Among many factors that potentially affect somatic cell nuclear transfer (SCNT) embryo development is the donor cell itself. Cloning potentials of somatic donor cells vary greatly, possibly because the cells have different capacities to be reprogrammed by ooplasma. It is therefore intriguing to identify factors that regulate the reprogrammability of somatic donor cells. Gene expression analysis is a widely used tool to investigate underlying mechanisms of various phenotypes. In this study, we conducted a retrospective analysis investigating whether donor cell lines with distinct cloning efficiencies express different levels of genes involved in epigenetic reprogramming including histone deacetylase-1 (HDAC1), -2 (HDAC2); DNA methyltransferase-1 (DNMT1), -3a (DNMT3a),-3b (DNMT3b), and the bovine homolog of yeast sucrose nonfermenting-2 (SNF2L), a SWI/SNF family of ATPases. Cell samples from 12 bovine donor cell lines were collected at the time of nuclear transfer experiments and expression levels of the genes were measured using quantitative polymerase chain reaction (PCR). Our results show that there are no significant differences in expression levels of these genes between donor cell lines of high and low cloning efficiency defined as live calving rates, although inverse correlations are observed between in vitro embryo developmental rates and expression levels of HDAC2 and SNF2L. We also show that selection of stable reference genes is important for relative quantification, and different batches of cells can have different gene expression patterns. In summary, we demonstrate that expression levels of these epigenome regulatory genes in bovine donor cells are not correlated with cloning potential. The experimental design and data analysis method reported here can be applied to study any genes expressed in donor cells.
We examined transgenic-cattle production by DNA microinjection into 1-, 2-, and 4-cell embryos, analyzing the impact on calf size and subsequent viability. Embryos were either collected at an abattoir by flushing oviducts from superovulated and artificially inseminated cows (in vivo-derived) or obtained by in vitro maturation and in vitro fertilization of oocytes aspirated from excised ovaries (in vitro-derived). A human serum albumin (hSA) milk-expression DNA construct was microinjected, either in one of the visible pronuclei of in vitro- and in vivo-derived 1-cell embryos or in the nuclei of two blastomeres of 2- and 4-cell in vivo-derived embryos. Microinjection-induced mortality (lysis and developmental block) was equivalent ( approximately 40%) for all microinjected embryos. Embryos were co-cultured with BRL cells in B-2 medium containing 10% fetal calf serum (FSC). Overall, embryo development to morulae/blastocysts was significantly greater for in vivo-derived ova (15.5%) than for in vitro-derived oocytes (9.3%). All morulae and blastocysts were transferred to synchronized recipient females on Days 6-8 post-fertilization. A total of 189 calves were delivered. Birth weights were significantly greater for calves generated from in vitro-derived oocytes compared with those generated from in vivo-derived oocytes. One transgenic bull calf was obtained from the microinjection of a 2-cell embryo. Fluorescence in situ hybridization (FISH) analysis of lymphocytes detected one transgenic integration site in all cells. Transmission frequency of the hSA transgene in embryos obtained through IVM/IVF/IVC utilizing the semen of the transgenic calf confirmed that it was not mosaic.
The use of IVF in horses has a limited efficiency, reflecting low oocyte developmental competence and inadequate sperm capacitation procedures. In a preliminary study, using carboxyfluorescein diacetate/propidium iodide staining, we determined that the freezing-thawing procedure left only 56.6 ± 3.4 % of the sperm cells with an intact membrane. The following incubation in TALP-IVF induced membrane damage at high rates with only 9.58 ± 1.8 % of them intact after 18 h. However, the presence of at least four cumulus-enclosed oocytes (CEO) in the medium significantly increased the number of membrane-intact spermatozoa at the end of incubation (53.87 ± 1.99%). This indicated that the sperm thawing and capacitating procedures can damage the cell membrane but the presence of four or more CEO in TALP-IVF could prevent further damages. The aim of the study was to investigate in detail the membrane damages and to analyze the differences induced by the presence of CEO. Spermatozoa were thawed in water at 37 • C, and centrifuged for 30 minutes at 600g in a 45-90% Percoll gradient made with modified Tyrode's medium. The sperm pellet was washed once in the same medium and diluted to a final concentration of 1 × 10 6 spermatozoa/ml TALP supplemented with 0.6% (w/v) BSA fatty acid free and 12 µg mL −1 heparin (TALP-IVF). Sperm cells were incubated with 0 or 4 in vitro-matured CEO. Sperm cells were examined after thawing, 0, 2 and 18 h from the beginning of incubation in TALP-IVF. Each experiment was replicated at least 3 times. Both scanning and transmission electron microscopy were performed on sperm samples fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, using standard procedures. Specimens for scanning electron microscopy were examined under a field emission gun JEOL JSM 6301 microscope. For transmission electron microscopy the samples were examined with a JEOL JEM 100 SX. A minimum of 25 cells were analyzed for each group. Immediately after thawing, damaged spermatozoa showed, on the surface of their heads, small vesicles correlated to a progressive process of vacuolisation and degeneration of membrane integrity. The same lesions were visible at all the successive time points taken into account. Moreover, a loss of the acrosome integrity with acrosomal swelling and a decrease of content homogeneity were observed particularly in the spermatozoa cultured for 18 h without CEO. When CEO were present in the IVF medium lesions were visible in a lower percentage of spermatozoa but the type of lesions did not differ from those observed in their absence. These observations confirmed our previous data and gave more details on the lesions that occur during the IVF procedures in the horse.Increasing the diary population and milk production is a goal of many tropical developing countries. We report in this paper an attempt to develop a system of intercontinental shipping for transfer of fresh crossbred Bos taurus × Bos indicus IVF embryos into local Laisind (Bos indicus) recipients as a way to produce tropical dairy calve...
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