Electrofusion for the pronuclear transplantation of mouse eggs

The present study was undertaken to determine the efficiency of HVJ treatment and electrofusion for pronuclear transplantation in the mouse. The output voltage and duration of the pulses were fixed to 200 μsec at 10 V or to 150 μsec at 15 V for electrofusion, because the maximum rates of blastomere fusion of 2-cell embryos and development of fused embryos in vitro were obtained under these conditions. Although the proportion of eggs with fused karyoplast (78%) and the fused eggs developed to morulae or blastocysts (67%) was significantly lower than those obtained after HVJ treatment (94% and 94%), the proportion of pregnant recipients and young obtained after treatment of fused eggs was not significantly different between these two procedures. It is advised that electrofusion can be used as a fusogenic procedure for pronuclear transplantation in the mouse in some cases where HVJ cannot be applied.     

First cleavage of enucleated rat eggs following transplantation of karyoplast removed from pronuclear eggs stored at 2 to 6 degrees C for various durations

Pronuclear rat eggs were cultured for 24 to 48 hours at 37 degrees C after storage at 2 to 6 degrees C for 0 to 216 hours in medium. Very high proportions (89 to 97%) of eggs cleaved to the two-cell stage after storage for 0 to 48 hours. The proportion, however, decreased rapidly in eggs stored for 72 hours (60%), and eggs stored for more than 120 hours cleaved poorly. However, when male and female pronuclei from stored eggs were transplanted into enucleated fresh eggs, 92 to 100% of the fused eggs with karyoplast stored for 0 to 144 hours cleaved. Although the cleavage rate was reduced to 50% when karyoplast from eggs stored for 168 hours was transplanted, this reduction was not significant. Complete loss of cleaving ability was observed in fused eggs with the karyoplast stored for 216 hours. These results clearly indicate that the pronuclei of rat eggs can be stored for a longer period than the cytoplasm at low temperatures (2 to 6 degrees C).     

Fusion and development rates of single blastomere pairs of mouse two- and four-cell embryos using the electrofusion method

The present study was undertaken to find suitable conditions for blastomere fusion of mouse two- and four-cell embryos using the electrofusion method to simplify the nuclear transfer procedure. Single blastomeres of ICR and F1 (C57BL/6J x CBA/N) two-cell embryos or ICR four-cell embryos and F1 two-cell embryos were paired and treated with electric stimulus under different fusion conditions. Two hours after electrofusion treatment, the fused blastomere pairs were encapsulated in alginate gel and cultured for 96 hours to observe their developmental potential. When the single blastomere pairs of two-cell embryos were exposed to electric pulses of 1.0, 1.5 and 2.0 kV/cm for 30, 60 and 90 mu sec, high fusion rates were obtained (84.6 to 100%). However, when two-cell blastomere were paired with four-cell blastomere and then treated under the same conditions, the fusion rates (27.5 to 87.5%) were lower than that of single blastomere pairs of two-cell embryos regardless of the duration and strength of the d.c. pulses. The blastocyst developmental rate after in vitro culture of the fused blastomere pairs of two-cell embryos using the above electrofusion conditions was high (81.8 to 100%). Lower blastocyst developmental rates were obtained on the fused blastomere pairs of two- and four-cell embryos (46.4 to 76.2%). Based on the results of this study, a pulse duration of 60 mu sec and a pulse strength of 1.0kV/cm were the most suitable conditions for single blastomere pair fusion of two-cell or two- and four-cell embryos. The study further showed that alginate gel is a good substitute for zonae pellucidae for encapsulating zona-free embryos.     

Nuclear transplantation in early pig embryos

Nuclear transfer was evaluated in early porcine embryos. Pronuclear stage embryos were centrifuged, treated with cytoskeletal inhibitors, and subsequently enucleated. Pronuclei containing karyoplasts were placed in the perivitelline space of the enucleated zygote and fused to the enucleated zygote with electrofusion. The resulting pronuclear exchange embryos were either monitored for cleavage in vitro (9/13 cleaved and contained 2 nuclei after 24 h, 69%) or for in vivo development. In vivo development after 3 days resulted in 14/15 (93%) of the embryos transferred cleaving to the greater than or equal to 4-cell stage and after 7 days 6/16 (38%) reaching the expanded blastocyst stage. A total of 56 pronuclear exchange embryos were allowed to go to term, and 7 piglets were born. A similar manipulation procedure was used to transfer 2-, 4- or 8-cell nuclei to enucleated, activated meiotic metaphase II oocytes. Enucleation was effective in 74% (36/49) of the contemporary oocytes. Activation was successful in 81% (37/46) of nonmanipulated but pulsed oocytes versus 13% (4/31) of control oocytes (p less than 0.01). After 6 days in vivo, 9% (1/11) of the 2-cell nuclei, 8% (7/83) of the 4-cell nuclei, and 19% (11/57) of the 8-cell nuclei transferred to enucleated, activated meiotic metaphase II oocytes resulted in development to the compact morula or blastocyst stage (p less than 0.01). A total of 88 nuclear transfer embryos were transferred to recipient gilts for continued development. A single piglet was born after the transfer of a 4-cell nucleus to an enucleated, activated metaphase II oocyte and subsequent in vivo development.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrofusion-mediated transmission of cytoplasmic organelles through the in vitro fertilization process,fusion of sperm cells with synergids and central cells,and cell reconstitution in maize

Summary Fusion products were created by the electrofusion of single sperm cells with single synergids and central cells. The synergid was also fused with the sperm cell, occasionally in the presence of adhering second synergids, egg cells, and central cells. Single egg cells were fused with single sperm cells in the presence of adhering synergids and the central cell. Cytoplasmic organelles were transmitted through the fertilization process by electrofusion using cytoplasts of maize mesophyll cells. Cell reconstitution was achieved by fusion of one or two sperm cells with single enucleated protoplasts, thus creating a haploid or a diploid cell.     

Effect of aging of recipient oocytes on the development of bovine nuclear transfer embryos in vitro

The present study was undertaken to examine the effect of the length of in vitro maturation of oocytes on the efficiency of enucleation, parthenogenetic activation and blastomere fusion by electric stimulus. In vitro development of reconstituted oocytes receiving a blastomere from 8 to 16-cell bovine embryos fertilized in vitro was investigated to assess the effect of aging of the oocytes. The proportion of oocytes with a first polar body at 22 to 24 hours after maturation was high (80%) compared with those obtained at 16 to 18, 28 to 30 or 42 to 44 hours (50 to 75%). The success rate of enucleation significantly decreased with aging (88, 85, 74 and 55%). The activation rate significantly increased with the length of maturation in vitro (P<0.01) (1 to 4, 24 to 41, 57 to 70 and 80 to 87%). The proportion of oocytes fused with a blastomere from 8- to 16-cell embryos was not dependent on the age of the oocytes (54 approximately 59%). The ability of the reconstituted oocytes to develop to the 2-cell and the 8- to 16-cell stage increased with the length of maturation of recipient oocytes. When oocytes enucleated and a blastomere at 22 to 24 hours were incubated further for 22 to 23 hours until electrofusion. The proportions of oocytes which developed to the 2-cell and the 8- to 16-cell stages (74 and 17%) were similar to those obtained at 42 to 44 hours after maturation. However, only 1 to 6% of reconstituted eggs receiving a blastomere from 8- to 16-cell embryos fertilized in vitro developed into a blastocyst in vitro.     

Age dependence of bovine oocyte activation

The ability of bovine oocytes to undergo parthenogenetic activation using either a Ca⁺⁺-Mg⁺⁺-H⁺ ionophore (A23187) or electric shock was investigated, as a prelude to understanding activation potential following nuclear transfer into ooplasm. Oocytes were collected from slaughterhouse ovaries by aspiration of 1–5-mm follicles. The time of placement into maturation medium was noted, and maturational age (time in culture) measured from that point. After exposure to activating conditions eggs were cultured for a further 12–16 hours, fixed, and stained with aceto-orcein. Oocytes that progressed to telophase or pronuclear formation were considered activated. Concentrations of A23187 ranging from 100 pM to 100 μM showed that 1–100 μM levels resulted in 94–100% activation at 30 hours maturation. Frequency of activation differed from controls (no ionophore) at 100 nM (49%; P < 0.05). With A23187 maximum response occurred between 26 and 30 hours of maturation (77% and 92%, respectively). A short pulse electric shock, capable of causing oocyte membrane fusion, gave similar results relative to maturational age (82% and 90% activation for 26 and 30 hours, respectively). Therefore, maximum response to the two activating stimuli occurred in oocytes at similar maturational ages. Exposure to activating conditions prior to onset of activating ability (18 hours) followed by another exposure at 26 hours showed that the oocytes were still fully able to activate upon reaching maturational activation competence. Because cytochalasin B is present in the medium used for nuclear transfer, oocytes were incubated with cytochalasin B prior to exposure to an activating stimulus. Frequency of activation was similar to the control treatment (61% and 73%). The effect of mechanical stress of cytoplasm removal and replacement by electrofusion on activation was also not significant. Overall, maturational age of the oocyte was the main determinant of activation ability.     

Preimplantation development of rabbit embryos after transfer of embryonic nuclei into different cytoplasmic environment

The development of nuclear-transfer oocytes and zygotes was tested in the rabbit. Metaphase II oocytes and zygotes in the early pronuclear stage were treated with a cytoskeletal inhibitor (cytochalasin D), enucleated, and subsequently fused either with single blastomeres from eight- and 16-cell stages (oocytes and zygotes) or with pronuclei-containing karyoplasts (zygotes only). Also, nonenucleated zygotes were fused with 1/8 blastomeres. Fusion was performed by means of an electric field. Development of reconstituted embryos was monitored mainly in vitro, but a certain number of embryos developed from oocytes and zygotes receiving nuclei from eight-cell stages were also transferred into pseudopregnant does. Development of nuclear-transfer oocytes was distinctly better than that of nuclear-transfer zygotes, since 16.9% and 9.5% oocytes vs. 8.1% and 3.7% zygotes carrying eight- and 16-cell nuclei, respectively, developed to the blastocyst stage. Two advanced but already dead fetuses were found after transfer of 27 four-cell embryos obtained after fusion of oocytes with 1/8 blastomeres. No implantations were observed after transfer of 25 four-cell embryos developed from enucleated zygotes receiving eight-cell nuclei. These findings indicate that, in the rabbit, some nuclei from 16-cell embryos are still capable of promoting at least preimplantation development. Comparison between the developmental abilities of oocyte- and zygote-derived nuclear-transfer embryos also suggests that the cytoplasmic environment of recipient cell is more crucial for the development of reconstituted embryos than the stage of introduced nuclei (at least up to the 16-cell stage). The majority of pronuclear exchange embryos (69.9%) and 40% of nonenucleated zygotes receiving eight-cell nuclei were able to develop to the blastocyst stage. This latter observation indicates, similarly as with mouse, a supporting role of residual pronuclei for participation of an eight-cell nucleus in the development of reconstituted zygotes.     

Influence of cell cycle stage at nuclear transplantation on the development in vitro of mouse embryos

Nuclei were transplanted from embryos of mice at different stages of the 1st and 2nd cell cycle to oocytes enucleated at various times after fertilization. After transfer of pronuclei, a greater proportion of embryos developed to blastocysts if donor and recipient embryos were at the same stage of the cell cycle (synchronous transfer = 94%, asynchronous transfer = 76%). By contrast, when 2-cell blastomere nuclei were fused to the cytoplasm of enucleated zygotes, there was a significant effect of both cytoplast and karyoplast cell cycle stage on the development of the reconstituted embryos. Karyoplasts and cytoplasts derived from embryos at later stages of the cell cycle had greater potential to support development to blastocysts in vitro. It is suggested that the secretion of stage-specific messengers and the timing of nuclear membrane breakdown are the main factors causing the karyoplast and cytoplast effects, respectively.     

Developmental ability of enucleated bovine oocytes matured in vitro after fusion with single blastomeres of eight-cell embryos matured and fertilized in vitro

Single blastomeres from eight-cell stage bovine embryos matured and fertilized in vitro were electrically fused with enucleated oocytes matured in vitro. In experiment 1, The percentage of these reconstituted embryos developed to the two- to eight-cell stage 48 hr after electrofusion was increased when both the eight-cell embryos and the enucleated oocytes were derived from oocytes cultured with granulosa cells (14% vs. 38%). In experiment 2, the relationship between activation of oocytes and developmental ability of reconstituted embryos was examined. Although both ethanol and electrical stimulation efficiently induced parthenogenetic activation of oocytes matured in vitro for 26–28 hr (ethanol, 89%; electrical stimulation, 73%), the ratio of the second polarbody extrusion differed (80% vs. 22%). Ethanol-treated enucleated oocytes, however, were not significantly different from the early cleavage of the reconstituted embryos 48 hr after electrofusion (nontreated, 38%; treated, 43%). In experiment 3, reconstituted embryos at the two- to eight-cell stage 48 hr after the electrofusion were cocultured with granulosa cells for 6–7 days. Of 69 embryos, one developed to a morula and three developed to blastocysts. © 1993 Wiley-Liss, Inc.     

电脉冲介导金鱼囊胚细胞融合及其发育能力的研究

本实验首次成功地利用电脉冲介异法使金鱼的囊胚细胞融合,融合率高于95％,并通过细胞核移植方法,将融合细胞的细胞核移入金鱼成熟未受精的去核卵内,以了解融合后细胞核的发育能力。实验中共移植111个细胞核,得44个囊胚、7个原肠胚和1条活了8天的幼鱼(因不进食而死亡)。并对移核后发育至囊胚的胚胎用静态光度计测定了DNA含量,共测定了11个移核胚胎的细胞,其中9个移核囊胚细胞核的DNA含量增加,这一结果证明:利用电脉冲介导法能有效地转移外源染色体,供体核有促进个体发育的能力。为人工干与鱼类染色体组的组成,进一步研究鱼类个体发育对染色体倍性的依赖关系以及体细胞遗传提供了一条新途径。     

Development of reconstituted mouse embryos produced from bisected and electrofused pronuclear-stage embryos

The present study was designed to investigate the in vitro and in vivo development potential of reconstituted mouse embryos produced by bisection and electrofusion of pronuclear stage embryos (PN-E). Pronuclear-stage ICR and F1 (C57BL x CBA) strain mouse embryos were bisected manually with a fine glass needle under the dissecting microscope to produce karyoplasts (KP) and cytoplasts (CP). The KP of ICR PN-E and CP of F1 PN-E (KP: ICR + CP:F1) or the KP of F1 PN-E and CP of F1 PN-E (KP:F1 + CP:ICR) were attached using phytohemagglutinin-P (PHA-P) and then electrofused. High fusion rates of the KP and CP of PN-E were obtained (93.5%). The fused embryos were encapsulated in alginate gel and cultured for 72 or 96 hours. The cleavage rates of reconstituted embryos were also high (98.8%). Developmental rates to the blastocyst stage in vitro for the 96-hour culture of reconstituted embryos were 68.9% (KP:ICR + CP:F1) and 78.4% (KP:F1 + CP:ICR). Furthermore, the developmental ability of reconstituted embryos in vivo was investigated, and some live young were obtained (KP:ICR + CP:F1, 7.5% and KP:F1 + CP:ICR, 10.8%). In this study, it was confirmed that reconstituted embryos produced by bisection and electrofusion of pronuclear stage embryos were able to develop into blastocysts in vitro and into live young in vivo.     

Haploid maternal genome derived from early diplotene oocytes can substitute for the female pronucleus in preimplantation mouse development

We describe the preimplantation development of mouse embryos that have received the haploid maternal genome derived from early diplotene nuclei of primordial oocytes (PO). Two generations of recipient egg-cells were used. Induction of two meiotic divisions of the PO nucleus and the reduction of the number of chromosomes to the haploid level were achieved in preovulatory oocytes (primary recipients). The developmental potential of the obtained haploid genome was examined in zygotes (secondary recipients). The nuclei of PO obtained from newborn mice were transferred by cell electrofusion to in vitro maturing (IVM) and enucleated preovulatory mouse oocytes. The reconstructed oocytes which had completed maturation, i.e., reached metaphase II, were artificially activated (8% ethanol + CHX). Activated oocytes were used as donors of haploid pronuclei of PO origin which were transferred (by karyoplast fusion) to partially enucleated zygotes containing only the male pronucleus. Thus, reconstituted zygotes were transplanted to the ligated oviducts of the cycling mice and 27% of them developed to the blastocyst stage. Our experiments demonstrate that 1) the nucleus of PO can be induced to premature meiotic divisions in an IVM enucleated preovulatory oocyte; 2) in the presence of a normal male pronucleus, the haploid pronucleus of PO origin can substitute for a female pronucleus during preimplantation development. Mol. Reprod. Dev. 48:488–495, 1997. © 1997 Wiley-Liss, Inc.     

Karyoplast-cytoplast volume ratio in bovine nuclear transfer embryos: Effect on developmental potential

To evaluate the effect of karyoplast-cytoplast ratio on the development of nuclear transfer embryos, karyoplasts from day 4, day 5, and day 6 embryos were transferred to oocytes enucleated with different volumes of cytoplasm: Type 1, removal of a small volume of cytoplasm equivalent to the first polar body, Type 2, removal of a volume of cytoplasm approximately equal to the volume of the respective karyoplast, and Type 3, removal of half of the oocyte volume. In addition, the effect of experimental reduction of karyoplast cytoplasm was investigated in day 4 and day 5 karyoplasts. Intact day 4 karyoplasts fused to Type 3 cytoplasts did not support development to blastocysts, whereas these karyoplasts yielded blastocysts in combination with Type 1 (7%) and Type 2 cytoplasts (12%). After experimental reduction of cytoplasmic volume in day 4 karyoplasts, blastocysts (10%) were also obtained after fusion with Type 3 cytoplasts, probably due to reduction of cytoplasmic chimerism. With day 5 karyoplasts, blastocyst rate was higher in combination with Type 2 (34%) than with Type 1 (19%) and Type 3 cytoplasts (16%; P < 0.05). The use of day 6 intact karyoplasts resulted in a significantly (P < 0.05) higher proportion of blastocysts when fused with Type 2 (38%) or Type 1 cytoplasts (34%) than with Type 3 cytoplasts (16%). These results suggest that enucleation of oocytes with a volume similar to that of the respective karyoplast creates better conditions for cell cycle interactions with all types of karyoplasts than enucleation with minimal or large volume of cytoplasm. Mol. Reprod. Dev. 48:332–338, 1997. © 1997 Wiley-Liss, Inc.     

Effect of volume of oocyte cytoplasm on embryo development after parthenogenetic activation, intracytoplasmic sperm injection, or somatic cell nuclear transfer

Animal cloning methods are now well described and are becoming routine. Yet, the frequency at which live cloned offspring are produced remains below 5%, irrespective of the nuclear donor species or cell type. One possible explanation is that the reprogramming factor(s) of each oocyte is insufficient or not properly adapted for the receipt of a somatic cell nucleus, because it is naturally prepared only for the receipt of a gamete. Here, we have increased the oocyte volume by oocyte fusion and examined its subsequent development. We constructed oocytes with volumes two to nine times greater than the normal volume by the electrofusion or mechanical fusion of intact and enucleated oocytes. We examined their in vitro and in vivo developmental potential after parthenogenetic activation, intracytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT). When the fused oocytes were activated parthenogenetically, most developed to morulae or blastocysts, regardless of their original size. Diploid fused oocytes were fertilized by ICSI and developed normally and after embryo transfer, we obtained 12 (4-15%) healthy and fertile offspring. However, enucleated fused oocytes could not support the development of mice cloned by SCNT. These results suggest that double fused oocytes have normal potential for development after fertilization, but oocytes with extra cytoplasm do not have enhanced reprogramming potential.     

Compaction in preimplantation mouse embryos is regulated by a cytoplasmic regulatory factor that alters between 1- and 2-cell stages in a concentration-dependent manner.

Present studies were performed to investigate what factors affect the morphogenesis of preimplantation mouse embryos, and to find the action mechanism of that factor by using cytoplasm removal and its reconstitution from a different developmental stage embryo. Half (HP group) or one-third of cytoplasm (TP group) was removed from 1-cell mouse embryos by micromanipulation, and their morphogenesis and genome expression were compared with sham-operated embryos (SP group). The compaction and blastocoel formation of embryos in both the HP and TP groups were accelerated in time and cell stage when compared with those of the SP group. However, the total activity and time of RNA synthesis, and gene expression of ZO-1alpha+ isoform were not different. To change the cytoplasm composition without altering the nucleus/cytoplasmic ratio, half a 1-cell embryo with both pronuclei was reconstituted with the half enucleated cytoplasm of 1-cell embryo (P + P group), 2-cell (P + 2 group) or 4-cell (P + 4 group) by electrofusion. Embryonic compaction, timing of RNA synthesis, and stage-specific gene expression of the ZO-1alpha(+) isoform in the P + 2 and P + 4 groups were accelerated in time and cell stage than that in the P + P group, but not different between the P + 2 and P + 4 groups. In addition, a blastomere of 2-cell embryo was reconstituted with the enucleated cytoplasm of 1-cell embryo (2 + P group) or 2-cell (2 + 2 group) in equal volume by electrofusion. Also, the karyoplast of 2-cell was fused with the enucleated 1-cell embryo (2 + PP group). Embryonic development, total activity of RNA synthesis, and gene expression of the ZO-1alpha(+) isoform of embryos in the 2 + P and 2 + PP groups were delayed when compared with those of the 2 + 2 group. Also, the phenomena of compaction and blastocoel formation were delayed in the development time and cell stage. From these results, the nucleus/cytoplasm ratio was found to have no direct effect on the regulation of embryonic morphogenesis, although it accelerated compaction and blastocoel formation. However, cytoplasmic factors that altered between 1- and 2-cell stages regulate embryonic morphogenesis, especially compaction, of preimplantation mouse embryos in concentration-dependent manner.     

Effect of activation time on the nuclear remodeling and in vitro development of nuclear transfer embryos derived from bovine somatic cells

This study was conducted to investigate the effect of recipient activation time on the chromatin structure and development of bovine nuclear transfer embryos. Serum-starved skin cells were electrofused to enucleated oocytes, activated 1-5 hr after fusion, and cultured in vitro. Some fused eggs were fixed at each time point after fusion without activation, or 3 or 7 hr after activation. Some nocodazole treated zygotes were fixed to analyze their chromosome constitutions. The proportion of eggs with a morphologically normal premature chromosome condensation (PCC) state increased 1-2 hr after fusion. Whereas eggs with elongated chromosome plate increased as activation time was prolonged to 3 hr, and 5 hr after fusion, 58.1% of eggs showed more than two scattered chromosome sets. The proportion of eggs with a single chromatin mass (40.6-56.7%) significantly increased when eggs were activated within 2.5 hr after fusion (P < 0.05). Only 23.3% of reconstituted embryos activated 5 hr after fusion formed one pronucleus-like structure (PN), whereas, 64.5-78.3% of embryos activated 1-2.5 hr after fusion formed one PN. The proportion of embryos with normal chromosome constitutions decreased as activation time was prolonged. Development rates to the blastocyst stage were higher in eggs activated within 2 hr after fusion (17.3-21.7%) compared to those of others (0-8.6%, P < 0.05). The result of the present study suggests that activation time can affect the chromatin structure and in vitro development of bovine nuclear transfer embryos.     

Effects of recipient oocyte age and interval from fusion to activation on development of buffalo (Bubalus bubalis) nuclear transfer embryos derived from fetal fibroblasts

The objective was to investigate the effect of recipient oocyte age and the interval from activation to fusion on developmental competence of buffalo nuclear transfer (NT) embryos. Buffalo oocytes matured in vitro for 22 h were enucleated by micromanipulation under the spindle view system, and a fetal fibroblast (pretreated with 0.1 μg/mL aphidicolin for 24 h, followed by culture for 48 h in 0.5% fetal bovine serum) was introduced into the enucleated oocyte, followed by electrofusion. Both oocytes and NT embryos were activated by exposure to 5 μM ionomycin for 5 min, followed by culture in 2 mM 6-dimethyl-aminopurine for 3 h. When oocytes matured in vitro for 28, 29, 30, 31, or 32 h were activated, more oocytes matured in vitro for 30 h developed into blastocysts in comparison with oocytes matured in vitro for 32 h (31.3 vs 19.9%, P < 0.05). When electrofusion was induced 27 h after the onset of oocyte maturation, the cleavage rate (78.0%) was higher than that of electrofusion induced at 28 h (67.2%, P < 0.05), and the blastocyst yield (18.1%) was higher (P < 0.05) than that of electrofusion induced at 25 or 26 h (7.4 and 8.5%, respectively). A higher proportion of NT embryos activated at 3 h after electrofusion developed to the blastocyst stage (18.6%) in comparison with NT embryos activated at 1 h (6.0%), 2 h (8.3%), or 4 h (10.6%) after fusion (P < 0.05). No recipient was pregnant 60 d after transfer of blastocysts developed from NT embryos activated at 1 h (0/8), 2 h (0/10), or 4 h (0/9) after fusion. However, 3 of 16 recipients were pregnant following transfer of blastocysts developed from the NT embryos activated at 3 h after fusion, and two of these recipients maintained pregnancy to term. We concluded that the developmental potential of buffalo NT embryos was related to recipient oocyte age and the interval from fusion to activation.     

Electrofusion parameters for nuclear transfer predicted using isofusion contours produced with bovine embryonic cells

Electrofusion is a valuable technique for the nuclear transfer procedure. An enucleated oocyte is electrofused with a blastomere to create a nuclear transfer embryo. The present study constructed isofusion contours after the electrofusion of identical coupled cells that characterized all the bovine embryonic cell types used in nuclear transfer. The intersection of isofusion contours for enucleated oocytes and blastomeres provided the parameters for electrofusion during nuclear transfer. Blastomeres isolated from in vitro produced embryos 3–6 days after (in vitro fertilization) were electrofused with oocytes enucleated by centrifugation (85, 87, 89, and 73% electrofusion, respectively). The cleavage (46, 40, 37, and 28%, respectively) of the nuclear transfer embryos produced a trend that decreased as the age of the blastomeres increased. The isofusion contours provided information about the interaction between different cell types in an electric field, and gave precise electrofusion parameters for a range of bovine embryonic cell types used in nuclear transfer. © 1996 Wiley-Liss, Inc.