Il-Keun Kong

Comparison of open pulled straw (ops) vs glass micropipette (gmp) vitrification in mouse blastocysts

The purpose of this study was to investigate the use of a glass micropipette (GMP) as a vessel for vitrification of mouse blastocysts, and to compare the post-thaw survival of these blastocysts with those cooled in open pulled straws (OPS). The GMP vessel permits higher freezing and warming rates than OPS due to the higher heat conductivity of glass and lower mass of the solution containing the embryos. Groups of 6 mouse blastocysts were sequentially placed into 2 vitrification solutions before being loaded into either the OPS or GMP vessels and immersed into LN2 within 20 to 25 sec. Post-thaw blastocysts were serially washed in 0.25 and 0.15 M sucrose in holding medium (HM) and modified human tubal fluid medium (mHTF), each for 5 min, and then cultured in mHTF supplemented with 10% FCS for 24 h. The rate of blastocyst re-expansion did not differ significantly for OPS (93.5%) and GMP (95.0%) methods (P<0.05). The hatching rate in OPS (88.7%) was similar to that in GMP (90.0%) but was lower than for the unvitrified control embryos (98.3%, P<0.05). To determine the optimal embryo population per GMP vessel, the pipettes were loaded with 2 to 10 embryos. The rate of blastocyst re-expansion after vitrification was significant for 2 to 4 embryos than for 6 to 10 embryos per vessel. In addition, the rate of blastocyst re-expansion was significantly lower if blastocysts were vitrified in the wide rather than the narrow portion of the micropipette (100 vs 87.5%; P<0.05) even when only 4 blastocysts were loaded per vessel. These results indicate that both vitrification vessels can provide high rates of embryo survival. However, the GMP vessel does not need a cap to protect the vessel from floating after immersion in LN2. The number and location of the embryos (narrow versus wide portion of capillary) were considered to be limiting factors to the viability of mouse embryos.

Improvement in post-thaw viability of in vitro-produced bovine blastocysts vitrified by glass micropipette (GMP)

The purpose of this study was to investigate the use of a glass micropipette (GMP) as a vessel for vitrification of in vitro-produced (IVP) bovine blastocysts and to compare the results with post-thaw survival rate of bovine blastocysts frozen in GMP with those frozen in open pulled straw (OPS) that have been previously investigated. The GMP vessel permitted higher freezing and warming rates than the OPS due to the higher heat conductivity of the glass and the lower mass of the solution that contained the embryos. Groups of three bovine IVP blastocysts were sequentially placed into vitrification solution before being loaded into either the OPS or GMP vessels and they immersed into LN(2) within 20-25 s. Post-thaw blastocysts were serially washed in 0.25 and 0.15 M sucrose in a holding medium (HM: D-PBS supplemented with 5% FCS) and then in TCM-199 for 5 min in both cases. They was then cultured in TCM 199 supplemented with 10% FCS for 24 or 48 h. The rate of blastocyst re-expansion was significantly different between OPS (79.6%) and GMP (90.4%) methods. Neither was the hatching rate significantly different among OPS (51.8%), GMP (57.1%) methods and non-vitrified group (67.3%). Only the rate of post-thaw re-expanding of blastocysts loaded in narrow column was significantly higher than that of the wide column (83.3% versus 56.7%) (P < 0.05), although the GMP straw was loaded with three blastocysts per vessel. These results indicated that the GMP vessels provided high survival rates of bovine IVP blastocysts. The location of the embryos loaded into a narrow or wide portion was considered to be a limiting factor to the viability of bovine IVP embryos.

Chromatin, microtubule and microfilament configurations in the canine oocyte

In the present study, we observed chromatin, microtubule and microfilament distribution in canine oocytes. The germinal vesicle (GV) chromatin of canine oocytes was classified into four configurations (GV-I, -II, -III and -IV) based on the degree of chromatin separation and condensation. Oocytes recovered from follicular phase ovaries had a greater amount (68%, P < 0.05) of GV-III or GV-IV chromatin than did those from non-follicular phase ovaries (35%). The majority (86.7%) of in vivo ovulated oocytes were at GV-IV. The rates of development to GV breakdown/metaphase I/metaphase II were higher in oocytes recovered from follicular ovaries than from non-follicular ovaries. Immunostaining results revealed cytoplasmic microtubules present in all GV-stage oocytes. Following GV breakdown, microtubular asters were produced from condensed chromatin. The asters appeared to be elongated, and encompassed condensed chromatin particles to form meiotic metaphase chromatin. Microfilaments were located in the cortex and around the GV. During meiotic maturation, a microfilament-rich area, in which the chromatin is allocated, was observed in the oocyte. Our results indicate that oocytes recovered from follicular ovaries were in an advanced stage of GV, and were more competent to complete maturation compared to those from non-follicular phase ovaries. Both microtubules and microfilaments are closely associated with reconstruction of chromatin during meiotic maturation in canine oocytes.

Effect of crude canine pituitary extract (cCPE) on the in vitro production of progesterone and nuclear maturation of canine oocytes

We examined the effect of crude canine pituitary extract (cCPE) on the in vitro nuclear maturation of canine oocytes and production of progesterone by cumulus cells. cCPE was extracted from canine pituitaries and the concentrations of canine follicle stimulating hormone (FSH) and luteinizing hormone (LH) were determined. Cumulus oocyte complexes (COCs) were harvested from anestrus cycle ovaries and matured in NCSU-37 supplemented with 10% estrus bitch serum, 50 µg/ml gentamycin and 0, 40 or 400 µg/ml cCPE at 38°C in a humidified atmosphere of 5% CO2 for 72 h. The nuclear maturation of the oocytes and the level of progesterone in the culture medium were evaluated. Development to metaphase I (MI) - metaphase II (MII) of canine oocytes in 400 µg/ml cCPE (15.4%) was significantly higher than in 0 and 40 µg/ml cCPE (4.3 and 8.7%), respectively. Treatment with 40 and 400 µg/ml cCPE also generated 0.33 and 0.65 ng/ml progesterone in the culture medium, respectively. Thus, the addition of cCPE to the culture medium promotes the nuclear maturation of canine oocytes and elevates the production of progesterone by cumulus cells.