In Vitro Culture of Late Stage Pig Embryos in a Chemically Defined Medium, Porcine Blastocyst Medium (PBM).

In vitro production (IVP) of porcine preimplantation embryos is an important technique not only for basic and biomedical research purposes but also for animal biotechnology application such as transgenesis, cloning, and embryo transfer. In this chapter, we demonstrate a superior IVP procedure of porcine embryos derived from cumulus-oocyte complexes (COCs) of slaughtered pig ovaries which are cultured sequentially in different defined media. Porcine blastocyst medium (PBM) particularly designed for the late stage embryo culture could improve the potential of morulae or blastocysts to develop into hatching and hatched blastocysts with good quality.

The relationship between oxygen consumption rate and viability of in vivo-derived pig embryos vitrified by the micro volume air cooling method.

The aim of this study was to assess the viability of vitrified-warmed in vivo-derived pig embryos after measuring the oxygen consumption rate. Six days after artificial insemination, blastocysts were collected from gilts and vitrified by the micro volume air cooling method. The oxygen consumption rate was measured in 60 vitrified-warmed embryos, which were then cultured for 48h to assess the viability.

Birth of piglets from in vitro-produced porcine blastocysts vitrified and warmed in a chemically defined medium.

We examined the effect of different embryo developmental stages, culture periods, and media on the cryotolerance of in vitro-produced porcine blastocysts. All media used for in vitro production, vitrification, and warming were chemically defined. When in vitro-produced embryos at the blastocyst and expanded blastocyst stages were vitrified using the Cryotop method on Day 5 or 6 (Day 0 = the day of IVF), the survival rate and hatching rate of expanded blastocysts after warming were higher than those of blastocysts.

Relationships between oxygen consumption rate, viability, and subsequent development of in vivo-derived porcine embryos.

Oxygen consumption rate of in vivo-derived porcine embryos was measured, and its value as an objective method for the assessment of embryo quality was evaluated. Embryos were surgically collected 5 or 6 days after artificial insemination (AI), and oxygen consumption rate of embryos was measured using an embryo respirometer. The average oxygen consumption rate (F × 10(14)/mol s(-1)) of the embryos that developed to the compacted morula stage on Day 5 (Day 0 = the day of artificial insemination) was 0.

Recombinant human follicle-stimulating hormone and transforming growth factor-alpha enhance in vitro maturation of porcine oocytes.

The biological functions of recombinant human follicle-stimulating hormone (FSH) and transforming growth factor-α (TGF-α) on in vitro maturation of porcine oocytes were investigated. Cumulus-oocyte complexes were matured in defined porcine oocyte medium containing 0-0.1 IU/ml FSH in the presence or absence of 10 ng/ml TGF-α.

Glucose and glycine synergistically enhance the in vitro development of porcine blastocysts in a chemically defined medium.

In the present study, the effects of glucose and/or glycine on the in vitro development of Day 5 (Day 0=IVF) porcine blastocysts were determined. The addition of 2.5-10 mM glucose to the chemically defined culture medium porcine zygote medium (PZM)-5 significantly increased blastocyst survival rates compared with those of blastocysts cultured in the absence of glucose.

Transforming growth factor-alpha in a defined medium during in vitro maturation of porcine oocytes improves their developmental competence and intracellular ultrastructure.

We examined the effects of transforming growth factor-alpha (TGF-alpha) to develop a defined medium for in vitro maturation (IVM) of porcine (Sus scrofa domesticus) oocytes. Cumulus-oocyte complexes (COCs) were matured in porcine oocyte medium containing 3mg/mL polyvinyl alcohol (POM) and TGF-alpha (0, 1, 10, or 100 ng/mL) in the presence or absence of the gonadotropins equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG). In the absence of gonadotropins, adding 10 ng/mL TGF-alpha increased (P<0.

alpha1,3-Galactosyltransferase-gene knockout in cattle using a single targeting vector with loxP sequences and cre-expressing adenovirus.

Background: Gene targeting in large animals has the potential to be useful in medicine as well as in agriculture. Previously, we reported the first successful targeting of the bovine alpha1,3-galactosyltransferase (alpha1,3GT) gene and establishment of a heterozygous knockout cell line. In this report, we generated both heterozygous and homozygous knockout bovine cell lines, and alpha1,3GT-gene knockout cattle.

Evaluation of bovine embryos produced in high performance serum-free media.

This review evaluates the quality of bovine embryos developed from in vitro-matured (IVM) and -fertilized (IVF) oocytes cultured in either serum-free or serum-containing media. Bovine embryos cultured in serum-supplemented medium contain numerous cytoplasmic lipid droplets and immature mitochondria compared to those cultured in serum-free medium. The accumulation of cytoplasmic lipids in embryos developed in serum-containing medium may be a result of incorporation of lipoproteins from the serum and may result in impaired function of mitochondria.

In vitro production of bovine embryos and their application for embryo transfer.

This review introduces newly developed serum-free media (IVD101 and IVMD101), that are effective for producing high yields of transferable embryos of good quality from in vitro-matured and -fertilized oocytes. Both serum-free media produced better results than serum-containing medium, including increased rates of blastocyst formation, post-thaw embryo viability, and pregnancy after transfer. In addition, reduced risks of calf mortality and large calf syndrome were also observed for the serum-free-derived embryos.

Growth, antrum formation, and estradiol production of bovine preantral follicles cultured in a serum-free medium.

The objective of this study was to identify factors that would allow the establishment of a serum-free culture system that could support follicular and oocyte growth, antrum formation, and estradiol-17beta (E(2)) production in preantral follicles of bovine ovaries. Large preantral follicles (145-170 micro m in diameter) were microsurgically dissected from ovaries, embedded in 0.15% type I collagen gels, and maintained in a serum-free medium for up to 13 days at 38.

Ultrastructural differences in bovine morulae classified as high and low qualities by morphological evaluation.

Bovine morulae collected from superovulated cows were classified into four groups (excellent, good, fair, poor) using morphological evaluation under a phase contrast microscope. The number of blastomeres in morulae classified as excellent and good (high quality) was significantly higher than the number in fair and poor (low quality) morulae. The ultrastructure of morulae in each group was compared.

Molecular cloning of a cDNA encoding a bovine growth differentiation factor-9 (GDF-9) and expression of GDF-9 in bovine ovarian oocytes and in vitro-produced embryos.

Growth differentiation factor-9 (GDF-9), a member of the transforming growth factor-beta superfamily, is known to be expressed specifically in ovaries of various mammalian species and to be important for normal follicular development in mice. In the present study, a cDNA encoding for bovine GDF-9 was isolated and characterized, and expression of GDF-9 mRNA in ovarian oocytes and in-vitro-derived embryos was examined. Isolation of bovine GDF-9 was achieved using the polymerase chain reaction (PCR) with primers based on an ovine GDF-9 cDNA sequence.

Accumulation of cytoplasmic lipid droplets in bovine embryos and cryotolerance of embryos developed in different culture systems using serum-free or serum-containing media.

In this study, the quantitative fluctuation of cytoplasmic lipid droplets (LD) and cryotolerance were investigated in bovine embryos derived from in vitro-matured (IVM) and in vitro-fertilized (IVF) oocytes developed in different culture systems using serum-free or serum-containing media. The serum-free cultures were grown using IVMD101 medium in conjunction with bovine cumulus/granulosa cell (BCGC) cocultures or IVD101 medium without BCGC cocultures, and the serum-containing cultures were grown in the presence of BCGC cocultures using HPM199 medium supplemented with 5% calf serum (HPM199 + CS). Large numbers of sudanophilic LD were present in the cytoplasm of bovine embryos from 2-cell to hatched blastocyst stages, and the number and size differed between the embryos cultured in serum-free and serum-supplemented media.

Efficient isolation and long-term viability of bovine small preantral follicles in vitro.

A comparison of isolation techniques for small preantral follicles (30-70 microm) from bovine ovaries using a mechanical method with a grating device or collagenase treatment was performed. The mean number (157.0) of intact follicles per ovary isolated by the mechanical method was significantly greater (P < 0.

Mouse oviduct-specific glycoprotein gene: genomic organization and structure of the 5'-flanking regulatory region.

A member of the chitinase protein family, oviduct-specific glycoprotein (OGP), can directly associate with gametes or with the early embryo in the oviduct. Although the glycoprotein is widely distributed among mammalian species and there is indirect evidence concerning the involvement of the molecule in the fertilization process, its physiological functions are far from completely understood. To understand the fundamental mechanisms that direct gene expression as well as to know the physiological significance of OGP, we have isolated and characterized a mouse OGP gene (mogp-1).

Ultrastructural features of goat oviductal secretory cells at follicular and luteal phases of the oestrous cycle.

The aim of the present study was to investigate the ultrastructure of secretory cells in the various regions of the goat oviduct during the follicular and luteal phases of the oestrous cycle. During the follicular phase in the fimbriae, the secretory cells contained small secretory granules with electron-dense matrices. In the luteal phase, the secretory granules disappeared and cytoplasmic protrusions, extending beyond the luminal border of the ciliated cells and often containing the nucleus, were predominant.

Ultrastructure of bovine embryos developed from in vitro-matured and -fertilized oocytes: comparative morphological evaluation of embryos cultured either in serum-free medium or in serum-supplemented medium.

The ultrastructure of bovine embryos developed from in vitro-matured and -fertilized oocytes, cocultured with bovine cumulus/granulosa cells either in a serum-free medium (IVMD101) or in a serum-containing medium (TCM199+CS) was compared. Embryos up to the eight-cell stage had many cellular organelles and cytoplasmic components that were randomly distributed in the cytoplasm. Mitochondria were spherical or ovoid and had only a few peripheral cristae.

Fine structure of bovine morulae and blastocysts in vivo and in vitro.

The ultrastructure of bovine morulae and blastocysts developed from in vitro-matured and -fertilized oocytes in a serum-supplemented medium was compared with that of morulae and blastocysts collected non-surgically from superovulated cows. In the in vivo-derived morulae, two characteristic cells types could be identified by the electron-density of their cytoplasm and by their ultrastructural features. One type appeared light in color with low electron-dense cytoplasm.

Primary modulation by oestradiol of the production of an oviduct-specific glycoprotein by the epithelial cells in the oviduct of newborn golden hamsters.

The effects of steroid hormones (oestradiol and progesterone) on the appearance of a golden hamster oviduct-specific glycoprotein (GHOGP) in the epithelium of the oviduct of the newborn golden hamster were investigated by immunoblotting and immunohistochemical staining with a GHOGP-specific monoclonal antibody. Newborn golden hamsters (1.5 days old) were injected daily with oestradiol (1 microgram) or progesterone (10 microgram).

Isolation from fetal bovine serum of a fragment b of complement factor B-like protein improving a long-term survival of human endothelial cells.

It is known that serum is a most important factor supporting cell survival and growth. Particularly, the deprivation of serum would result in the death of human endothelial cell. Our previous paper reported an endothelial cell-viability maintaining factor (EC-VMFa) purified from fetal bovine serum and identified as an apolipoprotein.

Bovine oviductal epithelial cells: their cell culture and applications in studies for reproductive biology.

Epithelial cells of the mammalian oviduct play an important role in reproductive and developmental events that occur there. Oviductal epithelial cells from several mammalian species can be isolated and cultured in serum or serum-free medium in vitro and cell culture of bovine oviductal epithelial cells (BOEC) has been described by many investigators. Cultured BOEC show a wide variety of secretory activities and these secretory factors may influence early embryonic development or sperm function.

The mammalian oviductal epithelium: regional variations in cytological and functional aspects of the oviductal secretory cells.

The secretory cells in the epithelium of mammalian oviducts produce and release various secretory materials into the lumen. Secretions from such cells provide a suitable environment for the events that occur in the oviductal lumen. This review focuses on the regional differentiation of the secretory cells in mammalian oviducts.

Immunocytochemical evidence that a specialised region of the rat oviduct secretes an oviductal glycoprotein.

The immunocytochemical localisation in the rat oviduct of an oviductal glycoprotein was investigated by light and electron microscopy. Using a monoclonal antibody (MAb) that cross-reacted with the rat oviductal glycoprotein ( > 330 kDa), we examined the epithelium of 4 regions (fimbriae, ampulla, isthmus and uterotubal junction) of the rat oviduct during the oestrous cycle. The MAb reacted specifically with the epithelial cells of the rat oviduct and not with the stromal cells.

Purification and characterization of an endothelial cell-viability maintaining factor from fetal bovine serum.

Serum is an essential requirement for the growth and long-term survival of human endothelial cells, even in the presence of such defined elements such as polypeptide growth factors and hormones. A polypeptide from fetal bovine serum was isolated and characterized on the basis of long-term survival of human endothelial cells in serum-free culture. The endothelial cell viability maintaining factor has been purified to homogeneity by a combination of polyethylene glycol precipitation, hydroxylapatite, gel permeation and reverse-phase high performance liquid chromatography.