cryotop (CT). Freshly collected COCs (Control), COCs exposed to vitrification and dilution solutions without cryopreservation (EC) and vitrified-warmed COCs were matured in vitro for 27h. The viability of vitrified-warmed COCs 2 h post warming and in vitro maturation was similar for CL, HS and CT. The proportion of oocytes that extruded a 1st polar body and reached TI/MII was significantly higher with CT and HS followed by CL, OPS and CS. Gene expression of GDF9, BMP15, BMPR2, BAX and P53 were comparable to control levels for OPS, CL, HS and CT. The gene expression pattern in CS vitrified COCs was by contrast changed in that GDF9, BMP15, TGFBR1 and BAX were up regulated and BMPR2, BCL2 and P53 down regulated. In conclusion immature goat COCs vitrified using CT and HS showed that viability, maturation rates and expression of genes coding for oocyte secreted factors and apoptosis were similar to non-vitrified controls.     

Developmental competence of frozen-thawed yak (Bos grunniens) oocytes followed by in vitro maturation and fertilization

In the present study, we examined the ability of immature germinal vesicle (GV) and subjected to in vitro matured (MII) yak oocytes to survive after cryopreservation as well as their subsequent development following in vitro maturation and fertilization. Both GV and MII oocytes were cryopreserved by using two different vitrification solutions (VS); VS-I contained 10% ethylene glycol (EG) and 10% dimethylsulfoxide (DMSO) in TCM-199 + 20% (v/v) fetal calf serum (FCS) whereas VS-II contained 40% EG + 18% Ficoll + 0.5 M sucrose in TCM-199 + 20% FCS. The percentage of oocytes found to be morphologically normal was greater (P < 0.01) in VS-I group than in VS-II group. Rates of cleavage (30.6–42.2%) and blastocyst formation (2.9–8.9%) did not differ among groups, but were lower than in unfrozen control (55.7% and 25.4%, P < 0.01). These results show that a combination of EG and DMSO or EG, Ficoll and sucrose can be used to cryopreserve yak oocytes in French straws.     

Protein phosphorylation patterns during in vitro maturation of the goat oocyte

Protein phosphorylation patterns were studied by radiolabelling goat cumulus oocyte complexes with [³²P]orthophosphate for various periods of time. The radiolabelled denuded oocytes were assessed for nuclear status and were used individually for gel electrophoresis. This study demonstrated that specific changes in protein phosphorylations were programmed during goat oocyte maturation. One of the most prominent changes was a general increase in the phosphorylation rate at germinal vesicle breakdown (GVBD). From 8 hr of culture, dominant phosphoprotein bands with apparent molecular weights of 27, 31, 40, and 50 kD were observed; they remained at this level until the metaphase II stage. In the molecular weight range of 65–80 kD, the protein phosphorylation pattern exhibited characteristic differences, with a complex series of phosphoproteins appearing and disappearing, during maturation. Addition of 6-dimethylaminopurine (6-DMAP) at the onset of culture blocked the maturation process after GVBD and induced a dramatic condensation of chromatin. When added at different times after GVBD, 6-DMAP invariably induced chromosome condensation. This inhibition was partly reversible; i.e., after removal of the drug, oocytes were able to progress only until metaphase l. © 1993 Wiley-Liss, Inc.     

Cryopreservation of human ovarian tissue: Comparison of novel direct cover vitrification and conventional vitrification

Objective

To compare the effect of novel direct cover vitrification (DCV) and conventional vitrification (CV) for human ovarian tissue.

Study design

Ovarian biopsy specimens obtained from 12 patients were randomly allocated into five groups: Fresh, DCV1, DCV2, DCV3 and CV. Three concentrations of cryoprotectants were used in DCV group. The equilibration solution of DCV1, DCV2, DCV3 was 5% EG + 5% DMSO + DPBS, 7.5% EG + 7.5%DMSO + DPBS, 10% EG + 10% DMSO + DPBS, respectively. And the vitrification solution of DCV1, DCV2, DCV3 was 10% EG + 10% DMSO + DPBS, 15%EG+15% DMSO + DPBS, 20% EG + 20% DMSO + DPBS, respectively. The equilibration solution and the vitrification solution of CV group was same as DCV3 group. The effects of cryopreserved procedure on human ovarian tissue were studied by histology, TUNEL assay, transmission electron microscopy (TEM) and heterotopic allograft.

Results

The percentages of morphologically normal and viable follicles of DCV2 were significantly higher than DCV1, DCV3 and CV groups (P < 0.05). TUNEL assay demonstrated that the incidence of apoptotic cell in vitrification ovarian tissue was significantly higher than fresh tissue (P < 0.05), but there were no difference in various groups with cryopreservation. TEM showed that less damage was detected in DCV2 group. After grafting, the follicle density of DCV2 was greater than DCV1, DCV3 and CV groups (P < 0.05).

Conclusions

The novel cover vitrification with optimal concentration of cryoprotectants is superior to conventional vitrification. It is suitable for human ovarian tissue fragments with high efficiency and facility.     

A chronologic review of mature oocyte vitrification research in cattle, pigs, and sheep

Vitrification as a means of cryopreservation has become a standard approach for oocytes from livestock. This paradigm shift occurred primarily as a result of the demonstration in 1996 that bovine oocytes are extremely susceptible to chilling injury. Since that early work, numerous devices have been used as supports for oocytes during so-called “ultra-rapid cooling”, and occasionally, trials involving the deposition of small volumes of media containing oocytes directly into liquid nitrogen to facilitate cooling have been reported. Results reporting blastocyst development exceeding 10% are common, but variability remains high, and a standard method for bovine oocytes remains to be established. Oocytes from pigs are particularly difficult to cryopreserve, even with the use of ultrarapid cooling approaches. Few reports have demonstrated blastocyst development exceeding 5%. The application of hydrostatic pressure before vitrification appears to impart stress tolerance to porcine oocytes, as the results of some treatments have shown development to blastocysts at proportions >10%. Work on sheep oocyte vitrification is relatively new, and a few articles have reported blastocyst development at 10% or more. Messenger RNA levels are reportedly altered in sheep oocytes as a result of vitrification, and damage to the cytoskeleton is common across species.     

Bovine oocyte vitrification using the Cryotop method: Effect of cumulus cells and vitrification protocol on survival and subsequent development

The ability to successfully cryopreserve mammalian oocytes has numerous practical, economical and ethical benefits, which may positively impact animal breeding programs and assisted conception in humans. However, oocyte survival and development following vitrification remains poor. The aim of the present study was (1) to evaluate the effect of the presence of cumulus cells on the outcome of vitrification of immature (GV) or mature (MII) bovine oocytes, (2) to compare empirical and theoretical vitrification protocols, and (3) to assess the effect of adding ice blockers to vitrification media on survival and development competence of bovine oocytes following vitrification using the Cryotop method. In Experiment 1, cumulus-enclosed and partially-denuded GV and MII oocytes were vitrified in 15% EG + 15% Me₂SO + 0.5 M sucrose in two steps. In Experiment 2, GV oocytes were vitrified either as above or using theoretical modeling based on permeability and osmotic tolerance characteristics in 30% EG + 11.4% trehalose in three steps or 40% EG + 11.4% trehalose in four steps. In Experiment 3, GV oocytes were vitrified in media supplemented or not with 1 of 2 ice blockers (21st Century Medicine, Fontana, CA) 1% X-1000, 1% Z-1000 or both in three steps. In Experiment 1, the survival, cleavage and blastocyst rate of cumulus-enclosed oocytes was significantly higher than those of partially-denuded oocytes when vitrified at the GV stage (93.8% vs. 81.3%, 65.8% vs. 47.3%, 11.3% vs. 4.0%, respectively, P < 0.05). However, no significant effect of cumulus cover was detected between the two groups when vitrified at MII (93.0% vs. 91.8%, 35.2% vs. 36.8%, 5.0% vs. 4.4%, respectively). Furthermore, cumulus-enclosed oocytes vitrified at the GV stage exhibited significantly higher developmental competence than those vitrified at the MII stage (P < 0.05). In Experiment 2, there were no significant differences in the survival, cleavage and blastocyst rate among three protocols (86.0% vs. 92.8% vs. 91.2%, 44.8% vs. 54.4% vs. 45.6%, 5.0% vs. 5.4% vs. 4.0%, respectively). However, cleavage and blastocyst rate were significantly lower (P < 0.05) than non-vitrified control oocytes. In Experiment 3, the presence of ice blockers did not alter the cleavage rate or blastocyst development (P > 0.05). In conclusion, cumulus-enclosed GV bovine oocytes survived vitrification and subsequently developed at higher rates than MII oocytes using Cryotop method and conventional IVF procedure. Theoretical analysis of permeability characteristics and tolerance limits could not explain the low developmental competence of vitrified oocytes.     

Role of germinal vesicle on protein synthesis in rat oocyte during in vitro maturation

To investigate the role of the germinal vesicle (GV) on in vitro maturation (IVM) of rat oocytes, we examined protein synthesis during IVM by comparing polypeptide patterns in control and enucleated oocytes using one and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Separation of polypeptides extracted from the cytoplasm of GV by one-dimensional SDS-PAGE revealed that a 55 kDa polypeptide was present only in the GVs of rat oocytes. At 0, 12, 24, 36, and 44 hr after PMSG injection, prior to the initiation of maturation, enucleated oocytes synthesized the same major polypeptides as cumulus intact (CI) oocytes. During meiotic maturation, no major changes were detected in protein synthesis from prophase (GV stage) to prometaphase I (0–6 hr IVM). However, after entry into prometaphase I (7 hr IVM), striking changes were seen; a 24 kDa polypeptide disappeared and expression of a 34 kDa polypeptide became stronger. This pattern lasted until metaphase II. We detected no major differences in the pattern of protein synthesis between CI and enucleated oocytes using two-dimensional PAGE. These results indicate that protein synthesis in the maturing rat oocyte is controlled by cytoplasmic regulators rather than intrinsic nuclear components. © 1996 Wiley-Liss, Inc.     

Mammalian oocyte growth and development in vitro

This paper is a review of the current status of technology for mammalian oocyte growth and development in vitro. It compares and contrasts the characteristics of the various culture systems that have been devised for the culture of either isolated preantral follicles or the oocyte-granulosa cell complexes from preantral follicles. The advantages and disadvantages of these various systems are discussed. Endpoints for the evaluation of oocyte development in vitro, including oocyte maturation and embryogenesis, are described. Considerations for the improvement of the culture systems are also presented. These include discussions of the possible effects of apoptosis and inappropriate differentiation of oocyte-associated granulosa cells on oocyte development. Finally, the potential applications of the technology for oocyte growth and development in vitro are discussed. For example, studies of oocyte development in vitro could help to identify specific molecules produced during oocyte development that are essential for normal early embryogenesis and perhaps recognize defects leading to infertility or abnormalities in embryonic development. Moreover, the culture systems may provide the methods necessary to enlarge the populations of valuable agricultural, pharmaceutical product-producing, and endangered animals, and to rescue the oocytes of women about to undergo clinical procedures that place oocytes at risk. © 1996 Wiley-Liss, Inc.     

Analysis of protein synthesis in morphologically classified bovine follicular oocytes before and after maturation in vitro

Bovine cumulus oocyte complexes (COCs) were isolated from antral ovarian follicles (4-8 mm). Immature COCs were classified into four categories, based on the homogeneity and clearness of the ooplasm and the transparency and compactness of the cumulus investment. In this study, the incorporation of TCA-precipitable 35S-methionine and the protein synthesis patterns of oocytes of these four categories were examined. Before maturation in vitro, similar incorporation rates and identical protein synthesis patterns were observed between oocytes of categories 1-3. Immature oocytes of category 4 showed reduced incorporation rates and exhibited aberrant protein synthesis patterns. After maturation in vitro, the patterns of category 4 oocytes were identical with the patterns of those in categories 1-3. The incorporation of 35S-methionine into in vitro matured oocytes was lower (P less than .001) in all categories. Based on these results, it is concluded that the initial classification of oocytes into four categories can be reduced to two categories.     

Expression of CD9 and CD81 in bovine germinal vesicle oocytes after vitrification followed by in vitro maturation

The present study aimed to investigate the effect of vitrification on the expression of fertilization related genes (CD9 and CD81) and DNA methyl transferases (DNMT1 and DNMT3b) in bovine germinal vesicle (GV) oocytes and their resulting metaphase Ⅱ (MⅡ) stages after in vitro maturation culture. GV oocytes were vitrified using the open-pulled straw method; after warming, they were cultured in vitro. The vitrified-warmed GV oocytes and more developed MII oocytes were used to calculate the maturation rates (first polar body extrusion under a stereomicroscopy), and to detect mRNA expression (qRT-PCR). Fresh GV oocytes and their in vitro-derived MII oocytes served as controls. The results showed that both the maturation rate (54.23% vs. 42.93%) and the relative abundance of CD9 mRNA decreased significantly (p < 0.05) in bovine GV oocytes after vitrification, but the expression of CD81 and DNMT3b increased significantly. After in vitro maturation of vitrified GV oocytes, the resulting MII oocytes showed lower (p < 0.05) mRNA expression of genes (CD9, CD81, DNMT1 and DNMT3b) when compared to the control group (MII oocytes). Altogether, vitrification decreased the maturation rate of bovine GV oocytes and changed the expression of fertilization related genes and DNA methyl transferases during in vitro maturation.     

Cryopreservation of mouse embryos by vitrification: A meta-analysis

A meta-analysis of cryopreservation studies vitrifying mouse embryos was undertaken to determine the treatment effect of vitrification. Treatment by vitrification decreased embryo viability compared with controls: the odds ratio was 9.02 (CI: 3.73-21.78; P < 0.001), a 24.90% (CI: 14.88-34.91; P < 0.001) reduction in risk was associated with embryos in the control group, and for every 4.00 (CI: 3.91-4.09) embryos treated by vitrification, one does not survive. A multiple regression analysis evaluated covariates of embryo survival. For each hour increase post-hCG treatment when embryos were cryopreserved, there was a decrease of 0.36% (SEM ± 0.01) in survival (P < 0.001). The number of embryos surviving vitrification decreased 0.25% (SEM ± 0.02) per day increase in age of the female mouse (P < 0.001), whereas there was no significant difference for control group embryos. For each 1 h increase post-hCG treatment after cryopreservation when blastocysts were assessed for viability, there was a decrease of 0.13% (SEM ± 0.01) in survival. The later interval post-hCG treatment when blastocysts were assessed, the less viable they were compared with earlier blastocysts, independent of the vitrification protocol. This effect was not observed for control embryos. A high percentage of variability in the treatment effect for vitrification was likely due to underlying heterogeneity among studies. A portion of the risk associated with vitrification could be attributed to the general effects of cryopreservation. Future research should identify effects in a cryopreservation protocol specific to vitrification that affect viability of mouse embryos.     

Response of porcine oocytes to electrical and chemical activation during maturation in vitro

These studies were conducted to examine activation of in vitro-matured porcine oocytes in response to an electrical stimulus or to an ionophore. Cumulus-enclosed porcine oocytes were incubated in maturation medium supplemented with either FSH and LH (MM:Exp.1) or pregnant mare serum gonadotropin (PMSG; MM-P: experiments 2-4) at 39 degrees C in 5% CO2:95% air with high humidity. In experiment 1, groups of oocytes were stripped of cumulus and then shampulsed (control) or electrically pulsed with a Zimmerman Cell Fusion unit at 24, 31, 41, 48, and 65 h of incubation. Control oocytes were exposed to the activation medium for 20 sec, whereas oocytes to be pulsed were subjected to a single activation pulse (120 V, 30 microseconds). Oocytes were cultured for an additional 24 h and then fixed and examined. For oocytes pulsed at 24, 31, 41, 48, and 65 h, the proportions which activated were 0, 0, 87, 88, and 83%, respectively. In experiment 2, oocytes were electrically or sham-pulsed with a BTX 200 Embryomanipulation System at 24, 30, and 40 h of incubation and respective proportions of oocytes activating were 27%, 39%, and 72%. In experiment 3, oocytes were subjected to 0, 1, or 2 activation pulses after 41 h of incubation in MM-P. Double-pulsing halved the proportion of activated oocytes (P less than .0001). In experiment 4, oocytes were subjected to 0, 25, 50, or 100 microM ionophore at 48 h of incubation. Proportions of oocytes activated by ionophore were greater than for control (P less than .05), but activation was not increased by increasing dose of ionophore.(ABSTRACT TRUNCATED AT 250 WORDS)     

Study of newly synthesized proteins during bovine oocyte maturation in vitro using image analysis of two-dimensional gel electrophoresis

To investigate protein synthesis during bovine oocyte maturation in vitro, oocytes were put in culture with 35S-methionine for 4 hr periods from time zero to 28 hr. Pools of 10 oocytes were then prepared for two-dimensional gel electrophoresis (2-DE). For each time interval, three gels were obtained, digitalized, and analyzed to detect proteins. Then, the gel containing the most proteins was chosen as the reference gel and compared with the others. An averaged gel was created with proteins present in at least two gels of the three. Our results indicate that the rate of protein synthesis is higher at the beginning of maturation until the appearance of metaphase I (MI, 8-12 hr) and then it decreases and stays relatively constant. Percentages of initial proteins (0-4 hr) and remaining present during the progression decrease progressively from 100% to 53%. In contrast, when we compare proteins synthesized from the 4 to 8 hr period with proteins from the 8 to 12 or the 12 to 16 hr intervals, percentages of overall protein matching are stable with values of 81 and 79%, respectively. Comparison of proteins from 20 to 24 hr with proteins from 16 to 20 or 24 to 28 hr intervals also gives stable percentages of overall protein matching with values of 83 and 84%, respectively. Furthermore, a higher number of new proteins is observed at 4-8 hr (n=130) and 16-20 hr (n=136) of maturation. Thus, three major patterns of protein synthesis were observed during bovine oocyte maturation in vitro: one at the beginning of maturation (0-4 hr), another one in the middle (4-16 hr), and the last one after the completion of MI stage (16-28 hr).     

The presence of synthetic polymers in the maturation medium affects the cryotolerance and developmental capacity after parthenogenic activation of vitrified goat oocytes

The purpose of this present study is to assess if addition of the synthetic polymers in maturation medium can influence cryotolerance and subsequently embryonic development of mammalian oocytes. We examined the roles of two polymers, including polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP), on in vitro maturation (IVM), embryonic developmental capacity, and cryotolerance of goat oocytes. The present study includes two parts. At first, goat cumulus−oocyte complexes (COCs) were matured in a medium supplemented with 10% fetal bovine serum (FBS), 3 mg/ml PVP, or 1 mg/ml PVA, respectively. Data of oocyte with first polar body, cleavage, and blastocyst following parthenogenetic activation (PA) were recorded. Secondly, after maturation in the above medium, oocytes were vitrified using the Cryotop technique and then the morphology, cleavage and blastocyst formation of vitrified oocytes have been checked. The results demonstrated that the adding of PVP or PVA in maturation medium can't affect IVM of goat oocytes in comparison with FBS, as concern cumulus cell expansion, first polar body formation, and embryonic development. Additionally, without plunging into liquid nitrogen, only exposure to the vitrification and warming solutions cannot also influence the quality of oocytes, in terms of morphology, cleavage, and blastocyst formation. However, after IVM with synthetic polymers and vitrification, the ratio of oocytes with standard morphology in PVP or PVA group was only 59.47% ± 3.56% or 54.86% ± 5.19%, respectively, and was significantly less than that in the FBS group (89.37% ± 4.52%, P < 0.05). Furthermore, the cleavage ratio of oocytes in PVP or PVA group was 37.41% ± 4.17% or 27.71% ± 3.91% and was considerably less than that in the FBS group (64.97% ± 4.69%, P < 0.05). In addition, the cleavage ratio in PVP group was statistically higher than that in PVA group (P < 0.05). In terms of blastocyst development, a significant difference was observed between the synthetic polymer group and the FBS group (24.96% ± 3.62%, P < 0.05). However, the blastocyst ratio in the PVA group (7.51% ± 1.68%) was statistically less than the PVP groups (13.20% ± 4.59%, P < 0.05) and the FBS group (P < 0.05). In conclusion, two potential serum replacements, either PVP or PVA, can support IVM and embryonic development of goat oocytes at the concentration used in this study. But IVM with synthetic polymers supplemented to maturation medium may reduce the cryotolerance of oocytes. Additionally, the supportive function of PVP on embryonic development of vitrified oocytes might be better than that of PVA.     

The dominance of warming rate over cooling rate in the survival of mouse oocytes subjected to a vitrification procedure

The formation of more than trace amounts of ice in cells is lethal. The two contrasting routes to avoiding it are slow equilibrium freezing and vitrification. The cryopreservation of mammalian oocytes by either method continues to be difficult, but there seems a slowly emerging consensus that vitrification procedures are somewhat better for mouse and human oocytes. The approach in these latter procedures is to load cells with high concentrations of glass-inducing solutes and cool them at rates high enough to induce the glassy state. Several devices have been developed to achieve very high cooling rates. Our study has been concerned with the relative influences of warming rate and cooling rate on the survival of mouse oocytes subjected to a vitrification procedure. Oocytes suspended in an ethylene glycol–acetamide–Ficoll–sucrose solution were cooled to −196 °C at rates ranging from 37 to 1827 °C/min between 20 and −120 °C, and for each cooling rate, warmed at rates ranging from 139 to 2950 °C/min between −70 and −35 °C. The results are unambiguous. If the samples were warmed at the highest rate, survivals were >80% over cooling rates of 187–1827 °C/min. If the samples were warmed at the lowest rate, survivals were near 0% regardless of the cooling rate. We interpret the lethality of slow warming to be a consequence of it allowing time for the growth of small intracellular ice crystals by recrystallization.