海洋脊椎與無脊椎動物雄性配子之超低溫保存—以六斑刺河魨與白蝦為例

Cryopreservation of Male Gamete of Marine Vertebrate and Invertebrate — Studies in Porcupine Fish (Diodon holocanthus) and White Shrimp (Litopenaeus vannamei)

陳雨農(Yu-Nong Chen)

指導教授 ： 謝文陽

共同指導教授 ： 趙乃賢(Nai-Hsien Chao)

國立臺灣大學/理學院/海洋研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.01330

摘要

超低溫冷凍保存係採用液態氮 (-196℃) 為冷媒維護生物樣品功能性之長期超低溫保存法。已知動物精子的冷凍保存會因不同物種、精子的品質及精液濃度使得抗凍劑 (cryoprotectant) 對精子之生化或生理反應有所差別而表現出不同的結果。稀釋液 (extender) 的種類及濃度、抗凍劑的種類及濃度、平衡時間 (equilibration time) 、降溫速率 (freezing rate) 、冷凍方法 (freezing method) 及解凍速率 (thawing rate) 為影響精子冷凍保存的主要因素。
本研究進行海水脊椎與無脊椎動物雄性配子之超低溫冷凍保存試驗，以六斑刺河魨及白蝦為試驗材料，一一考慮各種可能產生影響之因子並加以測試。因為兩種試驗對象生物之精子運動方式不同，故將六斑刺河魨具游動性精子的試驗前後結果以活動力指數表示，而白蝦的精子為不具游動性其試驗前後結果以活存率百分比表示。
實驗結果顯示，六斑刺河魨精子最佳超低溫冷凍保存效果之稀釋液、降溫流程、抗凍劑種類及抗凍劑濃度為：以海水魚生理食鹽水做為稀釋液，以濃度10%之DMSO當作抗凍劑，將精液使用可程式冷凍儀 (PLANER，KRYO 10 Series II) 之MF1(自室溫以-5℃/min降至4℃後，以-10℃/min降至-30℃再以-20℃/min降至-140℃後，用LN2保存)方式降溫，樣品在25℃水浴中解凍，解凍後第2 min之活動力指數為2.10。其次佳者為：以海水魚生理食鹽水做為稀釋液，精液以MF3(自室溫持續以-20℃/min降至-100℃後，用LN2保存)方式降溫，使用濃度15%之DMSO當作抗凍劑，樣品在25℃水浴中解凍，解凍後第2 min之活動力指數為2.05。
白蝦精莢之最佳超低溫冷凍保存效果之稀釋液、降溫流程、抗凍劑種類及抗凍劑濃度為：以Ca-F saline作為稀釋液，濃度5%之DMSO當作抗凍劑，室溫 (25℃) 平衡30 min後，以-2℃/min之速率，將精莢降溫至-80℃，維持2 min，之後再投入液態氮保存。樣品在30℃水浴中解凍，活存率為最高，34.4±3.4%。其次佳者為：以-1℃/min之速率降溫，活存率亦可達33.3±3.9%，與最佳之分組並無明顯差異。在白蝦精莢長期保存試驗中，以濃度5%之DMSO當作抗凍劑，室溫 (25℃) 平衡30 min之分組為例，其前處理後活存率為49.5±8.3%；保存1天後之活存率為44.3±6.6%，其後隨著保存時間每10天觀察一次，直到第70天之前其活存率介於33%~37%之間，尚稱穩定，並無顯著差異(P < 0.05)。
經由本實驗，除了針對此二對象物種之雄性配子超低溫冷凍保存，建立目前已知最佳條件組合，並可提供作為日後其餘海洋物種作超低溫冷凍保存雄性配子模式時之參考依據。

關鍵字

超低溫冷凍保存 ； 雄性配子 ； 刺河魨 ； 白蝦

並列摘要

Cryopreservation is a well-known technology in which liquid nitrogen (-196℃) is used as refrigerant to maintain functional biomaterial sample during long-term preservation. Different species, various quality and concentration of sperm may cause different result of biochemical or physiology function of cryoprotectants. Kinds and concentration of extender, kinds and concentration of cryoprotectant, equilibration time, freezing method, freezing rate and thawing rate are major factors to cause impact on the performance of cryopreserved-thawed of sperm.
In this study, experiments on cryopreservation of male gamete of marine vertebrate and invertebrate, porcupine fish (Diodon holocanthus) and white shrimp (Litopenaeus vannamei) were conducted by examining the effect of major factors one after one. Due to the fact of different viability patterns of sperm in two experimental species, viability index for movable sperm in porcupine fish and viability percentage for nonmovable sperm in white shrimp were recorded and compared between pre- and post freezing milt respectively.
The first part of this study resulted in establishment of the optimal cryopreservation method of milt in porcupine fish including favorable kind and concentration of extender, cryoprotectant and functional freezing protocol. Milt was treated with normal saline extender, 10% DMSO as cryoprotectant, frozen in program freezer (PLANER，KRYO 10 Series II) using protocol MF1 (sample was frozen at -5℃/min till 4℃, then at -10℃/min to -30℃, finally at -20℃/min to -140℃ before quenching in LN2) and thawed at 25℃ water bath. Post-thawing viability index 2 minutes after such a treatment was 2.10. The second best was in the group that milt was treated with normal saline extender, 15% DMSO as cryoprotectant, frozen using protocol MF3 (sample was frozen at -20℃/min to -100℃ before quenching in LN2), and thawed at 25℃ water bath. Post-thawing viability index after 2 minutes was 2.05.
The second part of this study resulted in establishment of the optimal cryopreservation method of spermatophores in white shrimp including favorable kind and concentration of extender, cryoprotectant and feasible freezing protocol. In the best group, spermatophores were treated with Ca-F saline as extender, 5% DMSO as cryoprotectant, equilibrated for 30 minutes at room temperature, frozen at the rate of -2℃/min to -80℃ followed by holding for 2 minutes before quenching in liquid nitrogen (-196℃), and finally thawed at 30℃ water bath. The sperm viability percentage in such a group averaged 34.4±3.4%. The second best was in the group that spermatophores was frozen at the rate of -1℃/min, the sperm viability percentage reached 33.3±3.9%. With the purpose to carry out long-term storage of spermatophores of white shrimp, the viability percentage of sham control group using 5% DMSO as cryoprotectant and being equilibrated for 30 minutes at room temperature was 49.5±8.3%. The viability percentage on the first day was 44.3±6.6% and decreased slightly as time elapsed. It ranged between 33% and 37% from the second to 70th day.
According to this study, the optimal protocols to cryopreserve sperm in these two experimental species were established respectively and could be referred as the ideal model of cryopreservation of sperm of other marine species.

並列關鍵字

cryopreservation ； male gamete ； porcupine fish ； white shrimp

參考文獻

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