Nuclear transfer of porcine embryos using cryopreserved delipated blastomeres as donor nuclei

Abstract

Nuclear transfer protocol for the pig using cryopreserved delipated four‐ to eight‐cell and morula stage embryos as nucleus donors was developed. Donor embryos, which had been delipated by micromanipulation following centrifugation for polarizing cytoplasmic lipid droplets, were cryopreserved with 1.5 M 1,2‐propanediol and 0.1 M sucrose. Recipient cytoplasts were prepared from ovulated oocytes. Activation of oocytes could be induced more efficiently when electric stimulation was given 53 hr after the hCG injection or later (66–83%), compared with 52 hr or earlier (11–16%, P < 0.05), suggesting that aging after ovulation may be required for in vivo matured porcine oocytes to be activated by electric stimuli. Membrane fusion rates between donor blastomeres and enucleated oocytes were 88% (127/144) and 97% (56/58, P > 0.05) for the four‐ to eight‐cell and morula stage embryos, respectively. In vitro developmental rates to the two‐cell (53/100 vs. 35/65), four‐cell (34/100 vs. 26/65), and morula stage (17/100 vs. 18/65) were the same between the nuclear transfer embryos with four‐ to eight‐cell and morula nuclei. However, more embryos reconstituted with morula nuclei developed to blastocysts (15% vs. 6%, P < 0.05). These data demonstrated that blastomeres of cryopreserved, delipated porcine embryos can be used as donor nuclei for nuclear transfer. Frozen‐thawed, delipated blastomeres can be efficiently isolated and fused, and therefore provide a useful source of donor nuclei. Mol. Reprod. Dev. 48:339–343, 1997. © 1997 Wiley‐Liss, Inc.

Establishment of a Porcine Cell Line from In Vitro-Produced Blastocysts and Transfer of the Cells into Enucleated Oocytes1

“…In general, a chamber consisting of parallel wires has been used for fusion [10,11,14,17,27,28]. It was reported that a chamber consisting of parallel wires with a separation of 1 mm is effective for fusion with blastomeres of embryos and enucleated oocytes in the pig [23]. Therefore, we used the same chamber for fusion initially, but few fused embryos were obtained (data not shown).…”

“…It has been shown that porcine nuclear transfer embryos reconstituted with 8-to 16-cell stage nuclei and enucleated mature oocytes cleaved in vitro at rates of 36-43%, but developmental rates beyond the morula stage were only 4.5-7% [29,30]. Recently, Nagashima et al [23] reported that 28% of nuclear transfer embryos reconstituted with morula-stage nuclei developed to morulae, and 15% formed blastocysts, with use of preactivated oocytes as recipient cytoplasts and induction of fusion of blastomeres and cytoplasts at 6 h after the electric activation. However, when porcine primordial germ cells were transferred to preactivated or nontreated oocytes, reconstructed embryos could not develop beyond the 4-cell stage in vitro [31].…”

Development of pig cloning studies: past, present and future

“…Thus, for many years, the effectiveness of the cloning method, measured both by the rate of reconstructed embryo development to compact morula/blastocyst stages and by percentage of born piglets in relation to the number of nuclear transfers remained at a constant, comparatively low level. Occasionally reports of the production of porcine morulae and blastocysts in vitro appeared (Saito et al, 1992;Terlouw et al, 1992;Nagashima et al, 1997). However, they did not exert a significant influence on increasing either the postimplantation developmental potential of reconstructed clonal embryos or the rate of foetal development to the end of gestation.…”

Effects of Recloning on the Efficiency of Production of .ALPHA.1,3-Galactosyltransferase Knockout Pigs

“…As soon as the cumulus cells were removed, the oocytes were enucleated by aspirating the first polar body and adjacent cytoplasm (about 10% of the ooplasm) using a bevelled pipette (35 µm in diameter) [24] in 10 mM Hepes-buffered Tyrode's lactose medium (Hepes-TL) containing 0.3% (w/v) polyvinylpyrrolidone (PVP), 10% (v/v) FCS and 7.5 µg/ml cytochalasin B (CB). Enucleation was confirmed by staining the cytoplasts with 5 µg/ml Hoechst 33342.…”