Fujino Yukihiro, Kikuchi Kazuhiro, Nakamura Yoshiyuki, Kobayashi Hiroshi, Yonemura Isao, Suzuki Misae, Misumi Koji, Nagai Takashi
Saitama Prefectural Agriculture and Forestry Research Center, Livestock Research Center, Kohnan Saitama 360-0102, Japan.
Theriogenology. 2007 Jan 15;67(2):413-22. doi: 10.1016/j.theriogenology.2006.08.008. Epub 2006 Sep 20.
The viability or developmental ability of porcine embryos after slow-freezing and thawing differs depending on the embryonic stage or the batch, which is defined as a group of embryos obtained from one donor at one time. We froze porcine blastocysts in batches and assessed their cryotolerance by using two expanded blastocysts (EBs) as samples to predict the developmental potential of other blastocysts from the same batch at different stages. Two EBs from the same batch that had been separately frozen were thawed and cultured in vitro for 48 h to examine their in vitro ability to develop to the hatched blastocyst stage. Thereafter, each batch was assigned to Grade A, B, or C according to the viability of the two EBs, i.e., 100% viability (2/2: number of hatched blastocysts/number of cultured EBs) was Grade A; 50% (1/2) was Grade B; and 0% (0/2) was Grade C. The viability of EBs after freeze-thawing and in vitro culture varied depending on the batch and was lower (31.0+/-10.2%, mean+/-S.E.M.; P<0.01) than that of unfrozen controls (96.8+/-2.3%). The viability of frozen-thawed hatched blastocysts (HBs) did not differ among the graded batches, but the blastocyst diameter decreased (from 409 to 326 microm) as the batch grade decreased (from A to C). When both EBs and HBs from batches of the same grade were transferred to recipients (average 11.7 EBs and 16.0 HBs per recipient), the rate of pregnancy and farrowing in recipients decreased (from 77.8% to 0%) and the number of piglets obtained decreased (from 15.3 to 0) as the batch grade decreased. However, when not only frozen-thawed EBs from Grade B or C batches, but also four helper embryos at the morula to early blastocyst stage (which were expected to support the pregnancy) were transferred, the number of piglets generated was higher from EBs from Grade B batches (16.0) than from EBs from Grade C batches (0.0). When frozen-thawed HBs and helper embryos were transferred, the number of piglets generated was higher from HBs from Grade B batches (12.7) than that from HBs from Grade C batches (1.9). After slow-freezing of porcine blastocysts, their rate of survival to the piglet stage differs batchwise, and in vitro viability assessment of sample EBs after freezing and thawing may help in assessing the post-freezing and post-thawing developmental potential of other blastocysts at different stages from the same batch.
猪胚胎经慢速冷冻和解冻后的活力或发育能力因胚胎阶段或批次而异，批次是指一次从一个供体获得的一组胚胎。我们将猪囊胚分批冷冻，并以两个扩张囊胚（EBs）作为样本评估其耐冻性，以预测同一批次不同阶段其他囊胚的发育潜力。将同一批次分别冷冻的两个EBs解冻并在体外培养48小时，以检查它们在体外发育至孵化囊胚阶段的能力。此后，根据两个EBs的活力将每个批次分为A、B或C级，即活力为100%（2/2：孵化囊胚数/培养的EBs数）为A级；50%（1/2）为B级；0%（0/2）为C级。冻融和体外培养后EBs的活力因批次而异，且低于未冷冻对照组（96.8±2.3%）（31.0±10.2%，平均值±标准误；P<0.01）。冻融后孵化囊胚（HBs）的活力在分级批次之间没有差异，但随着批次等级从A降至C，囊胚直径减小（从409微米降至326微米）。当将同一等级批次的EBs和HBs都移植到受体中时（每个受体平均11.7个EBs和16.0个HBs），随着批次等级降低，受体的妊娠率和产仔率下降（从77.8%降至0%），获得的仔猪数量减少（从15.3头降至0头）。然而，当不仅移植B级或C级批次的冻融EBs，还移植四个桑椹胚至早期囊胚阶段的辅助胚胎（预期可支持妊娠）时，B级批次EBs产生的仔猪数量（16.0头）高于C级批次EBs（0.0头）。当移植冻融的HBs和辅助胚胎时，B级批次HBs产生的仔猪数量（12.7头）高于C级批次HBs（1.9头）。猪囊胚经慢速冷冻后，其存活至仔猪阶段的比率因批次而异，冻融后样本EBs的体外活力评估可能有助于评估同一批次不同阶段其他囊胚冻融后的发育潜力。
