High Throughput Cryopreservation of In Vivo-Derived Swine Embryos

描述（申请人提供）：目前的猪胚胎冷冻保存方法耗时且麻烦。猪胚胎冷冻保存最成功的方法之一是将细胞离心使脂质极化，然后使用显微操作器从胚胎中去除脂质（Nagashima et al., 1995; Nagashima et al., 1994）。有趣的是，去除脂质不仅可以使胚胎在低温下存活（Nagashima et al., 1999），而且在某些情况下，它们实际上以更高的速率发育到囊胚阶段（Lai et al., 2006; Li et al., 2006）。不幸的是，目前的手术会导致透明带破裂。国际胚胎移植协会的指导方针指出，如果要保持健康状态，就不能损害透明带（Stringfellow， 1998年；Wrathall和Sutmoller， 1998年）。这对于胚胎被冷冻和运输到另一个设施尤其重要，因为猪通常在特定的病原体环境中饲养，病原体的移动会对接收猪群产生破坏性影响。在此背景下，我们制定了以下论文。

DESCRIPTION (provided by applicant): The current methods for cryopreservation of swine embryos are time consuming and troublesome. One of the most successful methods of cryopreservation of swine embryos is to centrifuge the cells to polarize the lipids and then use a micromanipulator to remove the lipids from the embryo (Nagashima et al., 1995; Nagashima et al., 1994). Interestingly, removal of the lipids not only permits the embryos to survive low temperatures (Nagashima et al., 1999), but in some cases they actually develop to the blastocyst stage at a higher rate (Lai et al., 2006; Li et al., 2006). Unfortunately with current procedures this results in a break in the zona pellucida. The International Embryo Transfer Society guidelines state that the zona pellucida cannot be compromised if the health status is to be maintained (Stringfellow, 1998; Wrathall and Sutmoller, 1998). This is especially important for embryos that are to be frozen and transported to another facility, as swine are generally raised in specific pathogen environments and the movement of pathogens can have a devastating effect on the recipient herd. With this background we have formulated the following thesis. Thesis: Lipids can be efficiently removed from swine embryos without damaging the zona pellucida. Such embryos can survive the rigors of cryopreservation. This leads to a single Specific Aim, with multiple sub-Aims. Specific Aim. Develop a non-invasive method for relatively high-throughput cryopreservation of swine embryos. Our aim is to develop a non-invasive "relatively" high-throughput method for lipid removal prior to cryopreservation. Our first sub-aim is to confirm that the amount of space in the zona pellucida relative to the zygote cytoplasm can be increased so that complete separation of the lipids and the zygote cytoplasm can be achieved by centrifugation. We propose to determine how sensitive the in vivo-produced embryo is to various osmolality conditions for a short duration. The second sub-aim will be to use the added space within the zona pellucida, caused by high osmolality treatment, to aid in completely separating the lipids within the zona pellucida (via centrifugation) prior to cryopreservation. Viability after warming will be determined. Finally, as an extension of these in vitro studies embryo transfers will be performed to assess the true viability of these cryopreserved embryos. PUBLIC HEALTH RELEVANCE (provided by applicant): The results from these studies will help to improve the resources that are needed to understand human health by making it easier to work with swine as models of human disease.