Developmental capacity of vitrified immature porcine oocytes following ICSI: effects of cytochalasin B and cryoprotectants

In the present study, effects of concentration and pretreatment time of cytochalasin B (CB), and of two types of cryoprotectant solutions on the nuclear maturation of vitrified-warmed porcine oocytes were examined. Also, the developmental capacity of vitrified immature porcine oocytes following intracytoplasmic sperm injection (ICSI) was investigated. The nuclear maturation rate (46.8%) of the vitrified-warmed oocytes treated with 7.5 microg/mL CB for 30 min was significantly higher (P < 0.05) than those (13.9-39.2%) of the vitrified-warmed oocytes treated with 0, 2.5, or 5.0 microg/mL CB for 10 or 30 min. Additionally, the nuclear maturation rate of oocytes treated with CB and vitrified in ethylene glycol (EG) (37.1%) was significantly higher (P < 0.05) than that of EG + dimethyl sulfoxide (Me(2)SO) (23.9%). However, no significant differences were observed in the cleavage and blastocyst development rates among the control (45.2 and 20.0%, respectively), the EG group (37.8 and 13.5%, respectively) and the EG + Me(2)SO group (39.3 and 14.3%, respectively). These results demonstrated that: (1) pretreatment with 7.5 microg/mL CB was beneficial for the vitrification of immature porcine oocytes; (2) the combination of EG and Me(2)SO as a cryoprotectant was not advantageous for in vitro maturation (IVM) of vitrified immature porcine oocytes; and (3) vitrified-warmed porcine oocytes matured after IVM, developed to the blastocyst stage without distinct differences compared to fresh oocytes following ICSI.     

Successful pregnancy following transfer of feline embryos derived from vitrified immature cat oocytes using 'stepwise' cryoprotectant exposure technique

Oocyte cryopreservation is the desired tool for the ‘long-term’ storage of female genetic potential especially for endangered/valuable species. This study aims at examining the ability of different cryoprotectant (CPA) and CPA exposure techniques to protect immature feline oocytes against cryoinjury during vitrification. Immature oocytes were submitted to different CPA exposure techniques: 1) 2-step DMSO, 2) 4-step DMSO, 3) 2-step EG, 4) 4-step EG, 5) 2-step EG plus DMSO and 6) 4-step EG plus DMSO. Non-CPA treated, non-vitrified oocytes served as controls. The oocytes were then submitted either to in vitro maturation (Experiment 1, n = 334) or to vitrification/warming (Experiment 2, n = 440). The stage of nuclear maturation was subsequently determined. In Experiment 3, the vitrified immature oocytes (n = 254) were matured and fertilized in vitro, and their developmental competence was assessed. A total of 424 embryos derived from vitrified immature oocytes were transferred into the oviduct of 6 recipient queens (Experiment 4).Vitrification reduced significantly the meiotic and developmental competence of immature cat oocytes compared with the non-vitrified controls. The EG alone or a combination of EG and DMSO yielded higher maturation rates than DMSO, irrespective of the CPA equilibration techniques used. The 4-step EG vitrification resulted in the highest maturation rate (37.6%) but cleavage and blastocyst rates were significantly lower than the non-vitrified controls (24.8% and 30.2% vs 62.5% and 49.3%, respectively). Pregnancy was established in recipients receiving embryos derived from non-vitrified and vitrified/warmed immature oocytes. It is concluded that the stepwise CPA exposure technique can be successfully applied for vitrification of immature cat oocytes, in terms of in vitro development but it is likely to affect in utero development.     

Nonequilibrium cryopreservation of rabbit embryos using a modified (sealed) open pulled straw procedure

The study was designed to evaluate the efficiency of a modified (sealed) open pulled straw (mOPS) method for cryopreserving rabbit embryos by vitrification or rapid freezing. An additional objective was to determine whether the mOPS method could cause the vitrification of a cryoprotectant solution generally used in rapid freezing procedures. Two consecutive experiments of in vitro and in vivo viability were performed. In Experiment 1, the in vitro viability of rabbit embryos at the morula, compacted morula, early blastocyst and blastocyst stages was assessed after exposure to a mixture of 25% glycerol and 25% ethylene glycol (25GLY:25EG: vitrification solution) or 4.5 M (approximately 25% EG) ethylene glycol and 0.25 M sucrose (25EG:SUC: rapid freezing solution). Embryos were loaded into standard straws or mOPS and plunged directly into liquid nitrogen. The mOPS consisted of standard straws that were heat-pulled, leaving a wide opening for the cotton plug and a narrow one for loading embryos by capillarity. The embryos were aspirated into the mOPS in a column positioned between two columns of cryoprotectant solution separated by air bubbles. The mOPS were then sealed with polyvinyl-alcohol (PVA) sealing powder. The vitrification 25GLY:25EG solution became vitrified both in standard straws and mOPS, whereas the rapid freezing 25EG:SUC solution crystallized in standard straws, but vitrified in mOPS. The total number of embryos cryopreserved was 1695. Embryos cryopreserved after exposure to each solution in mOPS showed higher rates (88.2%) of survival immediately after thawing and removal of the cryoprotectant than those cryopreserved in 0.25 ml standard straws (78.8%; P < 0.0001). After culture, the developmental stage of the cryopreserved embryos significantly affected the rates of development to the expanded blastocyst stage. Regardless of the cryoprotectant used, lower rates of in vitro development were obtained when the embryos were cryopreserved at the morula stage, and higher rates achieved using embryos at blastocyst stages. Based on the results of Experiment 1, the second experiment was performed on blastocysts using the mOPS method. Experiment 2 was designed to evaluate the in vivo viability of cryopreserved rabbit blastocysts loaded into mOPS after exposure to 25GLY:25EG or 25EG:SUC. Embryos cryopreserved in mOPS and 25GLY:25EG solution gave rise to rates of live offspring (51.7%) not significantly different to those achieved using fresh embryos (58.5%). In conclusion, the modified (sealed) OPS method allows vitrification of the cryoprotectant solution at a lower concentration of cryoprotectants than that generally used in vitrification procedures. Rabbit blastocysts cryopreserved using a 25GLY:25EG solution in mOPS showed a similar rate of in vivo development after thawing to that shown by fresh embryos.     

Developmental capacity of Antarctic minke whale ( Balaenoptera bonaerensis ) vitrified oocytes following in vitro maturation, and parthenogenetic activation or intracytoplasmic sperm injection

The present study investigated the effects of the sexual maturity of oocyte donors on in vitro maturation (IVM) and the parthenogenetic developmental capacity of fresh minke whale oocytes. The effects of cytochalasin B (CB) pretreatment and two types of cryoprotectant solutions (ethylene glycol (EG) or ethylene glycol and dimethylsulfoxide (EG + DMSO)) on the in vitro maturation of vitrified immature whale oocytes were compared, and the developmental capacity of vitrified immature whale oocytes following IVM and intracytoplasmic sperm injection examined (ICSI). The maturation rate did not differ significantly with sexual maturity (adult, 60.9%; prepubertal, 53.1%), but the parthenogenetic activation rate of oocytes from adult donors (76.7%) was significantly higher (p < 0.05) than that of oocytes from prepubertal donors (46.4%). The maturation rates after vitrification and warming were not significantly different between the EG (22.2%) and EG + DMSO groups (30.2%), or between the CB-treated (30.4%) and non-CB-treated groups (27.3%). These results indicate that parthenogenetic activation of in vitro matured oocytes from adult minke whales was superior to that from prepubertal whales, but that the developmental capacity of the whale oocytes after parthenogenetic activation or ICSI was still low. The present study also showed that CB treatment before vitrification and two kinds of cryoprotectants did not improve the IVM rate following the vitrification of immature whale oocytes.     

Preliminary studies on the vitrification of red sea bream (Pagrus major) embryos

The objective was to identify an appropriate cryoprotectant and protocol for vitrification of red sea bream (Pagrus major) embryos. The toxicity of five single-agent cryoprotectants, dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), glycerol (GLY), and methyl alcohol (MeOH), as well as nine cryoprotectant mixtures, were investigated by comparing post-thaw hatching rates. Two vitrifying protocols, a straw method and a solid surface vitrification method (copper floating over liquid nitrogen), were evaluated on the basis of post-thaw embryo morphology. Exposure to single-agent cryoprotectants (10% concentration for 15 min) was not toxic to embryos, whereas for higher concentrations (20 and 30%) and a longer duration of exposure (30 min), DMSO and PG were better tolerated than the other cryoprotectants. Among nine cryoprotectant mixtures, the combination of 20% DMSO+10% PG+10% MeOH had the lowest toxicity after exposure for 10 min or 15 min. High percentages of morphologically intact embryos, 50.6+/-16.7% (mean+/-S.D.) and 77.8+/-15.5%, were achieved by the straw vitrifying method (20.5% DMSO+15.5% acetamide+10% PG, thawing at 43 degrees C and washing in 0.5M sucrose solution for 5 min) and by the solid surface vitrification method (40% GLY, thawing at 22 degrees C and washing in 0.5M sucrose solution for 5 min). After thawing, morphological changes in the degenerated embryos included shrunken yolks and ruptured chorions. Furthermore, thawed embryos that were morphologically intact did not consistently survive incubation.     

Cryopreservation of immature bovine oocytes by vitrification in straws

The aim of this study was to cryopreserve by vitrification by ethylene glycol (EG) and dimethyl sulfoxide (DMSO) immature bovine oocytes in straws and to investigate the effects of vitrification on post-thaw oocyte maturation. A total of 575 cumulus oocyte complexes were obtained by follicle aspiration from 238 ovaries of cows slaughtered at a local abattoir. Following selection, oocytes with compacted cumulus cells and evenly granulated ooplasm were vitrified using one of the three different solutions with a non-vitrified group served as control. The first step vitrification solution contained 20% EG while the second step solution contained 40% EG+1M sucrose in a basic media used in group EG. Oocytes were matured in N-2-hidroxyethyl piperazine-N-2-ethanosulfonic acid (HEPES) buffered tissue culture medium (TCM) 199 for 24h at 39 degrees C in a humidified atmosphere of 5% CO2 in air. Oocytes were fixed following evaluation for polar body formation, stained with Giemsa solution and nuclear maturation was examined. The numbers of oocytes which were observed at Metaphase II (MII) stage were 41 (34.1%), 17 (14.9%), 29 (20.7%) and 78 (79.6%) in groups EG, DMSO, Mix and Control, respectively. Maturation rate distribution in group Mix was not statistically different when compared to maturation rate distributions in groups EG and DMSO (p>0.05). Differences between other groups were significant (p<0.001). However, better results were obtained in EG group compared to DMSO and mix groups. Maturation rates were lower in all treatment groups than the control group. The lowest maturation result was obtained in DMSO group. Maturation rate in group Mix was between maturation rates of EG and DMSO groups. Immature bovine oocytes can be vitrified in straws, but maturation success differs with the cryoprotectant and it seems that to obtain better maturation rates, new cryopreservation techniques specific for immature bovine oocytes are needed.     

Osmotic behavior of in vitro produced bovine blastocysts in cryoprotectant solutions as a potential predictive test of survival

The osmotic behavior of bovine blastocysts produced in vitro was filmed during exposure to and dilution of cryoprotectant solutions used for vitrification. The relationship between the changes in the diameter of embryos and their subsequent survival was assessed. Embryos collected on Day 6 and Day 7 postinsemination were exposed to 10% glycerol (GLY) for 5 min, 10% GLY + 20% ethylene glycol (EG) for 5 min, and 25% Gly + 25% EG for 30 s, before dilution in 0.85 M galactose and finally in embryo transfer freezing medium (ETF). Embryos that had a higher probability of survival behaved as perfect osmometers, shrinking, reexpanding, or swelling according to an identical pattern, whereas embryos that deviated from this standard usually did not survive. The initial embryo diameter, duration of shrinkage and expansion in 10% glycerol, duration of reexpansion in ETF, and final embryo diameter were clearly predictive of the ability to hatch after culture in vitro. On a given day postinsemination, larger blastocysts were more likely than smaller blastocysts to survive and hatch after exposure to cryoprotectants with or without vitrification.     

Cryopreservation of immature and in vitro matured porcine oocytes by solid surface vitrification

Cryopreservation of normal, lipid-containing porcine oocytes has had limited practical success. This study used solid surface vitrification (SSV) of immature germinal vesicle (GV) and mature meiosis II (MII) porcine oocytes and evaluated the effects of pretreatment with cytochalasin B, cryoprotectant type (dimethylsulfoxide (DMSO), ethylene glycol (EG), or both), and warming method (two-step versus single-step). Oocyte survival (post-thaw) was assessed by morphological appearance, staining (3',6'-diacetyl fluorescein), nuclear maturation, and developmental capacity (after in vitro fertilization). Both GV and MII oocytes were successfully vitrified; following cryopreservation in EG, more than 60% of GV and MII stage porcine oocytes remained intact (no significant improvement with cytochalasin B pretreatment). Oocytes (GV stage) vitrified in DMSO had lower (P<0.05) nuclear maturation rates (31%) than those vitrified in EG (51%) or EG+DMSO (53%). Survival was better with two-step versus single-step dilution. Despite high survival rates, rates of cleavage (20-26%) and blastocyst formation (3-9%) were significantly lower than for non-vitrified controls (60 and 20%). In conclusion, SSV was a very simple, rapid, procedure that allowed normal, lipid-containing, GV or MII porcine oocytes to be fertilized and develop to the blastocyst stage in vitro.     

Cryopreservation of Cattle Oocytes: Effects of Meiotic Stage, Cycloheximide Treatment, and Vitrification Procedure

Different parameters likely to influence the survival of bovine oocytes after a vitrification procedure were evaluated: oocyte meiotic stage, cycloheximide treatment at the beginning or the end of maturation, and three vitrification procedures using conventional straws, open pulled straws (OPS), or microdrops. For each procedure a mixture of cryoprotectants (25% ethylene glycol and 25% glycerol) was used. After the oocytes were warmed and subjected to in vitro maturation and fertilization, the number that developed into blastocysts was determined. Results show that cryoprotectant exposure reduced embryo development and that cycloheximide treatment had no beneficial effect on oocytes vitrified in conventional straws. Among the three vitrification procedures, only the OPS method yielded blastocysts (approximately 3% of vitrified oocytes) irrespective of their initial meiotic stage. This result highlights the major influence of the cooling rate in an oocyte vitrification protocol.     

Comparison of different vitrification protocols on viability after transfer of ovine blastocysts in vitro produced and in vivo derived

We compare different vitrification protocols on the pregnancy and lambing rate of in vitro produced (IVP) and in vivo derived (IVD) ovine embryos. Ovine blastocysts were produced by in vitro maturation, fertilization and culture of oocytes collected from slaughtered ewes or superovulated and inseminated animals. Embryos were cryopreserved after exposure at room temperature either for 5 min in 10% glycerol (G), then for 5 min in 10% G + 20% ethylene glycol (EG), then for 30 s in 25% G + 25% EG (glycerol group), or for 3 min in 10% EG + 10% dimethyl sulphoxide (DMSO), then for 30s in 20% EG + 20% DMSO + 0.3 M sucrose (DMSO group). One group of in vitro produced embryos was cryopreserved similarly to the DMSO group, but with 0.75 M sucrose added to the vitrification solution (DMSO 0.75 group). Glycerol group embryos were then loaded into French straws or open pulled Straws (OPS) while the DMSO group embryos were all loaded into OPS and directly plunged into liquid nitrogen. Embryos were warmed with either a one step or three step process. In the one step process, embryos were placed in 0.5 M sucrose. The three-step process was a serial dilution in 0.5, 0.25 and 0.125 M sucrose. The embryos of DMSO 0.75 group were warmed directly by plunging them into tissue culture medium-199 (TCM-199) + 20% foetal bovine serum (FBS) in the absence of sucrose (direct dilution). Following these manipulations, the embryos were transferred in pairs into synchronised recipient ewes and allowed to go to term. The pregnancy and the lambing rate within each group of IVP and IVD embryos indicated that there was no statistical difference among the vitrification protocols.     

Effect of cryoprotectants and their concentration on in vitro development of vitrified-warmed immature oocytes in buffalo (Bubalus bubalis)

Experiments were conducted to study the effect of cryoprotectants, dimethyl sulfoxide (DMSO), ethylene glycol (EG), 1,2-propanediol (PROH), and glycerol at different concentrations (3.5, 4, 5, 6, and 7 M each with 0.5 M sucrose and 0.4% BSA in DPBS) on survival, in vitro maturation, in vitro fertilization, and post-fertilization development of vitrified-thawed immature buffalo oocytes. The COCs were harvested from the ovaries by aspirating the visible follicles. The recovery of post-thaw morphologically normal oocytes was lower in 3.5 and 4 M DMSO, EG, and PROH compared to 5, 6, and 7 M. In all the concentrations of glycerol, an overall lower numbers of oocytes recovered were normal compared to other cryoprotectants. Less number of oocytes reached metaphase-II (M-II) stage from the oocytes cryopreserved in any of the concentrations of DMSO, EG, PROH, and glycerol compared to fresh oocytes. Among the vitrified groups, highest maturation was obtained in 7 M solutions of all the cryoprotectants. The cleavage rates of oocytes vitrified in different concentrations of DMSO, EG, PROH, and glycerol were lower than that of the fresh oocytes. The cleavage rates were higher in oocytes cryopreserved in 6 and 7 M DMSO, EG, PROH, and glycerol compared with oocytes cryopreserved in other concentrations. However, the percentage of morula and blastocyst formation from the cleaved embryos did not vary in fresh oocytes and vitrified oocytes. In conclusion, this report describes the first successful production of buffalo blastocysts from immature oocytes cryopreserved by vitrification.     

Effects of vitrification medium composition on the survival of bovine in vitro produced embryos, following in straw-dilution, in vitro and in vivo following transfer

This study examined the effects of adding a macromolecule, polyvinylpyrrolidone (10% PVP) and a sugar (0.3 M trehalose) to vitrification solutions (VS) containing either one (40% ethylene glycol [EG], two (25% EG+25% DMSO) or three (20% EG+20% DMSO+10% 1, 3-butanediol [BD]) permeable cryoprotectants on the survival and hatching of IVP bovine embryos, following vitrification, warming and in-straw cryoprotectant dilution. Grade 1 and 2 compact morulae and blastocysts were selected on Day 7 (Day 0=IVF) of culture in SOFaaBSA and equilibrated for 10 min at room temperature in 10% EG. Following exposure, for up to 1 min at 4 degrees C, to one of the above VS (with or without PVP+trehalose), the embryos were loaded into straws and immersed in liquid nitrogen. Following warming and in-straw cryoprotectant dilution, the embryos were cultured for 48 h to assess hatching. There was no effect of VS on the survival of embryos after 24 h, however fewer compact morulae than blastocysts survived after 24 h (24% vs. 75%; P<0.001) or hatched after 48 h (15% vs. 59%; P<0.001). When blastocysts only were considered, an interaction between VS and additional PVP+trehalose was also observed (P<0.01). Hatching was reduced when they were added to 25% EG+25% DMSO (70% vs. 45%) but was not affected for either 40% EG (44 and 49%) or to 20% EG+20% DMSO+10% BD (72 and 72%). Pregnancy rates (Day 90 ultrasound) of recipients that were transferred either two non-vitrified or two vitrified (20% EG+20% DMSO+10% BD) blastocysts, did not differ (3/6 [50%] and 11/20 [55%]). However, significantly (P<0.02) fewer recipients that received compact morulae maintained pregnancy to Day 90 although this was not affected by vitrification (fresh vs. vitrified; 1/5 [20%] vs. 3/18 [17]). These data demonstrate that a VS comprising three cryoprotectants, rather than one, enables more embryos to hatch during post-thaw culture and that the survival, following direct transfer of these vitrified embryos, is not different to non-vitrified embryos.     

Development of OPS vitrified pig blastocysts: effects of size of the collected blastocysts, cryoprotectant concentration used for vitrification and number of blastocysts transferred

Unhatched blastocysts from Large White hyperprolific gilts (n=103) were identified, measured and vitrified using the Open Pulled Straw (OPS) technique to evaluate the effects of the collected blastocyst size and cryoprotectant concentrations used for vitrification, and the number of embryos transferred per recipient. Vitrified/warmed blastocyst viability was estimated in vitro, as the percentage of embryos developing after 72h, and in vivo, on pregnancy Day 30. In the in vitro study, we compared the use of three cryoprotectant concentrations (16.5, 18, or 20% DMSO+16.5, 18, or 20% EG+0.4M sucrose). Survival rates differed significantly between the control (98.3%) and the three cryoprotectant concentrations (67, 62.3, and 57%, respectively). Blastocyst size at vitrification determined the further in vitro development of embryos (26% survival for blastocysts 126-144microm versus 100% for blastocysts >199microm). For the in vivo study, blastocysts were vitrified using cryoprotectant concentrations of 16.5 or 18% DMSO+EG and transferred surgically in groups of 20 or 30 per recipient (n=40). Recipients were slaughtered on pregnancy D30. No significant differences were detected in gestation rates (50-70%) and embryo survival rates (14.7-25%), although survival was higher (P=0.0003) when 20 blastocysts were transferred compared to 30 (24.7% versus 15.5%). Our findings indicate that best results, in terms of subsequent in vivo embryo survival, were achieved after transferring 20 embryos at the blastocyst or expanded blastocyst stage, previously vitrified using cryoprotectant concentrations of 16.5 or 18%.     

Maturation rates of vitrified-thawed immature buffalo (Bubalus bubalis) oocytes: effect of different types of cryoprotectants

Cryopreservation of oocytes collected from slaughtered animals of high genetic value, their subsequent utilisation for production of embryos for transfer may provide an opportunity to replenish the valuable germplasm lost. Experiments were conducted to study the effect of cryoprotectants, dimethyl sulfoxide (DMSO), ethylene glycol (EG), 1,2-propanediol (PROH) and glycerol at different concentrations (3.5, 4, 5, 6 and 7 M each with 0.5M sucrose and 0.4% BSA in DPBS) on morphological survival and in vitro maturation of vitrified-thawed immature buffalo oocytes. The cumulus oocyte complexes were harvested from the ovaries obtained from a local slaughterhouse by aspirating the visible follicles. Less number of oocytes reached metaphase-II stage from the oocytes cryopreserved in any of the concentrations of DMSO, EG, PROH and glycerol compared to fresh oocytes. Among the vitrified groups, highest maturation (40.3, 42.5, 40.4 and 23.5%) was obtained in 7 M DMSO, EG, PROH and glycerol, respectively. Oocytes reaching to M-II stage from the oocytes cryopreserved in 7 M glycerol were significantly lower than that of the oocytes vitrified in 7 M DMSO, EG and PROH. It can be concluded that 7 M solutions of DMSO, EG and PROH can be used for vitrification of immature buffalo oocytes for subsequent utilisation of these oocytes in IVM/IVF and embryo production for transfer.     

New technology for vitrification and field (microscope-free) warming and transfer of small ruminant embryos

This study was designed to test the efficiency of recently developed vitrification technology followed by microscope-free thawing and transfer of sheep embryos. In a first set of experiments, in vivo derived embryos at the morula to blastocyst stage were frozen in an automated freezer in ethylene glycol, and after thawing and removal of cryoprotectants, were transferred to recipient ewes according to a standard protocol (control group). A second group of embryos were loaded into open-pulled straws (OPS) and plunged into liquid nitrogen after exposure at room temperature to the media: 10% glycerol (G) for 5 min, 10% G+20% ethylene glycol (EG) for 5 min, 25% G+25% EG for 30s; or 10% EG+10% DMSO for 3 min, 20% EG+20% DMSO+0.3M trehalose for 30s. The OPS were thawed by plunging into tubes containing 0.5M trehalose. After this rapid thawing, the embryos were directly transferred using OPS as the catheter for the transplantation process. In a second set of experiments, in vivo derived and in vitro produced expanded blastocysts were vitrified in OPS and then transferred as described above. The lambing rates recorded (59% for the conventionally cryopreserved in vivo derived embryos, 56% for the vitrified in vivo derived embryos, and 20% for the vitrified in vitro produced embryos), suggest the suitability of the vitrification technique for the transfer of embryos obtained both in vivo and in vitro. This simple technology gives rise to a high embryo survival rate and will no doubt have applications in rearing sheep or other small ruminants.     

Bovine oocyte vitrification: effect of ethylene glycol concentrations and meiotic stages

Success in oocyte cryopreservation is limited and several factors as cryoprotectant type or concentration and stage of oocyte meiotic maturation are involved. The aim of the present study was to evaluate the effect of maturation stage and ethylene glycol (EG) concentration on survival of bovine oocytes after vitrification. In experiment 1, kinetics of oocyte in vitro maturation (IVM) was evaluated. Germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), and metaphase II (MII) oocytes were found predominantly at 0, 0–10, 10–14, and 18–24 h of IVM, respectively. In experiment 2, in vitro embryo development after in vitro fertilization (IVF) of oocytes exposed to equilibrium (ES) and vitrification solution VS-1 (EG 30%), or VS-2 (EG 40%) at 0, 12 or 18 h of IVM was evaluated. Only blastocyst rate from oocytes vitrified in SV-2 after 18 h of IVM was different from control oocytes. Hatched blastocyst rates from oocytes vitrified in VS-1 after 12 and 18 h, and SV-2 after 18 h of IVM were different from unvitrified oocytes. In experiment 3, embryo development was examined after IVF of oocytes vitrified using VS-1 or VS-2 at 0, 12 or 18 h of IVM. Rates of blastocyst development after vitrification of oocytes in VS-1 at each time interval were similar. However, after vitrification in VS-2, blastocyst rates were less at 18 h than 0 h. Both cleavage rates and blastocyst rates were significantly less in all vitrification groups when compared to control group and only control oocytes hatched. In conclusion, both EG concentration and stage of meiotic maturation affect the developmental potential of oocytes after vitrification.     

In-straw cryoprotectant dilution of IVP bovine blastocysts vitrified in hand-pulled glass micropipettes

The aim of this study was to determine the influence of two ethylene glycol-based vitrification solutions on in vitro and in vivo survival after in-straw cryoprotectant dilution of vitrified in vitro-produced bovine embryos. Day-7 expanded blastocysts were selected according to diameter (> or = 180 microm) and osmotic characteristics and randomly assigned to one of three groups (i) VSa: vitrification in 40% EG+17.1% SUC+0.1% PVA; (ii) VSb: vitrification in 20% EG+20% DMSO; (iii) control: non-vitrified embryos. Vitrification was performed in hand-pulled glass micropipettes (GMP) and cryoprotectant dilution in 0.25 ml straws after warming in a plastic tube. Embryo viability was assessed by re-expansion and hatching rates after 72 h of IVC and by pregnancy rates after direct transfer of vitrified embryos. No differences in re-expansion rates were observed between vitrified groups after 24 h in culture (VSa=84.5%; VSb=94.8%). However, fewer VSa embryos (55.2%, P<0.05) hatched after 72 h than the VSb (75.8%) and control embryos (80.0%). To evaluate in vivo viability, vitrified embryos (VSa=20; VSb=21) were warmed under field conditions and individually transferred to synchronous recipients. Pregnancy rates (day 60) were similar between groups (VSa=20%; VSb=19%). Greater hatching rates occurred after 72 h of IVC for EG+DMSO than EG+SUC+PVA vitrification solutions. However, using a GMP vitrification container and in-tube warming, both solutions provided similar pregnancy rates after the in-straw cryoprotectant dilution and direct embryo transfer.     

Effect of different vitrification solutions and cryodevices on viability and subsequent development of buffalo oocytes vitrified at the germinal vesicle (GV) stage

The cryopreservation of immature oocytes would generate a readily available, non-seasonal source of female gametes for research and reproduction. In domestic animals, the most promising results on oocyte cryopreservation have been reported in cattle, few studies have been conducted on buffalo. The aim of the present study was to compare the use of different vitrification solutions and various cryodevices on viability and developmental competence of buffalo oocytes vitrified at the germinal vesicle (GV) stage. Cumulus oocyte-complexes (COCs) obtained at slaughterhouse from mature buffalo ovaries were randomly divided into three main groups and vitrified by using either straw or open pulled-straw (OPS) or solid surface vitrification (SSV) in a solution composed of either 20% ethylene glycol (EG) + 20% glycerol (GLY); VS1 or 20% EG + 20% dimethylsulfoxide (DMSO); VS2, respectively. Following vitrification and warming, viable COCs were matured in vitro for 22 h. Some COCs were denuded and stained with 1.0% aceto-orcein to evaluate nuclear maturation, whereas the others were fertilized and cultured in vitro for 7 days to determine the developmental competence. Although the recovery rate (64.9%) was the lowest in the oocytes vitrified by SSV using 20% EG + 20% DMSO as compared to the other groups, the best survival rate of the COCs was achieved in the same treatment (96.7%), which was significantly higher (P < 0.05) than those vitrified using traditional straws (71.8% in VS1 and 73.6% in VS2) or those vitrified using OPS and VS1 (73.9%). Furthermore, in the nuclear maturation test, the highest maturation rate (75.5%) was achieved in SSV vitrified COCs using 20% EG + 20% DMSO (VS2), which was similar to the controls (77.1%). Post IVF and embryo culture, the highest cleavage and blastocyst development rates were obtained in COCs vitrified in 20% EG + 20% DMSO using SSV (47.1% and 24.0%, respectively), which showed no difference from the controls (61.2% and 46.9%, respectively). Our results clearly show that the combination of SSV and 20% EG + 20% DMSO could be used effectively to vitrify GV stage buffalo COCs.     

Vitrification of pre-pubertal ovine cumulus-oocyte complexes: effect of cytochalasin B pre-treatment

The aim of this study was to evaluate the effect of cytochalasin B (CCB) pre-treatment before vitrification on ability of immature oocytes from lamb ovaries to progress until metaphase II (MII) stage after vitrification/warming procedure. Cumulus-oocyte complexes (COCs) were obtained from ovaries of lambs, from 80 to 90 days old, collected from a local slaughterhouse. Before vitrification, COCs were randomly distributed in two experimental groups corresponding to the incubation with or without 7.5 microg/ml CCB for 30 min. In order to study cryoprotectant and CCB pre-treatment toxicity (toxicity test), oocytes were exposed to cryoprotectants, with or without CCB pre-treatment, but without plunging into N2 liquid. Vitrification solution was composed by 4.48 M EG plus 3.50 M DMSO supplemented with 0.25 M sucrose. Two-step addition was performed. After vitrification or toxicity test, COCs were matured in bicarbonate-buffered TCM 199 containing 10% foetal calf serum and 10 ng/ml epidermal growth factor. A sample of COCs was directly in vitro matured (control group). Rates of MII oocytes of toxicity groups both, with or without CCB pre-treatment were lower than control group (41.1-50.0 versus 79.9, respectively; P<0.05). After vitrification, a lower number of oocytes progressed to MII stage in comparison with non-vitrification groups (P<0.05). In vitrified groups both with or without CCB pre-treatment 8.0 and 12.7%, respectively, of immature oocytes reached MII stage by the end of in vitro maturation culture. No effect of CCB was observed, either in the toxicity or vitrified groups. In conclusion, no effect of CCB pre-treatment before vitrification was detected in this study with immature oocytes of pre-pubertal sheep. More studies are needed in order to increase ovine oocyte survival after vitrification.     

Effect of different cryoprotectants and vitrificant solutions on the hatching rate of turbot embryos (Scophthalmus maximus)

Vitrification could provide a promising tool for the cryopreservation of fish embryos. However, in order to achieve a vitrifiable medium, a high concentration of permeable cryoprotectants must be employed, and the incorporation of high molecular weight compounds should also be considered. The toxicity of these permeable and non-permeable agents has to be assessed, particularly when high concentrations are required. In the present study, permeable and non-permeable cryoprotectant toxicity was determined in turbot embryos at two development stages (F stage-tail bud and G stage-tail bud free). Embryos treated with pronase (2mg/ml, 10 min at 22 degrees C) were incubated in dimethyl sulfoxide (Me2SO), methanol (Meth.) or ethylene glycol (EG) in concentrations ranging from 0.5 to 6M for periods of 10 or 30 min, and in 5, 10, and 15% polyvinylpyrrolidone (PVP), 10, 15, and 20% sucrose or 0.1, 1, and 2% X-1000 for 2 min. The embryos were then washed well and incubated in seawater until hatching. The toxicity of permeable cryoprotectants increased with concentration and exposure time. There were no significant differences between permeable cryoprotectants. However, embryos tolerated higher concentrations of Me2SO than other cryoprotectants. Exposure to permeable cryoprotectants did not affect the hatching rate except at G stage with X-1000 treatment and 20% sucrose. Taking into account the cryoprotectant toxicity and the vitrification ability of cryoprotectant mixtures, three vitrification solutions (V1, V2, and V3), and one protocol for stepwise incorporation were designed. The tested solutions contained 5M Me2SO+2M Meth+1M EG plus 5% PVP, 10% sucrose or 2% X-1000. The hatching rate of embryos that had been exposed to the the vitrification solutions was analyzed and no significant differences were noticed compared with the controls. Our results demonstrate that turbot embryos can be subject to this cryoprotectant protocol without deleterious effect on the hatching rate.