Development of early bovine embryos to the blastocyst stage in serum-free conditioned medium from bovine granulosa cells

Summary Bovine granulosa cell — conditioned medium (BGC-CM) was prepared in a serum-free medium consisting of TCM 199, 5μg/ml insulin, and 0.5μg/ml aprotinin (TCM 199 IAP). Granulosa cells surrounded with embryos were denuded 24 to 30 h after in vitro fertilization. The proportion of denuded granulosa cell-free embryos that developed to the blastocyst stage in BGC-CM (43/219; 20%) as well as in the co-culture system (43/178; 24%) was significantly greater (P<0.001) than in fresh TCM 199 IAP medium (FM: 10/191; 5%), whereas the proportion of embryos that developed to the eight-cell stage was similar (P>0.05) in all three culture systems (95/178; 53% in co-culture, 111/219; 51% in BGC-CM, and 86/191; 45% in FM, respectively). Higher rates of hatching and hatched blastocysts 8.5 days after in vitro fertilization were observed in co-culture (13/44; 29.5%) and in conditioned medium (8/39; 20.5%). On the other hand, no hatching or hatched blastocysts were obtained in the fresh medium (0.7; 0%). Cell numbers per blastocyst in BGC-CM (178.3 cells/blastocyst) were approximately two-fold higher than those in FM (97.1 cells/blastocyst). However, higher cell numbers (249.3 cells/blastocyst) were observed in co-culture with BGC than in BGC-CM. The embryotrophic activity in BGC-CM was stable upon freezing and thawing, lyophilization, and heating at 56° C whereas activity was reduced by dilution in fresh medium, dialysis, pronase digestion, and heating at 80° C. These results suggest that BGC cultured in a serum-free medium can synthesize and secrete an embryotrophic factor(s) that supports blastocyst formation in vitro beyond the 8- to 16-cell stage.     

Changes in morphology and cell number of inner cell mass of porcine blastocysts during freezing

Changes in the morphology and cell number of the inner cell mass (ICM) of porcine blastocysts at the expanded and hatched stages during freezing (-6.8 degrees C, -35 degrees C and -196 degrees C) were studied by differential fluorochrome staining. The shape of each ICM cell from fresh blastocysts at the expanded and hatched stages was sharply delineated but that of ICM cells from frozen blastocysts was partially distorted. The cell-to-cell contact of the ICM from fresh blastocysts was tight, while that from frozen blastocysts was loose or scattered. The percentages (18 to 38%) of expanded and hatched blastocysts with tight-contact ICM cells from frozen groups at each step were significantly lower (P<0.05) than that (100%) from fresh blastocysts. The number of live ICM cells and their proportion from frozen expanded blastocysts (10.9, 12,4% at -36 degrees C) were significantly lower (P<0.05) than those from fresh embryos (18.4, 19.1%) and at -196 degrees C (20.6, 18.4%). At the hatched stage, the number of live ICM cells and their proportion were not significantly different between each freezing step. These results show that the ICM of porcine embryos at both the expanded- and hatched-blastocysts stages survived even after freezing at -196 degrees C and that the degree of ICM damage was lower at the hatched stage than at the expanded stage.     

Effects of Nu-Serum® on in vitro development of goat (Capra hircus) embryos

To evaluate the effects of a commercially produced serum substitute on the in vitro development of caprine embryos, registered Nubian doelings were synchronized with norgestomet-impregnated implants (Synchromate-B®: CEVA) and superovulated with descending doses of FSH-p® (Schering). A total of 246 embryos was collected and placed in Tissue Culture Medium 199 (TCM 199, Gibco Laboratories) containing Nu-Serum® (NuS) at concentrations of 2.5%, 5.0%, 10%, or 20%. Control treatments consisted of TCM 199 alone or TCM 199 plus 10% heat-inactivated fetal bovine serum (FBS). Embryos developed in all concentrations of NuS to the morula, blastocyst, and expanded blastocyst stages. The TCM 199 plus 10% NuS had significantly higher percentages of embryos developing to the expanded blastocyst stage than TCM 199 plus 10% FBS. Time to expanded blastocyst development in NuS was shorter than in the TCM 199 plus FBS. No stage-specific block to development was observed with embryos collected and cultured in vitro for any of the treatments. These results demonstrate that NuS, when compared to FBS, allowed a higher percentage (P < 0.05) of caprine embryos to develop to the expanded blastocyst stage, thus providing a valuable substitute for FBS.     

Ultrastructure of bovine embryos developed from in vitro–matured and –fertilized oocytes: Comparative morphological evaluation of embryos cultured either in serum‐free medium or in serum‐supplemented medium

The ultrastructure of bovine embryos developed from in vitro‐matured and ‐fertilized oocytes, cocultured with bovine cumulus/granulosa cells either in a serum‐free medium (IVMD101) or in a serum‐containing medium (TCM199+CS) was compared. Embryos up to the eight‐cell stage had many cellular organelles and cytoplasmic components that were randomly distributed in the cytoplasm. Mitochondria were spherical or ovoid and had only a few peripheral cristae. There were no obvious differences in the ultrastructure between embryos developed in IVMD101 and TCM199+CS up to the eight‐cell stage. However, conspicuous differences in the ultrastructural features between the embryos cultured in IVMD101 and TCM199+CS were observed at the morula and blastocyst stages. At the morula stage, embryos cultured in IVMD101 had cells containing elongated mitochondria, well‐developed Golgi apparatus, lipid droplets, and large vesicles resembling lysosomes. The lysosome‐like vesicles were partially filled with electron‐dense materials and were frequently fused with lipid droplets. The blastomeres of morulae cultured in TCM199+CS contained numerous large lipid droplets and fewer lysosome‐like vesicles than those cultured in IVMD101. In blastocysts cultured in IVMD101, lysosome‐like vesicles were frequently observed in the trophoblast cells and lipid droplets were present in the cytoplasm of trophoblast and inner cell mass (ICM)‐cells, but they were not abundant. On the other hand, the blastocysts developed in TCM199+CS contained fewer lysosome‐like vesicles and large numbers of lipid droplets. This accumulation of lipid droplets was higher in the trophoblast cells than in the ICM‐cells. This study showed major differences in the ultrastructural features between the morulae and blastocysts from serum‐free and serum‐supplemented cultures, suggesting that the ultrastructural differences may reflect physiological characteristics of embryos. Mol. Reprod. Dev. 53:325–335, 1999. © 1999 Wiley‐Liss, Inc.     

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.     

Effect of post-thaw cell rehydration at 4 degrees C on survival of frozen and vitrified IVP-derived bovine embryos.

In vitro-produced bovine embryos (IVP) were either frozen in 10% glycerol in a phosphate-buffered saline solution (PBS) using conventional slow freezing or vitrified in 25% glycerol and 25% ethylene glycol in PBS. The results of viability and hatching rates were compared between frozen and vitrified embryos after thawing and dilution using one of three different protocols: (A) a three-step dilution procedure, (B) a one-step dilution procedure or (C) a procedure in which embryos were kept in situ inside the straw at 4 degrees C for 10 min during a one-step dilution procedure. No significant differences in embryo survival were found among protocols A, B and C for frozen embryos and between protocols A and B for vitrified embryos. Viability and hatching rates of vitrified embryos thawed and diluted by protocol C (73 and 62%) were significantly enhanced (P < 0.05) in comparison to those obtained with protocol A (55 and 41.6%) or protocol B (54.5 and 35.3%). These results indicate that for vitrified IVP bovine embryos, direct in-straw rehydration at 4 degrees C for 10 min improves embryo survival and it could be a practical procedure for use under field conditions where there is sometimes a longer interval between thawing and transfer.     

The human blastocyst: cell number, death and allocation during late preimplantation development in vitro

The development of 181 surplus human embryos, including both normally and abnormally fertilized, was observed from day 2 to day 5, 6 or 7 in vitro. 63/149 (42%) normally fertilized embryos reached the blastocyst stage on day 5 or 6. Total, trophectoderm (TE) and inner cell mass (ICM) cell numbers were analyzed by differential labelling of the nuclei with polynucleotide-specific fluorochromes. The TE nuclei were labelled with one fluorochrome during immunosurgical lysis, before fixing the embryo and labelling both sets of nuclei with a second fluorochrome (Handyside and Hunter, 1984, 1986). Newly expanded normally fertilized blastocysts on day 5 had a total of 58.3 +/- 8.1 cells, which increased to 84.4 +/- 5.7 and 125.5 +/- 19 on days 6 and 7, respectively. The numbers of TE cells were similar on days 5 and 6 (37.9 +/- 6.0 and 40.3 +/- 5.0, respectively) and then doubled on day 7 (80.6 +/- 15.2). In contrast, ICM cell numbers doubled between days 5 and 6 (20.4 +/- 4.0 and 41.9 +/- 5.0, respectively) and remained virtually unchanged on day 7 (45.6 +/- 10.2). There was widespread cell death in both the TE and ICM as evidenced by fragmenting nuclei, which increased substantially by day 7. These results are compared with the numbers of cells in morphologically abnormal blastocysts and blastocysts derived from abnormally fertilized embryos. The nuclei of arrested embryos were also examined. The number of TE and ICM cells allocated in normally fertilized blastocysts appears to be similar to the numbers allocated in the mouse. Unlike the mouse, however, the proportion of ICM cells remains higher, despite cell death in both lineages.     

Actin filament distribution in blocked and developing pig embryos

Actin filaments play an important role in cell division. The present study was designed to examine the relationship between actin filament distribution and pig embryo development. When in vivo matured and fertilised pig oocytes were cultured in TCM 199 or NCSU 23, in various proportions, 45-65% of inseminated oocytes developed to the 2- to 4-cell stages but blastocyst development was observed only in NCSU 23 (34%) or NCSU 23 containing 10% TCM 199 (7%). Supplementation of NCSU 23 medium with 20% or more TCM 199 resulted in no blastocyst formation. Examination of actin filaments indicated that microfilaments were distributed in the cortex, at the junction of blastomeres and in the perinuclear area in the embryos cultured in NCSU 23, but perinuclear actin filaments were not observed in embryos cultured in TCM 199. When 2- to 4-cell stage embryos obtained from TCM 199 were transferred to NCSU 23 medium at 36 h after in vivo fertilisation, 57% of the cleaved embryos developed to blastocysts, which was no different from the proportion obtained after culture in NCSU 23 alone (56%). In addition, when 2- to 4-cell stage embryos obtained from TCM 199 were transferred to NCSU 23, most embryos showed perinuclear actin filaments within 6h. The results indicate that the composition of the culture medium plays an important role in the polymerisation of actin filaments, which in turn influences embryo development. It is possible that pig embryo development was blocked by some components in TCM 199 which prevented actin filament polymerisation.     

Morphological development,cell number,and allocation of cells to trophectoderm and inner cell mass of in vitro fertilized and parthenogenetically developed buffalo embryos: The effect of IGF-I

The morphology and number of cells in the trophectoderm (TE) and inner cell mass (ICM) of buffalo blastocysts derived from in vitro fertilization and cultured in the presence or absence of insulin-like growth factor-I (IGF-I) were analyzed by differential fluorochrome staining technique. The total cell number (TCN), TE number, and ICM cell number were significantly higher in blastocysts developed in vitro in the presence of IGF-I as compared to blastocysts developed without IGF-I (P < 0.01). It was observed that the buffalo blastocyst took 5–9 days postfertilization to develop in vitro. In order to correlate the time required for blastocyst development and the allocation of cells to TE and ICM, blastocysts were designated as fast (developing on or before day 7) or slow (developing after day 7). The TCN, TE, and ICM cells of fast-developing blastocysts cultured in the presence of IGF-I were significantly higher than slow-developing blastocysts (P < 0.01). The blastocysts developed on day 6 had a mean total cell number 118.6 ± 21.4, which significantly decreased to 85.6 ± 17.4, 62.0 ± 14.5, and 17.0 ± 4.0 on days 7, 8, and 9, respectively (P < 0.05). Normal development of buffalo embryo showed that, on average, embryos reached compact morula stage at the earliest between days 4.5–5.5. Blastocysts developed, at the earliest, between days 5.0–6.0, and it took them, on average, 6.5 days to hatch from the zona pellucida. TCN, TE, and ICM increased three times from morula to blastocyst; however, the proportion of ICM to TCN remained the same, in both embryonic stages. TE approximately doubled in hatched blastocysts, as compared to unhatched blastocysts (P < 0.05). However, ICM cells were decreased. The time required for development of parthenogenetic blastocysts was observed to be greater as compared to in vitro fertilized (IVF) blastocysts. The total cell number of parthenogenetic blastocysts was 100.8 ± 11.3, including 59.2 ± 8.4 cells of TE and 42.1 ± 6.9 cells of ICM. © 1996 Wiley-Liss, Inc.     

A paucity of structural integrity in cloned porcine blastocysts produced in vitro

The structural integrity of blastocyst stage embryos, consisting of the inner cell mass (ICM) and trophectoderm (TE) cells, is a prerequisite for normal development after implantation in mammals. In this study, allocation of nuclear transfer (NT)-derived porcine blastocysts to the ICM and to the TE cells was examined and compared with IVF- and in vivo-derived embryos. NT-derived embryos had a lower developmental competence to the blastocyst stage than IVF-derived embryos (P < 0.05). Total cell number of NT-derived blastocysts was inferior to that of IVF-derived embryos (P < 0.05), although no difference was detected between the two groups in the ratio of ICM to total cells. However, in vivo-derived blastocysts had a higher proportion of ICM to total cells compared with in vitro-produced embryos (P < 0.01). To investigate what proportions of in vitro-produced porcine embryos represent normal structural integrity, differentially-stained blastocysts were individually classified into three presumptive groups (I: <20%; II: 20-40%; III: >40%) according to the ratio of ICM to total cells. Low proportions of NT- (12.5%, 7/56) and IVF-derived blastocysts (15.8%, 9/57) were assigned to Group II, presumptively having a normal range of structural integrity, whereas, almost all in vivo-derived embryos (97.5%, 39/40) were allocated to Group II. In conclusion, limited structural integrity may lead to the poor survival to term of NT- or IVF-derived porcine embryos produced in vitro.     

High survival of mouse embryos after rapid freezing and thawing inside plastic straws with 1-2 propanediol as cryoprotectant

A method for obtaining a high survival rate of frozen-thawed mouse embryos is presented. Eight-cell mouse embryos were frozen inside small plastic straws in the presence of 1-2 propanediol and stored at -196 C. After thawing, the embryos were diluted for only 5 min in a 1.0 M sucrose solution to remove the 1-2 propanediol from the cells. At high rate of thawing (is equivalent to 2500 C/min) more than 88% of the embryos survived in vitro to the blastocyst stage provided that the dilution of propanediol was performed rapidly during thawing. At a lower rate of thawing (is equivalent to 300 C/min), survival tended to be higher (94.7%) when dilution was done 5 min after thawing. When the frozen-thawed embryos were transferred to the oviducts of day 1 pseudopregnant recipients either directly after the dilution of 1-2 propanediol or after 24 or 48 hr of culture, a high proportion of them (65.9%) develop normally to viable fetuses.     

A serum-free culture system for efficient in vitro production of bovine blastocysts with improved viability after freezing and thawing

The aim of this study was to evaluate whether two completely serum-free media (IVMD101 and IVD101) could improve the yield and quality of bovine blastocysts from in vitro matured and fertilized oocytes. The media were evaluated in the presence (IVMD101) or absence (IVD101) of bovine cumulus/granulosa cell (BCGC) cocultures. The proportion of embryos developing to the blastocyst stage in IVMD101 medium with BCGC cocultures (36.5%) and IVD101 medium without BCGC cocultures (37.1%) was significantly higher than in serum-supplemented medium (TCM199 + 5% calf serum) with BCGC cocultures (25.1%). Furthermore, the mean cell numbers per blastocyst on Day 7 developed in IVMD101 medium (179.5 cells) and IVD101 medium (177.1 cells) were greater than in the serum-supplemented medium (145.7 cells). The survival rates of blastocysts derived in IVMD101 medium (73.3%) and IVD101 medium (60.0%) based on hatching after 72 h of post-thaw culture were superior to that of blastocysts derived in the serum-supplemented medium (48.1%). Under microscopic observation, bovine blastocysts derived in the serum-supplemented medium showed abundant lipid droplets, largely into the trophectoderm cells. This morphological difference may partly explain the sensitivity of serum-derived embryos after freezing and thawing. In conclusion, these new serum-free culture media are useful, not only to study the mechanisms of early embryogenesis, but also for mass production of good quality embryos for embryo transfer, cloning and transgenesis. This revised version was published online in August 2006 with corrections to the Cover Date.     

A serum-free, cell-free culture system for development of bovine one-cell embryos up to blastocyst stage with improved viability

With the aim of developing a serum-free, cell-free culture system for embryo development, in vitro-matured (IVM) and -fertilized (IVF) bovine oocytes were cultured in TCM 199 with the following supplements: 1) BSA alone (10 mg/ml); 2) BSA with ITS (5 mug/ml insulin, 5 mug/ml transferrin and 5 ng/ml selenium; BSAITS medium); 3) estrous cow serum alone (ECS; 10%); or 4) ECS with BOEC (bovine oviduct epithelial cells) (Experiment 1). In Experiment 2, embryos were cultured in BSAITS medium with or without feeding with fresh medium on Day 4 (day of insemination = Day 0). Embryos were evaluated on Day 2 for first cleavage, on Day 7 for morulae and blastocysts, and on Day 8 for blastocysts. Blastocysts from Experiment 1 were frozen in 10% glycerol in PBS, thawed and further cultured in ECS medium with BOEC for 48 h, and evaluated for formation of a distinct blastocoel, or expansion and hatching of blastocysts. In vivo-developed, Grade-1 and Grade-2, 7-d-old embryos served as control for the freezing, thawing and subsequent culture procedures. The percentage of first cleavage did not differ between the treatments (74 to 79% in Experiment 1 and 80 to 83% in Experiment 2). The percentage of blastocysts developed in BSAITS medium did not differ from that in ECS medium whether BOEC were present or not. However, medium with BSA alone had fewer blastocysts than any other culture system (P<0.05). Feeding embryos with fresh BSAITS medium on Day 4 did not lead to any further increase in the proportion of blastocysts. The culture systems had a significant effect on the post-thaw viability of blastocysts developed in them (P<0.001). Blastocysts developed in BSAITS medium had better (P<0.05) viability (14/38) than those from medium with ECS alone (1/27) or with ECS and BOEC (3/37). The post-thaw survival of control embryos was 80% (n=30). One of the three transfers of BSAITS-treated, frozen-thawed blastocysts resulted in a pregnancy. The results indicate that a serum-free, cell-free culture system can support the development of IVM-IVF bovine oocytes up to the blastocyst stage with better viability than a complex co-culture system.     

Rabbit blastocyst: Allocation of cells to the inner cell mass and trophectoderm

The proportion of total cells in the blastocyst allocated to the inner cell mass (ICM) and trophectoderm (TE) is important for future development and may be a sensitive indicator to evaluate culture conditions. The number of cells and their distribution within the two primary cell lineages were determined for the rabbit embryo developing in vivo after superovulation or nonsuperovulation or embryo transfer and compared with embryos developing in vitro. Comparisons were made with cultured embryos or embryos grown in vivo until 3.5, 4.0, and 4.5 days of age. Embryos from superovulated rabbits developed in vivo for 3.5, 4.0, and 4.5 days, respectively, had 361, 758, and 902 total cells (P<0.05), and in nonsuperovulated rabbits 130, 414, and 905 total cells (P<0.05), with increasing proportions of ICM cells over time (P<0.05). One-cell embryos recovered from superovulated females and transferred to nonsuperovulated recipients developed more slowly with 70, 299, and 550 total cells after 3.5, 4.0, and 4.5 days of culture (P<0.05), respectively. The proportion of ICM cells increased with age of the embryo. Corresponding values for one-cell embryos cultured in vitro resulted in 70, 299, and 550 total cells (P<0.05). However, in vitro culture of morula-stage embryos in the presence of fetal bovine serum for 24 hr did not delay growth. In addition, the proportions of ICM/total cells were 0.17, 0.25, and 0.29 for embryos developing in vitro at 3.5, 4.0, and 4.5 days, respectively, similar to those for embryos developing in vivo at each of the three recovery times. These data establish for the first time the number and proportion of cells allocated to the ICM of the rabbit embryo developing in vivo or under defined conditions in vitro. © 1995 Wiley-Liss, Inc.     

Effects of hyaluronic acid on the development of 1- and 2-cell porcine embryos to the blastocyst stage in vitro

The purpose of this study was to evaluate the ability of hyaluronic acid to improve the development of 1- and 2-cell porcine embryos to the blastocyst stage in a simple medium. In Experiment 1, we confirmed the ability of Whitten's medium supplemented with 15 mg/ml BSA to support the development of porcine embryos to the blastocyst stage under our experimental conditions. Embryos collected from oviducts were cultured at 38.5 degrees C in an atmosphere of 5% CO(2) in humidified air up to 6 d. After 2 d of culture, 82 and 78% of embryos reached the 4-cell stage or beyond in TCM199 supplemented with 10% fetal calf serum (FCS) and in Whitten's medium with BSA, respectively. However, no embryo developed to the morula stage in TCM199 after 6 d of culture. On the other hand, 26 and 15% of embryos developed to the morula and the blastocyst stage in Whitten's medium, respectively. In Experiment 2, we determined whether supplementation of hyaluronic acid in Whitten's medium would improve the development of porcine embryos to the blastocyst stage. After 6 d of culture, development of the embryos to the blastocyst stage was best supported in Whitten's medium with 4 mg/ml BSA and 0.5 mg/ml hyaluronic acid (70%). The proportion of degenerated embryos was lower in the presence than in the absence of hyaluronic acid. These results indicate that the supplementation of Whitten's medium with hyaluronic acid improves the development of 1- and 2-cell porcine embryos to the blastocyst stage.     

Increasing the rate of blastocyst formation and hatching from in vitro-produced bovine zygotes

This study was designed to identify parameters that would facilitate early selection of superior embryos, as well as to define culture conditions that could increase the proportion of embryos proceeding to the blastocyst stage. In the first experiment, the developmental potential of bovine embryos that had reached different stages of development after 60 h of culture following insemination was assessed. No 2-cell embryos underwent further cleavage. Of the 4-cell embryos (n = 188) only 12.2% progressed to the blastocyst stage, while 62.5% of 8-cell embryos (n = 480) did so (P < 0.01). In a further experiment, the effects of conditioning the culture medium (TCM 199) either with Buffalo rat liver cells (BRLC) or bovine oviductal epithelial cells (BOEC) and the effects of co-culture with either of these 2 cell types were examined. The percentage of 8-cell embryos proceeding to the morula and blastocyst stages was independent of cell type and culture system. However, BOEC-conditioned medium supported significantly lower production of blastocysts than any of the other culture methods. Only 24.1% of the former proceeded to the blastocyst stage after the full 10 d of culture, and only 3% hatched, values that were significantly lower than in the other 3 groups (P < 0.01). Among the latter, 44% progressed to the blastocyst stage in BRLC-conditioned medium while 44 and 46% reached that endpoint after co-culture with BOEC or BRL cells, respectively. The percentages that hatched among these were 28.2, 31 and 28.5%, respectively.     

Development and viability of frozen-thawed cattle embryos

Embryos recovered 7 to 8 days after estrus were frozen from -7 to -30 degrees C at 0.3 degrees C/min, from -30 to -33 degrees C at 0.1 degrees C/min, and then plunged into liquid nitrogen. They were thawed in a 25 degrees C waterbath. In a preliminary study, 15 of 18 embryos continued to develop during the 24-hour culture post-thaw in either Ham's F-10 or modified Dulbecco's phosphate buffered saline (PBS). In the main study, 5 of 20 embryos developed to 60-day pregnancies when embryos were transferred within 5 hours after thawing. The incidence of extended estrous cycles (pregnancy or presumed embryonic mortality) was 10 of 14, when the zona pellucida was intact after thawing, and 0 of 6, when it was ruptured or absent (P<.05). Embryos cultured in PBS tended to develop more readily than those in Ham's F-10 (15 of 20 vs 9 of 20, respectively, P reverse similar.1). Quality of the embryos, at recovery from the donor and after thawing, affected development in culture (19 of 27 embryos excellent at recovery developed vs 5 of 13 poor to very good, P reverse similar.1; 23 of 33 embryos good to excellent after thawing developed vs 1 of 7 poor, P<.05). The proportion of pyknotic nuclei in embryos which were cultured ranged from 18 to 100%. The pregnancy rate from embryos which were cultured was low (2 of 20). Thirty percent of frozen and thawed embryos had damaged zonae pellucidae. The study showed that: the pregnancy rate from frozen embryos was approximately half that achieved with unfrozen embryos; culturing embryos for 24 hours before transfer was not beneficial; the PBS culture system appears to be the system of choice for assessing embryo viability in vitro .     

Morphology and proportion of inner cell mass of bovine blastocysts fertilized in vitro and in vivo

The morphology and proportion of inner cell mass (ICM) of bovine blastocysts cultured in vitro or in vivo in rabbit oviducts after in-vitro fertilization of in-vitro matured follicular oocytes were compared with those of blastocysts fertilized in vivo by a differential fluorochrome staining technique. The delineation of each ICM cell was improved by the transfer of embryos derived from in-vitro fertilization to a rabbit oviduct although the cell-cell contacts of ICM cells were not as tight as those from in-vivo fertilization. The proportions (15.8 and 14.9%) of ICM in blastocysts cultured in vitro at early and expanded stages were significantly lower than those cultured in rabbit oviducts after in-vitro fertilization and fertilized in vivo. These results show that the transfer of bovine embryos derived from in-vitro fertilization to the rabbit oviduct increased the proliferation of ICM cells to the level of embryos fertilized in vivo although the cell-cell contact of ICM cell is not improved by the process.     

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

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

Nuclear reprogramming in embryos generated by the transfer of yak (Bos grunniens) nuclei into bovine oocytes and comparison with bovine-bovine SCNT and bovine IVF embryos

Although inter-species SCNT may be useful for increasing and preserving populations of endangered species, there are many reports that inter-species nuclear transfer embryos only develop to the blastocyst stage. In this study, yak-bovine SCNT blastocysts were successfully implanted in the surrogate bovine uterus but failed to develop to term or aborted. To clarify the reasons, we examined yak-bovine SCNT blastocyst development, total cell number, inner cell mass (ICM) number, trophoblast (TE) cell number and relative gene expression in yak fibroblast cells and yak-bovine SCNT embryos at various stages. The potential for development of yak-bovine SCNT embryos to blastocysts was 30+/-5.7% (mean+/-S.E.M.); the total cell number was 85.3+/-16.3, fewer than in IVF bovine embryos (106.2+/-18.2) but within the reported range (60-300). The yak-bovine SCNT blastocysts had a lower ratio of TE cells to total cells (43.9+/-8.7%) than bovine IVF embryos (59.4+/-3.4%; P<0.05) or bovine-bovine SCNT (69.5+/-5.4%; P<0.05). Also, several yak-bovine SCNT embryos had abnormal initiation of expression of both Mash2 and IL6. However, expression of vimentin, collagen, Cx43 and PSMC3 were normal in yak fibroblast cells and yak-bovine SCNT embryos. In conclusion, we inferred that the normal allocation of ICM and TE cells in yak-bovine SCNT embryos and embryo-specific gene reprogramming may be important for successful inter-species animal cloning.