Improvement in post-thaw viability of in vitro-produced bovine blastocysts vitrified by glass micropipette (GMP)

The present work was designed to study the in vitro and in vivo viability, as assessed by blastocyst formation, pregnancy rate and term delivery of bovine embryos produced under completely defined conditions with or without insulin-like growth factor I (IGF-I) following direct transfer after cryopreservation. Slaughterhouse-derived bovine oocytes were matured for 24h, fertilized with frozen-thawed spermatozoa and cultured in vitro under completely defined conditions with or without exposure to IGF-I (5 ng/ml). Only those embryos classified as excellent or good quality blastocysts were frozen. Each blastocyst was individually loaded into a straw, seeded and pre-cooled to -7 degrees C. After 10 min at -7 degrees C straws were frozen further to -30 degrees C at a rate of 0.3 degrees C/min and then plunged into liquid nitrogen. Synchronized recipient cows received one embryo in the horn ipsilateral to the corpus luteum (CL). Pregnancies were diagnosed by ultrasonography 35-45 days after embryo transfer (ET). IGF-I failed to improve cleavage rate, as well as blastocyst production, when added during in vitro culture (IVC). Pregnancy outcome was not significantly improved in cows that received an IGF-I-treated embryo compared with controls (4/10 versus 3/10, respectively). Five out of six calves delivered to date were born alive and healthy. We have shown that it is possible to obtain healthy live offspring from frozen-thawed embryos produced under chemically defined conditions after direct transfer.     

Normal calves from transfer of biopsied, sexed and vitrified IVP bovine embryos

Data on biopsied, sexed and cryopreserved in vitro produced (IVP) bovine embryos, and their in vivo developmental competence are very limited. Two preliminary studies were conducted before the primary study. In Experiment 1, post-thaw in vitro developmental competence of biopsied and vitrified IVP embryos was evaluated using re-expansion as an endpoint. In Experiment 2, the pregnancy rates of biopsied fresh, frozen or vitrified embryos following single embryo transfer were compared. Since vitrified embryos resulted in a higher pregnancy rate than frozen-thawed embryos, in the primary study (Experiment 3), all IVP embryos were vitrified following biopsy and sexing (by DNA fingerprinting). In Experiment 3, we compared pregnancy initiation and calving results of heifers in the following treatments: 1) artificial insemination (AI); 2) AI plus contralateral transfer of a single embryo (AI + SET); 3) ipsilateral transfer of single embryo (SET); or 4) bilateral transfer of two embryos (DET). Birth weights, gestation lengths and dystocia scores were recorded. In Experiment 1, post-thaw re-expansion rate of biopsied and vitrified embryos was 85% (70/82). In Experiment 2, pregnancy rates (90 d) were 44% (7/16), 23% (3/13), and 50% (7/14) for vitrified, frozen and fresh embryos, respectively (P < 0.10). In Experiment 3, pregnancy rates of AI and SET were 65% (20/31) and 40% (16/40), respectively (P < 0.05). The pregnancy rate of AI + SET was 75% (27/36) with 11 carrying twins, and the pregnancy rate of DET was 72% (26/36) with 10 carrying twins. All AI fetuses were carried to term, but only half the SET fetuses were carried to term. Similar calving rates were observed in the AI + SET and DET groups, 76 and 70%, respectively, of those pregnant at Day 40. Mean birth weight, dystocia score and gestation length of AI calves were not different from those of SET calves. Mean birth weight and dystocia score of single-born calves were greater than those of twin born calves (P < 0.05). These data demonstrate that biopsied IVP bovine embryos can be successfully cryopreserved by vitrification and following post-thaw embryo transfer, acceptable rates of offspring with normal birth weights can be obtained without major calving difficulties.     

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.     

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 sugars-addition on the survival of vitrified bovine blastocysts produced in vitro

We investigated the effect of addition of sugars to a vitrification solution on the survival rate of bovine blastocysts produced in vitro. In vitro-matured (IVM) and in vitro-fertilized (IVF) bovine Day-6 to Day-8 bovine blastocysts were classified into 3 developmental stages: early blastocysts, blastocysts and expanded blastocysts. The blastocysts were cryopreserved in 1 of 3 vitrification solutions: 1) 25% glycerol25% ethylene glycol (GE); 2) 20% glycerol20% ethylene glycol3/4 M sucrose (GES); and 3) 20% glycerol20% ethylene glycol3/8 M sucrose3/8 M dextrose (GESD). The basic solution was Dulbecco's PBS supplemented with 20% of fetal calf serum. Embryos were exposed to each vitrification solution in 3 steps, and after loading into 0.25-ml straws, were plunged into liquid nitrogen. After warming in water bath at 20 degrees C, cryoprotectants were diluted in 1/2 M and 1/4 M sucrose each for 5 min. Equilibration and dilution procedure except warming were conducted at room temperature (23 to 27 degrees C). After dilution, the embryos were cultured in Ham's F10 medium0.1 mM beta-mercaptoethanol20% fetal calf serum. Survival rates of embryos at 48 h of incubation of each of the 3 developmental stages (early blastocysts, blastocysts and expanded blastocysts) exposed to the 3 types of the vitrification solutions (GE, GES and GESD) were 23.5, 33.3, 65.8% (early blastocysts, blastocysts and expanded blastocysts respectively) in GE, 55.6, 71.9, 90.5% in GES and 84.6, 83.3, 95.8% in GESD respectively. These results indicate that a mixture of 25% glycerol25% ethylene glycol is not suitable for vitrification of early bovine blastocysts; however, addition of sugars to the solution significantly (P<0.01) improved the survival rate of the vitrified blastocysts, independently of their stage of development.     

In-straw warming protocol improves survival of vitrified embryos and allows direct transfer in cattle

Vitrification is a superior method for cryopreservation of IVF embryos, but due to complicated warming protocols, it is not commonly used for commercial bovine embryos routine. To overcome the need of laboratory embryo preparation during warming, we developed an in-straw warming protocol compatible with most vitrification devices for embryo transfer without sucrose gradient steps and embryo evaluation. Surprisingly, one of the tested protocols improved embryo survival (95.0%* vs 83.1% expansion rate and 74.2%* vs 51.5% hatching rate) compared to conventional in-plate warming. Embryo quality was also increased, taken by the higher total cell numbers (160.7 ± 8.6* vs 99.0 ± 7.9) and lower apoptosis index (4.9 ± 0.6* vs 11.5 ± 2.4) 48 h after warming. Pregnancy rates were similar between vitrified-warmed embryos and fresh embryos (40% vs 43%). Based on our results, we suggest in-straw warming should always be used for vitrified embryos due to beneficial effects. Direct transfer can be safely performed using this protocol.     

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.     

Growth factors and growth hormone enhance in vitro embryo production and post-thaw survival of vitrified bovine blastocysts

The objective of this study was to assess the influence of specific growth factors and growth hormone (GH) in the culture medium on in vitro embryo production and post-thaw survival of vitrified blastocysts. In total, 1673 bovine oocytes were used for evaluating the nuclear status of the oocytes after in vitro maturation (n=560) or for in vitro fertilization (IVF, n=1113) and distributed in five treatment groups: (1). medium only control; (2). activin (10 ng/ml); (3). epidermal growth factor (EGF) (10 ng/ml); (4). insulin 5 microg/ml and (5). GH (100 ng/ml). There was an increase (P<0.05 and P<0.01, respectively) in the percentage of oocytes that reached meta phase II, developed to blastocysts and hatched, as well as in the blastocyst cell number in the groups treated with activin, EGF and GH compared to controls. There was no significant difference between insulin and control groups. A total of 465 blastocysts were vitrified in a three-step protocol using ethylene glycol and polyvinylpyrrolidone. After thawing, embryos were cultured in five treatments groups as described above. Groups EGF and GH had higher (P<0.05) survival rates with a mean blastocyst survival of 95.0+/-1.5 and 93.1+/-3.5%, respectively, while mean hatching rate was higher for EGF and activin groups (75.3+/-3.4 and 62.0+/-3.2%, respectively). Thawed control blastocysts had a mean cell count of 52.7+/-3.3%. With the exception of insulin, all growth factors and GH tested showed higher (P<0.01) total cell numbers when compared to controls. In conclusion, addition of growth factors and GH in the culture media has favorable effects on in vitro maturation, in vitro embryo production, and post-thaw survival of vitrified blastocysts.     

Prolonging bovine sperm-oocyte incubation in modified medium 199 improves embryo development rate and the viability of vitrified blastocysts

This study was conducted to compare the efficacy of four in vitro fertilization (IVF) media: Bracket and Oliphant's medium (BO), modified medium 199 (IVF-M199), modified Tyrode's medium (MTM), and modified KSOM (m-KSOM) on fertilization efficiency and blastocyst formation rate. In addition, we wanted to investigate the benefit of prolonging the IVF period (from 6 to 18 h) using the two most effective IVF media determined in our initial experiment; subsequently, blastocyst viability was assessed following vitrification. A higher incidence of polyspermic fertilization was observed in the MTM (6%) and in BO, in both the 6 and 18 h (7% and 11%, respectively) groups, than in the m-KSOM (1%) or in the IVF-M199 6 or 18 h (1 and 3%, respectively) groups. Cleavage rates were similar in BO, IVF-M199, and MTM 48 h post-fertilization; however, the lowest cleavage rate was observed for m-KSOM. A greater proportion of zygotes developed into 8-cell embryos in IVF-M199 than in other IVF media. Subsequently, a greater proportion of blastocyst formation and hatching was achieved in IVF-M199 (40% and 79%, respectively) or BO (35% and 74%, respectively) than in m-KSOM (18% and 58%, respectively) or MTM (22% and 66%, respectively). Prolonging IVF to 18 h did not alter cleavage rates; however, the highest rate of overall blastocyst formation was achieved in the IVF-M199 18 h (49%), rather than in the BO 18 h (20%) group. Vitrified/thawed blastocysts from IVF-M199 groups re-expanded and developed better, as compared to the BO 18 h group, and hatching rate and total cell number in IVF-M199 18 h group was comparable to the control groups (non-vitrified). Vitrification reduced survival compared to controls. In conclusion, IVF-M199 was successfully used for IVF, compared favorably to BO medium, and offered the advantage of an extended IVF period for up to 18 h that requires only one-half a dose of semen, and resulted in better quality blastocysts that endured vitrification with a hatching rate comparable to that of control groups.     

Comparison of the interferon-tau expression from primary trophectoderm outgrowths derived from IVP, NT, and parthenogenote bovine blastocysts

The expression of interferon-tau (IFN-tau) is essential for bovine embryo survival in the uterus. An evaluation of IFN-tau production from somatic cell nuclear transfer (NT)-embryo-derived primary trophectoderm cultures in comparison to trophectoderm cultured from parthenogenote (P) and in vitro matured, fertilized, and cultured (IVP) bovine embryos was performed. In Experiment 1, the success/failure ratio for primary trophectoderm colony formation was similar for IVP and NT blastocysts [IVP = 155/29 (84%); NT 104/25 (81%)], but was decreased (P = .05) for P blastocysts [54/43 (56%)]. Most trophectoderm colonies reached diameters of at least 1 cm within 3-4 weeks, and at this time, 72 hr conditioned cell culture medium was measured for IFN-tau concentration by antiviral activity assay. The amount of IFN-tau produced by IVP-outgrowths [4311 IU/mL (n = 155)] was greater (P < .05) than that from NT- [626 IU/mL (n = 104)] and P - [1595 IU/mL (n = 54)] derived trophectoderm. Differential expression of IFN-tau was confirmed by immunoblotting. In Experiment 2, colony formation was again similar for IVP and NT blastocysts [IVP = 70/5 (93%); NT 67/1 (99%)] and less (P < .05) for P blastocysts [65/27 (70%)]. Analysis of trophectoderm colony size after 23 days in culture showed a similar relationship with P-derived colonies being significantly smaller in comparison to IVP and NT colonies. A differential expression of IFN-tau was also observed again, but this time as measured over time in culture. Maximal IFN-tau production was found at day-14 of primary culture and diminished to a minimum by the 23rd day.     

Autograft of vitrified mouse ovaries using ethylene glycol as cryoprotectant.

Ovaries from 8 to 10-week-old N MRI mice were vitrified using RPMI solution containing 30% (W/V) ficoll 70, 0.5 M sucrose, 10.7% (V/V) acetamide and 40% (V/V) ethylene glycol (EGFS40%), and were stored in liquid nitrogen. After warming at 25 degrees C in 1 M sucrose solution and equilibration with RPMI medium, the vitrified and fresh ovarian tissues were autografted intraperitoneally. After one and two estrus cycles the animals were sacrificed and the recovered grafts were examined histologically. Five days after transplantation the vitrified ovaries they were invaded by fat and fibrous cells and the large preantral and antral follicles were degenerated. At 11 days postgrafting the stroma was devoid of necrotic cells and contained normal primordial and primary follicles, suggesting that the vitrification is a simple, useful and efficient procedure for cryopreservation of murine ovarian tissues.     

Higher survival rate of vitrified and thawed in vitro produced bovine blastocysts following culture in defined medium supplemented with beta-mercaptoethanol

The present study was conducted to compare bovine embryo developmental quality, after culture in different defined culture media, up to blastocyst stage, and subsequently cultured in media supplemented with beta-mercaptoethanol (beta-ME) following blastocyst vitrification and thawing. In part one of this study, presumptive zygotes were randomly allocated into the following media: (1) CR1, (2) KSOM, (3) SOF, and (4) sequential KSOM-SOF. In the second part of the study, blastocysts derived from four different culture media were subjected to a solid surface vitrification (35% (v/v) ethylene glycol+0.5M Sucrose+5% (w/v) Polyvinylpyrrolidone (PVP), and tested for the effect of beta-ME on their post-vitrification survival. Following thawing, blastocysts were cultured with or without beta-ME. Culture medium had no effect on cleavage rates; however, a significantly greater number of zygotes cultured in KSOM, KSOM-SOF, or SOF developed to the 8-cell stage, compared with those cultured in CR1. A greater proportion of the zygotes cultured in SOF or KSOM-SOF reached blastocysts, than did those cultured in CR1 or KSOM. The use of sequential KSOM-SOF significantly increased total cell numbers of Day 7 expanded-blastocysts when compared to those cultured in CR1, KSOM, or SOF. Addition of beta-ME into culture media after vitrification and thawing improved blastocyst survival, hatching rates, and total cell numbers of blastocysts. In conclusion, supplementation of beta-ME into culture medium after vitrification and thawing significantly increased blastocyst survival, hatching rates, and their total cell numbers. These results suggest that vitrified IVF embryos should be thawed and briefly cultured in beta-ME medium prior to embryo transfer.     

Protocols for thawing and cryoprotectant dilution of heart valves

PURPOSE: To reduce the time taken for thawing and removal of cryoprotectant from heart valves. METHODS: Three sets of experiments were carried out using porcine heart valves. The valves in all three experiments were first exposed to 10% (v/v) dimethyl sulphoxide (DMSO) by a 2-step protocol. Outcome was determined after the various experimental treatments by monitoring the outgrowth of cells from valve leaflet explants. Experiment 1-Dilution protocol. Valves exposed to 10% DMSO were subjected to 4-, 2- or 1-step dilution to remove the DMSO. Experiment 2-Warming rate. The rate of warming was increased by reducing the volume of cryoprotectant medium in which the valves were frozen. Valves were exposed to 10% DMSO, frozen in different volumes (100, 50, 25 or 0 ml) of cryoprotectant medium, and warmed in a 37 degrees C water bath. The DMSO was removed by 4-step dilution. Experiment 3-Standard vs. Modified protocol. Valves were either frozen in 100 ml 10% DMSO, thawed, and subjected to 4-step dilution (Standard) or frozen in 50 ml 10% DMSO, thawed, and the DMSO removed by single-step dilution (Modified). RESULTS: Neither the rate of warming nor the rate of dilution of DMSO had any influence on the subsequent outgrowth of valve leaflet fibroblasts. There were no differences in the outgrowth of cells from valve leaflets cryopreserved by the Standard or Modified protocols. CONCLUSION: The time taken for thawing and dilution of heart valves could be reduced from >20 min to <10 min without detriment to the viability of the leaflet fibroblasts. This should have a positive impact on valve replacement surgery as the thawing and dilution of valves are typically carried out while the patients are on cardiopulmonary bypass.     

Piglets born after non-surgical deep intrauterine transfer of vitrified blastocysts in gilts

The aims of this study were: (1) to evaluate the effect of the number of previous estrus of recipient gilts on effectiveness of intrauterine insertion of a flexible catheter designed for non-surgical deep intrauterine catheterization during diestrus in pigs; and (2) to determine the farrowing rate and the litter size after non-surgical deep intrauterine embryo transfer (ET) of porcine blastocysts vitrified by the open pulled straw (OPS) method. In experiment 1, 27 large white hyperprolific gilts (LWh) with 2-6 previous estrus were used. Intrauterine insertions of the flexible catheter were carried out at day 5.5-6 of the estrous cycle (D0=onset of estrus). During insertions, no or only moderate reactions were observed in 88.9% of gilts and was not related (P >0.05) to the number of estrus prior to the insertion periods. The number of the estrus had a significant effect (P <0.05) on the difficulties found during the procedure. In the 100% of gilts with two estrus (N=6) it was not possible to insert the flexible catheter through the cervix. In gilts with three or more estrus, it was possible to pass the cervix and to progress along a uterine horn in 80.9% of the cases. In 86.7% of the gilts, the tip of the flexible catheter achieved the second or third quarter of the uterine horn. In experiment 2, following non-surgical deep intrauterine transfer of 20 vitrified/warmed blastocysts, 9 Meishan recipients (42.9%) farrowed an average of 5.4 +/- 0.8 piglets (range 3-9) of which 0.6 +/- 0.3 piglets (range 0-2) were born dead. In conclusion, this study shows that it is possible to obtain birth of piglets following non-surgical deep intrauterine embryo transfer (ET) of vitrified/warmed blastocysts. Non-surgical deep intrauterine ET and OPS vitrification methods are promising procedures to be used together for the introduction of new genetic material in a farm.     

Ultrastructure and cell death of in vivo derived and vitrified porcine blastocysts

Morphological and molecular signs of injury and cell death and subsequent regeneration following vitrification of porcine blastocysts were evaluated by light (LM) and transmission electron microscopy (TEM) as well as TUNEL/propidium iodide (PI) nuclear staining followed by confocal microscopy (CSM). In vivo derived blastocysts were assigned to one of the following four groups: Controls-(1) fixed immediately after collection (C0h) and (2) after 24 hr culture in vitro (C24h) and vitrified embryos-(3) fixed immediately after vitrification and warming (V0h), and (4) after 24 hr of culture upon warming after vitrification (V24h). Observation by LM and TEM showed that the V0h embryos displayed collapse of the blastocoele cavity (BC) and cell swelling, a general distension or shrinkage of mitochondria and massive increase in the amount of vesicles, vacuoles, and secondary lysosomes (SLs). Approximately 2/3 of the V24h embryos had recovered, whereas the remaining 1/3 were degenerated. Recovered embryos displayed almost normal blastocyst morphology, except for a widening of the perivitelline space, accumulation of debris and partial distension of mitochondria, whereas degenerated embryos were disintegrated into a poorly defined mass of cells and debris including cells with abundant degeneration of mitochondria and other organelles. Both recovered and degenerated embryos displayed a persistent abundance of presence of small membrane bounded vesicles, vacuoles, and SLs. Evaluation of TUNEL/PI stained embryos showed only occasional appearance of TUNEL positive nuclei with typical apoptotic morphology in controls (C0h 0.67%, C24h 1.22%) and in the V0h embryos (0.93%). The percentage of apoptotic nuclei in embryos at V24h was significantly higher than in all other groups (2.64%). Vitrified embryos showed significantly increased appearance of DNA fragmented nuclei without typical morphological features of apoptosis (V0h 1.43%, V24h 4.30%) compared with controls (C0h 0.26%, C24h 0.45%). The observed morphological changes and increased DNA fragmentation observed immediately after vitrification and warming probably reflects a direct damaging effect of vitrification. During 24 hr of culture a portion of the embryos was able to regenerate and along with the regenerative process, apoptosis--a possible pathway for elimination of damaged cells--became evident.     

Survival and viability of vitrified in vitro and in vivo produced ovine blastocysts

Ovine blastocysts were produced by maturation, fertilization and in vitro culture (IVM/IVF/IVC) of oocytes from slaughtered adult and prepubertal ewes and collection from superovulated and inseminated adult animals. Dulbecco's PBS supplemented with 0.3 mM Na Pyruvate and 20% FCS was used as the basic cryopreservation solution. The embryos were exposed to the vitrification solution as follows: 10% glycerol (G) for 5 min, then 10% G +20% ethylene glycol (EG) for 5 min. Embryos were placed into 25% G + 25% EG in the center of 0.25- mL straws and plunged immediately into LN2. Warming was done by placing the straws into a water bath at 37 degrees C for 20 sec, and their contents were expelled into a 0.5 M sucrose solution for 3 min; the embryos were then transferred into 0.25 M and 0.125 M sucrose solution for 3 min each. Warmed blastocysts were transferred to the culture medium for 24 h. Survival was defined as the re-expansion of the blastocoele. All surviving blastocysts were transferred to synchronized recipient ewes, and the pregnancy was allowed to go to term. Of 68 vitrified in vitro produced blastocysts, 46 re-expanded (67.6%) and 10 lambs were born (14.7%). From the 62 in vivo derived and vitrified embryos, 52 re-expanded (83.8%) and 39 lambs were born (62.9%). The lambing rate of in vitro produced fresh transfer embryos was 40% (20 lambs/50 blastocysts transferred), and of the 32 in vivo derived blastocysts and transferred fresh, 26 lambs were born (81.2%). The results indicate that in vitro produced embryos can be successfully cryopreserved by vitrification.