Sex preselection by use of X- and Y- chromosome bearing spermatozoa has been recognized to be more efficient. A flow cytometric sperm sorting based on the difference of their DNA content is the best method for separation of X- and Y-sperm. To date, the flow cytometrically sorted sperm has been involved in the production of sex preselected offspring by surgical intratubal insemination, in vitro fertilization and embryo transfer and intracytoplasmic sperm injection. At first, flow cytometer was modified for DNA confirmation and sorting of sperm with high resolution. Especially, the beveled insertion tube could regulate orientation of flat-shaped sperm head. The forward fluorescent detector was essential for measuring DNA contents of sperm. Recently, the high-speed sperm sorting with the orienting nozzle resulted in production of 90% purity of X- and Y-sperm at rate of 6 million sperm per hour. This application can enable to accomplish more conventional technology for both artificial insemination and cryopreservation of X- or Y-sperm in farm animals.

View full abstract

In embryo transfer, the viability of the embryo collected in vivo or produced in vitro is important for success in the subsequent prepnancy. The present study was conducted to estimate the rate of embryo recovery in hormone-induced ovulated goats and to examine how embryo development would proceed in these animals. A series of FSH injections and a single injection of PMSG under asynchronization of estrus could induce ovulations 2 to 3 fold higher than in the unstimulated control independent of the breeding season. On the other hand, the rate of embryo recovery in FSH and PMSG injection groups differed, whereas the average number of the corpora lutea (CL) in both groups was similar. Profiles of embryo development showed that the embryos at the morula stage were collected from 6 to 8 days post coitus and at the blastocyst stage from 8 to 10 days. These results suggest that in hormonally treated goats healthy embryos could be obtained regardless of the breeding season.

View full abstract

We examined the relationship between the breakage modes occurring during intracytoplasmic sperm injection (ICSI) and survival, fertilization and embryonic development rates after ICSI, and the morphologic quality of the oocytes before ICSI. The tip of each injection pipette was flattened to exclude the factor of tip shape. Oolemma breakage modes were grouped into four types according to oolemma flexibility. Approximately 8% of the mature oocytes treated with ICSI featured an inflexible oolemma, and 50% of such oocytes resulted in cytolysis, but no significant differences were observed in fertilization rates among the surviving oocytes, and no relationship could be traced between breakage modes and morphologic oocyte quality. The selection of oocytes with flexible oolemma is believed to be crucial in obtaining satisfactory results with ICSI, but it is not yet possible to distinguish oocytes that have inflexible oolemma from other oocytes before ICSI.

View full abstract

Mammalian oocytes are surrounded by numerous layers of cumulus cells and the loss of gap junctional communication between cumulus cells and oocytes induces meiotic progression to the MII stage in oocytes. The closure of gap junctional communication was associated with the phosphorylation of connexin-43, gap junctional protein, in cumulus cells. In this study we investigated the effects of the phosphorylation of connexin-43 in cumulus cells on the cAMP level, MAP kinase activity and meiotic progression beyond the MI stage in the oocytes. The connexin-43 in cumulus cells was phosphorylated after 32-hr cultivation of COCs and up to 48 hr, whereas most of the connexin-43 was unphosphorylated in cumulus cells from COCs cultured for 24 hr. When the phosphorylation of connexin-43 in cumulus cells was suppressed by the addition of either PI 3-kinase inhibitor or PKC inhibitor, a significantly higher level of cAMP in the oocyte and a significantly lower proportion of oocytes at the MII stage were produced, as compared to those of oocytes cultured for 48 hr without these drugs. The activity of MAP kinase was also significantly inhibited by the addition of both drugs. It was therefore concluded that the closing of the gap junctional communication via the phosphorylation of connexin-43 might induce a decrease in the cAMP level, resulting in activation of MAP kinase and meiotic progression beyond the MI stage to the MII stage in porcine oocytes.

View full abstract

In nuclear transplantation, especially in enucleation, rotating the oocyte to a certain position has been an important work. Therefore, we developed an oocyte rotation system by using electrostatic force, which is known to be excellent in handling minute objects. This system has become the basic technology for the automated nuclear transplantation system. Mouse oocyles were rotated at a speed of about 60 deg/s by electrostatic phenomena (i.e. electrorotation and dielectrophoresis), and the direction of rotation could be reversed. In order to confirm the electrostatic effect on the fertilization and developmental ability of oocytes, we performed IVF and nuclear transplantation with rotated oocytes. The result was that the fertilization and developmental ability of the rotated oocytes was the same as that of the control, proving that rotation by electrostatic force does not influence the fertilization and development ability of oocytes.

View full abstract

Chromosomal analysis was accomplished in bovine embryos derived from in vitro fertilization (IVF) after insufficient in vitro maturation of oocytes. Bovine oocytes collected from different donors were pooled into two groups; oocytes matured in vitro for 12 hrs were classified as the 12-hr group (insufficient maturation group), and other oocytes matured in vitro for 26 hrs were classified as the 26-hr group. At forty-eight hrs after IVF, most of the embryos (185/223, 83%) in the 26-hr group developed into the 8- or 16-cell stages, while many embryos (125/165, 75.8%) in the 12-hr group were at the 2- or 4-cell stage. The incidence of chromosomally abnormal embryos in the 12-hr group, 66.7% (54/81), was significantly higher than that in the 26-hr group, 27.8% (20/72). These chromosomal anomalies were haploids, polyploids and mixoploids. It was presumed that the increase in these anomalies was caused by sperm penetration with dysfunction of zonae pellucida and/or membrane of the ooplasm of immature oocytes.

View full abstract

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) play important roles in bovine oocyte maturation. This study tested the effect of FSH and LH on bovine ZP gene expression during oocyte maturation in vitro with RNase protection assay. These gonadotropins had no effect on ZP gene expression in bovine oocytes cultured for 5 or 15 hours, but significant suppression of germinal vesicle breakdown (GVB) and metaphase II (MII) generation were observed with FSH. These data suggest that neither FSH nor LH has a significant effect on ZP gene expression in bovine oocytes. And the ZP gene expression could be independent of the mechanism of oocyte nuclear maturation in cattle.

View full abstract

The aim of this study was to determine the effectiveness of an intravaginal drug-releasing device commercially produced for use in goats and sheep (CIDR-G device) at inducing estrus in sows. Thirteen Landrace sows were used in the this study, and the sows were randomly divided into three groups: group A (n=5), group B (n=4) and group C (n=4). Two CIDR-G devices were inserted into the vagina of each sow in groups A and B, and one CIDR-G device was inserted into the vagina of each sow in group C. The device was removed from each sow on the seventh day after insertion, and 0.2625 mg of PGF_2α was applied to the vulva of each sow in groups A and C. All five sows in group A and all four sows in group C, the two groups of sows that were administered PGF_2α, showed onset of estrus 3-5 days and 2-4 days after removal of the CIDR-G device, respectively. Three of the four sows in group C showed onset of estrus on the second day after removal of the device. On the other hand, onset of estrus was observed (on day 3 after removal of the device) in only one of the four sows in group B, the group of sows that were not administered PGF_2α. Saliva progesterone concentrations continued to rise in both groups A and C after insertion of the device, and the saliva concentrations of progesterone on day 5 after insertion in these two groups were significantly higher than those just prior to insertion of the device (p<0.05). The number of devices inserted had no effect on the saliva progesterone concentration. The results of this study showed that insertion of a CIDR-G device for a period of seven days followed by application of PGF_2α to the vulva is effective for induction of estrus in the swine.

View full abstract

We attempted to produce chimeric mouse fetuses by using an embryonic stem (ES) cell subline, OM 3, which were transfected with pMGN(-4k)LacZ-neo transgene, and to analyze expression of the transgene in chimeric mouse fetuses. In embryonic days 11-13 (E11-13), no fetuses expressing the transgene were observed. In E14, three of 11 normal fetuses recovered showed the transgene expression. In these chimeric mouse fetuses, the regions expressing the transgene were found in a wide range of fetuses, including the temporal region of the head, trunk from neck to tail, and limbs. It was shown by histochemical analysis that these patterns of transgene expression were specific to skeletal muscles. These results indicated that OM 3 cells might be useful for analyses of mouse skeletal-myogenesis and for the development of basic techniques for skeletal muscles in regenerative medicine.

View full abstract

There have been very few reports about fertilized eggs of the Mongolian gerbil. This study was therefore designed to determine the chronological changes in fertilized eggs of the Mongolian gerbil. Superovulated females were monogamously paired with males and pregnant females were killed at 16, 19 and 22 hours after hCG injection. After slaughter, the presence of female and male pronuclei and sperm tail was examined. Rates of female and male pronuclei increased with the collection time for fertilized eggs (24.5, 58.9 and 67.9% at 16, 19 and 22 hr, respectively). Rates for a fertilizing sperm tail out of the total number of fertilization eggs decreased with the collection time for fertilized eggs (85.0, 58.0 and 28.0% at 16, 19 and 22 hr, respectively).

View full abstract