An Efficient Approach for Cryopreservation of the King Grouper (Epinephelus lanceolatus) Spermatozoa

龍膽石斑(Ep inephe lus Lanceo latus)精子之冷凍保存

10.29926/PLATAX.201012.0005

林家興(Chia-Hsin Lin)；劉紋君(Wen-Chun Liu)；蔡淑君(Su-June Tsai)

冷凍保存 ； SYBR-14 ； PI ； 抗凍劑 ； 龍膽石斑 ； cryopreservation ； SYBR-14 ； PI ； cryoprotectant ； grouper ； vapor freezing

Platax

2010卷7期（2010 / 12 / 01）

57 - 66

英文

龍膽石斑爲先雌後雄之變性魚種，雄性成魚通常需要三年至五年的養殖期，因此雄性成魚相對較少，魚苗的產出供不應求。魚類精子的冷凍保存除了可應用於遺傳育種的研究之外，此技術可持續供應所需的配子，在種魚的培育上扮演著重要的角色。本研究的目的在發展龍膽石斑冷凍保存的技術。本實驗利用螢光染劑SYBR-14及PI探討五種抗凍劑及三種平衡時間對於龍膽石斑精子的影響。20%DMSO在本研究中被發現其爲最適合精子冷凍保存的抗凍劑。本研究的結果也顯示在長時間的冷凍保存後，龍膽石斑精子的品質仍然維持不變。本研究發展之精子冷凍保存技術，將會對日後龍膽石斑人工繁殖技術的突破有實質上的幫助。

Supply of grouper fry has been inadequate to meet the heavy demand as functional males usually take at least 3-5 years of culture. Cryopreservation of spermatozoa play a crucial role in the improvement of broodstock management of cultured fish species and cryopreserved spermatozoa can continuously provide gametes for study of selective breeding. The present study aimed to investigate suitable cryoprotectants and develop a reliable spermatozoa quality method for grouper spermatozoa. Five penetrating cryoprotectants, dimethyul sulfoxide (DMSO), dimethylacetamide (DMA), glycerol, methanol and ethylene glycol (EG) were tested at three concentrations (10%, 20%, and 30%). The spermatozoa viability was assessed by the fluorescent dyes SYBR-14 and PI staining. Our results indicated that DMSO at 20% concentration was the most effective cryoprotectant for king grouper cryopreservation in comparison with methanol, EG, glycerol and DMA. The results also showed that no significance was found in viability and motility by storage for 3, 6 or 12 months in liquid nitrogen. The cryopreservation methods developed in this study are simple and this technique will assist in achieving artificial fertilization for the king grouper.