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Study on Sperm Cryopreservation for Paternal Germplasm Repository of Selected Indigenous Fishes in Taiwan
四種台灣原生種淡水魚之種原有關精液冷凍保存之研究
摘要
本文係探討台灣原生種淡水魚：鯝台灣鏟頜魚、台灣馬口魚、圓吻鯝和台灣石（魚賓）的精子生物學特性以及建立符合其精液冷凍保存最適程式以期建構相關種原庫。為逐步研究探知有效的整合型抗凍劑，先探討三種基礎抗凍劑dimethyl sulfoxide (DMSO), methanol和glycerol其不同濃度之效果，再進行選擇性添加0.03，0.06，0.09或0.12M的蔗糖或海藻糖，及更進一步添加CAPS、TAPS或牛乳加以比較。其次再配合三項預先設計之冷凍程式FW1，FW2和FW3逐一的測試。基礎抗凍劑中10-15%DMSO對鯝台灣鏟頜魚、台灣馬口魚、圓吻鯝和台灣石（魚賓）精液冷凍保存皆為適用，而理想的整合型抗凍劑研究結果指出0.06M濃度之海藻糖優於蔗糖。進一步添加TAPS，則適合提昇鯝台灣鏟頜魚與台灣馬口魚的冷凍保存後精子活力，而CAPS的添加則適合用於圓吻鯝與台灣石（魚賓）的精子提昇其冷凍保存後精子活力。綜上結果，除了加強冷凍保護用之整合型抗凍劑，另外在冷凍程式方面，此四種台灣原生種淡水魚亦表現種別特異性。使用個別最理想的整合型抗凍劑合併最佳冷凍程式，已經將四種魚類的精子以0.5 ml麥管冷凍保存於-196℃的液態氮中。上述實作經驗、研究模式和技術成果有助於建構台灣原生魚類相關精液保存和資料庫時之重要參考。
並列摘要
This study aimed to investigate the biological characteristics and to establish the related cryopreservation protocol of sperm of selected indigenous freshwater fishes-Onychostoma barbatulus, Candidia barbata, Distoechodon tumirostris and Acrossocheilus paradoxus in Taiwan. The enhanced cryoprotectants were progressively studied using basic dimethyl sulfoxide (DMSO) with optional addition of 0.03, 0.06, 0.09 or 0.12 M sucrose or trehalose, and further addition of CAPS (3-Cyclohexylamino-1-propanesulfonic acid), TAPS (tris-hydroxymethyl methylamino-propane sulfonic acid) or milk. Milt mixture with various cryoprotectant combinations were frozen using freezing laboratory-designed protocols FW1, FW2, or FW3. Results indicated that 10 to 15% DMSO were favorable for four experimental fishes and trehalose was better than sucrose with overall optimal concentration of 0.06 M. Addition of TAPS was found favorable for milt of S. barbatulus and C. barbata while CAPS was suitable for D. tumirostris and A. paradoxus. There was obvious species-specific preference of enhanced cryoprotectant and freezing protocol in each species. The optimal combination of enhanced cryoprotectant and freezing protocol for respective species were further studied. Using the optimal enhanced cyoprotectant combinations and freezing protocol, 0.5-ml straws containing cryopreserved sperm of these fishes have been stored in liquid nitrogen at -196℃ as an example of paternal germplasm repository and related databank.