Vitrification Solution Research Articles

C-Phycocyanin improves the quality of goat oocytes after in vitro maturation and vitrification

In vitro maturation (IVM) and cryopreservation of goat oocytes are important for establishing a valuable genetic bank for domesticated female animals and improving livestock reproductive efficiency. C-Phycocyanin (PC) is a Spirulina extract with antioxidant, antiinflammatory, and radical scavenging properties. However, whether PC has positive effect on goat oocytes IVM or developmental competence after vitrification is still unknown. In this study, we found that first polar body extrusion (n = 293), cumulus expansion index (n = 269), and parthenogenetic blastocyst formation (n = 281) were facilitated by adding 30 μg/mL PC to the oocyte maturation medium when compared with the control groups and that supplemented with 3, 10, 100 or 300 μg/mL PC (P < 0.05). Although PC supplementation did not affect spindle formation or chromosome alignment (n = 115), it facilitated or improved cortical granules migration (n = 46, P < 0.05), mitochondria distribution (n = 39, P < 0.05), and mitochondrial membrane potential (n = 46, P < 10−4). Meanwhile, supplementation with 30 μg/mL PC in the maturation medium could significantly inhibit the reactive oxygen species accumulation (n = 65, P < 10−4), and cell apoptosis (n = 42, P < 0.05). In addition, PC increased the oocyte mRNA levels of GPX4 (P < 0.01), and decreased the mRNA and protein levels of BAX (P < 0.01). Next, we investigated the effect of PC supplementation in the vitrification solution on oocyte cryopreservation. When compared with the those equilibrate in the vitrification solution without PC, recovered oocytes in the 30 μg/mL PC group showed higher ratios of normal morphology (n = 85, P < 0.05), survival (n = 85, P < 0.05), first polar body extrusion (n = 62, P < 0.05), and parthenogenetic blastocyst formation (n = 107, P < 0.05). Meanwhile, PC supplementation of the vitrification solution increased oocyte mitochondrial membrane potential (n = 53, P < 0.05), decreased the reactive oxygen species accumulation (n = 73, P < 0.05), promoted mitochondria distribution (n = 58, P < 0.05), and inhibited apoptosis (n = 46, P < 10−3). Collectively, our findings suggest that PC improves goat oocyte IVM and vitrification by reducing oxidative stress and early apoptosis, which providing a novel strategy for livestock gamete preservation and utilization.

Fast and furious: successful survival and resumption of meiosis in immature human oocytes vitrified and warmed using a short protocol

RESEARCH QUESTIONCan immature oocytes vitrified and warmed using a short protocol survive and resume meiosis? DESIGNThis study examined modifications of oocyte vitrification and warming protocols that shorten the time of exposure to vitrification and warming solutions. A total of 561 germinal vesicles (GV), and 218 metaphase I (MI) oocytes that were immature at oocyte retrieval were vitrified at room temperature in 2 minutes. Warming was done at 37°C in 2 minutes. Resumption of meiotic activity was evaluated after 24 and 48 hours of culture. Two different commercially available vitrification and warming kits were used for comparison. RESULTSNinety five percent of GV oocytes survived with no difference observed between kits. The survival of MI oocytes was on average 95.4% and not significantly different between the two kits. Of the 533 GV that survived, 491 converted to MI (92.1%). After culture for 48 hours, 54.4% converted to MII oocytes. In addition, of the 208 MI that survived the warming, 84.1% converted to MII oocytes after culture for 24 hours. CONCLUSIONSVitrification and warming of oocytes at different nuclear maturation stages can be done with a 2-minute exposure time to hypertonic and a 2-minute exposure time to hypotonic solutions. With this approach, the oocytes are less exposed to room temperature during dehydration and rehydration. Warming in 0.5M sucrose helps to maintain and support the potential to resume nuclear meiotic activity of oocytes and conversion from GV to MI and to MII oocytes.

Successful vitrification of equine embryos >300 microns without puncture or aspiration.

Equine embryos >300 μm require puncture before vitrification. Protocols that do not require pre-puncture would make vitrification easier and allow for its widespread use. To design a successful vitrification protocol for embryos >300 μm without puncture as a pre-treatment. Experimental in vivo study. Thirty-eight embryos were divided into 3 groups (G1: ≤300 μm, n = 11; G2: >300-500 μm, n = 20; G3: >500 μm, n = 7). Embryos were vitrified using a human vitrification kit. Following a 15 min exposure to equilibration solution (ES; 7.5% DMSO +7.5% ethylene glycol [EG] in a base medium [BM] of M199 HEPES-buffered medium [H199] + hydroxypropyl cellulose + gentamycin), embryos were exposed for ≤90 s to a vitrification solution (15% DMSO +15% EG + 0.5 M trelahose in BM), loaded onto a Cryolock and plunged into LN2. Warming was undertaken by plunging the Cryolock tip into 1 mL of H199 + 20% FBS + pen/strep +1 M sucrose at 42°C for 1 min. The embryos were then moved to a 0.5 M sucrose solution for 4 min, then placed in Vigro Hold for 4 min prior to transfer to a recipient. Pregnancy rates were 81.8% (9/11) for G1, 80% (16/20) for G2, and 0% (0/7) for G3. The largest embryo to survive was 480 μm. Limited numbers and only one pregnancy was followed to term. Equine embryos ≤480 μm can be successfully vitrified using a protocol with a longer exposure time to the ES. This does not appear to have a negative effect on early embryonic development.

Improvement of survivability and developmental ability in vitrified rat oocytes

Oocyte cryopreservation is useful for human fertility treatment and strain preservation in both experimental and domestic animals. However, the embryonic development of vitrified rat oocytes was lower than that of vitrified embryos. To increase the viability of vitrified oocytes, intracellular ice formation during cooling and warming must be prevented. Rapid warming is important to prevent ice formation. Furthermore, suppressing the spontaneous activation of oocytes is also important because vitrification promotes the spontaneous activation of rat oocytes, and thus compromise developmental competence of the gametes. MG132, a proteasome inhibitor, suppresses the spontaneous activation of rat oocytes. Here, we examined the effects of rapid warming and MG132 treatment on the survival and embryonic development of vitrified rat oocytes. The warming rate was adjusted by changing the vitrification solution volume and warming solution temperature. The survival rate of oocytes vitrified in 10 μL solution and warmed at 50 °C (94%) was significantly higher than that of oocytes vitrified in 100 μL and 10 μL solution and warmed at 37 °C (49% and 81%, respectively). Furthermore, the rate of embryonic development of vitrified oocytes treated with MG132 during vitrification, warming, and intracytoplasmic sperm injection (ICSI) (44%) was significantly higher than that of untreated gametes (10%). Offspring were obtained after transferring embryos derived from MG132-treated vitrified oocytes (14%). Altogether, the survivability of vitrified rat oocytes increased by rapid warming, and MG132 improved embryonic development after ICSI.

Model biological systems demonstrate the inducibility of pathways that strongly reduce cryoprotectant toxicity

Cryoprotectant toxicity is a limiting factor for the cryopreservation of many living systems. We were moved to address this problem by the potential of organ vitrification to relieve the severe shortage of viable donor organs available for human transplantation. The M22 vitrification solution is presently the only solution that has enabled the vitrification and subsequent transplantation with survival of large mammalian organs, but its toxicity remains an obstacle to organ stockpiling for transplantation. We therefore undertook a series of exploratory studies to identify potential pretreatment interventions that might reduce the toxic effects of M22. Hormesis, in which a living system becomes more resistant to toxic stress after prior subtoxic exposure to a related stress, was investigated as a potential remedy for M22 toxicity in yeast, in the nematode worm C. elegans, and in mouse kidney slices. In yeast, heat shock pretreatment increased survival by 18-fold after exposure to formamide and by over 9-fold after exposure to M22 at 30 °C; at 0 °C and with two-step addition, treatment with 90% M22 resulted in 100% yeast survival. In nematodes, surveying a panel of pretreatment interventions revealed 3 that conferred nearly total protection from acute whole-worm M22-induced damage. One of these protective pretreatments (exposure to hydrogen peroxide) was applied to mouse kidney slices in vitro and was found to strongly protect nuclear and plasma membrane integrity in both cortical and medullary renal cells exposed to 75–100% M22 at room temperature for 40 min. These studies demonstrate for the first time that endogenous cellular defenses, conserved from yeast to mammals, can be marshalled to substantially ameliorate the toxic effects of one of the most toxic single cryoprotectants and the toxicity of the most concentrated vitrification solution so far described for whole organs.

Improvement of Mouse Preantral Follicle Survival and Development following Co-Culture with Ovarian Parenchyma Cell Suspension.

The parallel and continued improvements in both infertility treatment and the management of malignancy cases have brought to the forefront the potential for fertility preservation. Using ovarian follicular resources can effectively improve reproductive capacity and prevent infertility. The primary aim of this research was to try to generate an appropriate in vivo environment for the growth of the mouse follicles. Hence, the possible effects of the ovarian parenchyma cell suspension were explored on the growth and maturation of preantral follicles in vitro. In this experimental study, ovarian parenchymal cells were mechanically dissociated from preantral follicles of 12-14 days-old NMRI mice and then divided into 5 experimental groups (G1: Control, G2: Fresh follicle with fresh parenchyma cell suspension, G3: Vitrified-warmed follicle with fresh parenchyma cell suspension, G4: Fresh follicle with frozen-thawed parenchyma cell suspension, and G5: Vitrified-warmed follicle with frozenthawed parenchyma cell suspension). The diameter of the follicles and immature oocytes, viability, antrum formation, resumption of meiosis, in vitro fertilization (IVF), and Gdf9, Bmp6, and Bmp15 gene expression were examined on different periods. The diameter of the follicles and the oocytes on days 4 and 8, as well as the survival rate of the follicles up to day 12, were significantly higher in G2 and G4 compared to the Ctrl group (G1: 73.66%, G2:87.99%, G3: 82.70%, G4: 94.37%, and G5: 78.59%). Expression of growth marker genes for G3, and G5 groups was significantly higher than other groups, which indicated the protective effects of parenchyma cell suspension on follicles damaged by vitrification solutions. The growth, survival, and maturation of preantral follicles could be enhanced by co-culturing them with ovarian parenchyma cells. Further studies are needed to optimize the conditions for a successful parenchyma cell suspension-induced in vitro maturation (IVM) to occur in infertility clinics.

Conservation of Green and White Ash Germplasm Using the Cryopreservation of Embryogenic Cultures

Green ash (Fraxinus pennsylvanica) and white ash (F. americana) populations are currently experiencing major declines across their native ranges in North America due to infestation by the exotic insect pest emerald ash borer (Agrilus planipennis). The development of a reliable method for the long-term storage of green and white ash germplasm in the form of embryogenic cultures using cryopreservation would be a considerable aid to ash conservation efforts. We compared recovery percentages of cryopreserved green and white ash embryogenic cultures using vitrification versus slow cooling methods. Three Plant Vitrification Solution 2 (PVS2) exposure durations (40, 60, and 80 min) for vitrification and three DMSO concentrations (5%, 10%, and 15%) for slow cooling were tested for their effects on the percentage of cultures that regrew following cryostorage. Vitrification resulted in a higher overall culture recovery percentage (91%) compared to cultures that were cryostored using the slow cooling approach (39%), and a more rapid initiation of regrowth (5 days versus 2–3 weeks) resulted. Recovery from cryostorage by cultures using the slow cooling approach varied significantly (p &lt; 0.05) between experiments and with genotype (p &lt; 0.05). The recovery of vitrified tissue from cryostorage did not vary with genotype, species, or PVS2 exposure duration (p &gt; 0.05). The vitrification cryopreservation protocol provides a reliable and versatile alternative to the traditional slow cooling method, strengthening our ability to preserve valuable ash germplasm for conservation and restoration.

Cryopreservation of highbush blueberry, strawberry, and saskatoon using V and D cryo-plate methods and monitoring of multiplication ability of regenerated shoots

AbstractThe purpose of this work was to apply V and D cryo-plate methods for cryopreservation of Vaccinium corymbosum ʻToroʼ, Fragaria × ananassa ʻCleryʼ, and Amelanchier alnifolia (Nutt.) M. Roem. and to monitor the multiplication capacity of shoots regenerated from cryopreserved explants. Shoot tips pre-cultured for 1 d at 23°C in the dark on medium containing 0.3 M sucrose were used as explants. Loading was performed in a solution containing 2 M glycerol and 0.8 M sucrose (30 min at room temperature). In the V cryo-plate, dehydration was carried out at room temperature (20 to 50 min) using the following plant vitrification solutions: original PVS2, 90% PVS2 solution, and PVS3. Regarding the D cryo-plate, dehydration was performed in closed glass containers over silica gel for 2, 2.5, or 3 h. In both protocols, rewarming was carried out in a 1.0 M sucrose solution (15 min at 25°C). Regenerated shoots were multiplied and multiplication parameters were monitored after the second subculture. Using the V cryo-plate method, the highest regrowth in highbush blueberry was obtained following 50-min treatment with all three VSs (61.7 to 80.9%). The D cryo-plate method was even more suitable with maximum regrowth of 89.4% achieved after 2.5 h of desiccation. For strawberry, 62.5% was the highest regrowth recorded using PVS3-based V cryo-plate method while 83.3% of regrowth was observed using D cryo-plate protocol. Regrowth of saskatoon reached a maximum of 50% after 50-min treatment with PVS3 while it did not exceed 40% in other treatments. By the second subculture, shoots regenerated from cryopreserved explants regained and even exceeded the multiplication capacity of shoots regenerated from non-cryopreserved explants. This study is the first to present the successful application of the V cryo-plate method in highbush blueberry, as well as the utilization of both V and D cryo-plate methods in saskatoon.

First Cryorepository for Coral Larvae: Safeguarding Corals for Future Generations

Research on the development of innovative cryobanking techniques can aid coral restoration and conservation. This study applied a customized freezing device and “cryojig” along with vitrification and laser warming techniques to create the first cryorepository for pelagic‐phase larvae of the corals Seriatopora caliendrum, Pocillopora verrucosa, Stylophora pistillata, and Pocillopora acuta. Three vitrification solutions (VSs) were prepared, with each composed of two of four cryoprotective agents. These included VS1: 2 M ethylene glycol (EG) and 1 M propylene glycol; VS2: 2 M EG and 1 M glycerol; VS3: 2 M dimethyl sulfoxide and 1 M EG. Larvae were then mixed with the VSs, 40% (w/v) Ficoll, and 10% (v/v) gold nanoparticles (GNPs) at a final concentration of 1.2 × 1018 particles/m3 and using our customized device for vitrification and laser warming. Over a thousand coral larvae (576 S. caliendrum, 376 P. verrucosa, 238 S. pistillata, and 524 P. acuta) were successfully stored in the cryorepository and the proportions of coral larvae that were noted to be actively swimming or settled after vitrification and laser warming. This is the first project to establish a coral larvae cryorepository, and the methods applied could represent a critical tool for reef restoration and maintenance of marine biodiversity.

Indução de calos, regeneração de brotos e conservação de tamareiras (Phoenix dactylifera) cultivadas in vitro

Abstract Date palm (Phoenix dactylifera( cv. Medjool is a significant plant, grown in Jordan. In vitro propagation gives operative resources for the significant propagation of date palms. Maximum callus induction was achieved from MS media supplemented with benzyl amino purine (BA) and naphthalene acetic acid (NAA). The highest plant regeneration was recorded on MS medium supplemented with dichlorophenoxyacetic acid (2,4-D) at 3.0 mg/L, and BA at 2.0 mg/L. A significant positive impact on shoot formation was recorded on MS medium supplemented with 1.0 mg/L BA with 0.5 to 1.5 mg/L NAA in both liquid and solid MS medium. To maintain survival and regrowth capacity, sucrose could be used for medium-term conservation at lower concentrations (0.1 - 0.2 M). In addition, sorbitol might be used at 0.1 M to maintain the quality of explants. The vitrification technique for long-term preservation was experimented. Embryogenic callus was used as explants for conservation. The survival as well as regrowth percentages of non-cryopreserved and cryopreserved tissue cultures were affected by their duration of treatment with the vitrification solution plant vitrification solution 2 (PVS2) and modified plant vitrification solution 2 (MPVS2). Results showed that using PVS2 for 60 minutes for cryopreserved calli was more effective than other treatments. After storage in liquid nitrogen, the highest survival rate (65%) and regrowth rate (40%) were achieved.

Assessment of Apoptosis-Related Genes in Autografted/ Vitrified Ovarian Tissue in Mice

Background: One of the new emerging techniques to preserve reproductive potential of cancer patients is cryopreservation of ovarian fragments prior to medical treatment and their re-transplantation after healing. In order to investigate and compare apoptosis in ovarian tissue, the current study has done while using different vitrification solutions. Methods: The ovaries of Balb/c mice were removed from the body. R ovaries autotransplanted and L ovaries vitrified and thawed in different solutions. Apoptosis-related genes (Bax, Bcl-2, Bid, P53) assessed by RT-PCR. Results: The results have shown that Bax and P53 expression showed significant changes in DCV3, but in DCV2 only p53 expression decreased considerably in vitrified-thawed group. Conclusion: We concluded that cryoprotectant solution is important for preserving tissue in vitrification process and in our study DCV2 showed better results than DCV3.

Automation in vitrification and thawing of mouse oocytes and embryos.

Vitrification is a common technique for cryopreserving oocytes or embryos. However, manual vitrification is tedious and labor-intensive, and can be subject to variations caused by human factors. To address these challenges, we developed an automated vitrification-thawing system (AVTS) based on a cryo-handle. Our study firstly assessed the efficiency of cryoprotectant exchange through comparing the osmolalities of fresh and collected solutions during automated vitrification and thawing, and evaluated the cooling and warming rates of the cryo-handle. We also compared mouse oocyte survival, fertilization and embryo development after thawing and ICSI, and the development of re-frozen cleavage embryos between manual operation and automated system. The results showed that the osmolalities of collected samples were within normal range and comparable to fresh solutions. Furthermore, the automated system could obtain the reliable cooling and warming rates. Particularly, there were no significant differences in oocyte survival rates, fertilization rates, and subsequent embryo development and its quality between two procedures. Our findings suggest that AVTS has no impact on osmolalities of vitrification and thawing solutions, ensuring the proper exchange of cryoprotectants. The cryo-handle also shows the ability to achieve reliable cooling and warming rates, which benefits for the cryopreservation and thawing process. Moreover, the results from mouse oocytes and embryos indicate that automated system has effectively maintained the survival and fertilization of frozen oocytes and supported subsequent embryo development. Therefore, the automated vitrification and thawing system will inevitably represent a superior alternative to manual operation.

Direct comparison of isobaric and isochoric vitrification of two aqueous solutions with photon counting X-ray computed tomography

Vitrification is a promising approach for ice-free cryopreservation of biological material, but progress is hindered by the limited set of experimental tools for studying processes in the interior of the vitrified matter. Isochoric cryopreservation chambers are often metallic, and their opacity prevents direct visual observation. In this study, we introduce photon counting X-ray computed tomography (CT) to compare the effects of rigid isochoric and unconfined isobaric conditions on vitrification and ice formation during cooling of two aqueous solutions: 50 wt% DMSO and a coral vitrification solution, CVS1. Previous studies have only compared vitrification in isochoric systems with isobaric systems that have an exposed air-liquid interface. We use a movable piston to replicate the surface and thermal boundary conditions of the isochoric system yet maintain isobaric conditions. When controlling for the boundary conditions we find that similar ice and vapor volume fractions form during cooling in isochoric and isobaric conditions. Interestingly, we observe distinct ice and vapor cavity morphology in the isochoric systems, possibly due to vapor outgassing or cavitation as rapid cooling causes the pressure to drop in the confined systems. These observations highlight the array of thermal-fluid processes that occur during vitrification in confined aqueous systems and motivate the further application of imaging techniques such as photon counting X-ray CT in fundamental studies of vitrification.

Influence of viscosity on dynamics of microalgae saturation with plant vitrification solutions

Influence of viscosity on dynamics of microalgae saturation with plant vitrification solutions

Re-Biopsy and Biopsy of Post-vitrified Blastocyst

In the last 45 years embryology lab has witnessed immense scientific and technological advancements. Implementation of new scientific methods is often questioned due to lack of evidence. Though Pre-Implantation Genetic Testing (PGT) and Vitrification have been applied in the field of embryology for more than three decades, lack of evidence about their efficacy still needs further research. Inconclusive results after PGT, might necessitate the need for re-biopsy and there after re-vitrification. Such an intervention is thought to be detrimental to embryo viability and reproductive outcomes. Recent advances in commercial vitrification solutions and an improved skill matrix have enabled 100% embryo survival possible. Application of LASER for PGT seem to be a safer intervention with minimal damage to embryos. Both these advanced embryology interventions, Vitrification, and use of LASER for PGT allow re-biopsy of embryos and re-vitrification of embryos with optimal survival and reproductive outcomes. However, such an intervention has limited role with poor quality blastocysts. Pre-caution needs to be exercised with vitrified-warmed embryos as the trophectoderm tissue gets very stick at biopsy and there is a chance of complete embryo hatching from zona-pellucida if excessive pressure is applied in the biopsy pipette. Re-biopsy and Re-Vitrification both seem to be possible in reference to the published literature and do seem to offer optimal outcomes.

Optimizing the Droplet-Vitrification Procedure by Balancing the Cryoprotection and Cytotoxicity of Alternative Plant Vitrification Solutions Based on the Nature of Donor Plant Vigor

Over 30 years of plant vitrification, droplet vitrification (DV) of in vitro propagules and slow freezing of dormant buds are typical methods of large-scale cryobanking worldwide. One-step sucrose preculture and Plant Vitrification Solution 2 (PVS2) cryoprotection in solution-based vitrification often face unacceptably low regeneration, and the results are on a case-by-case basis depending on the plant species, like a blind test. The absence of a universal protocol applicable across all plant diversity is considered one of the limiting factors. For wild flora, limits of source material available and difficulties in in vitro propagation make it worse to re-optimize the protocol steps for new species. Since cryoprotectant toxicity is the most crucial barrier to the vitrification of organized explants, selecting alternative plant vitrification solutions (PVS) based on the cytotoxicity of cryoprotectants is vital. This review proposes the concept of donor plant vigor (DPV), which refers to the donor plant properties that determine the potential to regenerate normal plantlets under various cryopreservation procedures. DV is a multi-stage procedure with many factors from stage (1) material preparation to (2) pre-liquid nitrogen (pre-LN) (preculture, osmoprotection, cryoprotection), (3) LN (cooling), (4) warming conditions (rewarming, unloading), and (5) regrowth. Since the cytotoxicity of PVS is a primary limiting factor in DV approaches, DPV is crucial for coping with the toxicity of PVS. The DPV is innate and can be maximized with appropriate material preparations, i.e., vigorously growing in subcultures aided by a liquid overlay on top of the gelled medium, selecting proper explants, optimizing the two-step preculture conditions, and media supplements. Developing the DV protocol starts with testing the material with a tentative standard protocol, which includes a two-step preculture (10% sucrose for 31 h and 17.5% sucrose for 16 h), osmoprotection with C4-35%, cryoprotection with A3-80% (60 min at 0 °C), cooling, and rewarming using aluminum foil strips. Using a three-step regrowth initially with ammonium-free regrowth medium, regrowth of shoot tips in one plate following the successive stages of the tentative standard protocol for shoot tips, i.e., fresh, PC, OP, CP (LNC), and LN, is a valuable tool to characterize the sensitivity of the material and to standardize the procedure by tuning the cryoprotection and cytotoxicity of cryoprotectants. A-series PVS (A3-90%, A3-80%, A3-70%) and B-series PVS (PVS3, B5-85%) can be tested based on the DPV. These alternative PVSs have been applied in over 30 pieces of literature with an 8.5~67.3% increase in LN regeneration compared to PVS2 and Plant Vitrification Solution 3 (PVS3) treatments. Using this approach as an alternative to blind condition screening would be influential in broadening the cryopreservation of diverse wild species and problem materials.

Effect of proline in comparison to PVS2 in cryopreservation of somatic embryo and callus cultures of date palm

In this work, the amino acid proline was investigated as cryoprotectant in cryostorage of date palm, Phoenix dactylifera L., tissue cultures. Among various levels of proline, it was found that 15% is the most effective one on survival. This concentration was compared to Plant Vitrification Solution 2 (PVS2). Generally, the maximum viability percentages of the two types of cryoprotectants were registered at 60 min exposure period. Proline was superior to PVS2 on callus viability. Moreover, healthy and good appearance calli were observed with the proline treatments. In contrast, the highest viability percentages of somatic embryos were observed with PVS2 solution. Regarding regrowth, the recovery parameters of both callus and somatic embryos increased as exposure period increased till 60 min and then decreased. The maximum callus fresh mass and growth value were registered when callus was cryoprotected by 15% proline. With respect to somatic embryos, proline gave good differentiation percentages, but they were slightly lower than PVS2. The highest differentiation percentage (70%) was recorded with PVS2 for 60 min in cryoprotection. However, the highest numbers of proliferated shootlets were recorded with proline in cyoprotection. Biodivers. Conserv. Bioresour. Manag. 2023, 9(2): 11-20

Cryopreservation of Bovine Oocyte using Vitrification Solution and Cryotop Techniques

Cryopreservation is used to preserve biological samples over an extended period at ultra-low temperatures. This process evolved into vitrification, a more advanced and superior technology in which fluids or water molecules form a glass-like structure without forming ice crystals. Unlike fresh cells, cryopreservation is reported to reduce oocyte viability and developmental competency. This study employed two vitrification techniques, vitrification solution (VS) and Cryotop, to investigate the meiotic resumption in bovine. Oocytes were extracted from cow ovaries collected from slaughterhouses in Banting and Shah Alam, Selangor, Malaysia. The oocytes were grouped (A, B, and B’) based on cumulus morphology and matured in vitro in a culture dish (humidified 5% carbon dioxide incubator at 38.5°C) for 20 to 24 hr. Oocytes were vitrified after maturation using straws or aids of Cryotop sheets, then submerged in liquid nitrogen and stored for five days before defrosting for cryoprotectant elimination. By using Giemsa staining, the maturation state of fresh and vitrified bovine oocytes was evaluated through five parameters: zygotene, pachytene, diakinesis, metaphase I, and metaphase II. The maturation rate demonstrated only slight differences in the three groups of oocytes treated with VS (A: 44.79%; B: 30.97%; B’: 20.70%) and Cryotop (A: 39.42%; B: 37.27%; B’: 28.97%), which were significantly lower than fresh oocytes (A: 55.83%; B: 44.82%; B’: 56.17%). Both VS and Cryotop methods were viable options for cryopreserving oocytes, but the Cryotop technique was more effective in increasing the meiotic competence of poor-quality oocytes.

Advances in cryopreservation of Syzygium maire (swamp maire, maire tawake) zygotic embryos, a critically endangered tree species endemic to New Zealand

IntroductionSyzygium maire is a threatened Myrtaceae tree species endemic to New Zealand. Due to its highly recalcitrant seed, cryopreservation is the only viable long-term ex situ conservation option for this species. Our previous attempts to cryopreserve the embryonic axis (EAs) of S. maire were unsuccessful but did provide a better understanding of desiccation behavior, biochemical composition, oxidative status, and ultrastructural changes associated with desiccation in EAs.MethodsWe incorporated this knowledge with biophysical information to investigate two advanced cryopreservation technologies: a droplet vacuum infiltration vitrification (DVIV) method and a novel metal-mesh vacuum infiltration vitrification (MVIV) method using Plant Vitrification Solution 2 (PVS2) for cryopreservation of the EAs.ResultsThe PVS2 treatment at room temperature (~20°C) proved phytotoxic with extended PVS2 incubation significantly reducing EA survival. No EAs survived cryopreservation using DVIV, however MVIV resulted in post-cryopreservation survival of up to 19% following PVS2 incubation for 20 min. Biophysical thermal analysis using Differential Scanning Calorimetry revealed a 15-fold reduction in ice crystallization following incubation in PVS2 for 20 min or more, with all freezable water removed after 60 min incubation.DiscussionThese results present a significant advance in being able to successfully cryopreserve S. maire EAs. The findings from this study will aid the development of cryopreservation protocols for other extremely recalcitrant seeded species, many of which are threatened with extinction due to climate change, plant pathogens, and habitat destruction.

EFFECT OF CRYOPROTECTANTS AND CRYOPRESERVATION METHODS ON THE QUALITY OF BUFFALO OOCYTES

This study was conducted to evaluate the effect of two types of cryoprotectants and two cryopreservation methods on viability and normal morphology of the immature and in vitro mature of post-thawed buffalo oocytes. The in vitro mature and immature oocytes were kept in the vitrification solution (VS1) for 5 min and the vitrification solution (VS2) for 15 sec. The results revealed that the proportion of buffalo oocytes found to be viable and normal morphology were significantly (P&lt; 0.01) higher in using glycerol which is 76.14% and 55.1%; respectively. We conclude from the present study that the viability and normal morphology of post-thawed buffalo vitrified in vitro matured oocytes were significantly (P &lt; 0.01) higher in glycerol than those obtained in dimethyl sulfoxide (DMSO) and in vitrification technique than rapid technique.