臺灣博碩士論文加值系統

珊瑚礁由於人類活動和全球性氣候變化而正遭受到前所未有的破壞及退化，甚至原地保育也無法有效減緩珊瑚礁的衰退，然而異地保護則可以提供保護措施來進行珊瑚礁復育，並利用冷凍保存技術可安全保存現有的物種和基因多樣性並重新引入到重建的棲息地，也因為珊瑚與共生藻的緊密共生關係，使得共生藻成為珊瑚是否冷凍保存成功的重要關鍵。然而，在冷凍保存的過程中，可能會對細胞造成冷凍傷害，因此找出適合共生藻之冷凍保存流程及相關條件是相當重要的。本次研究利用玻璃化及可程式冷凍保存進行比較，並確認適合共生藻clade G之冷凍保護劑種類、濃度、平衡時間及降溫速率，建立對其最佳化的冷凍流程和條件。實驗結果顯示在可程式冷凍法中，1M Glycerol在5分鐘平衡時間下有著最佳之抗凍效果，而在不同的降溫速率下並不影響Glycerol之抗凍能力；而在玻璃化冷凍保存中，利用DMSO、Methanol 及Glycerol的冷凍保護劑組合在平衡溶液之平衡時間10 分鐘及玻璃化溶液平衡時間2.5分鐘下有最佳之冷凍保存效果，玻璃化冷凍法比兩段式及可程式冷凍法有著更高的共生藻存活率。Adenosine triphosphate之測定能準確幫助了解共生藻能量之狀態，並對共生藻之存活進行評估。在兩種冷凍法之共生藻培養實驗中顯示，共生藻在冷凍過程中可能受到冷凍傷害的影響，進而造成細胞死亡，而在後續的培養中共生藻之狀態可維持平穩，此推斷共生藻clade G能對冷凍損傷進行修復。本次研究可幫助了解共生藻之冷凍條件，並協助未來相關共生藻冷凍保存技術的發展。

Coral reefs are suffering from unprecedented damage and degradation due to human activities and global climate change. Even with in situ conservation can not effectively mitigate the degradation of coral reefs. However, ex-situ conservation can provide a conservation measures for coral reef rehabilitation. Cryopreserving the existing species and preserving genetic diversity could later be reintroduced and reconstructed in natural habitat. Symbiodinium cryopreservation is important for the success of coral rehabilitation due to the close symbiotic relationship between coral and Symbiodinium.
However, cells might be damage during cryopreservation. Therefore, it is important to find the best cryopreservation protocol and conditions of Symbiodinium. This study used vitrification and programmable freezing to compare effects, also to confirm the appropriate CPAs, concentration, equilibrium time and cooling rate to establish the optimal freezing process and conditions for Symbiodinium. The experimental results showed that in the programmable freezing method, 1M Glycerol had the best antifreeze effect at 5 min equilibrium time. Different cooling rates did not affect antifreeze ability for Glycerol. In the vitrification cryopreservation, the used of DMSO, Methanol and Glycerol cryoprotectant combination had the best cryopreservation effect at 10 min in equilibration solution and 2.5 min in vitrification solution. Compared with the past research in our laboratory, the vitrification had higher survival rate in Symbiodinium clade G than the two-step and programmable freezing method. The determination of adenosine triphosphate level could accurately help understand the state of Symbiodinium vitality and assess the survival rate. In the algae culture experiment of vitrification and programmable freezing, the algae was affected by freezing injury during the cryopreservation process, and even caused cell death. The state of Symbiodinium in the culture can be maintained stable, it can be inferred that the Symbiodinium clade G can repair the damage caused by freezing. This study will help to understand the frozen conditions of Symbiodinium and assist in the development of future related Symbiodinium cryopreservation techniques.

摘要
Abstract
目錄
表目錄
圖目錄
第一章 前言
1.1 共生藻
1.1.1 共生藻與珊瑚間之利益關係
1.1.2 共生藻之細胞週期
1.2共生藻之冷凍保存
1.3低溫冷凍保存技術
1.4冷凍保護劑(Cryoprotectant)
1.5藻類的冷凍保存
1.5.1淡水藻類
1.5.2海水藻類
1.6低溫生物學之應用
1.6.1畜牧與水產養殖
1.6.2微生物之生物技術工程
1.6.3低溫保存瀕臨絕種之生物
第二章 材料與方法
2.1 採集地點
2.2 共生藻之分離
2.3 Adenosine triphosphate (ATP)之檢測
2.4 共生藻之鑑種
2.5 共生藻之低溫冷凍
2.5.1可程式慢速冷凍法
2.5.2玻璃化法
2.5.2.1 載體及VS、ES玻璃化檢測
2.5.2.2 毒性測試
2.5.2.3 玻璃化實驗
2.6 共生藻之培養
2.6.1 人工海水配製
2.6.2 人工海水之更換
2.7 實驗數據統計
第三章 可程式慢速冷凍法之冷凍結果
3.1 介紹
3.2 結果
3.2.1 抗凍保護劑毒性及降溫速率測試
3.2.2 降溫速率與冷凍保護劑平衡時間毒性之測試
3.2.3 植冰
3.2.4 共生藻之解凍培養
3.3 討論
3.3.1 抗凍保護劑選擇與濃度
3.3.2 抗凍保護劑平衡時間
3.3.3 冷凍速率之變化
3.3.4 冷凍後藻細胞之培養
3.4 結論
第四章 玻璃化冷凍法之冷凍結果
4.1 介紹
4.2 結果
4.2.1 三種載體與抗凍保護劑濃度之玻璃化測試
4.2.2三種載體與抗凍保護劑混合濃度之玻璃化測試
4.2.3 抗凍保護劑平衡時間之毒性測試
4.2.4 平衡溶液及玻璃化溶液之平衡時間毒性測試
4.2.5 玻璃化冷凍與共生藻培養
4.2.6 Cryotop玻璃化冷凍保存之比較
4.3 討論
4.3.1 載體與抗凍保護劑混合濃度之玻璃化
4.3.2 平衡溶液及玻璃化溶液之毒性測試
4.3.3 玻璃化冷凍與共生藻培養
4.4 結論
第五章 總結
參考文獻

Ali, J. and Shelton, J.N. (1993) Design of vitrification solutions for the cryopreservation of embryos. Journal of Reproduction and Fertility, 99:471-477

Anthony, K. R. N., Kline, D. I., Diaz-Pulido, G., Dove, S. and Hoegh-Guldberg, O. (2008) Ocean acidification causes bleaching and productivity loss in coral reef builders. National Academies of Sciences, 45: 17442-17446

Beaty. M. H. (1991) Cryopreservation of eukaryote algae. Unpublished Masters Thesis, Virginia Polytechnic Institute and State University

Beaty, M. and Parker, B. (1992) Cryopreservation of eukaryotic algae. Virginia Journal of Science, 43(4): 405-410

Ben-Amotz, A. and Gilboa, A. (1980) Cryopreservation of marine unicellular algae. A survey of algae with regard to size, culture age, photosynthetic activity and chlorophyll to cell ratio. Marine Ecology Progress Series, 2: 157-161

Benson, E. E. (2008) Cryopreservation Theory. Plant Cryopreservation: A Practical Guide, Chapter 2: 15-32

Bernard, A. and Fuller, B. J. (1996) Cryopreservation of human oocytes: a review of current problems and perspectives. Human Reproduction Update, 2(3): 193-207

Bertolini, M. C., Laramee, L., Thomas, D. Y., Cygler, M., Schrag, J. D., Vernet, T. (1994) Polymorphism in the lipase gene of geotrichumcandidum strains. European Journal of Biochemistry, 219(1-2): 119-125

Bogliolo, L., Ariu, F., Rosati, I., Zedda, M. T., Pau, S. and Naitana, S. (2006) Vitrification of immature and in vitro matured horse oocytes. Reproduction Fertility and Development, 18: 149-150

Bui, T. V. L., Ross, I. L., Jakob, G. and Hankamer, B. (2013) Impact of procedural steps and cryopreservation agents in the cryopreservation of chlorophyte microalgae. PLoS ONE, 8(11): 1-9

Cabrita, E., Robles, V., Chereguini, O., Wallace, J.C., and Herra´ez, M.P. (2003) Effect of different cryoprotectants and vitrificant solutions on the hatching rate of turbot embryos (Scophthalmus maximus). Cryobiology, 47: 204-213

Cañavate, J. P. and Lubian, L. M. (1995a) Some aspects on the cryopreservation of microalgae used as food for marine species. Aquaculture, 136: 277-290

Cañavate, J. P. and Lubian, L. M. (1995b) Relationship between cooling rates, cryoprotectant concentrations and salinities in the cryopreservation of marine microalgae. Marine Biology, 125: 325-334

Charoensub, R., Phansiri, S., Sakai, A. and Yongmenitchai, W. (1999) Cryopreservation of cassava in vitro-grown shoot tips cooled to −196 °C by vitrification. CryoLetters, 20(1999): 89-94

Cheng, Y. W. (2014) Symbiosis establishment mechanism between different clades of Symbiodinium in sea anemone (Aiptasia pulchella) - infection test and ultra-structural analysis. National Dong Hwa University

Chevalier, F. (1991) Essais de cryoconservation d'algues unicellulaires d'interet aquacole. Institut National des Techniques de la Mer de Cherbourg, France

Chian, R., Kuwayama, M., Tan, L., Tan, J., Kato, O. and Nagai, T. (2004) High survival rate of bovine oocytes matured in vitro following vitrification. Journal of Reproduction and Development, 50: 685-696

Chong, G., Tsai, S., Wang, L. H., Huang, C. Y. and Lin, C. (2016) Cryopreservation of the gorgonian endosymbiont Symbiodinium. Sci Rep., 6: 18816

Chong, W. (2013) Ultrastural observation of oocytes from six coral species for use in cryopreservation studies. National Dong Hwa University

Craig, P. (2009) Natural history guide to American Samoa. National Park of American Samoa

Crutchfield, A., Diller, K. and Brand, J. (1999) Cryopreservation of Chlamydomonas reinhardtii (Chlorophyta). European Journal of Phycology, 34(1): 43-52

Cunning, R., Gillette, P., Capo, T., Galvez, K. and Baker, A. C. (2015) Growth tradeoffs associated with thermotolerant symbionts in the coral Pocillopora damicornis are lost in warmer oceans. Coral Reefs, 34: 155-166

Day, J. G. and DeVille M. M. (1995) Cryopreservation of algae. Methods in Molecular Biology, 38: 81-89

Day, J. and Brand, J. (2005) Cryopreservation methods for maintaining cultures. In: R. Andersen (ed.). Algal culturing techniques. Academic Press, New York, 165-87

Day, J. G. and Harding, K. (2008) Cryopreservation of Algae. Plant Cryopreservation: A Practical Guide, Chapter 6: 95-116

Diaz, F., Bondiolli, K., Paccamonti, D. and Gentry, G. T. (2016) Cryopreservation of Day 8 equine embryos after blastocyst micromanipulation and vitrification. Theriogenology, 85: 894-903

Dunn, S. R. and Weis, V. M. (2009) Apoptosis as a post-phagocytic winnowing mechanism in a coral–dinoflagellate mutualism. Environmental Microbiology, 11(1): 268-276

Elliott, J. K. and Mariscal, R. N. (2001) Coexistence of nine anemonefish species: differential host and habitat utiliztion, size and recruitment. Marine Biology, 138: 23-36

Elnahas, A., Alcolak, E., Marar, E. A., Elnahas, T., Elnahas, K., Palapelas, V., Diedrich, K. and Al-Hasani, S. (2010) Vitrification of human oocytes and different development stages of embryos: An overview. Middle East Fertility Society Journal, 15: 2-9

Fahy, G. M. (1984) Cryoprotectant toxicity reduction: specific or nonspecific? CryoLetters, 5: 287-294

Fahy, G. M. (1986) The relevance of cryoprotectant “toxicity” to cryobiology. Cryobiology, 23: 1-13

Fenwick, C and Day, J. (1992) Cryopreservation of Tetraselmis suecica cultured under different nutrients regimes. Journal of Applied Phycology, 4(2): 105-109

Fischer, G., Prieler, S., van Velthuizen, H., Berndes G., Faaij, A., Londo, M. and de Wit, M. (2010) Biofuel production potentials in Europe: Sustainable use of cultivated land and pastures, Part II: Land use scenarios. Biomass Bioenergy, 34: 173-187

Fitt, W. K. (2000) Cellular growth of host and symbiont in a cnidarian-zooxanthellar symbiosis. Biology Bulletin, 198: 110-120

Fitt, W. K. and Trench, R. K. (1983) The relation of diel patterns of cell division to diel patterns of motility in the symbiotic dinoflagellate Symbiodinium microadriaticum Freudenthal in culture. New Phytologist, 94:421-432

Fleck, R. A., Pickup, R. W., Day, J. G. and Benson, E. E. (2006) Characterisation of cryoinjury in Euglena gracilis using flow-cytometry and cryomicroscopy. Cryobiology, 52: 261-268

Fornari, D. C., Ribeiro, R. P., Streit, D., Godoy, L. C., Neves, P. R., de Oliveira, D. and Sirol, R. N. (2011) Effect of cryoprotectants on the survival of cascudo preto (Rhinelepis aspera) embryos stored at –8℃. Zygote, 22(1): 58-63

Gates, R. D., Baghdasarian, G. and Muscatine, L.(1992) Temperature Stress Causes Host Cell Detachment in Symbiotic Cnidarians: Implications for Coral Bleaching. The Biological Bulletin, 182:324-332

Gao, D. and Critser, J. K. (2000) Mechanisms of cryoinjury in living cells. Institute Laboratory Animal Research Journal, 41(4): 187-196

Guermazi, W., Sellami-Kammoun, A., Elloumi, J., Drira, Z., Aleya, L., Marangoni, R., Ayadi, H. and Maalej, S. (2010) Microalgal cryo-preservation using dimethyl sulfoxide(Me2SO) coupledwith two freezing protocols: Influence on the fatty acid profile. Journal of Thermal Biology, 35(4): 175-181

Gwo, J. C. Chiu, J. Y., Chou, C.C. and Cheng, H.Y. (2005) Cryopreservation of a marine microalga, Nannochloropsis oculata (Eustigmatophyceae). Cryobiology, 50:338-343

Hagedorn, M., Carter, V. L., Leong, J. C. and Kleinhans, F. W. (2010) Physiology and cryosensitivity of coral endosymbiotic algae (Symbiodinium). Cryobiology, 60(2): 147-58

Hagedorn, M., Farrell, A. and Carter, V.L. (2013) Cryobiology of coral fragments. Cryobiology, 66: 17-23

Hagedorn, M. and Spindler, R. (2014) The reality, use and potential for cryopreservation of coral reefs. Advances in Experimental Medicine and Biology, 753: 317-329

Hagedorn, M. and Carter, V. L. (2015) Seasonal preservation success of the marine dinoflagellate coral symbiont, Symbiodinium sp. PLoS ONE, 10(9): e0136358

Hajarian, H., Wahid, H., Rosnina, Y., Daliri, M., Dashtizad, M., Mirzapour, T., Yimer, N., Bukar, M. M., Iswadi, M. I. and Mazni, O. A. (2011) Cryotop device enhances vitrification outcome of immature bovine oocytes. Journal of Animal and Veterinary Advances, 10(19): 2541-2545

Harding, K., Day, J. G., Lorenz, M., Timmermann, H., Friedl, T., Bremner, D. H. and Benson, E. E. (2004) Introducing the concept and application of vitrification for the cryo-conservation of algae - a mini-review. Nova Hedwigia, 79(1-2): 207-226

Heesch, S., Day, J., Yamagishi, T., Kawai, H., Müller, D. G. and Küpper, F. C. (2012) Cryopreservation of the model alga ectocarpus (Phaeophyceae). CryoLetters, 33(5): 327-336

Hirose, M. Reimer, J. D., Michio, H. and Suda, S. (2008) Phylogenetic analyses of potentially free-living Symbiodinium spp. isolated from coral reef sand in Okinawa, Japan. Marine Biology, 155: 105-112

Hochi, S., Terao, T., Kamei, M., Kato, M., Hirabayashi, M. and Hirao, M. (2004) Successful vitrification of pronuclear-stage rabbit zygotes by minimum volume cooling procedures. Theriogenology, 61: 267-275

Hoegh-Guldberg, O., Mumby, P. J., Hooten, A. J., Steneck, R. S., Greenfield, P., Gomez, E., Harvell, C. D., Sale, P. F., Edwards, A. J., Caldeira, K., Knowlton, N., Eakin, C. M., Iglesias-Prieto, R., Muthiga, N., Bradbury, R. H., Dubi, A. and Hatziolos, M. E. (2007) Coral Reefs Under Rapid Climate Change and Ocean Acidification. Science, 318: 1737

Horvath, A., Wayman, W. R., Urbanyi, B., Ware, K. M. Dean, J. C. and Tiersch, T. R. (2005) The relationship of the cryoprotectants methanol and dimethyl sulfoxide and hyperosmotic extenders on sperm cryopreservation of two North-American sturgeon species. Aquaculture, 247: 243-251

Hubálek, Z. (2003) Protectants used in the cryopreservation of microorganisms. Cryobiology, 46: 205-229

Hughes, T. P. (1994) Catastrophes, phase shifts, and large-scale degradation of a Caribbean coral reef. Science, 265(5178): 1547-1551

Iwamoto, K., Fukuyo, S., Okuda, M., Kobayashi, M. and Shiraiwa, Y. (2012) Cryopreservation of the chlorophyll d-containing cyanobacterium Acaryochloris marina. Procedia Environmental Sciences, 15: 118-125

Jackson, J. B., Kirby, M. X., Berger, W. H., Bjorndal, K. A., Botsford, L. W., Bourque, B. J., Bradbury, R. H., Cooke, R., Erlandson, J., Estes, J. A., Hughes, T. P., Kidwell, S., Lange C. B., Lenihan, H. S., Pandolfi, J. M., Peterson, C. H., Steneck, R. S., Tegner, M. J. and Warner, R. R. (2001) Historical overfishing and the recent collapse of coastal ecosystems. Science, 293(5530): 629-637

Jo, Y., Kang, S., Seo, T., Choi, S., Kho, K., Kuwano, K., Saga, N., Kim, M. and Shin, J. (2003) Cryopreservation of Sporothalli of the Genus Porphyra (Bangiales, Rhodophyta) from Korea. Algae, 18(4): 321-331

Joseph, I., Panigrahi, A. and Chandra, P. K. (2000) Tolerance of three marine microalgae to cryoprotectants dimethyl sulfoxide, methanol and glycerol. Indian Journal of Marine Sciences, 29: 243-247

Kirsop, B. E. and Doyle, A. (1991) Maintenance of Microorganisms and Cultured Cells. Academic Press Books, London.

Kegler, P., Baum, G., Indriana, L. F., Wild, C. and Kunzmann, A. (2015) Physiological response of the hard coral Pocillopora verrucosa from Lombok, Indonesia, to two common pollutants in combination with high temperature. PLoS ONE 10(11): e0142744

Knight, M. R., Campbell A. K., Smith S. M. and Trewavas A. J. (1991) Transgenic plant aequorin reports the effects of touch and cold-shock and elicitors on cytoplasmic calcium. Nature, 352(6335): 524-526

Kuleshova, L. L. and Lopata, A. (2002) Vitrification can be more favorable than slow cooling. Fertility and Sterility, 78: 449-454

Kumari, N., Gupta, M. K. and Singh, R. K. (2016) Open encapsulation-vitrification for cryopreservation of algae. Cryobiology, 73: 232-239

Kuwano, K., Aruga, Y. and Saga, N. (1992) Preliminary study on cryopreservation of the conchocelis of Porphyra yezoensis. Fisheries Science, 58: 1793-1798

Kuwayama, M., Vajta, G., Kato, Osamu. and Leibo, S. P. (2005) Highly efficient vitrification method for cryopreservation of human oocytes. Reproductive BioMedicine Online, 11(3): 300-308

Lambardi, M., Fabbri, A. and Caccavale, A.(2000) Cryopreservation of white poplar (Populus alba L.) by vitrification of in vitro-grown shoot tips. Plant Cell Reports, 19(2000): 213-218

Lane, M., Schoolcraft, W. B. and Gardner, D. K. (1999) Vitrification of mouse and human blastocysts using a novel cryoloop container-less technique. Fertility and Sterility, 72(6): 1073-1078

Langis, R. and Steponkus, P. L. (1990) Cryopreservation of Rye Protoplasts by vitrification. Plant Physiology, 92(3): 666-671

Larson, D. J., Middle, L., Vu, H., Zhang, W., Serianni, A. S., Duman, J. and Barnes, B. M. (2014) Wood frog adaptations to overwintering in Alaska: new limits to freezing tolerance. Journal of Experimental Biology, 217(12): 2193-2200

Levitt, J. (1980) Responses of plants to environmental stress, 2nd Edition, Volume 1: Chilling, Freezing, and High Temperature Stresses. Academic Press, NewYork

Li, Y. (2014) Ultrastructural observations of chilling injury in gorgonian (Junceella fragilis) oocytes. National Dong Hwa University

Li, M., Wang, S. Y. and Dong, S. R. (2016) Cryopreservation of Spirulina. Microbiology China, 43(3): 679-686

Lin, C. and Tsai, S. (2009) Review: Factors affecting cryopreservation of cells. Platax, 6: 93-106

Lin, C., Spikings, E., Zhang, T. and Rawson, D. M. (2009) Effect of chilling and cryopreservation on expression of Pax genes in zebrafish (Danio rerio) embryos and blastomeres. Cryobiology, 59 (1): 42-47

Lin, C., Liu, W. C. and Tsai, S. (2010) An efficient approach for cryopreservation of the king grouper (Epinephelus lanceolatus) spermatozoa. Platax, 7: 56-66

Lin, C., Zhang, T., Kuo, F. W. and Tsai, S. (2011) Gorgonian coral (Junceella juncea and Junceella fragilis) oocyte chilling sensitivity in the context of adenosine triphosphate response (ATP). CryoLetters, 32(2): 141-147

Lin, C. and Tsai, S. (2012) The effect of chilling and cryoprotectants on hard coral (Echinopora spp.) oocytes during short-term low temperature preservation. Theriogenology, 77: 1257-1261

Lin, C., Wang, L. H., Fan, T. Y. and Kuo, F. W. (2012) Lipid content and composition during the oocyte development of two gorgonian coral species in relation to low temperature preservation. PLoS ONE, 7(7): e38689

Lin, C. H., Kuo, F. W., Chavanich, S. and Viyakarn, V. (2014) Membrane lipid phase transition behavior of oocytes from three gorgonian corals in relation to chilling injury. PLoS ONE, 9(3): e92812

Liu, H. Q., Yu, W., Dai, J. X., Gong, Q. H., Yang, K. F. and Lu, X. Z. (2004) Cryopreservation of protoplasts of the alga Porphyra yezoensis by vitrification. Plant Science, 166(1): 97-102

Lovelock, J. E. (1953) The haemolysis of human red blood-cells by freezing and thawing. Biochimica et Biophysica Acta, 10: 414-426

Ludvigsen, S. (2003) Speech by the Norwegian Minister of Fisheries - seminar hosted by the Brazilian Ministry of Fisheries Brasilia.

Mahmoud, K. Gh. K., El-Sokary, M. M. M., Scholkamy, T. H., Abou El-Roos, M. E. A., Sosa, G. A. M. and Nawito, M. (2013) The effect of cryodevice and cryoprotectant concentration on buffalo oocytes vitrified at MII stage. Animal Reproduction Science, 10(4): 689-696

Mazur, P. (1970) Cryobiology: the freezing of biological systems. Science, N.Y. 168: 939-946

Mazur, P., Leibo, S. P. and Chu, E. H. (1972) A two-factor hypothesis of freezing injury. Evidence from Chinese hamster tissue culture cells. Experimental Cell Resesearch, 71, 345-355

Mazur, P. (1981) Fundamental cryobiology and preservation of organs by freezing, organ preservation for transplantation. Marcel Dekker, NewYork, 2: 143-175

Mazur, P. (1984) Freezing of living cells: mechanisms and implications. American Journal of Physiology, 247(16): 125-142

Mazur, P. (1990) Equilibrium, quasi-equilibrium, and nonequilibrium freezing of mammalian embryos. Cell Biochemistry and Biophysics, 17: 53-91

McClanahan, T., Polunin, N. and Done, T. (2002) Ecological states and the resilience of coral reefs. Conservation Ecology, 6(2): 18

Meryman, H. T. (2007) Cryopreservation of living cells: principles and practice. Transfusion, 47: 935-945

Moran, N. A. (2006) Symbiosis. Current Biology, 16(20): 1977-2080

Morris, G. J. (1978) Cryopreservation of 250 strains of Chlorococcales by the method of two-step cooling, British Phycological Journal, 13(1): 15-24

Morris, G. J. (1981) Cryopreservation: An introduction to cryopreservation in culture collections. Institute of Terrestrial Ecology, Cambridge

Morris, G. J. and Canning, C. E. (1978) The cryopreservation of Euglena gracilis. Journal of general microbiology, 108: 27-32

Morris, G. J. and Watson, P. F. (1984) Cold shock injury- a comprehensive bibliography. CryoLetter, 5: 352-372

Morris, G. J. and Clarke, A. (1984) The effects of low temperature on biological systems- Cell at low temperature. London: Edward Arnold, 72-188

Morris, G. J., Coulson, G. E. and Clarke, K. J. (1988) Freezing injury in Saccharomyces cerevisiae: the effect of growth conditions. Cryobiology, 25: 471-482

Morris, G. J. (1976) The cryopreservation of Chlorella. 1. Interactions of rate of cooling, protective additive and warming rate. Archives of Microbiology, 107: 57-62

Muldrew, K. and McGann, L. E. (1994) The osmotic rupture hypothesis of intracellular freezing injury. Biophysical Journal, 66(2): 532-541

Muscatine, L.,Falkowski, P., Porter, J. and Dubinsky, Z. (1984) Fate of photosynthetically-fixed carbon in light and shade adapted colonies of the symbiotic coral Stylophora pistillata. Proceedings of the Royal Society of London, 222: 18l-202

Nakanishi, K., Deuchi, K. and Kuwano, K. (2012) Cryopreservation of four valuable strains of microalgae, including viability and characteristics during 15 years of cryostorage. Journal of Applied Phycology, 24(6): 1381-1385.

Nascimento, I. A., Leite, M. B. N. L., de Araujo, M. M. S., Sansone, G., Pereira, S. A. and Santo, M. E. (2005) Selection of cryoprotectants based on their toxic effects on oyster gametes and embryos. Cryobiology, 51: 113-117

Radhakrishna T. G., Ramanatha C. L., Gupta, C. and Reddy, N. C. (1983) Economics of artificial insemination and calves born from liquid semen and frozen semen. Indian Journal of Animal Reproduction., 3: 44-45

Pandolfi, J. M., Connolly, S. R., Marshall, D. J. and Cohen, A. L. (2011) Projecting coral reef futures under global warming and ocean acidification. Science, 22(333): 418-422

Pasternak, Z., Blasius, B., Abelson, A. and Achituv, Y. (2006) Host-finding behaviour and navigation capabilities of symbiotic zooxanthellae. Coral Reefs, 25: 201-207

Pasquinelli, G., Foroni, L., Buzzi, M., Tazzari, P. L., Vaselli, C., Mirelli, M., Gargiulo, M., Conte, R. and Stella, A. (2006) Smooth muscle cell injury after cryopreservation of human thoracic aortas. Cryobiology, 52: 309-316

Pearl, M. and Arav, A. (2000) Chilling sensitivity in zebrafish (Brachydanio rerio) oocytes is related to lipid phase transition. CryoLetters, 21(3): 171-178.

Pegg, D. E. and Diaper, M. P. (1991) The effect of initial tonicity on freeze-thraw injury to human red cells suspended in solutions of NaCl. Cryobiology, 28: 18-35

Pennycooke, J. C. and Towill, L. E. (2000) Cryopreservation of shoot tips from in vitro plants of sweet potato (Ipomoea batatas (L) Lam.) by vitrification. Plant Cell Reports, 19(2000): 733-737

Pereira, R. M. and Marques, C. C. (2008) Animal oocyte and embryo cryopreservation. Cell Tissue Banking, 9: 267-277

Petersen, D., Carl, M., Borneman, E., Brittsan, M., Hagedorn, M., Laterveer, M. and Schick, M. (2008) Noah’s Ark for the threatened Elkhorn coral Acropora palmata. Coral Reefs, 27: 715

Polge, C., Smith, A. U. and Parkes, A. S. (1949) Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature, 164: 666

Poncet, J. M. and Veron, B. (2003) Cryopreservation of the unicellular marine alga, Nannochloropsis oculata. Biotechnology Letters, 25(23): 2017-2022

Rall, W. F. (1987) Factors affecting the survival of mouse embryos cryopreserved by vitrification. Cryobiology, 24:387-402

Rall, W. F. and Fahy, G. M. (1985) Ice-free cryopreservation of mouse embryos at -196℃ by vitrification. Nature, 313:573-575

Rodriguez-Lanetty, M., Krupp, D. A. and Weis, V. M. (2004) Distinct ITS types of Symbiodinium in Clade C correlate with cnidarian/dinoflagellate specificity during onset of symbiosis. Marine Ecology Progress Series, 275: 97-102

Sakai, A. and Engelmann, F. (2007) Vitrification, encapsulation-vitrification and droplet-vitrification: a review. Cryoletters, 8(3): 151-72

Sandin, S. A., Smith, J. E., DeMartini, E. E., Dinsdale, E. A., Donner, S. D., Friedlander, A. M., Konotchick, T., Malay, M., Maragos, J. E., Obura, D., Pantos, O., Paulay, G., Richie, M., Rohwer, F., Schroeder, R. E., Walsh, S., Jackson, J. B. C., Knowlton, N. and Sala, E. (2008) Baselines and degradation of coral reefs in the Northern Line Islands. PLoS ONE, 3(2): e1548

Santiago-Vazquez, L. Z, Newberger, N. C. and Kerr, R. G. (2007) Cryopreservation of the dinoflagellate symbiont of the octocoral Pseudopterogorgia elisabethae. Marine Biology, 152(3): 549-556

Sorek, M., Diaz-Almeyda, E. M., Medina, M. and Levy, O. (2014) Marine Genomics, 14: 47-57

Stat, M., Carter, D. and Hoegh-Guldberg, O. (2006) The evolutionary history of Symbiodinium and scleractinian hosts— Symbiosis, diversity, and the effect of climate change. Perspectives in Plant Ecology, Evolution and Systematics, 8: 23-43

Storey, K. B. and Storey, J. M. (2005) Freeze tolerance. Extremophiles, 2

Swanson, R. and Hoegh-Guldberg, O. (1998) Amino acid synthesis in the symbiotic sea anemone Aiptasia pulchella. Marine Biology, 131(1): 83-93

Széll, A. and Shelton, J. N. (1986) Sucrose dilution of glycerol from mouse embryos frozen rapidly in liquid nitrogen vapour. Journal of Reproduction and Fertility, 76(1): 401-408

Taylor, R. and Fletcher, R. L. (1999) Cryopreservation of eukaryotic algae: a review of methodologies. Journal of Applied Phycology, 10: 481-501

Thinh, N. T., Takagi, H. and Yashima, S. (1999) Cryopreservation of in vitro grown shoot tips of banana (Musa sp.) by vitrification method. CryoLetter, 20(1999): 163–174

Tonus, C., Connan, D., Waroux, O., Vandenhove, B., Wayet, J., Gillet, L., Desmecht, D., Antoine, N., Ectors, F. J. and Grobet, L. (2017) Cryopreservation of chicken primordial germ cells by vitrification and slow freezing: A comparative study. Theriogenology, 88: 197-206

Tsai, S. and Lin, C. (2009) Effects of cryoprotectant on the embryos of banded coral shrimp (Stenopus hispidus), preliminary studies to establish freezing protocols. CryoLetters, 30(5): 373-381

Tsai, S. and Lin, C. (2012) Advantages and applications of cryopreservation in fisheries science. Brazilian Archives of Biology and Technology, 55(3): 425-433

Tsai, S., Chen, J. C., Spikings, E., Li, J. J. and Lin, C. (2015) Degradation of mitochondrial DNA in cryoprotectant-treated hard coral (Echinopora spp.) oocytes. Mitochondrial DNA, 26(3): 420-425

Tsai, S., Kuit, V., Lin, Z. G. and Lin, C. (2014) Application of a functional marker for the effect of cryoprotectant agents on gorgonian coral (Junceella juncea and Junceella fragilis) Sperm Sacs. Cryoletters, 35(1): 1-7

Tsai, S., Rawson, D. M. and Zhang, T. (2009) Development of cryopreservation protocols for early stage zebrafish (Danio rerio) ovarian follicles using controlled slow cooling. Theriogenology, 71(8): 1226-1233

Tsai, S., Spikings, E. and Lin, C. (2010) Effects of the controlled slow cooling procedure on freezing parameters and ultrastructural morphology of Taiwan shoveljaw carp (Varicorhinus barbatulus) sperm. Aquatic Living Resources, 23: 119-124

Tsai, S., Spikings, E., Huang, I. C. and 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. CryoLetters, 32(1): 1-12

Tsai, S., Spikings, E., Kuo, F. W., Lin, N. C. and 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., Yen, W., Chavanich, S., Viyakarn, V. and Lin, C. (2015) Development of cryopreservation techniques for gorgonian (Junceella juncea) oocytes through vitrification. PLoS ONE, 10(5): e0123409

Turner, S., Krauss, S. L., Bunn, E., Senaratna, T., Dixon, K., Tan, B. and Touchell, D. (2001) Genetic fidelity and viability of Anigozanthos viridis following tissue culture, cold storage and cryopreservation. Plant Science, 161: 1099-1106

Tzovenis, I., Triantaphyllidis, G., Naihong, X., Chatzinikolaou, E., Papadopoulou, K., Xouri, G. and Tafas, T. (2004) Cryopreservation of marine microalgae and potential toxicity of cryoprotectants to the primary steps of the aquacultural food chain. Aquaculture, 230: 457-473

Vajta, G., Holm, P., Kuwayama, M., Booth, P. J., Jacobsen, H., Greve, T. and Callesen, H. (1998) Open pulled straw (OPS) vitrification: A new way to reduce cryoinjuries of bovine ova and embryos. Molecular Reproduction and Development, 51: 53-58

Valojerdi, M. R., Eftekhari-Yazdi, P., Karimian, L., Hassani, F. and Movaghar, B. (2009) Vitrification versus slow freezing gives excellent survival, post warming embryo morphology and pregnancy outcomes for human cleaved embryos. Journal of Assisted Reproduction and Genetics, 26: 347-354

van der Meer, J and Simpson, F. J. (1984) Cryopreservation of Gracilaria tikvahiae (Rhodophyta) and other macrophytic marine algae. Phycologia, 23(2): 195-202

Xu, P. (2007) Application of cryobioloby and reproductive engineering techniques in laboratory animals. Chinese Journal of Comparative Medicine, 17(8): 30-37

Wang, L. H., Liu, Y. H., Ju, Y. M., Hsiao, Y. Y., Fang, L. S. and Chen, C. S. (2008) Cell cycle propagation is driven by light–dark stimulation in a cultured symbiotic dinoflagellate isolated from corals. Coral Reefs, 27:823-835

Wang, B., Zhang, E., Gu, Y., Ning, S., Wang, Q. and Zhou, J. (2011) Cryopreservation of brown algae gametophytes of Undaria pinnatifida by encapsulation–vitrification. Aquaculture, 317 (1–4): 89-93

Whitehead, L. F. and Douglas, A. E. (2003) Metabolite comparisons and the identity of nutrients translocated from symbiotic algae to an animal host. Journal of Experimental Biology, 206(18): 3149-3157

Zhang, E., Zhang, L., Wang, B., Yan, B. and Wang, Q. (2009) Cryopreservation of marine diatom algae by encapsulation-vitrification. Cryoletters, 30(3): 224-231