Presence Of Embryonic Cells Research Articles

In vivo tissue sampling of embryonic resorption sites using ultrasound guided biopsy

In the polytocous European brown hare (Lepus europaeus) more than 23% of all successful implantations undergo embryonic resorption. The objective of the study was to establish a minimally invasive ultrasound guided biopsy technique to collect embryonic resorption tissue in vivo. The sampled material was genetically analysed to determine paternity and the sex of the embryo. Female hares were either mated or artificially inseminated and pregnancy was confirmed by ultrasound on day six post ovulation. Subsequent embryonic development was ultrasonographically monitored on a regular basis to detect embryos undergoing resorption. Cell material of the resorption site was collected under ultrasonographic control via transabdominal biopsy of the placenta or aspiration of resorption fluid. To avoid breathing movements during the biopsy, the animals were intubated and a short apnoea was evoked by assisted ventilation. The presence of embryonic cells in the biopsy material was confirmed by microsatellite analysis in 11 of the fluid samples (n = 28) and six of the placental samples (n = 8). The lower success rate in the fluid samples was attributed to the abundance of maternal cells which was confirmed by the analysis of fluid sample smears. Male sex of the embryos undergoing resorption was detected by SRY analysis for ten of the fluid samples and for one of the placental samples. The two biopsy techniques did not have any negative impact on the prenatal development of the healthy siblings nor did it influence the future breeding performance of the females that were biopsied.

Evaluation of an internal control in a multiplex-PCR assay for sex determination of <i>in vitro</i>-produced bovine embryos

The use of an internal control in a multiplex-PCR assay for sex determination of In Vitro-produced bovine embryos was evaluated in biopsies of groups of 54 fresh and 44 frozen embryos. The internal controls used were the primers BOV 1 and BOV 2, which amplify a product with 626 base pairs (bp) of bovine mitochondrial DNA ND5 gene. The primers BRY.4aF and BRY.4aR were used for bovine Y chromosome sequence amplification. The specificity of multiplex- PCR reactions realized in biopsies corresponding to about 20% of each fresh embryo (10 male and 10 female) by means of confirming the sexing in the remaining embryo content (~80%) presented 100% specificity. Amplicons of the internal control and Y chromosome were both amplified until dilution corresponding to 6.25% of total extracted DNA from a male embryo. Sex determination was possible in 53 (98.1%) fresh embryos and 40 (90.9%) frozen embryos. The products related to the Y chromosome and mitochondrial DNA were simultaneously amplified in 34 (63%) fresh embryos and 27 (61.4%) frozen embryos, showing a male embryo. The female sex, distinguished by internal control amplification only, was detected in 19 (35.2%) and 13 (29.5%) biopsies, respectively, of fresh and frozen embryos. In one (1.8%) and four (9.1%) biopsies of fresh and frozen embryos, respectively, neither product was amplified, most likely due to the absence of embryonic cells or the presence of embryonic cells going through apoptosis. The multiplex-PCR assay developed in this work showed avoided the limitation of a lack of an internal standard, and was also sensitive, specific, and efficient in reaction failure identification. This technique shows great potential for use on a commercial scale in routine sex determination of In Vitro-produced embryos.

Growth of Paenibacillus larvae, the causative agent of American foulbrood in honey bees and Bacillus popilliae, the causative agent of milky disease in beetle larvae in lepidopteran cell cultures

AbstractTNM‐FH Lepidopteran insect cell culture medium containing 10% fetal bovine serum (FBS), while allowing limited vegetative growth of Paenibacillus larvae (wild‐type strain), the causative agent of American foulbrood, contained no viable vegetative cells upon subculture, nor were any heat resistant spores produced in this medium alone. However, TNM‐FH medium cotaining embryonic or midgut cells from Trichoplusia ni, hemocytes from Estigmene acrea, ovarian and embryonic cells from Spodoptera frugiperda, embryonic cells from Plutella xylostella, Spodoptera exigua and Pseudaletia unipuncta or ovarian cells from Lymantria dispar, supported both heavy vegetative cell growth and moderate production of heat resistant spores. EX‐CELL 405 serum‐free insect cell culture medium alone appeared to contain the appropriate nutrients required for both vegetative growth and sporulation of P. larvae. However, in the presence of embryonic cells from T. ni, limited vegetative growth occurred and the P. larvae cells appeared to die off. This was confirmed by the fact that no colony growth occurred upon subculture, nor were any heat resistant spores detected. This was true also in the presence of fat body cells from T. ni, except that a limited number of spores (4,000/ml) were detected in the form of cology‐forming units (CFU) on plates following heating to 80°C for 20 minutes. In a parallel study with a wild‐type strain of Bacillus popilliae, vegetative cells grew only in TNM‐FH medium in the presence of mid‐gut BTI‐Tn‐MG and ovarian (Tn‐368) cells of T. ni. No heat resistant spores, however, were detected in any of the cultures. When BTI‐Tn‐MG and Tn‐368 cells were further challenged with four variant cultures of B. popilliae, vegetative growth and limited sporulation were achieved. The BTI‐Tn‐MG cell line in TNM‐FH medium produced as many as 12,000 spores/ml after 21 days in culture.

DNA replication may be uncoupled from nuclear and cellular division in temperature-sensitive embryonic lethal mutants of Caenorhabditis elegans

The nuclear and DNA contents of 18 temperature-sensitive embryonic lethal mutants of Caenorhabditis elegans have been determined at the time of arrest. After each mutant was shifted to restrictive temperature, the embryonic arrest stage was recorded by the number of nuclei counted in embryonic squashes and as DNA nuclear equivalents recorded by flow cytometry. Together the two methods complemented each other and provided qualitative and quantitative information concerning nuclear number, DNA content, morphological stage of arrest, and presence of anucleate embryonic cells. The arrest stage for most of the embryonic lethal mutants demonstrated their potential to continue nuclear division and DNA replication beyond their respective temperature-sensitive periods. These results suggested that the mutants' primary defect did not reside on a pathway closely coupled to DNA replication or nuclear division. For example, B65 contained up to 650 DNA nuclear equivalents beyond the end of its temperature-sensitive period at the 20- to 30-cell stage. B65 is of additional interest because it continued DNA synthesis beyond the normal 550-cell stage and at the same time failed to progress beyond the morphogenetic period. The presence of enlarged nuclei that contained extra DNA demonstrated that DNA replication was independent of nuclear and cellular division. For example, B1 and B244 arrested with 1 to 50 and 1 to 200 nuclei, respectively. However, they both contained 250 to 400 nuclear equivalents of DNA. Conversely, the presence of embryonic cells without nuclei suggested that cell division may also be independent of nuclear division or nuclear migration. The enlarged nuclear and anucleate embryonic cells were observed only in those mutants requiring a normal parental function.