低溫刺激下雜交吳郭魚（ Oreochromis niloticus × O. aureus ）能量代謝途徑的探討

Abstract

吳郭魚為台灣重要養殖魚種之一,其產業對台灣漁業發展上是相當重要的。惟因吳郭魚為熱帶魚種，適於高溫養殖，每在冬季寒流來襲之際，因其耐寒性欠佳而大量凍斃，產業損失甚為慘重，有鑑於此，就吳郭魚對低溫之生理反應與能量代謝的調節進行全盤性研究，探討本省主要養殖經濟魚種吳郭魚在低溫適應下能量代謝型態及途徑，為本研究之主要目標。 馴化於 25℃ 下的吳郭魚，分別以不同的低溫 25℃ 、 20℃ 、 15℃、 10℃ 進行刺激，探討其在低溫壓迫下能量利用情形，包括溫度耐受性、耗氧量、 24 小時內的血液葡萄糖、乳酸以及碳水化合物相關代謝酵素之時程變化，包括肌肉及肝臟組織中醣解作用的中磷酸果糖啟動酵素 (phosphofructokinase ）、丙酮酸啟動酵素（pyruvate kinase ）、乳酸去氫酵素（ lactate dehydrogenase ) ，糖質新生作用中的果糖－1 , 6－雙磷酸化酵素（fructose－1 , 6－biphosphatease ) ，肝醣代謝酵素肝醣合成酵素（ glycogen synthase ）、磷酸水解酵素 a ( phosphorylase a ) ，檸檬酸循環酵素中的檸檬酸合成酵素（citrate synthase ) ，五碳糖代謝中的葡萄糖－6－磷酸去氫酵素 ( glucose－6 －phosphate dehydrogenase ）。且酵素活性的變動，可反應出體內能量代謝的狀態。 魚類在不同棲息環境下生存繁衍，經由適應演化發展出對水溫變動的調節機能，出現耐溫性不同的魚種，因而，每一魚種對於水溫環境變動，具有與生俱來的適應潛力，以維持生理狀態的恆定性。魚類遭遇到低溫壓迫時，就低溫衝擊之幅度，魚類立即產生行為反應及生理上的反應與調節，以抒解低溫對生理機能產生的影響。本研究中吳郭魚之耐寒性欠佳，在 15℃ 低溫之急速刺激下，血糖量的增加非常明顯，僅僅在 6hr 內即由63.91 mg / dL 遽增至132.04m mg / dL，增加幅度達 2 倍之多。吳郭魚暴露於 10℃ 水溫中，血糖量僅由 41 . 74mg/dL ，在 1 小時內增加到 57 . 15 mg / dL ，雖然血糖量增加幅度不甚顯著，主要原因在於吳郭魚對 10℃ 之刺激反應，生理已失去調節能力，呈現衰減的結果。血漿中乳酸變動的情形，移入溫度為 15℃ 者，隨著移入時間的延長，乳酸濃度有呈現下降的趨勢，由開始血乳酸濃度 16 . 54 士 0 . 92 mg / dL 下降至 24 hr 的 12 . 11 士 0 . 28 mg / dL ，而移入溫度為 10℃ 之個體，濃度移入後 30 分鐘內仍維持在 16 . 54 士 0 . 92 mg / dL ，與移入前之測定值變化不大，但在移入後一小時，呈現衰減之現象，乳酸量則下降至 9.47 士 2 . 24mg/dL ，隨即死亡。至於氧氣消耗量之下降、是與水溫之高低及刺激持續時間呈相關性。 耐寒性較差的吳郭魚，其肝臟組織在低溫壓迫初期的適應策略為增加糖質新生及無氧代謝，尤其在臨界致死溫度下，為了應付劇烈的刺激，在壓迫時間 lhr 以內進行無氧代謝、有氧代謝及糖質新生作用，然而在低溫壓迫後期的適應策略則增加有氧代謝及肝醣分解作用。在肌肉組織中於不同壓迫溫度的適應方面，吳郭魚在低溫壓迫初期的適應策略為增加無氧代謝及糖質新生為主，尤其在 10℃ 的處理組方面，在壓迫時間 lhr 內主要進行無氧代謝、有氧代謝。至於在低溫壓迫後期的適應策略為增加五碳糖代謝作用，這顯示了吳郭魚在低水溫環境下表現不同的生理適應策略。

Tilapia, an economically important cultured teleost in Taiwan, has played a significant role in the fisheries development in the past. As a tropical species, they are suitable for culture in the warm waters. However, mass mortality of these cultured tilapia resulted from less tolerable attributes of this species to the cold, has often caused tremendous loss for the tilapia culture industry, when the severe cold current approaches in the winter season. This study is therefore aimed to comprehend the physiological responses and energy metabolism of tilapias under the cold shock in order to delineate the pattern of energy metabolism and pathways under the cold. The acclimated tilapias at 25°C were rapidly exposed to varying temperatures of 25°C , 20°C, 15°C and 10°C, and the physiological responses and energy metabolism were monitored under various cold shocks. The parameters monitored included temperature tolerance, oxygen consumption, time-course changes in plasma glucose and lactate, and the enzymes involved in carbohydrate metabolism. The monitored enzymes included phosphofructokinase, pyruvate kinase and lactate dehydrogenase in glycolysis, fructose-1,6- biphosphatase in glyconeogenesis, glycogen synthase and phosphorylase a in glycogen metabolism, citrate synthase in Kreb’s cycle, and glucose-6-phosphate dehydrogenase in pentose shunt pathway. The kinetics of energy metabolism is quantitatively reflected by the changes in the enzyme activities. Adaptability to fluctuating environmental temperatures developed through adaptation and evolution, enable fish to survive and propagate in a variety of habitat niches. In consequence, fish have also developed mechanisms for capacity adaptation, by which physiological processes can be maintained homeostasis. Under the cold shock, fish will display behavioral responses and physiological regulations, by which fish are able to compensate their physiological processes to the environmental temperature changes. Remarkable hyperglycemic responses were detected in tilapias under the cold shock at 15°C, the plasma glucose rapidly increased from 63.91 mg/dl to 132.04 mg/dl in 6 hr, about two-fold increase over the original level. However, a slight elevation in plasma glucose, from 41.74 mg/dl to 57.15 mg/dl in 1 hr, was observed, when the tilapias were exposed acutely to 10°C The hyperglycemic response at this lethal temperature was found less pronounced as compared to the previous. The observations suggested that the tilapias under 10°C cold shock were unable to perform physiolozical regalation and compensation properly. The stress responses and the exhaustion of regulatory capability were indicated. With respect to the lactocemic responses under 15°C, the plasma lactate concentration depressed gradually from the initiation of cold shock treatment, decreased from 16.54±0.92 g/dl down to 12.11±0.28 mg/dl in 24 hr. At the extreme temperature of 10°C, the plasma lactate maintained at the constant level for 30 min, followed by a notable depression down to 9.47±2.24 mg/dl at the end of 1 hr, and eventually physical exhaustion and death . The changes in the oxygen consumption, which is a reliable biological indicator for the stress responses, was found highly correlated with the temperatures employed and sustained duration of cold shock. The pathway of energy metabolism shifted with persistence of cold shock stimulation. In hepatic tissues, glyconeogenesis and anaerobic pathway were dominant in the initial phase of cold shock at sublethal temperature, and aerobic metabolism was supplemented at the lethal temperature, while in the later phase, the energy were derived primarily through glycogenolysis and aerobic pathway. Similar observations were obtained in the muscular tissues in the initial cold shock period, but the pentose shunt pathway was the most important source of energy towards the later period of cold shock. Evidences suggested that the tilapias display temporal changes in the compensation strategy under cold temperatures.