Yoshinori Fukuda

Developmental ability of porcine ova matured in porcine follicular fluid in vitro and fertilized in vitro

Developmental ability of porcine ova matured in porcine follicular fluid (pFF) with FSH in vitro and fertilized in vitro was examined by culturing in BMOC-2. Forty-eight hours after insemination, 35.6% of ova cleaved normally, and this rate was significantly higher (13.0%) than that of the ova matured in a modified Krebs-Ringer bicarbonate solution. Twenty-four percent (29 120 ) of ova matured in pFF with FSH developed to the four-cell stage and two of them developed to the eight-cell stage 66 h after insemination. Most cleaved embryos stopped developing at the four-cell stage and neither the morula nor blastocyst stage was observed throughout the culture period as reported in the in vivo matured ova. In culture at 37 degrees C, the appearance of two-cell and four-cell embryos was delayed from that of in vivo embryos, but their development was significantly accelerated by culturing at 39 degrees C. These results show that pFF is an excellent maturation medium for porcine oocytes, and the developmental capacity of the ova matured in pFF seems to be similar to that of in vivo matured ova. Culturing at 39 degrees C was found to be more suit-able for the development of ova than 37 degrees C.

Vitrification of Bovine Blastocysts on a Membrane Filter Absorbing Extracellular Vitrification Solution

ABSTRACT The purpose of this study was to test the hypothesis that an extracellular vitrification solution might be unnecessary after equilibration of bovine blastocysts with a vitrification solution. We assessed hatching in culture and the development to normal calves of bovine blastocysts vitrified on a nitrocellulose membrane filter that absorbed extracellular vitrification solution. Twenty in vitro produced bovine blastocysts were vitrified. All embryos were collected after warming and classified as excellent at the start of culture and all hatched between 48–96 h of culture. Transfer of three vitrified blastocysts derived in vivo given to three recipients resulted in the birth of one healthy calf on December 3, 2004. These results indicate that an extracellular vitrification solution is unnecessary after equilibration of embryos and that this new method of vitrification of embryos on a membrane filter is useful for the cryopreservation of bovine blastocysts.

Effects of Fractions of Bovine Follicular Fluid and Fetal Bovine Serum as Supplements to Maturation Medium on In Vitro Development of In Vitro Fertilized Bovine Embryos

Abstract: The purpose of this study was to examine the effects of different fractions of bovine follicular fluid (bFF) and fetal bovine serum (FBS) as maturation medium supplements on development to the blastocyst stage of bovine oocytes matured and fertilized in vitro. Three bFF fractions obtained by ultracentrifugation were designated as the 1st, 2nd and 3rd fractions, and four FBS fractions, also obtained by ultracentrifugation, were designated as the 1st, 2nd, 3rd and 4th fractions. Cumulus-oocyte complexes (COCs) were divided into two groups: those with homogeneous ooplasm and those with heterogeneous ooplasm. COCs were cultured in basic maturation medium with a bFF fraction or FBS fraction. In oocytes with homogeneous ooplasm, the rates of development to the blastocyst stage with bFF-1st and bFF-2nd were significantly higher than that with bFF. In oocytes with heterogeneous ooplasm, the rate of development to the blastocyst stage with bFF-2nd was significantly higher than that with bFF. In oocytes with homogeneous ooplasm, the rate of development to the blastocyst stage with FBS-1st was significantly lower than that with FBS. In oocytes with heterogeneous ooplasm, the rate of development to the blastocyst stage with FBS-3rd was significantly higher than that with FBS. The results indicate that bFF-2nd and FBS-3rd, obtained by ultracentrifugation, are effective maturation medium supplements, as they promoted the development of highquality matured bovine oocytes.

In vitro Development of Porcine In Vitro Matured Oocytes Vitrified After Removal of Cytoplasmic Lipid Droplets

Abstract: The objective of the present study was to investigate the in vitro developmental competence of porcine in vitro matured (IVM) oocytes vitrified after removal of cytoplasmic lipid droplets (delipation). After vitrification and warming, the delipated porcine IVM oocytes were inseminated and subsequently cultured in vitro. The rate of development to the blastocyst stage of delipated, vitrified oocytes (5.9%) was significantly lower than that of control oocytes (untreated oocytes) (26.2%). We also examined the influence of delipation of porcine IVM oocytes on development to the blastocyst stage following in vitro fertilization (IVF). Delipated porcine IVM oocytes (not vitrified) were inseminated and subsequently cultured in vitro. The rates of development to the blastocyst stage were similar for delipated and undelipated oocytes (21.1% and 26.2%, respectively). The results of the present study showed that delipated, vitrified porcine IVM oocytes can develop to the blastocyst stage following IVF, though blastocyst formation rate was low, and that delipation of porcine IVM oocytes did not negatively affect their development to blastocyst stage.

In vitro developmental capacity of mouse follicular oocytes recovered at various times after hCG injection and matured in vitro

Abstract The study was designed to examine in vitro developmental capacity of mouse follicular oocytes recovered at various times after hCG injection and matured in vitro. Jc1/ICR mice were treated with 5 IU PMSG and 5 IU hCG at 48-h intervals. Oocytes were recovered from follicles at 0, 4, 8 and 12 h after hCG injection and from the oviduct at 12 and 16 h after that (F-0, F-4, F-8, F-12, T-12 and T-16 oocytes, respectively). They were cultured in modified Krebs-Ringer solution (TYH) with or without 5% fetal calf serum (FCS) for the remaining periods to total 16 h before insemination. These matured oocytes were transferred into modified Whittens medium and cultured for up to 120 h after insemination. The TYH medium with FCS increased the proportion of development into blastocysts in F-4 to F-12 oocytes to 80 to 86%. Development of F-8 and F-12 oocytes to blastocysts without FCS was 83 and 89%. These rates were not significantly different from those of T-12 (83%) and T-16 (87%) oocytes. The maturation stages of most F-4, F-8 and F-12 oocytes at recovery were prometaphase-I, metaphase-I plus anaphase-I, and metaphase-II. The process of oocyte maturation in vitro was similar to in vivo. These results suggest that mouse oocytes which resumed meiotic division until prometaphase-I or further stages in vivo, and were cultured in TYH with or without FCS, have a developmental capacity as high as that of ovulated oocytes. In addition, FCS seemed to have most beneficial effect on the fertilization ability.