Solid Surface Vitrification Method Research Articles

Boric Acid Improved Cryopreserved Mouse Embryo Development.

Boric acid (BA) is an essential trace element that is required to support the metabolic pathways in plants, humans, and animals. The present study investigates the in vitro development and quality of single-cell mouse embryos in a BA-added culture medium after cryopreservation using the solid-surface vitrification method. For this purpose, the pronuclear-stage embryos derived from superovulated C57Bl/6j mouse strains and the one-cell embryos were then cryopreserved using the solid-surface vitrification (SSV) method. After thawing, the embryos were cultured in a BA-added medium at 37 °C in a 5% CO2 environment until the blastocyst stage. The resulting in vitro development rates of the embryos in the control group, SSV group, and SSV + 1.62 × 10-4 μM BA group were 68.11% (36/59), 40.16% (16/48), and 64.92% (28/48) respectively, indicating that the BA supported the in vitro development of the embryos cryopreserved using the SSV method. Our results suggest that the addition of boric acid to the culture media increased the development rate of the embryos that were vitrified using the SSV method.

Microbiological load and preantral follicle preservation using different systems for ovarian tissue vitrification in the red-rumped agouti

We evaluated the effect of open and closed systems used for ovarian tissue vitrification on the microbiological load and preservation of preantral follicles (PAFs) in the red-rumped agoutis. The ovaries from eight females were recovered and fragmented, with four cortexes fragments immediately fixed and evaluated (fresh group). The other fragments were processed for the solid-surface vitrification method (SSV) or an ovarian tissue cryosystem (OTC) using fetal calf serum, ethylene glycol, and sucrose as cryoprotectants, stored for two weeks, and rewarmed. Subsequently, fragments were subjected to a 24-h in vitro culture and assessed for microbiological load, PAF morphology, and DNA integrity. There was no fungal contamination; however, the vitrified samples from two individuals showed bacterial contamination of 79 200 colony forming units per milliliter (CFU)/mL for SSV and 3120 CFU/mL for OTC. From those samples, a total of eight different types of bacterial colonies were isolated and identified as coagulase-negative Staphylococci and Gram-positive bacilli. Regarding PAF morphology, both systems provided adequate preservation, with values higher than 70% normal follicles observed before and after culture. The TUNEL assay revealed that both SSV (52.39%) and OTC (41.67%) could preserve DNA integrity after vitrification and after 24 h of culture. In summary, both open and closed systems were equally efficient in preserving agouti ovarian tissues, especially concerning the preantral follicle morphology and DNA integrity; however, the OTC seems to provide a less adequate environment for bacterial proliferation.

Vitrification of collared peccary ovarian tissue using open or closed systems and different intracellular cryoprotectants

This study aimed to evaluate different vitrification methods using distinct cryoprotectants (CPAs) for the preservation of collared peccary ovarian preantral follicles (PFs). Ovarian pairs from six females were fragmented and three fragments (fresh control group) were immediately evaluated for morphology, viability, cell proliferation capacity (assessed by quantifying the number of argyrophilic nucleolus organizer regions – NORs), and apoptosis (by the identification of activated caspase-3 expression). The remaining 18 fragments were vitrified using the solid surface vitrification (SSV) method or the ovarian tissue cryosystem (OTC) with 3 M ethylene glycol (EG), 3 M dimethylsulfoxide (DMSO), or a combination of the two (1.5 M EG/1.5 M DMSO). After two weeks, samples were rewarmed and evaluated as described previously. The OTC with any of the CPAs provided a similar conservation of morphologically normal PFs as the fresh control group (75.6 ± 8.6%); however, the SSV was only efficient with DMSO alone (63.9 ± 7.6%). Regarding the viability or cell proliferation, all tested groups provided post rewarming values similar to those observed for the fresh control group, 84.0 ± 2.9% viable cells with 2.0 ± 0.2 NORs. Related to apoptosis analysis, only the OTC with EG (46.7%) and the SSV method with EG (43.4%) or the combination of EG and DMSO (33.4%) provided similar values to those found for the fresh control group (36.7%). Our findings indicate the utilization of a closed system, the OTC, with 3 M EG as the CPA for the vitrification of collared peccary ovarian tissue.

Effects of cryopreservation techniques on the preservation of ear skin – An alternative approach to conservation of jaguar, Panthera onca (Linnaeus, 1758)

Currently, it has been observed that a considerable segment of the jaguar population is declining mainly because of hunting, and destruction and fragmentation of habitat. Given this scenario, efforts of the scientific community have been concentrated on the development of conservation strategies, such as the formation and use of somatic sample banks. We aimed to assess the effects of cryopreservation techniques of the ear skin of jaguar [slow freezing (SF) or direct vitrification in cryovials (DVC) or solid-surface vitrification (SSV)] on the morphological analysis and cell ability during the culture. All cryopreserved fragments regardless of the technique used, showed a reduction in the dermis and total thickness of the skin. Although a collagen matrix similar to the control group (fresh) has been observed only for the fragments from SF and SSV groups, all cryopreserved techniques were able to maintain normal patterns of the fibroblasts. Moreover, DVC and SSV methods maintained the proliferative activity of the tissues even after warming. After the culture, SF and SSV techniques were efficient for the recovery of the somatic cells according to most of the evaluated parameters, especially with regard to the duration of culture and cell metabolic activity. In conclusion, SSV was found to be a more efficient technique for cryopreserving jaguar skin when compared to DVC and SF. These results are relevant for the formation of somatic resource banks of this species, directed at cryopreserving adequate samplings of different individuals and generations for future applications in regenerative medicine, and assisted reproductive technologies.

Impact of cryopreservation method on dromedary camel ovary structure, viability, and development of antral follicular oocytes

The objectives of this study were, a) to compare two different vitrification techniques, the solid surface vitrification (SSV) and direct vitrification (DV) method, b) to evaluate the effect of four cryoprotectant agents and their toxicity on the morphological appearance and ultrastructural of camel ovarian cortex, c) to examine the development of oocytes recovered from the vitrified ovarian cortex. Fragments of ovarian cortex were exposed to equilibration solution consisting of TCM- 199 with 10% fetal camel serum (FCS); 0.10M sucrose and including one of the following cryoprotectants; 20% glycerol (GLY); 3.5M ethylene glycol (EG); 3.5M propanediol (PROH) or 3M dimethylsulphoxide (DMSO). After vitrification of ovarian fragments, they were warmed and evaluated by histological and transmission electron microscope. The oocytes isolated from vitrified ovarian cortex were cultured in TCM-199 at 38.5°C under 5% CO2 for 44h. Maturation was indicated through cumulus expansion and calculated by oocytes reaching first telophase and second metaphase (TI+MII). The percentage of morphologically normal and viable follicles of SSV was significantly higher P<0.05 than DV group (52.9 vs. 38.1, respectively). In conclusion, viability, histological and ultrastructural observations revealed that SSV method and ethylene glycol-based freezing solution were able to remain morphology better follicle and oocyte. Additionally, most organelles of oocytes are able to recover their normal morphology in camel ovarian cortex following cryopreservation and thawing processes, and oocytes isolated from vitrified ovarian cortex can exhibit maturation and reaches to (TI+MII).

Successful vitrification of pronuclear-stage pig embryos with a novel cryoprotective agent, carboxylated ε-poly-L-lysine.

Vitrification is a powerful tool for the efficient production of offspring derived from cryopreserved oocytes or embryos in mammalian species including domestic animals. Genome editing technologies such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated (Cas)9 are now available even for domestic species, suggesting that the vitrification of embryos at the pronuclear stage (PN) will be more important because they could provide genomic host cells to be targeted by TALENs or CRISPR/Cas9. Although we reported the successful production of piglets derived from vitrified PN embryos by a solid-surface vitrification method with glutathione supplementation, further improvements are required. The cryoprotective agent (CPA) carboxylated ε-poly-L-lysine (COOH-PLL) was introduced in 2009. COOH-PLL reduces the physical and physiological damage caused by cryopreservation in mammalian stem cells and the vitrification of mouse oocytes and embryos. Those results suggested that vitrification of COOH-PLL may help improve the developmental ability of pig embryos vitrified at the PN stage. However, it remains unclear whether COOH-PLL is available as a CPA for the vitrification of embryos in domestic species. In this study, we evaluated COOH-PLL as a CPA with ethylene glycol (EG) and Cryotop as a device for the vitrification of PN pig embryos. Exposure to vitrification solution supplemented with COOH-PLL up to 30% did not decrease developmental ability to the 2-cell stage and the blastocyst stage. After warming, most of the vitrified embryos survived regardless of the concentration of COOH-PLL (76.0 ± 11.8% to 91.8 ± 4.6%). However, the vitrified embryos without COOH-PLL showed a lower development rate up to the blastocyst stage (1.3 ± 1.0%) compared to the fresh embryos (28.4 ± 5.0%) (p<0.05). In contrast, supplementation of 20% (w/v) COOH-PLL in the vitrification solution dramatically improved the developmental ability to blastocysts of the vitrified embryos (19.4 ± 4.6%) compared to those without COOH-PLL (p<0.05). After the transfer of embryos vitrified with 30% (v/v) EG and 20% (w/v) COOH-PLL, we successfully obtained 15 piglets from 8 recipients. Taken together, our present findings demonstrate for the first time that COOH-PLL is an effective CPA for embryo vitrification in the pig. COOH-PLL is a promising CPA for further improvements in the vitrification of oocytes and embryos in mammalian species.

Appendix B: Solid Surface Vitrification.

Solid surface vitrification method involves direct contact of carrier loaded with droplet containing gametes or embryos with precooled metal surface. Over the years, following certain modifications, solid surface vitrification has emerged as an efficient method for vitrifying human gametes and embryos. Here, we describe the principle and methodology of solid surface vitrification.

32 EFFECTS OF HIGH HYDROSTATIC PRESSURE ON EXPRESSION PROFILES OF IN VITRO-PRODUCED, VITRIFIED BOVINE BLASTOCYSTS

High hydrostatic pressure (HHP) has been used to enhance stress tolerance and to promote embryo survival before they are subjected to insulting procedures such as cryopreservation. However, the molecular mechanisms of the beneficial effects of HHP are poorly understood. Here in vitro-produced bovine blastocysts were treated with 40, 60, and 80 MPa of HHP for 1 h at either 25 or 37°C, followed by 3 different recovery periods (0, 1, and 2 h) after HHP before vitrification by the solid surface vitrification method (Dinnyes et al. 2000). The re-expansion rates after vitrification-warming were significantly (P &lt; 0.05) higher in embryos treated with 40 or 60 MPa than controls, demonstrating that HHP promotes the in vitro developmental competence of vitrified bovine embryos. However, 80 MPa resulted in significantly reduced re-expansion rates, suggesting that this pressure started to be lethal to bovine blastocysts. In addition, no significant difference was found on re-expansion rates between 25 and 37°C; data were therefore combined for the 2 temperatures. Microarray analysis revealed a total of 399 differentially expressed transcripts, representing 254 unique genes, among different treatment groups. Gene ontology analysis revealed that HHP at 40 and 60 MPa promoted embryo competence through down-regulation of genes involved in cell death and apoptosis, and up-regulation of RNA processing, cellular growth, and proliferation. Moreover, gene expression was also changed by the length of the recovery time after HHP. The significantly over-represented groups are apoptosis and cell death in the 1-h group, and protein folding, response to unfolded protein, and cell cycle in the 2-h group. Although 80 MPa also up-regulated expression of genes for apoptosis, but it also significantly down-regulated genes for protein folding and cell cycle, which may explain why these embryos stopped developing. Taken together, these data suggest that HHP induces specific responses in vitrified bovine blastocysts and promotes their developmental competence through modest transcriptional reprogramming.

Cytochalasin B efficiency in the cryopreservation of immature bovine oocytes by Cryotop and solid surface vitrification methods

The present study was undertaken to compare the efficacies of Cryotop (CT), solid surface vitrification (SSV) methods and cytochalasin B (CB) treatment for the cryopreservation of immature bovine oocytes, in terms of survival, nuclear maturation, and in vitro development. Solution exposed oocytes were in vitro maturated and fertilized. No difference was found in the rates of survival, nuclear maturation and blastocyst among solution exposed groups and fresh control group, except blastocysts rates in oocytes exposed to CB, cryoprotectant (CPA) and fluorescein diacetate (FDA) group (CB–CPA–FDA) (23%) significantly lower than that of control group (32%). CB pretreated ((+)CB) or non-pretreated ((−)CB) COCs were vitrified either by SSV or CT. Among four vitrified groups the nuclear maturation rates (CT(−)CB: 58%, CT(+)CB: 57%, SSV(−)CB: 60%, SSV(+)CB: 63%), cleavage (CT(−)CB: 36%, CT(+)CB: 24%, SSV(−)CB: 34%, SSV(+)CB: 26%) and blastocysts rates (CT(−)CB: 6%, CT(+)CB: 7%, SSV(−)CB: 4%, SSV(+)CB: 6%) did not differ, but the rates of the four vitrified groups were significantly lower than those of non-vitrified group (81%, 71% and 26%, respectively). We thus conclude that CT and SSV perform equally in vitrification of bovine immature oocytes, and CB did not increase the viability, nuclear maturation, or in vitro development of vitrified oocytes.

Forskolin improves the cryosurvival of in vivo-derived porcine embryos at very early stages using two vitrification methods

This study was aimed to determine the effect of forskolin on the viability of in vivo-derived porcine embryos vitrified by the superfine open pulled straw (SOPS) or solid surface vitrification (SSV) methods at the 2-cell, 4-cell, and blastocyst stages. Zygotes, 2- to 4-cell embryos, and morulae were obtained from superovulated sows. After collection, embryos were cultured for 24h with 0 or 10μM forskolin and then vitrified using the SOPS and SSV method, or not vitrified (fresh controls). Fresh and vitrified-warmed 2-cells, 4-cells, and blastocysts were cultured for additional 96h, 72h and 24h, respectively. At the end of the culture, embryos were evaluated for progression to the blastocyst stage and total cell number. The vitrification method did not affect any of the parameters evaluated for any embryo stage. Forskolin increased (P<0.01) the blastocyst formation and the final developmental stage of vitrified 2- and 4-cell embryos. However, these embryos exhibited lower (P<0.003) blastocyst formation rates than their fresh counterparts. The total cell number and hatching rate were similar in both groups (vitrified and fresh) of 2- and 4-cell embryos. Vitrified blastocysts exhibited viabilities, final developmental stages, hatching rates, and total cell numbers that were similar to those of their fresh counterparts, regardless of the addition of forskolin. In conclusion, the SOPS and SSV methods are suitable for the cryopreservation of in vivo-derived 2- to 4-cell porcine embryos. Pre-treatment with forskolin for 24h before vitrification improves the cryotolerance of 2- and 4-cell porcine embryos.

Cryopreservation of immature buffalo oocytes: Effects of cytochalasin B pretreatment on the efficiency of cryotop and solid surface vitrification methods

The aim of the present study was to compare the efficiency of the solid surface (SSV), cryotop (CT) vitrification methods and cytochalasin B (CB) pretreatment for cryopreservation of immature buffalo oocytes. Cumulus-oocyte complexes (COCs) were placed for 1 min in TCM199 containing 10% dimethylsulfoxide (DMSO), 10% ethylene glycol (EG), and 20% fetal bovine serum, and then transferred for 30 s to base medium containing 20% DMSO, 20% EG and 0.5 mol/L sucrose. CB pretreated ((+)CB) or non-pretreated ((-)CB) COCs were vitrified either by SSV or CT. Surviving vitrified COCs were selected for in vitro maturation (IVM) and in vitro fertilization (IVF). The rate of viable oocytes after vitrification in CT groups (82%) was significantly lower (P < 0.05) than that in a fresh control group (100%), but significantly higher (P < 0.05) than those in SSV groups (71-72%). Among vitrified groups, the highest maturation rate was obtained in the CT (-)CB group (32%). After IVF, the cleavage and blastocyst formation rates were similar among vitrified groups but significantly lower than those of the control group. In conclusion, a higher survival rate of oocytes after vitrification and IVM was obtained in the CT group compared with that in the SSV group, indicating the superiority of the CT method. Pretreatment with CB did not increase the viability, maturation or embryo development of vitrified oocytes.

83 EFFECTS OF CYTOCHALASIN B AND VITRIFICATION PROCEDURE ON SURVIVAL AND IN VITRO MATURATION OF SWAMP BUFFALO OOCYTES CRYOPRESERVED AT THE IMMATURE STAGE

Cryopreservation of oocytes from swamp buffaloes of high genetic and individual values is important for conservation of animal genetic resources. This study investigated the effect of cytochalasin B (CB) pretreatment and the efficiencies of solid surface vitrification (SSV) and cryotop (CT) vitrification methods to cryopreserve immature buffalo oocytes. Cumulus oocyte complexes harvested from abattoir-derived ovaries were randomly separated into 5 groups: 1) CT method with pretreatment by 7.5 μg mL–1 CB in base medium (BM, TCM-199 HEPES+20% FBS) for 15 min [CT(+)CB]; 2) CT method without CB pretreatment [CT(–)CB]; 3) SSV method with pretreatment by 7.5 μg mL–1 CB in BM for 15 min [SSV(+)CB]; 4) SSV method without CB pretreatment [SSV(–)CB]; and 5) fresh control. Immature oocytes were partially denuded, and those with 2 layers of cumulus cells were placed in 10% dimethylsulfoxide (DMSO) + 10% ethylene glycol (EG) for 1 min followed by exposure to 20% DMSO + 20% EG + 0.5 M sucrose in BM for 30 s. The oocytes were then vitrified either by SSV or CT method and stored in liquid nitrogen. Vitrified oocytes were thawed by placing them directly in warming solution (0.5 M sucrose in BM) for 5 min at 38.5°C and then washed in BM for 5 min. Oocyte viability was assessed by fluorescein diacetate staining. Surviving oocytes were in vitro matured (IVM) for 23 h (Parnpai et al. 1999 Buffalo J. 15, 371–384) and their meiotic progression was investigated by nuclear staining. Data were analysed by 1-way ANOVA. The survival rate of oocytes in CT groups [CT(+)CB, 82%; CT(–)CB, 82%] was significantly lower (P &lt; 0.05) than that of the control group (100%), but significantly higher (P &lt; 0.05) than those in the SSV groups [SSV(+)CB, 71%; SSV(–)CB, 72%]. After IVM, the proportions of viable oocytes were similar among vitrified groups [CT(+)CB, 84%; CT(–)CB, 86%; SSV(+)CB, 80%; SSV(–)CB, 79%], but significantly lower than those of the control group (96%). Pretreatment with CB did not increase oocyte survival. Among vitrified groups the highest maturation rate was obtained in CT(–)CB (32%), which was significantly higher than that of SSV(+)CB (13%). Vitrified oocytes in all groups reached metaphase-II at significantly lower rates compared with the control group (55%). In conclusion, CB did not improve the survival and maturation of vitrified oocytes. Higher survival and maturation rates in CT groups indicate the superiority of this method. This work was supported by Thailand Research Fund, Suranaree University of Technology and Japan Society for the Promotion of Science – National Research Council of Thailand (JSPS-NRCT) scientific cooperation program.

Vitrification of bovine ovarian tissue by the solid-surface vitrification method.

Exposure time and addition of sucrose to the vitrification medium as well as the solid-surface vitrification (SSV) on the morphology of bovine preantral follicles were evaluated. Ovarian tissue was exposed for 1, 5, or 10 min to 4.0 M ethylene glycol with or without the addition of 0.5 M sucrose. Subsequently, the tissue was washed out from cryoprotectants or vitrified by the SSV method. Independently of the presence of sucrose, exposure to vitrification solution for 10 min did reduce the percentages of normal follicles when compared with control. However, the highest rates of normal follicles were attained when tissue was previously exposed to the vitrification solution, with sucrose added or not, for 10 min. Although the SSV is a promising procedure to be applied in ovarian tissue, an optimal vitrification solution for bovine ovarian tissue needs to be developed.

Comparison of Different Cryopreservation Techniques: Higher Survival and Implantation Rate of Frozen–Thawed Mouse Pronuclear Embryos in the Presence of Beta‐Mercaptoethanol in Post‐Thaw Culture

The objective of this study was to investigate the effects of beta-mercaptoethanol (β-ME) on post-thaw embryo developmental competence and implantation rate of mouse pronuclear (PN) embryos that were cryopreserved after slow freezing, solid surface vitrification (SSV) or open-pulled straw (OPS) vitrification methods. Mouse PN embryos were cryopreserved by using slow freezing, SSV and OPS methods. After cryopreservation, freeze-thawed PN embryos were cultured up to blastocyst stage in a defined medium supplemented without or with 50 μM β-ME. The blastocyst formation rate of embryos that were cryopreserved by slow freezing method (40.0%) or vitrified by OPS method (18.3%) were lower than those vitrified by SSV method (55.6%) and fresh embryos (61.9%) in the absence of 50 β-ME in the culture media (p < 0.05). The blastocyst formation rate of embryos that were cryopreserved by slow freezing method (53.1%) or by OPS method (41.9%) were lower than those vitrified by SSV method (79.5%) and that of fresh (85.7%) in the presence of β-ME in the culture media (p < 0.05). The embryos transfer results revealed that the implantation rate of blastocyst derived from mouse PN embryos vitrified by SSV method (31.9% vs 51.2%) was similar to that of the control (39.0% vs 52.5%), but higher than those cryopreserved by slow freezing (28.2% vs 52.0%) and by OPS method (0.0% vs 51.2%) (p < 0.05). In conclusion, supplementation of β-ME in an in vitro culture medium was shown to increase survival of embryo development and implantation rate of frozen-thawed mouse PN embryos after different cryopreservation protocols.

A Comparison of Cryotop and Solid Surface Vitrification Methods for the Cryopreservation of In Vitro Matured Bovine Oocytes

The aim of the present study was to compare the efficacies of the cooling systems of the solid surface (SSV) and Cryotop vitrification methods for cryopreservation of bovine oocytes at the metaphase II stage. The effects of vitrification on oocyte viability, in vitro fertilization (IVF), pronucleus formation and subsequent in vitro development were assessed. In vitro matured (IVM) bovine oocytes were subjected to equilibration and vitrification solutions according to the SSV method, and then the oocytes were vitrified either by dropping onto a cold dry metal surface (SSV group) or by plunging into liquid nitrogen on Cryotop sheets (Cryotop group). Warming was conducted according to the SSV method. Some oocytes were subjected to the cryoprotectants and warming regimen without cooling (Solution control group). The live/dead status of oocytes was evaluated by fluorescein diacetate staining. Live oocytes were subjected to IVF, and the resultant embryos were cultured in vitro. The rates of live oocytes were similar among the Fresh control, Solution control, SSV and Cryotop groups. There was no difference in the rates of fertilization, pronuclear formation and monospermy among these groups. The cleavage rates in the SSV and Cryotop groups (41.6 and 53.2%, respectively) were significantly lower than those in the Fresh control and Solution control groups (65.9 and 61.3%, respectively). The blastocyst rates in SSV and Cryotop groups did not differ (10.3 and 12.8%, respectively); however, they were significantly lower than those in the Fresh control and Solution control groups (36.4 and 24.8%, respectively). The inner cell mass, trophectoderm and total cell numbers in blastocysts did not differ significantly among the Fresh control, Solution control, SSV and Cryotop groups. Our results indicate that IVM bovine oocytes could be cryopreserved successfully using the cooling systems of the Cryotop and Solid Surface Vitrification methods with similar efficacy.

Effects of vitrification and post-thawing interval on the cytoskeleton and subsequent fertilization rate of &lt;i&gt;in vitro&lt;/i&gt; derived bovine oocytes

Vitrification may alter the cytoskeleton (microtubule, meiotic spindle, microfilament, etc.) and the subsequent fertilization rate of in vitro derived bovine oocytes. This study was conducted to evaluate the effect of vitrification and post-thawing incubation periods on the cytoskeleton and fertilization rate of in vitro matured (IVM) bovine oocytes. Following 22 h of IVM, 184 fresh matured oocytes (MO) were immediately fertilized in vitro and served as a control. The remaining MO (1009) were then vitrified by the solid surface vitrification method. Immediately after thawing, MO were incubated in maturation medium in 5% CO2 at 39 °C for 0, 30, 60, 90 and 120 min respectively. Following incubation, half of the MO from each vitrified-thawed treatment group (0, 30, 60, 90, and 120 min) was stained with fluorescein isothiocyanate conjugated (FITC) and propidium iodide (PI) to evaluate the microtubule and DNA or spindle under laser-scanning confocal microscopy. The remaining half from the vitrified-thawed MO treatment groups was washed three times in Brackett and Oliphant\'s fertilization medium and in vitro fertilized. Cleavage and blastocyst rates were recorded 48 h post-fertilization. Results demonstrated that vitrification damaged MO zona pellucida (ZP), microtubule (MT), meiotic spindle (MS), and caused chromosomal fragmentation. Both the cleavage (84%) and blastocyst rates (50%) of the control group were significantly higher compared to the vitrified-thawed treatment groups. However, extending the incubation period of vitrified MO to 120 min after thawing (prior to fertilization) improved cleavage (65%) and blastocyst (13%) rates 48 h post-fertilization. Fertilizing vitrified MO immediately (0 min group) after thawing resulted in the lowest cleavage (42%) and blastocyst (1.9%) rates. In conclusion, vitrification reduces the subsequent fertilization rate of MO, however, a prolonged post-thawing incubation period (120 min) improves survival, cleavage and blastocyst formation rates, and enhances the reorganization of MO\'s cytoskeleton (MT and MS). Keywords: Vitrification; cytoskeleton; bovine; oocytes; fertilization; in vitro matured South African Journal of Animal Science Vol. 36 (5) 2006: pp.42-45

Preliminary studies on the vitrification of red sea bream ( Pagrus major) embryos

The objective was to identify an appropriate cryoprotectant and protocol for vitrification of red sea bream ( Pagrus major) embryos. The toxicity of five single-agent cryoprotectants, dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), glycerol (GLY), and methyl alcohol (MeOH), as well as nine cryoprotectant mixtures, were investigated by comparing post-thaw hatching rates. Two vitrifying protocols, a straw method and a solid surface vitrification method (copper floating over liquid nitrogen), were evaluated on the basis of post-thaw embryo morphology. Exposure to single-agent cryoprotectants (10% concentration for 15 min) was not toxic to embryos, whereas for higher concentrations (20 and 30%) and a longer duration of exposure (30 min), DMSO and PG were better tolerated than the other cryoprotectants. Among nine cryoprotectant mixtures, the combination of 20% DMSO + 10% PG + 10% MeOH had the lowest toxicity after exposure for 10 min or 15 min. High percentages of morphologically intact embryos, 50.6 ± 16.7% (mean ± S.D.) and 77.8 ± 15.5%, were achieved by the straw vitrifying method (20.5% DMSO + 15.5% acetamide + 10% PG, thawing at 43 °C and washing in 0.5 M sucrose solution for 5 min) and by the solid surface vitrification method (40% GLY, thawing at 22 °C and washing in 0.5 M sucrose solution for 5 min). After thawing, morphological changes in the degenerated embryos included shrunken yolks and ruptured chorions. Furthermore, thawed embryos that were morphologically intact did not consistently survive incubation.

109 EFFECTS OF VITRIFICATION PROCEDURES ON SUBSEQUENT DEVELOPMENT AND ULTRASTRUCTURE OF IN VITRO-MATURED SWAMP BUFFALO (BUBALUS BUBALIS) OOCYTES

The purpose of this study was to investigate the effects of 2 vitrification procedures on the developmental capacity and ultrastructural changes of matured swamp buffalo (Bubalus bubalis) oocytes. In vitro-matured (IVM) oocytes were vitrified by using 35% and 40% ethylene glycol (EG) as vitrification solution (VS) for solid surface vitrification (SSV) and in-straw vitrification (ISV), respectively. Survival rate of vitrified–warmed oocytes was evaluated on the basis of homogeneous cytoplasm, membrane integrity, and complete zona pellucida. All developmental data were analyzed using chi-square analysis. P &amp;lt; 0.05 was considered significant. The blastocyst rates of parthenogenetic vitrified–warmed oocytes were significantly higher with SSV (89.3% and 13.6%, respectively) than with ISV (81.8% and 5.5%, respectively). However, they were still significantly lower than those of control (100% and 34.2%, respectively). For examining the ultrastructural changes, fresh VS-exposed (ISV and SSV), and vitrified–warmed oocytes were processed for transmission electron microscopy. In VS-exposed oocytes, reduction of microvilli abundance and damage of mitochondrial membrane were found only in the ISV group. In vitrified–warmed oocytes, however, it was clear that both methods of vitrification induced profound ultrastructural modifications to microvilli, mitochondria, oolemma, and cortical granules as well as to the size and position of vesicles. Damaged mitochondria were, however, more abundant in ISV vitrified oocytes than in SSV vitrified oocytes, which correlated with the developmental data, showing the superiority of the SSV method. This study demonstrated for the first time the feasibility of vitrification of IVM swamp buffalo oocytes.

Effects of vitrification procedures on subsequent development and ultrastructure of in vitro-matured swamp buffalo (Bubalus bubalis) oocytes

The purpose of the present study was to investigate the effects of two vitrification procedures on developmental capacity and ultrastructural changes of matured swamp buffalo oocytes. In vitro-matured oocytes were vitrified by using 35 and 40% ethylene glycol as vitrification solution for solid surface vitrification (SSV) and in-straw vitrification (ISV), respectively. Survival rate of vitrified-warmed oocytes, evaluated on the basis of ooplasm homogeneity, oolemma integrity and zona pellucida intactness, as well as parthenogenetic blastocyst rates of vitrified-warmed oocytes were significantly higher with SSV (89.3 and 13.6%, respectively) than ISV (81.8 and 5.5%, respectively). However, they were still significantly lower than that of control oocytes (100 and 34.2%, respectively). For examining the ultrastructural changes, fresh, VS-exposed (ISV and SSV), and vitrified-warmed (ISV and SSV) oocytes were processed for transmission electron microscopy. In VS-exposed oocytes, reduction of microvilli abundance and damage of mitochondrial membrane were found only in the ISV group. In vitrified-warmed oocytes, however, it was clear that both methods of vitrification induced profound ultrastructural modifications to microvilli, mitochondria, oolemma and cortical granules as well as to the size and position of vesicles. Damaged mitochondria were, however, more abundant in ISV vitrified oocytes than in SSV vitrified oocytes, which correlated with the developmental data, showing the superiority of the SSV method. The present study demonstrated the feasibility of vitrification of in vitro-matured swamp buffalo oocytes.

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.