CRYOPRESERVATION OF SPERM

这个项目的目标是开发一个可靠的协议，使用 冷冻保存的精子，以最低的成本生产最大数量的 每只雄鼠的幼鼠。以前已经分析过替代策略 选择我们的方法。冷冻保存中的一系列步骤 对循环进行了理论和实践的分析，以建立一个 可以用来测试实验变量的模板。同样， 分析了导致受精成功的步骤顺序 选择用于评估分层模式的精子状态的方法 检测冷冻保存引起的精子功能障碍。最终 试验性设计适应了早期的成功(即运气)但确保 我们将从最初的失败中吸取教训。响应面方法 将用于同时评估3-5个水平的 冻结率、解冻率、延伸剂及防冻剂组成 小鼠精子冷冻保存的成功率和重复性。这个 一个阶段的结果被用来计划更精细和更有针对性的 评估原始数据的子集(加上其他因素) 下一阶段。此方法提供了最有效的方法来 多精子冷冻保存最佳条件的建立 不同品系的老鼠。渐进式分析，选择遵循以下步骤 受精，包括：(A)通过化验初步筛选 精子活力和膜室(用流式细胞术)；(B) 一种测定松散精子-卵子结合的独特方法(C)精子穿透 透明带：(D)早期胚胎发育；(E)最终生产 一群幼崽。最佳协议的识别是交互的 流程，要求根据经验和化验结果做出是/否的决定 界定不明确的分界标准。我们(和其他任何人)都没有信心 “正确”的决定是在“截止值”是 已经成立了。我们的所有数据都将存储起来，以供定期检索和 项目期间的回顾分析。这个，我们可以退货 到我们的数据集(和我们的决策)，用总体知识武装起来 这一特定范例在保留受精潜力方面的成功。 这将允许我们在进化过程中学习，并做出更好的决定， 朝着成功的协议迈进。为这个项目组建的团队是 能够：(A)驱动将满足以下条件的交互程序 和(B)提供一个实验性的模板 有助于解决人类其他低温生物学问题的设计 医药和畜牧业。

The goal of this project is to develop a reliable protocol, using cryopreserved sperm, to produce, at minimum cost, the maximum number of mouse pups per male. Alternative strategies were analyzed before selecting our approach. The sequence of steps in a cryopreservation cycle, both theoretical and pragmatic, was analyzed to establish a template against which experimental variable can be tested. Likewise, the sequence to steps leading to successful fertilization was analyzed to select methods for assessment of sperm status for a hierarchal mode of detecting cryopreservation induced spermatozoal dysfunction. Final experimental design accommodates early success (ie.. luck) but assures that we will learn from initial failures. Response surface methodology will be used to simultaneously assess the effects of 3-5 levels of freezing rate, thaw rate, extender and composition of cryoprotectant on the success and reproducibility of mouse sperm cryopreservation. The results from one stage are used to plan more refined and focused assessment of a subset of the original (plus additional factors) at the next stage. This approach provides the most efficient method to establish optimal conditions for cryopreservation of sperm from multiple strains of mice. Progressive assays, chosen to follow the steps of fertilization, include: (a) preliminary screening via assay of spermatozoal motility and membrane compartments (by flow cytometry); (b) a unique assay for loose sperm-egg binding (c) sperm penetration into the zona pellucida: (d) early embryo development; and (e) final production of pups. Identification of an optimal protocol is an interactive process, requiring "yes/no" decisions, based upon experience, from assays with ill-defined cut off criteria. We (and no one else) have confidence that the "right" decision was made when "cut off" values were established. All of our data will be stored for periodic retrieval and retrospective analysis during the project period. This, we can return to our data sets (and our decisions) armed with knowledge of the overall success of that specific paradigm in retaining fertilization potential. This will allow us to learn as we evolve, and make ever better decisions, toward the successful protocol. The team assembled for this project is capable of: (a) driving an interactive program that will satisfy the stated goals of this RFA; and (b) providing a template of experimental design useful for solution of other cryobiological problems in human medicine and animal agriculture.