EFFICACY OF MOUSE SPERM CRYOPRESERVATION

描述：(改编自申请者？S描述)革命的进步 在基因组研究和创造基因特异性菌株的能力方面 老鼠正在导致菌株数量的指数增长 可用于生物医学研究。冷冻和储存精子最有可能 有效地保存了这些新产生的大多数菌株，但现有的 这些方法并不适用于所有品系的小鼠。长期的 本项目的目的是改进精子冷冻保存方法，用于 可靠地恢复无病原体和生殖正常的后代。这个 该项目的具体目标是： (1)建立有效的冷冻方案，以改进超低温保存 来自不同近交系的精子。的各种添加和替换 现有低温防护剂配方中的成分将在 小范围渗透压，并测试它们对 成功冷冻近交系和突变株精子。 (2)提高不同自交系配子的体外受精能力 菌株。体外受精的各种修饰，如添加自由基 清除剂对培养基中透明带的去除作用 将测试卵母细胞提高近交系受精的能力。 配子。 (3)确定通过冷冻精子传播病原体的风险 通过体外受精或卵胞浆内单精子注射获得后代 (ICSI)使用被送往杰克逊实验室重新衍生的受感染男性精子 以及输入一种将被诊断为存在 病原体。 (4)提供动物生殖功能的定量评估 从冷冻的精子中产生。从冷冻保存产生的雌性和雄性 精子将与ICR小鼠交配，以评估它们的生育能力，并将进行测试 用于生殖行为，测量血浆水平和体外释放 生殖相关激素，并评估精子发生。 神经内分泌机制可能是任何已发现的生殖缺陷的基础 将会被确认。

DESCRIPTION: (Adapted from Applicant?s Description) Revolutionary advancements in genome research and the ability to create genetically specific strains of mice are resulting in an exponential increase in the number of strains available for biomedical research. Freezing and storing sperm could most efficiently preserve the majority of these newly created strains, but existing methods do not work sufficiently well for all strains of mice. The long-term objective of this project is to improve methods of sperm cryopreservation for reliable recovery of pathogen free and reproductively normal offspring. The specific aims of this project are to: (1) Establish efficient freezing protocols for improved cryopreservation of sperm from different inbred strains. Various additions and substitutions of components in an existing cryoprotectant formulation will be made within a narrow range of osmolality and tested for their effect on the efficiency of successfully freezing inbred and mutant strain sperm. (2) Enhance in vitro fertilization (IVF) for gametes from different inbred strains. Various modifications of IVF such as addition of free radical scavengers to the culture medium and removal of the zona pellucida from oocytes will be tested for ability to enhance fertilization of inbred strain gametes. (3) Determine the risk of pathogen transmission through cryopreserved sperm upon recovery of offspring through IVF or intracytoplasmic sperm injection (ICSI) using sperm of infected males sent to The Jackson Laboratory for re-derivation and importation of a strain that will be diagnosed for presence of pathogens. (4) Provide quantitative assessment of reproductive function in the animals produced from frozen sperm. Females and males produced from cryopreserved sperm will be mated to ICR mice to evaluate their fertility and will be tested for reproductive behaviors, measured for plasma levels and in vitro release of reproduction-related hormones, and assessed for spermatogenesis. Neuroendocrine mechanisms that may underlie any reproductive deficits found will be identified.