Establishment of goat embryonic stem cells from in vivo produced blastocyst-stage embryos.

Embryonic stem (ES) cells with the capacity for germ line transmission have only been verified in mouse and rat. Methods for derivation, propagation, and differentiation of ES cells from domestic animals have not been fully established. Here, we describe derivation of ES cells from goat embryos. In vivo-derived embryos were cultured on goat fetal fibroblast feeders. Embryos either attached to the feeder layer or remained floating and expanded in culture. Embryos that attached showed a prominent inner cell mass (ICM) and those that remained floating formed structures resembling ICM disks surrounded by trophectodermal cells. ICM cells and embryonic disks were isolated mechanically, cultured on feeder cells in the presence of hLIF, and outgrown into ES-like colonies. Two cell lines were cultured for 25 passages and stained positive for alkaline phosphatase, POU5F1, NANOG, SOX2, SSEA-1, and SSEA-4. Embryoid bodies formed in suspension culture without hLIF. One cell line was cultured for 2 years (over 120 passages). This cell line differentiated in vitro into epithelia and neuronal cells, and could be stably transfected and selected for expression of a fluorescent marker. When cells were injected into SCID mice, teratomas were identified 5-6 weeks after transplantation. Expression of known ES cell markers, maintenance in vitro for 2 years in an undifferentiated state, differentiation in vitro, and formation of teratomas in immunodeficient mice provide evidence that the established cell line represents goat ES cells. This also is the first report of teratoma formation from large animal ES cells.

Seasonal variation in preovulatory events associated with synchronization of estrus in dwarf goats.

This experiment was conducted to define the temporal relationships among estrus, the LH surge and ovulation after estrus synchronization in dwarf goats and to assess the effect of season on these parameters. In November (breeding season), March (transition period) and July (non-breeding season), estrus was synchronized in 12 dwarf goats by means of intravaginal sponges containing 60 mg medroxyprogesterone acetate (MAP) for 10 d, coupled with 125 microg cloprostenol i.m. 48 h before sponge removal and 300 IU eCG i.m. at sponge removal. A different group of animals was used during each time period. Onset of estrus was monitored using two males, and blood samples for the measurement of plasma LH were collected at 2-h intervals from 24 to 60 h after sponge removal. Ovulation was confirmed by laparoscopy at 54 and 72 h after sponge removal. A seasonal shift was detected in the intervals to onset of estrus, LH surge, and ovulation after sponge removal (P<0.05), with sponge removal to onset of estrus being shorter (P<0.05) in November (25.0 +/- 1.56 h) and July (28.9 +/- 2.43 h) than in March (40.9 +/- 3.27 h). The intervals between onset of estrus and the LH surge and between the LH surge and ovulation were found to be constant throughout the different seasons. An optimal time for breeding, artificial insemination, oocyte and embryo recovery, and embryo transfer may be predicted using information gained from these studies.