大鼠精子化学限定冷冻保存液的开发及通过体外受精产生后代

摘要(中文译文)

※ 中文译文由 AI 辅助生成,仅供学术参考,请以英文原文为准。

体外受精（IVF）和使用冷冻解冻大鼠精子的人工授精（AI）已经建立，能够产生可存活的后代。然而，目前的标准冷冻保存液含有鸡蛋黄和Orvus ES Paste（OEP；十二烷基硫酸钠及其他未定义成分），存在微生物污染的风险，并限制了IVF和AI的实验可重复性。在本研究中，我们开发了一种不含鸡蛋黄或OEP的化学成分明确的冷冻保存液，并评估了其在IVF中的效果。在考察了各种冷冻保护剂对精子活力的影响后，我们发现OptiPrep（一种化学成分明确的试剂）和乙二醇在乳糖溶液中能最好地保持冻融后的精子活力。此外，丝胶蛋白作为一种细胞膜保护剂，有效减少了对精子头部质膜和顶体膜的损伤。尽管在化学成分明确溶液中冷冻保存的精子的受精率（71.4%）显著低于新鲜精子（90.5%），但仍成功获得了受精卵。约60%的受精卵在体外发育至囊胚阶段。此外，当将两细胞期胚胎移植到假孕雌性的输卵管中时，36%发育为后代。综上所述，我们的结果提出了一种利用化学成分明确的精子冷冻液对大鼠品系进行冷冻保存的新策略，能够安全且可重复地产生后代。

Abstract

In vitro fertilization (IVF) and artificial insemination (AI) using frozen-thawed rat sperm have been established to enable the production of viable offspring. However, the current standard cryopreservation solution, which contains chicken egg yolk and Orvus ES Paste (OEP; sodium lauryl sulfate and other undefined components), poses the risk of microbial contamination and limits the experimental reproducibility of IVF and AI. In this study, we developed a chemically defined cryopreservation solution that does not contain chicken egg yolk or OEP and evaluated its efficacy in IVF. After examining the effects of various cryoprotective agents on sperm motility, we found that OptiPrep (a chemically defined reagent) and ethylene glycol in a lactose solution best preserved sperm motility after freezing and thawing. In addition, sericin, a cell membrane protective agent, effectively reduced damage to the plasma and acrosomal membranes of the sperm head. Although the fertilization rate of the sperm cryopreserved in a chemically defined solution (71.4%) was significantly lower than that of fresh sperm (90.5%), fertilized oocytes were successfully obtained. Approximately 60% of the fertilized oocytes developed to the blastocyst stage in vitro. Furthermore, when two-cell embryos were transferred to the oviducts of pseudopregnant females, 36% developed into offspring. Taken together, our results suggest a new strategy for the cryopreservation of rat strains using a chemically defined sperm-freezing solution, enabling the safe and reproducible production of offspring.

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