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精子。 每只雄鼠的鼠仔数。 之前分析了替代策略 选择我们的方法。 低温保存的步骤顺序 周期，理论和务实，分析，以建立一个 可以测试实验变量的模板。 同样地， 分析了导致成功受精步骤的顺序 选择分级模式的精子状态评估方法 检测冷冻引起的精子功能障碍。 最终 实验设计适应早期的成功（即...运气），但保证 我们将从最初的失败中吸取教训。 响应面法 将用于同时评估3-5个水平的 冷冻速率、解冻速率、稀释剂和冷冻保护剂的组成 小鼠精子冷冻保存的成功性和可重复性。 的 一个阶段的结果用于更精确和更集中的计划 评估的一个子集的原始（加上额外的因素）， 下一阶段。 这种方法提供了最有效的方法， 建立多个精子冷冻条件 小鼠品系。 渐进式测定，选择遵循以下步骤 施肥，包括：（a）初步筛选，通过测定 精子运动性和膜区室（通过流式细胞术）;（B） 一个独特的测定松散的精子-卵子结合（c）精子渗透到 透明幼体：（d）早期胚胎发育;和（e）最终生产 的小狗。 最佳协议的识别是一个交互式的 过程，需要根据试验经验做出“是/否”决定 没有明确的截止标准。 我们（没有其他人）有信心 当“截止”值被 确立了习 我们所有的数据都将被存储以供定期检索， 项目期间的回顾性分析。 这个，我们可以退回去 我们的数据集（和我们的决策）武装与知识的整体 保持施肥潜力具体范例的成功。 这将使我们能够在进化中学习，并做出更好的决定， 走向成功的协议。 这个项目的团队是 能够：（a）驱动将满足用户的交互式程序， 本RFA的既定目标;以及（B）提供一个实验性的 一种可用于解决人体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.