Gene expression profiles of vitrified in vitro- and in vivo-derived bovine blastocysts

Vitrification is becoming a preferred method for pre‐implantation embryo cryopreservation. The objective of this study was to determine the differentially expressed genes of in vivo‐ and in vitro‐produced bovine embryos after vitrification. In vitro‐ (IVF) and in vivo‐derived (IVV) bovine blastocysts were identified as follows: in vitro‐produced fresh (IVF‐F), in vitro‐produced vitrified (IVF‐V), in vivo‐derived fresh (IVV‐F), in vivo‐derived vitrified (IVV‐V). The microarray results showed that 53 genes were differentially regulated between IVF and IVV, and 121 genes were differentially regulated between fresh and vitrified blastocysts (P < 0.05). There were 6, 268, 962, and 17 differentially regulated genes between IVF‐F × IVV‐F, IVF‐V × IVV‐V, IVF‐F × IVF‐V, and IVV‐F × IVV‐V, respectively (P < 0.05). While gene expression was significantly different between fresh and vitrified IVF blastocysts (P < 0.05), it was similar between fresh and vitrified IVV blastocysts. Significantly up‐regulated KEGG pathways included ribosome, oxidative phosphorylation, spliceosome, and oocyte meiosis in the fresh IVF blastocyst samples, while sphingolipid and purine metabolisms were up‐regulated in the vitrified IVF blastocyst. The results showed that in vitro bovine blastocyst production protocols used in this study caused no major gene expression differences compared to those of in vivo‐produced blastocysts. After vitrification, however, in vitro‐produced blastocysts showed major gene expression differences compared to in vivo blastocysts. This study suggests that in vitro‐produced embryos are of comparable quality to their in vivo counterparts. Vitrification of in vitro blastocysts, on the other hand, causes significant up‐regulation of genes that are involved in stress responses. Mol. Reprod. Dev. 79: 613–625, 2012. © 2012 Wiley Periodicals, Inc.     

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

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

Sheep embryo cryopreservation by vitrification and conventional freezing

The survival of ovine embryos (morulae and blastocysts) either frozen by a conventional method or vitrified was investigated in culture. In Experiment I, embryos were vitrified using a solution containing 25% propylene glycol and 25% glycerol. A group of embryos (simulated control) was processed without freezing to evaluate the toxicity of the vitrification solution. In Experiment II, embryos were exposed to a solution of PBS containing 10% glycerol and 0.25 M sucrose placed horizontally in a programmable freezer. Automatic seeding was applied at -7 degrees C in 2 positions on straws and cooled at -0.3 degrees C/min to -25 degrees C and then stored in liquid nitrogen. In vitro development rates of vitrified embryos were 12% (morulae) and 19% (blastocysts). Simulated embryos showed a higher rate of survival than embryos cryopreserved by vitrification (67 and 63%, morulae and blastocysts respectively). In conventional cooling, the blastocysts showed the highest viability percentage (67%) of all the experimental groups but these values decreased significantly in morulae (31%). Differences in temperature between straws placed in distinct positions in the freezing chamber and thermic deviation were observed when automatic seeding was applied. Embryo viability differed from 51 to 75% according the relative position of the embryos within the chamber. Survival was higher when automatic seeding was applied on the meniscus of the embryo column versus the central point of this column (65 vs 21%). The damage of both cryopreservation methods on zona pellucida integrity (27 and 35% in vitrified and conventionally frozen embryos, respectively) had no effect on the in vitro survival.     

Comparison of open pulled straw (OPS) vs glass micropipette (GMP) vitrification in mouse blastocysts

The purpose of this study was to investigate the use of a glass micropipette (GMP) as a vessel for vitrification of mouse blastocysts, and to compare the post-thaw survival of these blastocysts with those cooled in open pulled straws (OPS). The GMP vessel permits higher freezing and warming rates than OPS due to the higher heat conductivity of glass and lower mass of the solution containing the embryos. Groups of 6 mouse blastocysts were sequentially placed into 2 vitrification solutions before being loaded into either the OPS or GMP vessels and immersed into LN2 within 20 to 25 sec. Post-thaw blastocysts were serially washed in 0.25 and 0.15 M sucrose in holding medium (HM) and modified human tubal fluid medium (mHTF), each for 5 min, and then cultured in mHTF supplemented with 10% FCS for 24 h. The rate of blastocyst re-expansion did not differ significantly for OPS (93.5%) and GMP (95.0%) methods (P<0.05). The hatching rate in OPS (88.7%) was similar to that in GMP (90.0%) but was lower than for the unvitrified control embryos (98.3%, P<0.05). To determine the optimal embryo population per GMP vessel, the pipettes were loaded with 2 to 10 embryos. The rate of blastocyst re-expansion after vitrification was significant for 2 to 4 embryos than for 6 to 10 embryos per vessel. In addition, the rate of blastocyst re-expansion was significantly lower if blastocysts were vitrified in the wide rather than the narrow portion of the micropipette (100 vs 87.5%; P<0.05) even when only 4 blastocysts were loaded per vessel. These results indicate that both vitrification vessels can provide high rates of embryo survival. However, the GMP vessel does not need a cap to protect the vessel from floating after immersion in LN2. The number and location of the embryos (narrow versus wide portion of capillary) were considered to be limiting factors to the viability of mouse embryos.     

A new vitrification device that absorbs excess vitrification solution adaptable to a closed system for the cryopreservation of mouse embryos

Several closed vitrification devices that avoid contact with liquid nitrogen have been reported. Recently, based on the Kitasato Vitrification System (KVS), we developed the Closed-KVS, which is a closed vitrification device. The KVS is an open vitrification device that can absorb excess vitrification solution. In this study, we performed two experiments to evaluate the efficacy of the Closed-KVS as a vitrification device for the cryopreservation of mouse embryos at the blastocyst and two-cell stage. In the first experiment, the blastocysts were vitrified using either the Closed-KVS or the KVS (control device). The survival, re-expansion, and hatching rates were not significantly different between embryos vitrified using the Closed-KVS and those vitrified using the KVS. In the second experiment, we evaluated the embryonic development of the two-cell stage embryos vitrified using the Closed-KVS. There were no significant differences in the survival, blastocyst formation, or hatching rates between vitrified or non-vitrified embryos. Additionally, we evaluated the cooling and warming rates of these devices using a numerical simulation method. The cooling rates of the Closed-KVS were similar regardless of whether the outer cap was pre-cooled and were lower than those of the KVS. However, the warming rates of the Closed-KVS (irrespective of cap pre-cooling) were the same as those of the KVS (612,000 °C/min). In summary, the Closed-KVS is a novel closed vitrification device for the cryopreservation of mouse embryos at the blastocyst and two-cell stage.     

Factors affecting the success rate of porcine embryo vitrification by the Open Pulled Straw method

The objectives of this study were: (1) to evaluate the influence of porcine embryo developmental stage on in vitro embryo development after vitrification, (2) to study the efficiency of the one-step dilution procedure, compared with conventional warming, for vitrified embryos at different stages of development, and (3) to determine the influence of the embryo donor on the in vitro survival of vitrified embryos at morulae and blastocyst stages. Two to four cell embryos, morulae and blastocysts were collected by laparotomy from weaned crossbred sows (n=55). Vitrification and conventional warming were performed using the OPS procedure with Superfine Open Pulled Straws (SOPS). For one-step dilution, embryos were placed in 800 microl TCM199-HEPES containing 20% of new born calf serum and 0.13 M sucrose for 5 min. To evaluate development, two to four cell embryos, morulae and blastocysts were cultured in vitro for 120, 48 and 24h, respectively. Some fresh embryos from each developmental stage were not vitrified and cultured as controls. Embryos were morphologically evaluated for their developmental capacity during the in vitro culture by stereomicroscopy. The total cell number of embryos was assessed by Hoechst-33342 staining and fluorescence microscope observation. There was a significant effect of the stage of development on the in vitro survival, perihatching rate and the number of cells of embryos after vitrification and warming (Experiment 1; p<0.001). The survival and perihatching rates of two to four cell embryos were lower than those obtained for morulae and blastocysts (p<0.001). No differences (p>0.05) in survival rates were found between vitrified and fresh blastocysts. The warming procedure did not affect the development and total cell number of vitrified two to four cell embryos, morulae or blastocysts (Experiment 2). However, donor had a significant effect (p<0.001) on the in vitro development and the number of cells of morulae and blastocysts after vitrification and warming (Experiment 3). In conclusion, the embryo developmental stage and the embryo donor were important factors that affected the development of porcine embryos after OPS-vitrification and warming. OPS-vitrification and the one-step dilution are efficient procedures to be used with intact porcine morulae and blastocysts.     

Piglets produced from in vivo blastocysts vitrified using the Cryologic Vitrification Method (solid surface vitrification) and a sealed storage container

The objective was to develop a simple successful porcine cryopreservation protocol that prevented contact between embryos and liquid nitrogen, avoiding potential contamination risks. In vivo-derived blastocysts were collected surgically from donor pigs, and two porcine embryo vitrification protocols (one used centrifugation to polarize intracytoplasmic lipids, whereas the other did not) were compared using the Cryologic Vitrification Method (CVM), which used solid surface vitrification. The CVM allowed embryos to be vitrified, without any contact between embryos and liquid nitrogen. Both protocols resulted in similar in vitro survival rates (90% and 94%) and cell number (89 ± 5 and 99 ± 5) after 48 h in vitro culture of vitrified and warmed blastocysts. The protocol that did not use centrifugation was selected for continued use. To protect vitrified embryos from contact with liquid nitrogen and potential contamination during storage, a sealed outer container was developed. Use of this sealed outer container did not affect in vitro survival of cryopreserved blastocysts. In vivo blastocysts (n = 151) were collected, vitrified, and stored using the selected protocol and sealed container. These embryos were subsequently warmed and transferred to six recipients; five became pregnant and farrowed a total of 26 piglets. This embryo vitrification method allowed porcine embryos to be successfully vitrified and stored without any contact with liquid nitrogen.     

Vitrification of mouse embryos at various stages by open-pulled straw (OPS) method

This study was performed to pursue the optimal condition for the cryopreservation of mouse morulae by a two-step OPS method and to investigate the feasibility of the optimal condition for vitrification of embryos at other developmental stages. First, the mouse morulae were vitrified in OPS using one-step procedure-that is, embryos were vitrified after direct exposure to EDFS30 (15% ethylene glycol (EG), 15% dimethyl sulfoxide (DMSO), Ficoll and sucrose), or two-step method-that is, embryos were first pretreated in 10%E + 10%D (10% EG and 10% DMSO in mPBS) for 30 sec, then exposed to EDFS30 for 15 to 60 sec, respectively. After vitrification and warming, the embryos were morphologically evaluated and assessed by their development to blastocysts, expanded/hatched blastocysts, or to term after transfer. The result showed that all the vitrified-warmed morulae had similar blastocyst rate compared to that of control (91.7% vs. 100%), and the highest developmental rate to expanded blastocysts (100%) or hatched blastocysts (62.3%) was observed when the morulae were pretreated with 10%E + 10%D for 0.5 min, exposed to EDFS30for 25 sec before vitrification and warming in 0.5 M sucrose for 5 min. After transfer, the survival rate (33.1%) in vivo of the vitrified morulae was higher (P > 0.05) than that of the fresh embryos (24.6%). Secondly, embryos at different stages were cryopreserved and thawed following the above program. Most (93.4 to 100%) of the embryos recovered after vitrification were morphologically normal at all the developmental stages. The blastocyst rates of the vitrified one-cell (52.5 to 66.7%) and the two-cell (63.3 to 68.9%) embryos were lower (P < 0.05) than those of the vitrified four-cell embryos (81.7 to 86.4%), the eight-cell embryos (90.0 to 93.3%), morulae (96.7 to 100%), and the expanded blastocysts rate (98.3 to 100.0%) of the vitrified early blastocysts. The highest survival rate in vivo of vitrified embryos were from the early blastocysts (40.4%), which was similar to that of fresh embryos (48.6%). The data demonstrate that the optimal protocol for the cryopreservation of morulae was suitable for the four-cell embryos to early blastocyst stages and that the early blastocyst stage is the most feasible stage for mouse embryo cryopreservation under our experimental conditions.     

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

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

Evaluation of sheep ovarian tissue cryopreservation with slow freezing or vitrification after chick embryo chorioallantoic membrane transplantation

The aim of our investigations was to compare the effectiveness of two methods for cryopreservation of sheep ovarian tissue, slow freezing and vitrification. The quality of cryopreserved tissues was evaluated after 5 days of thawing and chorioallantoic membrane (CAM) transplantation. Follicular structure, stromal integrity and neovascularization were assessed. The areas of fibrosis and necrosis were measured using MICROVISIBLE software, and proliferation was assessed with Ki-67 immunostaning. After 5 days of culture, the proportion of primordial follicles decreased, whereas the primary and intermediary follicles increased insignificantly (p > .05). Only necrosis in the vitrified culture group increased significantly (p < .05). It was established also that 5 days CAM culture was not suitable methodology for detection of folliculogenesis. Follicular quality decreased after culture, but was better in fresh and slow frozen tissues than after vitrification (p < .05). Cellular proliferative activity fell, but it preserved to some extent in all groups. In conclusion, follicles was preserved better in grafted tissue after slow freezing than vitrification and stroma was more susceptible to ischemia in vitrified rather than conventional freezing in this view. Vitrification may not be a suitable alternative to the slow freezing.     

Comparison on in vitro fertilized bovine embryos cultured in KSOM or SOF and cryopreserved by slow freezing or vitrification

The objectives of this study were to identify an improved in vitro cell-free embryo culture system and to compare post-warming development of in vitro produced (IVP) bovine embryos following vitrification versus slow freezing. In Experiment 1, non-selected presumptive zygotes were randomly allocated to four medium treatments without co-culture: (1) SOF + 5% FCS for 9 days; (2) KSOM + 0.1% BSA for 4 days and then KSOM + 1% BSA to Day 9; (3) SOF + 5% FCS for 4 days and then KSOM + 1% BSA to Day 9; and (4) KSOM + 0.1% BSA for 4 days and then SOF + 5% FCS to Day 9. Treatment 4 (sequential KSOM-SOF culture system) improved (P > 0.05) morulae (47%), early blastocysts (26%), Day-7 blastocysts (36%), cell numbers, as well as total hatching rate (79%) compared to KSOM alone (Treatment 2). Embryos cultured in KSOM + BSA alone developed slowly and most of them hatched late on Day 9, compared to other treatments. In Experiment 2, the sequential KSOM-SOF culture system was used and Day-7 blastocysts were subjected to following cryopreservation comparison: (1) vitrification (VS3a, 6.5 M glycerol); or (2) slow freezing (1.36 M glycerol). Warmed embryos were cultured in SOF with 7.5% FCS. Higher embryo development and hatching rates (P < 0.05) were obtained by vitrification at 6h (71%), 24h (64%), and 48h (60%) post-warming compared to slow freezing (48, 40, and 31%, respectively). Following transfer of vitrified embryos to synchronized recipients, a 30% pregnancy rate was obtained. In conclusion, replacing KSOM with SOF after 4 days of culture produced better quality blastocysts. Vitrification using VS3a may be used more effectively to cryopreserve in vitro produced embryos than the conventional slow freezing method.     

Vitrification of Mouse Embryos at Various Stages by Open-Pulled Straw (OPS) Method

This study was performed to pursue the optimal condition for the cryopreservation of mouse morulae by a two-step OPS method and to investigate the feasibility of the optimal condition for vitrification of embryos at other developmental stages. First, the mouse morulae were vitrified in OPS using one-step procedure—that is, embryos were vitrified after direct exposure to EDFS30 (15% ethylene glycol (EG), 15% dimethyl sulfoxide (DMSO), Ficoll and sucrose), or two-step method—that is, embryos were first pretreated in 10%E+10%D (10% EG and 10% DMSO in mPBS) for 30 sec, then exposed to EDFS30 for 15 to 60 sec, respectively. After vitrification and warming, the embryos were morphologically evaluated and assessed by their development to blastocysts, expanded/hatched blastocysts, or to term after transfer. The result showed that all the vitrified-warmed morulae had similar blastocyst rate compared to that of control (91.7% vs. 100%), and the highest developmental rate to expanded blastocysts (100%) or hatched blastocysts (62.3%) was observed when the morulae were pretreated with 10%E+10%D for 0.5 min, exposed to EDFS30 for 25 sec before vitrification and warming in 0.5 M sucrose for 5 min. After transfer, the survival rate (33.1%) in vivo of the vitrified morulae was higher (P > 0.05) than that of the fresh embryos (24.6%). Secondly, embryos at different stages were cryopreserved and thawed following the above program. Most (93.4 to 100%) of the embryos recovered after vitrification were morphologically normal at all the developmental stages. The blastocyst rates of the vitrified one-cell (52.5 to 66.7%) and the two-cell (63.3 to 68.9%) embryos were lower (P < 0.05) than those of the vitrified four-cell embryos (81.7 to 86.4%), the eight-cell embryos (90.0 to 93.3%), morulae (96.7 to 100%), and the expanded blastocysts rate (98.3 to 100.0%) of the vitrified early blastocysts. The highest survival rate in vivo of vitrified embryos were from the early blastocysts (40.4%), which was similar to that of fresh embryos (48.6%). The data demonstrate that the optimal protocol for the cryopreservation of morulae was suitable for the four-cell embryos to early blastocyst stages and that the early blastocyst stage is the most feasible stage for mouse embryo cryopreservation under our experimental conditions.     

Ultrastructural characterization of fresh and cryopreserved in vivo produced ovine embryos

Controlled slow freezing and vitrification have been successfully used for ovine embryo cryopreservation. Selection of embryos for transfer is based on stereomicroscopical embryo scoring after thawing, but the subjectivity inherent to this selection step has been demonstrated by ultrastructural studies of controlled slow frozen, in vivo produced ovine morulae and blastocysts. These studies have shown that certain abnormalities remain undetected by stereomicroscopy only. In the present study, using ovine in vivo produced morulae and blastocysts, we have studied the ultrastructural alterations induced by open pulled straw vitrification (OPS) and controlled slow freezing, compared stereomicroscopical embryo scoring with light microscopy evaluation of embryo's semithin sections, and related the ultrastructural cellular damage with the embryo classification by stereomicroscopical embryo scoring of embryos’ and semithin section evaluation by light microscopy. The ultrastructural lesions found for OPS-vitrified and controlled slow frozen embryos were similar, independently of embryo stage. A significant higher number of grade 3 embryos was found at stereomicroscopical scoring after controlled slow freezing (P = 0.02), and a significant higher number of grade 3 blastocysts was found at semithin sectioning after OPS vitrification (P = 0.037). The extension of ultrastructural damage, especially of mitochondria and cytoskeleton, was related to the semithin classification but not to stereomicroscopical scoring at thawing. This suggests that semithin scoring is a useful tool for predicting ultrastructural lesions and new improvements in cryopreservation and thawing methods of ovine embryos are still warranted, including in the case of blastocysts cryopreserved by OPS vitrification.     

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%.     

Effect of estrous cow serum during bovine embryo culture on blastocyst development and cryotolerance after slow freezing or vitrification

The present study investigated the effect of estrous cow serum (ECS) during culture of bovine embryos on blastocyst development and survival after cryopreservation by slow freezing or vitrification. Embryos were derived from in vitro maturation (IVM) and in vitro fertilization (IVF) of abbatoir-derived oocytes. At Day 3, embryos were cultured in three different media: Charles Ronsenkrans medium + amino acids (CR1aa; without bovine serum albumin (BSA)) + 5% estrous cow serum (CR1-ECS), CR1aa + 3 mg/mL BSA (CR1-BSA) or CR1aa + 5% ECS + 3 mg/mL BSA (CR1-ECS-BSA). At 7.5 d post-insemination (PI), blastocyst yield and quality were evaluated; blastocysts and expanded blastocysts from each media were cryopreserved by Open Pulled Straw (OPS) vitrification method or slow freezing (1.5 M ethylene glycol, EM). Total blastocyst yield did not differ among CR1-ECS, CR1-BSA and CR1-ECS-BSA (30.9, 33.1 and 32.9%, respectively, P < 0.05). Embryo survival (hatching rate) was higher in vitrified versus slow-frozen embryos (43% versus 12%, respectively, P < 0.01), and in embryos cultured in CR1-BSA (40.3%) compared with those cultured in serum-containing media (CR1-ECS, 21.5% and CR1-ECS-BSA, 19.8%; P < 0.01). In conclusion: (a) it was possible to produce in vitro bovine embryos in serum-free culture medium without affecting blastocyst yield and quality; (b) serum-free medium produced the best quality embryos (in terms of post-cryopreservation survival); and (c) vitrification yielded the highest post-cryopreservation survival rates, regardless of the presence of serum in the culture medium.     

Factors affecting nuclear maturation, cleavage and embryo development of vitrified bovine cumulus-oocyte complexes

The objective was to investigate the effects of cryodevice, vitrification solutions, and equilibration time on in vitro maturation, cleavage, and embryo development of vitrified bovine oocytes. In Experiment 1, the nuclear maturation (MII) rate of immature bovine COCs vitrified was compared between two equilibration times (0 vs 10 min) in vitrification solution 1 (VS1) and two cryodevices (cryotop vs 0.25 mL straw). The MII rate was higher in the non-vitrified control group than in vitrified groups (61 vs 16%, P < 0.0001). Equilibration time did not affect MII rate (P = 0.964); however, the MII rate was higher for COCs vitrified on cryotops than in straws (23 vs 9%, P = 0.007). In Experiment 2, bovine COCs were vitrified on cryotops using two equilibration times (0 vs 5 min) in VS1 and two kinds of vitrification solutions (freshly prepared vs frozen). Cleavage and blastocyst rates were higher (P < 0.0001) in the non-vitrified control group than vitrified groups (cleavage rate 93 vs 42% and blastocysts rate 31 vs 0.4%). Cleavage rate of COCs vitrified using frozen solutions with 5 min equilibration was higher (P = 0.05) than other treatment groups. However, blastocyst rate did not differ (P = 0.993) among treatment groups. In conclusion, cryotop was a better cryodevice than 0.25 mL straw for vitrification of bovine COCs. Furthermore, 5 min equilibration in VS1 improved cleavage. Compared with control, the vitrification procedure per se damaged bovine COCs, resulting in poor nuclear maturation and embryo development. However, vitrification did not immediately kill oocytes, as the cleavage rate was acceptable.     

Expression of miR-Let-7a,miR-15a,miR-16-1, and their target genes in fresh and vitrified embryos and its surrounding culture media for noninvasive embryo assessment

microRNAs (miRNAs) play a critical role in implantation and development of mouse embryos. In this study, we aim to evaluate the possibility of miRNAs as potential biomarkers in the blastocyst culture to assess embryo quality. We also intend to investigate whether improved clinical outcomes of vitrified embryos agree with altered miRNA expressions. Mouse embryos from in vitro fertilization were vitrified at the two-cell stage. After thawing, the embryos were individually cultured and developed to the blastocyst stage. We used quantitative real-time polymerase chain reaction to evaluate miRNA expression levels in both vitrified and fresh groups, and culture medium (CM). The fibronectin binding assay was performed to examine for blastocyst attachment. The findings showed reduced expressions of miR-16-1 (0.2 ± 0.06) and miR-Let-7a (0.65 ± 0.1) after vitrification compared to fresh embryos. We observed significant upregulation of the target genes Vav3 (4.33 ± 0.25), integrin β-3 (Itg β3; 4.73 ± 0.2), and Bcl2 (2.29 ± 0.16) in the vitrified embryos compared to the fresh groups. Evaluation of blastocyst CM showed upregulation of miR-Let-7a (15.68 ± 0.89), miR-16-1 (16.18 ± 0.75), and miR-15a (13.36 ± 0.73) in the vitrified group in comparison to the fresh blastocysts (P < .05). The expression levels of miR-16-1 (3.28 ± 0.63), miR-15a (5.91 ± 0.38), and miR-Let-7a (9.07 ± 0.6) in CM of the vitrified blastocysts conducted on fibronectin were significantly higher than the fresh group (P < .05).This study showed that vitrification of embryos changes implantation and proliferation biomarkers. In addition, upregulated miRNAs in CM could be potentially used for noninvasive early assessment of embryo quality.     

Open pulled straw vitrification of goat embryos at various stages of development

This investigation addresses the question whether it is possible to apply the open pulled straw (OPS) vitrification method, found to be effective for cryopreserving caprine (Capra aegagrus hircus) blastocysts, to other embryonal stages. Morulae, blastocysts and hatched blastocysts were cryopreserved by way of OPS vitrification and blastocysts and hatched blastocysts by conventional freezing. Morulae were not included with conventional freezing because in our experience the survival rate is very low. To assess the viability of the cryopreserved embryos, they were transferred to synchronized does; in most cases, two embryos per doe. After OPS vitrification, of nine does receiving morulae, not a single one became pregnant; of 11 does receiving blastocysts, nine (82%) became pregnant (all of which kidded and gave birth to, on average, 1.8 kids); and of nine does receiving hatched blastocysts, three (33%) became pregnant (two of which [22%] kidded, giving birth to a single kid each). After conventional freezing, of 10 does receiving blastocysts, five became pregnant (four of which [40%] carried to term and gave birth to a pair of twins each); and of nine does receiving hatched blastocysts, three (33%) became pregnant (and gave birth to a single kid each). Embryo survival (kids born/embryos transferred) after vitrification for morulae, blastocysts, and hatched blastocysts was 0, 70% (16 of 23), and 13% (2 of 16), respectively, and after conventional freezing for blastocysts and hatched blastocysts was 42% (8 of 19) and 19% (3 of 16), respectively. The difference in pregnancy and kidding rate between vitrified and conventionally frozen blastocysts was significant, and so was the difference in pregnancy rate between hatched and nonhatched blastocysts, regardless whether OPS-vitrified or conventionally frozen. The results of the current study indicate that OPS vitrification is a very effective means of cryopreserving caprine blastocysts. Unfortunately, the superiority of OPS vitrification over conventional freezing does not apply to caprine morulae and hatched blastocysts.     

Aberrant expression of miR-29a/29b and methylation level of mouse embryos after in vitro fertilization and vitrification at two-cell stage

Proper epigenetic modifications during preimplantation embryo development are important for a successful pregnancy. We aim to investigate the putative influence of in vitro fertilization (IVF) and vitrification on DNA methylation in mouse preimplantation embryos. The study groups consisted of blastocyst-derived vitrified two-cell embryos, nonvitrified embryos, and a control group of in vivo derived blastocysts. We assessed developmental competence, global DNA methylation, relative expression levels of miR-29a/29b, and their target genes, Dnmt3a/3b. Vitrified embryos had a lower developmental rate as compared with nonvitrified embryos. There was no significant decrease in blastocyst cell numbers among studied groups, whereas there was a steady decline in DNA methylation after IVF and vitrification. The levels of miR-29a/29b upregulated in the experimental groups as compared with the control group. IVF and vitrification caused Dnmt3a/3b downregulations in blastocysts. The results of this study have suggested that a relationship exists between IVF and embryo vitrification with methylation interruptions in the blastocysts.     

The effect of equilibration time on survival and development rates of mouse pronuclear-stage embryos vitrified in solid surface (SSV) and convential straws: in vitro and in vivo evaluations

The objective of this study was to improve the efficiency of cryopreservation of pronuclear-stage (PN) mouse embryos. A novel vitrification technique (solid surface vitrification, SSV) was compared with a convential one in straws both for cryosurvival and obtaining progeny from cryopreserved PN mouse embryos. In the SSV method, 15-20 PN embryos were exposed to vitrification solutions for approximately 20 sec after equilibration, and then they were dropped in 2 microl drops onto a pre-cooled (-150 to -180 degrees C) metal surface. In the straws method, groups of 5-10 PN embryos were loaded in a single straw after equilibration. In experiment I, it was compared the effect of the vitrification solutions alone, without vitrification. No reduction was detected in survival, cleavage and blastocysts rates and the lowest development rate was obtained from hatched blastocyst for 20 min equilibration (24.5%). In experiment II, SSV method resulted in significantly higher survival and cleavage rates than that of in-straw vitrified 15-20 min group (87% vs. 60%, 83% vs. 67%, respectively; P < 0.05). There were no statistical differences among any of the blastocyts groups. However, there was a statistical difference in hatched blastocysts between 15 to 5, 10, and 20 min (P < 0.05). In experiment III, it was found no major effect among equilibration time periods in toxicity groups according to the mean cell number of blastocysts developed from PN embryos. But, there was a significant differences between 15 min SSV and 10 min in straw vitrified according to the mean cell number of blastocysts developed from PN embryos following vitrification (P < 0.05). The good results were obtained from 15 min equilibration group for SSV and 10 min equilibration group for straw vitrification. In the last experiment, embryo transfer after vitrification and toxicity was investigated. There were significant differences between SSV and straw just on the rate of pups born (30% and 20.5% respectively; P < 0.05). In conclusion, vitrification of PN mouse embryos by SSV can result in high rates of in vitro development to expanded and hatched blastocyst stage and in vivo development to live pups.