Development Of Frozen-thawed Embryos Research Articles

In vitro production of embryos from high performance cows and the development of frozen-thawed embryos after transfer: a field study

In this field study, embryos were derived from genetically highly valuable cows excluded from breeding due to reproductive disorders. Cows, 5 to 10 years old, of Czech Siemmental, Holstein Dairy and Beef Cattle breeds were used as oocyte donors. Oocytes were obtained either in the growth phase of the first follicular wave from cows with synchronized oestrus or in any other phases of follicular development from cows without oestrus synchronization. The embryos were prepared by a standard protocol described previously. The mean number of usable oocytes, transferable and freezable embryos per donor, and the mean percentage of usable, transferable and freezable embryos were assessed. The results were analyzed by Student’s-<I>t</I> and Chi-squared tests. The embryos were frozen according to a slow freezing protocol. After thawing, they were transferred to recipients on Day 7 after oestrus. Irrespective of the breed, the mean numbers of usable oocytes and transferable and freezable embryos collected per donor were significantly higher (<I>P</I> < 0.01) for the synchronized than for the nonsynchronized donors (20.4 vs 11.7, 4.3 vs 1.0 and 3.2 vs 0.8, respectively). Similarly, the mean percentages of usable oocytes, transferable and freezable embryos were significantly higher (<I>P</I> < 0.01) for the synchronized than for the nonsynchronized donors (28.5% vs 20.5%, 20.9% vs 9.0% and 15.8% vs 6.5%, respectively). On comparison of the synchronized and nonsynchronized donors of each breed, the difference in the mean percentage of usable oocytes was significant (<I>P</I> < 0.01) in cows of all three breeds, the difference in the mean percentage of transferable embryos was significant in Czech Siemmental and Holstein Dairy cows (<I>P</I> < 0.01) and the difference in the mean percentage of freezable embryos was significant only in Holstein Dairy cows (P < 0.01). After the transfer of 41 frozen-thawed embryos and 43 fresh embryos, 20 heifers and 24 heifers became pregnant, respectively. In conclusion: (a) higher number of oocytes from infertile, genetically valuable cows was recovered in the growth phase compared with the other phases of follicular development; (b) greater development of these oocytes resulted in more embryos for transfer and cryopreservation; (c) the transfer of frozen-thawed and fresh embryos resulted in pregnancy rates of 48.8% and 55.8% , respectively.

In vitro maturation--n vitro fertilization--embryo transfer--frozen-thawed of human oocytes

To investigate the influence of different cycles, ovarian follicle size and IVM culture media on the number of retrieved immature oocytes, maturation rate, fertilization rate, embryo quality and implantation rate, pregnancy rate, delivery rate, survival and development of frozen-thawed embryos from IVM. The oocytes were obtained by follicular aspiration from 19 women undergoing oocyte retrieval for in vitro maturation due to the possible risk of ovarian hyperstimulation in IVF-ET program. One patient was in natural cycle, four patients were in ovulation induction cycles with gonadotropine and fourteen patients is controlled ovarian stimulated cycles. All the oocytes retrieved from follicles with 10.0 - 13.5 mm in maximumdiameter were allowed to culture in medium M-199 (TCM 199) or HTF supplemented with other substance. When there were nonuniform diameters of follicles and the diameter of largest oocyte exceeded 12 mm, the retrieval rate of oocytes, fertilization rate, and the number of high-quality embryos decreased. The high-quality embryos formation rate was higher for the oocytes cultured in TCM 199 medium than in HTF medium (P<0.01). After being frozen-thawed, the IVM embryos could achieve the same outcome when compared with the conventional IVF treatment. In addition, the offspring were healthy. When the nonuniform diameters of follicles and the diameter of largest oocyte exceeds 12 mm,the retrieval rate of oocytes, fertilization rate, and the number of high-quality embryos decreased. TCM199-based medium is better to improve the developmental potential and implantation rate of embryos derived from in vitro matured oocytes. After being frozen-thawed, the IVM embryos could achieve the same outcome when compared with the conventional IVF treatment. In addition, the offspring are healthy.

Rapid freezing of the mouse blastocyst: effects of cryoprotectants and of time and temperature of exposure to cryoprotectant before direct plunging into liquid nitrogen

This study investigates the effects of time and temperature of exposure to a high concentration (4.5 M) of dimethyl sulfoxide (DMSO), glycerol, 1,2-propanediol (PROH), or a mixture of DMSO and glycerol (DG) in a solution containing 0.25 M sucrose, on the survival and development of rapidly frozen mouse blastocysts. Embryos had significantly (P less than 0.01) higher rates of survival and development when exposed to cryoprotectant at 0 degree C compared with room temperature. The time of exposure to cryoprotectant at either 0 degree C or room temperature before being plunged into liquid nitrogen significantly (P less than 0.01) affected the survival and development of frozen-thawed embryos. Survival and development of blastocysts in vitro and in vivo was significantly (P less than 0.05) higher when exposed at 0 degree C for 10 min to DG, DMSO and glycerol than to PROH. It is concluded that, unlike early-cleavage stage embryos, blastocysts need to be equilibrated at a low temperature (0 degree C) with high concentrations of cryoprotectant before rapid freezing. Exposure of blastocysts to 4.5 M cryoprotectant and 0.25 M sucrose at room temperature either was toxic or else markedly reduced their viability after freezing and thawing, depending on the duration of the initial exposure.

Cryopreservation of strains and mutant genes in mice.

Three kinds of freezing methods were tested with embryos of DNI strain. The survival rate after thawing was 47.5%, 66.7% and 77.8% in the 2-step method, modified slow freezing method and modified 2-step method, respectively. Then, the modified 2-step method was applied to the embryos from 7 strains and a pair of interstrain crosses. PMSG treatment at the beginning of diestrus following HCG treatment after 48 hrs resulted in much yield of 8-16-cell embryos in all strains. The average number for each strain was as follows: DNI; 18.9, DDN; 13.0, BS; 20.4, C57BL/6; 12.9, DBA/2; 17.5, CRN; 19.8, PAN; 13.7 and DNI x C57BL/6-Ay; 21.7. Development of frozen-thawed embryos in culture varied among strains. Proportion of embryos that developed to the morula or blastocyst stage was as follows: DNI; 64.6%, DDN; 71.9%, BS; 53.6%, C57BL/6; 57.3%, DBA/2; 65.0%, CRN; 52.5%, PAN; 17.4% and DNI x C57BL/6-Ay; 44.1%. These results indicate that the ability of embryos to survive freezing and thawing is influenced by their genetic background. Live young were produced from DNI, DDN, BS and DNI x C57BL/6-Ay embryos after transfer to recipients. Comparative assessment of the developmental ability of frozen-thawed embryos after transfer among strains should be performed in further study.