Various sample containers have been developed for ultra-rapid vitrification of oocytes and embryos to minimize chilling and other injuries. In this study, the survival rates of in vitro-derived porcine oocytes were compared after cryopreservation using open pulled straw (OPS), electron microscope grid (EMG), and nylon loop system (NLS). In addition, the
post-thaw survival of porcine morulae and blastocysts was assessed after vitrification by the OPS method. In vitro maturation and fertilization were performed according to the procedures of Funahashi et al., 1994 Theriogenology 41, 1425–1433). Fertilized oocytes were cultured in glucose-free NCSU 23
supplemented with 5mM sodium pyruvate, 0.5mM sodium lactate and 4mgmL−1 bovine serum albumin for 2 days at 39°C; 10% fetal bovine serum was added to the culture medium thereafter. Oocytes and embryos were treated with 7.5μgmL−1 cytochalasin B for 30min, centrifuged at 13,000g for 13min and then exposed sequentially to an ethylene glycol (EG) vitrification solution (0M for 1min, 2M for 5min and 8M plus 7% polyvinylpyrrolidone for 1min).
Oocytes (n=100per treatment group) were placed in or on sample containers and plunged into liquid nitrogen. Porcine morulae and blastocysts (n=137) were similarly treated, aspirated into OPSs, and plunged into liquid nitrogen. Oocytes and embryos were thawed rapidly by transferring the sample container into 1M EG for 2min, and then the embryos were serially diluted by transfer into 0.5M for 2min, and medium for 5min. Post-thaw survival of vitrified oocytes was assessed by development after IVF and culture to morulae or blastocysts. Post-thaw survival of vitrified morulae and blastocysts was assessed by their ability to continue development to, respectively, blastocysts and expanded blastocysts. After oocytes were thawed, fertilized and cultured in vitro, the rates of development to the morula- or blastocyst-stage were 8%, 0% and 6% for, respectively, the OPS, EMG and NLS groups. The rate of development of non-cryopreserved control oocytes was 38% (38/100). Although significantly fewer oocytes developed after vitrification compared to the control (P<0.05), OPS and NLS containers were superior to EMG containers (P<0.05). Morula- or blastocyst-stage embryos vitrified using OPS yielded a significantly higher rate of survival than did oocytes (48%, 66/137; P<0.05). This study indicates that very few in vitro-matured porcine oocytes survive vitrification using OPS, EMG and NLS methods. Considerably higher rates of survival were obtained by in vitro-produced porcine morulae and blastocysts following vitrification by the OPS method. Additional research is needed to identify and develop methods to overcome the factors limiting the cryopreservation of porcine oocytes for practical uses. Supported by KOSEF (R05-2002-000-00388-0) grant.
The fibrillar substructure of keratin filaments unraveled.
