Lipid content and cryotolerance of porcine embryos cultured with phenazine ethosulfate

The addition of phenazine ethosulfate (PES) to culture medium was investigated for its effect on pig embryo development, apoptosis, cytoplasmic lipid content and survival after OPS vitrification. Porcine zygotes were cultured in NCSU-23 medium supplemented with 0 (control) or 0.05 microM PES up to the blastocyst stage and were vitrified using OPS technology. Culture of embryos with PES reduced the cytoplasmic lipid content, as measured by fluorescence of blastocysts stained with Nile Red. The survival rate of vitrified blastocysts was slightly enhanced, although not significantly, in the presence of PES compared to the PES-free group (45.2 and 37.9 percent, respectively). These results showed that culturing porcine embryos in medium with PES increased the proportion of morula and blastocyst formation and reduced the index of DNA fragmentation and the cytoplasmic lipid content of cultured blastocysts. However, the use of PES during in vitro culture had limited effect on porcine blastocyst survival after vitrification.     

Histone deacetyltransferase1 expression in mouse oocyte and their in vitro-fertilized embryo: effect of oocyte vitrification

This study was conducted to investigate the expression of Histone Deacetyltransferase1 (HDAC1) in mouse embryos derived from the vitrified-warmed oocytes. Firstly, the mouse oocytes at metaphaseII (MII) stage were randomly allocated into three groups: A untreated (control), B exposed to vitri?cation solution (VS) without being plunged into liquid nitrogen (toxicity), or C vitri?ed by open-pulled straw (OPS) method (vitri?cation). After warming, they were fertilized in vitro. Fresh oocytes were used as control. Expression of HDAC1 was then examined in MII mouse oocytes and embryos by immunofluorescence with anti-HDAC1 polyclonal antibody and fluorescein isothiocyanate-conjugated goat anti-rabbit IgG. Results showed that after in vitro fertilization (IVF), developmental rates to two-cell embryos (39%), 4-cell embryos (35%), morula (32%) and blastocysts (26%) in cryopreserved oocytes were all significantly lower than those of fresh oocytes (P < 0.01). In addition, HDAC1 expression in the vitrified group was significantly lower (P< 0.05) than that in the control and toxicity groups at all developmental stages except for the blastocyst. Moreover, the vitrified-warmed oocytes showed significantly lower (P < 0.05) HDAC1 expression compared with that of control and toxicity groups. In conclusion, HDAC1 was expressed both in oocytes and in their in vitro-fertilized embryos. This decreased expression of HDAC1 in mouse oocytes and the embryos due to the cryopreservation may have a negative impact on embryo development.     

Effect of cytochalasin B pre-treatment of in vitro matured porcine oocytes before vitrification

The study tested the efficacy of pre-treating mature porcine oocytes with cytochalasin B before vitrification by the open pulled straw method (OPS) in a low toxicity solution containing ice blockers. The effects of pre-treating the oocytes with 7.5 micrograms per ml cytochalasin B before vitrification on membrane integrity, chromosome organisation and cortical granule distribution were evaluated. When oocytes pre-treated with cytochalasin B before vitrification were compared with control oocytes, similar membrane integrity was observed. In contrast, when both vitrified oocytes groups (treated and untreated with cytochalasin B) were compared with fresh oocytes, significantly lower proportions of oocytes with normal chromosomes aligned regularly on the metaphase plate and peripheral cortical granule distribution were observed. The percentages of oocytes with normal chromosomes aligned regularly on the metaphase plate were similar between those treated or untreated with cytochalasin B before vitrification. Similar results were found for normal cortical granules distribution. Irrespective of previous cytochalasin B exposure, vitrification gave rise to higher abnormal cortical granule distribution percentages. Cytochalasin B pretreatment of oocytes before vitrification does not help to reduce the damage induced by the cryopreservation process of porcine oocytes.     

In vitro and in vivo survival of mouse blastocysts after repeated vitrification with the open pulled straw (OPS) method

This study addresses the in vitro and in vivo survival of mouse embryos repeatedly vitrified by the OPS-technique of Vajta et al. (24). Of 225 vitrified blastocysts, after warming one third was cultured in vitro, the other two-thirds were re-vitrified. After these were warmed, the second third was put to culture. With the remaining third the procedure was repeated. Of embryos vitrified once, 97% developed to expanded blastocysts and 81% proceeded to hatching. Corresponding values for embryos re-vitrified once were 93% and 72%, respectively (P > 0.05). After another re-vitrification, expansion and hatching rates were reduced to 76% and 35%, respectively (P < 0.01). Of 10 recipients provided with 10 embryos each that had been vitrified once, 80% remained pregnant with 5.5 +/- 0.3 fetuses. Corresponding values for re-vitrified embryos were 80% and 5.0 +/- 0.3. Of all embryos transferred, 44% became vital fetuses after a single vitrification, compared to 40% after revitrification.     

Vitrification of cultured and non-cultured expanded and hatched porcine blastocysts

The aim of this experiment was to examine the survival of porcine embryos following exposure or vitrification in EFS solution. The work was carried out on cultured and non-cultured expanded and hatched blastocysts. The viability of treated embryos was assessed by their ability to develop in in vitro culture. The results showed a higher viability of cultured expanded blastocyst (35.1 percent) vitrified in EFS medium compared to those non-cultured (12.5 percent). The proportion of cultured hatched blastocyst vitrified in this medium was 13.2 percent, while none of the non-cultured hatched blastocyst survived vitrification. It is concluded that: 1. The EFS solution was more toxic to hatched blastocyst than expanded blastocyst 2. The expanded blastocyst was a more suitable developmental stage for vitrification than hatched blastocyst. Moreover, we demonstrated that the survival rate of vitrified cultured pig blastocyst was higher compared to non-cultured.     

Changes in intracellular potassium and sodium content of 2-cell mouse embryos induced by exposition to vitrification concentrations of ethylene glycol

Intracellular concentration of potassium and sodium in two-cell mouse embryos in G1/S phase after exposition to vitrification solutions containing ethylene glycol (EG) and sucrose or after incubation in Dulbecco solution were measured by electron probe microanalysis (EPMA). The embryos at room temperature were treated in 10 percent EG for 10 min, transferred into mixture of EG and 1.0 M sucrose in ratio of 3:7 (v/v) for 3 min, then to 0.5 M sucrose for 10 min followed by washing the cells with Dulbecco;s solution for 10 min prior to analysis. The cytoplasmic concentration of potassium and sodium in controlled untreated with EG embryos were in a range of 116-130 mM of potassium and 120 mM of sodium, with good concordance in two identical experiments. After exposition that mimicked vitrification protocols, the intracellular potassium dropped almost two-three-fold (47 + 3 mM in one experiment and to 70 mM in the second experiment. The intracellular sodium concentration also decreased two-fold in range 60-70 mM after treatment with EG. Possible mechanisms of changes in the intracellular elemental concentrations including the high intracellular sodium observed in intact embryos are discussed.     

Aseptic minimum volume vitrification technique for porcine parthenogenetically activated blastocyst

Minimum volume vitrification may provide extremely high cooling and warming rates if the sample and the surrounding medium contacts directly with the respective liquid nitrogen and warming medium. However, this direct contact may result in microbial contamination. In this work, an earlier aseptic technique was applied for minimum volume vitrification. After equilibration, samples were loaded on a plastic film, immersed rapidly into factory derived, filter-sterilized liquid nitrogen, and sealed into sterile, pre-cooled straws. At warming, the straw was cut, the filmstrip was immersed into a 39 degree C warming medium, and the sample was stepwise rehydrated. Cryosurvival rates of porcine blastocysts produced by parthenogenetical activation did not differ from control, vitrified blastocysts with Cryotop. This approach can be used for minimum volume vitrification methods and may be suitable to overcome the biological dangers and legal restrictions that hamper the application of open vitrification techniques.     

Pellet freezing of in vitro produced bovine embryos using dry ice

In vitro produced bovine embryos were frozen by pellet freezing or vitrification method. In the pellet freezing method, the embryos were cooled on the dry ice and then frozen as pellets. At warming, the pellets were immersed directly into 0.5 M sucrose. The survival rates of blastocysts frozen by the pellet freezing method were higher (P<0.01) in 40% ethylene glycol (EG) than those in the lower concentrations (20 and 30% EG). Higher survival rates of blastocysts frozen by the pellet freezing method were obtained but the development rates did not differ, as compared with those by the vitrification method. There were no significant differences between the pellet freezing and vitrification method in the frequencies of post-thaw survival of hatched blastocysts. These results demonstrate that the pellet freezing method using dry ice can be used successfully for the cryopreservation of blastocysts.     

Ultra rapid freezing and vitrification of human embryos derived from abnormally fertilised zygotes

The present study was undertaken to compare the developmental capacity of human embryos derived from abnormally fertilised zygotes (1 PN, > 3 PN; 16-18 hours after ICSI) cryopreserved using two techniques: ultra rapid freezing and vitrification. At 2-4 cell stage, (48 hours after ICSI), these abnormally fertilised embryos were then distributed in three groups: a) embryos that were cryopreserved by ultra rapid freezing (URF Group), b) embryos cryopreserved by vitrification (V Group) and c) embryos that were not cryopreserved (Control group). Survival rates and embryo development after 24 hours of in vitro culture (72 hours after ICSI) were compared. 42 embryos were cryopreserved by ultra rapid freezing in 0.5 mL straws, using a mixture of dimethyl sulphoxide (3M) and sucrose (0.25M) in a base solution consisting of IVF medium plus 20 percent (v/v) of Human Serum Albumin (HSA), and 24 embryos were vitrified in 0.25 ml straws, using a two step protocol with an equilibration solution consisting of 10 percent ethylene glycol (1.79 M) and 10 percent dimethyl sulphoxide (1.41 M) in a base solution of modified phosphate buffered saline (PBS) with 20 percent of HSA and a vitrification solution consisting of 20 percent ethylene glycol (3.58 M), 20 percent dimethyl sulphoxide (2.82 M) and 0.5 M sucrose in base solution. The recovery rate after thawing/warming was lower for the vitrification group (75 percent V; 83 percent URF). The number of embryos with less than 50 percent of intact blastomeres after cryopreservation was significantly higher for the URF group (0 percent V; 34 percent URF). After in vitro culture, the rate of embryos not cryopreserved (Control group) that developed in vitro (72 hours after ICSI) was the highest (86 percent), followed by group V (50 percent), while group URF was the lowest (13 percent). These differences were statistically significant. This straw method of vitrification is successful and safe.     

Sheep ovarian tissue vitrification by two different dehydration protocols and needle immersing methods

The present research investigated the effects of two vitrification methods on sheep ovarian tissue. The base medium (BM) of the vitrification solutions contains 60% HEPES tissue culture medium (HTCM), 15% ethylene glycol (EG) and 15% dimethyl sulfoxide (DMSO). Ovarian cortex pieces were dehydrated by two different regimens, the 2-step which consisted of 50% BM and a 90% solution of 0.25 mol/L sucrose in BM for 10 minutes each at 4 degree C and the 4-step method which utilized: a) 25% BM, b) 50% BM, c) 75% BM and d) a 90% solution of 0.25 mol/L sucrose in BM for 5 minutes each at 4 degree C. After warming, the proportion of intact antral follicles in the control (non-vitrified) and 2-step vitrification groups was significantly higher than in the 4-step vitrification group. The number of apoptotic follicles in the ovarian pieces was significantly different between the control and 4-step vitrification groups. These results indicated that sheep ovarian tissue vitrification by the 2-step method was simpler and more effective than the 4-step method.     

Survival of pig morula and blastocyst after exposure to vitrification media or vitrification

The aim of this experiment was to examine the survival of porcine embryos following exposure to vitrification solutions and vitrification. The work was carried out on non-cultured and cultured morulae and blastocysts. The viability of treated embryos was assessed by their ability to develop in in vitro culture. The results showed that the most detrimental step in the vitrification of pig embryos is exposing them specifically to a vitrification medium rather than the vitrification process itself. Moreover, we demonstrated the beneficial effect of culture on the viability of pig embryos after vitrification     

Cryopreservation of human ovarian tissue by direct plunging into liquid nitrogen: negative effect of disaccharides in vitrification solution

Given that it has been possible to successfully cryopreserve human ovarian tissue by direct plunging into liquid nitrogen, this study was designed to establish the future direction to be taken in this line of research. Bovine oviductal epithelial fragments (as a tissue model) and large biopsy fragments (approximately 2.0 cubic mm) of human ovarian tissue were used for cryopreservation. Two protocols were tested: with permeable cryoprotectants (dimethyl sulphoxide, propylene glycol, glycerol) + egg yolk + sucrose or trehalose + a synthetic blocker of ice nucleation, Supercool X-1000; and using only permeable cryoprotectants (glycerol and ethylene glycol) + egg yolk + Supercool X-1000. The cryopreserved tissue specimens were subsequently thawed and the cryoprotectants removed by dilution in graded sucrose solutions. Both the dynamic growth and hormonal activity of the ovarian tissue pieces, vitrified using only permeable cryoprotectants, were greater than after vitrification in a mixture of permeable cryoprotectants and sucrose. Unlike the case for other reproductive tissue (spermatozoa, oocytes, embryos), these findings suggest that the cryopreservation of ovarian tissue by direct plunging into liquid nitrogen must be achieved by vitrification using only permeable cryoprotectants and agents that prevention ice formation.     

Influence of exposure to vitrification solutions on 2-cell mouse embryos: I. Intracellular potassium and sodium content

Intracellular concentrations of potassium and sodium in two-cell mouse embryos in G1/S phase after exposure to vitrification solutions containing vitrificant agents (VFAs) ethane-1,2-diol (ethylene glycol, EG), propane-1,2-diol (propylene glycol, PG), or dimethyl sulfoxide (DMSO), and sucrose (S) after incubation in Dulbecco solution were measured by electron probe microanalysis (EPMA) as described earlier (CryoLetters, 2006, 27: 87-98). The 4-step protocol was as followed: 10% VFA for 10 min => 30% VFA + 0.7 M S for 1.5 min ==> 0.5 M S for 10 min ==> 10 min pure Dulbecco's. The cytoplasmic concentration of potassium and sodium in immediately flashed out from the oviduct embryos was in range of 120 +/- 2 mM, with good concordance with the previous data (CryoLetters, 2006, 27:87-98). Exposure in Dulbecco's for 30 min did not alter elemental composition, neither did exposure in PG or DMSO for 1.5 min. In contrast, exposure for 1.5 min in EG dropped the level of potassium to 96 +/- 2 mM while elevating level of cytoplasmic sodium to 136 +/- 3 mM. Further exposure to 30%-EG for 3 min led to a two-fold decrease of both elements (60 +/- 3 mM and 66 +/- 2 mM for K and Na, respectively).     

Cryopreservation of coconut (Cocos nucifera L.) zygotic embryos by vitrification

The present study investigates the effect of preculture conditions, vitrification and unloading solutions on survival and regeneration of coconut zygotic embryos after cryopreservation. Among the seven plant vitrification solutions tested, PVS3 was found to be the most effective for regeneration of cryopreserved embryos. The optimal protocol involved preculture of embryos for 3 days on medium with 0.6 M sucrose, PVS3 treatment for 16 h, rapid cooling and rewarming and unloading in 1.2 M sucrose liquid medium for 1.5 h. Under these conditions, 70-80 survival (corresponding to size enlargement and weight gain) was observed with cryopreserved embryos and 20-25 percent of the plants regenerated (showing normal shoot and root growth) from cryopreserved embryos were established in pots.     

Survival of bovine fibroblasts and cumulus cells after vitrification

The aim of the experiment was to investigate the effect of vitrification on viability and the cell cycle of bovine cumulus cells and fibroblasts after culture with or without serum starvation. In all vitrified-thawed bovine somatic cells, the number of samples that reached the confluence stage was high (50 to 100%). The viability of vitrified somatic cells depended on the concentration of the cells. The viability was higher for cells vitrified at the concentration of 10 x 10(6) per ml than for cells vitrified at a concentration of 1 x 10(6) per ml (p < 0.05; for cumulus cell, and fibroblast). Time of cell starving has had no impact on their susceptibility to vitrification in case of vitrified cumulus cells. Starving time caused shifts in proportions of subsequent cell cycle phases of vitrified fibroblasts and cumulus cells. In conclusion, the bovine cumulus and fibroblast cells can be cryopreserved successfully by vitrification procedure.     

Cryopreservation of embryogenic cultures from mature Quercus suber trees using vitrification

Recent progress in somatic embryogenesis from selected mature trees of Quercus suber, has led to a demand for maintenance of a large number of selected embryogenic lines. To facilitate the management of this material a protocol for the long-term storage of this germplasm should be defined. This study reports on the use of a simple vitrification procedure for the successful cryopreservation of three cork oak embryogenic lines. High embryo recovery levels (88-93 percent) were obtained by first preculturing 2-4 mg clumps of two or three globular embryos on semisolid medium containing 0.3 M sucrose for three days, followed by incubation in PVS2 vitrification solution at 0 degree C for 60 min before direct immersion in liquid nitrogen. The mean number of embryos produced per explant was significantly greater for cryostored embryos than for untreated stock cultures, but the productivity of the latter was recovered in subsequent subcultures of the material produced by cryostored embryos. The germination and plant regeneration rates achieved by cultures derived from cryostored embryos, around 60 percent, were similar to those of non-cryopreserved stock cultures.     

Vitrification of zebrafish embryo blastomeres in microvolumes

Cryopreservation of fish embryos may play an important role in biodiversity preservation and in aquaculture, but it is very difficult. In addition, the cryopreservation of fish embryo blastomeres makes conservation strategies feasible when they are used in germ-line chimaerism, including interspecific chimaerism. Fish embryo blastomere cryopreservation has been achieved by equilibrium procedures, but to our knowledge, no data on vitrification procedures are available. In the present work, zebrafish embryo blastomeres were successfully vitrified in microvolumes: a number of 0.25 microl drops, sufficient to contain all the blastomeres of an embryo at blastula stage (from 1000-cell stage to oblong stage), were placed over a 2.5 cm loop of nylon filament. In this procedure, where intracellular cryoprotectant permeation is not required, blastomeres were exposed to cryoprotectants for a maximum of 25 sec prior vitrification. The assayed cryoprotectants (ethylene glycol, propylene glycol, dimethyl sulphoxide, glycerol and methanol) are all frequently used in fish embryo and blastomere cryopreservation. Methanol was finally rejected because of the excessive concentration required for the vitrification (15M). All other cryoprotectants were prepared (individually) at 5 M in Hanks' buffered salt solution (sigma) plus 20% FBS (vitrification solutions: vs). After direct thawing in Hanks' buffered salt solution plus 20% FBS, acceptable survival rates were obtained with ethylene glycol: 82.8%, propylene glycol: 87.7%, dimethyl sulphoxide: 93.4%, and glycerol: 73.9% (p < 0.05). Dimethyl sulphoxide showed the highest blastomere survival rate and allowed the rescue of as much as 20% of the total blastomeres from each zebrafish blastula embryo.     

Vitrification-based cryopreservation of shoot-tips of Pinus kesiya Royle ex. Gord

The present investigation was aimed at developing a protocol for long-term preservation of germplasm of Pinus kesiya Royle ex. Gord. through vitrification. Some of the critical components affecting explant tolerance to cryopreservation, such as effects of preculture, vitrification solutions, exposure time to vitrification solutions, volume of vitrification solution and its toxicity, washing of vitrified tissues after thawing, were analysed. The results showed that shoot regrowth of P. kesiya shoot-tips was considerably affected when exposed to cryoprotectants for longer periods of time (longer than 10 min). Among different vitrification solutions studied, maximum survival (76 percent) of shoot-tips was achieved with mVSL (using 0.6 ml of the solution) in MS basal medium containing 4.0 mg l-1 N6-benzyladenine (BA).     

Influence of exposure to vitrification solutions on 2-cell mouse embryos: II. Osmotic effects or chemical toxicity?

In the companion paper (CryoLetters, 2007, 28:403-408), we reported effects of exposure of two-cell mouse embryos to vitrification solutions containing different vitrificants (EG, PG and DMSO) on the intracellular potassium and sodium content. We also compared exposure of 30% (v:v) ethylene glycol for 1.5 min to the similar experiments with 3-min exposure reported previously (CryoLetters, 2006, 27:87-98). In all experiments, four step protocols (2 steps of addition and 2 steps of washing) were used. Here we present mathematical modeling of the cell osmotic response using the relativistic permeability (RP) approach, which allows calculation of the osmotic curves without using simulation software but by direct calculations of the cell volume, intracellular concentration, and amount of the permeable vitrificants (Cryobiology, 2006, 53:402-3). Magnitude of the maximum cell volume excursion and other important osmotic characteristics were calculated for each step of the protocol, and the results of the mathematical modeling were superimposed onto the experimental data reported and discussed in the companion paper. The osmotic damage vs. specific chemical toxicity of the vitrificants as the major cause of the elemental disturbance of intracellular potassium and sodium content are discussed.