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題 名 | 兔卵經急速冷凍後粒線體分佈的螢光染色=Fast Freezing of Rabbit Ova and Fluorescent Assay for Mitochondrial Distribution |
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作 者 | 吳明哲; 李秀美; 駱亞欣; 黃瓊姿; | 書刊名 | 畜產研究 |
卷 期 | 29:2 1996.06[民85.06] |
頁 次 | 頁157-167 |
分類號 | 437.68 |
關鍵詞 | 兔; 卵; 玻璃化冷凍; 大小; 螢光染色; Rabbit; Ovum; Vitrification; Size; Fluorescent staining; |
語 文 | 中文(Chinese) |
中文摘要 | 本試驗用的兔卵取自成熟的白色紐西蘭種母觸11頭和毛皮生產用的雷克斯種母兔四頭之卵巢。卵巢先以組織培養液mDPBS來洗滌三次,再放入含有18%胎牛血清的培養液中,以刀尖刺破卵巢上濾泡,平均每頭兔可回收到15個卵,數目1至78不等。兔卵的急速冷凍用保護劑為Ethylene Glycol(EG)和Ficoll 70(F70),分別先以18%胎牛血清的mDPBS來調配成40% EG和18% F70溶液,兩種溶液再以2:1容積比調和成最終冷凍溶液。共計有182個卵被吸置入冷凍溶液中,每頭兔卵分別吸填入0.25ml麥管,封口後至已浸置10分鐘時,就把麥管直接投入液態氮,完成急速冷凍步驟。解凍時,冷凍麥管自液態氮桶取出就直接投入20℃水浴槽,一分鐘後取出再擠出解凍卵於1.0M蔗糖溶液中,經10分鐘後檢視外觀完整的卵,完整率為82.4±20.2%。完整的解凍卵經檢出並培養於18%胎牛血清的mDPBS中20分鐘,存活率為77.5±20.3%。存活卵的直徑為145.2±17.1μm,透明帶厚度為18.5±2.8μm,卵細胞直徑。為108.0±14.9μm。存活卵經活性Rhodamine 123螢光染色後,觀察其粒線體分佈均勻等級,有2.8%的存活卵仍具有如新鮮卵般的均勻分佈相,餘依序有11.2、23.4、14.0、22.4和26.2%的存活卵之粒線體分佈呈次均勻分佈、粉圓狀聚集、油滴狀散佈、蜂巢式孔洞和片塊狀。 |
英文摘要 | Rabbit ova were collected from excised ovaries of 11 mature New Zealand White does and four fur breed Rex does. Rabbit ovaries were washed three times in mDPBS tissue medium prior to transferring into the culture solution containing 18% fetal calf serum. Ovarian follicles were punctured with shape blade tip for collection of follicular ova. A mean of 15 follicular ova were collected from each doe with a range of 1 to 78 ova. Cryoprotectants for fast freezing were Ethylene glycol (EG) and Ficoll 70 (F70). EG and F70 were stocked by a version of mDPBS medium containing 18% fetal calf serum to be mixed into the 40% EG and 18% F70 solution, respectively. The fast freezing medium EGF4018 was then prepared from the 40% EG and 18% F70 stock solutions in a volume ratio of 2:1. A total of 182 ova were put in the EGF4018 medium for 10 minutes and were then pipetted into a 0.25ml straw. Each straw containing only ova from the same doe was directly plunged into liquid nitrogen for vitrification preservation. For thawing, the straw was removed from the liquid nitrogen tank and was immediately plunged into a water bath held at 20℃ for one minute, and then the cryoprotectants were diuluted with 1.0M sucrose solution for 10 minutes. Frozen-thawed ova were evaluated for their integrality based upon morphological appearance. The intact rate was 82.4±20.2%. Intact ova were further cultured in mDPBS medium containing 18% fetal calf serum for 20 minutes and the survival rate of frozen ova was 77.5±20.3%. The diameter of surviving ovum was 145.2±17.1μm, the thickness of zona pellucida was 18.5±2.8μm, and the diameter of oocyte was 108.0±14.9μm. Surviving ova were then assayed by the mitochondria-specific vital fluorescent dye rhodamine 123 to examine the degree of homogeneous distribution of mitochondria. A total of 2.8% of surviving ova had a homogeneous distribution throughout the cytoplasm comparable to that of the fresh ova. Surviving ova had partially homogeneous distribution, bead-like distribution, oil droplets aggregation, bee-nest vesicle distribution, or severe clumping aggregation in percentages of 11.2, 23.4, 14.0, 22.4, and 26.2%, respectively. |
本系統中英文摘要資訊取自各篇刊載內容。