Bovine Nuclear Transfer Embryos Research Articles

Nuclear-Cytoplasmic Interactions during the First Cell Cycle of Nuclear Transfer Reconstructed Bovine Embryos: Implications for Deoxyribonucleic Acid Replication and Development1

The present study investigated the decay of maturation-promoting factor (MPF) activity in electrically activated in vitro-matured bovine oocytes and examined the influence of the cell cycle stage of both the donor nucleus and the recipient cytoplasm upon the morphology and DNA synthesis potential of the donor nucleus in reconstructed embryos. The decay of MPF activity was studied both biochemically in electrically activated in vitro-matured oocytes and by morphological examination of nuclear structure in reconstructed bovine embryos. As measured by H1 kinase activity in groups of 10 oocytes, the level of MPF declined rapidly to 30 +/- 4% (of the maximum level in unactivated control oocytes) at 60 min and reached a basal level of 20 +/- 6% at 120 min. This level of activity was then maintained until at least 9 h postactivation. In contrast, when MPF activity was assayed by morphological examination of nuclei in individual reconstructed embryos, the decline in activity occurred over a period of 9 h postactivation. DNA synthesis of nuclei arrested at the G1/S border and in G2 phases of the cell cycle was examined in embryos reconstructed at the time of oocyte activation, i.e., when MPF levels were maximal, and by fusion 10 h postactivation, when no MPF activity could be detected. All nuclei transferred at the time of oocyte activation underwent nuclear envelope breakdown (NEBD) and subsequent DNA synthesis. However, when nuclei were transferred 10 h after activation, no NEBD was observed in any nuclei. Nuclei arrested at the G1/S border or nuclei in S phase when transferred in the absence of NEBD underwent DNA synthesis, while no DNA synthesis was observed in G2 nuclei transferred into this cytoplasmic environment. The results presented show that all nuclei, regardless of cell cycle stage, undergo DNA replication when transplanted into metaphase II (MeII) cytoplasts in which MPF activity is high. From these observations we would suggest that one factor that may contribute to the present low frequency of development of bovine nuclear transfer embryos is the ploidy of the reconstructed embryo after the first cell cycle. In order to maintain correct ploidy of the reconstructed embryo, only G1 nuclei should be transferred at the time of activation, when MPF levels are high. In contrast, when the integrity of the nuclear membrane is maintained by transfer at 10 h postactivation, when MPF activity is absent, the rereplication of G2 nuclei is prevented and correct ploidy of the reconstructed embryo may be maintained.

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 ∼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.

Donor blastomere cell cycle stage affects developmental competence of bovine nuclear transfer embryos

The system operator is responsible for maintaining a constant balance between generation and load to keep frequency at the nominal value. This fundamental objective is achieved with upward (e.g., synchronized and nonsynchronized generation units) and downward (e.g., demand response, storage) reserve capacity. The system operator needs to define, in advance, the reserve capacity requirements that mitigate the risk of imbalances due to forecast errors and unplanned outages of generation units. The research trend is to apply probabilistic methodologies for setting the reserve requirements based on uncertainty forecasts for renewable generation and load, as well as a probabilistic modeling of units' outages. This chapter describes two probabilistic methods, which share a common modeling framework, for quantifying risk and reserve requirements in two types of electricity markets: (1) sequential markets with the reserves market after the energy market clearing and (2) cooptimization (or joint market clearing) of energy and reserves. Two case studies with real data are presented to illustrate the application of both methodologies.

In vitro culture of bovine nuclear transfer embryos in synthetic oviduct fluid medium (SOFM)

In vitro culture of bovine nuclear transfer embryos in synthetic oviduct fluid medium (SOFM)

Development of bovine nuclear transfer embryos in in vivo versus in vitro culture systems

At various times after mating, fertilized eggs were flushed from the ampulla of the oviduct of estrus hamsters. Cumulus cells were removed with hyaluronidase before fixation. After fixation, the zonae pellucidae were removed mechanically. The initial association between the fertilizing spermatozoon and the oocyte surface appears to be between the tips of oocyte microvilli and the equatorial segment. As the sperm head is incorporated, it undergoes a pronounced bend such that the anterior tip is the last portion of the sperm to be incorporated. A small incorporation cone forms at the site where the sperm head was incorporated into the ooplasm. As the sperm tail is incorporated, it forms numerous bends which distort the oolemma into a row of small elevations. From these observations, we have been able to propose a model for the mechanics of sperm entry into the ovum in vivo.

Effect of cytoskeletal inhibitor on fusion and development of bovine nuclear transfer embryos

Effect of cytoskeletal inhibitor on fusion and development of bovine nuclear transfer embryos