High Throughput Cryopreservation of In Vivo-Derived Swine Embryos

描述（申请人提供）：目前猪胚胎的冷冻保存方法耗时且麻烦。冷冻保存猪胚胎的最成功的方法之一是离心细胞以除去脂质，然后使用显微操作器从胚胎中除去脂质（Nagashima等人，1995; Nagashima等人，1994年）。有趣的是，脂质的去除不仅允许胚胎在低温下存活（Nagashima等人，1999），但在某些情况下，它们实际上以更高的速率发育到胚泡阶段（Lai等人，2006; Li等人，2006年）。不幸的是，在目前的程序中，这会导致透明膜破裂。国际胚胎移植学会指南指出，如果要保持健康状态，则不能损害透明卵母细胞（Stringfellow，1998; Wrathall和Sutmoller，1998）。这对于要冷冻并运输到另一个设施的胚胎尤其重要，因为猪通常在特定的病原体环境中饲养，病原体的移动可能对受体猪群产生破坏性影响。在这一背景下，我们提出了以下论点。 论文：脂质可以有效地从猪胚胎中去除，而不损害透明细胞。这样的胚胎可以在冷冻保存的严酷条件下存活下来。这导致了一个单一的具体目标，多个子目标。具体目标。开发一种非侵入性的猪胚胎高通量冷冻保存方法。 我们的目标是开发一种非侵入性的“相对”高通量的方法，用于在冷冻保存前去除脂质。我们的第一个子目标是确认相对于受精卵细胞质的透明囊中的空间量可以增加，使得可以通过离心实现脂质和受精卵细胞质的完全分离。我们建议确定体内产生的胚胎在短时间内对各种渗透压条件的敏感程度。第二个子目标将是使用由高渗透压处理引起的透明膜内的增加的空间，以帮助在冷冻保存之前完全分离透明膜内的脂质（通过离心）。将测定升温后的活力。最后，作为这些体外研究的延伸，将进行胚胎移植以评估这些冷冻保存胚胎的真实存活力。 公共卫生相关性（由申请人提供）：这些研究的结果将有助于改善了解人类健康所需的资源，使猪更容易作为人类疾病模型。

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.