Cryopreservation of germ cells from bovine fetal ovaries.

Bovine fetuses at stages required for studies of female germ cells (primordial germ cells and oogonia) become available from the abattoir at unpredictable times. To alleviate this logistical problem, a procedure to cryopreserve these ovarian germ cells has been devised. Fetal ovarian cells were dispersed and suspended in 1.5 M dimethyl sulfoxide (DMSO) prepared in modified TCM 199 medium. The suspensions were aspirated into plastic semen straws, cooled, seeded to induce ice formation at -7 degrees C, and then cooled at 1 degree C min-1 to -70 degrees C before being plunged into liquid nitrogen at -196 degrees C for storage. The straws were thawed at a moderate rate of approximately 250 degrees C min-1, the DMSO was diluted 28-fold with culture medium, and then the cells were cultured for > 2 h before their viability was tested or they were used for nuclear transfer. No statistically significant difference in viability before and after cryopreservation was detected by vital staining with fluorescein diacetate (P > 0.05). When frozen-thawed germ cells were fused to cytoplasts, the cleavage rate of the resultant reconstructed embryos 44 h after fusion was 31%, although none developed into blastocysts. It is concluded that cryopreservation of bovine fetal ovarian germ cells is feasible and can play a major role in facilitating future experimentation.

Rate of abnormalities in lambs from in vitro produced embryos transferred on Day 2 compared with Day 6 postfertilization.

The effect of transferring ovine IVP embryos on Day 2 versus Day 6 postinsemination was investigated. Oocytes were collected from 35 cull ewes and cultured separately for each donor. Embryos were exposed to serum in the maturation and fertilization media, and then cultured in a serum-free SOF system under serum-conditioned silicone oil. Cleaved Day 2 postfertilization embryos were randomly allocated for immediate transfer versus Day 6 transfer. Parturition was induced (with 15 mg dexamethasone) between Days 141 and 143 of pregnancy; those failing to initiate parturition by Day 146 had a Caesarean section. Stillborn lambs and those dying within several days of birth were necropsied. Lambs were weighed at birth; lambs (n = 132) derived from the same flock (from natural breeding or AI) were used as a control. An estimate of the 99th percentile of the normal population was used as the point above which lambs from three litter-size categories were classified as abnormally large (analysis was repeated for 95% confidence and median estimates of this value). Thirty-eight lambs were born from early transfer, 45 from late transfer and 12 from unobserved lambings (parentage could not be determined). Lambs from early transfer were heavier at birth (P = 6.0 x 10 (-11)). The early treatment group had fewer lambs produced per embryo transferred (P = 0.0002), fewer live lambs per embryo transferred (P = 0.00009) and fewer normal lambs per embryo transferred (P= 0.0002). There was an effect of donor on the number of dead lambs per embryo transferred (P = 0.017). The number of dystocias per transfer was not significantly different for early versus late transfer groups. However, the probabilities of survival for cleaved embryos were 27.3% and 13.1% in the early and late transfer treatment, respectively. There were more lambs produced per cleaved embryo with early transfer (P = 0.004) and there was an effect of donor within ram and replicate (P = 0.04) on the number of lambs produced. The odds ratio for early/late treatment was 2.449 with a confidence interval of 1.368-4.382. Day 2 transfer of cleaved embryos did not prevent the production of oversized or abnormal offspring; however, there was an increased chance of a cleaved embryo producing a lamb when transferred on Day 2 rather than Day 6 postfertilization.

Effect of ambient temperatures during oocyte recovery on in vitro production of bovine embryos.

Recovery of oocytes from ovaries collected at slaughter was carried out at three ambient temperatures (25 degrees, 30 degrees and 35 degrees C) to assess the effect on subsequent embryonic production in vitro. Oocytes recovered at each temperature were thereafter maintained at temperatures > or =35 degrees C as they were subjected to in vitro maturation, fertilization and culture (IVM/IVF/IVC). The oocytes and resulting embryos within each temperature group were subsequently evaluated for their rates of fertilization, cleavage and development to blastocysts, as well as for the number of cells/blastocyst. The results demonstrate that exposure of cumulus-ocyte-complexes (COCs) to temperatures below 35 degrees C during oocyte recovery is detrimental to optimal embryo production. Although the fertilization and cleavage rates of oocytes recovered at temperatures below 35 degrees C were not significantly lower than that of the controls, the percentage of oocytes recovered at 35 degrees C that developed to the blastocyst stage following fertilization and culture (33.7%) was significantly greater than those from oocytes recovered at either 25 degrees C (22.4%) or 30 degrees C (19.5%). The mean numbers of blastomeres/embryo were significantly lower in embryos derived from oocytes collected at either 25 degrees or 30 degrees compared with those collected at 35 degrees C. The results of this study suggest that exposure of COCs to temperatures below 35 degrees C during oocyte recovery may significantly decrease both the quantity and quality of embryos produced by in vitro methods.

Transcription and translation in bovine nuclear transfer embryos.

The development of bovine embryos reconstructed by nuclear transfer (NT) is poor compared to that of embryos produced by in vitro fertilization. One reason for this could be incomplete reprogramming of the transferred nucleus. Therefore, with a view to optimizing the conditions for NT, the reprogramming of blastomere nuclei from 16- to 32-cell-stage in vitro-fertilized (IVF) embryos was investigated following NT by fusion of individual blastomeres with cytoplasts prepared from oocytes at two different stages of maturation. Heterogeneous RNA (hnRNA) production, nucleolar ultrastructure, and protein profiles of the NT embryos up to the 8-cell stage were analyzed. In all NT embryos analyzed for their hnRNA production (n = 133), [3H]uridine incorporation was higher at the 1-, 2-, and 4-cell stages than in control IVF embryos (n = 50). Ultrastructural examination of 11 NT embryos revealed evidence of transcriptional activity; fibrillar and granular components were seen in the nucleolus at the 1-cell stage. At the 2-, 4-, and 8-cell stages, fibrillar components were still evident but granular components had become scarce. The hnRNA synthesis, however, was not reflected in the one-dimensional electrophoretic patterns of protein production in the NT embryos (n = 56); these were largely similar to those of IVF embryos (n = 34) of corresponding stages. Thus, NT embryos made in this way do not behave like equivalent IVF embryos, suggesting that reprogramming of the transferred nucleus is absent or incomplete.

Development of bovine nuclear transfer embryos made with oogonia.

The pluripotency of embryonic germ cells in the mouse suggests that mitotic bovine fetal germ cells might also be a source of pluripotent cells. To investigate the pluripotency of bovine oogonia, the development in vitro of bovine embryos reconstructed by fusing oogonia with enucleated oocytes was compared with that of embryos made similarly with either blastomeres or granulosa cells. The donor cells (fresh oogonia, cryopreserved oogonia, 16- to 32-cell-stage blastomeres, or granulosa cells) were fused to the enucleated oocytes electrically. The proportions of reconstructed embryos that had cleaved at 40 h after fusion using these types of donor cells were not significantly different (37%, 33%, 56%, and 31%, respectively; p > 0.05). However, the proportions of cleaved reconstructed embryos that developed to the blastocyst stage were 9%, 13%, 36%, and 3%, respectively, significantly higher (p < or = 0.05) with blastomeres than with the other three types of donor cells. After transfer of 3 morulae and 4 blastocysts made with oogonia into three recipient heifers, embryonic and extra-embryonic tissues developed in one animal. On recovery after 43 days gestation, this conceptus was shown to be genetically identical, at 11 microsatellite loci, to the fetus that had provided the oogonia. Cytological analysis of the embryos made with oogonia at 40-44 h after fusion and at the morula and blastocyst stages revealed that aberrant cytokinesis and nucleokinesis had given rise to multinucleated, anucleate, and polyploid cells in the reconstructed embryos. It is concluded that limited pluripotency of bovine oogonia has been demonstrated, warranting further study in this area.