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.     

In vivo development of vitrified rabbit embryos: Effects on prenatal survival and placental development

The aim of this work is to study the effect of the vitrification procedure on prenatal survival and on placental development at the end of gestation in rabbits (Oryctolagus cuniculus). One hundred eighty-one females were slaughtered at 72 h of gestation. Morphologically normal embryos recovered at 72 h of gestation were kept at room temperature until transfer or vitrification. Vitrified embryos (320 embryos) were transferred into a total of 24 does and fresh embryos (712 embryos) were transferred into a total of 43 does. Females were induced to ovulate 72 h before transfer when fresh embryos were transferred and 60 to 63 h before transfer when vitrified embryos were transferred. Each recipient doe received eight embryos into the left oviduct and eight embryos into the right oviduct. The number of implanted embryos was estimated by laparoscopy as number of implantation sites at Day 14 of gestation. Recipient females were slaughtered by stunning and exsanguination 25 d after the transfer, and fetuses were classified according to their status. Live fetuses and fetal and maternal placenta were weighed Pregnancy rate was defined as the total number of females having at least one live fetus at Day 28 of gestation divided by the total number of females. Prenatal survival was estimated as live fetuses at Day 28 of gestation divided by the number of transferred embryos. The pregnancy rate after transfer of vitrified embryos (92%) was similar to that achieved with fresh embryos (86%), but prenatal survival was lower for vitrified than for fresh embryos (53% vs. 34%). We did not find differences in embryo survival from 72 h to implantation. Transfer of vitrified embryos reduced fetal survival from implantation to Day 28 (57% vs. 82%). Differences in the number of live fetuses at Day 28 of gestation were mainly due to the higher fetal mortality observed soon after implantation in pregnancies resulting from the transfer of vitrified embryos. A higher percentage of decidual reactions and atrophic maternal placentas (27.5% vs. 8.3%) and also of atrophic fetal and maternal placentas (12.1% vs. 5.3%) were observed after transfer of vitrified embryos. Both treatments showed similar percentage of dead fetuses (3.3% vs. 4%). Maternal placenta of the fetuses from fresh embryos was 15% heavier than maternal placenta of fetuses from vitrified embryos.     

Effects of Different Maturation Systems on Bovine Oocyte Quality,Plasma Membrane Phospholipid Composition and Resistance to Vitrification and Warming

The objective of this study was to evaluate the effects of different maturation systems on oocyte resistance after vitrification and on the phospholipid profile of the oocyte plasma membrane (PM). Four different maturation systems were tested: 1) in vitro maturation using immature oocytes aspirated from slaughterhouse ovaries (CONT; n = 136); 2) in vitro maturation using immature oocytes obtained by ovum pick-up (OPU) from unstimulated heifers (IMA; n = 433); 3) in vitro maturation using immature oocytes obtained by OPU from stimulated heifers (FSH; n = 444); and 4) in vivo maturation using oocytes obtained from heifers stimulated 24 hours prior by an injection of GnRH (MII; n = 658). A sample of matured oocytes from each fresh group was analyzed by matrix associated laser desorption-ionization (MALDI-TOF) to determine their PM composition. Then, half of the matured oocytes from each group were vitrified/warmed (CONT VIT, IMA VIT, FSH VIT and MII VIT), while the other half were used as fresh controls. Afterwards, the eight groups underwent IVF and IVC, and blastocyst development was assessed at D2, D7 and D8. A chi-square test was used to compare embryo development between the groups. Corresponding phospholipid ion intensity was expressed in arbitrary units, and following principal components analyses (PCA) the data were distributed on a 3D graph. Oocytes obtained from superstimulated animals showed a greater rate of developmental (P<0.05) at D7 (MII = 62.4±17.5% and FSH = 58.8±16.1%) compared to those obtained from unstimulated animals (CONT = 37.9±8.5% and IMA = 50.6±14.4%). However, the maturation system did not affect the resistance of oocytes to vitrification because the blastocyst rate at D7 was similar (P>0.05) for all groups (CONT VIT = 2.8±3.5%, IMA VIT = 2.9±4.0%, FSH VIT = 4.3±7.2% and MII VIT = 3.6±7.2%). MALDI-TOF revealed that oocytes from all maturation groups had similar phospholipid contents, except for 760.6 ([PC (34:1) + H]⁺), which was more highly expressed in MII compared to FSH (P<0.05). The results suggest that although maturation systems improve embryonic development, they do not change the PM composition nor the resistance of bovine oocytes to vitrification.     

Cryotops versus open-pulled straws (OPS) as carriers for the cryopreservation of bovine oocytes: effects on spindle and chromosome configuration and embryo development

Two experiments were designed to assess the effectiveness of cryopreserving bovine MII oocytes using cryotops as the carrier system for vitrification. In the first experiment, we examined the developmental competence of oocytes after: (i) vitrification in open-pulled straws (OPS method); or (ii) vitrification in <0.1 μl medium droplet on the surface of a specially constructed fine polypropylene strip attached to a plastic handle (Cryotop method). In the second experiment, warmed oocytes that had been vitrified in OPS or cryotops were fixed to analyze spindle and chromosome configuration. In all experiments both cow and calf oocytes were used. Significantly different fertilization rates were observed between the vitrification groups: 31.5% and 20.2% for the cow and calf oocytes vitrified in OPS, respectively, versus 46.1% and 46.4% for the oocytes vitrified using cryotops. After in vitro fertilization, 3.8% of the calf oocytes and 5.3% of the cow oocytes developed to the blastocyst stage. All blastocysts from vitrified oocytes resulted from the Cryotop method. A significantly lower percentage of the OPS-vitrified calf oocytes showed a normal spindle configuration (37.8%) compared to control fresh oocytes (69.9%), while normal spindle and chromosome configurations were observed in a significantly higher proportion of the cryotop-vitrified calf oocytes (60.2%). For the cow oocytes, 60.6% in the OPS group and 60.3% in the Cryotop group exhibited a normal morphology after warming. These findings suggest the cryotop system is a more efficient carrier for vitrification than OPS for the cryopreservation of bovine oocytes.     

Successful vitrification of early-stage porcine cloned embryos

The objective of this study was to investigate the survival and development of porcine cloned embryos vitrified by Cryotop carrier at the zygote, 2- and 4-cell stages. The quality of resultant blastocysts was evaluated according to their total cell number, apoptotic cell rate, reactive oxygen species (ROS) production, glutathione (GSH) content and mRNA expression levels of genes related to embryonic development. The survival rates of zygotes, 2- and 4-cell embryos after vitrification did not differ from those of their fresh counterparts. Vitrification still resulted in significantly decreased blastocyst formation rates of these early-stage embryos. Moreover, the total cells, apoptotic rate, ROS and GSH levels in resultant blastocysts were unaffected by vitrification. The mRNA expression levels of PCNA, CPT1, POU5F1 and DNMT3B in the blastocysts derived from vitrified early-stage embryos were significantly higher than those in the fresh blastocysts, but there was no change in expression of CDX2 and DNMT3A genes. In conclusion, our data demonstrate that the early-stage porcine cloned embryos including zygotes, 2- and 4-cells can be successfully vitrified, with respectable blastocyst yield and quality.     

Transfer of fresh and cryopreserved IVP bovine embryos: normal calving, birth weight and gestation lengths

In vitro and in vivo developmental competence of fresh and cryopreserved in vitro produced (IVP) bovine embryos was evaluated up to birth. Three experiments were done. The objective in the first experiment was to develop an optimal vitrification procedure for IVP bovine embryos by determining effects of exposure time (2, 5, 10, 20 min) and temperature (4, 22, 27 degrees C) in cryoprotective agents prior to vitrification on their post-thaw viability. The best combination was used in Experiments 2 and 3. In the second experiment, the importance of post-thaw morphologic selection on pregnancy rates was determined by transferring either selected or unselected single embryos. In the third experiment, pregnancy initiation, maintenance and calving results of vitrified embryos were compared with fresh and conventionally frozen embryos. Fetal losses, birth weights, gestation lengths and frequency of dystocia in the third experiment were monitored. The interaction of exposure time and temperature on both post-thaw re-expansion and hatching rates was significant (P < 0.01). Five minute exposure at 27 degrees C was optimal. In the second experiment, post-thaw selected vitrified embryos had higher pregnancy rates than unselected embryos (P < 0.05). In the third experiment, the pregnancy rate of vitrified embryos did not differ from that of fresh embryos (P > 0.05). However, pregnancy rate of conventionally frozen embryos was lower than that of fresh or vitrified embryos (P < 0.05). Of 92 calves born, 53 were male and 39 were female. Birth weights and dystocia scores of single-born calves did not differ between sexes (P > 0.05). Twin-born calves were lighter than single-born calves (P < 0.05). Overall, the data demonstrate that the transfer of vitrified IVP bovine embryos can result in healthy, apparently normal calves similar to those derived from transfer of fresh and conventionally frozen IVP bovine embryos.     

Effect of the cryoprotectant concentration on the in vitro embryo development and cell proliferation of OPS-vitrified porcine blastocysts

Our objective was to study the effect of the concentration of ethylene glycol (EG) and dimethyl sulfoxide (Me₂SO) during vitrification on the development of porcine blastocysts. Vitrification was performed with 0.4 M sucrose and either a Me₂SO and EG mixture (15%, 16% and 17% v/v of each) or EG alone (40% v/v), using superfine open pulled straws. Fresh and vitrified blastocysts were cultured for 48 h and the survival and hatching rates were evaluated. Some vitrified and fresh embryos were processed for Hoechst 33342 staining and proliferation cell nuclear antigen (PCNA) inmunolocalization to determine the proliferation index. The survival rate was similar for fresh and vitrified blastocysts, except for blastocysts vitrified using 15% of cryoprotectants, which displayed lower (P < 0.05) survival than fresh blastocysts. Vitrified and fresh blastocysts had a similar cell proliferation index (range: 75.8 ± 3.2 to 83.7 ± 3). When only hatched blastocysts among groups were compared, the proliferation rate decreased (P < 0.05) after vitrification with 17% of EG–Me₂SO. In conclusion, the concentration of EG–Me₂SO could be decreased to 16% in the vitrification medium with no reduction of the in vitro developmental ability of the blastocysts. In addition, a 40% EG-based medium can be used for vitrification with similar results to those achieved with a medium containing 16% EG–Me₂SO.     

Normal calves from transfer of biopsied, sexed and vitrified IVP bovine embryos

Data on biopsied, sexed and cryopreserved in vitro produced (IVP) bovine embryos, and their in vivo developmental competence are very limited. Two preliminary studies were conducted before the primary study. In Experiment 1, post-thaw in vitro developmental competence of biopsied and vitrified IVP embryos was evaluated using re-expansion as an endpoint. In Experiment 2, the pregnancy rates of biopsied fresh, frozen or vitrified embryos following single embryo transfer were compared. Since vitrified embryos resulted in a higher pregnancy rate than frozen-thawed embryos, in the primary study (Experiment 3), all IVP embryos were vitrified following biopsy and sexing (by DNA fingerprinting). In Experiment 3, we compared pregnancy initiation and calving results of heifers in the following treatments: 1) artificial insemination (AI); 2) AI plus contralateral transfer of a single embryo (AI + SET); 3) ipsilateral transfer of single embryo (SET); or 4) bilateral transfer of two embryos (DET). Birth weights, gestation lengths and dystocia scores were recorded. In Experiment 1, post-thaw re-expansion rate of biopsied and vitrified embryos was 85% (70/82). In Experiment 2, pregnancy rates (90 d) were 44% (7/16), 23% (3/13), and 50% (7/14) for vitrified, frozen and fresh embryos, respectively (P < 0.10). In Experiment 3, pregnancy rates of AI and SET were 65% (20/31) and 40% (16/40), respectively (P < 0.05). The pregnancy rate of AI + SET was 75% (27/36) with 11 carrying twins, and the pregnancy rate of DET was 72% (26/36) with 10 carrying twins. All AI fetuses were carried to term, but only half the SET fetuses were carried to term. Similar calving rates were observed in the AI + SET and DET groups, 76 and 70%, respectively, of those pregnant at Day 40. Mean birth weight, dystocia score and gestation length of AI calves were not different from those of SET calves. Mean birth weight and dystocia score of single-born calves were greater than those of twin born calves (P < 0.05). These data demonstrate that biopsied IVP bovine embryos can be successfully cryopreserved by vitrification and following post-thaw embryo transfer, acceptable rates of offspring with normal birth weights can be obtained without major calving difficulties.     

Survival rate of preantral follicles derived from vitrified neonate mouse ovarian tissue by Cryotop and conventional methods

The aim of this study was to investigate the growth and survival rate of preantral follicles isolated from vitrified ovarian tissue by Cryotop and conventional methods. The ovaries of 14-day-old mice were separated and divided into four groups as following: Cryotop group, vitrified by Cryotop; CV (Conventional; CV) group, vitrified by conventional straw; toxicity test group and control group. After warming the vitrified ovaries, isolated preantral follicles from four groups were cultured for 4 days to compare survival rate and follicular growth between above-mentioned groups. Survival rate (97.3%) in toxicity test group alike the control group (98.7%) were significantly higher (P<0.05) than the Cryotop (92.7%) and CV (47.7%) groups. Increase in follicle diameters after 4 days in Cryotop and CV groups was significantly lower (P<0.05) than the control and toxicity test groups, but growth and survival rate of follicles in Cryotop group was significantly higher (P<0.05) than the CV group. These results demonstrated that ovarian tissue vitrification by Cryotop highly preserves the viability rate of preantral follicles.     

Embryo survival and birth rate after minimum volume vitrification or slow freezing of in vivo and in vitro produced ovine embryos

The objective was to evaluate pregnancy outcomes and birth rate of in vivo derived vs. in vitro produced ovine embryos submitted to different cryopreservation methods. A total of 197 in vivo and 240 in vitro produced embryos were cryopreserved either by conventional freezing, or by vitrification with Cryotop or Spatula MVD methods on Day 6 after insemination/fertilization. After thawing/warming and transfer, embryo survival rate on Day 30 of gestation was affected by the source of the embryos (in vivo 53.3%, in vitro 20.8%; P < 0.05) and by the method of cryopreservation (conventional freezing 26.5%, Cryotop 52.0%, Spatula MVD 22.2%; P < 0.05). For in vivo derived embryos, survival rate after embryo transfer was 45.6% for conventional freezing, 67.1% for Cryotop, and 40.4% for Spatula MVD. For in vitro produced embryos, survival rate was 7.3% for conventional freezing, 38.7% for Cryotop, and 11.4% for Spatula MVD. Fetal loss from Day 30 to birth showed a tendency to be greater for in vitro (15.0%) rather than for in vivo produced embryos (5.7%), and was not affected by the cryopreservation method. Gestation length, weight at birth and lamb survival rate after birth were not affected by the source of the embryo, the cryopreservation method or stage of development (average: 150.5 ± 1.8 days; 4232.8 ± 102.8 g; 85.4%; respectively). This study demonstrates that embryo survival and birth rate of both in vivo and in vitro produced ovine embryos are improved by vitrification with the minimum volume Cryotop method.     

Successful vitrification of pronuclear-stage rabbit zygotes by minimum volume cooling procedure

Rabbit zygotes at the pronuclear-stage were cryopreserved by vitrification using a gel-loading tip (GL-tip), Cryoloop or Cryotop. In GL-tip and Cryoloop methods, zygotes were first exposed to 10% ethylene glycol (EG)+10% DMSO in TCM199+20% fetal bovine serum (FBS) for 2 min, and then equilibrated for 30 s in a vitrification solution composed of 20% EG+20% DMSO+0.6 M sucrose in TCM199+20% FBS. In Cryotop method, zygotes were first exposed to 7.5% EG+7.5% DMSO+20% FBS in TCM199 for 3 min, and then equilibrated for 1 min in a vitrification solution composed of 15% EG+15% DMSO+0.5 M sucrose+20% FBS in TCM199. In vitro culture of vitrified-warmed zygotes using GL-tip and Cryoloop resulted in low cleavage rates (2 and 5%, respectively) and no development into blastocysts. In contrast, zygotes vitrified-warmed using Cryotop exhibited higher proportions of cleavage (58%) and development into blastocysts (24%). When compacted morulae or early blastocysts were vitrified by these three procedures, 80-93% of them exhibited blastocoele expansion or zona hatching during the subsequent 48 h of culture. Use of Cryotop instead of GL-tip or Cryoloop for zygote vitrification, without changing conditions of solutions and periods for exposure, equilibration and post-warm dilution, resulted in cleavage and blastocyst development rates of 88 and 45%, respectively. A longer exposure time (10 min) of zygotes to 7.5% EG+7.5% DMSO+20% FBS in TCM199 resulted in higher proportions of zygotes cleaving (94%) and developing into blastocysts (51%) after Cryotop vitrification. Proportions of post-warm zygotes (10-min exposure group) and fresh control zygotes developing into newborn offspring were 36 and 53%, respectively. Pronuclear-stage rabbit zygotes were successfully cryopreserved by vitrification using the Cryotop method.     

Effects of cryodevice type and donors' sexual maturity on vitrification of minke whale (Balaenoptera bonaerensis) oocytes at germinal vesicle stage

Germinal-vesicle-stage oocytes enclosed with compact cumulus cell layers (COCs) were recovered from adult or prepubertal minke whale ovaries, and were vitrified in a solution containing 15% ethylene glycol, 15% DMSO and 0.5 M sucrose using either a Cryotop or an open-pulled straw (OPS) as the cryodevice. The post-warm COCs with normal morphology were cultured for 40 h in a 390 mosmol in vitro maturation medium, and oocytes extruding the first polar body were considered to be matured. The proportion of morphologically normal COCs after vitrification and warming was higher when the COCs were cryopreserved by Cryotop (adult origin, 88.4%; prepubertal origin, 80.8%) compared with the OPS (adult origin, 67.7%; prepubertal origin, 64.2%). The oocyte maturation rate was higher in the adult/Cryotop group (29.1%) compared with those of the prepubertal/Cryotop group (14.4%), the adult/OPS group (14.3%) and the prepubertal/OPS group (10.6%). These results indicate that the Cryotop is a better device than the OPS for vitrification of immature oocytes from adult minke whales.     

Effect of coculture with oviduct epithelial cells on viability after transfer of vitrified in vitro produced goat embryos

This study evaluates the effect of coculture with goat oviduct epithelial cells (GOEC) on the pregnancy rate, embryo survival rate and offspring development after direct transfer of vitrified/thawed caprine in vitro produced (IVP) embryos. Oocytes were recovered from slaughterhouse goat ovaries, matured and inseminated with frozen/thawed capacitated semen, and presumptive zygotes were randomly cultured in synthetic oviduct fluid (SOF) (n=352) or GOEC (n=314). The percentage of cleaved embryos reaching the blastocyst stage was 28% and 20% in SOF and GOEC, respectively (P<0.05). Overall, 26 blastocysts of SOF were transferred freshly in pairs to recipient goats, whereas 58 of SOF and 36 of GOEC were vitrified and transferred directly in pairs to recipient goats after thawing without removal of cryoprotectants or morphological evaluation. The kidding rate was 92% for SOF fresh, 14% for SOF vitrified (P<0.001) and 56% for GOEC vitrified (P<0.05); the difference was also significant between vitrified groups (P<0.01). The embryo survival rate was 62% for SOF fresh, 9% for SOF vitrified (P<0.001) and 33% for GOEC vitrified (P<0.05) with a significant difference between vitrified groups (P<0.01). The results showed that the coculture of IVP goat embryos with GOEC significantly improves the pregnancy and embryo survival rates and leads to the birth of healthy offspring. However, further research using more defined GOEC coculture is required to confirm its capacity to increase the success rate of IVP embryo technology in goat.     

Expression of miR-Let-7a,miR-15a,miR-16-1, and their target genes in fresh and vitrified embryos and its surrounding culture media for noninvasive embryo assessment

microRNAs (miRNAs) play a critical role in implantation and development of mouse embryos. In this study, we aim to evaluate the possibility of miRNAs as potential biomarkers in the blastocyst culture to assess embryo quality. We also intend to investigate whether improved clinical outcomes of vitrified embryos agree with altered miRNA expressions. Mouse embryos from in vitro fertilization were vitrified at the two-cell stage. After thawing, the embryos were individually cultured and developed to the blastocyst stage. We used quantitative real-time polymerase chain reaction to evaluate miRNA expression levels in both vitrified and fresh groups, and culture medium (CM). The fibronectin binding assay was performed to examine for blastocyst attachment. The findings showed reduced expressions of miR-16-1 (0.2 ± 0.06) and miR-Let-7a (0.65 ± 0.1) after vitrification compared to fresh embryos. We observed significant upregulation of the target genes Vav3 (4.33 ± 0.25), integrin β-3 (Itg β3; 4.73 ± 0.2), and Bcl2 (2.29 ± 0.16) in the vitrified embryos compared to the fresh groups. Evaluation of blastocyst CM showed upregulation of miR-Let-7a (15.68 ± 0.89), miR-16-1 (16.18 ± 0.75), and miR-15a (13.36 ± 0.73) in the vitrified group in comparison to the fresh blastocysts (P < .05). The expression levels of miR-16-1 (3.28 ± 0.63), miR-15a (5.91 ± 0.38), and miR-Let-7a (9.07 ± 0.6) in CM of the vitrified blastocysts conducted on fibronectin were significantly higher than the fresh group (P < .05).This study showed that vitrification of embryos changes implantation and proliferation biomarkers. In addition, upregulated miRNAs in CM could be potentially used for noninvasive early assessment of embryo quality.     

Effect of vitrification on mitochondrial distribution and membrane potential in mouse two pronuclear (2‐PN) embryos

The present study was designed to investigate the effect of vitrification on mitochondrial distribution, membrane potential (Δψ) and microtubule distribution in mouse 2‐PN embryos, as well as to document the relationship between mitochondrial distribution and developmental ability of those embryos. Mitochondrial distribution was examined by fluorescence microscopy technology. Results indicated that: (1) The rate of mitochondrial ring formation around pronuclei in vitrified 2‐PN embryos was significantly lower than in fresh ones (67.3 ± 3.0% vs. 84.9 ± 3.1%) (P < 0.05). (2) Blastocyst development rate of vitrified 2‐PN embryos without mitochondrial rings (61.7 ± 4.5%) was significantly lower than that of vitrified embryos with mitochondrial rings (82.1 ± 2.8%). (3) Following staining by 5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethyl‐imidacarbo‐cyanine iodide (JC‐1), most red‐colored mitochondria (high Δψ) were distributed peripherally around pronuclei and along cell membranes of fresh 2‐PN embryos. Conversely, red‐colored mitochondria were greatly diminished in vitrified embryos, with green mitochondria (low Δψ) evenly distributed throughout the cytoplasm. The proportion of fresh 2‐PN embryos with obvious aggregation of high Δψ mitochondria (84.2 ± 2.2%) was significantly higher than that of vitrified embryos (26.7 ± 3.0%) (P < 0.05). (4) The proportion of fresh embryos with microtubules distributed around pronuclei (83.5 ± 3.4%) was similar to that of vitrified embryos (74.7 ± 2.5%). In conclusion, vitrification affected mitochondrial distribution and decreased the mitochondrial membrane potential in mouse 2‐PN embryos, events which may affect subsequent developmental viability of such embryos. Mol. Reprod. Dev. 76: 1056–1063, 2009. © 2009 Wiley‐Liss, Inc.     

Vitrification of Mouse Embryos at Various Stages by Open-Pulled Straw (OPS) Method

This study was performed to pursue the optimal condition for the cryopreservation of mouse morulae by a two-step OPS method and to investigate the feasibility of the optimal condition for vitrification of embryos at other developmental stages. First, the mouse morulae were vitrified in OPS using one-step procedure—that is, embryos were vitrified after direct exposure to EDFS30 (15% ethylene glycol (EG), 15% dimethyl sulfoxide (DMSO), Ficoll and sucrose), or two-step method—that is, embryos were first pretreated in 10%E+10%D (10% EG and 10% DMSO in mPBS) for 30 sec, then exposed to EDFS30 for 15 to 60 sec, respectively. After vitrification and warming, the embryos were morphologically evaluated and assessed by their development to blastocysts, expanded/hatched blastocysts, or to term after transfer. The result showed that all the vitrified-warmed morulae had similar blastocyst rate compared to that of control (91.7% vs. 100%), and the highest developmental rate to expanded blastocysts (100%) or hatched blastocysts (62.3%) was observed when the morulae were pretreated with 10%E+10%D for 0.5 min, exposed to EDFS30 for 25 sec before vitrification and warming in 0.5 M sucrose for 5 min. After transfer, the survival rate (33.1%) in vivo of the vitrified morulae was higher (P > 0.05) than that of the fresh embryos (24.6%). Secondly, embryos at different stages were cryopreserved and thawed following the above program. Most (93.4 to 100%) of the embryos recovered after vitrification were morphologically normal at all the developmental stages. The blastocyst rates of the vitrified one-cell (52.5 to 66.7%) and the two-cell (63.3 to 68.9%) embryos were lower (P < 0.05) than those of the vitrified four-cell embryos (81.7 to 86.4%), the eight-cell embryos (90.0 to 93.3%), morulae (96.7 to 100%), and the expanded blastocysts rate (98.3 to 100.0%) of the vitrified early blastocysts. The highest survival rate in vivo of vitrified embryos were from the early blastocysts (40.4%), which was similar to that of fresh embryos (48.6%). The data demonstrate that the optimal protocol for the cryopreservation of morulae was suitable for the four-cell embryos to early blastocyst stages and that the early blastocyst stage is the most feasible stage for mouse embryo cryopreservation under our experimental conditions.     

Determination of sex and scrapie resistance genotype in preimplantation ovine embryos

The aim of this study was to test the accuracy of genotype diagnosis after pre-amplification of DNA extracted from biopsies obtained by microblade cutting of ovine embryos and to evaluate the viability of biopsied embryos after vitrification/warming and transfer to recipients. Sex and PrP genotypes were determined. Sex diagnosis was done by PCR amplification of ZFX/ZFY and SRY sequences after PEP-PCR while PrP genotype determination was performed after specific pre-amplification of specific target including codons 136, 154 and 171. Embryos were collected at Day 7 after oestrus. Blastocysts and expanded blastocysts were biopsied immediately after collection whereas compacted morulae were biopsied after 24 hr of in vitro culture. Eighty-nine biopsied embryos were frozen by vitrification. Fresh and vitrified whole embryos were kept as control. DNA of biopsies was extracted and pre-amplified. Sex diagnosis was efficient for 96.6% of biopsies and PrP genotyping was determined in 95.8% of codons. After embryo transfer, no significant difference was observed in lambing rate between biopsied, vitrified control and fresh embryos (54.5%, 60% and 66.6%, respectively). Embryo survival rate was not different between biopsied and whole vitrified embryos (P = 0.38). At birth, 96.7% of diagnosed sex and 95.4% of predetermined codons were correct. Lamb PrP profiles were in agreement with parental genotype. PEP-PCR coupled with sex diagnosis and nested PCR coupled with PrP genotype predetermination are very accurate techniques to genotype ovine embryo before transfer. These original results allow planning of selection of resistant genotype to scrapie and sex of offspring before transfer of cryopreserved embryo.     

Vitrification of porcine embryos at various developmental stages using different ultra-rapid cooling procedures

In this study, three different vitrification systems (open pulled straw: OPS; superfine open pulled straw: SOPS; and Vit-Master technology using SOPS: Vit-Master-SOPS) were compared in order to investigate the influence of cooling rate on in vitro development of vitrified/warmed porcine morulae, early blastocysts, or expanded blastocysts. Embryos were obtained surgically on Day 6 of the estrous cycle (D0 = onset of estrus) from weaned crossbred sows, classified and pooled according their developmental stage. A subset of embryos from each developmental stage was cultured to evaluate the in vitro development of fresh embryos; the remaining embryos were randomly allocated to each vitrification system. After vitrification and warming, embryos were cultured in vitro for 96 h in TCM199 with 10% fetal calf serum at 39 degrees C, in 5% CO(2) in humidified air. During the culture period, embryos were morphologically evaluated for their developmental progression. The developmental stage of embryos at collection affected the survival and hatching rates of vitrified/warmed embryos (P < 0.001). The vitrification system or the interaction of vitrification system and developmental stage had no effect on these parameters (P > 0.05). Vitrified expanded blastocysts showed the best development in vitro (P < 0.001), with survival and hatching rates similar to those of fresh expanded blastocysts. The hatching rate of fresh morula or early blastocyst stage embryos was higher than their vitrified counterparts. In conclusion, under our experimental conditions, cooling rates greater than 20,000 degrees C/min, as occurs when SOPS or Vit-Master-SOPS systems are used, do not enhance the efficiency of in vitro development of vitrified porcine embryos.     

Production of transgenic mice from vitrified pronuclear-stage embryos.

Cryopreservation of pronuclear-stage embryos would be useful for transgenic technology and genome preservation purposes. We compared a novel vitrification technique (solid surface vitrification, SSV) with another vitrification method in straws for cryosurvival and to generate transgenic progeny from cryopreserved mouse zygotes following microinjection. The SSV solution consisted of 35% ethylene glycol (EG), 5% polyvinyl-pyrrolidone (PVP), and 0.4 M trehalose in M2 supplemented with 4 mg/ml BSA; the in straw vitrification solution was 7 M EG in M2 plus BSA. In experiment I, we compared the effect of the vitrification solutions alone, without cooling. No reduction was detected in survival and cleavage rates. In experiment II, SSV yielded a significantly higher percentage of morphologically normal zygotes (96%) that also cleaved at significantly higher rates (80%) when compared to that following "in straw" vitrification (68 and 66%, respectively). Cleavage rate in the non-vitrified control group (93%) was significantly higher than that of both vitrified groups. Following embryo transfer, there was no difference in the rate of pups obtained from the SSV, "in straw" vitrified, and control groups (97/457, 21%; 15/75, 20% and 56/209, 27%, respectively). In experiment III, SSV vitrified and fresh embryos were used for pronuclear DNA injection. Survival rate of vitrified embryos after microinjection was reduced compared to nonvitrified ones (64 vs. 72%, respectively; P < 0.05); however, development to two-cell stage was not different (76 vs. 72%, respectively). Following embryo transfer of vitrified vs. fresh microinjected embryos, in both cases 10% live pups were generated, including transgenic pups. The results demonstrated that the efficiency of generating transgenic pups from SSV vitrified pronuclear zygotes is comparable to that from fresh embryos.