In vitro development up to hatching of bovine in vitro-matured and fertilized oocytes with or without support from somatic cells

To verify the importance of somatic cells upon in vitro embryo development, in vitro-matured (IVM) and -fertilized (IVF) bovine oocytes were cultured in TCM 199 supplemented with estrous cow serum (10% v/v) and 0.25 mM sodium pyruvate (ECSTCM) under the following treatments: 1) ECSTCM alone; 2) together with bovine oviduct epithelial cells (BOEC); 3) with cumulus cells (CC); 4) in fresh BOEC conditioned ECSTCM; or 5) in frozen-thawed BOEC conditioned ECSTCM. Culturing zygotes encased in cumulus cells significantly reduced the cleavage rate (P<0.05). There was no difference between culture systems in the proportions of embryo development through the 8-cell stage (P=0.42) up to the morula/blastocyst stages (P=0.50) at Day 7 post insemination. However, co-culture with BOEC yielded the highest percentage (21.2% of zygotes; P<0.05) of quality Grade-1 and Grade-2 embryos with the number of blastomeres per embryo (114.4) comparable to that of 7-day-old in vivo-developed embryos of similar grades (102.5), and higher (P<0.05) than those of the other treatments. The ratio of blastocysts to total morulae/blastocysts obtained from frozen-thawed conditioned medium was lower (P<0.05) than that from ECSTCM or after co-culture with BOEC at Day 7 post insemination. On average, 7.5 to 17.5% of the zygotes developed to blastocyst, expanded blastocyst and hatched blastocyst stages by Day 10 post insemination, depending upon the culture system. The difference between treatments, however, was not significant (P=0.68). The results indicate that chronological development up to hatching of bovine IVM-IVF embryos is not favored by somatic cells; however, the presence of viable oviduct epithelial cells in culture significantly improves the quality of 7-day-old embryos.     

Development of in vitro matured and fertilized bovine oocytes co-cultured with Buffalo Rat Liver cells

This study was designed to evaluate the efficacy of Buffalo Rat Liver cells (BRLC) monolayers in supporting the development of in vitro matured and fertilized (IVM/IVF) bovine oocytes through to the hatched blastocyst stage compared to the commonly used co-culture system of bovine oviduct epithelial cells (BOEC). Cumulus oocyte complexes (COCs) obtained from 2- to 6-mm ovarian follicles at slaughter were matured for 24 h in TCM-199 supplemented with FBS and hormones (FSH, LH and estradiol 17-beta). In vitro fertilization (IVF) was performed using 1 x 10(6) percoll separated frozen-thawed spermatozoa in 1 ml of IVF-TL medium containing 18 to 20 matured oocytes. After 20 to 22 h of sperm exposure, 584 presumptive zygotes in 2 separate trials were randomly assigned to 3 treatment groups (BRLC co-culture, BOEC co-culture and control, consisting of medium alone). Zygotes were cultured in CZB media, a simple semi-defined medium, without glucose for the first 2 d, transferred to M199/FBS (TCM-199-HEPES supplemented with 20% HTFBS, 1 mM Sodium pyruvate), and cultured for an additional 8 days. Cleavage and development to morula and various blastocyst stages were recorded between d 3 and 11 after the start of IVF. Overall average cleavage rate was 75% (440 584 ) and did not vary across the treatments or trials. The proportion of embryos that reached the morula stage in both co-culture systems did not differ (P > 0.05) and was significantly higher (P > 0.05) compared to the control group. However, the percentage of the number of blastocysts, expanded blastocysts and hatched blastocysts varied across the treatment groups (P < 0.05), with the highest results obtained in the BRLC co-culture system. The production of blastocysts in BOEC co-culture was inconsistent between the 2 trials where a significant difference (40.6 vs 53.0%; P > 0.05) was observed. Rate of development to the blastocyst stage was similar between the 2 co-culture systems, with most of the embryos reaching the blastocyst stage by d 8 post insemination. The results of this study show that BRLC from a commercially available established cell line offer a more reliable alternative to a BOEC co-culture system for in vitro maturation, fertilization and culture of bovine embryos.     

Addition of penicillamine, hypotaurine and epinephrine (PHE) or bovine oviductal epithelial cells (BOEC) alone or in combination to bovine in vitro fertilization medium increases the subsequent embryo cleavage rate

The cleavage rate of in vitro-matured bovine oocytes was compared after fertilization in 1) TALP medium alone (control); 2) in TALP + BOEC; 3) in TALP + PHE; or 4) in TALP + BOEC and PHE. The overall cleavage rate at 45 h post insemination was greater for embryos in Treatments 2 (52%), 3 (55%) and 4 (66%) than for Treatment 1 (32%). The oocyte cleavage rates for Treatments 2 and 3 were similar, but were lower than that of Treatment 4. Addition of PHE or BOEC, alone or in combination, to the fertilization medium resulted in more embryos at the 3- or 4-cell stage than the 2-cell stage by 45 h post insemination. After 5 d of co-culture with BOEC in M-199 medium, 21, 28, 25 and 35% of the cleaved embryos in Treatments 1, 2, 3 and 4, respectively, developed to the morula or blastocyst stage. The rate of development to morulae and blastocysts was similar among Treatments 1, 2 and 3, and between Treatments 2 and 4. Across treatments, a correlation of 0.98 was noted between the portion of embryos that had reached the 3- or 4-cell stage by 45 h post insemination and the percentage of embryos in each treatment that continued to develop to the morula or blastocyst stage in vitro.     

Bovine 1-2-cell embryo development using a simple medium in three oviduct epithelial cell coculture systems

These studies were designed to develop a coculture system using a simple medium to promote development of 1-cell bovine embryos through the 8-16-cell stage to morula and blastocyst stages. Monolayers for coculture were prepared from bovine oviduct epithelial cells (BOEC). In vivo-fertilized 1-2-cell embryos and ova (384) were surgically collected from superovulated cows. In Experiment 1, embryos cocultured in a simple glucose-free and serum-free medium (CZB) developed with superior scores of embryo quality than embryos cocultured in Ham's F-10 with serum, and a greater percentage developed past 8-16 cells than embryos cocultured in CMRL-1066 with serum (p less than 0.05). In Experiment 2, embryos cocultured with fresh BOEC monolayers averaged more (p less than 0.05) cells than did embryos in coculture with frozen-thawed BOEC monolayers or in BOEC-conditioned medium. Without glucose in the simple medium for the first 48 h of culture, more embryos blastulated (p less than 0.01) by Day 5.5 of culture (Day 6.5 of donor's estrous cycle) than embryos in the same medium with glucose present throughout. In Experiment 3, more embryos tended to hatch in BOEC coculture (p less than 0.10) than in conditioned medium. These results show that a chemically simple medium with fresh BOEC monolayers can provide a significant benefit for coculture of early bovine embryos.     

Improvement of in vitro co-culture systems for bovine embryos using a low concentration of carbon dioxide and medium supplemented with beta-mercaptoethanol

Two experiments were conducted to investigate the effect of carbon dioxide (CO2) gas atmosphere and beta-mercaptoethanol on the development of bovine embryos in an in vitro co-culture system. In Experiment 1, in vitro-matured bovine oocytes were inseminated and then co-cultured with cumulus cells in culture medium (CM; 25 mM HEPES buffered TCM-199 supplemented with 5% superovulated cow serum and 0.5 mM sodium pyruvate). Oocytes matured and fertilized in 2 or 5% CO2 in air exhibited similar cleavage rates, but the proportion of embryos that developed to the blastocyst stage was higher for embryos co-cultured in 2 versus 5% CO2 in air. In Experiment two, 4- to 8-cell embryos produced under the condition of 2% CO2 in air were co-cultured with cumulus cells in CM supplemented with various levels of beta-mercaptoethanol (0, 5, 10, 50 microM). The percentage of embryos that developed to the blastocyst stage in CM with 10 microM beta-mercaptoethanol was higher (P<0.05) than that of embryos co-cultured with 0 or 50 microM beta-mercaptoethanol. These results indicate that cumulus cell co-culture in an atmosphere of 2% CO2 in air has a marked stimulatory effect on in vitro development of bovine embryos and that addition of beta-mercaptoethanol to the co-culture medium 2 d after insemination improved the in vitro development of bovine 4- to 8-cell embryos to the blastocyst stage.     

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.     

Oviduct Epithelial Cell Co-culture of early Porcine Embryos

One- to 16-cell porcine embryos were cultured in either Whittens medium supplemented with bovine serum albumin and fetal calf serum (WM) or in the same medium with porcine oviduct epithelial cell co-culture (WM-Poec). All stages of embryos cultured in WM-POEC had higher cell counts after 144–168 h of development than did embryos in WM. There was however, no significant difference in blastocyst formation rate of embryos cultured in WM-POEC over those cultured in WM. A high proportion of the embryos entering culture at the 1-2-cell were able to pass the 4-cell block stage in both WM and WM-POEC, 81% and 77%, respectively. In both media, most of the 1-2-cell embryos arrested their development at the compacted morula stage and failed to blastulate while embryos initiating culture at the 4-and 8-16-cell embryos formed blastocysts in culture at a rate of 80–90%.     

Effects of protein source and co-culture on bovine embryo development in synthetic oviductal fluid medium

Experiment 1 compared the development of 2- to 4-cell bovine embryos cultured in synthetic oviductal fluid with 20% fetal calf serum or 3.2% BSA and in the presence of oviductal cells, cumulus cells, or medium alone. More embryos developed in medium with serum, regardless of culture method (P = 0.063). Oviductal cell co-culture resulted in more embryos developing to at least the morula stage (P /= 0.400). Addition of serum to oviductal cell co-culture medium increased the number of excellent or good quality embryos (P = 0.019). Experiment 2 further compared the development of 2-cell or 3- to 4-cell embryos co-cultured with oviductal cell suspensions in serum-supplemented synthetic oviductal fluid or M-199 medium. More 3- to 4-cell than 2-cell embryos developed to at least the morula stage (P < 0.001). More embryos developed to at least the morula stage in synthetic oviductal fluid (P = 0.083). Neither initial embryo cell stage nor medium type influenced the percentage of developing embryos that achieved the blastocyst stage or final morphological quality of embryos (P >/= 0.535).     

Development of in vitro matured and fertilized bovine embryos cocultured with bovine oviductal epithelial cells obtained from oviducts ipsilateral to cystic follicles.

The present experiment was conducted to clarify the effect of bovine oviductal epithelial cells (BOEC) collected from oviducts ipsilateral to cystic follicles (CFs) using an in vitro coculture system on the development of in vitro matured/fertilized (IVM/IVF) bovine embryos. In the first comparison, the effect of the presence of CF on the development of the embryos cocultured with BOEC derived from the cows with CF (n = 18) and corpus hemorrhagicum (CH, n = 10) was examined. In the second comparison, the effect of the type of cyst [progesterone (P4)-dominant; n = 9, estradiol-17beta (E2)-dominant; n = 5] on the development of the embryos cocultured with BOEC derived from the cystic cows was examined. No difference was observed between CF and CH (control) groups in the mean developmental rates of embryos developed to > or =2-cell (86.3% vs. 86.4%), 8-16 cells (53.0% vs. 56.2%), blastocyst (24.2% vs. 24.8%) and hatched blastocyst (12.0% vs. 14.6%). However, the blastocyst production rate was significantly different (P<0.05) between the P4-dominant (19.8%) and E2-dominant (32.6%) groups. The rate of development from cleavage stage embryo to blastocyst was significantly different between P4-dominant (22.9%) and E2-dominant (37.9%) groups. Moreover, the blastocyst rate from 8-16 cells of E2-dominant group (61.6%) was significantly higher than that of P4-dominant one (39.5%). These results indicate that the effects of BOEC collected from oviduct ipsilateral to CFs on embryo development are variable, and the variability is closely associated with the steroid hormone profiles of the follicular fluid.     

Porcine oviductal cells support in vitro bovine embryo development

This study was designed to investigate the developmental competency of in vitro-matured and in vitro-fertilized bovine embryos co-cultured with a) medium alone, b) bovine oviductal cells (BOC), c) bovine conditioned medium (BCM), d) porcine oviductal cells (POC), and porcine conditioned medium (PCM). Follicular oocytes collected from cattle at local slaughterhouses were matured and fertilized in vitro. Epithelial cells were scraped from the luminal surface tissue of either bovine or porcine oviducts collected after ovulation, cultured in TALP + 10% heat-treated fetal calf serum, and the conditioned media were collected following a 3- to 5-d incubation period. After 18 to 22 h of sperm-ova co-incubation, the fertilized and/or cleaved ova were randomly assigned to 1 of 5 co-culture groups. The results revealed that the efficiency of medium alone in supporting embryo development from the 16- to 32-cell stage up to the blastocyst stage was significantly (P<0.01) lower than of embryos co-cultured with either bovine or porcine epithelial cells, or with conditioned media from such cells. Epithelial cell co-culture, regardless of cell source, was more effective (P<0.01) than culture with conditioned medium. Co-culture in medium containing or conditioned by porcine cells was more effective in supporting bovine embryo development than co-culture with bovine-derived cells or conditioned medium. These data support the concept that oviductal cells produce a soluble component which enhances embryo development to the blastocyst stage in vitro and that the effect is not species-specific.     

Culture of bovine embryos to the blastocyst stage using Buffalo rat liver (BRL) cells

A comparison was made between the development of in vitro matured and fertilized bovine oocytes in co-culture with bovine oviduct epithelial (BOE) cells or with Buffalo rat liver (BRL) cells. Both cell types supported development from the 1-cell to the blastocyst stage with equal efficiencies (4.4% for BRL cells, 4.0% for BOE cells). Medium conditioned by either cell type supported development to the blastocyst stage as efficiently as co-cultures (6.4 and 7.3% blastocysts for BOE and BRL conditioned medium, respectively). A higher percentage of blastocyst development was found when embryos were cultured closely apposed in small drops of BRL-conditioned medium compared with larger volumes (20.5 versus 7.0%). The ability of BRL-conditioned medium to support embryonic development was dependent on the duration of the conditioning period (optimum 24 to 48 h), and was not lost when the medium was stored at -20 degrees C for extended periods. The effects were independent of the conditions used to promote maturation in vitro and the procedure for fertilization. With 2 different methods to produce embryos in culture, both the BRL cell co-culture and BRL-conditioned medium in microdrops supported embryo development to the blastocyst stage. The use of the BRL cell line reduces the variability associated with primary BOE cell cultures.     

Survival rates and sex ratio of bovine IVE embryos frozen at different developmental stages on day 7

Two experiments were designed to determine the effects of stage of development on Day 7 of in vitro-produced bovine embryos on survival after deep freezing and on sex ratio. Bovine IVF embryos and bovine oviductal epithelial cells (BOEC) were co-cultured in TCM-199 and, on Day 7 after insemination (Day 0), were morphologically evaluated and divided into groups by developmental stage. In Experiment 1, embryos classified as early blastocysts, blastocysts and full-expanding blastocysts were randomly subdivided into 2 groups by replicate: 50% of the embryos were placed immediately in a new BOEC co-culture (fresh group), while the other 50% were frozen, thawed and placed in a new BOEC co-culture (frozen/thawed group). Embryos were frozen in 1.5 M glycerol using a standard slow cooling technique. Fresh and frozen/thawed embryos were compared for survival rate (embryos hatching/hatched) in BOEC co-culture over the following 3 d (i.e., Days 7 to 10). The overall survival of the 425 embryos (early to full-expanding blastocysts) was 33% and was not different between fresh (35%) and frozen/thawed (30%) embryos. Survival of embryos cultured fresh or after freezing/thawing was higher for full-expanding blastocysts than for early blastocysts or for blastocysts, both of which were not different. In Experiment 2, all frozen/thawed embryos used in Experiment 1 plus all morulae and hatched blastocysts collected and frozen on Day 7 without regard to survival were sexed utilizing the polymerase chain reaction (PCR) technique. Sex of the embryos, by stage of development on Day 7, was determined in order to compare the rate of development in BOEC co-culture with the sex ratio (percentage of males). A total of 235 embryos was sex-determined with an overall percentage of males of 51%, which was not different from the expected 1:1 sex ratio. Both full-expanding blastocysts and hatched blastocysts had a significantly higher (P < 0.05) proportion of males (68 and 100%, respectively), while morulae had a significantly lower proportion of males (24%). Early blastocysts and blastocysts did not differ from a 1:1 sex ratio. The results indicate that male embryos develop faster in vitro than female embryos. The higher survival rate of full-expanding blastocysts after freezing/thawing, and the production of a higher number of males than females among embryos of this developmental stage suggest that a greater number of male fetuses may result from the successful freezing and transfer of in vitro-produced bovine embryos.     

Comparison of sex ratio and cell number of IVM-IVF bovine blastocysts co-cultured with bovine oviduct epithelial cells or with Vero cells

The influence of 2 co-culture systems (BOEC and Vero cells) on the development rates, quality grades and sex ratios of IVM-IVF bovine embryos were studied. Zygotes obtained after IVF were co-cultured in each co-culture system for 7 and 8 d (Day 0 = day of insemination) in B2 medium. No effect of the co-culture system was observed on development rates measured on Days 7 and 8. However, Vero cell co-culture had a positive influence on embryo quality. Irrespective of their sex, embryos produced on Vero cells showed higher cells number than those co-cultured on BOEC (103.4 +/- 3.8 and 97 +/- 8.12 for BOEC vs 113.7 +/- 3.5 and 114 +/- 5.9 for Vero cells at Days 7 and 8, respectively; P < 0.05). The percentage of male embryos was increased in the two co-culture systems (60.7% males for BOEC; P < 0.05 vs 63% males for Vero cells; P < 0.01) on Day 7. In both co-culture systems the increase in the percentage of males was more obvious for embryos reaching the most advanced stage (expanded blastocysts). The results show that Vero cells improved the quality grade of bovine embryos produced in vitro, and thus are recommended for use as a safe co-culture system that does not contain pathogens.     

Oviductal fluid and growth factors failed to enhance development of porcine embryos

Porcine embryos (1-, 2- and 4-cell) were cultured in a basal medium consisting of Krebs-Ringer bicarbonate buffer supplemented with oviductal fluid and several growth factors and observed for further development. Oviducts were flushed at either 48 h (Experiment 1) or 96 h (Experiment 2) after the onset of estrus. Observations were made every 48 h (Experiment 1) or 12 h (Experiment 2) until failure of the embryos to develop for 2 consecutive observations. Embryos were scored 0 = no development, 1 = cleavage, 2 = morula, 3 = blastocyst, or 4 = hatched blastocyst. In the first experiment, development of 1-, 2- and 4-cell embryos (n=282) in the basal medium supplemented with oviductal fluid (4:1) or 3 sets of growth factors, was less or equal to one cleavage stage. Those embryos cultured in the basal medium supplemented with bovine serum albumin (fatty acid free) (BSA) advanced to the blastocyst stage. In the second experiment, 96 h aged embryos (n=142) were cultured in the basal medium supplemented with IGF-1 and - 2 and EGF, or with BSA alone or with BSA and the three growth factors. In the treatments without BSA, the embryonic development was less than one cleavage, whereas in those treatments with BSA, embryos advanced beyond hatching and began to expand. We conclude that for culture of porcine embryos, supplementation with several growth factors or with oviductal fluid, in the concentration used in this study, was of little benefit at this stage of development. However, the type of BSA significantly affected development. More than 90% of the embryos reached the morula and blastocyst stages in medium than included BSA (fatty acid free).     

Open-pulled straw vitrification differentiates cryotolerance of in vitro cultured rabbit embryos at the eight-cell stage

The objective was to determine cryotolerance of in vitro cultured rabbit embryos to the open-pulled straw (OPS) method. Overall, 844 rabbit embryos at pronuclear, 2- to 4-cell, 8-cell, and morula/blastocyst stages were vitrified, and ≥ 1 mo later, were sequentially warmed, rehydrated, and subjected to continuous culture (n = 691) or embryo transfer (ET, n = 153). Embryos vitrified at the 8-cell stage or beyond had greater survival, expanded blastocyst and hatched blastocyst rates in vitro, and better term development than those vitrified at earlier stages. The 8-cell group had 70.1% expanded blastocysts, 63.7% hatched blastocysts, and 25.7% term development, as compared to 1.5-17.7%, 1.5-4.3% and 2.8-3.7% in the pronuclear, 2-cell and 4-cell embryos, respectively (P < 0.05). The expanded and hatched blastocyst rates in vitrified morula/blastocyst post-warming were higher than that in the 8-cell group; however, their term development after ET was similar (8-cell vs morula/blastocyst: 25.7 vs 19.4%, P > 0.05). Development after ET was comparable between vitrified-warmed embryos and fresh controls at 8-cell and morula/blastocyst stages (19.4-25.7 vs 13.7-26.6%, P > 0.05). For embryos at pronuclear or 2- to 4-cell stages, however, term rates were lower in the vitrified-warmed (2.8-3.7%) than in fresh controls (28.6-35.6%, P < 0.05). Therefore, cultured rabbit embryos at various developmental stages had differential crytolerance. Under the present experimental conditions, the 8-cell stage appeared to be the critical point for acquiring cryotolerance. We inferred that for this OPS cryopreservation protocol, rabbit embryos should be vitrified no earlier than the 8-cell stage, and stage-specific protocols may be needed to maximize embryo survival after vitrification and re-warming.     

Effect of retinoids and growth factor on in vitro bovine embryos produced under chemically defined conditions

Experiments were conducted to investigate the beneficial effects of adding retinol (RT) and retinoic acid (RA) to bovine oocyte maturation media and insulin-like growth factor-I (IGF-I) to embryo culture under chemically-defined conditions. In Experiment 1.1, in vitro maturation (IVM) was performed in basic maturation media (bMM) and supplemented with 0.3microM RT or 0.5microM RA. For embryo development presumptive zygotes and embryos were placed in droplets of potassium simplex optimized medium (KSOM). Addition of RT and RA to bMM improved (p<0.05) blastocyst formation as compared with control treatments. In Experiment 1.2, using embryos originating from oocytes previously treated with RT and RA, the presumptive zygotes were placed in droplets of KSOM and embryos (2-4 cells) in droplets of fresh KSOM supplemented or not with IGF-I. The number of 2-4-cell stage embryos developing to the blastocyst and expanded blastocyst stages were greater (p<0.05) when embryo culture media was supplemented with IGF-I. In Experiment 2.1, IVM was conducted with bMM+FSH containing 0.3microM RT or 0.5microM RA. For embryo development, presumptive zygotes were placed in droplets of KSOM. Addition of RT or RA to IVM medium also enhanced (p<0.05) blastocyst formation. The supplementation of embryo culture media with IGF-I resulted in a greater number (p<0.05) of 2-4-cell stage embryos developing into blastocysts, expanded blastocysts and hatched blastocysts. In Experiment 2.2, using embryos originating from oocytes previously treated with RT and RA, presumptive zygotes were also placed in droplets of KSOM and embryos (2-4 cells) in droplets of fresh KSOM supplemented or not with IGF-I. The supplementation of embryo culture media with IGF-I resulted in a greater (p<0.05) number of 2-4-cell stage embryos developing to the blastocyst, expanded blastocyst and hatched blastocyst stages.     

Nonspecies-specific effects of mouse oviducts on the development of bovine IVM/IVF embryos by a serum free co-culture

In Experiment 1, development of bovine embryos derived from in vitro-matured (IVM) and in vitro-fertilized (IVF) oocytes was examined under 4 culture conditions: 1) co-culture with mouse ampullae continuously for 8 d, 2) co-culture with mouse ampullae that were replaced with fresh ampullae at 48-h intervals, 3) co-culture with bovine granulosa cell monolayers, and 4) culture in medium alone. Culture medium consisted of tissue culture medium 199 (TCM-199) supplemented with 1% fetal calf serum (FCS). Inseminated oocytes were transferred to each of the culture treatment 24 h after insemination and were cultured for 8 d. The number of blastocysts per number of cleaved ova obtained after co-culture with mouse ampullae (42.9%) was significantly (P<0.05) higher than that obtained after co-culture with granulosa cell monolayers (28.3%) or culture without cells (4.2%). In Experiment 2, the developmental ability of bovine IVM/IVF embryos co-cultured with mouse ampullae supplemented with or without serum was examined. When serum was excluded from the culture medium, 26.4% (33 125 ) of the total number of embryos cultured were able to develop to the blastocysts stage using this co-culture system. This value was comparable to that obtained in a serum-supplemented co-culture system (30.7%; 39 125 ). In addition, the developmental ability of embryos that reached to the 4-cell stage or beyond at 46 to 48 h after insemination was not significantly different when the embryos were co-cultured with mouse ampullae with (38.5 vs 44.6%) or without (37.0 vs 33.8%) serum.     

Gene expression and in vitro development of inter-species nuclear transfer embryos

This study examined the chromatin morphology, in vitro development, and expression of selected genes in cloned embryos produced by transfer of mouse embryonic fibroblasts (MEF) into the bovine ooplasm. After 6 hr of activation, inter-species nuclear transfer (NT) embryos (MEF-NT) had one (70%) or two pronuclei (20%), respectively. After 72 hr of culture in vitro, 62.6% of the MEF-NTs were arrested at the 8-cell stage, 31.2% reached the 2- to 4-cell stage, and only 6.2% had more than eight blastomeres, but none of these developed to the blastocyst stage. Whereas, 20% of NT embryos derived from bovine embryonic fibroblast fused with bovine ooplasm (BEF-NT) reached the blastocyst stage. Donor MEF nuclei expressing an Enhanced Green Fluorescent Protein (EGFP) transgene resulted in 1- to 8-cell stage MEF-NT that expressed EGFP. The expression of selected genes was examined in 8-cell MEF-NTs, 8-cell mouse embryos, enucleated bovine oocytes, and MEFs using RT-PCR. The mRNA for heat shock protein 70.1 (Hsp 70.1) gene was detected in MEF-NTs and MEF, but not in mouse embryos. The hydroxy-phosphoribosyl transferase (HPRT) mRNA was found in normal mouse embryos and MEF but not in MEF-NTs. Expression of Oct-4 and embryonic alkaline phospatase (eAP) genes was only detected in normal mouse embryos and not in the inter-species NT embryos. Abnormal gene expression profiles were associated with an arrest in the development at the 8-cell stage, but MEF-NT embryos appeared to have progressed through gross chromatin remodeling, typical of intra-species NT embryos. Therefore, molecular reprogramming rather than chromatin remodeling may be a better indicator of nuclear reprogramming in inter-species NT embryos.