H.R. Tervit

Fertilized sheep and cattle ova have not been reported to develop readily during culture in vitro. Up to 60% of sheep morulae develop normally during culture (Moor & Cragle, 1971) but earlier cleavage stages undergo limited development (Hancock, 1963; Kraemer, 1966; Tervit & McDonald, 1969; Moore, 1970) and it has been suggested that there is a block to development in vitro at the eight- to twelve-cell stage (Wintenberger, Dauzier & Thibault, 1953). Only the early cleavage stages of cattle ova have been cultured and these have not been reported to develop beyond the twenty-four-cell stage in vitro (Thibault, 1966; Brinster, 1968; Sreenan, 1968; Sreenan, Scanlon & Gordon, 1968). This communication describes the successful culture of one-cell to eight-cell sheep ova and one-cell and eight-cell cattle ova to the morula and blastocyst stages and reports a high embryo survival after transfer of cultured ova to recipient animals. Welsh mountain ewes were induced to superovulate and were mated at the

Adult somatic cell nuclear transfer was used to determine the totipotent potential of cultured mural granulosa cells, obtained from a Friesian dairy cow of high genetic merit. Nuclei were exposed to oocyte cytoplasm for prolonged periods by electrically fusing quiescent cultured cells to enucleated metaphase II cytoplasts 4-6 h before activation (fusion before activation [FBA] treatment). Additionally, some first-generation morulae were recloned by fusing blastomeres to S-phase cytoplasts. A significantly higher proportion of fused embryos developed in vitro to grade 1-2 blastocysts on Day 7 with FBA (27.5 +/- 2.5%) than with recloning (13.0 +/- 3.6%; p < 0. 05). After the transfer of 100 blastocysts from the FBA treatment, survival rates on Days 60, 100, 180, and term were 45%, 21%, 17%, and 10%, respectively. Ten heifer calves were delivered by elective cesarean section; all have survived. After the transfer of 16 recloned blastocysts, embryo survival on Day 60 was 38%; however, no fetuses survived to Day 100. DNA analyses confirmed that the calves are all genetically identical to the donor cow. It is suggested that the losses throughout gestation may in part be due to placental dysfunction at specific stages. The next advance in this technology will be to introduce specific genetic modifications of biomedical or agricultural interest.

It has previously been reported that ovine embryos cultured in Synthetic Oviduct Fluid medium supplemented with 20% human serum (SOF+HS) develop into lambs with a high birth weight. We have investigated this phenomenon by culturing ovine zygotes in SOF+HS or a serum-free version of Synthetic Oviduct Fluid with BSA and amino acids (SOFaaBSA) in place of serum. Zygotes were either obtained from superovulated and naturally mated ewes or produced in vitro. Embryos were subsequently transferred to synchronized recipient ewes (n = 63). An additional group of ewes (n = 16) served as flock fertility and lambing controls. Development of zygotes to stages suitable for transfer (i.e., good to excellent compact morulae or blastocysts) was not affected by medium (SOFaaBSA = 53 +/- 5% vs. SOF+HS = 59 +/- 5%) but was affected by source (in vivo-derived = 74 +/- 5% vs. in vitro-derived = 35 +/- 5%, p < 0.001). Embryos incubated in SOF+HS were morphologically different from those incubated in SOFaaBSA, having abundant lipid droplets. Pregnancy rate (65%) and embryo survival (48%) of recipients determined by ultrasonography on approximately Day 60 of pregnancy did not differ between medium treatments or source of embryo. Mean weight of lambs from embryos cultured in SOF+HS (4.2 +/- 0.2 kg) was significantly heavier than that of controls (3.4 +/- 0.2 kg, p < 0.01) or of lambs from embryos cultured in SOFaaBSA (3.5 +/- 0.2 kg, p < 0.05). Furthermore, mean gestation length was longer in recipients receiving embryos incubated in SOF+HS (147 +/- 1 days) than in SOFaaBSA (145 +/- 1 day, p < 0.05). Reasons for this birth weight and gestation length difference are unclear, but our data suggest that different culture conditions can produce embryos with differing morphology, apparent chemical composition, and rate of development, resulting in lambs with differing gestation length and birth weight.

Two-cell sheep embryos and 2-4-cell and 8-cell cow embryos were cultured for 5 days in stoppered test-tubes in Synthetic Oviduct Fluid supplemented with 32 mg BSA/ml. The medium had been previously equilibrated with one of the following O2 concentrations (sheep: 0, 2, 4, 6, 8, 10, 12, 17, 20%; cow: 0, 4, 8, 12, 17, 20%). At the end of culture embryos were examined for morphology and stained to assess numbers of nuclei. Mean (+/- s.e.m.) nuclei/embryo was highest at 8% O2 for sheep embryos (23.6 +/- 3.1), 4% for 2-4-cell cow embryos (23.2 +/- 6.1) and 8% for 8-cell cow embryos (29.6 +/- 5.2). The minimum number of nuclei/embryo occurred at 20% O2 in each case (10.3 +/- 0.9, 10.3 +/- 2.7, 14.5 +/- 2.4, respectively) with similar values also recorded at 0% O2 (10.8 +/- 1.9, 16.5 +/- 6.0, 14.6 +/- 2.4, respectively). Analysis of the proportion of embryos reaching at least the morula stage demonstrated a significant quadratic component for the different oxygen concentrations for sheep (P less than 0.01) and cow (P less than 0.05) embryos. A number of sheep and cow embryos showed abnormalities, suggesting that the culture conditions require further refinement. The results confirm that, under lowered oxygen levels, development of sheep and cattle embryos can occur through the 8- to 16-cell block in a simple defined medium without somatic cell support.

Nuclear transfer procedures were used to determine the in vivo developmental potential of an ovine embryonic cell line isolated from the inner cell mass of a Day 8 blastocyst-stage embryo. This cell line possessed a differentiated epithelial-like cell morphology. In this study, a comparison was made between in vivo- and in vitro-derived oocytes used as recipient cytoplasts in the nuclear transfer procedure. Cultured cells were induced to quiesce and enter presumptive G0 before being used as donor karyoplasts between passages 8 and 16 of culture. After cell fusion, reconstructed embryos were cultured for 6 days in vitro in embryo culture medium. Blastocyst-stage embryos were subsequently transferred to synchronized recipient ewes (n = 37), and development was allowed to proceed to term. There was a significant effect of source of recipient cytoplast, with development being consistently greater with in vivo compared to in vitro cytoplasts in terms of, respectively, blastocysts produced (24.2 +/- 3.8% vs. 17.1 +/- 2.3%; p = 0.1), Day 35 pregnancy rate (40.0% vs. 9.1 %; p < 0.05), and Day 35 embryo survival (19.4% vs. 4.5%; p < 0.05). A high proportion of fetuses died during late gestation (5 of 8). The major abnormalities were associated with the urogenital tract. However, three lambs were delivered alive following cesarean section on Day 147. One lamb, derived from an in vitro-matured oocyte, died after 10 min, while the remaining two from in vivo-ovulated oocytes are apparently normal and healthy. DNA microsatellite markers conclusively show that the three lambs are genetically identical and were derived from the embryonic cell line. In conclusion, some cells from this blastocyst-derived embryonic cell line are totipotent by nuclear transfer and can produce viable offspring.