因現今人類過度捕撈與生態之破壞，海洋物種正面臨嚴重的生存危機。美人蝦（Stenopus hispidus）是一種非常受歡迎的海洋水族館與清潔蝦的物種。擁有觀賞及貿易的經濟價值，而低溫冷凍保存美人蝦胚胎或早期幼體階段將顯著減少野生美人蝦的採集及珊瑚礁破壞並供應市場，降低對生態的影響從而提高珊瑚礁的保育。本研究的目的在了解不同發育期美人蝦胚胎對低溫、醣類、脂肪酸抗凍劑及不同海水鹽度對於胚胎孵化率的影響關係，並利用低溫顯微鏡分析不同胚胎冷凍保存過程及不同抗凍劑濃度對於胚胎顯微結變化的影響關係。
研究結果顯示雖然添加抗凍劑後，可增加美人蝦的胚胎細胞在低溫下其存活率，但低溫的處理對於胚胎仍具有傷害，且隨著胚胎在低溫下的暴露時間增長，胞器發生融合現象並產生不規則液泡，而利用抗凍劑處理後發現胚胎胞器的融合現象降低，本研究結果也顯示醣類抗凍劑在低溫處理後對於美人蝦胚胎的孵化率並無顯著提升的效果，脂肪酸抗凍劑及調整海水濃度作為抗凍劑則提升孵化率，且在脂肪酸抗凍劑及海水加入1M Meth後進一步提升美人蝦胚胎孵化率，本研究深入探討美人蝦胚胎低溫保存過程中造成死亡之機制，以做為未來最佳冷凍保存程序建立的參考。

Banded coral shrimp (Stenopus hispidus), a very popular species in the marine aquarium, is current under the pressure of extinction due to over-collection. The application of cryopreserving S. hispidus embryos or early stage larva will provide a respite to the wild collection of this species and reduce the destruction of coral reefs. The present study aimed to investigate the effect of low temperature, carbohydrate, lipid cryoprotectant, and seawater salinity on the hatching rate of S. hispidus at different stages of embryo. The results showed that S. hispidus with the addition of cryoprotectants were able to keep the survival rate during low-temperature treatments, but they still sustained injuries during chilling. Moreover, prolonged incubation at a low temperature was detrimental, resulting in that organelles were fused together and irregular vesicles in the developing embryo. The use of cryoprotectants during cooling showed an effective reduction of these two phenomena. The results also showed that the use of lipids as a cryoprotectant and increasing seawater salinity could improve the hatching rate of the shrimp embryo, and the addition of Methanol further increased the rate of embryo hatching. However, the addition of carbohydrates as a supplementary cryoprotectant failed to show an increment in the embryo hatching rate. This study implies that the choice of cryoprotectant supplement in cryopreservation of S. hispidus embryo needs to be exemplified.

Contents
Fig. Contents 3
Table. Contents 4
Introduction 5
Materials and methods 9
Materials 9
Breeding 9
Embryo collection 9
Cryoprotectant 10
Effect of antifreeze on embryos at a low temperature 10
Hatching rate assessment 14
Cryomicroscopy 14
Statistical Analysis 15
Results 16
Effect of sugar antifreeze on the hatching rate of Stenopus hispidus embryos 16
Effect of fatty acids antifreezer on the hatching rate of Stenopus hispidus heartbeat-stage embryos 17
Effect of fatty acids antifreezer added with 1 M Meth on the hatching rate of Stenopus hispidus heart-beat stage embryos 18
Effect of seawater salinity with 1 M Meth on the hatching rate of Stenopus hispidus heart-beat stage embryos 18
Effect of fatty acids antifreezer on the formation of embryonic ice crystals in the Stenopus hispidus heart-beat stage embryos by low-temperature microscopy 19
Discussion 22
Effect of sugar antifreezer on the hatching rate of Stenopus hispidus embryos 22
Effect of fatty acids antifreezer on the hatching rate of Stenopus hispidus heart-beat stage embryos 23
Effect of fatty acids antifreezer added with 1 M Meth on the hatching rate of Stenopus hispidus heart-beat stage embryos 24
Effect of seawater salinity with 1 M Meth on the hatching rate of Stenopus hispidus heart-beat stage embryos 24
Effect of fatty acids antifreezer on the formation of embryonic ice crystals in the Stenopus hispidus heart-beat stage embryos by low-temperature microscopy 25
Conclusion 28
REFERENCES 30
Appendix 49
References FOR TABLES S1–s5 75

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