Hagedorn M, Pan R, Cox E F, Hollingsworth L, Krupp D, Lewis T D, Leong J C, Mazur P, Rall W F, MacFarlane D R, Fahy G, Kleinhans F W
Department of Reproductive Sciences, Smithsonian National Zoological Park, Washington, DC 20008, USA.
Cryobiology. 2006 Feb;52(1):33-47. doi: 10.1016/j.cryobiol.2005.09.008. Epub 2005 Dec 6.
Coral species throughout the world's oceans are facing severe environmental pressures. We are interested in conserving coral larvae by means of cryopreservation, but little is known about their cellular physiology or cryobiology. These experiments examined cryoprotectant toxicity, dry weight, water and cryoprotectant permeability using cold and radiolabeled glycerol, spontaneous ice nucleation temperatures, chilling sensitivity, and settlement of coral larvae. Our two test species of coral larvae, Pocillopora damicornis (lace coral), and Fungia scutaria (mushroom coral) demonstrated a wide tolerance to cryoprotectants. Computer-aided morphometry determined that F. scutaria larvae were smaller than P. damicornis larvae. The average dry weight for P. damicornis was 24.5%, while that for F. scutaria was 17%, yielding osmotically inactive volumes (V(b)) of 0.22 and 0.15, respectively. The larvae from both species demonstrated radiolabeled glycerol uptake over time, suggesting they were permeable to the glycerol. Parameter fitting of the F. scutaria larvae data yielded a water permeability 2 microm/min/atm and a cryoprotectant permeability = 2.3 x 10(-4) cm/min while modeling indicated that glycerol reached 90% of final concentration in the larvae within 25 min. The spontaneous ice nucleation temperature for F. scutaria larvae in filtered seawater was -37.8+/-1.4 degrees C. However, when F. scutaria larvae were chilled from room temperature to -11 degrees C at various rates, they exhibited 100% mortality. When instantly cooled from room temperature to test temperatures, they showed damage below 10 degrees C. These data suggest that they are sensitive to both the rate of chilling and the absolute temperature, and indicate that vitrification may be the only means to successfully cryopreserve these organisms. Without prior cryopreservation, both species of coral settled under laboratory conditions.
全球海洋中的珊瑚物种正面临着严峻的环境压力。我们致力于通过冷冻保存来保护珊瑚幼虫，但对它们的细胞生理学或低温生物学却知之甚少。这些实验研究了冷冻保护剂的毒性、干重、水和冷冻保护剂的渗透性（使用冷的和放射性标记的甘油）、自发冰核形成温度、冷敏感性以及珊瑚幼虫的附着情况。我们的两种珊瑚幼虫测试物种，鹿角杯形珊瑚（花边珊瑚）和盾形陀螺珊瑚（蘑菇珊瑚）对冷冻保护剂表现出广泛的耐受性。计算机辅助形态测量法确定，盾形陀螺珊瑚幼虫比鹿角杯形珊瑚幼虫小。鹿角杯形珊瑚的平均干重为24.5%，而盾形陀螺珊瑚的平均干重为17%，其非渗透体积（V(b)）分别为0.22和0.15。两个物种的幼虫都显示出随着时间推移对放射性标记甘油的摄取，这表明它们对甘油具有渗透性。对盾形陀螺珊瑚幼虫数据进行参数拟合得出水渗透性为2微米/分钟/大气压，冷冻保护剂渗透性 = 2.3×10(-4)厘米/分钟，而模型表明甘油在25分钟内达到幼虫最终浓度的90%。盾形陀螺珊瑚幼虫在过滤海水中的自发冰核形成温度为 -37.8±1.4摄氏度。然而，当盾形陀螺珊瑚幼虫以不同速率从室温冷却至 -11摄氏度时，它们的死亡率为100%。当从室温立即冷却至测试温度时，它们在低于10摄氏度时会出现损伤。这些数据表明它们对冷却速率和绝对温度都很敏感，并表明玻璃化可能是成功冷冻保存这些生物的唯一方法。在没有预先冷冻保存的情况下，这两种珊瑚在实验室条件下都会附着。
