Non-vitrified Control Group Research Articles

High survival of bovine mature oocytes after nylon mesh vitrification, as assessed by intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) is an alternative technique to in vitro fertilization (IVF) for producing transferable blastocysts, especially in combination with cryopreserved oocytes, when the IVF system does not work sufficiently. The present study was conducted to directly compare the efficacy of producing bovine blastocysts by ICSI and IVF from vitrified-warmed and fresh oocytes. Denuded oocytes with a detectable first polar body were vitrified-warmed using a nylon mesh device. In the non-vitrified control group, blastocyst yields 8 days after IVF and ICSI were 32.0 and 26.8%, respectively. Oocyte vitrification and subsequent IVF resulted in an impaired blastocyst yield (15.0%); however, such a loss of efficacy due to vitrification was not observed in the ICSI group (blastocyst yield, 25.2%). The alignment of cortical granules beneath the oolemma was comparable between the fresh control and vitrified-warmed oocytes. Here, we report the high survival of vitrified-warmed bovine oocytes, as assessed by ICSI.

26 Effect of different cryoprotectant concentrations on vitrification of invitro-matured bovine oocytes in paper containers

A great challenge for successful oocyte vitrification is the development of a low-cytotoxic cryoprotectant solution in a safe device allowing ultra-rapid cooling. This study compared different concentrations of cryoprotectants for bovine IVM-oocyte vitrification in a safe paper container device on oocyte survival and cleavage rates. Abattoir ovaries were obtained and cumulus–oocyte complexes (COCs) were recovered by aspirating follicles of 3 to 6mm in diameter. A total of 470 COCs with homogeneous cytoplasm oocytes, surrounded by several layers of cumulus cells were selected, in 5 replicates. Groups of ∼50 COCs were matured in 500µL of semi-defined IVM medium for 22h at 38.8°C in a humidified atmosphere with 5% CO2. After IVM, COCs were allocated to 1 of 3 groups of 20 to 30 COCs, differing only in final concentration of cryoprotectants. A nonvitrified control group (CG) was also tested, totalling 4 groups. Before vitrification, each group was transferred to 500µL of TCM-199 HEPES with 20% fetal bovine serum (FBS) (Base medium, BM) for 5min at 34°C, and COCs were partially denuded by gentle pipetting. Vitrification followed a 3-step protocol at room temperature and groups of 4 to 5 COCs were transferred to BM solution drops containing (1) 5% ethylene glycol (EG) + 5% dimethyl sulfoxide (DMSO) for 30 s; (2) 10% EG + 10% DMSO + 0.25M sucrose for 30 s; and (3) vitrification solution (VS), according to each group: high (HG), 20% EG + 20% DMSO + 0.5M sucrose; medium (MG), 15% EG + 15% DMSO + 0.5M sucrose; or low (LG), 10% EG + 10% DMSO + 0.5M sucrose for 30s. Afterwards, COCs were loaded in <1µL of solution and placed in a homemade paper container device, and immediately plunged in liquid nitrogen. Warming was performed placing the paper container in 3mL of 1M sucrose in BM for 2min. After warming, a 3-step protocol was conducted and COCs were transferred to (1) 500µL of 0.5M sucrose in BM for 2 min; (2) 500µL of 0.25M sucrose for 2 min; (3) 500µL of BM for 2min. Then, COCs from each group were transferred to 250µL of semi-defined IVF medium. Motile sperm were recovered by Percoll washing from one bull and added to IVF medium (Day 0) at final concentration of 106 sperm mL−1 for 18h. At Day 1, all presumptive zygotes were cultured in 25µL of SOF medium with 5% FBS under mineral oil at 38.8°C with 5% CO2 and 5% O2. Normal data were subjected to ANOVA and post hoc Tukey test. Cleavage rate was recorded at Day 2 after IVF. Oocyte survival rate was similar (P>0.05) among vitrified groups (HG, 80%; MG, 86%; LG,87%). Cleavage rate differed (P<0.05) in all vitrified groups compared with control (CG, 82%; HG, 10%; MG, 16%; LG, 16%). Although no difference (P>0.05) was observed among vitrified groups, MG and LG showed a slightly increased oocyte survival and cleavage rates compared with HG. In conclusion, the use of either medium or low concentrations of cryoprotectants may be a less toxic alternative for vitrification of IVM bovine oocytes on paper device. This research was funded by CAPES/COFECUB (#88881.142966/2017-01).

Effect of ovum pick-up medium on blastocyst rate

In this study, we compared two staining protocols assessing the nuclear chromatin stage of equine oocytes after vitrification using permeable and nonpermeable cryoprotectants. Slaughterhouse-derived oocytes (n = 155) were obtained from a total of 32 mares and in vitro matured in M199 medium for 42 hours at 38.5°C in 5% CO2. In the first experiment, two concentrations of Hoechst 33342 (HO) were tested (10 μg/mL; P1 and 2.5 μg/mL; P2) combined with 50 μg/mL of propidium iodide as staining protocols to evaluate the visibility of matured oocytes (n = 44). In the second experiment, 111 oocytes were evaluated using the staining protocol P2, before (C, control) and after vitrification following a two-step conventional protocol with (15% dimethyl sulfoxide, 15% ethylene glycol, and 0.5 M sucrose; V1) or without (1 M sucrose; V2) using permeable cryoprotectants. Our results showed that P2 provided a higher percentage of oocytes with outstanding visibility of the nuclear chromatin stage (52.17%; P < .05) in comparison with P1 (19.04%). In the second experiment, no cryoprotectant-free vitrified oocytes reached the metaphase II maturation stage. This result was significantly lower (P < .05) than conventional vitrification (15.38%) and both lower in comparison with the nonvitrified control group (42.11%). In conclusion, permeable cryoprotectant-free vitrification of equine oocytes obtained poor results and therefore cannot be considered an alternative to vitrification using permeable cryoprotectants. In addition, a staining protocol with a low concentration of HO is recommended to evaluate the nuclear chromatin stage of equine oocytes after in vitro maturation.

The Effect of Different Vitrification and Staining Protocols on the Visibility of the Nuclear Maturation Stage of Equine Oocytes

In this study, we compared two staining protocols assessing the nuclear chromatin stage of equine oocytes after vitrification using permeable and nonpermeable cryoprotectants. Slaughterhouse-derived oocytes (n = 155) were obtained from a total of 32 mares and in vitro matured in M199 medium for 42 hours at 38.5°C in 5% CO2. In the first experiment, two concentrations of Hoechst 33342 (HO) were tested (10 μg/mL; P1 and 2.5 μg/mL; P2) combined with 50 μg/mL of propidium iodide as staining protocols to evaluate the visibility of matured oocytes (n = 44). In the second experiment, 111 oocytes were evaluated using the staining protocol P2, before (C, control) and after vitrification following a two-step conventional protocol with (15% dimethyl sulfoxide, 15% ethylene glycol, and 0.5 M sucrose; V1) or without (1 M sucrose; V2) using permeable cryoprotectants. Our results showed that P2 provided a higher percentage of oocytes with outstanding visibility of the nuclear chromatin stage (52.17%; P < .05) in comparison with P1 (19.04%). In the second experiment, no cryoprotectant-free vitrified oocytes reached the metaphase II maturation stage. This result was significantly lower (P < .05) than conventional vitrification (15.38%) and both lower in comparison with the nonvitrified control group (42.11%). In conclusion, permeable cryoprotectant-free vitrification of equine oocytes obtained poor results and therefore cannot be considered an alternative to vitrification using permeable cryoprotectants. In addition, a staining protocol with a low concentration of HO is recommended to evaluate the nuclear chromatin stage of equine oocytes after in vitro maturation.

Faster, cheaper, defined and efficient vitrification for immature porcine oocytes through modification of exposure time, macromolecule source and temperature

Vitrification reduces the developmental competence of porcine immature oocytes. We investigated the effects of modifying various factors on the viability and development of oocytes after vitrification. These factors included: 1) exposure to the vitrification solution, 2) macromolecule addition (bovine serum albumin (BSA) or polyvinyl pyrrolidone (PVP)), 3) treatment with cytochalasin B, 4) equilibration temperature, and 5) vitrification method (microdrop or Cryotop). Oocytes were equilibrated and vitrified using medium containing ethylene glycol and propylene glycol. After warming, oocytes were subjected to in vitro maturation, stimulated parthenogenetically, and cultured in vitro. Survival rate, nuclear maturation, cleavage, development to the blastocyst stage and their quality were compared between the vitrified groups and the non-vitrified control group. It was found that 1) exposure to the vitrification solution for longer than 30 s was detrimental to embryo development; 2) replacement of BSA with PVP improved embryo development; 3) cytochalasin B treatment reduced the survival rates, but did not affect the blastocyst development rates, 4) equilibration at room temperature (25 °C) was the most beneficial, and 5) the microdrop method improved survival rates. With these adjustments, we were able to establish a simplified and defined cryopreservation system for porcine immature oocytes with improved efficacy.

Effect of antioxidant on in-vitro maturation of vitrified bovine oocytes

The present study was undertaken to investigate the effect of -tocopherol on in-vitro maturation of vitrified bovine oocytes. The cumulus-oocyte-complexes (COCs) recovered from follicles (3–5 mm diameter) by aspiration and slicing techniques were equally divided into 5 (five) groups consisting 66 COCs in each. The vitrified bovine oocytes were sub-grouped into control and treatment group. The vitrified- and non-vitrified- control group of oocytes were in-vitro matured in TCM-199 media without supplementation of vitamin E. In three vitrified treatment groups, the oocytes were in-vitro cultured in TCM-199 media supplemented with vitamin E (-tocopherol) @ 50 μM, 100 μM and 200 μM, respectively. The mean percentage of cumulus cell expansion and polar body formation in non-vitrified control and vitrified control groups were 85.18±2.57 and 65.38±1.83, and 51.67±1.94 and 30.26±0.16, respectively. The rate of cumulus cell expansion and polar body formation in the oocytes of three vitrified treatment groups media supplemented with vitamin E @ 50 μM, 100 μM and 200 μM were 56.39±3.49 and 34.62±1.83, 69.95±3.20 and 56.23±1.61, and 54.44±4.73 and 31.62±4.50% respectively. Mean percentage of both cumulus cell expansion and polar body formation in the non-vitrified control group were significantly higher than that in the vitrified control group. In the treatment group supplemented with vitamin E @ 100 μM, both cumulus cells expansion and polar body formation were significantly higher than that in media supplemented with 50 μM and 200 μM vitamin E as well as in vitrified control group.

Embryonic survival, development and cryoinjury of repeatedly vitrified mouse preimplantation embryos

ObjectiveThe aim of this study is to investigate the embryonic survival, development, and expressions of cryoinjury- or antioxidant-related genes in once, twice, or three-time vitrified mouse preimplantation embryos. Study desisgn: Six hundred 8-cell stage embryos were obtained from 60 female mice and randomly assigned to control and three experimental groups. Embryos were vitrified by indirect methods The developmental outcomes such as survival rate, blastocyst-forming rate, and the percentage of hatching/hatched blastocyst were assessed. The cell numbers of hatching/hatched blastocyst were counted after nuclear staining. From hatching/hatched blastocysts, the mRNA expressions for Cirbp, Casp3, Sod1, Gpx3, and Cat were quantified by real-time quantitative RT-PCR. ResultsIn once, twice, or three-time vitrified mouse 8-cell stage embryos, survival rates, blastocyst-forming rates, the percentages of hatching/hatched blastocyst, and the cell counts were all similar when compared with non-vitrified control group. The mRNA expression levels of Cirbp, Casp3, Sod1, Gpx3 and Cat were not affected. ConclusionRepeatedly vitrified mouse 8-cell stage embryos well developed up to blastocyst stage without cryoinjury and without decrease of antioxidant-related genes.

Open pulled straw vitrification of in vitro matured sheep oocytes using different cryoprotectants

The aim of this study was to compare the survival and in vitro development of sheep oocytes after open pulled straw vitrification and different final concentrations of permeable cryoprotectants. In 5 identical replicates of two experiments, in vitro matured (IVM) oocytes were vitrified by the Open Pulled Straw (OPS) method, then warmed, and the surviving ones were subjected to parthenogenetic activation. In Experiment 1, survival rate of oocytes after vitrification in 33% ethylene glycol was higher than in 33% DMSO or a mixture of 17.5% ethylene glycol and 17.5% DMSO (87.64 vs. 77.43 vs. 69.39%, respectively). The cleavage and blastocyst rates were higher after vitrification in mixture group than in ethylene glycol and DMSO (46.81 and 15.5 vs. 37.55 and 9.12 vs. 29.51 and 6.40%, for cleavage and blastocyst rates in different groups, respectively). In Experiment 2, elevated concentrations of vitrification solutions were used. The survival rate was higher after vitrification in 40% ethylene glycol and in the mixture of 20% ethylene glycol and 20% DMSO than in 40% DMSO (90.22 vs. 87.56 vs. 75.34%, respectively). Cleavage and blastocyst rates were also higher in the ethylene glycol and ethylene glycol – DMSO mixture group than in DMSO alone group (50.67 and17.60 vs. 49.13 and 14.45 vs. 33.86 and 9.81% for cleavage and blastocyst rates in different groups, respectively). The survival rates between the two experimental groups was higher in 40% ethylene glycol group, 40% mixture group and 33% ethylene glycol group than in 40% DMSO group, 33% mixture group and 33% DMSO group. Cleavage and blastocyst rates were higher in 40% ethylene glycol group, 40% mixture group and 33% mixture group than in 40% DMSO group, 33% ethylene glycol group and 33% DMSO group. All cleavage and blastocyst rates in both the experiments were lower than those of the non-vitrified control group (87.00 and 45.00, respectively). In conclusion, although ethylene glycol group and ethylene glycol – DMSO mixture group gave better survival and cleavage – blastocyst rates than DMSO group, the survival rates were lower than the control group and hence the technique could be further improved to get better results after OPS vitrification of IVM sheep oocytes.

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.

Effect of cryotop vitrification on preimplantation developmental competence of murine morula and blastocyst stage embryos

Vitrification is an effective method for the cryopreservation of mammalian embryos. Nevertheless, it is unclear which embryonic developmental stage is the most suited for vitrification and would ensure maximal developmental competence upon subsequent warming. This study, therefore, compared the effects of cryotop vitrification on the developmental competence of murine morula and blastocyst stage embryos. Additionally, trophectoderm (TE) and inner cell mass (ICM) cell numbers were compared in two hatched blastocyst groups derived from vitrified morulae and blastocysts, respectively. The post-vitrification survival rates for mouse embryos at the morula and blastocyst stage were 95.4% (186/195) and 96.5% (195/202), respectively. The blastocyst formation rate was significantly lower for vitrified morulae (90.3%) compared with the non-vitrified control group (98.4%) (P < 0.05). The hatching rates were similar between the vitrified morula (79.6%) and the vitrified blastocyst (81.0%) groups. When further development to the fully hatched blastocyst stage was compared, fully hatched blastocysts derived from vitrified morulae had significantly higher cell counts for both the ICM and TE lineage, as compared with hatched blastocysts derived from vitrified blastocysts (P < 0.001). Cryotop vitrification of mouse embryos at the morula stage rather than blastocyst stage would thus ensure a higher degree of post-warming developmental competence.

Calves Born after Direct Transfer of Vitrified Bovine In Vitro‐produced Blastocysts Derived from Vitrified Immature Oocytes

Vitrification has been the method of choice for the cryopreservation of bovine oocytes, as rapid cooling decreases chilling sensitivity. The aim of this study was to determine the in vitro and in vivo survival and the viability of immature oocytes vitrified using super-cooled liquid nitrogen. Immature oocytes were randomly allocated to three groups: (i) non-vitrified control group, (ii) vitrified in normal (-196 degrees C) liquid nitrogen (LN(2)) and (iii) vitrified in super-cooled LN(2) (< or =-200 degrees C). Open-pulled glass micropipettes were used as vitrification containers. Immature oocytes were in vitro-matured, fertilized and cultured to the blastocyst stage. In vitro viability was assessed by cleavage and blastocyst rates on days 2 and 7 of culture respectively. Vitrified blastocysts derived from the immature vitrified oocytes were directly transferred to synchronous recipients. The in vitro embryo development of vitrified immature oocytes was not influenced by the LN(2) state. After direct transfer (one embryo per recipient) of 16 embryos obtained from immature vitrified oocytes (eight from each vitrified group), two healthy calves were born in each group. These results indicated that vitrification of immature bovine oocytes using glass micropipettes under normal or super-cooled LN(2), resulted in viable blastocysts and live calves following in vitro embryo production.

The CryoLoop facilitates re-vitrification of embryos at four successive stages of development without impairing embryo growth

Vitrification has been shown to be an effective method of cryopreservation, but little is known about re-vitrification of embryos. This study investigated the effect of re-vitrification on mouse embryo preimplantation development and viability post-transfer. Mouse embryos at the 1-cell stage were vitrified using the CryoLoop technique. Embryos were warmed and then re-vitrified successively at the 2-, 8-cell and blastocyst stages. The effects of multiple rounds of vitrification on development, differentiation and viability were assessed and compared with non-vitrified embryos. Development to the 8-cell stage on day 3 and blastocyst on day 5 were not affected by re-vitrification. However, better hatching rates were observed in the non-vitrified control group. Total cell number and the number of cells allocated to the inner cell mass (ICM) were not different between treatments. The percentage of ICM development was also not different between treatments. Implantation rate and fetal weights were the same between treatments. However, overall there were fewer fetuses per embryo transferred in the re-vitrified group. Re-vitrification of mouse embryos has minimal effect on preimplantation embryo development or implantation potential.

116 EFFECTS OF l-GLUTAMINE ON CRYOPRESERVATION OF IMMATURE BOVINE OOCYTES

The cryopreservation of bovine oocytes remains a challenge despite significant reported progress. Immature bovine oocytes have a complex structure and the conventional cryoprotectants (penetrating cryoprotectants, sugars, and macromolecules) appear to be not sufficient to preserve them efficiently during freezing. Studies on semen and fibroblast cryopreservation indicate that amino acids, particularly l-glutamine, protect enzymes during freezing and increase the post-thaw viability. Therefore, the amino acids may optimize oocyte cryopreservation when associated with conventional cryoprotectants. This work evaluated the effect of l-glutamine on cryopreservation of immature bovine oocytes after in vitro maturation. Oocytes with homogeneous cytoplasm and several cumulus cell layers from slaughterhouse ovaries were distributed randomly in three groups: non-vitrified control, vitrified control, and vitrified with l-glutamine. Oocytes from vitrified groups were exposed for 10 min to PBS + 10% FCS + 10% ethylene glycol (EG) + 0.25 m trehalose (T), and for 30 s to PBS + 10% FCS + 25% EG + 25% dimethylsulfoxide + 0.5 m T at room temperature, adding 80 mm l-glutamine for the third group. Oocytes were loaded into OPS and plunged in liquid nitrogen. For thawing, OPS were immersed in PBS + 10% FCS + 10% EG + 1 m T for three min. Oocytes werethen placed in PBS + 10% FCS + 0.5 m T and in PBS + 10% FCS, remaining three min in each solution. For in vitro maturation, oocytes were washed three times on holding medium (TCM-HEPES + FCS + pyruvate + gentamycin), washed three times in maturation medium (TCM-bicarbonate + FCS + pyruvate + gentamycin + hCG + FSH + estradiol), and cultured in microdrops (90 μL) of maturation medium covered with mineral oil at 38.5°C under 5% CO2 in air and high humidity for 24 h. Oocytes were denuded, fixed in paraformaldehyde and triton, stained with Hoechst 33342, and evaluated under epifluorescence microscopy. Oocytes at metaphase II were considered matured. The group vitrified with l-glutamine had a significantly higher maturation rate than the group vitrified without l-glutamine; however, both had significantly lower maturation rates than the non-vitrified control group. In conclusion, l-glutamine improved the viability of vitrified oocytes. Table 1. Oocyte maturation rates of non-vitrified control, vitrified control, and vitrified with glutamine groups This work was supported by FAPESP 03/08543-1.

Factors affecting cryosurvival of nuclear-transferred bovine and swamp buffalo blastocysts: effects of hatching stage, linoleic acid–albumin in IVC medium and Ficoll supplementation to vitrification solution

The objective was to determine whether the hatching stage of cattle and swamp buffalo somatic cell nuclear-transferred (SCNT) blastocysts affected cryosurvival after vitrification, and whether addition of linoleic acid–albumin (LAA) to the IVC medium and Ficoll to the vitrification solution improves cryosurvival. Fused couplets were activated with ethanol and cycloheximide-cytochalasin D (day 0), and were allowed to develop in the presence of 0.3% BSA or 0.1% LAA + 0.2% BSA. Hatching blastocysts were harvested at day 7.0 (cattle) or day 6.5 (buffalo), and classified into one of three categories, according to the ratio of extruding embryonic diameter from zona to embryonic diameter inside the zona. The blastocysts were vitrified in 20% DMSO + 20% ethylene glycol + 0.5 M sucrose, with or without 10% Ficoll in TCM199 + 20% FBS, using Cryotop as a cryodevice. The post-thaw survival of the blastocysts was assessed by in vitro culture for 24 h. In cattle, when the LAA-supplemented IVC medium and the Ficoll-free vitrification solution were used, cryosurvival of the early-hatching blastocysts (77%) was not different from those of middle- and late-hatching blastocysts (74 and 80%, respectively). Inclusion of Ficoll in the vitrification solution did not improve the cryosurvival of SCNT blastocysts (54 to 68%). Early-hatching SCNT blastocysts produced in the absence of LAA were sensitive to the vitrification procedure (cryosurvival 56%; P < 0.05 versus 80% in the late-hatching blastocysts). The full-term developmental potential of SCNT blastocysts was proven only in the non-vitrified control group. In buffalo, the mean cryosurvival of hatching SCNT blastocysts produced with LAA (89%) was not different from that of those produced without LAA (87%). In conclusion, bovine SCNT blastocysts, regardless of their hatching stage, were relatively resistant to vitrification by the ultra-rapid cooling procedure when the blastocysts were produced in the presence of LAA. Furthermore, swamp buffalo SCNT blastocysts were more tolerant of vitrification than bovine SCNT blastocysts.

Vitrification of goat, sheep, and cattle skin samples from whole ear extirpated after death and maintained at different storage times and temperatures

Proper tissue preservation from a wide range of animals of different species is of paramount importance, as these tissue samples could be used to reintroduce lost genes back into the breeding pool by somatic cloning. We aim to study the temporal and thermal post-mortem limits, tested in rabbits and pigs, within which there will be guarantees of obtaining living skin cells in goat, sheep, and cattle. We also intend to study the effect of vitrification on the ability of ear skin cells, stored at different times and temperatures, to attach to the substratum and grow in vitro after warming. Ears were stored either at 4 °C for 12, 252, and 348 h post-mortem (hpm), or at room temperature (22–25 °C) for 60 and 96 hpm. In all cases, skin samples from these ears were sorted into two groups: one group was in vitro cultured immediately after storage, and the other group was vitrified after storage and further in vitro cultured. In goat and sheep, no differences in attachment (100%: goat; 90–100%: sheep) or subconfluence (75–100%: goat; 70–100%: sheep) rates were observed between experimental groups. However, in days of culture to reach subconfluence, significant differences between non-vitrified and vitrified groups were observed when ears were stored at 4 °C for 12 and 252 hpm. In cattle, with respect to attachment rate, vitrified samples from ears stored at 22–25 °C for 60 hpm were different from non-vitrified control group (60 vs. 100%, respectively; P < 0.05). Also, days of culture to reach subconfluence were analysed by a non-parametric Cox Survival Analysis. In general, results from ANOVA and Survival Analysis were similar, because the proportion of censored data was quite low (9%), so the bias when using ANOVA is not too high. In spite of all the above, the lowest survival rates (75%: goat; 70%: sheep; and 40%: cattle) were sufficiently high to enable collection of skin samples from the majority of dead animals and their cryopreservation.

Successful production of blastocysts following ultrarapid vitrification with step-wise equilibriation of germinal vesicle-stage mouse oocytes.

The purpose of this study was to evaluate the viability and subsequent developmental ability of murine germinal vesicle (GV) oocytes ultrarapidly vitrified after step-wise exposure to cryoprotectants (CPAs). Oocytes were transferred to a vitrification solution composed of 15% ethylene glycol, 15% dimethyl sulfoxide and 0.5 M sucrose in a direct manner (non-preequilibrium) or in a step-wise manner (single-, two-, or ten-step preequilibrium). After ultrarapid vitrification and storage in liquid nitrogen, the oocytes were thawed, washed by diluting the CPAs in five steps, and then subjected to in vitro maturation, fertilization and culture. In the non-preequilibrium group, the rates of post-thawed oocytes surviving, maturing to metaphase-II, developing to blastocysts and to hatching/hatched blastocysts were 91.8, 87.1, 15.9 and 2.3%, respectively. In the single- and two-step groups, the corresponding rates were 97.0-98.2%, 92.2-95.0%, 22.0-29.4% and 8.8-15.6%, whereas in the ten-step group they were 98.2, 91.8, 38.6 and 22.8%, respectively. In the non-vitrified control group, the rates of oocytes maturing to metaphase-II, developing to blastocysts and to hatching/hatched blastocysts were 90.2, 75.2 and 51.5%, respectively. The present study shows that the ultrarapid vitrification of murine GV oocytes by a step-wise manner involving 10 steps preequilibrium may have an advantage in maintaining the viability and subsequent production of blastocysts.