No abstract

Abstract. We conducted two experiments to investigate the effects of storage temperature and period for cat ovaries on the meiotic and developmental competence of oocytes collected from the ovaries. In Experiment 1, ovaries were stored in physiological saline for 24 h at 4 C, 23-25 C, or 38 C (cold, room, and incubator temperature groups, respectively), and then oocytes were collected from the ovaries in each group. Morphologically intact oocytes were then selected and cultured in maturation medium for 24 h. Significantly more oocytes reached metaphase II (MII) in the cold temperature group (53.4%) than in the room and incubator temperature groups (20.0 and 2.4%, respectively). In Experiment 2, ovaries were stored in physiological saline at room temperature for 0, 6, 12 or 18 h, and then they were stored at 4 C (cold storage) until reaching a total storage period of 24 h. After storage of the ovaries, morphologically intact oocytes were matured, fertilized with frozen-thawed spermatozoa, and cultured in vitro. The rates of morphologically intact oocytes obtained from the ovaries stored at room temperature for 0, 6, 12 or 18 h were 35.3, 30.0, 26.4 and 14.7%, respectively, and the rates of intact oocytes that reached MII were 63.2, 36.4, 26.5 and 11.9%, respectively. The results suggested that the numbers of morphologically intact oocytes and intact oocytes that reached MII after in vitro maturation decrease gradually as the period of storage at room temperature before cold storage increases. Only oocytes from ovaries stored for 6 h developed to the blastocyst stage after in vitro maturation and fertilization when ovaries were stored at room temperature before cold storage. These results indicate that 24 h storage of ovaries at high temperatures (>23 C) decreases the meiotic competence of oocytes. The quality and developmental competence of oocytes are influenced by the length of storage at room temperature before cold storage.

Abstract. In this study the efficacy of the combination of glycerol (GLY) and ethylene glycol (EG) as cryoprotectants in a vitrification method developed for direct embryo transfer was evaluated by in vitro development of in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos after vitrification. The IVF and SCNT blastocysts were vitrified in either 40% GLY, 30% GLY + 10% EG, or 20% GLY + 20% EG using French straws. After warming, the straws were held vertically for 1 min without shaking and were then placed horizontally for 5 min to dilute the cryoprotectants. After washing, the vitrified-warmed embryos were cultured in vitro for 72 h. There were no differences among the vitrification solutions with respect to the rates of vitrified-warmed IVF and SCNT embryos surviving and developing to the hatched blastocyst stage. However, the rates of development to the hatched blastocyst stage of the SCNT embryos vitrified with 40% GLY tended to be higher than those vitrified with 30% GLY + 10% EG or 20% GLY + 20% EG (26% vs. 7-8%, respectively). The development rates to the hatched blastocyst stage of the IVF and SCNT embryos vitrified with solution containing EG were significantly lower (P<0.05) than those of non-vitrified embryos. These results suggest that use of the combination of GLY and EG as cryoprotectants had no beneficial effect on the viability of embryos after in-straw dilution. However, this method is so simple that it can be used for practical direct transfer of vitrified embryos in the field.

Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis) and is known for its antioxidant effects. The objective of the present study was to examine the effects of EGCG during in vitro fertilization (IVF) on the sperm quality and penetrability into oocytes. In the first experiment, the effects of concentration and incubation period of EGCG on the motility and penetrability of spermatozoa were examined. When frozen-thawed spermatozoa were incubated in IVF medium supplemented with 0 (control), 1, 50 and 100 μm EGCG for 1, 3 and 5 h, supplementation with 50 and 100 μm EGCG improved motility of the spermatozoa (p < 0.05), but not viability, as compared with the control group. When frozen-thawed spermatozoa were co-incubated with in vitro-matured (IVM) oocytes in IVF medium supplemented with 50 and 100 μm EGCG for 5 h, supplementation of EGCG had positive effects on sperm penetration rates. In the second experiment, the effects of supplementation of EGCG in IVF medium on penetrability of sperm from different boars and development of fertilized oocytes were evaluated. When frozen-thawed spermatozoa from six boars were co-incubated with IVM oocytes in IVF medium supplemented with 50 μm EGCG, the effect of EGCG on sperm penetration and development of oocytes after fertilization was found to vary with individual boar. Our results indicate that motility and penetrability of boar spermatozoa are improved by co-incubation with 50 μm EGCG, but the effects vary with individual boars.

The present study was conducted to examine the meiotic competence of canine oocytes embedded in collagen gel, and to investigate the effects of timed exposure of the oocytes embedded in collagen gel to gonadotrophins during maturation culture, on their nuclear maturation. Cumulus-oocyte complexes (COCs) were collected from bitches at the anoestrous and dioestrous stages of the reproductive cycle. In the first experiment, half of the COCs were embedded in collagen gels. The COCs with or without collagen-gel embedding were cultured in a TCM-199 medium supplemented with 0.1 IU/ml human menopausal gonadotropin (hMG) and 10 IU/ml human chorionic gonadotropin (hCG) for 72 h. In the second experiment, the COCs embedded in collagen gels were cultured in TCM-199 medium with gonadotrophins (hMG and hCG) for various periods (0, 24, 48 and 72 h) and then cultured in the medium without gonadotrophins until reaching total culture period (72 h). The percentage of the oocytes reaching metaphase I and metaphase II (MI/MII) was significantly higher (p < 0.05) in COCs with collagen-gel embedding than in COCs without collagen-gel embedding. The percentage of oocytes that were arrested at the germinal vesicle stage was significantly lower (p < 0.05) in oocytes cultured with gonadotrophins than in oocytes cultured without gonadotrophins. However, there were no significant differences in the percentages of oocytes that reached each stage of meiosis among the groups, irrespective of the duration of exposure to gonadotrophins. These observations indicate that embedding of COCs by collagen gel enhances the meiotic competence of canine oocytes, but removal of hormone supplement from maturation medium does not improve the ability of the oocytes to reach MII stage.