Development of embryos from in vitro ovulated and fertilized oocytes of the quail (Coturnix coturnix japonica)

Abstract

The development of quail embryos obtained after in vitro fertilization of oocytes ovulated in vitro was investigated. About 40% of the specimens, after 18-20 hr of incubation, had undergone cleavage to reach stages IV-VI when viewed under a stereo microscope. However, only 36% of these embryos contained normal, DAPI-stained nuclei when observed under a fluorescent microscope; the other 64% showing a morphologically normal cleavage pattern did not contain nuclei. Control unfertilized oocytes, ovulated in vitro and cultured for the same time, also sometimes attained the morphologically correct stages IV-VI but their "blastomeres" were always devoid of nuclei. Therefore, it is advisable to monitor early avian embryos for the presence of nuclei when assessing development in culture. The results demonstrate, for the first time, that cytoplasmic segmentation can occur in the absence of nuclear divisions in the germinal disc of the quail and show the existence and significance of ooplasmic maternal information in birds. This phenomenon is also known for sea urchin and frogs. It is indicative of the role of maternal information in early development. The in vitro method presented here links the steps of ovulation and fertilization with the early cleavage stages under in vitro conditions and may be useful in studying mechanisms of fertilization and differentiation in birds as well as in obtaining transgenic birds by DNA injection or application of foreign, DNA-carrying sperm.

Post-testicular sperm maturation and identification of an epididymal protein in the Japanese quail (Coturnix coturnix japonica)

“…These studies (Figs 2 and 3) support earlier work (Howarth 1970, Olszanska et al 2002 that concluded that avian spermatozoa do not require a period of capacitation. Indeed, we have demonstrated that the potential to undergo tyrosine phosphorylation, one of the key hallmarks of the capacitation process in mammals, is not overtly influenced by epididymal transit in the quail (Fig.…”

Establishment of Intracytoplasmic Sperm Injection Technique in Japanese Quail and its Possible Application for Poultry Resources and Transgenic Birds

“…The rate of fertilization is around 16%, which is comparable to that of those after conventional IVF using 1-2 ×10 4 semen of quail (Olszańska et al, 2002). The developmental stage (stages VI classified according to EyalGiladi and Kochav, 1976) is also comparable between ICSI and IVF.…”

“…Some oocytes were continuously cultured for 48 hr at 37.5℃ and 70% relative humidity, with rocking at a 90°angle at 30 min intervals. To select successfully fertilized eggs, we chose the following criteria: (1) cell division under stereomicroscope observation; (2) the presence of nuclei of blastomere of germinal disc by 4,6-diamidino-2-phenylinedole, a DNA-specific dye and (3) cleavage furrows and nuclei by hematoxylin and eosin (HE) staining, because Olszańska et al (2002) demonstrates that in vitro experimental procedures induce the normal cleavage without any nuclei in some eggs (parthenogenetic cleavage).…”

“…Presently, it is possible to carry out in vitro several separate steps of avian reproduction such as in vitro fertilization (Howarth, 1971;Nakanishi et al, 1990Nakanishi et al, , 1991Olszańska et al, 2002;Batellier et al, 2003), and the complete embryo culture from single-cell stage of in vivo fertilized egg to hatching (Perry, 1988;Ono et al, 1994Ono et al, , 1996. However, a continuous in vitro system covering fertilization, embryo development and hatching is not yet available.…”

Cell number and sex ratio in unfertilized chicken eggs (Gallus gallus domesticus)

“…They did not achieve reliable results in the samples of unfertilized eggs. Moreover, Olszanska et al (2002) reported the observation of cleaved germinal disc cells without nuclei in in vitro ovulated, unfertilized quail eggs after 24 hr. For these reasons, in the first experiment of our study we counted the number of cell nuclei in the germinal discs of virgin hens and in the discs of unfertilized eggs from inseminated hens after 3 days of incubation to see if there were enough cells for subsequent sex analysis.…”