Addition of cholesterol loaded cyclodextrin prior to GV-phase vitrification improves the quality of mature porcine oocytes in vitro

The purpose of this study was to determine whether the mitochondrial membrane potential, pro-apoptotic gene expression, and ubiquitylation status of zona pellucida proteins (ZP1, ZP2, and ZP3) of vitrified GV-stage mature oocytes could be protected by treatment with cholesterol-loaded methyl-β-cyclodextrin (CLC) prior to vitrification. Porcine GV oocytes were treated with CLC prior to the vitrification process, and the effects on the mitochondrial membrane potential and ZP ubiquitylation status were determined by JC-1 single staining and western blot assays. We found that porcine GV-stage oocytes were treated with CLC at different concentrations (0.5, 5, and 10 mg/mL) prior to vitrification improved in vitro maturation of these oocytes (P < 0.05). The mitochondrial membrane potential of matured oocyte without vitrification or treated with 5 mg/mL CLC vitrification treatment was higher than that of the 0 mg/mL CLC group and other treatment groups (vitrified) (P < 0.05). The expression of Caspase 3, Caspase 8, and Caspase 9 genes in the high concentration CLC treatment groups (5 and 10 mg/mL) was significantly lower than that in the 0 (vitrified) mg/mL CLC group (P < 0.05). ZPs protein and ZP3 protein ubiquitylation were also higher in the non-vitrified controls, 5 and 10 mg/mL CLC-treated oocytes than in the 0 (vitrified) and 0.5 mg/mL vitrified groups (P < 0.05). Whereas the sperm–oocyte binding capacity was improved in the CLC treatment groups (P < 0.05) but the embryonic development rate was not improved. In conclusion, pretreatment with CLC can improve the survival rate and maturation rate of oocytes and protect their mitochondria and zona pellucida of porcine oocytes from cryodamage during the vitrification process.     

Vitrification of bovine oocytes and its application to intergeneric somatic cell nucleus transfer

We determined the efficacy of a microdrop vitrification procedure for cryopreservation of bovine oocytes, using vitrified oocytes as cytoplasts for intraspecies and intergeneric somatic cell nucleus transfer (NT). In vitro matured bovine MII oocytes were vitrified in microdrops with a vitrification solution containing 35% ethylene glycol, 5% polyvinyl pyrrolidone, and 0.4 M trehalose. After warming, approximately 80% of the vitrified oocytes were morphologically normal, and their enucleation rate was similar to that of fresh oocytes. The NT embryos constructed with bovine cumulus cells and the vitrified oocytes developed similar to blastocysts constructed with fresh oocytes, although the cell number of NT blastocysts originating from vitrified oocytes was lower than that of the fresh control. In a second experiment, we examined the development of NT embryos constructed with vitrified bovine oocytes and bovine fibroblasts (intraspecies NT embryos) or swamp buffalo fibroblasts (intergeneric NT embryos). There were no differences between the intraspecies and intergeneric NT embryos in fusion, cleavage and development to blastocysts, except for lower cell numbers in the intergeneric NT blastocysts. In conclusion, the efficacy of this microdrop vitrification procedure and the production of swamp buffalo NT blastocysts using vitrified bovine oocytes was demonstrated.     

Improved development of ovine matured oocyte following solid surface vitrification (SSV): effect of cumulus cells and cytoskeleton stabilizer

The objective of the present study was to examine the effects of cumulus cells, cytochalasin B (CB), and taxol on the development of ovine matured oocyte following solid surface vitrification (SSV). In experiment 1, effects of cumulus cells during the vitrification were examined. Survival rates after warming were not different between ovine mature oocytes with cumulus cells and without cumulus cells. After in vitro fertilization, rates of embryonic cleavage and development to blastocyst were not different between these two groups. In experiment 2, the effects of cytochalasin B (CB) on vitrification of ovine matured oocytes were examined. The rates of survived ovine matured oocytes were not significantly different among the treatment with 0, 2.5, 5.0, 7.5 and 10.0 microg/mL CB. After in vitro fertilization, the rate of cleavage was not different between the five treatment groups. However, vitrified oocytes treated with 7.5 or 10.0 microg/mL CB resulted in a higher (8.1+/-4.6% and 7.8+/-2.4% respectively, P<0.05) blastocyst development rate than those of oocytes treated with lower CB concentrations. In Experiment 3, the effects of taxol on vitrification of ovine matured oocytes were examined. The rate of survived oocytes was not significantly different among the taxol treatment group with 0, 0.5, 1.0, and 5.0 microM taxol. After in vitro fertilization, the rates of embryos that reached cleavage were not different between the four treatment groups. However, vitrified oocytes treated with 0.5 microM taxol resulted in a higher blastocyst (10.1%+/-6.3, P<0.05) development rate compared to other treatment groups. In conclusion, no effect of cumulus cells on vitrification of ovine matured oocytes was detected in this study. Pretreatment of ovine matured oocytes with cytoskeletal inhibitor cytochalasin B or taxol have a positive effect and helps to reduce the damage induced by vitrification and is a potential way to improve the development of vitrified/warmed ovine matured oocytes.     

Effect of polyvinyl alcohol (PVA) concentration during vitrification of in vitro matured bovine oocytes

Polyvinyl alcohol (PVA) was used as a substitute for serum in a vitrification solution for in vitro matured bovine oocytes. In vitro matured bovine oocytes were cryopreserved in various vitrification solutions (VS) supplemented with different concentrations (0.05, 0.1, 0.5, and 1%) of PVA, 20% fetal calf serum (FCS) or without macromolecule supplementation in a gel-loading tip (GL-tip). After warming, vitrified oocytes were examined for effects on survivability, fertilizability, and embryonic development in vitro. At 18 h in vitro fertilization after vitrifying and warming, the number of surviving mature oocytes vitrified in VS without macromolecule supplementation was significantly (P < 0.05) lower than those with macromolecule supplementation. For fertilizability after vitrification, there was no significant difference in the penetration rate of oocytes among fresh oocytes (98.7%); oocytes vitrified in VS supplemented with 0.1 (76.8%), 0.5 (70.2%), or 1% (80.3%) PVA; 20% (84.1%) FCS; or without supplementation (61.7%). Also, the normal fertilization rate was not significantly different in oocytes vitrified with 0.1 (56.5%), 0.5 (43.5%), or 1% (49.7%) PVA and 20% (60.6%) FCS, compared with fresh oocytes (84.0%). Subsequently, vitrified oocytes were examined for embryonic development effects in vitro. The highest proportion of cleaved oocytes after vitrification was obtained in VS supplemented with 0.1% (18.8%) PVA. Additionally, the proportion of development to morula stage (7.7%) in the oocytes vitrified in a VS supplemented with 0.1% PVA was significantly (P < 0.05) superior to that of the 0, 0.5, and 1% PVA-vitrified groups. However, the beneficial effect of PVA addition was not found in blastocyst development. Embryonic development of vitrified oocytes was significantly lower than that of fresh oocytes. In conclusion, the present results indicate that 0.1% PVA supplementation in VS results in a significantly higher rate of morula stage embryos than 0, 0.5, and 1% PVA supplementation, and could replace FCS in VS for vitrification of in vitro matured bovine oocytes.     

Production of live piglets following cryopreservation of embryos derived from in vitro-matured oocytes

We have successfully produced healthy piglets following cryopreservation of embryos derived from oocytes matured and fertilized in vitro. The appropriate timing of cryopreservation pretreatment (removal of cytoplasmic lipid droplets [delipation] and vitrification) was initially determined using parthenogenetic embryos derived from in vitro-matured (IVM) oocytes. Viable embryos were obtained at the highest rate when embryos were delipated at the four- to eight-cell stages (Day 2 of embryo culture) and were vitrified approximately 15 h later (Day 3) by means of the minimum volume cooling method. After cryopreservation of embryos derived from oocytes matured and fertilized in vitro under the most appropriate conditions, 401 embryos were transferred to five recipient gilts, and the recipients all became pregnant. At autopsy of one of the recipients, which had received 47 embryos, eight fetuses (17.0%) were found. Three recipients each gave birth to two to four piglets (1.4%-6.0%). These results demonstrate that normal offspring can be produced from vitrified porcine embryos derived from IVM oocytes by a strategic combination of delipation and vitrification at the early cleavage stages. This approach has great potential in the reproduction of micromanipulated porcine embryos, such as cloned and sperm-injected embryos, produced from IVM oocytes.     

Morphological and biochemical analysis of immature ovine oocytes vitrified with or without cumulus cells

The cryopreservation of oocytes is an open problem as a result of their structural sensitivity to the freezing process. This study examined (i) the survival and meiotic competence of ovine oocytes vitrified at the GV stage with or without cumulus cells; (ii) the viability and functional status of cumulus cells after cryopreservation; (iii) the effect of cytochalasin B treatment before vitrification; (iv) chromatin and spindle organization; (v) the maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK) activity of vitrified oocytes after in vitro maturation. Sheep oocytes were vitrified at different times during in vitro maturation (0, 2, and 6 h) with (COCs) or without cumulus cells (DOs). After warming and in vitro maturation, oocytes denuded at 0 h culture showed a significantly higher survival and meiotic maturation rate compared to the other groups. Hoechst 33342/propidium iodide double staining of COCs and microinjection of Lucifer Yellow revealed extensive cumulus cell membrane damage and reduced oocyte-cumulus cell communications after vitrification. Cytochalasin B treatment of COCs before vitrification exerted a negative effect on oocyte survival. After in vitro maturation, the number of vitrified oocytes with abnormal spindle and chromatin configuration was significantly higher compared to control oocytes, independently of the presence or absence of cumulus cells. The removal of cumulus cells combined with vitrification significantly decreased the MPF and MAPK levels. This study provides evidence that the removal of cumulus cells before vitrification enhances oocyte survival and meiotic competence, while impairing the activity of important proteins that could affect the developmental competence of oocytes.     

Vitrification of immature and mature equine and bovine oocytes in an ethylene glycol, ficoll and sucrose solution using open-pulled straws

Studies were conducted to compare viability of immature and mature equine and bovine oocytes vitrified in ethylene glycol. Ficoll using open-pulled straws. Oocytes from slaughterhouse ovaries (N=50/group) with >2 layers of compact cumulus cells were vitrified immediately after collection (immature groups) or vitrified after 36 to 40 (equine) or 22 to 24 (bovine) h of maturation (mature groups). Immature oocytes were matured after thawing. Before vitrification, oocytes were exposed to TCM-199 + 10 FCS + 2.5 M ethylene glycol + 18% Ficoll + 0.5 M sucrose (EFS) for 30 sec and then to 5 M ethylene glycol in EFS for 25 to 30 sec at 37 degrees C. Oocytes were loaded into straws in approximately 2 microL of cryoprotectant and plunged directly into LN2. Warming straws and dilution of cryoprotectant was at 37 degrees C in TCM-199 + 10% FCS + 0.25 M sucrose for 1 min and then TCM-199 + 10% FCS + 0.15 M sucrose for 5 min. Non-vitrified oocytes undergoing the same maturation protocol for both species were used as controls. Oocytes were stained with orcein for nuclear maturation and live/dead status was determined using Hoechst 33342. Maturation of oocytes to MII after thawing was similar (P>0.05) among groups within species. All equine treatment groups had lower (P<0.01) maturation rates than bovine groups. Live/dead status did not differ among vitrification treatments within species. The percentage of oocytes that survived and reached MII did not differ (P>0.05) within treatment groups of each species. Rates of mature cortical granule distribution did not differ (P>0.05) within species; however, more bovine oocytes (P<0.05) had mature cortical granule distribution and nuclear maturation than equine oocytes. When concurrent cortical granule distribution and nuclear maturation were examined, there was no difference within species; however, only 30% of equine oocytes had nuclear and cytoplasmic maturation compared with 70% of bovine oocytes (P<0.05). In summary, both immature and mature equine and bovine oocytes survived cryopreservation using vitrification in open-pulled straws. However, survival rates were lower for equine than for bovine oocytes.     

Cryopreservation of in vitro matured buffalo (Bubalus bubalis) oocytes by minimum volumes vitrification methods

The aim of this study was to evaluate the efficiency of the solid surface vitrification (SSV) and the cryoloop vitrification (CLV) methods to cryopreserve in vitro matured buffalo oocytes. Another objective of the work was to investigate whether the presence of cumulus cells affects the efficiency of oocyte vitrification in this species. In the SSV method, oocytes were vitrified in a solution of 35% ethylene glycol, 5% polyvinyl-pyrrolidone and 0.4% trehalose and they were warmed in a 0.3M trehalose solution. In the CLV method, oocytes were vitrified in 16.5% ethylene glycol and 16.5% dimethyl sulfoxide and warmed in decreasing concentrations of sucrose. The oocytes that survived vitrification were fertilized and cultured in vitro up to the blastocyst stage. Although high survival rates were recorded in all groups, when the oocytes were vitrified by the CLV method in the absence of cumulus cells, the survival rate was significantly (P<0.05) lower. However, the CLV gave a significantly higher cleavage rate compared to the SSV with the denuded oocytes (45% versus 26%, respectively; P<0.05), whereas no differences were found between methods with the cumulus-enclosed oocytes (14% versus 15%, respectively). Blastocysts were produced for the first time from in vitro matured oocytes that were vitrified-warmed in buffalo. Nevertheless, vitrification significantly decreased blastocyst yield, regardless of both the method employed and the presence or absence of cumulus cells.     

Vitrification of early-stage bovine and equine embryos

The objectives of this study were to: (1) determine an optimal method and stage of development for vitrification of bovine zygotes or early embryos; and (2) use the optimal procedure for bovine embryos to establish equine pregnancies after vitrification and warming of early embryos. Initially, bovine embryos produced by in-vitro fertilization (IVF) were frozen and vitrified in 0.25 mL straws with minimal success. A subsequent experiment was done using two vitrification methods and super open pulled straws (OPS) with 1- or 8-cell bovine embryos. In Method 1 (EG-O), embryos were exposed to 1.5 M ethylene glycol (EG) for 5 min, 7 M ethylene glycol and 0.6 M galactose for 30 s, loaded in an OPS, and plunged into liquid nitrogen. In Method 2 (EG-DMSO), embryos were exposed to 1.1 M ethylene glycol and 1.1 M dimethyl sulfoxide (DMSO) for 3 min, 2.5 M ethylene glycol, 2.5 M DMSO and 0.5 M galactose for 30 s, and loaded and plunged as for EG-O. Cryoprotectants were removed after warming in three steps. One- and eight-cell bovine embryos were cultured for 7 and 4.5 d, respectively, after warming, and control embryos were cultured without vitrification. Cleavage rates of 1-cell embryos were similar (P > 0.05) for vitrified and control embryos, although the blastocyst rates for EG-O and control embryos were similar and higher (P < 0.05) than for EG-DMSO. The blastocyst rate of 8-cell embryos was higher (P < 0.05) for EG-O than EG-DMSO. Therefore, EG-O was used to cryopreserve equine embryos. Equine oocytes were obtained from preovulatory follicles. After ICSI, injected oocytes were cultured for 1-3 d. Two- to eight-cell embryos were vitrified, warmed and transferred into recipient's oviducts. The pregnancy rate on Day 20 was 62% (5/8) for equine embryos after vitrification and warming. In summary, a successful method was established for vitrification of early-stage bovine embryos, and this method was used to establish equine pregnancies after vitrification and warming of 2- to 8-cell embryos produced by ICSI.     

Bovine oocyte vitrification before or after meiotic arrest: effects on ultrastructure and developmental ability

The nuclear stage at which oocytes are cryopreserved influences further development ability and cryopreservation affects ultrastructure of both cumulus cells and the oocyte. In this work, we analyze the effects of vitrification at different nuclear and cytoplasmic maturation stages on the oocyte ultrastructure and developmental ability. Culture in TCM199 + PVA with roscovitine 25 M during 24h led to meiotic arrest (MA) in cumulus-oocyte complexes (COCs), while permissive in vitro maturation (IVM) was performed in TCM199, 10% FCS, FSH-LH and 17beta-estradiol for 24 h. Oocytes were vitrified using the open pulled straw method (OPS) with minor modifications. Fresh and vitrified/warmed COCs were fixed as immature, after IVM, after meiotic arrest (MA) and after MA + IVM. Vitrification combined with MA followed by IVM produced the highest rates of degeneration, regardless of the vitrification time. As a consequence, lower proportions of embryos cleaved in these groups, although differences were eliminated at the five-eight cell stage. Development rates up to day 8 were similar in all experimental groups, being significantly lower than those in fresh controls. Only oocytes vitrified after IVM were able to give blastociysts. The morphological alterations observed can be responsible for compromised development. More research is needed to explain the low survival rates of the bovine oocyte after vitrification and warming.     

Relationship between equilibration times and the presence of cumulus cells, and effect of taxol treatment for vitrification of in vitro matured porcine oocytes

The effects of the presence or absence of cumulus cells and equilibration times (1 and 4 min) with cryoprotectant, and Taxol treatment before vitrification (0.5-5.0 microM) on development of in vitro matured porcine oocytes after vitrification were examined. Ethylene glycol (30%) and sucrose (0.5M) was used as a vitrification solution (39 degrees C), and cryotop was used for cryo-container. There was a significant relationship (F value: 6.077, P<0.05) in the rate of morphologically normal oocytes after vitrification between the equilibration times and the presence or absence of cumulus cells. The blastocyst rates were not significantly different between Taxol (1.4-5.5%) and non-treated control (8.8%). The results show that the optimal exposure time to achieve survival after vitrification depends on the presence or absence of cumulus cells, and that Taxol has no positive effect on the developmental capacity of vitrified, in vitro matured porcine oocytes.     

Vitrification of mouse oocytes and embryos at various stages of development in an ethylene glycol-based solution by a simple method

Mouse oocytes and embryos at various developmental stages were exposed directly to an ethylene glycol-based vitrification solution (EFS) for 2 or 5 minutes at 20 degrees C. They were then vitrified at -196 degrees C and were warmed rapidly. At the germinal vesicle stage, the proportion of morphologically normal oocytes was 36 to 39% if they had cumulus cells, whereas in cumulus-removed immature oocytes and in ovulated oocytes it was only 2 to 4%. This low survival was attributed to the harmful action of ethylene glycol. After fertilization, on the other hand, the post-warming survival rate of 1-cell zygotes, as assessed by cleavage to the 2-cell stage, increased markedly (62%). As the developmental stage proceeded, higher proportions of vitrified embryos developed to expanded blastocysts; the rates increased up to 77 and 80% in 2-cell and 4-cell embryos, respectively. For embryos at the 8-cell, morula and early blastocyst stages, the proportion of embryos developed after vitrification (90 to 95%) was not significantly different from that of the untreated embryos (95 to 100%) when the period of exposure to EFS solution was 2 minutes. As the blastocoel began to enlarge, however, survival began to decrease again, with rates of 79 and 57% in blastocysts and expanded blastocysts, respectively. After the cryopreserved 2-cell, 4-cell and 8-cell embryos as well as morulae and blastocysts were transferred to recipients, 43 to 57% of the recipients became pregnant, and 48 to 60% of these various stage embryos developed into live young.     

Treatment with cholesterol-loaded methyl-β-cyclodextrin increased the cholesterol in rabbit oocytes,but did not improve developmental competence of cryopreserved oocytes

Membrane cholesterol:phospholipids ratio is an important determinant of cell chilling sensitivity. At low temperatures, major membrane destabilisation occurs when the membrane undergoes a phase transition. To increase membrane fluidity and stability during cooling and thus increase oocyte cryoresistance, cholesterol has been added to the plasma membrane. This study was conducted to determine if cholesterol could be incorporated into rabbit oocytes by incubation with cholesterol-loaded methyl-β-cyclodextrin (CLC) and if added cholesterol could improve the developmental ability of cryopreserved oocytes after parthenogenetic activation or intracytoplasmic sperm injection. Fresh, frozen and vitrified oocytes incubated with CLC containing 20% NBD-labelled cholesterol (NBD-CLC) were evaluated using confocal microscopy. Fluorescence intensity was higher in fresh oocytes than in cryopreserved ones. Pre-treating rabbit oocytes with 1 mg of NBD-CLC/mL did not improve cleavage and developmental rates after cryopreservation. Results showed that treatment with CLC increased the cytoplasmic cholesterol content, but did not improve cleavage rate and developmental competence of cryopreserved oocytes.     

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.     

Ultra-structural changes and developmental potential of porcine oocytes following vitrification

This study evaluated the effects of exposure and/or vitrification of porcine metaphase II (MII) oocytes on their in vitro viability and ultra-structural changes with two experiments. Experiment 1 examined the effect of vitrified oocytes on microtubule localization, mitochondrial morphology, chromosome organization and the developmental rate in IVF control and vitrified oocytes. Oocytes matured for 44 h were subjected to IVF (IVF control). Oocytes matured for 42 h were exposed to cryoprotectants (CPA control), followed by 2h culture, and subjected to IVF. Oocytes vitrified at 42 h post-maturation were warmed, cultured for 2h, and subjected to IVF (vitrified). Experiment 2 evaluated the effect of oocytes freezing on development of ICSI with and without activation and parthenotes. Fresh and vitrified oocytes were subjected to ICSI with and without electrical activation. Cleavage and blastocyst rates were significantly (P<0.05) lower in vitrified IVF, parthenote and ICSI embryos than those in fresh counterparts. Between ICSI embryos from fresh oocytes and vitrified oocytes, the rates of blastocyst were significantly higher (P<0.05) in activated group than the group without activation. Significant differences (P<0.05) were observed in normal spindle configuration of vitrified (43.5%) compared to control (81.0%) oocytes, but no significant difference was observed between CPA exposed and control groups. In conclusion, porcine oocytes at MII stage are very sensitive to vitrification with altered microtubule localization and mitochondrial organization thus resulting in impaired fertilization and embryo development.     

Effect of vitrification on the zona pellucida hardening and follistatin and cathepsin B genes expression and developmental competence of in vitro matured bovine oocytes

The purpose of our study was to assess the effect of vitrification with or without the presence of calcium in the vitrification solution on the: 1) diameter of oocytes and thickness of the zona pellucida, 2) zona pellucida hardening, 3) expression of mRNA follistatin (FST) and cathepsin B (CTSB) in oocytes and 4) developmental competence of embryos derived from in vitro matured and vitrified oocytes.The results of our study demonstrate, that vitrification did not alter thickness of the zona pellucida and diameter of the oocytes, however it triggered hardening of the zona pellucida. The presence of calcium in the vitrification solutions intensified hardening of zona in immature and mature oocytes (P < 0.04, P < 0.001, respectively) and provoked increased mRNA FST expression in oocytes matured in vitro compared to immature oocytes (P < 0.01) and those vitrified without calcium (P < 0.004). CTSB mRNA expression was increased in immature oocytes and oocytes vitrified with calcium compare to mature oocytes (P < 0.02). The developmental potential of vitrified oocytes was impaired compared to non-vitrified oocytes, being more evident in oocytes vitrified with calcium.In summary, vitrification did not change the oocyte diameter and thickness of the zona pellucida and expression of FST and CTSB mRNA. It diminished developmental potential of the vitrified oocytes. The presence of calcium in the vitrification solutions increased hardening of zona pellucida as well as affected the level of FST and CTSB mRNA in oocytes and developmental potential of these oocytes.     

Cytoplasmic changes and developmental competence of bovine oocytes cryopreserved without cumulus cells

The cryopreservation of female gametes is still an open problem because of their structural sensitivity to the cooling-and-freezing process and to the exposure to cryoprotectants. The present work was aimed to study the effect of vitrification on immature bovine oocytes freed of cumulus cell investment before freezing. To verify the feasibility and efficiency of denuded oocyte (DO) cryopreservation, the cytoplasmic alterations eventually induced either by cell removal or by the vitrification process were analyzed. In particular, the migration of cortical granules and Ca++ localization were studied. In addition, the localization and distribution of microtubules and microfilaments in immature fresh and vitrified DOs were evaluated. Finally, to establish whether the removal of cumulus cells influenced developmental competence, DOs were thawed after vitrification, matured in vitro and fertilized; then presumptive zygotes were cultured to reach the blastocyst stage. The results indicate that mechanical removal of cumulus cells from immature bovine oocytes does not affect their maturation competence but reduces the blastocyst rate when compared with intact cumulus oocyte complexes (COCs). The findings indicate further that the vitrification process induces changes of cytoplasmic components. However, the composition of the manipulation medium used to remove cumulus cells plays a crucial role in reducing the injuries caused by cryopreservation in both cytoplasmic and nuclear compartments. In fact, the presence of serum exerts a sort of protection, significantly improving both oocyte maturation and blastocyst rates. In conclusion, we demonstrate that denuded immature oocytes can be vitrified after cumulus cells removal and successfully develop up, after thawing, to the blastocyst stage, following in vitro maturation and fertilization.     

Cryopreservation of goat oocytes and in vivo derived 2- to 4-cell embryos using the cryoloop (CLV) and solid-surface vitrification (SSV) methods

This study evaluated the efficiency and toxicity of two cryopreservation methods, solid-surface vitrification (SSV) and cryoloop vitrification (CLV), on in vitro matured oocytes and in vivo derived early stage goat embryos. In the SSV method, oocytes were vitrified in a solution of 35% ethylene glycol (EG), 5% polyvinyl-pyrrolidone (PVP), and 0.4% trehalose. Microdrops containing the oocytes were cryopreserved by dropping them on a cold metal surface that was partially immersed in liquid nitrogen. In the cryoloop method, oocytes were transferred onto a film of the CLV solution (20% DMSO, 20% EG, 10mg/ml Ficoll and 0.65 M sucrose) suspended in the cryoloop. The cryoloop was then plunged into the liquid nitrogen. In vivo derived embryos were vitrified using the same procedures. The SSV microdrops were warmed in a solution of 0.3M trehalose and those vitrified with CLV were warmed with incubation in 0.25 and 0.125 M sucrose. Oocytes and embryos vitrified by the SSV method had a significantly lower survival rate than the control (60 and 39% versus 100%, respectively; P<0.05), while the survival rate of CLV oocytes and embryos (89 and 88%, respectively) did not differ from controls. Cleavage and blastocyst rates of the surviving vitrified oocytes (parthenogenetically activated) and embryos (cultured for 9 days) were not significantly different (P>0.05) from the control nor did they differ between vitrification methods. Embryos vitrified with the CLV method gave rise to blastocysts (2/15). Our data demonstrated that the two vitrification methods employed resulted in acceptable levels of survival and cleavage of goat oocytes and embryos.     

Effects of culture systems on development of in vitro fertilized bovine ova into blastocysts

We examined the effects of co-culture with oviductal epithelial cells, cumulus cells, trophoblastic vesicles or amniotic sac cells on the development of bovine eight-cell embryos derived from in vitro maturation and fertilization into blastocysts. Frozen-thawed spermatozoa were treated with caffeine plus Ca-ionophore A23187 for capacitation and were then co-incubated for 4 h with oocytes matured in vitro. Ova resulting from this in vitro fertilization were cultured in HEPES-buffered TCM-199 + 10% fetal calf serum(FCS) for 68 h and then removed from the cumulus cell mass. The eight-cell embryos were cultured using four co-culture systems either without cells(controls) or within rabbit oviducts. The co-culture of oviductal epithelial cells, trophoblastic vesicles or amniotic sac cells significantly (P<0.05) increased development into blastocysts (39.0 to 50.7%) when compared with co-culture with cumulus cells, control or rabbit oviducts(1.9 to 29.3%). Six of 16 recipients became pregnant with frozen embryos derived from co-culture with oviductal epithelial cells(1/2), trophoblastic vesicles(2/7) or amniotic sac cells(3/7). Eight calves, including two sets of twins, were obtained.     

Vitrification of mouse oocytes results in aneuploid zygotes and malformed fetuses

Vitrification of mouse oocytes adversely affected the subsequent developmental potential of embryos and fetuses derived from the fertilization of such oocytes after thawing. Only 5% of oocytes vitrified formed viable fetuses on the 15th day of gestation as compared to 47% in the controls. The incidence of chromosomally aneuploid zygotes, derived from cryopreserved oocytes, was approximately threefold higher than the controls irrespective of whether the oocytes were cryopreserved by vitrification or DMSO slow-freezing. Malformed fetuses were obtained from oocytes that had been vitrified as well as those that had been exposed to vitrification solutions only, whereas no malformed fetuses were obtained in oocytes slow-frozen by DMSO or fresh controls--thus demonstrating that the exposure of oocytes to the vitrification chemicals was responsible for the fetal malformations. The data in this study suggest that the vitrification technique should be cautiously applied to human oocyte cryopreservation. Furthermore, the data also demonstrate that the exposure of female gametes to carcinogenic and/or teratogenic chemicals may result in malformed embryos when such oocytes are subsequently fertilized.