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中國畜牧學會會誌

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篇名 豬未成熟卵母細胞玻璃化冷凍技術之建立
卷期 46增刊
並列篇名 Vitrification of immature oocytes for pigs
作者 康定傑陳裕信曲鳳翔劉振發陳立人
頁次 207-207
關鍵字 未成熟卵母細胞玻璃化冷凍Immature oocytesSwineVitrification
出刊日期 201712

中文摘要

試驗共使用採集自屠宰場卵巢之 330 顆豬未成熟卵母細胞進行 microdrop 及 cryotopz 方法之玻璃化冷凍。選取外圍包覆 2-3 層卵丘細胞之卵母細胞,以含有 2% (v/v)ethylene glycol(EG)與 2%(v/v)propylene glycol(PG)之第一階段平衡劑 平衡 12-15 分鐘後,將卵母細胞移入由17.5%(v/v) PG 及 17.5%(v/v)EG 組成之第 二階段玻璃化冷凍保護劑中,於 30 秒內置入液態氮液面下完成玻璃化冷凍。完成玻 璃化冷凍之卵母細胞收集至 2 mL 的冷凍管中,最後移入液態氮桶中保存,待後續使 用。玻璃化冷凍之未成熟卵母細胞以攝氏 42 度之加熱板解凍後,進行 48 小時之體 外成熟(in vitro maturation, IVM)培養。IVM 培養後,以玻尿酸酶將卵丘細胞移除, 續進行 fluorescein diacetate (FDA)染色,並利用螢光顯微鏡以 UV 及 GFP-II 濾 鏡進行觀察,判定死活。活細胞在細胞質處,會顯現明亮的綠色螢光。試驗結果顯示, 利用 microdrop 與 cryotop 方法進行之豬未成熟卵母細胞冷凍解凍後之平均存活率 分別為 87.3±7.5% 和 87.65±6.2%。

英文摘要

There are total 330 swine immature oocytes collected from ovaries obtained from local slaughter house that vitrified with microdrop and cryotop method in this study. The oocytes surrounded by 2-3 layers of cumulus cell were used. The oocytes were treated with an equilibration medium for 12-15 min, which was supplemented with 2% (v/v), ethylene glycol(EG), 2% (v/v) propylene glycol(PG), then move the oocytes to vitrification solution supplemented with 17.5% (v/v) EG, 17.5% (v/v) PG and place the oocytes into liquid nitrogen in 30 sec. The vitrified oocytes were transferred to 2 ml cryotubes and then stored in LN until use. The oocytes were warmed with 42 degrees Celsius warm plate and then in vitro maturation incubated for 48 h. Thereafter, the cumulus cells of oocytes were removed with hyaluronidase treatment and the oocytes were stained with fluorescein diacetate (FDA). Living oocytes exhibited a bright green color under UV. The result showed the survival rate of oocytes vitrifed with microdrop and cryotop methods were 87.3±7.5% and 87.65±6.2%, respectively。

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