Co-culture of day-5 to day-7 equine embryos in medium with oviductal tissue

Oviductal and uterine embryos were collected from mares at 5 to 7 days following ovulation 1) to evaluate the effects of oviductal tissue explants on in vitro growth and development of equine embryos and 2) to study the morphologic development of equine embryos in culture. Embryos were incubated for 5 days in a medium (control group) or in medium supplemented with oviductal tissue explants (co-culture group). Embryos were evaluated and the media changed daily. Following 5 days in culture, 10 10 (100%) control embryos and 27 29 (93%) co-cultured embryos had doubled in diameter. All embryos that were recovered as morulae developed to the blastocyst stage in culture. By 5 days in culture, 6 10 (60%) control embryos and 19 29 (66%) co-cultured embryos had reached the hatching blastocyst stage of development. By 3 days in culture, significantly more (P<0.05) control embryos versus co-cultured embryos had degenerated (4 10 vs 2 29 , respectively). By 5 days in culture, significantly more (P<0.01) control embryos versus co-cultured embryos had degenerated (6 10 vs. 3 29 , respectively). Embryos cultured with oviductal tissue were sustained longer than embryos cultured in medium alone. Hatching was characterized by the blastocyst squeezing through a small opening in the zona pellucida or by the zona pellucida thinning over approximately half of the blastocyst surface and subsequently disappearing entirely.     

Commercial freezing of bovine embryos in straws

Bovine embryos were frozen commercially in clear double length $\text{1}\text{2}$ cc French straws with the wick and powder plug in the center of the straw. One-half of the double length straw serves as a handle and contains a color coded $\text{1}\text{4}$ cc straw around which an adhesive backed label has been applied. After plunging into liquid nitrogen, straws are transferred into goblets on canes while under liquid nitrogen. The straws are stored in the liquid phase of a nitrogen tank and canes containing straws are not transferred from one container to another unless the goblet containing the straws is full of liquid nitrogen.Embryos held for longer than 4 hours after collection prior to freezing showed a steady decline in pregnancy rate related to the length of time held prior to freezing. The percentage of embryos thawed and then evaluated as being transferrable was related to the quality of the embryos prior to freeze (Grade 1–93.6%, Grade 2–87.0%, Grade 3–63.8%). There was no statistical difference in pregnancy rates obtained from prefreeze Grade 1 embryos when comparing advanced blastocysts (45.2%), blastocysts (38.7%), early blastoclyst (43.1%) and advanced morula (41.6%).     

Prostaglandin E2 secretion by day-6 to day-9 equine embryos.

Prostaglandin E2 (PGE2) secreted by Day-6, Day-7, Day-8 and Day-9 equine embryos (ovulation = Day 0) during in vitro incubation was measured by radioimmunoassay. Embryonic PGE2 secretion (ng/embryo/24 hr) was detectable on Day 6 (0.27 +/- 0.39), tended to increase (P less than 0.1) on Day 7 (0.57 +/- 0.88), and increased significantly (P less than 0.05) on Day 8 (2.23 +/- 0.86) and Day 9 (4.13 +/- 0.71). Embryo diameter at the start of the incubation period was linearly correlated (P less than 0.01) to embryonic PGE2 secretion.     

Pluripotency of bovine embryonic cell line derived from precompacting embryos

We report herein the establishment of three bovine pluripotent embryonic cell lines derived from 8-16-cell precompacting embryos. Two cell lines were cultured for 10 passages and underwent spontaneous differentiation. One cell line (Z2) has been cultured continuously for over 3 years and has remained undifferentiated. These cells express cell surface markers that have been used routinely to characterize embryonic stem (ES) and embryonic germ (EG) cells in other species such as stage-specific embryonic antigens SSEA-1, SSEA-3, and SSEA-4, and c-Kit receptor. In the absence of a feeder layer, these cells differentiated into a variety of cell types and formed embryoid bodies (EBs). When cultured for an extended period of time, EBs differentiated into derivatives of three EG layers - mesoderm, ectoderm, and endoderm - which were characterized by detection of specific cell surface markers. Our results indicate that the Z2 cell line is pluripotent and resembles an ES cell line. To our knowledge, this is the first bovine embryonic cell line that has remained pluripotent in culture for more than 150 passages.     

Effects of freezing of bovine preimplantation embryos derived from oocytes fertilized in vitro on survival of their inner cell mass cells

The morphology of the inner cell mass (ICM) cells and the proportion of dead ICM cells in frozen-thawed bovine preimplantation embryos were investigated by differential fluorochrome staining. Embryos at the blastocyst stage of development were frozen and thawed by two different techniques (three-step and one-step) in two different basic salt solutions (PBS and TCM 199) containing 1.36M glycerol. After thawing and glycerol removal, embryos were co-cultured in a cumulus cells monolayer in TCM 199 for 48 hr (morula) or 24 hr (blastocysts). Differential cell counts of the ICM and trophectoderm were then done using differential fluorochrome staining. Overall, there was no significant difference in the viability of embryos frozen in the two basic salt solutions. Low proportions of dead ICM cells were observed in embryos frozen at the morula stage in both PBS (19.1%) or TCM 199 (18.0%). However, blastocyst stage embryos frozen by the three-step technique had a higher (P < 0.05) proportion of dead ICM cells in TCM 199 (37.7%) than in PBS (18.2%). Blastocysts frozen by the one-step technique had a higher (P < 0.05) proportion of dead ICM cells (42.2%) than those frozen by the three-step technique (18.2%), regardless of basic salt solutions. Results indicate that freezing and thawing damages ICM cells in morphologically normal embryos and that the degree of damage depended on the basic salt solution and the freezing method. © 1994 Wiley-Liss, Inc.     

Prostaglandin E2 secretion by day-6 to day-9 equine embryos

Prostaglandin E₂ (PGE₂) secreted by Day-6, Day-7, Day-8 and Day-9 equine embryos (ovulation = Day 0) during in vitro incubation was measured by radioimmunoassay. Embryonic PGE₂ secretion (ng/embryo/24 hr) was detectable on Day 6 (0.27±0.39), tended to increase (P <0.1) on Day 7 (0.57±0.88), and increased significantly (P <0.05) on Day 8 (2.23±0.86) and Day 9 (4.13±0.71). Embryo diameter at the start of the incubation period was linearly correlated (P <0.01) to embryonic PGE₂ secretion.     

Secretion of interferon-tau by bovine embryos in long-term culture: comparison of in vivo derived, in vitro produced, nuclear transfer and demi-embryos.

Interferon-tau (IFNtau) is the pregnancy recognition signal of bovine embryos, inhibiting luteolysis. We studied trophoblastic growth and IFNtau secretion of embryos with different developmental potential, i.e., in vivo derived and in vitro produced embryos, cloned embryos and demi-embryos, to evaluate if the ability of secreting IFNtau might be responsible for differences in pregnancy rates after transfer of these categories of embryos to recipients. Day 8 embryos of excellent quality were individually placed in microdrops of buffalo rat liver cell-conditioned medium and maintained for up to 23 days. Embryos were observed on Days 11, 15, 19 and 23, the mean diameter (2r) of attached and spherical embryos was measured, and their trophoblastic area was calculated as r2pi or 4r2pi, respectively. Simultaneously, medium was changed and the IFNtau levels of conditioned media were determined using a bioassay of antiviral activity. Trophoblastic area was smaller (P < 0.05) in demi-embryos than in all other groups, which exhibited similar trophoblastic growth until Day 19. However, on Day 23 trophoblastic area of in vivo derived embryos was more than twice (P < 0.05) as large as those of in vitro produced and nuclear transfer (NT) embryos. IFNtau levels increased only slowly with time in culture of demi-embryos. By contrast, the level of IFNtau doubled from Day 11 to Day 15 in conditioned media from all other groups of embryos. The linear increase in IFNtau production of vivo and in vitro derived embryos continued until the end of the culture period, whereas conditioned media from NT embryos contained significantly (P < 0.05) less IFNtau activity on Days 19 and 23 than those of the former two groups. Our results demonstrate different capabilities of secreting IFNtau for in vivo derived and in vitro produced embryos vs. NT and demi-embryos, which may--at least part--be responsible for the differences in pregnancy rates after transfer to recipients.     

Survival of biopsied and sexed bovine demi-embryos

The viability of sex-diagnosed bovine demi-embryos was investigated after transfer. Day-7 morulae and blastocysts were subjected to splitting and biopsy in PBS + 4mg/ml polyvinylpyrrolidone + 200mM sucrose using a microblade. The biopsy (approximately 2 to 8 blastomeres) was transferred to a tube, and its presence in the tube was verified by examination under a stereomicroscope. After proteinase K treatment, repetetive male-specific DNA was amplified by the polymerase chain reaction (PCR). No autosomal control primers were used in the PCR. Instead, the absence of a characteristic Y-specific product together with the amplification of non-specific products was considered an indication of a female sample. The biopsied demi-embryos were transferred either singly or in pairs to synchronous heifer or cow recipients 6 to 10 h after flushing. Sex diagnosis was carried out within 6 to 7 h. Of 19 original embryos, 7 were diagnosed as males and 5 as females. The DNA of the biopsies of the remaining 7 embryos did not result in any amplification products. Since 5 of these samples were seen in the tubes prior to PCR, the corresponding embryos were considered "potential females." The sex of the last 2 samples could not be determined. Nine of 10 embryos were correctly sexed as revealed by calving data. Of the 38 transferred demi-embryos, 16 had developed to live fetuses as detected by ultrasonography on Day 65 of pregnancy. Eleven live calves and three stillborn calves were delivered. After bisection, biopsy and single transfer, 6 live calves were born from 7 original embryos (86%). After transfer of both halves into the same recipient, only 5 live calves from 12 original embryos were produced (42%). None of the 4 manipulated Grade-2 embryos survived to term, nor did any of the 4 manipulated blastocysts. Of the 14 original Grade-1 morulae manipulated and transferred, 15 were live fetuses at Day 65, and 11 live calves were born.     

Transfer of inner cell mass cells derived from bovine nuclear transfer embryos into the trophoblast of bovine in vitro-produced embryos

Presence of placental tissues from more normal noncloned embryos could reduce the pregnancy failure of somatic cloning in cattle. In this study, inner cell mass (ICM) cells of in vitro-produced (IVP) embryos was replaced with those of nuclear transfer (NT) embryos to reconstruct bovine blastocysts with ICM and trophoblast cells from NT and IVP embryos, respectively. A total of 65 of these reconstructed embryos were nonsurgically transferred to 20 recipient beef females. Of those, two females were diagnosed pregnant by ultrasonography on day 30 of gestation. One pregnancy was lost at 60-90 days of gestation, and the other recipient cow remained pregnant at day 240 of gestation; however, this female died on day 252 of gestation. Gross pathology of the internal organs of the recipient female, a large fetus, and a large placental tissue mass suggested the massive size of the fetus and placental tissue were likely involved in terminating the life of the recipient female. Biopsy samples were harvested from the skin of the dead recipient cow, the fetus and from cotyledonary tissue. Microsatellite DNA analysis of these samples revealed that the genotype of the fetus was the same as that of the NT donor cells and different from that of the recipient cow. Correspondingly, neither the fetus nor recipient cow had the same genotype with that of the fetal cotyledonary tissue. These results present the first known documented case of a bovine somatic NT pregnancy with nonclone placental tissues after transfer of a blastocyst reconstructed by a microsurgical method to exchange of ICM cells and trophoblast tissue between NT and IVP blastocysts.     

The monitoring of bovine pregnancies derived from transfer of in vitro produced embryos

Both an increased rate of embryonic, foetal and perinatal losses, and the occurrence of deviations in foetal and placental development are associated with bovine pregnancies obtained from in vitro produced embryos. This thus requires for a more accurate and frequent monitoring of foetal and maternal functions during pregnancies. Such approaches will enable to establish the period during which these losses and deviations in development occur and to plan possible clinical interventions. This paper reviews some recent data on return rates, late embryonic and foetal losses in recipients after the transfer of either MOET, IVF or nuclear transfer embryos. Special attention is paid to the diagnostic value of measurements of pregnancy specific/associated proteins and progesterone in maternal plasma. Possibilities to measure foetal body sizes, size of placentomes and foetal heart rate by means of transrectal or transabdominal ultrasonography are illustrated with data from the literature and with recent results from our own large field study with MOET, IVP-co-culture and IVP-SOF embryos.     

Incidence of chromosomal anomalies in early bovine embryos derived from in vitro fertilization

The incidence of chromosomal anomalies in early bovine embryos derived from follicular oocytes fertilized in vitro using sperm separated by Percoll density gradient centrifugation was investigated. Overall, chromosomal anomalies were observed in 13.7% (138/1005) of embryos. There were 14 haploids (1.4%), 2 hypodiploids (0.2%), 6 hyperdiploids (0.6%), 101 triploids (10.0%), 12 tetraploids (1.2%), 2 diploid/triploid mosaics (0.2%), and 1 diploid/tetraploid mosaic (0.1%). The frequency of triploidy was caused mainly by polyspermy. There was a significant difference in the frequency of embryos with abnormal chromosomes between the two bulls used (P < 0.005), but Percoll centrifugation did not affect the observed incidence of anomalies. The frequency of chromosomal anomalies in embryos at each stage increased with delay or arrest of development. These results suggest that the incidence of chromosomal anomalies depended on the conditions of in vitro fertilization and the arrest of development.     

Washing and trypsin treatment of in vitro derived bovine embryos exposed to bovine viral diarrhea virus

Gametes, somatic cells and materials of animal origin in media are potential sources for introducing bovine viral diarrhea virus (BVDV) into systems for production of IVF bovine embryos. Further, the efficacy of washing and trypsin treatment for removal of BVDV from IVF embryos is questionable. Washing and trypsin treatments recommended by the International Embryo Transfer Society for in vivo-derived embryos were applied to in vitro-derived, virus-exposed, bovine embryos in this side-by-side comparison of treatments. Embryos for the study were produced in a virus-free system in which follicular oocytes were matured and fertilized in vitro and presumptive zygotes were co-cultured with bovine uterine tubal cells for 7 d. A total of 18 trials was performed, 9 using a noncytopathic BVDV and 9 using a cytopathic BVDV. In each trial, 4 equal groups of 10 or less, zona pellucida-intact embryos/ova were assembled, including 2 groups of morulae and blastocysts (M/B) and 2 groups of nonfertile or degenerated ova (NFD). Each group was prewashed and exposed to 10(4) to 10(6) TCID50/mL of either noncytopathic (SD-1) or cytopathic (NADL) BVDV for 2 h. Following in vitro viral exposure, one group of M/B and one group of NFD were washed. The other groups of M/B and NFD were trypsin-treated. Both treatments were consistent with IETS guidelines. After in vitro exposure to noncytopathic BVDV and washing, viral assays of 100% (9/9) and 78% (7/9) of the groups of M/B and NFD ova, respectively, were positive. After in vitro exposure to cytopathic BVDV and washing, viral assay of 33% (3/9) of the groups of both M/B and NFD ova were positive. After in vitro exposure to noncytopathic BVDV and trypsin treatment, viral assay of 44% (4/9) of groups of M/B and 67% (6/9) of groups of NFD ova were positive. Finally, after in vitro exposure to cytopathic BVDV and trypsin treatment, viral assay of 22% (2/9) of the groups of M/B and 44% (4/9) of the groups of NFD ova were positive. Contingency table analysis, in which data was stratified by embryo type and virus biotype, was used to compare results. While a difference existed between results of the 2 treatments of groups of M/B within the noncytopathic biotype (P = 0.01, Mantel Haenszel Chi-square), no difference was observed between comparison of treatment between all groups in both biotypes (P > 0.05).