珊瑚礁擁有生物多樣性，對人類極其爲重要。然而這些脆弱的珊瑚礁生態正受到巨烈的自然及人爲壓力威脅，許多珊瑚物種正面臨瀕臨絕種的問題。冷凍保存是一種長期保存技術，利用降低溫度来保持细胞、胚胎及組織使其處於生理静止狀態中減少能量的耗損。本研究利用兩種鞭珊瑚（Junceella fragillis and J. juncea）發展可程式冷凍保存技術對其卵母细胞進行低溫冷凍保存之相關測試，例如：平衡時間、抗凍劑及其輔助劑濃度與種類、降溫速率、共晶溫度、解凍時間等。結果顯示，10 ⁰C 的降溫速率及- 50 ⁰C的共晶溫度雖然有最佳之J.fragillis卵母細胞活性但其活性卻小於16 %，而5 ⁰C 的降溫速率及- 40 ⁰C共晶溫度卻對J.juncea有著明顯的冷凍保存效益（≥ 80 %）。當降溫速率超過該物種的臨界點時，會對該物種造成冷凍傷害，因此，有效的的降溫速率及共晶溫度能提升冷凍保存的效果。在配合加入糖類抗凍輔助劑後，0.2 M蔗糖和1 M甲醇的混合型抗凍劑對於J. juncea的卵母細胞的存活率及卵母細胞完整百分比有最佳的效果。在本次實驗也發現0.5 M的甲醇以脂質球帶入油酸或芥酸可有效的提升卵母細胞的存活率以及提升其完整百分比。最後，本實驗發現J.juncea的卵母細胞無論在低溫冷凍保存還是抗凍劑的耐受性都遠比J.fragillis卵母細胞來得高。這是因爲兩種珊瑚共存生活環境不同所造成，這也和其脂質的含量有關。本實驗爲第一篇探討利用可程式冷凍保存珊瑚卵母細胞之相關研究。本實驗結果可爲未來的珊瑚保育及復育提供實際上的幫助。

Coral reefs demonstrate considerable biological diversity and are critical to humans. However, these fragile ecosystems are currently threatened by the onslaught of anthropogenic stressors; this may result in the extinction of numerous coral species in the near future. Cryopreservation is a long-term storage technique that uses low temperatures to keep cells and tissues alive in a quiescent state. In this study, the gorgonian corals Junceella fragillis and J. juncea were target species; their oocytes were cryopreserved using a controlled, slow-cooling protocol. Results revealed that the effective cooling rate and eutectic temperature were respectively 10 °C/min and −50 °C for J. fragillis oocytes and 5 °C/min and −40 °C for J. juncea oocytes. Regarding sugar supplementation, for J. fragillis oocytes, 1 M methanol with 0.4 M sucrose resulted in higher viability and 1 M methanol with 0.2 M sucrose led to better morphology. For J. juncea, 1 M methanol with 0.2 M sucrose led to both higher viability and better morphology. Moreover, 0.5 M methanol supplemented with two lipids (oleic acid and erucic acid) improved both oocyte viability and reduced the average percentage of damaged oocytes. This study is the first to effectively cryopreserve coral oocytes by using a controlled, slow-cooling method; in the future, the fertilisation of thawed and viable oocytes (which were cryopreserved using the current approach) will be studied.

CHAPTER 1 LITERATURE REVIEW
1.0 Introduction 1
2.0 Cryopreservation 1
2.1 Cold shock 2
2.2 Ice crystal formation 2
2.3 Solution effect 2
3.0 Freezing technique 2
3.1 Slow freezing 3
3.2 Two-step freezing 3
3.3 Vitrification 3
4.0 Cryopreservation protocol 4
4.1 Cooling rate 4
4.2 CPA 4
4.3 Equilibration time 5
4.4 Thawing method 6
4.5 Viability assay 6
5.0 Current status 12
5.1 Sperm 13
5.2 Oocytes 15
5.3 Embryos, larvae, fragment and tissue ball 17
5.4 Symbiodinium 19

CHAPTER 2 MATERIAL AND METHODS
2.1 Coral collection 21
2.2 Oocyte isolation 24
2.3 Preparation of MeOH 24
2.4 Preparation of sugar 24
2.5 Preparation of lipid 24
2.6 Freezing 26
2.7 Viability assay 29
2.8 Statistical analysis 29

CHAPTER 3 RESULTS
3.1 Introduction 31
3.1.1 Results
3.1.1.1 Effect of cooling and eutectic temperature 35
3.1.1.2 Effect of using cryoprotectant and supplement (sugar) 37
3.1.1.3 Effect of cryoprotectant with lipids on oocytes 39
3.1.1.4 Effect of cryoprotectant and lipids with cold hardening on oocytes 41

CHAPTER 4 DISCUSSION 43
CHAPTER 5 CONCLUSION 49
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