Ultra-structural changes and developmental potential of porcine oocytes following vitrification.

This study evaluated the effects of exposure and/or vitrification of porcine metaphase II (MII) oocytes on their in vitro viability and ultra-structural changes with two experiments. Experiment 1 examined the effect of vitrified oocytes on microtubule localization, mitochondrial morphology, chromosome organization and the developmental rate in IVF control and vitrified oocytes. Oocytes matured for 44 h were subjected to IVF (IVF control). Oocytes matured for 42 h were exposed to cryoprotectants (CPA control), followed by 2h culture, and subjected to IVF. Oocytes vitrified at 42 h post-maturation were warmed, cultured for 2h, and subjected to IVF (vitrified). Experiment 2 evaluated the effect of oocytes freezing on development of ICSI with and without activation and parthenotes. Fresh and vitrified oocytes were subjected to ICSI with and without electrical activation. Cleavage and blastocyst rates were significantly (P<0.05) lower in vitrified IVF, parthenote and ICSI embryos than those in fresh counterparts. Between ICSI embryos from fresh oocytes and vitrified oocytes, the rates of blastocyst were significantly higher (P<0.05) in activated group than the group without activation. Significant differences (P<0.05) were observed in normal spindle configuration of vitrified (43.5%) compared to control (81.0%) oocytes, but no significant difference was observed between CPA exposed and control groups. In conclusion, porcine oocytes at MII stage are very sensitive to vitrification with altered microtubule localization and mitochondrial organization thus resulting in impaired fertilization and embryo development.

Influence of in vitro oxygen concentrations on preimplantation embryo development, gene expression and production of Hanwoo calves following embryo transfer.

This study evaluated the effects of two different oxygen (O2) concentrations on in vitro embryo development, embryo quality, and gene expression and the in vivo development following embryos transfer to recipients of natural and synchronized estrus in bovines. Cumulus oocyte complexes were in vitro matured in TCM199 supplemented with FSH (10 microg/ml), LH (10 microg/ml), and 10% (v/v) FBS. Presumptive zygotes were cultured in SOF medium either under 5% (low) or 20% (high) O2 in air. Cleavage rates did not differ between groups. Blastocyst and hatched blastocyst development in 5% O2 were significantly (P < 0.05) higher than in 20% O2. Total cell number of in vivo blastocyst was significantly (P < 0.05) higher than that of in vitro blastocyst. ICM ratio and apoptosis of in vivo blastocyst were significantly (P < 0.05) lower than that of in vitro blastocyst. Using real time PCR, we have found that for the set of genes (GLUT-1, MnSOD, VEGF, Bax, and Bcl-2) analyzed, there were differences in mRNA expression between in vitro produced (IVP) and in vivo produced embryos. Interestingly, the abundance of transcript for IFN-tau in IVP embryos produced under 5% O2 concentration was similar to in vivo counterparts. The pregnancy and twin rates of natural recipients were significantly (P < 0.05) higher than those of synchronized counterparts. No significant difference in the offspring sex was observed. In conclusion, low (5%) O2 concentration during IVC was beneficial for enhancing the embryo quality and recipients of natural estrus were more suitable than synchronized estrus for stable production of Hanwoo calves.

Development of cloned pig embryos by nuclear transfer following different activation treatments.

This study compared the effects of activation treatments on the development and ploidy of nuclear transferred (NT) pig embryos. After in vitro maturation of oocytes collected from the slaughterhouse, oocytes were enucleated and reconstructed by transfer of donor cells and fusion with three DC pulses (1.4 kV/cm, 20 musec). Oocytes were pulsed thrice electrically with 1.4 kV/cm for 20 musec and NT eggs were divided into three treatment groups: Group 1 (no further treatment), Group 2 (10 mug/mL cycloheximide [CHX], 3 hr), and Group 3 (1.9 mM 6-dimethylaminopurine [DMAP], 3 hr). All the eggs were cultured in sets of 30 in 60 muL drops of NCSU-23 supplemented with 4 mg/mL fatty acid free BSA, and compared for the rates of development and ploidy. The rates of cleavage, development, and total cell number of parthenotes in Group 3 were significantly (P < 0.05) higher than those in Groups 1 and 2. Cleavage rates of NT embryos in Group 1 were significantly (P < 0.05) lower than those in Groups 2 and 3 (73% vs. 81% and 82%, respectively). Development into blastocyst stage and total cell number of NT embryos in Group 3 were significantly (P < 0.05) higher than those in Groups 1 and 2. Although the embryos in Group 3 had higher development, approximately 58% of NT embryos evaluated were abnormal ploidy (6% haploidy, 9% polyploidy, and 42% mixoploid). The results suggested that although DMAP enhanced development and higher cell number, due attention should be paid to abnormal ploidy in pig NT embryos.

Intracytoplasmic sperm injection of frozen-thawed bovine oocytes and subsequent embryo development.

Oocyte cryopreservation and intracytoplasmic sperm injection (ICSI) are advantageous to expand their usefulness in genetic engineering. Oocytes matured for 22 hr were vitrified in droplets of cryoprotectants (3.2 M ethylene glycol (EG), 2.36 M dimethyl sulfoxide (DMSO), 0.6 M sucrose) on copper electron microscope (EM) grids. After being warmed, the oocytes were cultured in IVM medium for an additional 2 hr. Sperm treated with dithiothreitol were utilized for ICSI. Oocytes injected with sperm were activated by combination of ionomycin with cycloheximide (CHX). The ICSI oocytes were compared for the rates of pronuclear formation, development, cell number, and the ratio of ICM to those of fresh ICSI and IVF control. The proportion of 2PN formation was significantly higher in IVF control (Group 1) than those in other treated groups. Among the treated groups a significant lower 2PN formation was observed in IVF-frozen-thawed than in ICSI-fresh and frozen-thawed groups. Cleavage rates in IVF-frozen-thawed and ICSI-frozen-thawed groups were significantly lower than those of IVF control and ICSI-fresh groups. In ICSI groups, the rates of cleavage and blastocyst in fresh oocytes were significantly higher than in frozen-thawed. Development rates into blastocysts in the ICSI-fresh and frozen-thawed groups were significantly lower than that of IVF control. Total cell number was significantly lower in both frozen-thawed IVF and ICSI groups than those in IVF-control and ICSI-fresh groups. However, the rates of the remaining cells that were found in the ICM were significantly higher in both frozen-thawed IVF and ICSI than in the IVF-control and ICSI-fresh groups. The results indicated that frozen-thawed bovine oocytes were suitable for ICSI procedure.