臺灣博碩士論文加值系統

謝辭 I
摘要 II
Abstract III
目錄 V
圖目錄 VIII
緒論 1
一、 珊瑚的生活史 1
二、 珊瑚礁的重要性與面臨之危機 1
三、 珊瑚的復育方法與困難 2
四、 珊瑚的冷凍保存：一種新的珊瑚復育方法 2
五、 珊瑚的冷凍保存技術與目前研究狀況 3
六、 研究目的 4
第壹章 挑選萼形柱珊瑚冷凍保護劑條件與瞭解低溫對珊瑚之影響 5
一、 前言 5
1. 冷凍保護劑 5
2. 冷凍保存技術於水生生物的應用 5
3. 石珊瑚的凍保存技術與先前研究 6
二、 實驗目的 7
三、 分析方法及材料 7
1. 實驗物種：萼形柱珊瑚 (Stylophora pistillata) 7
2. 樣本採集 7
3. 海洋資源復育園區的珊瑚養殖池設計與照顧方式 8
4. 國立臺灣海洋大學珊瑚養殖缸設計與照顧方式 8
5. 實驗前準備 9
5-1 實驗樣本挑選方式與珊瑚片段使用大小 9
5-2 實驗前的樣本分析方式 9
6. 冷凍保護劑與糖類對於萼形柱珊瑚片段毒性測試 (實驗1-4) 9
6-1 四種1.3 M的冷凍保護劑對於萼形柱珊瑚片段毒性測試 (實驗1) 9
6-2 挑選對萼形柱珊瑚片段毒性較低的冷凍保護劑進行不同濃度測試 (實驗2) 10
6-3 不同糖類對於萼形柱珊瑚片段毒性測試 (實驗3) 10
6-4 同時添加1.3 M冷凍保護劑與糖類對於萼形柱珊瑚片段毒性測試 (實驗4) 11
7. 低溫 (4°C)對萼形柱珊瑚片段的影響 (實驗5) 11
8. 萼形柱珊瑚片段於 4°C低溫中冷凍保護劑和糖類毒性測試 (實驗6 –8) 11
8-1 於 4°C添加冷凍保護劑對於萼形柱珊瑚毒性測試 (實驗6)…………………………………………………………………….. 12
8-2 於4°C中同時添加1.3 M冷凍保護劑與不同濃度糖類對於萼形柱珊瑚毒性測試 (實驗7) 12
9. 實驗處理後樣本的分析方式 12
10. 珊瑚片段存活率計算方式 13
11. 珊瑚蟲數量變化計算方式 13
12. 珊瑚白化程度分析 (RGB色彩分析) 13
13. 統計方法 13
13-1 One-way ANOVA 統計分析 13
13-2 Student’s t-test 統計分析 14
四、 結果 14
1. 冷凍保護劑對於萼形柱珊瑚片段之影響 (實驗1) 14
2. 不同濃度冷凍保護劑對於萼形柱珊瑚片段毒性測試分析 (實驗2) 14
3. 不同糖類對於萼形柱珊瑚片段毒性測試分析 (實驗3) 15
4. 同時添加1.3 M冷凍保護劑與糖類對於萼形柱珊瑚片段毒性測試分析結果 (實驗4) 15
5. 低溫 (4°C)對萼形柱珊瑚片段的影響 (實驗5) 16
6. 於 4°C添加冷凍保護劑對於萼形柱珊瑚毒性測試分析結果 (實驗6) 16
7. 於 4°C同時添加1.3 M冷凍保護劑與不同濃度糖類對於萼形柱珊瑚毒性測試分析結果 (實驗7) 17
五、 討論 18
第貳章 開發萼形柱珊瑚片段冷凍保存技術 20
一、 前言 20
1. 冷凍保存技術發展 20
二、 實驗目的 21
三、 材料方法 21
1. 實驗物種：萼形柱珊瑚 (Stylophora pistillata) 21
2. 樣本採集 21
3. 實驗前準備 21
4. 萼形柱珊瑚片段冷凍保存實驗分析 (實驗1-實驗2) 21
4-1 冷凍條件與方法測試1 (實驗1) 21
4-2 冷凍條件與方法測試2 (實驗2) 22
5. 使用自製降溫盒用於萼形柱珊瑚片段冷凍保存 (實驗3) 22
6. 萼形柱珊瑚片段玻璃化冷凍保存實驗 (實驗4) 22
7. 實驗後樣本處理方式 23
8. 萼形柱珊瑚片段細胞冷凍分析 (實驗5) 23
8-1 萼形柱珊瑚片段細胞分散 24
8-2 萼形柱珊瑚細胞冷凍實驗 24
9. 萼形柱珊瑚細胞計算 24
10. 萼形柱珊瑚細胞ATP測定 25
11. 統計方法 25
四、 結果 26
1. 萼形柱珊瑚片段冷凍保存實驗結果 26
2. 使用自製降溫盒冷凍萼形柱珊瑚片段之結果 26
3. 萼形柱珊瑚片段玻璃化冷凍保存實驗結果 26
4. 萼形柱珊瑚片段細胞冷凍實驗 26
五、 討論 27
結論 29
參考文獻 74

戴昌鳳、洪聖雯，2009。台灣珊瑚礁圖鑑。貓頭鷹出版社。
段文宏，2010。珊瑚的秘密。科學發展，第445期，24-29頁。
林家興，2015。珊瑚諾亞方舟 ─ 珊瑚礁的未來不是夢。國立海洋生物博物館館訊，第78期，第6頁。
朱惠真、陳力豪、胡鄴方、蔡惠萍、張錦宜。2015。生殖細胞冷凍保存技術的現況。水試專訓。第50期。第48-50頁。
蔡雅如、施彤煒。2015。海洋環境與保育：東北角珊瑚礁的現況與復育策略，科學發展，第516期，12-18頁。
Best B. P. (2015). Cryoprotectant Toxicity: Facts, Issues, and Questions. Rejuvenation research, 18(5), 422–436.
Burke L, Reytar K, Spalding M and Perry A. (2004). Reefs at risk revisit. World Resources Institute,Washington, DC, 124.
Cai H, Tang B, Wu JY, et al. Enrichment and in vitro features of the putative gonocytes from cryopreserved testicular tissue of neonatal bulls. Andrology. 2016;4(6):1150-1158.
Cai, H., Wu, J. Y., An, X. L., Zhao, X. X., Wang, Z. Z., Tang, B., Yue, Z. P., Li, Z. Y., & Zhang, X. M. (2016). Enrichment and culture of spermatogonia from cryopreserved adult bovine testis tissue. Animal reproduction science, 166, 109–115.
Chiu, Y. L., Shikina, S., & Chang, C. F. (2019). Testicular somatic cells in the stony coral Euphyllia ancora express an endogenous green fluorescent protein. Molecular reproduction and development, 86(7), 798–811.
Chong, G., Tsai, S., Wang, L. H., Huang, C. Y., & Lin, C. (2016). Cryopreservation of the gorgonian endosymbiont Symbiodinium. Scientific reports, 6, 18816.
Cirino, L., Wen, Z. H., Hsieh, K., Huang, C. L., Leong, Q. L., Wang, L. H., Chen, C. S., Daly, J., Tsai, S., & Lin, C. (2019). First instance of settlement by cryopreserved coral larvae in symbiotic association with dinoflagellates. Scientific reports, 9(1), 18851.
Daly, J., Zuchowicz, N., Nuñez Lendo, C. I., Khosla, K., Lager, C., Henley, E. M., Bischof, J., Kleinhans, F. W., Lin, C., Peters, E. C., & Hagedorn, M. (2018). Successful cryopreservation of coral larvae using vitrification and laser warming. Scientific reports, 8(1), 15714.
De'ath, G., Fabricius, K. E., Sweatman, H., & Puotinen, M. (2012). The 27-year decline of coral cover on the Great Barrier Reef and its causes. Proceedings of the National Academy of Sciences of the United States of America, 109(44), 17995–17999.
Delbeek, JC. (2001) Coral farming: past, present and future trends. Aquarium Sciences and Conservation, 3, 171-181.
Epstein N, Bak RPM, and Rinkevich B. (2001). Strategies for gardening denuded coral reef areas: the applicability of using different types of coral material for reef restoration, Restoration Ecology, 9,(4) 1-11.
Feuillassier, L., Romans, P., Engelmann-Sylvestre, I., Masanet, P., Barthélémy, D., & Engelmann, F. (2014). Tolerance of apexes of coral Pocillopora damicornis L. to cryoprotectant solutions. Cryobiology, 68(1), 96–106.
Forsman, Z. H., Page, C. A., Toonen, R. J., & Vaughan, D. (2015). Growing coral larger and faster: micro-colony-fusion as a strategy for accelerating coral cover. PeerJ, 3, e1313.
Hagedorn M, Spindler R, Daly J. Cryopreservation as a Tool for Reef Restoration: 2019. Adv Exp Med Biol. 2019;1200:489-505.
Hagedorn, M., Daly, J. P., Carter, V. L., Cole, K. S., Jaafar, Z., Lager, C., & Parenti, L. R. (2018). Cryopreservation of Fish Spermatogonial Cells: The Future of Natural History Collections. Scientific reports, 8(1), 6149.
Hagedorn, M., Carter, V. L., Henley, E. M., van Oppen, M., Hobbs, R., & Spindler, R. E. (2017). Producing Coral Offspring with Cryopreserved Sperm: A Tool for Coral Reef Restoration. Scientific reports, 7(1), 14432.
Hagedorn, M., Farrell, A., & Carter, V. L. (2013). Cryobiology of coral fragments. Cryobiology, 66(1), 17–23.
Hagedorn, M., Carter, V., Martorana, K., Paresa, M. K., Acker, J., Baums, I. B., Borneman, E., Brittsan, M., Byers, M., Henley, M., Laterveer, M., Leong, J. A., McCarthy, M., Meyers, S., Nelson, B. D., Petersen, D., Tiersch, T., Uribe, R. C., Woods, E., & Wildt, D. (2012). Preserving and using germplasm and dissociated embryonic cells for conserving Caribbean and Pacific coral. PloS one, 7(3), e33354.
Hagedorn, M., van Oppen, M. J., Carter, V., Henley, M., Abrego, D., Puill-Stephan, E., Negri, A., Heyward, A., MacFarlane, D., & Spindler, R. (2012). First frozen repository for the Great Barrier Reef coral created. Cryobiology, 65(2), 157–158
Hagedorn, M., Carter, V. L., Leong, J. C., & Kleinhans, F. W. (2010). Physiology and cryosensitivity of coral endosymbiotic algae (Symbiodinium). Cryobiology, 60(2), 147–158.
Hagedorn, M., Carter, V. L., Steyn, R. A., Krupp, D., Leong, J. C., Lang, R. P., & Tiersch, T. R. (2006). Preliminary studies of sperm cryopreservation in the mushroom coral, Fungia scutaria. Cryobiology, 52(3), 454–458.
Hagedorn, M., Pan, R., Cox, E. F., Hollingsworth, L., Krupp, D., Lewis, T. D., Leong, J. C., Mazur, P., Rall, W. F., MacFarlane, D. R., Fahy, G., & Kleinhans, F. W. (2006). Coral larvae conservation: physiology and reproduction. Cryobiology, 52(1), 33–47.
Hatta M, Iwao K. (2002). Metamorphosis induction and its possible application to coral seedlings production, In: Saxena N (Ed.), Recent Advances in Marine Science and Technology, Japan International Marine Science and Technology Federation Akasaka, Minatoku, Tokyo.2003, pp.465-470.
Hirayama, K., Takayama, K., Haruta, S., Ishibashi, H., & Takeuchi, I. (2017). Effect of low concentrations of Irgarol 1051 on RGB (R, red; G, green; B, blue) colour values of the hard-coral Acropora tenuis. Marine pollution bulletin, 124(2), 678–686.
Hughes TP, Baird AH, Dinsdale EA, Moltschaniwskyj NA, Pratchett MS, Tanner JE, Willis BL. (2000). Supply-side ecology works both ways: The link between benthic adults, fecundity, and larval recruits, Ecology, 81, 2241-2249.
Hughes, T. P., Baird, A. H., Bellwood, D. R., Card, M., Connolly, S. R., Folke, C., Grosberg, R., Hoegh-Guldberg, O., Jackson, J. B., Kleypas, J., Lough, J. M., Marshall, P., Nyström, M., Palumbi, S. R., Pandolfi, J. M., Rosen, B., & Roughgarden, J. (2003). Climate change, human impacts, and the resilience of coral reefs. Science (New York, N.Y.), 301(5635), 929–933.
Hughes, T. P., Barnes, M. L., Bellwood, D. R., Cinner, J. E., Cumming, G. S., Jackson, J., Kleypas, J., van de Leemput, I. A., Lough, J. M., Morrison, T. H., Palumbi, S. R., van Nes, E. H., & Scheffer, M. (2017). Coral reefs in the Anthropocene. Nature, 546(7656), 82–90.
Hughes, T. P., Kerry, J. T., Baird, A. H., Connolly, S. R., Dietzel, A., Eakin, C. M., Heron, S. F., Hoey, A. S., Hoogenboom, M. O., Liu, G., McWilliam, M. J., Pears, R. J., Pratchett, M. S., Skirving, W. J., Stella, J. S., & Torda, G. (2018). Global warming transforms coral reef assemblages. Nature, 556(7702), 492–496.
Khosla, K., Wang, Y., Hagedorn, M., Qin, Z., & Bischof, J. (2017). Gold Nanorod Induced Warming of Embryos from the Cryogenic State Enhances Viability. ACS nano, 11(8), 7869–7878.
Lee, S., Iwasaki, Y., Shikina, S., & Yoshizaki, G. (2013). Generation of functional eggs and sperm from cryopreserved whole testes. Proceedings of the National Academy of Sciences of the United States of America, 110(5), 1640–1645.
Leonel, E., Corral, A., Risco, R., Camboni, A., Taboga, S. R., Kilbride, P., Vazquez, M., Morris, J., Dolmans, M. M., & Amorim, C. A. (2019). Stepped vitrification technique for human ovarian tissue cryopreservation. Scientific reports, 9(1), 20008.
Lin, C., & Tsai, S. (2012). The effect of chilling and cryoprotectants on hard coral (Echinopora spp.) oocytes during short-term low temperature preservation. Theriogenology, 77(6), 1257–1261.
Lin, C., Kuo, F. W., Chavanich, S., & Viyakarn, V. (2014). Membrane lipid phase transition behavior of oocytes from three gorgonian corals in relation to chilling injury. PloS one, 9(3), e92812.
Lin, C., Thongpoo, P., Juri, C., Wang, L. H., Meng, P. J., Kuo, F. W., & Tsai, S. (2019). Cryopreservation of a Thermotolerant Lineage of the Coral Reef Dinoflagellate Symbiodinium. Biopreservation and biobanking, 17(6), 520–529.
Lin, C., Zhang, T., Kuo, F. W., & Tsai, S. (2011). Gorgonian coral (Junceella juncea and Junceella fragilis) oocyte chilling sensitivity in the context of adenosine triphosphate response (ATP). Cryo letters, 32(2), 141–147.
MAZUR P. (1963). KINETICS OF WATER LOSS FROM CELLS AT SUBZERO TEMPERATURES AND THE LIKELIHOOD OF INTRACELLULAR FREEZING. The Journal of general physiology, 47(2), 347–369.
Nagy ZP, Varghese AC, Agarwal A. (2017). Cryopreservation of Mammalian Gametes and Embryos Methods and Protocols, Methods in Molecular Biology, Springer protocol, ISBN 978-1-4939-6826-8.
Nannou1 TK, Trembley J, Herrmann J, Jouhara1H. (2016). Cryopreservation: Methods, Equipment and Critical Concerns. 1st IIR International Conference of Cryogenics and Refrigeration Technology (ICCRT 2016), At Bucharest, Romania.
Nedimyer K, Gaines K, Roach S. (2011). Coral Tree Nursery©: An innovative approach to growing corals in an ocean-based field nursery. AACL Bioflux, 4.
Ohki, S., Morita, M., Kitanobo, S., Kowalska, A. A., & Kowalski, R. K. (2014). Cryopreservation of Acropora digitifera sperm with use of sucrose and methanol based solution. Cryobiology, 69(1), 134–139.
Petersen D, Wietheger A, Laterveer M. (2008). Influence of different food sources on the initial development of sexual recruits of reef-building corals in aquaculture, Aquaculture, 277, 174-178.
POLGE, C., SMITH, A. U., & PARKES, A. S. (1949). Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature, 164(4172), 666.
Rall, W. F., & Fahy, G. M. (1985). Ice-free cryopreservation of mouse embryos at -196 degrees C by vitrification. Nature, 313(6003), 573–575.
Richmond RH, Hunter CL. (1990). Reproduction and recruitment of corals: comparisons among the Caribbean, the Tropical Pacific, and the Red Sea. Marine Ecology Progress Series.
Seki, S., Kusano, K., Lee, S., Iwasaki, Y., Yagisawa, M., Ishida, M., Hiratsuka, T., Sasado, T., Naruse, K., & Yoshizaki, G. (2017). Production of the medaka derived from vitrified whole testes by germ cell transplantation. Scientific reports, 7, 43185.
Shafir S, Rijn JV, Rinkevich B. (2006). Steps in the construction of underwater coral nursery, an essential component in reef restoration acts, Marine Biology, 149, 679–687.
Tsai S, Chong G, Meng PJ, Lin C. (2017). Sugars as supplemental cryoprotectants for marine organisms, Reviews in Aquaculture, 0, 1–13.
Tsai, S., Spikings, E., Huang, I. C., & Lin, C. (2011). Study on the mitochondrial activity and membrane potential after exposing later stage oocytes of two gorgonian corals (Junceella juncea and Junceella fragilis) to cryoprotectants. Cryo letters, 32(1), 1–12.
Tsai, S., Spikings, E., Kuo, F. W., Lin, N. C., & Lin, C. (2010). Use of an adenosine triphosphate assay, and simultaneous staining with fluorescein diacetate and propidium iodide, to evaluate the effects of cryoprotectants on hard coral (Echinopora spp.) oocytes. Theriogenology, 73(5), 605–611.
Tsai, S., Yang, V., & Lin, C. (2016). Comparison of the cryo-tolerance of vitrified gorgonian oocytes. Scientific reports, 6, 23290.
Tsai, S., Yen, W., Chavanich, S., Viyakarn, V., & Lin, C. (2015). Development of Cryopreservation Techniques for Gorgonian (Junceella juncea) Oocytes through Vitrification. PloS one, 10(5), e0123409.
Van Oppen, M. J., & Gates, R. D. (2006). Conservation genetics and the resilience of reef-building corals. Molecular ecology, 15(13), 3863–3883.
Viyakarn, V., Chavanich, S., Chong, G., Tsai, S., & Lin, C. (2018). Cryopreservation of sperm from the coral Acropora humilis. Cryobiology, 80, 130–138.
Vutyavanich, T., Piromlertamorn, W., & Nunta, S. (2010). Rapid freezing versus slow programmable freezing of human spermatozoa. Fertility and sterility, 93(6), 1921–1928.
Wowk, B. (2007). How cryoprotectant work. Cryonics Third Quarter 3, 28, 3-5.
Yeemin T, Sutthacheep M, Pettongma R. (2006). Coral reef restoration projects in Thailand. Ocean & Coastal Management, 49, 562–57