Conservation of somatic tissue derived from collared peccaries (<Emphasis Type="Italic">Pecari tajacu</Emphasis> Linnaeus, 1758) using direct or solid-surface vitrification techniques

Cryopreservation of somatic tissue can be applied in biodiversity conservation, especially for wild species as collared peccary. We aimed to evaluate the effect of vitrification techniques of ear tissue of collared peccary [direct vitrification in cryovials (DVC) or solid-surface vitrification (SSV)] on the layers of epidermis and dermis by conventional histology and cell ability during the in vitro culture. Thus, both the vitrification methods were able to maintain normal patterns of the epidermis as the cornea and granular layers, furthermore the intercellular space and dermal–epidermal junction of the spinous layer when compared to fresh control. Nevertheless, DVC and SSV percentage of normality decreased in the morphological integrity of cytoplasm (37.5 and 25.0%) of spinous layer, respectively, as compared to the fresh fragments (100%, p < 0.05). Moreover, other differences between the fresh control (100%) and DVC tissues were verified in the intra-epidermal cleavage of the spinous (37.5%) and basal (37.5%) layers. In general, DVC and SSV techniques were efficient for the recovery of the somatic cells according to most of the evaluated parameters for the in vitro culture (p > 0.05). In addition, only at time of 72 h (D3), in the growth curve, DVC fragments showed a reduced cell concentration than fresh control. In conclusion, SSV was found to be a more efficient method for vitrifying collared peccary skin tissue when compared to DVC. These results are relevant for the tissue cryopreservation from collared peccary and could also be useful for mammals with phylogenetic relationships.     

Combination of intracellular cryoprotectants preserves the structure and the cells proliferative capacity potential of adult collared peccary testicular tissue subjected to solid surface vitrification

The objective was to evaluate different permeating cryoprotectants to vitrify testicular tissue biopsies from adult collared peccaries. Five pairs of testicles were dissected into fragments (9 mm³) that were allocated to non-vitrified (control) and vitrified groups using a solid-surface method following exposure to different cryoprotectants (3.0 M dimethyl sulfoxide (DMSO), 3.0 M ethylene glycol (EG) or 1.5 M DMSO + 1.5 M EG). After warming, samples were evaluated for histomorphology, ultrastructure, viability, and proliferative capacity potential. The appropriate conservation of the ultrastructural organization of the seminiferous tubule in terms of lumen presence and cell junctions was only observed at the use of DMSO/EG combination. Regardless of the cryoprotectant, the vitrification effectively preserved cell nuclear visualization and condensation similarly as observed at the non-vitrified group. Moreover, DMSO/EG combination provided a better preservation of basal membranes of seminiferous tubules than DMSO (P < 0.05). The occurrence of cell swelling was more evident in the use of DMSO than EG (P < 0.05), but both isolate cryoprotectants were similar to the DMSO/EG combination. Only the DMSO/EG combination maintained the proliferative capacity potential for spermatogonia (3.69 NORs/cell) and Sertoli cell (3.19 NORs/cell) similar to controls (3.46 and 3.31 NORS/cell, respectively). Moreover, ~40% cell viability was found after vitrification independent of cryoprotectant. In conclusion, DMSO/EG in combination is better than DMSO or EG alone for SSV of testicular tissue biopsies from adult collared peccaries.     

Vitrification of goat preantral follicles enclosed in ovarian tissue by using conventional and solid-surface vitrification methods

Caprine preantral follicles within ovarian fragments were exposed to or vitrified in the presence of sucrose, dimethyl sulfoxide (DMSO), ethylene glycol (EG), or various combinations thereof. The fragments were cryopreserved by using either a conventional (CV) or a solid-surface vitrification (SSV) protocol, and the cryoprotectants were removed by equilibrating vitrified ovarian fragments in “warming solution” consisting of minimum essential medium and heat-inactivated fetal calf serum (MEM⁺) followed by washes in MEM⁺ with or without sucrose. Histological analysis of follicle integrity showed that the percentages of normal follicles in ovarian fragments vitrified in sucrose mixed with EG and/or DMSO (CV method) or mixed with EG or DMSO (SSV method) followed by washes in MEM⁺ plus sucrose were similar to those of controls (ovarian fragments fixed without previous vitrification). Unlike for MEM⁺ (supplemented or unsupplemented by sucrose) and DMSO followed by washes in the absence of sucrose, the percentages of normal follicles found after exposure to cryoprotectant did not significantly differ from that found after vitrification, indicating that follicular degeneration was attributable to a toxic effect of cryoprotectants and not to the vitrification procedure. The viability of preantral follicles after the CV and SSV procedures was investigated by using calcein-AM and the ethidium-homodimer as “live” and “dead” markers, respectively. In both tested vitrification procedures, the highest percentages of viable follicles were observed when a mixture of sucrose and EG (70.3% for CV and 72.4% for SSV) was used. Preantral follicles were also vitrified (either by CV or SSV) in sucrose and EG and then cultured for 24 h, after which their viability was compared with that of cultured fresh and uncultured vitrified follicles. The viability of these follicles was maintained after SSV, but not after CV. Thus, the viability of caprine preantral follicles can be best preserved after SSV in a mixture of sucrose and EG, followed by washes in medium containing sucrose.CAPES/Brazil supported this work. Regiane Rodrigues dos Santos is a recipient of a grant from CAPES/Brazil.     

Production of transgenic mice from vitrified pronuclear-stage embryos.

Cryopreservation of pronuclear-stage embryos would be useful for transgenic technology and genome preservation purposes. We compared a novel vitrification technique (solid surface vitrification, SSV) with another vitrification method in straws for cryosurvival and to generate transgenic progeny from cryopreserved mouse zygotes following microinjection. The SSV solution consisted of 35% ethylene glycol (EG), 5% polyvinyl-pyrrolidone (PVP), and 0.4 M trehalose in M2 supplemented with 4 mg/ml BSA; the in straw vitrification solution was 7 M EG in M2 plus BSA. In experiment I, we compared the effect of the vitrification solutions alone, without cooling. No reduction was detected in survival and cleavage rates. In experiment II, SSV yielded a significantly higher percentage of morphologically normal zygotes (96%) that also cleaved at significantly higher rates (80%) when compared to that following "in straw" vitrification (68 and 66%, respectively). Cleavage rate in the non-vitrified control group (93%) was significantly higher than that of both vitrified groups. Following embryo transfer, there was no difference in the rate of pups obtained from the SSV, "in straw" vitrified, and control groups (97/457, 21%; 15/75, 20% and 56/209, 27%, respectively). In experiment III, SSV vitrified and fresh embryos were used for pronuclear DNA injection. Survival rate of vitrified embryos after microinjection was reduced compared to nonvitrified ones (64 vs. 72%, respectively; P < 0.05); however, development to two-cell stage was not different (76 vs. 72%, respectively). Following embryo transfer of vitrified vs. fresh microinjected embryos, in both cases 10% live pups were generated, including transgenic pups. The results demonstrated that the efficiency of generating transgenic pups from SSV vitrified pronuclear zygotes is comparable to that from fresh embryos.     

Influence of vitrification techniques and solutions on the morphology and survival of preantral follicles after in vitro culture of caprine ovarian tissue

The objective was to compare the efficiency of various vitrification techniques and solutions for preserving morphology and viability of preantral caprine follicles enclosed in ovarian tissue. Fragments of ovarian cortex were cryopreserved by conventional vitrification (CV) in French straws, vitrification in macrotubes (MTV), or solid-surface vitrification (SSV). Six solutions containing 6 M ethylene glycol, with or without sucrose (SUC; 0.25 or 0.50 M) and/or 10% fetal calf serum (FCS) were tested (Experiment I). After 1 wk, samples were warmed and preantral follicles were examined histologically. To evaluate follicular viability (Experiment II), ovarian fragments were vitrified with the three techniques listed above, in a solution containing 0.25 M SUC and 10% FCS. After warming, follicles were assessed by the trypan blue dye exclusion test. In Experiment III, preantral follicles enclosed in ovarian tissue were vitrified using the protocol which yielded the highest percentage of viable preantral follicles (SSV with 0.25 M SUC and 10% SFB). After warming, the preantral follicles enclosed in ovarian tissue were cultured in vitro and then, were analyzed by histology and fluorescence microscopy (calcein-AM and ethidium homodimer-1). Every vitrification protocol significantly reduced the percentages of morphologically normal follicles relative to the control (88.0%); however, the addition of 0.25 M SUC and 10% FCS to the vitrification solution improved preservation of follicular morphology (67.4, 67.4, and 72.0% for CV, MTV, and SSV, respectively). Although follicular viability after SSV (80.7%) did not differ from that in fresh (non-vitrified) ovarian tissues (88.0%), after in vitro culture, percentages of viable follicles were significantly reduced (70.0%). Percentages of morphologically normal follicles after in vitro culture of vitrified ovarian tissue were similar (76.0%) to those in ovarian cortex fragments cultured without previous vitrification (83.2%). In conclusion, SSV using a solution containing 0.25 M SUC and 10% FCS, was the most efficient method for vitrifying caprine ovarian tissue.     

Alginate beads as a tool to handle,cryopreserve and culture isolated human primordial/primary follicles

Background

One major concern of grafting cryopreserved ovarian tissue to restore fertility in cancer patients is the possibility of reintroducing tumor cells. Cryopreservation of isolated primordial/primary follicles (PFs) may circumvent this problem. The aim of our work was to compare dimethyl sulfoxide (ME₂SO) and ethylene glycol (EG) as cryoprotectants (CPAs) for slow-freezing of isolated human PFs in alginate.

Methods

Ovarian biopsies from four women were processed for follicle isolation. PFs were embedded in alginate (5–15 per group). Follicles were frozen-thawed using 1.4 M ME₂SO or 1.5 M EG as CPAs. Fresh and cryopreserved isolated follicles were in vitro cultured (IVC) for 7 days. At different time periods (after isolation, cryopreservation and IVC), follicles were evaluated with live/dead assay (using fluorescent probes) and diameter measurement. Follicle viability was calculated according to the percentage of dead follicular cells and the presence of a live/dead oocyte.

Results

A total of 841 PFs were isolated, embedded in alginate and cryopreserved with ME₂SO (n = 424) or EG (n = 259), or used as controls (n = 158). After 7 days of IVC, a significant increase in follicle size was observed in the fresh and ME₂SO groups, but not in the EG group. The percentage of totally viable PFs was not significantly different before or after seven days of culture in fresh (100% and 82%) or ME₂SO (93.2% and 85.1%) tissue. The EG group showed significantly lower viability before (63.9%) and after IVC (66.2%) than the fresh and ME₂SO groups.

Conclusions

Our results show that 1.4 M ME₂SO yields better preservation of isolated PF viability after thawing and 7 days of IVC than 1.5 M EG. Alginate constitutes an easy, safe hydrogel matrix to handle and cryopreserve isolated human follicles using ME₂SO as a CPA.     

Vitrification of domestic cat (Felis catus) ovarian tissue: Effects of three different sugars

We aimed to evaluate the effect of three extracellular cryoprotectants on the morphology of vitrified feline preantral follicles. Feline ovarian fragments (0.5 × 2 × 2 mm) collected from five domestic adult cats subjected to ovariohysterectomy for routine castration were vitrified with ethylene glycol (EG) 40% combined or not with sucrose (0.1 or 0.5 M), trehalose (0.1 or 0.5 M), or raffinose (0.1 M). After vitrification using the solid-surface method and warming of the tissues, cryoprotectants were washed out of the ovarian tissues, which were fixed for histological analysis. The percentages of normal follicles were similar to the control (fresh) (62.9 ± 4.1%) only for tissues exposed and cryopreserved with EG + trehalose at concentrations of 0.1 (35.8 ± 8.3%) and 0.5 M (33.4 ± 5.4%). All the other sugars decreased the percentages of morphologically normal follicles as compared to the control group and the trehalose groups. Based on the results of the present study, we recommend the use of trehalose as the extracellular cryoprotectant for the vitrification of feline ovarian tissue.     

Cryopreservation of goat oocytes and in vivo derived 2- to 4-cell embryos using the cryoloop (CLV) and solid-surface vitrification (SSV) methods

This study evaluated the efficiency and toxicity of two cryopreservation methods, solid-surface vitrification (SSV) and cryoloop vitrification (CLV), on in vitro matured oocytes and in vivo derived early stage goat embryos. In the SSV method, oocytes were vitrified in a solution of 35% ethylene glycol (EG), 5% polyvinyl-pyrrolidone (PVP), and 0.4% trehalose. Microdrops containing the oocytes were cryopreserved by dropping them on a cold metal surface that was partially immersed in liquid nitrogen. In the cryoloop method, oocytes were transferred onto a film of the CLV solution (20% DMSO, 20% EG, 10mg/ml Ficoll and 0.65 M sucrose) suspended in the cryoloop. The cryoloop was then plunged into the liquid nitrogen. In vivo derived embryos were vitrified using the same procedures. The SSV microdrops were warmed in a solution of 0.3M trehalose and those vitrified with CLV were warmed with incubation in 0.25 and 0.125 M sucrose. Oocytes and embryos vitrified by the SSV method had a significantly lower survival rate than the control (60 and 39% versus 100%, respectively; P<0.05), while the survival rate of CLV oocytes and embryos (89 and 88%, respectively) did not differ from controls. Cleavage and blastocyst rates of the surviving vitrified oocytes (parthenogenetically activated) and embryos (cultured for 9 days) were not significantly different (P>0.05) from the control nor did they differ between vitrification methods. Embryos vitrified with the CLV method gave rise to blastocysts (2/15). Our data demonstrated that the two vitrification methods employed resulted in acceptable levels of survival and cleavage of goat oocytes and embryos.     

Successful vitrification of pronuclear-stage rabbit zygotes by minimum volume cooling procedure

Rabbit zygotes at the pronuclear-stage were cryopreserved by vitrification using a gel-loading tip (GL-tip), Cryoloop or Cryotop. In GL-tip and Cryoloop methods, zygotes were first exposed to 10% ethylene glycol (EG)+10% DMSO in TCM199+20% fetal bovine serum (FBS) for 2 min, and then equilibrated for 30 s in a vitrification solution composed of 20% EG+20% DMSO+0.6 M sucrose in TCM199+20% FBS. In Cryotop method, zygotes were first exposed to 7.5% EG+7.5% DMSO+20% FBS in TCM199 for 3 min, and then equilibrated for 1 min in a vitrification solution composed of 15% EG+15% DMSO+0.5 M sucrose+20% FBS in TCM199. In vitro culture of vitrified-warmed zygotes using GL-tip and Cryoloop resulted in low cleavage rates (2 and 5%, respectively) and no development into blastocysts. In contrast, zygotes vitrified-warmed using Cryotop exhibited higher proportions of cleavage (58%) and development into blastocysts (24%). When compacted morulae or early blastocysts were vitrified by these three procedures, 80-93% of them exhibited blastocoele expansion or zona hatching during the subsequent 48 h of culture. Use of Cryotop instead of GL-tip or Cryoloop for zygote vitrification, without changing conditions of solutions and periods for exposure, equilibration and post-warm dilution, resulted in cleavage and blastocyst development rates of 88 and 45%, respectively. A longer exposure time (10 min) of zygotes to 7.5% EG+7.5% DMSO+20% FBS in TCM199 resulted in higher proportions of zygotes cleaving (94%) and developing into blastocysts (51%) after Cryotop vitrification. Proportions of post-warm zygotes (10-min exposure group) and fresh control zygotes developing into newborn offspring were 36 and 53%, respectively. Pronuclear-stage rabbit zygotes were successfully cryopreserved by vitrification using the Cryotop method.     

Membrane permeabilization of phosphatidylcholine liposomes induced by cryopreservation and vitrification solutions

Membranes are the primary site of freezing injury during cryopreservation or vitrification of cells. Addition of cryoprotective agents (CPAs) can reduce freezing damage, but can also disturb membrane integrity causing leakage of intracellular constituents. The aim of this study was to investigate lipid-CPA interactions in a liposome model system to obtain insights in mechanisms of cellular protection and toxicity during cryopreservation or vitrification processing. Various CPAs were studied including dimethyl sulfoxide (DMSO), glycerol (GLY), ethylene glycol (EG), dimethyl formamide (DMF), and propylene glycol (PG). Protection against leakage of phosphatidylcholine liposomes encapsulated with carboxyfluorescein (CF) was studied upon CPA addition as well as after freezing-and-thawing. Molecular interactions between CPAs and phospholipid acyl chains and headgroups as well as membrane phase behavior were studied using Fourier transform infrared spectroscopy. A clear difference was observed between the effects of DMSO on PC-liposomes compared to the other CPAs tested, both for measurements on CF-retention and membrane phase behavior. All CPAs were found to inhibit membrane leakiness during freezing. However, exposure to high CPA concentrations already caused leakage before freezing, increasing in the order DMSO, EG, DMF/PG, and GLY. With DMSO, liposomes were able to withstand up to 6 M concentrations compared to only 1 M for GLY. Cholesterol addition to PC-liposomes increased membrane stability towards leakiness. DMSO was found to dehydrate the phospholipid headgroups while raising the membrane phase transition temperature, whereas the other CPAs caused an increase in the hydration level of the lipid headgroups while decreasing the membrane phase transition temperature.     

Impacts of different synthetic polymers on vitrification of ovarian tissue

Type and concentration of cryoprotective agents (CPAs) are important factors which influence the likelihood of a successful ovarian tissue vitrification outcome. In an attempt to address this factor, the present study was conducted to evaluate the impacts of different synthetic polymers (Supercool X-1000, Supercool Z-1000 and PVP K-12) on vitrification of bovine ovarian tissue. From each ovarian pair, fragments were recovered and immediately fixed for analysis (fresh control) or submitted to vitrification, either or not followed by in vitro culture for one or five days. Vitrification was performed using the ovarian tissue cryosystem (OTC) system. The ovarian tissues were intended for histological and viability analysis [Reactive oxygen species (ROS) production and degenerate cells assay (Ethidium homodimer-1)], as well as immunolocalization of AQP3 and AQP9 were measured. The results showed that during almost all the periods after warming, in treatment groups which contain polymer (X-1000, Z-1000 and PVP), the percentage of morphologically normal follicles was the highest in the X-1000 samples. Furthermore, post-thawed X-1000 group revealed stronger labeling for AQP9 in primordial and transitional follicles, when compared with others. However, morphology after cryopreservation did not correlate with follicle viability and function where the levels of degeneration and tissue damage of PVP K-12 group were lower in comparison with X-1000 group and only in PVP K-12 group, ROS level was similar to that of the fresh control group. We believe that in addition to permeating CPAs, the addition of one (Supercool X-1000) or maybe a combination (Supercool X-1000 and PVP K-12) of non-permeating polymers could be useful to improve the outcome for vitrified bovine ovarian tissue.     

Vitrification of Yunnan Yellow Cattle oocytes: work in progress

The objective of this study was to provide a simple cryopreservation method for oocytes from Yunnan Yellow Cattle and facilitate preservation efforts in this native Chinese breed, which is threatened by agricultural modernization. Cumulus-oocyte complexes (COCs) were collected from slaughterhouse ovaries and matured in vitro for 22-24 h, then selected for cryopreservation. Vitrification in open pulled straws (OPS) or in microdrops on a cooled metal surface (solid surface vitrification, SSV) was compared. The OPS vitrification solution consisted of 20% ethylene glycol (EG) and 20% DMSO. The SSV solution was a mixture of 35% EG, 5% polyvinyl-pyrrolidon (PVP) and 0.4 M trehalose. Vitrified and warmed oocytes were either fertilized in vitro or parthenogenetically activated. The rates of cleavage and development to blastocysts of fertilized oocytes following OPS versus SSV were not statistically different (38.3 and 12.5% versus 35.8 and 6.0%, respectively). The corresponding rates of parthenogenetic development to blastocysts were also not different (8.2 versus 3.5%, respectively). Development to blastocysts of non-vitrified controls following fertilization was significantly higher than that of the vitrified oocytes (22.6%, P < 0.05). These results demonstrate for the first time, that although both OPS and SSV procedures reduced embryonic development, Yunnan Yellow Cattle oocytes are capable of developing to blastocysts following cryopreservation.     

Cryopreservation of immature bovine oocytes by vitrification in straws

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

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

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

Quantitative investigations on the effects of exposure durations to the combined cryoprotective agents on mouse oocyte vitrification procedures

Vitrification by using two-step exposures to combined cryoprotective agents (CPAs) has become one of the most common methods for oocyte cryopreservation. By quantitatively examining the status of oocytes during CPA additions and dilutions, we can analyze the degree of the associated osmotic damages. The osmotic responses of mouse MII oocyte in the presence of the combined CPAs (ethylene glycol, EG, and dimethyl sulfoxide, DMSO) were recorded and analyzed. A two-parameter model was used in the curve-fitting calculation to determine the values of hydraulic conductivity (L(p)) and permeability (P(s)) to the combined CPAs at 25°C and 37°C. The effects of exposure durations and the exposure temperatures on the cryopreservation in terms of frozen-thawed cell survival rates and subsequent development were examined in a series of cryopreservation experiments. Mouse MII oocytes were exposed to pretreatment solution (PTS) and vitrification solution (VS) at specific temperatures. The PTS used in our experiment was 10% EG and 10% DMSO dissolved in modified PBS (mPBS), and the VS was EDFS30 (15% EG, 15% DMSO, 3 × 10(-3) M Ficoll, and 0.35 M sucrose in mPBS).The accumulative osmotic damage (AOD) and intracellular CPA concentrations were calculated under the different cryopreservation conditions, and for the first time, the quantitative interactions between survival rates, subsequent development rates, and values of AOD were investigated.     

Successful vitrification of mouse ovaries using less-concentrated cryoprotectants with Supercool X-1000 supplementation

The purpose of our study was to investigate the feasibility of using less-concentrated cryoprotectants supplemented with ice blocker Supercool X-1000 to vitrify ovarian tissues. Mouse ovaries were cryopreserved in different concentrations of vitrification solution alone or with Supercool X-1000, and fresh non-frozen ovaries were used as control. The proportions of morphological normality of follicles, normal GCs in follicular fluids and developing to blastocysts were higher in 12.5% ethylene glycol (EG) + 12.5% dimethylsulfoxide (DMSO) with Supercool X-1000 than those of treated in 10% EG + 10% DMSO or 15% EG + 15% DMSO alone or with Supercool X-1000. In conclusion, the inclusion of Supercool X-1000 in less-concentrated vitrification solution was effective to improve the efficiency and efficacy of cryopreservation of ovarian tissues.     

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

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

Cryopreservation of murine testicular Leydig cells by modified solid surface vitrification with supplementation of antioxidants

Reports on the vitrification of somatic cells are scarce. Here, we show that Leydig cells (murine cell line TM3) could be successfully vitrified by both open vitrification [plastic straw (PS) and plastic vials (PV)] and closed ultravitrification [microdrop (MD) and solid surface vitrification (SSV)], after protocol optimization. However, open ultravitrification resulted in better post-warming viability than closed systems of vitrification with highest success obtained in modified SSV (84.8 ± 1.86%; p < 0.05). Leydig cells vitrified-warmed by modified SSV also showed superior (p < 0.05) cell growth, mitochondrial activity and cytoplasmic esterase enzyme activity, than MD, PS and PV, respectively. It was also observed that vitrified-warmed cells had higher level of ROS activity than non-vitrified control cells (41.6 ± 4.0 vs. 16.7 ± 1.0; p < 0.05). Treatment of cells with glutathione (GSH) or 2-mercaptoethanol (2-ME) (0, 10, 50, 100 μM) significantly (p < 0.05) reduced the ROS activity but had no significant (p > 0.05) effect on post-warm viability. Nevertheless, antioxidant-treated cells had improved mitochondrial activity, cytoplasmic esterase activity and cell growth during in vitro culture (p < 0.05). In conclusion, our results suggest that modified SSV offers a viable method for vitrifying single cell suspension of Leydig cells. To the best of our knowledge, this is the first report on cryopreservation of Leydig cells by vitrification.     

Immature bovine oocyte cryopreservation: comparison of different associations with ethylene glycol, glycerol and dimethylsulfoxide

Research on different cryoprotectants and their associations is important for successful vitrification, since greater cryoprotectant concentration of vitrification solution may be toxic to oocytes. The aim of the present research was to compare the efficiency of immature bovine oocyte vitrification in different associations of ethylene glycol (EG), glycerol and dimethylsulfoxide (Me(2)SO). In the first experiment, oocytes were exposed to the cryoprotectant for either 30 or 60s in final solutions of EG+DMSO1 (20% EG+20% Me(2)SO) or EG+DMSO2 (25% EG+25% Me(2)SO) or EG+GLY (25% EG+25% glycerol). In the second experiment, the oocytes were vitrified in open pulled straws (OPS) using 30s exposure of final solutions of EG+DMSO1 or EG+DMSO2 or EG+GLY. Maturation rates of 30s exposure groups were not different from the control, but 60s cryoprotectant exposure was toxic, decreasing maturation rates. The vitrification with EG+DMSO2 resulted in enhanced maturation rate (29.2%) as compared with EG+DMSO1 (11.7%) and EG+GLY (4.3%) treatments. These data demonstrate that concentration and type of cryoprotectant have important effects on the developmental competence of vitrified oocytes.     

Vitrification of in vitro cultured rabbit morulae

In the present work, we attempt to establish an efficient vitrification procedure for 32-cell rabbit embryos obtained in vitro. In experiment 1, both the effect of the composition of the vitrification solutions and the cryoprotectant addition (either in one or two steps) were studied. For one-step addition, straws with embryos in the final vitrification solution (total time 60s) were plunged into liquid nitrogen. For two-step addition, previously embryos were 2 min pre-equilibrated in 0.5 ml of (1:1) PBS plus 20% FCS: vitrification solution without sucrose. Different solutions of cryoprotectants were compared: 25 vol.% ethylene glycol supplemented with 0.25 M sucrose (25EG+S) and 20% ethylene glycol plus 20% dimethyl sulfoxide, alone (20EG+20DMSO-S) or supplemented with 0.25 M sucrose (20EG+20DMSO+S). Six percent (30/487) of the total of 32-cell embryos obtained by in vitro culture in each experimental session was slow-frozen by a classical method as a technical efficiency control. Only 30% slow-frozen embryos reached blastocyst stage. Significant differences in embryo development were detected between the one-step (25EG+S) and two-step (25EG+S) groups and the one-step (20EG-20DMSO+S) and two-step (20EG-20DMSO-S) groups (0-6% versus 36-50%, respectively). Consequently, in the following experiments only these two vitrification procedures were used. In experiment 2, we attempted to substitute the use of PBS by HEPES-buffered Ham's F-10 (h-CM) in all cryoprotective solutions or media. When h-CM was used, a significant reduction in the in vitro embryo development was observed when the HEPES-buffered groups were compared with one-step (20EG-20DMSO+S) group in s-PBS (35-45% versus 73%). In experiment 3, the one-step (20EG+20DMSO+S) and two-step (20EG+20DMSO-S) procedures were assayed using two FCS levels (20 and 40%) in the PBS-based media. Relative to in vitro development, the highest rates were reached with one step (20EG-20DMSO+S), using PBS plus 20% FCS, which was different from two steps (20EG-20DMSO-S), regardless of percentage of FCS in the PBS-based media (81% versus 41-45%; P<0.05). In conclusion, we propose either the one step (20EG-20DMSO+S) or two steps (20EG-20DMSO-S) prepared in PBS plus 20% serum for use in future works.