Development of Embryo Cryopreservation Procedures for Laboratory Rats

美国国立卫生研究院动物遗传资源（NIHAGR）保存了许多独特的大鼠基因型，可用作人类正常和疾病过程的模型。 为了建立实验大鼠冷冻保存的种质库，必须建立胚胎库和再衍生过程的每个步骤的方案。这些将包括胚胎供体雌性的准备和交配、胚胎收集、冷冻保存、解冻和胚胎移植。 以前的研究表明，为实验室小鼠开发的超排卵程序在应用于实验室大鼠时并不有效。 本项目的主要目的是评价促性腺激素释放激素（GnRH-a）类似物作为同步发情周期的替代方法进行胚胎收集和冷冻保存的治疗。迄今为止，我们的努力已经产生了世界上最大的冷冻保存大鼠胚胎库。总共有大约58，000个胚胎从109个大鼠品系和种群中冷冻保存。 70个大鼠品系和种群仅作为库存胚胎存在。胚胎收集的效率在模型之间以类似于模型的育种性能的方式变化。然而，迄今为止，在开发所有大鼠模型的冷冻胚胎库方面取得了进展。研究人员最近提出了四项要求，要求重新建立仅以冷冻胚胎存在的大鼠模型，这证明了大鼠胚胎库的有用性。对于LER/N、LEW/N-op（骨硬化症）和LEW/N-tl（无牙）三种模型，分别在储存75、128和179个胚胎后解散基础集落。在第四种情况下，M520/N-lr（瘦肉型），在基础集落解散之前建立了367个胚胎的“最小库”。然而，解冻和转移后分别获得了23、94、55和76个胚胎，以恢复育种者重新建立基础菌落。在每种情况下，建立一个银行的三个标准已经得到满足：幼崽是从银行的大鼠胚胎再衍生，再衍生的幼崽表现出所需的基因型，再衍生的幼崽已经繁殖和生产幼崽。这四个模型的胚胎库达到我们的最终标准，500个库存胚胎，几乎完成。

The NIH Animal Genetic Resource (NIHAGR) maintains many unique rat genotypes used as models of normal and disease processes in man. Effective management of this resource would be facilitated by the development of embryo cryopreservation procedures for long-term banking. In order to develop banks of cryopreserved germ plasm from laboratory rats, protocols for each step of the embryo banking and rederivation process must be established. These would include the preparation and mating of embryo donor females, embryo collection, cryopreservation, thawing, and embryo transfer. Previous studies suggest that superovulation procedures developed for laboratory mice are not as effective when applied to laboratory rats. The principal objective of this project is to evaluate treatment with an analog of gonadotropin releasing hormone (GnRH-a) as an alternative approach to synchronize estrus cycles for embryo collection and cryopreservation. Our efforts to date have yielded the world's largest bank of cryopreserved rat embryos. A total of approximately 58,000 embryos have been cryopreserved from 109 rat strains and stocks. Seventy rat strains and stocks exist solely as banked embryos. The efficiency of embryo collection varies between models in a manner similar to that of the breeding performance of the model. However, progress has been made in developing banks of cryopreserved embryos from all rat models examined to date. Four recent requests by investigators to reestablish rat models that existed only as frozen embryos demonstrate the usefulness of rat embryo banking. For three of the models, LER/N, LEW/N-op (osteopetrosis), and LEW/N-tl (toothless), the foundation colonies were disbanded after, respectively, 75, 128, and 179 embryos were banked. In the fourth case, M520/N-lr (leaner), a "minimal bank" of 367 embryos was established before the foundation colony was disbanded. Nevertheless, sufficient numbers of pups were obtained after thawing and transfer of, respectively, 23, 94, 55, and 76 embryos to recover breeders to reestablish foundation colonies. In each case, three of the criteria for establishing a bank have been met: pups were rederived from banked rat embryos, rederived pups exhibited the desired genotype, and the rederived pups have bred and produced pups. Embryo banking of these four models to reach our final criteria, 500 banked embryos, is nearly complete.