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既费时又麻烦。冷冻保存猪胚胎最成功的方法之一是对细胞进行离心，使脂肪极化，然后使用显微操作器从胚胎中去除脂肪(Nagashima等人，1995；Nagashima等人，1994)。有趣的是，去除脂质不仅可以使胚胎在低温下存活(Nagashima等人，1999)，而且在某些情况下，它们实际上以更高的速度发育到囊胚期(Lai等人，2006；Li等人，2006)。不幸的是，在目前的手术中，这会导致透明带破裂。国际胚胎移植协会的指导方针指出，如果要保持健康状态，透明带就不能受到损害(StringFloor，1998；Wrathall和Sutmoller，1998)。这对将被冷冻并运输到另一家机构的胚胎尤其重要，因为猪通常在特定的病原体环境中饲养，病原体的移动可能会对受体牛群产生毁灭性的影响。在此背景下，我们形成了以下论文。 论文：可以有效地去除猪胚胎中的脂肪，而不会破坏透明带。这样的胚胎可以在严酷的冷冻保存中存活下来。这导致了单一的具体目标，具有多个子目标。明确目标。发展一种相对高通量的猪胚胎非侵入性冷冻保存方法。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肪。我们的第一个次目标是确认透明带相对于受精卵细胞质的空间可以增加，从而通过离心法实现脂质和受精卵细胞质的完全分离。我们建议在短时间内确定体内产生的胚胎对各种渗透压条件的敏感性。第二个子目标将是利用高渗透压处理引起的透明带内增加的空间，在冷冻保存之前帮助完全分离透明带内的脂类(通过离心法)。变暖后的生存能力将被确定。最后，作为这些体外研究的延伸，将进行胚胎移植，以评估这些冷冻胚胎的真实生存能力。 公共卫生相关性(由申请者提供)：这些研究的结果将通过使将猪作为人类疾病模型进行研究变得更容易，从而有助于改善了解人类健康所需的资源。

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.