High survival of rabbit morulae after vitrification in an ethylene glycol-based solution by a simple method.

Rabbit morulae were exposed to a vitrification solution-modified PBS [PB1] medium containing 40% ethylene glycol + 18% Ficoll + 0.3 M sucrose (EFS) for 2, 5, or 10 min at 20 degrees C and were vitrified in liquid nitrogen. When morulae were rapidly warmed, 96% had an intact zona pellucida. When embryos were cultured after removal of the mucin coat, high proportions of them formed blastocoel (79-100%), but the percentage of embryos developed to fully expanded blastocysts decreased with increased exposure time 87%, 40%, and 17%). The survival rate of morulae vitrified after removal of the mucin coat was lower than that of mucin-intact embryos. To assess the development potential in vivo, 131 embryos were vitrified after 2 min of exposure to EFS solution; all the embryos were recovered and 120 were transferred to recipients without removal of the mucin coat, resulting in 78 (65%) full-term fetuses or young. This simple method, which yields high survival both in vitro and in vivo, will be of practical use for vitrifying rabbit embryos.     

In vivo survival rate of rabbit morulae after vitrification in a medium without serum protein

The in vivo survival rate of rabbit morulae after vitrification in a mixture of dimethyl sulphoxide and ethylene glycol solution without protein supplement (WPS) was compared with two types of protein supplements: rabbit serum (RS) and bovine serum albumin (BSA). Significant differences were observed in the percentage of transferable embryos (undamaged embryos after devitrification, 80.4% versus 93.2 and 92.1%, WPS, BSA and RS, respectively, P < 0.05) and live born rate (40.9% versus 56.1%, WPS and BSA, respectively, P < 0.05). Non-significant differences were, however, observed in the percentages of implanted embryos at 12 days post-ovulation induction (56.7, 69.7 and 68.6%), post-implantation survival rate (82.3, 74.2 and 77.2%) and live born rate in pregnant does (54.6, 56.1 and 50.5%) with different vitrification media (RS, BSA and WPS). We conclude that rabbit embryos can be vitrified and stored using protein-free vitrification medium with moderate losses of viability.     

Survival of bisected rabbit morulae transferred to synchronous and asynchronous recipients

The rabbit was used as a model to test the concept that temporal asynchrony is required to establish physiological synchrony when embryos are bisected to produce demiembryos. In preliminary studies with intact embryos it was confirmed that embryos harvested on days 2, 3, 4, or 5 (day 0 = day of breeding) can be transferred with +/- 1 day of asynchrony to the uteri of recipient rabbits. Three experiments were conducted with bisected embryos. In experiment 1, 192 bisected and 194 control day 3 embryos were transferred to uteri of day 2, 2.5, and 3 recipients (ovulated 0, 12, and 24 h after the donors), with 14% of the bisected and 39% of the intact embryos (P less than .05) resulting in young. Only 4% (2/48) of the day 3 bisected embryos vs. 39% (P less than .05) of the intact day 3 embryos survived in the uteri of day 2 recipients. In experiment 2, day 3 bisected and intact embryos were transferred to the oviducts of day 3, 3.5, or 4 recipients, the speculation being that the oviduct might provide a more neutral environment than the uterus. However, embryo survival was very low, except for the intact embryos transferred to synchronized recipients (42% young born). In experiment 3, 150 intact and 162 (81 pairs) bisected day 3 embryos collected from uteri were transferred to uteri of day 2.5, 3.0, and 3.5 recipient does. Significantly more pregnancies (100% vs. 47%, P less than .01) and young born (56% vs. 19%, P less than .01) resulted from intact embryos than from bisected embryos, irrespective of the uterine age.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sex-dependent loss of bisected bovine morulae after culture and freezing.

The objective of this study was to determine the post-transfer survival rate of bovine embryos cultured between the time of bisection and freezing. In this experiment 158 morulae were bisected and both portions were cultured for 24-44 h either in vivo after transfer to sheep oviducts (n = 80 morulae) or in vitro (n = 78 morulae) in Ham's F10 medium with 20% fetal calf serum, with bovine oviduct cells or in medium collected from oviduct cultures (conditioned medium). After culture, half of each morula was fixed for cytogenetic sex determination (n = 125) and the other half was frozen. The frozen halves were later thawed and transferred (n = 115) to recipients, who were, if pregnant, slaughtered to determine the sex of the fetus. The culture resulted in better pregnancy rates than those previously reported for embryos frozen immediately after bisection. The sex of 49 (33 males, 16 females) of the fixed demi-morulae was determined, and 38 of the transferred demi-morulae established pregnancies (23 males, 10 females and 5 fetuses that were not recovered). The male:female ratio in in vivo and in vitro culture groups was significantly different from the expected ratio of 1:1 and suggests that manipulation and culture of embryos results in a preferential loss of female embryos.     

The survival of in vitro shoot tips of Garcinia mangostana L. after cryopreservation by vitrification

This report highlights the first successful cryopreservation protocol for shoot tips of Garcinia mangostana L. achieved by using vitrification technique. We investigated the effects of different temperatures and exposure periods to a plant vitrification solution 2 (PVS2), sucrose concentrations and preculture periods, and unloading treatments in steps of the vitrification protocol on the survival of G. mangostana shoot tips after cryopreservation. Exposure to PVS2 for 25 min gave beneficial effects with 10.4 ± 1.8 % survival at 0 °C with average water content of 1.1 ± 0.3 g g^?1 dry mass. Survival was 13.7 ± 5.5 % when using preculture medium with full-strength Murashige and Skoog (MS) medium supplemented with 0.6 M sucrose for 2 days. A significant difference was observed in survival of shoot tips when treated with various sucrose concentrations in preculture which strengthens their importance towards enhancing survival of shoot tips after cryopreservation. MS with 0.4 M sucrose and 2 M glycerol applied as an unloading solution increased the survival of shoot tips to 44.1 ± 6.5 %. Experiments on the effect of ascorbic acid were also conducted for each step of vitrification. Our results showed higher survival of 45.8 ± 3.8 % but there were no significant effects compared with the control (without ascorbic acid). Further study on the recovery dark/light period was conducted. Survival of shoot tips significantly increased to 50.0 ± 16.7 % when subjected to 7 days in the dark before transferring to 16 h/8 h light/dark photoperiod. These studies strengthen suggestions that cryopreservation through vitrification is possible for ex situ conservation of germplasm of this tropical recalcitrant species.     

Development of infantile rat ovaries autotransplanted after cryopreservation by vitrification

We cryopreserved infantile rat ovaries by vitrification and assessed their viability by autotransplantation. Hemilateral ovarian transplantation was performed on rats on postnatal Days 10 to 12. The left ovary of each rat was dissected out, cryopreserved by vitrification using a modified vitrification solution (VS1), and then autotransplanted under the capsule of the right kidney. The right ovary of each rat was removed. For the control, the left ovary was dissected out from each rat and was immediately transplanted by the same procedure, without cryopreservation. Rats were nursed until weaning, and then the day of vaginal opening, estrous cyclicity from the day of vaginal opening until postnatal Day 84, and histology of ovarian grafts at postnatal Day 84 were examined. The time course of development of endocrine function of cryopreserved grafts was similar to that of fresh grafts. In ovarian transplants recovered on postnatal Day 84, antral follicles and corpora lutea (CL) were observed in addition to small follicles, although the number of antral follicles in cryopreserved grafts was smaller than in the fresh grafts. These results indicate that cryopreservation of ovarian tissue by vitrification can be used for the preservation of fertility and endocrine function of ovaries.     

Structural, metabolic and developmental evaluation of ovulated rabbit oocytes before and after cryopreservation by vitrification and slow freezing

The cryopreservation of oocytes is valuable for the preservation of women's fertility and might also be an interesting tool to preserve animal genetic biodiversity but it is not often used because of the very poor fertility recovered after thawing, especially in rabbit species. The objective of our study was to evaluate the effect of slow-freezing and vitrification on the structural integrity of ovulated rabbit oocytes, their ATP contents, and their developmental competence. Results show that, whatever the method is used, cryopreservation has a dramatic effect on the metabolic integrity, the structural integrity, and the developmental ability of the oocytes. Vitrification and slow freezing both impair the rabbit oocytes viability after thawing but the processes act differently. Further studies are needed to improve the cryopreservation techniques in rabbit species. Moreover, we underlined that morphology and maintenance of the structural integrity of the oocytes are not suitable enough to assess the potential for further development of cryopreserved M_II oocytes. The assessment of ATP metabolism allows efficient evaluation of the viability of the frozen or vitrified oocytes. It should be used in addition to parthenogenesis to better assess the potential for further development.     

Factors affecting the survival of one- and two-cell rabbit embryos cryopreserved by vitrification

The effects of equilibration time, glycerol (GLY), and 1,2-propanediol (PROH) concentration, and of vitrification and sucrose solution on the viability of 1- and 2-cell rabbit embryos were investigated. After collection, the embryos were equilibrated for 5 or 10 minutes in phosphate buffered saline (PBS) containing 10% GLY-20% PROH and were exposed for 30 seconds at 4 degrees C or were exposed and vitrified in one of two vitrification solutions 35% GLY-35% PROH or 20% GLY-50% PROH. The in vitro survival rates of 1-cell embryos equilibrated for both 5 and 10 minutes were lower (34.0 and 48.0%, respectively) than those of 2-cell embryos (78.8 and 68.5%, respectively; P<0.01). No differences were noted in the viability of embryos exposed to the 2 vitrification solutions. Following vitrification in a mixture of 35% GLY-35% PROH, the survival rates of 1- and 2-cell embryos were 18.3 and 13.7% and 19.6 and 10.4% for 5 and 10 minutes of equilibration, respectively. The survival rates of 1- and 2-cell embryos vitrified in a solution of 20% GLY-50% PROH were 25.7 and 35.4% and 26.2 and 21.3% for 5 and 10 minutes of equilibration, respectively. The survival rates of 1-and 2-cell embryos stored in 1M sucrose solution were 63.8 and 84.0%, respectively. In conclusion, the viability of vitrified 1- and 2-cell rabbit embryos was reduced as a consequence of their equilibration before vitrification, the exposure to vitrification solution and the dilution in a sucrose solution rather than of the vitrification process itself.     

Successful cryopreservation of mouse ovaries by vitrification

We developed a new method of cryopreservation of whole ovaries by vitrification using DAP213 (2 M dimethyl sulfoxide, 1 M acetamide, and M propylene glycol) as a cryoprotectant. Four-week-old C57BL/6 mice that underwent partial ovariectomy were orthotopically transplanted with cryopreserved or fresh ovaries (experimental or control group) isolated from 10-day-old green fluorescent protein (GFP)-transgenic mice (+/+). GFP-positive pups were similarly obtained from both groups by natural mating or in vitro fertilization (IVF) followed by embryo transfer, indicating that the cryopreserved ovaries by vitrification retain their fecundity. However, a statistically significant difference (P < 0.05) was found between both groups with respect to the following parameters: the number of GFP-positive pups born by natural mating/grafted ovary (0.8 +/- 0.3 for the experimental group versus 2.0 +/- 0.7 for the control group, mean +/- SEM), the number of collected oocytes by superovulation per mouse (7.0 +/- 1.7 for the experimental group versus 22.7 +/- 3.2 for the control group), the percentage of two-cell embryos obtained from GFP-positive oocytes by IVF (38.5% for the experimental group versus 90.0% for the control group). Histologically, normal development of follicles and formation of corpora lutea were observed in frozen-thawed grafts. However, estimated number of follicles decreased in frozen-thawed ovaries compared with fresh ovaries. Taken together, cryopreservation of the ovary by vitrification seems a promising method to preserve ovarian function, but further studies are required to overcome the possible inhibitory effects of this method on the growth of the ovarian graft.     

Post-thaw survival, cell death and actin cytoskeleton in gene-microinjected rabbit embryos after vitrification

The aim of this study was to compare two vitrification procedures (VPs), using either ethylene glycol (EG) in combination with dimethylsulfoxide (DMSO, vitrification protocol (VPI)) or Ficoll 70 (vitrification protocol II (VPII)), for rabbit embryo cryopreservation based on their post-thaw survival, cell death and actin cytoskeleton. The pronuclear stage eggs were flushed from the oviducts of the slaughtered New Zealand White rabbit does 19-20h post coitum (hpc) and randomly divided into two groups: intact (control) and microinjected (Mi). Mi embryos or intact embryos were cultured for up to 72hpc (morula stage), and then vitrified using either VPI (VPI+Mi, VPI) or VPII (VPII+Mi, VPII). After 2-3 days at -196 degrees C, the embryos were thawed and cultured until 96-100hpc to assess their development to blastocyst, apoptotic rate (TUNEL assay) and state of actin cytoskeleton (phalloidine-TRITC). Mi procedure reduced blastocyst yield, but it was higher than in either vitrified (VPI) or Mi vitrified (VPI+Mi) embryos. VPI compromised, whereas VPII did not significantly affect blastocyst development compared to intact embryos. Mi and VP both affected the embryo quality increasing TUNEL-index and decreasing the ratio of embryos with high quality actin cytoskeleton compared to control. A higher apoptotic index was recorded in VPI group. A combination of Mi and VP induced an increase in apoptotic rate (10.35% and 7.54% for VPI+Mi and VPII+Mi, respectively) as compared to Mi alone (5.7%). Ratio of embryos belonging to best actin quality (grade I) was different among groups and most of the embryos with grade I actin were in intact (84%), Mi (71%) or VPII (70%) groups. A significantly lower number of embryos with grade I actin quality was observed in VPI (58%), VPI+Mi (54%) or VPII+Mi (66%). These observations indicate that of the vitrification schemes tested, the VPII using EG and ficoll 70 as cryoprotectants, was less harmful than VPI (EG combined with DMSO in vitrification medium).     

Sheep embryo cryopreservation by vitrification and conventional freezing

The survival of ovine embryos (morulae and blastocysts) either frozen by a conventional method or vitrified was investigated in culture. In Experiment I, embryos were vitrified using a solution containing 25% propylene glycol and 25% glycerol. A group of embryos (simulated control) was processed without freezing to evaluate the toxicity of the vitrification solution. In Experiment II, embryos were exposed to a solution of PBS containing 10% glycerol and 0.25 M sucrose placed horizontally in a programmable freezer. Automatic seeding was applied at -7 degrees C in 2 positions on straws and cooled at -0.3 degrees C/min to -25 degrees C and then stored in liquid nitrogen. In vitro development rates of vitrified embryos were 12% (morulae) and 19% (blastocysts). Simulated embryos showed a higher rate of survival than embryos cryopreserved by vitrification (67 and 63%, morulae and blastocysts respectively). In conventional cooling, the blastocysts showed the highest viability percentage (67%) of all the experimental groups but these values decreased significantly in morulae (31%). Differences in temperature between straws placed in distinct positions in the freezing chamber and thermic deviation were observed when automatic seeding was applied. Embryo viability differed from 51 to 75% according the relative position of the embryos within the chamber. Survival was higher when automatic seeding was applied on the meniscus of the embryo column versus the central point of this column (65 vs 21%). The damage of both cryopreservation methods on zona pellucida integrity (27 and 35% in vitrified and conventionally frozen embryos, respectively) had no effect on the in vitro survival.     

Survival and ultrastructure of gene-microinjected rabbit embryos after vitrification

Morphological signs of injury and subsequent regeneration following vitrification of either rabbit gene microinjected (Gene-Mi) or intact in vitro cultured embryos derived from in vivo fertilized eggs were evaluated by post-warming recovery in culture and analysed by transmission electron microscopy (TEM). The percentages of vitrified/warmed Gene-Mi embryos that reached the blastocyst stage (69%) and hatched (57%) did not differ significantly from those of intact embryos (78% and 56%, respectively). In contrast, in vitro development of embryos to the blastocyst stage among non-vitrified intact (96%) and Gene-Mi (90%) embryos compared with both the intact vitrified (78%) and Gene-Mi vitrified (69%) groups, as well as hatching rate (94%, 90% vs 56%, 57%, respectively) varied significantly (p < 0.001). Observations by TEM showed that the vitrified/warmed intact or Gene-Mi embryos without post-culture displayed severe degenerative changes among their cells. During 24 h of culture a proportion of the embryos were able to regenerate and complete the compaction process. Nevertheless the signs of previous injury were retained, such as swollen cytoplasmic organelles and remaining cellular debris in the perivitelline space. These observations indicate that the procedure of gene Mi does not significantly compromise embryo tolerance to cryopreservation and post-warming developmental ability. Severe changes in embryo morphology, observed at the ultrastructural level, can be attributed to a direct influence of the vitrification process rather than to the Mi procedure itself.     

Highly efficient vitrification for cryopreservation of human oocytes and embryos: the Cryotop method

Vitrification is frequently referred to as a novel technology of cryopreservation in embryology, although some young embryologists were born after its first successful application. Unfortunately, in spite of the accumulated evidence regarding its enormous potential value, most domestic animal and human laboratories use exclusively the traditional slow-rate freezing with its compromised efficiency and inconsistency. The purpose of this paper is to clarify terms and conditions, to summarize arguments supporting or disapproving the use of vitrification, and to outline its role among assisted reproductive technologies. To provide evidence for the potential significance of vitrification, achievements with the Cryotop technology, an advanced version of the "minimal volume approaches" is analyzed. This technology alone has resulted in more healthy babies after cryopreservation of blastocysts than any other vitrification technique, and more successful human oocyte vitrification resulting in normal births than any other cryopreservation method. The value of this method is also demonstrated by achievements in the field of domestic animal embryology. A modification of the technique using a hermetically sealed container for storage may help to eliminate potential dangers of disease transmission and open the way for widespread application for cryopreservation at all phases of oocyte and preimplantation embryo development in mammals.     

Survival and genetic stability of Dendranthema grandiflora Tzvelev shoot apices after cryopreservation by vitrification and encapsulation-dehydration

The aim of this study was to compare the genetic stability of chrysanthemum (cv. Pasodoble) apices cryopreserved using two different methods: encapsulation-dehydration and vitrification. The assessment of the genetic stability was developed using RAPDs markers. Assessment of stability was evaluated in pot-cultivated mother plants (from which buds were excised for micropropagation), in shoots (leave tissue) from which apices were extracted for cryopreservation, and in shoots regenerated from cryopreserved apices 30 days after recovery and after further 3 months in culture. Throughout the process the origin of the apices (in vitro-shoot from which they were excised) was recorded. Twenty one regenerants cryopreserved by vitrification and 25 by encapsulation-dehydration were assessed. Only one cryopreserved regenerant from the encapsulation-dehydration method showed a different band pattern. These results support the necessity of monitoring the genetic stability of the regenerants obtained after cryopreservation, as this is a very useful technique for the conservation of plant genetic resources.     

Effects of cryopreservation on glucose metabolism and survival of bovine morulae and blastocysts derived in vitro or in vivo

Bovine morulae and blastocysts were either produced in vitro through maturation, fertilization and culture of immature oocytes recovered from slaughterhouse-derived ovaries, collected in vivo or obtained after 24 h in vitro culture of in vivo collected embryos. The morulae and blastocysts were classified into four categories of embryo quality and two stages of embryonic development. Embryos were frozen by a controlled freezing method using 10% glycerol as a cryoprotectant. The ability of individual embryos to withstand freeze/thawing was measured immediately before and after cryopreservation by changes in CO2 production from (U-14C)glucose during a 2 h incubation period in a non-invasive closed system immediately before and after cryopreservation. Post-thaw survival was assessed by development in vitro during a 48 h culture period. Survival rates and oxidative metabolism after freeze/thawing were similar in embryos of the two developmental stages. However, after freeze/thawing, the rate of CO2 production of in vitro produced embryos was reduced to one half of their pre-freeze levels and was associated with poor survival rates. In vivo collected embryos had a significantly better tolerance to freezing and higher survival rates. However, when in vivo embryos were exposed to in vitro culture conditions, the rates of CO2 production and survival were significantly reduced. Pre-freeze embryo quality affected post-thaw in vitro development and metabolic activity markedly in embryos produced in vitro or pre-exposed to in vitro culture conditions. While there was no relationship between pre-freeze levels of CO2 production and post-thaw in vitro embryo development, all embryos which developed in vitro after freezing/thawing retained at least 58% of the pre-freeze levels of CO2 production regardless of their origin. Results of the present study indicate that embryos produced in vitro or pre-exposed to in vitro culture conditions are more sensitive to cryo-injury. This sensitivity is affected by embryo quality and is similarly reflected at the biochemical level. Determination of oxidative metabolism offers a feasibility for selection of viable morulae/blastocysts after freezing/thawing.     

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.