Vitrification Medium Research Articles

Protective effect of the superoxide dismutase mimetic MnTBAP during sperm vitrification process.

Sperm cryopreservation is widely used in assisted reproduction and male infertility therapy; however, it induces oxidative stress affecting sperm quality. This work evaluated the effect of the antioxidant MnTBAP during vitrification steps in human spermatozoa. First, the effect of MnTBAP on viability and ROS production was evaluated. Then, the spermatozoa were vitrified in straws with the vitrification, warming and post-warming incubation media separately supplemented with MnTBAP. An untreated control was included. The sperm viability, ROS production, total and progressive motility were evaluated. The results showed that the direct exposure of spermatozoa to MnTBAP significantly decreases the ROS levels in comparison with the untreated control without affecting the viability. The supplementation of the vitrification medium with MnTBAP did not affect the parameters analysed. However, the supplementation of the warming and incubation post-warming media resulted in a decrease in ROS production and maintained viability and motility for 4hr after warming with concentrations up to 100μM of MnTBAP. Higher concentrations of MnTBAP caused a decrease in total motility. In conclusion, the use of MnTBAP during the warming or post-warming incubation media has beneficial effect decreasing ROS levels and maintaining the viability and motility during the vitrification procedure.

The harmful effect of removing the extracellular vitrification medium during embryo cryopreservation using a nylon mesh device in rabbit

During the last decades, many techniques have been developed to reduce sample volume and improve cooling and warming rates during embryo vitrification. The vast majority are based on the “minimum drop size” concept, in which the vitrification solution around embryos is reduced by aspiration, leaving a tiny part of volume surrounding embryos. However, novel cryodevices were aimed to remove the entire vitrification solution. This study was designed to compare the “minimum drop size” technique using Cryotop® with the nylon mesh as cryodevice on rabbit morula embryos. The outcomes assessed were the in vitro development rates (experiment 1) and the offspring rates at birth (experiment 2). Embryos were vitrified in a two-step procedure; equilibrium (10% EG + 10% Me2SO) for 2 min and vitrification (20% EG + 20% Me2SO) for 1 min. In experiment 1, embryos (n = 323) were warmed and subsequently in vitro cultured for 48 h to assess the embryo developmental capability to reach the hatching-hatched blastocyst stage. In experiment 2, embryos were transferred using the laparoscopic technique (n = 369) to assess the offspring rate at birth. In this context, rates of in vitro embryo development were similar between vitrified groups (0.73 ± 0.042% and 0.66 ± 0.047% for Cryotop® and nylon mesh device, respectively), but lower than in the fresh group (0.97 ± 0.016%, p < 0.05). In experiment 2, there were no significant differences in survival rates (offspring born/total embryos transferred) among the Cryotop® device group and fresh group (0.41 ± 0.049% and 0.49 ± 0.050%, respectively). But significantly lower value was obtained in the nylon mesh device group (0.18 ± 0.030%). These results indicate that nylon mesh is not suitable as cryodevice for rabbit morula vitrification, remaining those using the “minimum drop size” methodology as the best option.

Assessment of DNA Repair Gene Expressions in Vitrified Mouse Preantral Follicles.

Objective Vitrification of the ovarian tissue is one of the techniques recommended for preserving the fertility of women who are dealing with infertility. Despite its benefits, our information about the molecular aspects of ovarian follicles vitrification is somehow ambiguous. Therefore, the aim of this study was to evaluate the expression pattern of DNA repair genes in vitrified preantral follicles. Materials and Methods In this experimental study, the isolated preantral follicles (n=906) from 14-16 days old mice (n=12) were divided into three groups: fresh, toxic and vitrified which were cultured in vitro for 12 days. Preantral follicles were vitrified using cryotop followed by exposure to equilibration solution for five minutes and vitrification solution (VS) for 30 seconds. In the toxic group, preantral follicles were only placed in equilibration and vitrification media and they were then placed in the warming solutions without exposure to liquid nitrogen. On the second and sixth days of the culture period, real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was carried out to evaluate expression of the selected genes involved in DNA repair, including Msh6 (MutS homolog 6), Mre11 (Meiotic recombination 11), Brca1 (Breast cancer type 1), Rad51 (RAD51 recombinase), Pcna (Proliferating cell nuclear antigen) and Atm (ATM serine/threonine kinase). In addition, developmental parameters including growth, survival rate, antrum cavity formation and ovulation were analyzed. Results The relative mRNA expression of Msh6, Mre11, Brca1, Rad51, Pcna and Atm on the second and sixth days of the culture period in vitrified group was significantly higher than those of the control and toxic groups, but there was no significant difference between the toxic and control groups. In addition, developmental parameters of follicles were similar in both toxic and control groups, while both were significantly higher than that of vitrified group. ConclusionVitrification changes the expression pattern of DNA repair genes of the mouse preantral follicles.

Effects of MTG and GSH on Human Sperm Motility and DNA Integrity during Vitrification in the Presence of Trehalose

Limited information exists about the effects of antioxidants on sperm motility and DNA integrity during vitrification in humans. This study compared the effects of reduced glutathione (GSH) and monothioglycerol (MTG) at different concentrations on post-thaw sperm motility and DNA integrity after vitrification in humans using 0.25 M trehalose as a cryoprotective agent, and found that supplementation of MTG or GSH at 0.5 mM resulted in significantly higher (P < 0.05) recovery rates of post-thaw total and progressive motilities. GSH was more powerful than MTG at the same concentration in cryoprotecting sperm motility (38.9% ± 3.6% vs 32.8% ± 2.4% compared to the control 26.8% ± 2.1% in recovery rate of progressive motility), but both had no significant influence on sperm DNA integrity during vitrification. It was concluded that sperm motility is more sensitive to oxidative stress during vitrification than sperm nuclear DNA, and supplementation of MTG or GSH in vitrification medium is beneficial in cryoprotecting sperm motility.

42 Vitrification of invitro-produced feline embryos

Preservation of feline embryos is useful in propagating endangered species, preserving valuable genetics, and supporting biomedical research. Although a wide variety of cryoprotectants (CP) and protocols are successfully used for vitrification of invitro-produced (IVP) embryos, there are often species-specific differences in viability of embryos post-warming. The purpose of this study was to evaluate the viability of IVP feline embryos after vitrification using two common CPs, propanediol (PrOH) or ethylene glycol (EG). Embryos were produced with oocytes and frozen-thawed epididymal sperm collected from local spay-neuter clinics using a published IVP protocol developed for producing domestic feline embryos. Day 7 early blastocysts (stage 5), blastocysts (stage 6), and expanded blastocysts (stage 7) were evaluated for quality (grade 1 or 2) and randomly assigned to one of three treatments: vitrification with PrOH (n=32), vitrification with EG (n=31), or control (n=47), which was allowed to continue in culture until Day 8. The vitrification protocol was as follows. The base medium for all vitrification media was a HEPES-buffered feline optimized culture medium (FOCMH). Embryos were placed in 0.5mL of equilibration medium (7.5% dimethyl sulfoxide, 7.5% PrOH or EG, 0.5M sucrose, 10% Ficoll, and 20% fetal calf serum (FCS)) for 5min at room temperature. Individual embryos were then moved to 20-μL drops of vitrification medium (15% dimethyl sulfoxide, 15% PrOH or EG, 0.5M sucrose, 10% Ficoll, and 20% FCS) at room temperature for 30s before being loaded onto Cryolock devices and plunged into liquid nitrogen. Warming was done using a 3-step process for all vitrified embryos. First, embryos were moved from liquid nitrogen directly to 0.5mL of 1M sucrose, 10% Ficoll, and 20% FCS at 37°C for 1min. Next, embryos were moved to 0.5mL of 0.5M sucrose, 10% Ficoll, and 20% FCS at 20°C for 3min. Finally, embryos were transferred to 0.5mL of FOCMH for 5min at 37°C. All warmed embryos were cultured in medium, optimized for feline embryos, with 5% FCS and evaluated for re-expansion of the blastocoele and progression in development at 24 and 48h. Results are from five replicates. Embryos vitrified in EG exhibited higher percentages of viable embryos 24h after warming (84%) than embryos vitrified in PrOH (59%; P&amp;lt;0.05). The continued embryonic growth of viable embryos after culture for 48h showed equivalent developmental rates, at 87, 96, and 100% for control, EG-treated, and PrOH-treated embryos, respectively (P&amp;gt;0.05). Results indicate EG is a more successful CP treatment for vitrification of feline embryos when evaluating viability 24h post-warming. We report a higher viability of embryos post-thaw than previous studies using the same CPs (Pope et al. 2012 Reprod. Domest. Anim. 47, 125). This may be due to the shorter exposure time to the CPs we used during the vitrification process. We conclude that EG and PrOH are effective CPs for Day 7 feline IVP embryos using this protocol. Further research is needed to increase treatment numbers and evaluate pregnancy rates from embryos transferred post-warming.

The cryoprotective effect of Ficoll 70 on the post-warming survival and quality of Cryotop-vitrified donkey embryos

Many domestic donkey breeds are at risk of extinction, there is a critical urgency for genome resource banking. In the present study, we examined whether the use of Ficoll 70 added to the vitrification medium containing ethylene glycol (EG), dimethyl sulfoxide (DMSO) and sucrose improves the cryotolerance of donkey in vivo derived embryos. Day 7–8, grade 1–2 donkey embryos were measured and morphologically evaluated and then vitrified-warmed using the Cryotop technique. Before vitrification, embryos were randomly distributed into two groups: (i) VS1 (n = 14): vitrified using 15% EG + 15% DMSO + 0.5 M sucrose; and (ii) VS2 (n = 10): vitrified in the same medium supplemented also with 18% of Ficoll 70. After 24 h of warming, the embryos were measured and evaluated for their morphology, development and viability (Propidium Iodide-Hoechst 33342 dyes). Post-warming survival was numerically higher but not significantly different (P > 0.05) when embryos were vitrified in VS2 (70%) compared to VS1 (57.1%). Embryo rupture was only observed in the VS1 group (21.4%, 3/14). Higher embryo diameter was observed in all groups after 24 h culture (P < 0.05). No significant differences (P > 0.05) were observed among treatments in terms of percentages of cell death. These results demonstrate that the addition of Ficoll 70 to the vitrification medium is not a pre-requisite for successful vitrification of donkey embryos. However, its addition seems to enhance some of the post-warming embryo quality characteristics. Since no statistically significant evidence was found, further studies should be conducted in order to confirm our findings.

Kualitas, Kemampuan Implantasi dan Viabilitas in-vivo Embrio Mencit (Mus muculus) Galur Swiss Webster Setelah Pembekuan Dengan Metode Vitrifikasi

Reproductive technologies including in vitro fertilization (IVF), embryo manipulation, gamete and embryo freezing, thawing and embryo transfer were rapidly developed. Vitrification is an embryo freezing technique that is the most developed. In this experiment, we vitrified mouse embryos and then examined the embryos i.e: (i) the quality of the embryos after thawing, (ii) the implantation rate of the embryos and (iii) viability of the embryos in vivo. Morulae and blastocycsts were collected from female mice that were pregnant a day 3,5. The embryos were equilibraten in mPBS +10% etilene glycol. Vitrification was carried out by using VABEDS medium, containing 6-10 embryos that were dropped into a tip of a straw, then frozen in liquid nitrogen for 24 hours. Thawing was carried out by flushing the embryos using mPBS suplemented with 0.5, 0.25, 0.1 and 0 M sucrose. After being incubated in M2 medium at 37oC for 1-2 hours, the recovery embryos were then transferred into the uteri of day 2.5 of pseudopregnat females. The females were then sacrificed at day 16 of gestation and the total implantaion, total life and death fetuses, as well as resorpted embryos, were taken as data. The results showed that vitrification significantly (p&lt;0,05) reduced the quality of the embryos, as well as their implantation rate and the viability of the fetuses, which may be caused by the unoptimal combination of the cryoprotectant in the vitrification medium, temperature and exposure time during vitrification.

The effects of TETs on DNA methylation and hydroxymethylation of mouse oocytes after vitrification and warming

Oocyte vitrification has extensively been applied in the field of embryo engineering and in the preservation of genetic resources of fine livestock. Following our previous work in oocyte vitrification and the level change of DNA methylation, here we further explored the dynamic change of three active demethylation proteins: Ten-Eleven-Translocation 1/2/3(TET1/2/3), 5-methylcytosine (5 mC) and 5-hydroxymethycytosine (5hmC) after vitrification and warming. In order to observe the active demethylation in vitrified oocytes, two small molecular regulators, i.e. Vitamin C (VC) and dimethyloxaloylglycine (DMOG) were used to adjust activity and level of the TET 3 protein. The results showed that the levels of 5 mC and 5hmC were significantly decreased after 2 h of vitrification (P < 0.01). Moreover, the level of TET3 protein was significantly increased after 2 h warming (P < 0.01). And the relative gene expression of TET2/3 did not change in the first 2 h, but significantly increased after 2 h (P < 0.01). When VC was added to vitrification and recovery medium, it could not significantly improve the level of TET3 gene expression, and affect 5 mC and 5hmC expression (P > 0.05). When the DMOG was added to the solutions of vitrification, the level of 5hmC showed significantly increase (P < 0.01). In conclusion, the oocyte vitrification procedure reduced DNA methylation and hydroxymethylation in MII oocytes, but adding VC and DMOG to vitrification medium can prevent the reduction of DNA hydroxymethylation by increasing activity of TET3 methylation protein after vitrification and warming.

Improved embryology outcome and morphokinetic characteristics of embryos derived from frozen/thawed oocytes injected 3 versus 2 hours post-warming

To assess whether the duration of recovery following the warming of cryopreserved oocytes affects the morphological and morphokinetic properties of the developing embryo. This is a retrospective data analysis examining the effect of the post-warming recovery duration of vitrified oocytes prior to ICSI. Frozen oocytes from 36 patients (July 2018 to March 2019), either autologous or donor, were included in the study. The study examined cycles in which the oocytes were injected within a 2-hour recovery period and compared them to those in which the oocytes were injected within 3 hours or more within the same patient oocyte cohort. All injected oocytes were cultured individually in time-lapse incubators (TLM; EmbryoScope, Vitrolife, Sweden). Embryo transfers occurred on D3 or D5 regardless of the study groups. Standard oocyte freezing protocol included vitrification 2 hours after retrieval using a Kitazato-based (Japan) vitrification media. Oocyte warming was performed using Kitazato thaw media. ICSI was performed in the standard fashion, and embryos were annotated daily for their developmental hallmarks. Embryo selection for ET or freezing was performed by standard laboratory protocols. No significant differences were observed in 2PN and/or abnormal fertilization rates between the groups. Similarly, on day 3, no significant differences were found in the average cell numbers or in the average number of good (≥8 cells, <20% frag.), fair (5-7 cells, any abnormal cleavages), and poor (≤4 cells) embryos. Equally, the incidence of abnormal cleavage patterns was similar among the cohort. More embryos were selected for transfer from the 3-hour group than from the 2-hour group (51.1% vs. 48.9%; p=0.78). There were no significant differences in the utilization rate (the number of embryos transferred fresh plus blastocysts frozen) between the 3- and 2-hour groups (49.6% vs. 49.6%, respectively). The time-lapse morphokinetic data indicated faster-growing embryos in the 3- versus 2-hour group. This difference is apparent in late-stage morphokinetic parameters (t9 to tsB). The 3-hour group produced significantly better quality blastocysts in cycles that were either frozen upfront, fresh D5 ET, or PGT (p=0.03). The duration of the recovery time post-warming showed no significant differences in overall embryology outcomes prior to blastocyst formation. However, at the BL stage, the 3-hour group demonstrated improvement in morphokinetic parameters and in overall BL quality.

On the assessment of the stability of vitrified cryo-media by differential scanning calorimetry: A new tool for biobanks to derive standard operating procedures for storage, access and transport

When a vitrified sample is heated over the glass transition temperature it may start to devitrify endangering the sample. The ability to estimate the stability of the vitrified state can help in the development of new vitrification media as well as handling procedures. By employing differential scanning calorimetry, we can measure the ice crystallization rate in a vitrified sample and thus study the devitrification kinetics. Using this technique, we have studied samples comprised of PBS with cryoprotective additives (CPA) as dimethylsulfoxide (Me2SO), ethylene glycol (EG) and mixtures thereof, regarding the dependence of the devitrification kinetics on the CPA concentration. We found that already small concentration changes lead to significant changes in the devitrification times. Changing the CPA concentration by 4 wt% changed the devitrification time with a factor of 342 and 271 for Me2SO and EG, respectively. Concentration changes in EG/Me2SO mixtures was found to have a smaller impact on the devitrification kinetics compared to the pure CPA samples. Our data suggest that these significant increases in the devitrification times are primarily due to a relation between nucleation rates and the CPA concentration. Finally, we investigated an established vitrification medium used to preserve human embryonic stem cells. This medium was found to have the poorest glass stability in this study and reflects the tradeoff between stability and biocompatibility. The present work finally provides a tool to evaluate handling and storage procedures when employing vitrification as a cryopreservation method and underlines the importance of these.

The effect of adding Rosmarinic and Ascorbic acids to vitrification media on fertilization rate of the mice oocyte: An experimental study.

BackgroundOocytes vitrification is a pivotal step for the widespread and safekeeping of animal genetic resources. Oocytes endure notable morphological and functional damage during cryopreservation. Oxidative stress is one of the adverse effects that vitrification imparts on oocytes.Objective In the present study, we investigated the antioxidant effect of Rosmarinic and Ascorbic acids on the quality and fertilizing ability of frozen-thawed mice oocyte.Materials and MethodsIn this experimental study, germinal vesicle oocytes obtained from two-months-old (30–40gr) NMRI mice were randomly divided into four groups. The basic cryoprotectants were 7.5% (v/v) ethylene glycol+7.5% (v/v) Propanediol as an equilibration media. Vitrification medium contained 15% (v/v) ethylene glycol+15% (v/v) propanediol, and 0.5 M sucrose. In the first group (Control), nothing was added to vitrification mediums, whereas, in the second and third groups, 0.5 mmol/L of Ascorbic acid and 105 µmol/L of Rosmarinic acid were added into vitrification medium, respectively. The cumulative concentration of Rosmarinic and Ascorbic acids were added to group 4. Mouse oocytes were vitrified and preserved for one month. The thawed oocytes were transferred into the α-MEM medium (Alpha Minimum Essential Medium) and maintained in this medium for 24 hr, to be matured and reach the metaphase II stage.Results The addition of Rosmarinic and Ascorbic acids to the vitrification solution improved the survival, maturation of Germinal vesicles, fertilization rate, and finally development to 4-cell stage. Maturation rates to 4-cell stage for Ascorbic acid, Rosmarinic acid, and both of them together were 80%, 80.76%, and 86.61%, respectively.ConclusionThese results indicate that the addition of a cumulative concentration of 0.5 mmol/L Ascorbic acid and 105 µmol/L of Rosmarinic acid to the cryopreservation solution for the mouse immature oocytes would be of significant value (p 0.01).

Fluorescent Microscopy Analysis of Mammalian Oocyte Viability after Vitrification

The paper reports the results of a fluorescent microscopy analysis of the viability of oocytes from cattle and pigs after vitrification. Oocytes were frozen in a vitrification media containing varying concentrations of cryoprotectors in several steps with subsequent vitrification. After cryobank storage for 14 days, experimental samples were thawed and oocyte viability was analyzed by oocyte morphology assessment and fluorescent microscopy. Two different kits were used to stain oocytes, one specific for necrosis/apoptosis (Propidium iodide/Alexa Fluor 488 Annexin) and the other specific for live/dead cells (Calcein-AM/ethidium homodimer-1). Fluorescent microscopy of porcine and bovine oocytes has demonstrated that the fluorescent dye Calcein-AM should be chosen to assess oocyte viability, since Propidium iodide and ethidium homodimer-1 do not represent the oocyte cell death. Therefore, Propidium iodide and ethidium homodimer-1 cannot serve as indicators of real oocyte death.

Флуоресцентный микроскопический анализ жизнеспособности ооцитов млекопитающих после витрификации-=SUP=-*-=/SUP=-

AbstractThe paper reports the results of a fluorescent microscopy analysis of the viability of oocytes from cattle and pigs after vitrification. Oocytes were frozen in a vitrification media containing varying concentrations of cryoprotectors in several steps with subsequent vitrification. After cryobank storage for 14 days, experimental samples were thawed and oocyte viability was analyzed by oocyte morphology assessment and fluorescent microscopy. Two different kits were used to stain oocytes, one specific for necrosis/apoptosis (Propidium iodide/Alexa Fluor 488 Annexin) and the other specific for live/dead cells (Calcein-AM/ethidium homodimer-1). Fluorescent microscopy of porcine and bovine oocytes has demonstrated that the fluorescent dye Calcein-AM should be chosen to assess oocyte viability, since Propidium iodide and ethidium homodimer-1 do not represent the oocyte cell death. Therefore, Propidium iodide and ethidium homodimer-1 cannot serve as indicators of real oocyte death.

29 Development and survival of bovine vitrified sexed IVF-derived embryos in vitro matured with pituitary or human recombinant follicle-stimulating hormone

Improving in vitro production efficiency involves the development of effective oocyte in vitro maturation conditions. Although &amp;gt;80% of cumulus-oocyte complexes (COC) undergo nuclear maturation, only approximately 30 to 35% of immature bovine COC develop to the blastocyst stage. Also, animal-sourced FSH is typically used in IVF, so an effective alternative using recombinant DNA technology is desirable. The aim of this study was to compare the effect of porcine (p) and recombinant human (rh)FSH concentrations on in vitro performance and post-thaw survival. Ovaries were collected from an abattoir and oocytes were aspirated from 2- to 6-mm follicles. The COC with compact and complete cumulus cell layers were selected and matured in groups of 25 COC in 200µL of M199 medium supplemented with alanyl-glutamine (0.1mM), Na pyruvate (0.2mM), gentamicin (5µg mL−1), epidermal growth factor (50ng mL−1), pLH (5µg mL−1), cysteamine (0.1mM), and 10% FBS for 22 to 24h in humidified air and 5% CO2. Oocytes were divided into the following groups: 1× pFSH (2µg mL−1), 1× rhFSH (0.01 UI mL−1), 1× pFSH+1× rhFSH, 2× pFSH, and 2× rhFSH. After 22 to 24h, fertilization (Day 0) was carried out using female sexed-sorted semen selected with a mini single-continuous 80% layer (PureSperm, Nidacon International AB, Mölndal, Sweden) and diluted to 1×106 sperm mL−1. The SOF-FERT medium was supplemented with fructose (90µg mL−1), penicillamine (3µg mL−1), hypotaurine (11µg mL−1), and heparin (20µg mL−1). After 18h, presumptive zygotes were denuded and cultured under low oxygen tension in groups of 15 to 20 in 50-µL drops of SOF-BSA for 7 days. Also, 2% FBS was added post-fertilization on Day 3.5. Expanded blastocysts were selected based on IETS standards at Day 6.5 to 7 of culture. Only grade 1 expanded blastocysts were vitrified (Cryotop, Kitazato, Tokyo, Japan). Vitrification medium was 15% (vol/vol) ethylene glycol+propylene glycol. Vitrified embryos were thawed in a solution of H199+20% FBS and 0.25M sucrose at 39°C. Thawed embryos were cultured in SOF-BSA+10% FBS under cumulus/granulosa cell monolayer co-culture. Embryo assessment involved post-thaw survival (0h), re-expansion and development progress (24-48h), and hatching of the zona pellucida (72h). A minimum of 4 replicates were performed. Data were analysed using a generalized linear mixed model with logit-link binomial distribution. Media treatment differences were determined using Fisher’s least significant difference test with the Bonferroni correction (α-level=0.05). The FSH origin affected cleavage and embryo development rate but not cryotolerance (Table 1). The results support previous research on low dose versus high dose rhFSH effectiveness and interspecies interaction of FSH on follicular receptors. Table 1.Cleavage, embryo development rate, and cryotolerance of FSH of various origins

Oocyte vitrification using a new vitrification medium and a new closed vitrification device. A sibling oocyte study

Oocyte vitrification using a new vitrification medium and a new closed vitrification device. A sibling oocyte study

Single blastocyst transfer (SET) and pregnancy outcome of day 5 and day 6 human blastocysts vitrified using a closed device

This study investigates the utility of the Rapid-i closed device for vitrification of human blastocysts on day-5 (D5) and day-6 (D6) of development and the implantation and pregnancy rate following single blastocyst transfer (SBT) of warmed D5/D6 blastocysts. This retrospective cohort study was performed at Edinburgh Assisted Conception Programme, EFREC, Royal Infirmary of Edinburgh between January 2013 and January 2017. Good quality blastocysts were vitrified on D5 or D6 using Irvine Vitrification medium (Irvine Scientific-USA) and the Rapid-I closed Vitrification System™ (Vitrolife, Sweden). After warming, blastocysts were cultured in G-TL™ medium (Vitrolife) supplemented with 20% HSA-solution™ (Human Serum Albumin) for 2 h before the transfer. The survival, pregnancy and implantation rates were compared in relation to the day of culture at the time of vitrification (D5/D6) in 1090 cryopreserved cycles. The overall survival rate was 93.4% (1018/1090) with no significant difference between the D5 and D6 groups: 93.9% (712/758) and 92.2% (306/332) respectively. Single embryo transfers of D6 vitrified/warmed blastocysts resulted in a lower implantation and clinical pregnancy rate compared to D5 embryos. The implantation rate (IPR) and clinical pregnancy rate (CPR) were respectively 49.6% and 43.0% for the D5 and 37.0% and 33.0% for the D6 embryos, which was statistically significant. The multiple pregnancy rate was 1.08% (0.98% for D5 vs 1.3% for day 6).

Establishment of an improved vitrification protocol by combinations of vitrification medium for isolated mouse ovarian follicles

In vitro follicle growth (IVFG) is an emerging alternative option for fertility preservation in women instead of ovarian tissue cryopreservation and transplantation. To widen the application of this technique, follicle cryopreservation should be established prior to clinical use. In the present study, we tried to determine the optimal vitrification protocol of mouse ovarian follicle for in vitro culture and oocyte maturation by comparing four different compositions of cryoprotective agents (CPA). Secondary follicles were mechanically isolated from 2-week-old BDF-1 mice and randomly assigned to fresh control and four different groups by the composition of CPAs (ES, EDS, EFS and EPS groups; E: ethylene glycol, D: dimethyl sulfoxide, S: sucrose, F: ficoll, P: 1,2-propanediol (PROH)). After vitrification and warming procedures, the follicles were cultured in vitro for 10 days and then treated with human chorionic gonadotropin and epidermal growth factor to induce oocyte maturation. Fourteen to 16 h later, oocyte maturation and quality were assessed. Follicle viability was evaluated by Calcein-AM/ethidium homodimer-1 staining immediately after warming, and their survival and diameters were measured during follicle culture periods. Antral cavity formation was observed at the end of the culture period (on the 10th day of culture). Following oocyte maturation, its maturational ability and meiotic spindle formation were assessed to evaluate their competence. There was no significant difference in viability after warming among the vitrification groups. From the 8th day of culture, the survival rate of ES and EDS were significantly higher than those of other vitrification groups (EPS and EFS). The follicle diameter was largest in the fresh-control group from the 6th day, while smallest in the EFS with statistical significance. On the 10th day of culture, the antral-cavity formation rate of EDS was comparable to that of the fresh control group. However, the oocyte maturation was significantly decreased in all four vitrification groups when compared with control group; especially, the EFS showed a more marked reduction in the oocyte maturation. There were no significant differences in meiotic spindle formation among all of those groups. Our results suggest that EDS combination for mouse follicle vitrification are the most effective vitrification protocols for mouse follicle and evaluated by an in vitro culture and oocyte maturation after warming.

CRYOPRESERVATION OF OOCYTE-CUMULUS COMPLEXES OF COWS WITH DIFFERENT BIOACTIVE SUBSTANCES

Implementation of biotechnological process in livestock should be considered not only in terms of selection process intensification (obtain of embryos in vitro, their transplantation) and to a greater extent as development of effective methods of freezing and long storage of mammalian cells, including ova and embryos. Application of biotechnology in livestock breeding increases rate of genetic progress, preservation of gene pool of breeds via banks of sperm, embryos and cryobank of oocytes, obtaining and regulation of progeny of the desired sex, providing genetic evaluation of gametes and embryos, and it will enable to use genetic potential of animals after culling by age, replicate and create new genotypes with desired properties repeatedly. Solution to this problem is to improve medium and conditions of gametes and embryos freezing. Although the overall development of cryopreservation method is through simplification of the equilibration and vitrification solutions which would be able to ensure the full development frozen-thawed gametes. Addition of biologically active substances to the solution for cryopreservation contributes to protection of gametes during freezing and thawing, and determination of the consistent patterns of these substances will improve procedures of frozen-thawed oocyte cultivation outside the body. So it is necessary to deepen the fundamental research on the mechanisms of formation of a mature ovum of cows obtained from frozen-thawed oocytes to obtain embryos. The aim of the research is to conduct comparative analysis of different biologically active substances in equilibration and vitrification solutions at cryopreservation of oocyte-cumulus complexes of cows. Material and methods of the research. The objects of experimental studies were oocyte-cumulus complexes of black-and-white cows. The oocytes with homogeneous fine-grained ooplasm, undamaged pellucid zone, thick or partially loosened cumulus were used for freezing. The gametes of cows were treated by equilibration solution before freezing and then were transferred into vitrification solution. All the equilibration (10% glycerol + 20% propanediol) and vitrification (25% glycerol + 25% propanediol) solutions for cryopreservation of cows’ oocyte-cumulus complexes were prepared in Dulbecco phosphatebuffered saline with addition of 20% fetal serum of cows, 1х10-4 M unithiol, 1х10-6 M acetylcholine and without addition of bioactive substances. The research on adding some biologically active substances (fetal serum of cows – version A, unithiol – version B, acetylcholine – version C, without addition of biologically active substances – version D and not frozen cells of the control group (K)) in equilibration and vitrification solutions at freezing the oocyte-cumulus complexes of cows was carried out. It was found by the results of experimental studies that introduction of the above-mentioned components into the equilibration solution for freezing cows’ oocyte-cumulus complexes increased their cryoresistance, as evidenced by increasing indicator of maturation of frozen-thawed gametes outside the body to metaphase-2 of meiosis after 27-hour cultivation by 5,4-23,0% and decreasing indicator of number of oocytes with chromosomal abnormalities by 2,9-15,3%. The introduction of biologically active substances into the vitrification solution and subsequent cultivation during 27 hours after freezing and thawing cows’ oocyte-cumulus complexes showed that the indicator of maturation of frozen-thawed gametes outside the body to metaphase-2 of meiosis increased by 3,9-16,4% and the indicator of number of oocytes with chromosomal abnormalities decreased by 2,9-8,4%. Comparative analysis of the results of in vitro fertilization of cows’ frozen-thawed ova which were frozen using fetal serum of cows (version A) and without it (version - B) has shown a positive effect of adding it to equilibration and vitrification medium at freezing gametes of cows; it led to increase of obtaining bovine embryos in vitro by 11,5%. Thus, the analysis of experimental results showed different efficiency of using fetal serum of cows, unithiol, acetylcholine in the equilibration solution for freezing cows’ oocyte-cumulus complexes. The advantage of using these biologically active substances in the vitrification solution for cryopreservation of cow’s gametes by the indicator of maturation of frozen-thawed oocyte-cumulus complexes outside the body to metaphase-2 of meiosis wasn’t established. Conclusions. Introduction of fetal serum into cryopreservation solution increases cryoresistance of cow’s oocytes to cooling leading to increasing the indicator of the matured frozen-thawed gametes outside the body to metaphase-2 of meiosis by 23,0% and the obtained bovine embryos in vitro by 11,5%.

Sucrose ‘Versus’ Trehalose Cryoprotectant Modification in Oocyte Vitrification : A Study of Embryo Development

Numerous studies reported that vitrification, an ultra-rapid cooling technique, seems to be highly effective and could increase oocyte survival rate rather than slow freezing. The successful of oocyte vitrification depends on the proper combination of type and concentration of cryoprotectant. This study was addressed to determine the effects of the combination of type and concentration of cryoprotectants of vitrification media, notably in the embryo development. This experimental research was conducted by using oocyte obtained from thirty-two adult female Deutschland, Denken and Yoken (DDY) mice (7-8 weeks old). The MII mice oocytes were vitrified within 24 h after retrieval using the Cryotop method with cryoprotectants as follow : sucrose (16.5% EG, 16.5% DMSO, 0.5 mol/l sucrose), trehalose (16.5% EG, 16.5% DMSO, 0.5 mol/l trehalose) and Kitazato. The embryo development and morphological grading was observed at 2-cell and 8-cells under reverse phase light microscope and inverted microscope. This study demonstrated a good embryo development and morphological grading in sucrose and trehalose vitrification media. In embryo development, trehalose medium seems more superior compared to sucrose medium, even though Kitazato was the most superior compared to both. In the morphological grading, in 2-cells embryo, there were no significant differences between the three cryoprotectants, While, in 8-cells embryo, trehalose medium appeared to be superior compared to sucrose medium, even though seemed more inferior compared to Kitazato. The appropriate type and concentration of sugar as extracellular cryoprotectant was trehalose in oocyte vitrification based on embryo development, compared to sucrose.

Bovine oocyte membrane permeability and cryosurvival: Effects of different cryoprotectants and calcium in the vitrification media

The cryopreservation process must be improved to enhance oocyte cryosurvival and functionality. Two protocols with different cryoprotectants (CPAs), containing either ethylene glycol (EG), dimethyl sulfoxide (DMSO) and sucrose (EGDMSO) or 1,2-propanediol and sucrose (PrOH) were evaluated. In both protocols, calcium (Ca2+) free or -containing base media were tested. Oocytes were subjected to vitrification or only exposed to CPAs without immersion in liquid nitrogen. Oocyte's viability, cortical granules location and competence for development after fertilization were assessed. Finally, fatty acid composition and membrane permeability of oocytes exposed to CPAs were analyzed. Independently of Ca2+ concentration in the vitrification media, the development rates were higher in oocytes vitrified with EGDMSO protocols (p = 0.0005). After warming, higher cleavage rates were obtained in EGDMSO + Ca2+ compared to the PrOH without Ca2+ protocol (p = 0.02). Oocytes exposed to PrOH without Ca2+ presented lower cleavage rates compared to control (p = 0.04). An enhanced premature zona hardening in vitrified oocytes as well as lower concentrations of the fatty acids c11:18:1 and 20:4n-6 in cumulus oocyte complexes exposed to PrOH protocols were identified. The oocytes minimum volume and permeability were affected by the exposure to PrOH and Ca2+ (p ≤ 0.007). In conclusion, the most effective protocol for bovine oocytes cryopreservation combines EG and DMSO, independently of Ca2+ concentration in the media. A higher toxicity and an incomplete depletion of water during PrOH loading may hamper oocyte viability. The type of CPAs and Ca2+ interfered differentially on oocyte pathways to functionality, and this should be considered when choosing a cryopreservation protocol.