From the time of early follicular growth to some time after ovulation, the oocyte undergoes many critical changes in its biochemistry. In this paper the author describes different steps in oocytes maturation some of which, during the early period, will require support from the follicle cells, while later in development endogenous synthetic activity can be maintained in the absence of such support.

Oocyte maturation and fertilization in vitro.

Screening of biotechnical parameters for production of bovine inter-subspecies embryonic chimeras by the aggregation of tetraploid Bos indicus and diploid crossbred Bos taurus embryos

Electrofus on is a reliable method of inducing cell to cell fusion. It can be used for a variety of purposes, including nuclear transfer for the production of identical progeny and hybridoma production for monoclonal antibody production. Electrofusion involves the combination of alternating current (AC) and direct current (DC) pulses to induce membrane fusion. An AC pulse induces cell alignment and dielectrophoresis, which causes tight membrane contact (Mahaworasilpa et al., 1994). The DC pulse results in point membrane fusion and the formation of an unstable flat membrane diaphragm, where spectrin holds lipid fragments together. Although cytoplasm can move between the cells through the pores of a flat membrane diaphragm, large organelles such as mitochondria and nuclei remain contained in the original cell. Under favourable conditions, the flat diaphragm deteriorates to allow cell mixing, indicating true cell to cell fusion (Chernomordik and Sowers, 1991). Electrical parameters for successful electrofusion rely on the amplitude and duration of the DC pulse. Cell mixing occurs when these parameters are of sufficient magnitude to prevent the flat diaphragm from reforming the membrane lipid bilayers. Other factors that influence cell mixing are temperature, pH and molecules such as cholesterol and ethylmaleimide (A. Sowers, personal communication). An isofusion contour describes the electrofusion characteristics of a particular cell type and its membranes. A range of DC pulse amplitudes and durations are used to attempt electrofusion of identical cells of equal diameter and of a particular type. The maximum electrofusion result for each pulse duration can be used to construct an isofusion contour. The isofusion contours for different cell types can be compared to predict the optimal electrofusion parameters for fusion between them (Mahaworasilpa, 1992). This study investigated the electrofusion of identical cell types to produce isofusion contours for cells used for nuclear transfer. The cells used were bovine oocytes matured in vitro, oocytes enucleated using two different centrifugation treatments and blastomeres isolated from embryos 3, 4, 5 and 6 days after in vitro fertilization (IVF).

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Electrofusion of in vitro produced bovine embryonic cells for the production of isofusion contours for cells used in nuclear transfer

Tetraploid complementation has been used to improve the production of cloned animals. The main objective of this study was to improve the efficiency of bovine tetraploid embryo production and the development potential of bovine tetraploid embryos into blastocysts. We assessed early embryonic cleavage timing and established the defined time quantum to collect synchronous 2-cell stage bovine embryos for electrofusion. Different electrofusion protocols were also tested. The second aim was to monitor ploidy transition by fluorescence visualization to shed lights on the nuclear fusion process of the cytoplasmic membrane-fused 2-cell stage bovine embryos. Karyotypes of day 8 blastocysts (day 0: day of electrofusion) were determined by karyotyping analysis. We found that electrofusion of in vitro produced bovine 2-cell stage embryos results in both tetraploid and diploid embryos and that blastocyst formation rates from fused 2-cell stage embryos are affected by the number of electrofusion pulses and the timing of how soon the 2-cell stage embryos are formed post insemination. We identified two distinct nuclear configurations after electrofusion of 2-cell stage embryos, each of which is uniquely related to the formation of tetraploid or diploid embryos. Based on the observation that tetraploid and diploid embryos derived from fused 2-cell stage embryos undergo different timings to become 2-cell stage embryos again, diploid and tetraploid 2-cell embryos can be readily separated after electrofusion. Finally, our study established an experimental protocol for the effective production of bovine tetraploid embryos by electrofusion of 2-cell embryos.

https://www.longdom.org/open-access/efficient-production-of-bovine-tetraploid-embryos-by-electrofusion-of-invitro-produced-twocell-stage-embryos-2375-4508-1000167.pdf

Efficient Production of Bovine Tetraploid Embryos by Electrofusion of InVitro Produced Two-Cell Stage Embryos

Background: The values of embryonic stem cell and cloning are evident. Production of clone from embryonic stem cells can be achieved by introduction of stem cell into a tetraploid blastocyst. Tetraploid blastocyst can be produced in vitro by electrofusion of 2-cell embryos. Objective: The aim of this study was to assess the effect of different voltages and durations on fusion rate of bovine 2-cell embryos and their subsequent development in vitro. Material and Methods: The in vitro produced bovine 2-cell embryos were categorized into 3 groups: (1) fused group (FG); 2-cell embryos fused by exposure to different voltages (0.5, 0.75, 1, 1.25 and 1.5 kV/cm) and durations (20, 40, 60, 80 and 100 μs), (2) exposed control group (ECG); 2-cell embryos exposed to different voltages and durations but remained unfused and (3) unexposed control group (UCG); embryos cultured without exposure to any voltage. The embryos from each group were cultured and fusion, cleavage and developmental rates were compared in each group. Results: The results show that increased voltage, increases the fusion rate up to 88% for 1.5 kV/cm; however, the rate of cleavage and blastocyst formation decreases significantly to 18% and 10% respectively (p<0.05). Increased duration does not significantly increase fusion rate, however, in high voltage, increased duration decreases cleavage rate and blastocyst formation rate. Blastocyst formation rate in UCG showed a better development (32%) compared to FG (20%) or ECG (22.5%) (p<0.05). Conclusion: It can be concluded that for optimal fusion, cleavage and development, one pulse of 0.75 kV/cm for 60μs should be applied.

Mohamad Reza Darabi

Papers

Fusion and development of 2-cell bovine embryos to tetraploid blastocyst with different voltages and durations