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Cryopreservation of Small Abalone (Haliotis diversicolor) Sperm-Technique and Its Significance
冷凍保存九孔精液的技術與意義
摘要
九孔(Haliotis diversicolor)爲本省重要天然海產貝類之一，極具經濟價值，近年來更成爲本省重要的淺海養殖項目。鑑於省產九孔種貝於人工繁殖時，雄貝通常較雌貝提早成熟，是故對其精液保存期限之延長開始加以研究。本研究即針對此項目進行九孔精液之低溫及冷凍保存試驗。 九孔精液在室溫(25℃)下存放二小時後受精其卵之孵化率明顯降低，四小時後則孵化率爲零，但若在其精液中添加8% DMSO及5% Glucose則可增加其保存期限及孵化率。倘改爲存放於15℃、5℃中又添加8% DMSO及5% Glucose或5% Sucrose皆可進一步延長其存放時間，保存八小時後受精仍有88.21%之孵化率。唯時間一旦超過十二小時則以低溫保存的效果急遽下降，無法應用。 至於另外探討可供九孔精液更長期保存之超低溫冷凍保存法之各項因子，結果以8% DMSO爲抗凍劑，降溫係使用液態氮蒸氣採簡易式二段降溫法，於-30℃在液態氮蒸氣中20分鐘降溫後存入液態氮中長期保存，20天後解凍受精有94.80%孵化率，保存一年後則仍有89.10%之孵化率。本研究亦針對使用保存容器、受精量等進行試驗，以便供實際應用時參考。 展望冷凍保存九孔精液之未來應用方向，甚具下列幾點育種與保種的意義：一、在育種上可避免近親交配，以增加其遺傳强勢。二、建立九孔之種原庫。三、可與國內外育種單位進行合作，以覓得養殖對象種形質改良之最佳途徑。四、可與其它水產生物技術如誘發雌核生殖、誘發全雄性子代等配合使用研究。由此可見冷凍保存九孔精液確有其超越時空的價值與意義。
並列摘要
At room temperature small abalone eggs fertilized with sperm preserved for 2 h had a low hatching rate; for 4 h, the hatching rate was zero. Low-temperature preservation and cryopreservation of small abalone sperm are studied to determine species specific characteristics and to assure a steady supply of male gametes for artificial propagation. At low temperature (15, 10, and 5℃) addition of 8% DMSO and 5% glucose was found to increase the preservation period and hatching rate, e.g. , 88.21% in 8-h preserved sperm. The effect of preservation, however, greatly decreased when preserved at 5℃ or above for 12 h or longer, again in term of hatching rate. The experiments on the selection of optimum parameters to cryo preserve small abalone sperm on a long-term basis resulted in an efficient protocol. Fresh sperm was added with 8% DMSO as cryoprotectant and exposed to liquid nitrogen vapor with a sufficiently low starting temperature of -30℃, which was obtained using a closed styrofoam chamber filled with liquid nitrogen with a stand set at an optimal distance. Freezing the sperm in PE straws of 0.5, 1 or 5 ml for 20 minis adequate before quenching the straws in liquid nitrogen. The best fertility rates of the frozen-thawed sperm were 94.80% and 89.10% obtained from the samples frozen in liquid nitrogen for 20 and 365 days, respectively. The preservation of shellfish sperm has high potential in increasing genetic vigor without too much inbreeding; establishing a locally-based shellfish gene pool; providing a channel for cooperative projects with other national and international breeding units in the search for the best course toward improving characteristics of cultured species; and linking with other biological techniques used in aquatic animals, such as induction of gynogenetic, polyploid, and all-male off spring. In summary, the potential practical significance of the cryopreservation of small abalone sperm is high.