碩士 / 國立臺灣大學 / 海洋研究所 / 96 / 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.
| Identifer | oai:union.ndltd.org:TW/096NTU05274016 |
| Date | January 2008 |
| Creators | Yu-Nong Chen, 陳雨農 |
| Contributors | Wung-Yang Shieh, 謝文陽 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 105 |
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