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This study was conducted to examine the utility of vitrification for bovine embryos with low-quality grade, and simple cryoprotectants dilution method for practitioners. In Experiment 1, survival of frozen embryos was compared with that of vitrified embryos using minimum volume cooling (MVC). Then, vitrified embryos were used to confirm the optimum sucrose concentration in Experiment 2. The survival rates of embryos that had been vitrified following diluted cryoprotectants with the one-step in-straw method were compared with those of fresh control embryos in Experiment 3. Frozen-thawed or vitrified-warmed blastocysts were cultured with TCM-199 supplemented with 100 μmol/L beta-mercaptoethanol +5% fetal bovine serum at 38.5°C in an atmosphere of 5% CO 2 in air, their survival after 24 hr were compared. The development to term of fair quality in vivo embryos after vitrification was examined in Experiment 4. Results show that survival rates of frozen-thawed embryos were lower (p < .05) than that of vitrified-warmed ones. When vitrified embryos were warmed in 0.3 mol/L sucrose in straws, their survival rate was 100%. The total cell numbers of vitrified-warmed embryos were comparable to those of fresh control embryos. The six calves from 13 vitrified embryos were delivered in Experiment 4. These results indicate that MVC vitrification following one-step cryoprotectants dilution is utilized to preserve low-quality bovine embryos., (© 2018 Japanese Society of Animal Science.)

In the last 15-20 years, many centers are using vitrification as a method of choice for cryopreservation of human oocytes and embryos. As vitrification technologies have improved their success profiles, new applications seem to have emerged, making IVF treatments more successful and more flexible.This appendix describes the Cryotech ® method, which is the latest "minimal volume approach" method, suitable for cryopreservation of oocytes and embryos of any developmental stage, including blastocysts. Dr. Masashige Kuwayama, who has introduced major advances in oocyte and embryo cryopreservation, has developed this method. A detailed protocol has been described with finer tips for the accurate use of the method for perfect survival and safety.

Abstract: We evaluated: (1) cleavage rate after IVF or intracytoplasmic sperm injection (ICSI) of in vivo- and in vitro-matured oocytes after vitrification (experiment 1); and (2) fetal development after transfer of resultant ICSI-derived embryos into recipients (experiment 2). In vivo-matured cumulus-oocyte complexes (COCs) were recovered from gonadotropin-treated donors at 24 h after LH treatment. In vitro-matured oocytes were obtained by mincing ovaries (from local veterinary clinics) and placing COCs into maturation medium for 24 h. Mature oocytes were denuded and cryopreserved in a vitrification solution of 15% DMSO, 15% ethylene glycol, and 18% sucrose. In experiment 1, for both in vivo- and in vitro-matured oocytes, cleavage frequencies after IVF of control and vitrified oocytes and after ICSI of vitrified oocytes were not different (P > 0.05). After vitrification, blastocyst development occurred only in IVF-derived, in vitro-matured oocytes. In experiment 2, 18 presumptive zygotes and four two-cell embryos derived by ICSI of vitrified in vitro-matured oocytes and 19 presumptive zygotes produced from seven in vivo- and 12 in vitro-matured oocytes were transferred by laparoscopy into the oviducts of two recipients, respectively. On Day 21, there were three fetuses in one recipient and one fetus in the other. On Days 63 and 66 of gestation, four live kittens were born. In vivo viability of zygotes and/or embryos produced via ICSI of vitrified oocytes was established by birth of live kittens after transfer to recipients. [Copyright &y& Elsevier]

Abstract: Recently, a non-invasive delipation (lipid removal) method combined with ultrarapid vitrification has been used successfully for in vitro produced (IVP) porcine embryos. In the present study, this method was combined with parthenogenesis and a recent form of somatic cell nuclear transfer (SCNT)—handmade cloning (HMC)—to establish a simplified and efficient cryopreservation system for porcine cloned embryos. In Experiment 1, zonae pellucidae of oocytes were partially digested with pronase, followed by centrifugation to polarize lipid particles. Ninety percent (173/192) oocytes were successfully delipated in this way. Parthenogenetic activation (PA) after complete removal of zona resulted in similar blastocyst rates in delipated vs. control oocytes (28±7% vs. 28±5%, respectively). Subsequent vitrification of produced blastocysts with the Cryotop technique resulted in higher survival rates in the delipated group compared to the control group (85±6% vs. 32±7%, respectively; P <0.01). In Experiment 2, delipated oocytes were used for HMC with normal oocytes as control. Partial zona digestion was further applied before enucleation both in delipated and control groups, to bisect oocyte successfully. Although the blastocyst rate of reconstructed embryos was similar between groups derived from delipated vs. control oocytes (21±6% and 23±6%, respectively), after vitrification higher survival rates were achieved in the delipated groups than in controls (79±6% vs. 32±8%, respectively). Our results prove that porcine embryos produced from delipated oocytes by PA or HMC can be cryopreserved effectively by ultrarapid vitrification. Further experiments are required to assess the in vivo developmental competence of the cloned-vitrified embryos. [Copyright &y& Elsevier]