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自動化系統在魚類染色體操作之應用研究

  • 日期:98-04-17
  • 年度:2007
  • 領域:農漁牧產業自動化領域
  • 主持人:趙乃賢
  • 研究人員:趙乃賢

本計畫第二年度大量且多批次進行泥鰍染色體操作實驗並逐步比較與分析ㄧ系列較佳參數,經整合後鍵入系統軟體。熱擊法使用高溫,如40℃、42℃等誘發效果不佳,其受精率、成胚率均受影響,所得幼苗成長亦較不順利。冷擊法使用低溫如1℃、3℃、5℃等進行誘發效果明確,但連續40分鐘之受精率與成胚率兩者相乘參數偏低,遠落於30分鐘組。化學誘發法之參數相乘效果在水溫為24±1℃時,以從受精8-10分鐘開始以0.5 mg/1000ml Cytochalasin B 連續10-15分鐘組為佳。26±1℃時,以從受精5分鐘開始以0.5 mg/1000ml Cytochalasin B 連續10-15分鐘組亦佳。以上後二項之參數組合已鍵入自動化系統成內建程式。 使用自動化系統進行冷擊法,程序流暢,大量卵同步接受誘發之低溫法,快速進入與離開既定低溫,效果可由受精率、成胚率未有負面影響,三倍體率提高作為實證。至於同ㄧ系統用以進行化學處理誘發時,不僅操作者不必接觸易於滲透細胞膜之藥物,而且大量卵同步接受一系列需時間精準,攪拌均勻且加精、去精、加藥、去藥、洗卵、排水數道程序十分便捷,得力之至。綜合比較各種參數組合,化學法雖不如冷擊法的整體效果,唯二者佐以自動化系統已提升成效。

研究報告摘要(英)


Research on the Application of Automatic Apparatus for Chromosome Manipulation in FishTechnology from established platforms in the first year of the project was utilized to study the application of a patented automatic system for chromosome manipulation in cyprinid loach. Large amount of eggs obtained by induced maturation and fertilized with cryopreserved or fresh sperm were used for a series of experiment on chromosome manipulation in order to evaluate the best parameter combination and to construct the feasible protocol program in the software of the self-designed automatic apparatus. It was found that heat shock (HS) using 40 or 42℃ to manipulate chromosome resulted in comparatively non-acceptable overall outcome including low rates of fertilization, embryo formation, growth of fingerlings and survival. However, cold shock (CS) using 1, 3, or 5℃ to manipulate chromosome was much better in terms of satisfactory fertilization, embryo formation, and integrated propagation index. Especially, the 30-min cold shock duration gave much better results than that of 40 min. At water temperature of 24±1℃, chemical treatment with 0.5 mg/1000 ml cytochalasin B (CB) starting at 5 min after artificial fertilization and lasting for 10- 15 min also resulted in similar overall performance. At water temperature of 26±1℃, chemical treatment with 0.5 mg/1000 ml cytochalasin B (CB) starting at 8-10 min after artificial fertilization and lasting for 10- 15 min resulted in ideal overall performance. Parameter combination of cold shock and chemical treatment methods were collected and keyed into the software of the automatic apparatus.Judging the ploidy of mixed cells from cell suspension of crushed and ground larvae or erythrocyte of young fish using flowcytometer gave the index integrated with fertilization and embryo formation rates. From these widely distributed data, high-ranking index was found mainly with properly designed cold shock and chemical treatment groups.As for research on practical application of the automatic apparatus, the advantages in cold shock for chromosome manipulation included simultaneous exposure of the selected cold temperature in mass manipulation, quick-in and quick-out to and from manipulation temperature, favorable fertilization and embryo formation rates, and triploidy percentage, as proven in this study. The advantages in chemical treatment for chromosome manipulation included avoiding direct contact with trans-cell-membrane CB by manipulators, precise timing and sufficient exposure during ten procedures such as sperm addition, sperm rinsing-out, CB addition, CB rinsing-out by DMSO, water addition, and three times DMSO rinsing. Generally speaking, overall efficiency to conduct the cold shock in loach using the automatic apparatus leads to a satisfactory integrated index of embryo formation rate and triploidy rate, far above that of chemical treatment. However, further studies on both methods of chromosome manipulation using the automatic apparatus are needed. It is found that more parameters were closely involved with artificial chromosome manipulation in finfish than in shellfish, although both with potentially high feasibility.