Development of oocytes from synchronized follicles of early prepubertal goats under exotic hormone control.

The aim of this study was to establish whether early prepubertal Sannen dairy goat could provide a large source of ova for SCNT. The effects of different hormonal treatments including untreated control, FSH alone, estadiol plus progesterone (E2-P4), and E2-P4 and FSH (E2-P4-FSH) on ovary size, follicle number and size were studied using the early prepubertal goats aged 39-60 days. Then prepubertal goats aged 39-120 days were categorized into three groups to study the effect of age on recruited follicle number. The meiotic competence of oocytes derived from > or =3 mm follicles recovered from the early prepubertal goats treated with E2-P4-FSH was compared with those treated with FSH alone. Finally, the development competence of the ova from the early goats treated with E2-P4-FSH was evaluated by SCNT. The E2-P4-FSH treatment produced the largest ovaries, the highest numbers of total follicles and follicles > or =3 mm diameter compared with the other treatments. The number of > or =3 mm follicles per goat treated with E2-P4-FSH was significantly higher for those in the age groups 39-60 days than those in the age groups 61-90 days and 91-120 days. The FSH alone treatment resulted in a lower proportion matured ova in vitro within 27 hr than from those goats treated with E2-P4-FSH. Ova derived from the early prepubetal goats resulted in lower rate of blastocyst in SCNT (15.3% versus 22.1%, P < 0.01) than that of adult goat However, the number of ova recovered per goat was substantially greater for the prepubertal goats (108 +/- 10.3 Versus 28 +/- 5.0). Consequently, the younger goats produced significantly more blastocyst (7.1 +/- 2.7 versus 4.2 +/- 1.4) per head. It was concluded that early pubertal goats treated with E2-P4-FSH could provide a relatively high number of developmentally competent ova for SCNT studies.

Secondary SCNT doubles the pre-implantation development rate of reconstructed interspecies embryos by using cytoplasts of Sannen dairy goat ova.

The aim of this study was to investigate whether ova of Sannen goat could support the pre-implantation development of interspecies embryos constructed through somatic cell nucleus transfer (SCNT) embryos and whether secondary SCNT (SSCNT) could improve the pre-implantation development of those embryos. The primary SCNT (PSCNT) embryos were produced by using Sannen goat ovum cytoplasts as recipients and fibroblast cells, derived from human, rabbit and Boer goat skins, as nucleus donors. The blastomeres of 8 to 16 cells stage of PSCNT embryos were subsequently used as nucleus donor cells and Sannen goat ovum cytoplasts as recipients to evaluate the effect of SSCNT on the pre-implantation development rate of these reconstructed interspecies embryos. Our results indicate that the pre-implantation development rates of SSCNT embryos reconstructed using these three different blastomeres are almost twice of that of corresponding PSCNT embryos (human, 15.8% vs. 7.8%; rabbit, 27.9% vs. 12.5%; Boer goat 55.3% vs. 24.5%; P < 0.05 in all three cases). The time durations that embryos need for the serial events of remodeling and reprogramming to take place vary, depending on the animal species of nucleus donors. These data suggest that remodeling and reprogramming of donor nucleus may be enhanced by prolonged exposure of donor nucleus to maternal cytoplast. We conclude that Sannen goat cytoplast can support the pre-implantation development of embryos constructed with nuclei from various donors, including fibroblasts of human, rabbit and Boer goat; and the somatic nucleus derived from different species requires more time to achieve its reprogramming necessary for pre-implantation development.

Factors derived from mouse embryonic stem cells promote self-renewal of goat embryonic stem-like cells.

Goat embryonic stem (ES)-like cells could be isolated from primary materials-inner cell masses (ICMs) and remain undifferentiated for eight passages in a new culture system containing mouse ES cell conditioned medium (ESCCM) and on a feeder layer of mouse embryo fibroblasts (MEFs). However, when cultured in medium without mouse ESCCM, goat ES-like cells could not survive for more than three passages. In addition, no ES-like cells could be obtained when ICMs were cultured on goat embryo fibroblasts or the primary materials-whole goat blastocysts were cultured on MEFs. Goat ES-like cells isolated from ICMs had a normal karyotype and highly expressed alkaline phosphatase. Multiple differentiation potency of the ES-like cells was confirmed by differentiation into neural cells and fibroblast-like cells in vitro. These results suggest that mouse ES cells might secrete factors playing important roles in promoting goat ES-like cells' self-renewal, moreover, the feeder layers and primary materials could also influence the successful isolation of goat ES-like cells.

[Effect on development in NT embryos after transplantation of nuclei derived from transfected goat fetal fibroblasts suffering different treatments into enucleate eggs].

In order to improve the development rate of preimplantation nuclear transfer embryos (NT embryos) after transplanting nuclei derived from transgenic goat fetal cells, the donor fetal fibroblasts starved for 5 days in DMEM containing 0.5% FCS were divided into three groups and treated with different methods respectively before using as donor cell. Group 1 was frozen at -80 degrees C or in liquid nitrogen for several days or months. Group 2 was at first treated as the same as group 1, then cultured for 2-5 days in DMEM containing 10% FCS and starved for another 5 days subsequently. Group 3 was cultured for 2-5 days in DMEM containing 10% FCS and starved for another 5 days subsequently. The rate of G0/G1 phase cells from group 2 was 95.68% and significantly different from group 1's 88.66%. The rate of survival cells from group 2 was 99.9% and significantly different from group 1's 80.00% (P < 0.05).The morula- blastocyst stage NT embryos development rate of group 2 was 66.09% and significantly different from group 1's 22.00% and group 3's 50.51% (P < 0.05). All NT embryos of above three groups were transferred into synchronous oestrus recipients and the pregnant status of recipients was checked by B-mode ultrasound diagnosis after 35 days. The recipient pregnancy rate of group 2 was 45.83%, much higher than that of group 1(20.00%) and group 3 (29.58%). The result of this experiment showed that donor cells treated with freezing and two times starvation could significantly improve the rate of G0/G1 phase cells, the rate of survival cells, the NT embryos development rate and the recipient pregnancy rate.