Antifreeze protein from Anatolia polita (ApAFP914) improved outcome of vitrified in vitro sheep embryos

Embryo cryopreservation is an important tool to preserve endangered species. As a cryoprotectant for mouse oocytes, antifreeze protein from Anatolica polita (ApAFP914) has demonstrated utility. In the present study, the effects of controlled slow freezing and vitrification methods on the survival rate of sheep oocytes fertilized in vitro after freezing-thawing were compared. Different ApAFP914 concentrations were added to the vitrification liquid for exploring the effect of antifreeze protein on the warmed embryos. The results showed that the survival and hatching rates of in vitro derived embryos were significantly higher than that of the slow freezing method. Furthermore, among the cryopreserved embryos at different developmental stages, the survival and hatching rates of the expanded blastocyst were significantly higher than those of the blastocysts, early blastocysts and morula. The survival and the hatching rates of the fast-growing embryos were both significantly higher than that of the slow-growing embryos. Additionally, treatment of ApAFP914 (5–30 μg/mL) did not increase the freezing efficiency of the 6–6.5 d embryos. However, addition of 10 μg/mL of ApAFP914 significantly increased the hatching rate of slow-growing embryos. In conclusion, our study suggests that the vitrification is better than the slow freezing method for the conservation of in vitro sheep embryos, and supplementation of ApAFP914 (10 μg/mL) significantly increased the hatching rate of slow-growing embryos after cryopreservation.     

Conventional slow freezing, vitrification and open pulled straw (OPS) vitrification of rabbit embryos

Three different methods of cryopreservation viz., conventional slow freezing, vitrification and open pulled straw vitrification were compared for their ability to support post thaw in vitro and in vivo development of rabbit embryos. Morula stage rabbit embryos were collected from super-ovulated donor does. They were randomly allocated to different freezing methods and stored up to 3 months in liquid nitrogen. After thawing and removal of cryoprotectants, embryos exhibiting intact zona pellucida and uniform blastomeres were considered suitable for in vitro culture and/or transfer. Three to five cryopreserved embryos placed in approximately 1 ml of culture medium (TCM 199 supplemented with foetal calf serum and antibiotics) were incubated for up to 72 h under humidified atmosphere of 5% CO2 in air at 39 degrees C. Development to hatched blastocyst stage was considered the initial indicator of success of cryopreservation of embryos. Of the embryos cryopreserved by programmed freezing, open pulled straw vitrification, vitrification-55 h pc and vitrification-72 h pc 55, 71, 17 and 48%, respectively, developed into hatched blastocysts. Similarly 19, 29, and 4% of embryos cryopreserved by programmed freezing, open pulled straw vitrification and vitrification -72 h pc developed into live offspring on transfer to recipient does. This is the first report on open pulled straw vitrification of rabbit embryos. Present results, suggest that (a) open pulled straw vitrification supports better in vitro survival of frozen thawed rabbit morulae; (b) both programmed freezing and OPS are similar but superior to vitirification in supporting in vivo survival of frozen thawed rabbit embryos.     

Effects of reproductive aging and postovulatory aging on the maintenance of biological competence after oocyte vitrification: insights from the mouse model

Cryopreservation of female reproductive cells allows preservation of fertility and provides materials for research. Although freezing protocols have been optimized, and there is a high survival rate after thawing, the in vitro fertilization (IVF) pregnancy rate is still lower in cycles with cryopreserved oocytes, thus highlighting the importance of identifying intrinsic limiting factors characterizing the cells at time of freezing. The aim of the present study is to investigate in the mouse model the impact of reproductive aging and postovulatory aging on oocyte biological competence after vitrification. Metaphase II oocytes were vitrified soon after retrieval from young and reproductively old mice. Part of the oocytes from young animals was vitrified after 6 h incubation (in vitro aged oocytes). All classes of oocytes showed similar survival rate after vitrification. Moreover, vitrification did not alter chromosomal organization in young cells, whereas in vitro aged and old oocytes presented an increase of slightly aberrant metaphase configurations. Compared to fresh young oocytes, in vitro aged and old oocytes showed increased ROS levels which remained unchanged after vitrification. By contrast, cryopreservation significantly increased ROS production in young oocytes. Both the aging processes negatively impacted oocyte ability to undergo pronucleus formation and first cleavage after vitrification by stimulating cellular fragmentation. These results could be helpful for establishing the correct time table for cryopreservation in the laboratory routine and improving its application in reproductively old females. Moreover, our observations highlight the importance of oxidative stress protection during vitrification procedures.     

In vitro oocyte fertilization and subsequent embryonic development after cryopreservation of bovine ovarian tissue, using an effective approach for oocyte collection

This study was undertaken to assess dissection/puncture combined technique for collecting large number of oocytes from bovine ovaries and to determine the effect of ovarian tissue cryopreservation on the oocytes capability to undergo in vitro maturation, fertilization and subsequent embryonic development. Ovaries (n=31) of slaughtered cows were cut into small fragments using a scalpel blade and the ovarian tissues were randomly assigned to cryopreserved by slow freezing and vitrification and non cryopreserved (fresh) groups. Oocytes were collected from non-atretic follicles from fresh and post-thawing ovarian tissue by the puncture method. The advantage of this technique appeared through morphologically good quality cumulus-oocyte complex (COC) recovery rate from fresh tissue (31.7±2.0 oocytes/ovary). However, the cryopreservation affected the post thawing total and good quality COC recovery rates from slow freezing (26.6±2.0 and 23.5±2.3 oocytes/ovary, respectively) and vitrification groups (21.7±1.1 and 17.6±1.8 oocyte/ovary, respectively). The maturation rate resulted in significant differences between the fresh tissue (94.1±1.1%) and the two cryopreservation groups. Moreover, this rate was significantly higher in the slow freezing group (80.1±1.3%) than in the vitrification group (73.0±1.9%). No statistical differences were observed in the cleavage and the embryonic developmental rates between fresh tissue group and cryopreservation groups. Furthermore the number of embryos produced per animal was statistically higher for fresh tissues than for slow freezing and the vitrification groups (34.4±1.4, 27.8±3.1 and 22.0±0.7, respectively). In conclusion, dissection method followed by puncture of bovine ovaries greatly maximizes the number of good quality oocytes recovered, as well as the number of embryos obtained per animal. Ovarian tissue can be successfully cryopreserved by slow freezing and vitrification.     

Applications of cryopreserved unfertilized mouse oocytes for in vitro fertilization

Since the first successful reports into oocyte freezing, many papers concerning the cryopreservation of mouse oocytes have been published. However, a simple and practical cryopreservation method for unfertilized C57BL/6 mouse oocytes, and an IVF system using these cryopreserved oocytes have yet to be established, in spite of the fact that C57BL/6 is the prevalent inbred strain and is used for large-scale knockout programs. In this study, unfertilized C57BL/6 mouse oocytes were cryopreserved via a simple vitrification method. After warming, IVF was performed using cryopreserved unfertilized oocytes and fresh sperm, cryopreserved unfertilized oocytes and cold-stored sperm, cryopreserved unfertilized oocytes and frozen sperm (C57BL/6 strain sperm), and cryopreserved unfertilized oocytes and frozen sperm derived from GEM strains (C57BL/6 background GEM strains). Nearly all of the cryopreserved oocytes were recovered, of which over 90% were morphologically normal. Those oocytes were then used for in vitro fertilization, resulting in 72–97% of oocytes developing into 2-cell embryos. A portion of the 2-cell embryos were transferred to recipients, resulting in live young being produced from 32–49% of the embryos. In summary, we established the simple and practical method of mouse oocyte vitrification with high survivability and developmental ability and the IVF using the vitrified-warmed oocytes with fresh, cold-stored or cryopreserved sperm with high fertility.     

Cryopreservation of Bufotes viridis embryos by vitrification

Loss of biodiversity among amphibians is a current concern. Our hypothesis is that the embryos of amphibian species at risk of extinction could be cryopreserved by vitrification, using methods which have proved successful with fish oocyte. To test this hypothesis, samples of four cryoprotectants - methanol (MeOH), dimethyl sulphoxide (Me2SO), propylene glycol (PG) and polyethylene glycol (PEG), some singly, some in combination, were plunged in liquid nitrogen for 5 min to find the best solution for vitrification. To find the least toxic of these solutions, blastulae and stage G17 embryos of Bufotes Viridis, a typical amphibian, were exposed to solutions at different concentrations (0.5–10 M) for different lengths of time (15–30 min), with and without their normal protective jelly coats. In each case the number of survivors, which reached stage G25 was counted. Finally a series of embryos was vitrified in liquid nitrogen using the most efficient and least toxic cryoprotectants.Propylene glycol had the best vitrification characteristics, but MeOH vitrified at higher concentrations. The optimum regime, with the least toxic ctyoprotectants, consisted of 1M Me2SO for 15 min and a combination of 15% PEG_(w/v) + 3M PG + 2M Me2SO for 3 min, with the jelly coat intact, followed by vitrification. This gave a survival percentage of 87.6% immediately after vitrification. Methods designed for cryopreservation of fish embryos make a good starting point for cryopreservation of the embryos of amphibian.     

Cryopreservation of kunming mouse oocytes using slow cooling, ultrarapid cooling and vitrification protocols

The cryopreservation of oocytes has been only marginally successful with any of the current protocols, including slow cooling, rapid cooling and vitrification. We wished to test the hypothesis that oocytes from a single mouse strain would freeze successfully by 1 of the 3 mentioned protocols. Unfertilized Kunming mouse oocytes obtained 14 h after PMSG/hCG administration were randomly assigned to be cryopreserved after slow cooling, ultra rapid cooling and vitrification. Oocytes were thawed by straws being placed into 37 degrees C water, and their morphological appearance and in vitro fertilization capability were compared with that of oocytes that had not undergone cryopreservation. Survival of oocytes was indicated by the absence of darkened ooplasm or by broken membranes or zona pellucida. Functional integrity was evaluated by the formation of a 2-cell embryo after IVF. Survival rate of slow cooled oocytes did not differ from that seen in vitrified oocytes (55.1 vs 65.9%) but was significantly lower in the rapidly cooled oocytes (24.2%; P < 0.01). The results of IVF of slow cooled and vitrified oocytes were similar to those of the control group (72 and 73 vs 77%; P > 0.05). It appears that Kunming mouse oocytes can be successfully cryopreserved using the slow cooling method with 1,2-propanediol and vitrification, which contains both permeating and nonpermeating cryoprotectants.     

A comparative analysis of the efficacy of three cryopreservation protocols on the survival of in vitro-derived cattle embryos at pronuclear and blastocyst stages

The effectiveness of three cryopreservation protocols (slow freezing, short equilibration vitrification and long equilibration vitrification) on in vitro-derived cattle embryos at expanded blastocyst and pronuclear stages was compared. 199 expanded blastocysts of good quality were assigned randomly into four treatment groups [control, non-cryopreserved (fresh, unfrozen); and the three cryopreservation methods]. The re-expansion of the cryopreserved blastocysts after 24 h in vitro culture was similar to that of the fresh control group. However, the hatching rate of expanded blastocysts after 48 h culture was significantly less for the slow freezing group (31/47; 66.0%) than for both the short equilibration vitrification (46/51; 90.2%) and long equilibration vitrification groups (42/50; 84.0%). Denuded presumptive zygotes at the pronuclear stage (14–18 h post-insemination) were assigned randomly to the same four treatment groups and, following thawing, embryos were assessed for their capacity to cleave and to develop into a blastocyst. Overall, cleavage rates of cryopreserved zygotes were significantly less than those of the fresh control. The blastocyst formation rate of slow-frozen zygotes (4/81; 4.9%) was significantly less than that of zygotes subjected either to short equilibration vitrification (18/82; 22.0%) or long equilibration vitrification (16/74; 21.6%). All cryopreservation groups showed rates of blastocyst formation that were significantly less than that of the fresh control (51/92; 55.4%). Collectively, our findings indicate that vitrification is the preferred technology to cryopreserve in vitro-derived cattle embryos at expanded blastocyst and pronuclear stages. Moreover, short equilibration vitrification technology can improve outcomes and be more efficient by taking less time to perform.     

Factors affecting the survival of mouse embryos cryopreserved by vitrification

Preimplantation stage mouse embryos have been used to examine the response of a simple multicellular system to cryopreservation by the complete vitrification of the suspension. Successful vitrification requires the use of a solution of cryoprotectants that is sufficiently concentrated to supercool and solidify into a glass at practicable cooling rates. Factors that influence the survival of embryos include the concentration and composition of the vitrification solution, the procedure used to equilibrate embryos in this solution, the cooling and warming conditions, and the procedure used to dilute embryos from the vitrification solution. High rates of survival are obtained when embryos are dehydrated prior to vitrification in solutions composed of saline plus multimolar concentrations of either mixtures of permeating cryoprotectants (e.g. dimethyl sulphoxide-acetamide-propylene glycol) or single permeating cryoprotectants (propylene glycol or glycerol). Full permeation of cryoprotectants into the cells is not necessary and may lead to chemical toxicity and osmotic injury. Partial permeation and osmotic shrinkage concentrates the endogenous cytoplasmic macromolecules and greatly increases the likelihood of intracellular vitrification. Vitrification is a practical approach for embryo cryopreservation and offers new opportunities to examine fundamental aspects of cryoprotection and cryoinjury in the absence of freezing.     

Japanese flounder (Paralichthys olivaceus) embryos are difficult to cryopreserve by vitrification

The first successful cryopreservation of fish embryos was reported in the Japanese flounder by vitrification [Chen and Tian, Theriogenology, 63, 1207-1219, 2005]. Since very high concentrations of cryoprotectants are needed for vitrification and fish embryos have a large volume, Japanese flounder embryos must have low sensitivity to cryoprotectant toxicity and high permeability to water and cryoprotectants. So, we investigated the sensitivity and the permeability of Japanese flounder embryos. In addition, we assessed the survival of flounder embryos after vitrification with solutions containing methanol and propylene glycol, following Chen and Tian's report. The embryos were relatively insensitive to the toxicity of individual cryoprotectants at lower concentrations, especially methanol and propylene glycol as their report. Although their permeability to water and cryoprotectants could not be measured from volume changes in cryoprotectant solutions, the embryos appeared to be permeable to methanol but less permeable to DMSO, ethylene glycol, and propylene glycol. Although vitrification solutions containing methanol and propylene glycol, which were used in Chen and Tian's report, were toxic to embryos, a small proportion of embryos did survived. However, when vitrified with the vitrification solutions, no embryos survived after warming. The embryos became opaque during cooling with liquid nitrogen, indicating the formation of intracellular ice during cooling. When embryos had been kept in vitrification solutions for 60 min after being treated with the vitrification solution, some remained transparent during cooling, but became opaque during warming. This suggests that dehydration and/or permeation by cryoprotectants were insufficient for vitrification of the embryos even after they had been over-treated with the vitrification solutions. Thus, Chen and Tian's cryopreservation method lacks general application to Japanese flounder embryos.     

Cryopreservation of porcine embryos derived from in vitro-matured oocytes

This study describes a cryopreservation method for porcine in vitro-produced (IVP) embryos using as a model parthenogenetic embryos derived from in vitro-matured (IVM) oocytes. IVP embryos at the expanded blastocyst stage were cryopreserved by vitrification using the minimum volume cooling (MVC) method and exhibited an embryo survival rate of 41.2%. Survival was then significantly improved (83.3%, P < 0.05) by decreasing the amount of cytoplasmic lipid droplets (delipation) prior to vitrification. IVP embryos at the 4-cell stage also survived cryopreservation when vitrified after delipation (survival rate, 36.0%), whereas post-thaw survival of nondelipated embryos was quite low (9.7%). Furthermore, it was demonstrated that porcine IVP morulae can be cryopreserved by vitrification following delipation by a noninvasive method (survival rate, 82.5%). These results clearly confirm that porcine embryos derived from IVM oocytes can be effectively cryopreserved with high embryo survival using the MVC method in conjunction with delipation.     

Effect of cryoprotectants and their concentration on in vitro development of vitrified-warmed immature oocytes in buffalo (Bubalus bubalis)

Experiments were conducted to study the effect of cryoprotectants, dimethyl sulfoxide (DMSO), ethylene glycol (EG), 1,2-propanediol (PROH), and glycerol at different concentrations (3.5, 4, 5, 6, and 7 M each with 0.5 M sucrose and 0.4% BSA in DPBS) on survival, in vitro maturation, in vitro fertilization, and post-fertilization development of vitrified-thawed immature buffalo oocytes. The COCs were harvested from the ovaries by aspirating the visible follicles. The recovery of post-thaw morphologically normal oocytes was lower in 3.5 and 4 M DMSO, EG, and PROH compared to 5, 6, and 7 M. In all the concentrations of glycerol, an overall lower numbers of oocytes recovered were normal compared to other cryoprotectants. Less number of oocytes reached metaphase-II (M-II) stage from the oocytes cryopreserved in any of the concentrations of DMSO, EG, PROH, and glycerol compared to fresh oocytes. Among the vitrified groups, highest maturation was obtained in 7 M solutions of all the cryoprotectants. The cleavage rates of oocytes vitrified in different concentrations of DMSO, EG, PROH, and glycerol were lower than that of the fresh oocytes. The cleavage rates were higher in oocytes cryopreserved in 6 and 7 M DMSO, EG, PROH, and glycerol compared with oocytes cryopreserved in other concentrations. However, the percentage of morula and blastocyst formation from the cleaved embryos did not vary in fresh oocytes and vitrified oocytes. In conclusion, this report describes the first successful production of buffalo blastocysts from immature oocytes cryopreserved by vitrification.     

Effect of genotype on the efficiency of mouse embryo cryopreservation by vitrification or slow freezing methods

We examined possible genotype effects on the survival of 8- to 16-cell mouse embryos isolated from four inbred strains (C57BL/6N, BALB/cAnN, DBA/2N, and C3H/HeN), a outbred stock (ICR), and various crosses after cryopreservation by vitrification or conventional slow freezing using glycerol solutions. The rates of in vitro development of C57BL/6N, BALB/cAnN, C3H/HeN, and ICR embryos to expanded blastocysts ranged from 86% to 94% after slow freezing and 85% to 97% after vitrification. The cryopreservation method did not significantly influence in vitro embryo survival after thawing (P >0.05). Although genotype significantly influenced the in vitro survival of embryos (P = 0.008), this presumably resulted from an increased difficulty in assessing the quality grade of C3H/HeN embryos prior to cryopreservation. The rates in vivo development of C57BL/6N, BALB/cAnN, C3H/HeN, DBA/2N, and ICR embryos to normal day 18–19 fetuses ranged from 19% to 64% after slow freezing and from 18% to 63% after vitrification. The in vivo development of cryopreserved embryos was significantly influenced by cryopreservation method and genotype (P = 0.01 and P = 0.001, respectively). Vitrification yielded significantly higher rates of in vivo development than that after slow freezing (P > 0.05). In vivo development rates of DBA/2N and ICR♀ X B6D2F1 ♂ embryos after cryopreservation were significantly higher than that of embryos from BALB/cAnN and C3H/HeN mice (P < 0.05). These results indicate that parental genotype exerts little or no effect on the ability of embryos to develop in vitro after vitrification or slow freezing. Differences in the ability of cryopreserved embryos to develop normally in vivo may reflect inherent genotype related differences in their post-implantation developmental potential and not their sensitivity to cryoinjury. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Cryopreservation of porcine embryos by vitrification: A study of in vitro development

Until recently, attempts to preserve porcine embryos have been unsuccessful. Vitrification has been developed as a method of cryopreserving mammalian embryos by avoiding ice crystal formation, assuring a cryopreserved glass state during storage in liquid nitrogen. Vitrification may be a useful method of overcoming the deleterious effects of chilling injury when pig embryos are cryopreserved using conventional slow freezing procedures. In this study, we applied vitrification procedures for rodent and/or bovine embryos to cryopreserve porcine embryos. Following warming, survival was defined as normal development of embryos in culture, namely the formation or reexpansion of the blastocoelic cavity. Experiment 1 tested the relative toxicity of 3 vitrification procedures on Day-5, 6 and 7 porcine embryos. Embryos equilibrated in vitrification solution (VS3a) continued to develop in vitro at rates comparable to that of untreated control embryos. Experiment 2 was designed to evaluate embryonic development following cryopreservation by vitrification in VS3a. Day-5 porcine embryos did not survive cryopreservation while Day-6 and Day-7 embryos survived and continued development in vitro. In Experiment 3, we evaluated a period of culture prior to vitrification and its effect on cryosurvivability of porcine embryos. A 3-h culture period prior to vitrification had no effect on cryosurvivability over that of freshly recovered, immediately vitrified embryos. These studies indicate, for the first time, that porcine embryos can be successfully cryopreserved by vitrification based on morphology and subsequent development in vitro. However, survival following cryopreservation appears to depend upon embryonic age or stage of development.     

Ultrastructural characterization of fresh and cryopreserved in vivo produced ovine embryos

Controlled slow freezing and vitrification have been successfully used for ovine embryo cryopreservation. Selection of embryos for transfer is based on stereomicroscopical embryo scoring after thawing, but the subjectivity inherent to this selection step has been demonstrated by ultrastructural studies of controlled slow frozen, in vivo produced ovine morulae and blastocysts. These studies have shown that certain abnormalities remain undetected by stereomicroscopy only. In the present study, using ovine in vivo produced morulae and blastocysts, we have studied the ultrastructural alterations induced by open pulled straw vitrification (OPS) and controlled slow freezing, compared stereomicroscopical embryo scoring with light microscopy evaluation of embryo's semithin sections, and related the ultrastructural cellular damage with the embryo classification by stereomicroscopical embryo scoring of embryos’ and semithin section evaluation by light microscopy. The ultrastructural lesions found for OPS-vitrified and controlled slow frozen embryos were similar, independently of embryo stage. A significant higher number of grade 3 embryos was found at stereomicroscopical scoring after controlled slow freezing (P = 0.02), and a significant higher number of grade 3 blastocysts was found at semithin sectioning after OPS vitrification (P = 0.037). The extension of ultrastructural damage, especially of mitochondria and cytoskeleton, was related to the semithin classification but not to stereomicroscopical scoring at thawing. This suggests that semithin scoring is a useful tool for predicting ultrastructural lesions and new improvements in cryopreservation and thawing methods of ovine embryos are still warranted, including in the case of blastocysts cryopreserved by OPS vitrification.     

Evaluation of zebrafish (Danio rerio) PGCs viability and DNA damage using different cryopreservation protocols

Cryopreservation of primordial germ cells (PGCs) is a better alternative for the conservation of the diploid genome in fish until embryo cryopreservation is achieved. A good cryopreservation protocol must guarantee high survival rates but also absence of genetic damage. In this study, a cell toxicity test using several internal and external cryoprotectants was carried out. The best combination of cryoprotectants (DMSO 5 mol/L, ethylene glicol (EG) 1 mol/L, polyvinyl pyrrolidone (PVP) 4%) was used with and without antifreeze proteins (AFPs) at two different concentrations (10 mg/mL and 20 mg/mL) for cryopreservation trials. Different cryopreservation methods were used with single PGCs, genital ridges, and whole zebrafish embryos using cryovials, 0.5 mL straws, microcapsules, and microdrops. All embryos were obtained from the vasa EGFP zf45 transgenic line and viability was evaluated using trypan blue. High cell viability rates after cryopreservation in 0.5 mL straws were obtained (around 90%) and a decrease in viability was only observed when cells were cryopreserved in microcapsules and when AFP at 20 mg/mL was added to the freezing media. Genetic damage was determined by comet assay and was compared in cells cryopreserved in 0.5 mL straws and microcapsules (lowest viability rate). There were significantly more DNA strand breaks after cryopreservation in the cells cryopreserved without cryoprotectants and in those cryopreserved in microcapsules. Genetic damage in the cells cryopreserved with cryoprotectants in 0.5 mL straws was similar to fresh control samples, regardless of the concentration of AFP used. The decrease in PGC viability with the addition of AFP 20 mg/mL did not correlate with an increase in DNA damage. This study reported a successful method for zebrafish PGC cryopreservation that not only guarantees high cell survival but also the absence of DNA damage.     

Open pulled straw (OPS) vitrification: A new way to reduce cryoinjuries of bovine ova and embryos

Although cryopreservation of certain mammalian embryos is now a routine procedure, considerable differences of efficiency exist depending on stage, species and origin (in vivo or in vitro produced). Factors that are suspected to cause most of these differences are the amount of the intracellular lipid droplets and the different microtubular structure leading to chilling injury as well as the volume/surface ratio influencing the penetration of cryoprotectants. A new approach, the Open Pulled Straw (OPS) method, which renders very high cooling and warming rates (over 20,000°C/min) and short contact with concentrated cryoprotective additives (less than 30 sec over −180°C) offers a possibility to circumvent chilling injury and to decrease toxic and osmotic damage. In this paper we report the vitrification by the OPS method of in vitro produced bovine embryos at various stages of development. Embryos cryopreserved from Day 3 to Day 7 (Day 0 = day of fertilization) exhibited development into blastocysts at rates equivalent to those of control embryos; even those cryopreserved on Day 1 or 2 exhibited only somewhat reduced survival. Eighty-one percent of Day 8 hatched blastocysts also survived the procedure. The method was also successfully used for bovine oocytes; of 184 vitrified oocytes, 25% developed into blastocysts after fertilization and culture for 7 days. Pregnancies were achieved following transfer after vitrification at both the oocyte and blastocyst stage. The OPS vitrification offers a new way to solve basic problems of reproductive cryobiology and may have practical impact on animal biotechnology and human assisted reproduction. Mol. Reprod. Dev. 51:53–58, 1998. © 1998 Wiley-Liss, Inc.     

Microtubulin configuration and mitochondrial distribution after ultra-rapid cooling of bovine oocytes

Considerable attention has been focused on the cryopreservation of mammalian oocytes, as a consequence of poor development of cryopreserved bovine oocytes in vitro, in order to enhance the application of genetic engineering. Experiments were carried out to evaluate the viability and ultra-structural changes of bovine oocytes cryopreserved by ultra rapid cooling methods. Oocytes that had been allowed to mature for 22 hr were exposed to a mixture of cryoprotectants (3.2 M ethylene glycol, 2.36 M dimethyl sulfoxide (DMSO), 0.6 M sucrose), and were cryopreserved by very rapid cooling either within glass capillaries or as droplets on copper electron microscope grids. After being warmed, the oocytes were cultured in in vitro maturation (IVM) medium for an additional 2 hr. Viability was assessed by determining the development rate after fertilization with frozen-thawed semen from which motile sperm had been recovered using a Percoll density gradient, and by immunochemical evaluation of microtubule and mitochondrial morphology. Cleavage and development rates were significantly (P < 0.05) lower in oocytes cryopreserved by vitrification than in in vitro fertilization (IVF) control group, but did not differ in the open-pulled glass (OPG) or copper grid (CG) groups. In most oocytes cryopreserved by vitrification, the microtubules were partially or completely broken. Similarly mitochondria appeared to be abnormal compared to that of unfrozen oocytes. Oocytes cultured in IVM medium supplemented with both cytochalasin B (a protein synthesis inhibitor) and 2-mercaptoethanol (an antioxidant) showed less damage to microtubules, but not to mitochondria after cryopreservation. In conclusion, this study showed that bovine oocytes can be cryopreserved by vitrification within small droplets using CGs. While damage to microtubules and mitochondria may be involved in reduced viability, supplementation of IVM medium with cytochalasin B appears to enhance stabilization of microtubules during oocyte cryopreservation.     

Animal oocyte and embryo cryopreservation

Cryopreservation of oocytes and embryos is a crucial step for the widespread and conservation of animal genetic resources. However, oocytes and early embryos are very sensitive to chilling and cryopreservation and although new advances have been achieved in the past few years the perfect protocol has not yet been established. All oocytes and embryos suffer considerable morphological and functional damage during cryopreservation but the extent of the injury as well as differences in survival and developmental rates may be highly variable depending on the species, developmental stage and origin (for example, in vitro produced or in vivo derived, micromanipulated or not). Currently, there are two methods for gamete and embryos cryopreservation: slow freezing and vitrification. We have experienced both techniques but vitrification has become a viable and promising alternative to traditional approaches especially when dealing with in vitro produced or micromanipulated embryos and oocytes. Recently new strategies based on emerging studies in the field of lipid research have been used to reduce intracellular lipid content in bovine in vitro produced embryos and therefore increase their tolerance to micromanipulation and cryopreservation. The addition of a conjugated isomer of linoleic acid, the trans-10, cis-12 octadecadienoic acid to embryo culture medium more than twice improved embryo post-thawing viability after micromanipulation and vitrification. Vitrification was also used for the cryopreservation of embryos belonging to the Portuguese Animal Germplasm Bank project presently running at our facilities. Presented at the International Consensus Meeting “New Horizons in Cell and Tissue Banking” on May 2007 at Vale de Santarém, Portugal.     

Survival of vitrified sheep embryos in vitro and in vivo

The effects of the composition of vitrification media, the duration of exposure to the media and the stage of development were examined on the survival of vitrified Day-6 sheep embryos. Vitrification media that contained two cryoprotectants in equal molar concentrations were used. In Experiment 1, the effects of the types (glycerol + propylene glycol or glycerol + ethylene glycol) and concentrations (3.5 + 3.5 or 4.5 + 4.5 M) of cryoprotectants and the level of BSA supplementation (0.4 or 20%) were investigated in a 2 x 2 x 2 design. The embryos were exposed to vitrification media for 30 sec at 18 to 24 degrees C before vitrification. The in vitro survival rate was not affected by the level of BSA supplementation, but there was an interaction between the types and concentrations of cryoprotectants used (P<0.01). Embryos cryopreserved in mixtures of glycerol + propylene glycol survived better when the concentration of cryoprotectants was 3.5 M while the survival of embryos cryopreserved in mixtures of glycerol + ethylene glycol was higher at 4.5 M cryoprotectant concentration. In Experiments 2 and 3, the effect of the duration of exposure (15, 30, 60 or 120 sec) to vitrification media at 4 to 12 degrees C was investigated on the survival rate in vivo. Vitrification media contained 3.5 M glycerol + 3.5 M propylene glycol or 4.5 M glycerol + 4.5 M ethylene glycol in Experiments 2 and 3, respectively. The survival rate in vivo, increased when the duration of exposure to vitrification media was increased from 15 to 30 sec, but the viability declined when the duration of exposure was further increased to 60 (Experiment 3) or to 120 sec (Experiment 2). The effect of the stage of development was significant only in Experiment 1 (P = 0.032), but in all three experiments the rate of survival increased with advancing stages of development from late morulae to late blastocysts. The best result was achieved in Experiment 2, when embryos were exposed to a mixture of 3.5 M glycerol + 3.5 M propylene glycol for 30 or 60 sec. Under these conditions 52% (22 42 ) of rapidly cryopreserved sheep embryos developed into lambs. This result shows that a simple rapid procedure for the cryopreservation of sheep embryos can produce a survival rate comparable to that obtained using more complex traditional procedures.