In vitro culture of goat morulae to blastocysts before freezing

The results of transfer of frozen-thawed caprine embryos that were collected either as blastocysts or morulae and cultured to the blastocyst stage prior to freezing were compared. After thawing, the embryos collected as blastocysts appeared to be of marginally better quality than those that had been cultured from morulae (89 vs 72% rated as good; P > 0.05). The transfer of 24 frozen-thawed embryos collected as blastocysts to 12 recipients resulted in a pregnancy rate of 83% (10/12) and an embryo survival rate of 67%. Corresponding results for frozen-thawed blastocysts that had been cultured from morulae and were transferred to 11 recipients were 54% (6/11) and 41%, respectively. Since an earlier investigation had shown that the transfer of frozen caprine morulae yields very poor results, in our laboratory all morulae are now cultured to the blastocyst stage before being cryopreserved.     

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.     

Nuclear transfer in cattle using in vivo-derived vs. in vitro-produced donor embryos: Effect of developmental stage

To determine the best developmental stage of donor embryos for yielding the highest number of clones per embryo, we compared the efficiencies of nuclear transfer when using blastomeres from morulae or morulae at cavitation, or when using inner-cell-mass cells of blastocysts as nuclear donors. This comparison was done both on in vivo-derived and in vitro-produced donor embryos. In experiment 1, with in vivo-derived donor embryos, nuclei from morulae at cavitation supported the development of nuclear transfer embryos to the blastocyst stage (36%) at a rate similar to that of nuclei from morulae (27%), blastomeres from morulae at cavitation being superior (P < 0.05) to inner-cell-mass cells from blastocysts (21%). The number of blastocysts per donor embryo was significantly (P < 0.05) higher when using nuclei from morulae at cavitation (15.7 ± 4.1) rather than nuclei from morulae (9.8 ± 5.5) or blastocysts (6.3 ± 3.3). With in vitro-produced donor embryos (experiment 2), nuclei from morulae yielded slightly more blastocysts (32%) than nuclei from morulae at cavitation (29%), both stages being superior to nuclei from blastocysts (15% development to the blastocyst stage). Morulae at cavitation yielded a higher number of cloned blastocysts per donor embryo (11.5 ± 5.9) than did morulae (9.3 ± 3.2) and blastocysts (3.3 ± 1.4). Transfer of cloned embryos originating from in vivo-derived morulae, morulae at cavitation, and blastocysts resulted in four pregnancies (10%), three pregnancies (7%), and one (17%) pregnancy on day 45. The corresponding numbers of calves born were 3 (4%), 3 (7%), and 0, respectively. After transfer of blastocysts derived from in vitro nuclear donor morulae (n = 16) and morulae at cavitation (n = 7), two (20%) and two (50%) recipients, respectively, were pregnant on day 45. However, transfer of seven cloned embryos from in vitro donor blastocysts to three recipients did not result in a pregnancy. Using in vitro-produced donor embryos, calves were only obtained from morula-stage donors (13%). Our results indicate that the developmental stage of donor embryos affects the efficiency of nuclear transfer, with morulae at cavitation yielding a high number of cloned blastocysts. © 1996 Wiley-Liss, Inc.     

Viability of frozen-thawed bovine embryos bisected in sucrose: A preliminary report

A method for producing identical twin calves is described in which Day 7 frozen-thawed bovine embryos in 12.5% sucrose solution were bisected using a fine microsurgical blade. The resulting bisected embryos were transferred nonsurgically to the uterine horn ipsilateral to the corpus luteum of synchronous recipients (+/-1 d), two bisected embryos per recipient. The pregnancy rate when both halves remained in the same zona pellucida was 50% (5 10 ); the pregnancy rate was 1 5 for morulae and 4 5 for blastocysts. The pregnancy rate for unfrozen morulae bisected in PBS and transferred without zona pellucida was 27% (4 15 ). The in vitro survival rate of embryos bisected in 12.5% sucrose when both halves remained in the original zona pellucida was 82% (18 22 ), which was higher than when embryos were bisected in PBS (53%, 9 17 ).     

Developmental capacity,size and number of nuclei in pig embryos cultured in vitro

Twenty-five surgical embryo recoveries were made from 17 postpuberal gilts 3 to 6 days after mating. A total of 242 eggs was recovered. Recovery rate was 87.5%, fertilization rate was 97.5%, and 98.7% of the fertilized eggs were morphologically intact. The embryos were cultured in vitro in Krebs-Ringer-Bicarbonate (KRB) with 10% heat inactivated lamb serum for 72 or 96 h at +37°C in a humidified 5% CO₂ atmosphere. Of the cultured four-cell embryos 26.6% developed to expanded blastocysts, 16.7% to hatching blastocysts and 5.0% to hatched blastocysts. Of the eight-cell embryos 52.6% developed to hatching blastocysts, 10.5% to hatched blastocysts. When recovered as morulae, the percentage of hatching blastocysts subsequently obtained was 25.8% and 33.9% hatched. A total of 75.0% of the cultured early blastocysts were in the process of hatching (30.6%) or had hatched (44.4%). Significant differences in overall embryo diameter were determined between morulae (156.5 ± 3.94 μm) and early blastocysts (156.9 ± 3.72 μm) versus expanded (197.6 ± 12.57 μm), hatching (207.4 ± 15.86 μm) or hatched (270.0 ± 36.67 μm) blastocysts. The zona pellucida of expanded blastocysts was significantly thinner (5.5 ± 1.59 μm) than that of morulae (12.0 ± 1.01 μm). The number of nuclei was significantly higher for hatching (151 ± 49.8) and hatched (130 ± 17.9) blastocysts cultured as early blastocysts as compared to those cultured from the four-cell stage (88 ± 12.7 and 69 ± 3.6 respectively). Hatching blastocysts that had developed from early blastocysts also had significantly more nuclei than those cultured as eight-cell embryos (99 ± 32.5) or morulae (91 ± 21.2).By the culture method used in this study, a high percentage of pig embryos was capable of developing.     

Birth of piglets after OPS vitrification and transfer of compacted morula stage embryos with intact zona pellucida

In swine, five to six days post-insemination, morulae and blastocysts are collected together after uterine flushing. The purpose of this study was to vitrify zona pellucida-intact morulae with Open Pulled Straw (OPS) technology and obtain piglets after transfer. Morulae (200) were vitrified after a two-step equilibration in ethylene glycol, dimethyl sulfoxide and sucrose in Hepes-buffered TCM199 + 20% NBCS medium (TCM). 2-6 morulae were loaded into OPS and plunged into liquid nitrogen. At embryo warming, a three-step dilution with decreasing concentrations of sucrose was applied. In each of 10 recipients, 20 morulae were transferred surgically. Day 25, gestation rate and the farrowing rate were 80% and 70%, respectively. The pregnant recipients farrowed from 1 to 8 piglets and the survival of total transferred embryos was 13%. Although survival rates are still compromised, OPS technology is therefore appropriate to cryopreserve porcine morulae with intact zona pellucida.     

Blastomeres aggregation as an efficient alternative for trophoblast culture from porcine parthenogenetic embryos

Zona pellucida free (ZPF) oocytes were cultured after electrical activation to allow blastomeres aggregation and compared to ZP intact (ZPI) oocytes. In feeder‐dependent conditions, the trophoblast attachment and primary outgrowths were significantly higher in ZPF than in ZPI groups. In feeder‐free conditions, trophoblast attachment and typical morphological trophoblast primary outgrowths were observed in ZPF group. The primary colonies derived from the ZPF embryos in both culture conditions were able to establish secondary and tertiary colonies and showed mRNA expression of CDX2, TEAD4 and KRT8 as trophoblast markers, while outgrowths from the ZPI embryos could not grow beyond primary colonies.     

Effect of growth factors on morula and blastocyst development of in vitro matured and in vitro fertilized bovine oocytes

Growth factors were studied as a means of increasing the development of in vitro matured (IVM) and in vitro fertilized (IVF) oocytes into morulae or blastocysts. Cell numbers of blastocysts were also counted. In Experiment 1, 2- to 8-cell embryos derived from bovine IVM/IVF oocytes were randomly allotted to one of 3 culture groups: a) synthetic oviduct fluid (SOF); b) SOF + 10 ng/ml epidermal growth factor (EGF); or c) SOF + 100 ng/ml EGF; all 3 culture media contained 10% fetal bovine serum. Culture resulted in 12%, 23% and 14% (P>0.05), respectively, developing into morulae and blastocysts. In Experiment 2, 5 ng/ml of transforming growth factor B (1) (TGFB (1)) added to CR(1aa) medium containing BSA increased the percentage of blastocysts to 56% vs 40% for the control (P<0.05). In Experiment 3, EGF and TGFB(1), added singly and in combination to CR(1aa) did not produce a synergistic effect. More embryos developed into morulae and blastocysts (45%) in a bovine oviduct epithelial co-culture than in any other treatment except in CR(1aa) + EGF (34%; P>0.05). In Experiment 4, 0, 1 and 5 ng/ml of platelet derived growth factor (PDGF) added to CR(1aa) yielded 39%, 70% and 52% morulae and blastocysts, respectively (P<0.05). Cell number was not increased, indicating that growth factors can increase the proportion of embryos that develop into morulae and blastocysts without an increase in the cell number.     

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.     

Susceptibility of pig embryos to porcine circovirus type 2 infection

The aim of the present study was to determine if porcine circovirus type 2 (PCV2) is able to infect embryonic cells of in vivo produced porcine embryos with and without zona pellucida (ZP). ZP-intact and ZP-free morulae (6-day post-insemination) and early blastocysts (7-day post-insemination), and hatched blastocysts (8-day post-insemination) were exposed to 10(5.0) TCID50 PCV2 per ml (strain 1121, fifth passage PK15). At 48 h post-incubation, the percentage of infected embryos and the percentage of viral antigen-positive cells per embryo were determined by indirect immunofluorescence (IF). Significantly different percentages of infected embryos were detected: 15% for ZP-free morulae, 50% for ZP-free early blastocysts and 100% for hatched blastocysts. The percentage of cells that expressed viral antigens was similar for the three stages of development. PCV2 exposure did not affect the in vitro development of the embryos during the 48 h study period. All ZP-intact embryos remained negative for viral antigens. In an additional experiment the diameter of the channels in the porcine ZP was determined. After incubation of early blastocysts with fluorescent microspheres of three different sizes, beads with a diameter of 20 nm and beads with a diameter of 26 nm crossed the zona whereas beads with a diameter of 200 nm did not. In conclusion, it can be stated that PCV2 is able to replicate in in vivo produced ZP-free morulae and blastocysts and that the susceptibility increases during development. The ZP forms a barrier to PCV2 infection, but based on the size of the channels in the ZP the possibility that PCV2 particles cross the ZP cannot be excluded.     

Hypotaurine requirement for in vitro development of golden hamster one-cell embryos into morulae and blastocysts, and production of term offspring from in vitro-fertilized ova.

Almost 30 years after the first successful in vitro fertilization (IVF) in golden hamsters (Mesocricetus auratus), we report that IVF hamster embryos can develop in a chemically defined, protein-free culture medium into morulae and blastocysts, and produce normal offspring after transfer to recipients. When examined 96 h post-insemination, 82% (160/200) of IVF ova had cleaved to at least 2 cells, 55% (97/200) had developed beyond the 4-cell stage, and 22% (38/200) had developed into morulae/blastocysts. In vitro development of IVF embryos to greater than or equal to 8 cells was absolutely dependent on hypotaurine. Twenty living offspring were produced from transfer of IVF embryos to recipients, with an overall success rate of 5% and 17% for oviductal (2-cell) and uterine (8-cell/morulae) transfers, respectively. In vivo-fertilized pronucleate embryos collected 3 h after egg activation were less able to develop in vitro than embryos collected only 6 h later, revealing a critical influence of the oviduct within the first hours of embryo development. Hypotaurine partly compensated for the decreased oviductal exposure of early 1-cell embryos. Establishment of a key role for hypotaurine in hamster embryo development, support of IVF embryos to morula/blastocyst stages in vitro, and production of living offspring after IVF embryo transfer are significant steps towards the goal of obtaining comparative data on preimplantation embryogenesis.     

Factors affecting the success rate of porcine embryo vitrification by the Open Pulled Straw method

The objectives of this study were: (1) to evaluate the influence of porcine embryo developmental stage on in vitro embryo development after vitrification, (2) to study the efficiency of the one-step dilution procedure, compared with conventional warming, for vitrified embryos at different stages of development, and (3) to determine the influence of the embryo donor on the in vitro survival of vitrified embryos at morulae and blastocyst stages. Two to four cell embryos, morulae and blastocysts were collected by laparotomy from weaned crossbred sows (n=55). Vitrification and conventional warming were performed using the OPS procedure with Superfine Open Pulled Straws (SOPS). For one-step dilution, embryos were placed in 800 microl TCM199-HEPES containing 20% of new born calf serum and 0.13 M sucrose for 5 min. To evaluate development, two to four cell embryos, morulae and blastocysts were cultured in vitro for 120, 48 and 24h, respectively. Some fresh embryos from each developmental stage were not vitrified and cultured as controls. Embryos were morphologically evaluated for their developmental capacity during the in vitro culture by stereomicroscopy. The total cell number of embryos was assessed by Hoechst-33342 staining and fluorescence microscope observation. There was a significant effect of the stage of development on the in vitro survival, perihatching rate and the number of cells of embryos after vitrification and warming (Experiment 1; p<0.001). The survival and perihatching rates of two to four cell embryos were lower than those obtained for morulae and blastocysts (p<0.001). No differences (p>0.05) in survival rates were found between vitrified and fresh blastocysts. The warming procedure did not affect the development and total cell number of vitrified two to four cell embryos, morulae or blastocysts (Experiment 2). However, donor had a significant effect (p<0.001) on the in vitro development and the number of cells of morulae and blastocysts after vitrification and warming (Experiment 3). In conclusion, the embryo developmental stage and the embryo donor were important factors that affected the development of porcine embryos after OPS-vitrification and warming. OPS-vitrification and the one-step dilution are efficient procedures to be used with intact porcine morulae and blastocysts.     

Cellular composition and viability of demi- and quarter-embryos made from bisected bovine morulae and blastocysts produced in vitro

The cellular composition and viability of intact, IVP embryos were compared with those of demi- and quarter-embryos produced by bisection of IVP morulae and blastocysts. Embryos were produced by established techniques from oocytes harvested from slaughterhouse ovaries. In Experiment 1, morulae at Day 6 or blastocysts at Day 7 were bisected on an inverted microscope using a microsurgical steel blade. Demi-embryos were then cultured without a zona pellucida until Day 8, when they were morphologically assessed for quality (viability). A higher proportion of demi-embryos made from blastocysts than from morulae were classified as viable (381/420, 91% vs 164/267, 61%; P < 0.001). In Experiment 2, only Day 7 blastocysts were bisected, and some of the resulting demi-embryos were bisected a second time 24 h later to produce quarter-embryos. The remaining demi-embryos, the quarter-embryos, and control intact embryos were cultured until Day 9, at which time they were assessed for quality and subjected to immunosurgery and differential staining to count inner cell mass (ICM) and trophectoderm cells. A higher proportion of demi-embryos than quarter-embryos was classified as viable (408/459, 89% vs 223/319, 70%, respectively; P < 0.001). Total cell numbers decreased with successive bisections, but the proportion of surviving cells found in the ICM was significantly (P < 0.05) higher in the best quality demi- and quarter-embryos (35 and 32%, respectively) than in the controls (22%). Transfer of all 12 quarter-embryos derived from 3 blastocysts, in pairs, into 6 recipient heifers resulted in 2 pregnancies, each with a single viable fetus at 90 d of gestation. The fetuses originated from 2 different blastocysts. The results suggest that bisection of intact IVP embryos into demi-embryos and bisection of those into quarter-embryos can increase the number of transferable embryos by as much as 178 and 235%, respectively.     

Useful short-range transport of mouse embryos by means of a nonfreezing technique.

The survival of mouse morulae to be implanted on the uterine wall of a recipient after storage at 0 degree C in sucrose-containing medium has shown tendencies similar to those of blastocysts developing in vitro. The pregnancy rate, as defined by implantation sites per embryos transferred, has varied from 52.9 to 77.2% and decreased depending on the duration of storage (0 to 72 h), and was lower than the rate of blastocyst formation in vitro (57.2 to 87.3%). Regarding the birth rate, 56.0 to 59.4% of implanted embryos were delivered as live pups regardless of storage duration, with the exception of controls (78.9%, 0 h). Eventually, 37.0, 32.8, or 29.9% of embryos were obtained as live pups when preserved by our system for 24, 48, or 72 hours, respectively. This viable, highly reproducible nonfreezing technique is useful for embryo preservation. Embryos were transported to evaluate the efficacy of the nonfreezing method. Mouse morulae were kept in a thermos bottle filled with ice-water and packaged in polystyrene foam. The package was transported from Kochi to Sapporo, about 1300 km, within 48 hours during which it was possible to maintain 0 degree C in the bottle and deliver to most areas in Japan. After shipping, the survival of embryos was 46.8% for development to blastocysts in vitro, and 37.1% for the pregnancy rate, with a final birth rate of 14.1%. Transport factors other than storage may have contributed to the lower birth rate; however, our system is cheaper, more convenient, and more practical than others for short-range transport.     

Cold culture of different stage mouse embryos in bicarbonated and bicarbonate-free media

Mouse embryos of different stages of development were cultured to expanded blastocysts following storage (1 to 8 d) at 4 degrees C in the presence or absence of HCO(3)(-). The effect of oxygen tension on the cold storage of one- and two-cell mouse embryos at 4 degrees C was evaluated by 37 degrees C culture and transfer to pseudopregnant recipients. Survival at 4 degrees C of early, one- to four-cell mouse embryos was improved with HCO(3)(-) in the medium. The presence of HCO(3)(-) was not of benefit for morulae or blastocyst survival following cold storage. Reducing the oxygen atmosphere from 20 to 5% O(2) improved survival of one-cell mouse embryos stored at 4 degrees C. The survival of two- and four-cell embryos, morulae and blastocysts at 4 degrees C was similar in 90% N(2), 5% CO(2) and 5% CO(2) in air, but it was significantly poorer in air alone. The collapse of morulae and blastocysts during cold storage up to 5 d was reduced with HCO(3)(-) in the storage medium. Blastocysts stored for 6 d at 4 degrees C failed to survive following immediate transfer to pseudopregnant recipients. Blastocyst survival was improved compared to controls (direct transfer of unstored blastocysts to recipients) when cultured for 36 h at 37 degrees C following 6 d of cold storage. This result suggests that cold-stored mouse blastocysts may require a metabolic period of readjustment to survive following transfer to synchronized recipients.     

Effects of increased ambient temperature on the development of in vitro derived bovine zygotes

In this study, presumptive bovine zygotes were subjected to two consecutive 24-h cycles of heat treatment during the first 48 h (Experiment I) of in vitro culture (IVC) or 24h of heat treatment during the fourth day of IVC (Experiment II). In Experiment I, the percentage of heat treatment zygotes that developed to > or =8-cell stage embryos after 72 h IVC was 2.0% (n = 459) compared with 28.4% (n = 458) for the control zygotes (P<0.001). The subsequent yield of morulae or blastocysts after 144 h IVC for the heat treatment and control groups was 0.9% (n = 457) and 12.3% (n = 456) (P<0.001), respectively. These results demonstrate that heat treatment during the first 48 h of IVC significantly impaired embryo development. In Experiment II, the percentage of zygotes that developed into morulae and blastocysts following heat treatment during the fourth day of IVC was 4.5% (n = 468) compared to 10.5% (n = 456) for the control group (P<0.001). This study has demonstrated that in vitro heat stress during the critical stage of early embryo development significantly increases the incidence of early embryonic mortality.     

Survival of sheep demi-embryos in vivo and in vitro

Sheep embryos (morulae and blastocysts) were bisected either by microscalpel or by microneedle after dissolving the zona pellucida with acidified Tyrode's solution. Fourteen and 11 cryopreserved demi-embryos failed to develop when transferred to recipients or placed in culture, respectively. When fresh demi-embryos were cultured in Dulbecco's phosphate buffered saline (DPBS) plus fetal calf serum (FCS) or Whitten's medium, the survival rate was 26% compared to 68% for whole embryos (P<0.01), and there was a suggestion that the presence of a zona pellucida was beneficial to survival. When two demi-embryos each within a zona pellucida were transferred into each of 10 ewes, six of them lambed to produce a total of eight lambs, including two sets of identical twins. Of 10 ewes receiving two demi-embryos without zonae pellucidae, three lambed to produce a total of four lambs, including one set of identical twins. Of 10 ewes that each received two whole embryos, 10 lambed to produce a total of 16 lambs. There was a suggestion that the zona pellucida might enhance the survival of demi-morulae but not demi-blastocysts.     

Factors affecting survival rates of in vitro produced bovine embryos after vitrification and direct in-straw rehydration

The aim of this work was to investigate the possibilities of simplification, and to outline the limits of application, of a vitrification method for cow embryos. Morulae and blastocysts were produced by in vitro fertilization of slaughterhouse-derived, in vitro matured oocytes with frozen-thawed bull semen, and subsequent culture on a granulosa cell monolayer. Vitrification was performed by equilibration of embryos with 12.5% ethylene glycol and 12.5% dimethylsulphoxide at 20–22°C for 60 s, then with 25% ethylene glycol and 25% dimethylsulphoxide at 4°C for another 60 s. Embryos were then loaded in straws, placed in liquid nitrogen vapour for 2 min, and then plunged. Straws were thawed in a 22°C water-bath, the embryos were directly rehydrated and further incubated in straw, and were then expelled and cultured in vitro for 72 h. In the first experiment, embryos of different age and developmental stage (Day 5 compacted morulae, Day 6 early blastocysts, Days 6 and 7 blastocysts, Day 7 expanded blastocysts and Day 8 hatched blastocysts) as well as Days 7 and 5 blastocysts previously subjected to partial zona dissection were vitrified. After thawing, the re-expansion rates of blastocysts and zona-dissected embryos did not differ (67 and 87%, respectively), and hatching was more frequent for blastocysts frozen in advanced developmental stages (34, 47 and 63% for early blastocysts, blastocysts and expanded blastocysts, respectively). The re-expansion rate of morulae was lower (10%) and no hatching of these embryos was observed. In the second experiment, Day 7 expanded blastocysts were vitrified using PBS, PBS + albumin, TCM199 and TCM199 + calf serum as holding media. No differences in re-expansion and hatching rates were seen. However, when incubation with the concentrated cryoprotectant solution was performed at 20–22°C, the embryo survival rate decreased (PBS + albumin) or no embryo survived (TCM199 + calf serum) the vitrification procedure. In the third experiment, Day 7 expanded blastocysts were vitrified, thawed, cultured for 1 day, and then re-expanded embryos were again vitrified and thawed. Out of the 87% that survived the first cycle, 73% re-expanded and 47% hatched following the second vitrification and thawing. These observations prove that the vitrification procedure described is relatively harmless, that it can be used for blastocysts of different developmental stages and that an intact zona is not required to obtain high survival rates.     

Vitrification and warming of in vivo-derived porcine embryos in a chemically defined medium

The aim of this study was to design a protocol for vitrification and warming of porcine embryos in a chemically defined medium. A total of 663 morulae and blastocysts were collected from weaned crossbred sows (Large White-Landrace) 5 to 6 d after estrus and vitrified with the Superfine Open Pulled Straw method. In Experiment 1, embryos were vitrified using as a basic medium TCM-199-HEPES supplemented with 20% newborn calf serum (NBCS) or with 0, 0.1%, 0.5%, or 1% polyvinyl alcohol (PVA). Nonvitrified embryos were used as a fresh control group. Survival and hatching rates were evaluated after 72 h of in vitro culture to assess embryo viability. In addition, some hatched blastocysts derived from morulae and blastocysts were processed to determine the total cell number and the cell proliferating index as measures of their quality. Within each stage of embryo development, the different vitrification groups and the fresh control group showed similar high embryo survival (range, 70.5 ± 7.1% to 84.9 ± 8.1% and 85.3 ± 8.1% to 98.4 ± 8.2% for morulae and blastocysts, respectively) and hatching rate (range, 46.3 ± 10.1% to 66.7 ± 11.2% and 73.7 ± 11.3% to 89.4 ± 11.2% for morulae and blastocysts, respectively) and quality after in vitro culture. In Experiment 2, embryos were vitrified using 0.1% PVA and warmed with TCM-199-HEPES-0.13 M sucrose supplemented with 20% NBCS or either 0 or 0.1% PVA. Nonvitrified embryos were used as a fresh control group. As in Experiment 1, no significant differences were detected in embryo survival (range, 67.9 ± 6.6% to 74.5 ± 6.6% and 91.9 ± 7.0% to 99.5 ± 6.3% for morulae and blastocysts, respectively) and hatching rate (range, 47.0 ± 7.2% to 64.8 ± 9.9% and 89.4 ± 7.4% to 98.2 ± 6.9% for morulae and blastocysts, respectively) and quality among the warming groups or among vitrified and fresh control embryos. In both experiments, the developmental embryo stage influenced the survival and hatching rates, as well as the number of cells (P < 0.01), with the blastocyst stage yielding the best results. In conclusion, PVA can be used as a substitute for serum in vitrification and warming solutions without detrimental effects on the in vitro development of in vivo-derived porcine morulae and blastocysts.     

In vitro analysis of pre- and early postimplantation development of lethal yellow (Ay/Ay) mouse embryos

Preimplantation embryos from matings between yellow heterozygous (Ay/a) mice were recovered at 56 hours post coitum, cultured for five days, and compared with the development of embryos from three control matings (Ay/a female X a/a male, a/a female X Ay/a male, a/a female X a/a male). Most embryos were at the 8-cell stage at recovery; however fewer embryos from the experimental cross had developed to the 8-cell stage than embryos of control matings, indicating a developmental lag of experimental embryos (P less than 0.01). The yellow (Ay/a) uterus did not contribute (P = 0.05) to delayed development. Experimental and control embryos were equally capable of successful development in culture to the morula stage with no distinct morphological characteristics identifying the class of Ay/Ay mutants. However, significant differences were observed in the development from morulae to blastocysts; 9.4% (10/106) of the morulae in experimental crosses failed to undergo blastocyst formation as compared with 2.5% (10/398) of morulae in pooled control crosses (P = 0.010-0.025). In the experimental cross 25.0% (24/96) of embryos that developed successfully to the blastocyst stage failed to hatch from the zona pellucida; these are presumed to include the class of lethal yellow homozygotes. Abnormalities seen in cultured embryos consisted primarily of blastomere disintegration, blastomere arrest and exclusion, and embryo fragmentation.