Changes in the ovaries and plasma progesterone (P) and estradiol-17β (E2) levels were examined and compared to pregnancy and twinning rate in 17 Holstein-Friesian cows with twin ovulations after insemination. Plasma P and E2 levels of 8 cows that conceived following a single ovulation were used as normal controls. Of the 17 cows, 11 cows conceived; 5 cows delivered twins, 4 cows had a single, and remaining 2 cows aborted fetuses at 95 and 106 days of pregnancy, respectively. In 6 other non-pregnant cows, 2 cows ovulated again 20 & 21 days after insemination, and 4 cows ovulated 26-58 days after the service. Plasma P levels in all cows changed within the range of mean±2SD of the levels in control cows during the period of 12-13 days after insemination. The levels decreased coincident with the regression of either of the 2 corpora lutea in 1 cow which delivered a single, and in 4 non-pregnant cows which showed a prolonged estrous cycle. Plasma E2 levels in pregnant cows changed within the range of the control level, but the levels in non-pregnant cows changed above or over the control level. The results indicated that there was a high incidence of the embryonic death after twin ovulation, and an increase of plasma E2 level after ovulation may adversely affect embryo survival rate. No relationships between plasma P profiles and pregnancy rate were observed.
Four ES-like cell lines (M4, R1, DMR2 and DMR3) were established from the inner cell masses of Japanese White (M4, R1) and Dutch (DMR2, DMR3) rabbit blastocysts. Three and one cell lines of them can be maintained on primary mouse and rabbit fetal fibroblast feeder layers, respectively. All 4 lines (1 female and 3 male) were karyotypically normal with 44 chromosomes. They spontaneously differentiated into embryoid bodies on suspension culture, and endoderm-like, muscular-like, neuron-like cells, lipid vesicles and tubular structures on monolayer culture. An alkaline phosphatase (AP) activity of them decreased with passage times, indicating progressive differentiation status.
The effects of culture time for oocyte maturation and the injection conditions of capacitated sperm (with/without manipulation medium) on the subsequent morphological changes in male and female nuclei and the development of embryos derived from microfertilization of bovine oocytes were investigated. The rates of cleavage and development to blastocyst stage increased with the extension of maturation time of oocytes from 18h to 25h. The best results (2-cell: 71.4%, blastocysts: 11.4%) were obtained from the oocytes cultured for 25 h. Microinjection of sperm into the oocytes without manipulation medium, resulted in higher (P<0.05) developmental rates (2-cell: 28.8%, blastocysts: 9.6%) than those (19.5% and 4.9%) from sperm with manipulation medium. This result suggested that the injected manipulation medium was harmful to the ooplasm and affected the development of embryos. The development of embryos derived from microfertilization up to 3-to 7-cell stage and over 8-cell stage was delayed for 4 h and 8 h than those from in-vitro morphological observation of the male and female nuclei after the microinjection, the formation of male pronucleus was slower than that of female pronucleus.
The viability of embryos transferred to 37 cows were monitored by detecting early pregnancy factor (EPF) in the bovine serum. Embryos were transferred nonsurgically to the uterine horn ipsilateral to the ovary bearing the corpus luteum on day 7 to day 11 (day 0=day of estrus). The transferred embryos included 33 in vitro fertilized embryos; 3 fresh embryos, 24 freezed embryos, 6 freezed, thawed and cultured embryos, and 4 in vitro fertilized embryos which were freezed and thawed. Blood samples were collected weekly after the embryo transfer for the EPF assay and progesterone determination. Total pregnancy rate on day 42 to 43 obtained by ultrasonographic examination was 18.9%. Percentage of cows with viable embryo on 7 and 28 days after the transfer was 59.5% and 18.9%, respectively. This discrepancy indicated the occurrence of embryonic death. All 7 cows in which EPF was detected on 28 days after embryo transfer were diagnosed with ultrasonographic examination as pregnant on 35 days after the transfer. Determination of EPF in the serum is proved to be useful for monitoring the viability of the transferred embryo.
Bovine luteal tissue slices and separately isolated luteal cells were cultured in the presence of prostagrandin F2α (PGF2α) with an without other additives to determine whether inhibition of the luteal function, which is seen in vivo, could be induced. The luteal function was assessed with reference to the content of progesterone (P) released into the media. Samples of functional corpus luteum were collected at a slaughter house and cultured for 12 or 24 hours at 37 C using (1) a serum-free medium (basic medium) as a control and media containing (2) PGF2α alone, (3) PGF2α+filtrate of cultured bovine endometrium, (4) PGF2α+endometrial tissue, (5) serum from PGF2α+treated cattle or (6) HCG+PGF2α. The experiment produced no evidence of marked inhibition of P secretion such as seen in vivo in any of the media used, except for the contrarily accelerated production of P in the medium containing HCG and PGF2α. The results suggested the involvement of other delicate factors or mechanism in the inhibited luteal function in vivo.
The present study was conducted to establish a cloning system by nuclear transfer technique in mice in which nuclear transfer into 2-cell emrbyos and Metaphase II oocytes were examined and the developmental property of the reconstituted eggs was investigated in vitro and in vitro. The results were summerized as follows. 1) By transfer of a single nucleus from 2-and 4-cell embryos into one of blastomeres of the enucleated 2-cell embryo, identical triplet and twin mice were produced after in vitro culture and transfer to recipients.2) In the nuclear transfer into enucleated oocytes, the chromosomes of oocytes at telophase of the first meiotic division were successfully removed using standard micromanipulation and a differential interference micros-copy. The enucleated oocytes (ooplasts) wewe able to use as recipient ooplasm for nuclear transfer after culture for 4-6h in vitro.3) When the ooplasts received a nucleus from the late 2-cell embryos, 23% developed to blastocysts in vitro, and the offspring were obtained after transfer to recipients. Transfer of a inner cell mass (ICM) cell and a thymocyte from fetuses and new born mice into the ooplasts also resulted in development into blastocyst stage. 4) It was shown that the timing of fusion of donor nucleus with a ooplast and activation of it is essential factor for regulation of the remodelling of the donor nucleus in nuclear transferred oocytes. This study shows the possibility that not only early embryonic nucleui but also more advanced nuclei are reprogrammed when they are transferred into the ooplast in mice.