- 學位論文
日本鰻人工繁殖之階段性研究: 銀鰻性別評估以及胚胎和仔稚魚的營養代謝之探討
Phased Research on the Artificial Propagation of Japanese Eels: Assessment of the Sex of Silver Eels and Investigation on the Embryonic and Larval Nutrition Metabolism
摘要
日本鰻在東亞是一個重要的水產養殖物種，但由於人工繁殖的玻璃鰻數量稀少，使得養殖所需的種苗仍依賴野生資源。而導致人工繁殖的玻璃鰻無法量產主要有幾個因素，如在荷爾蒙注射之前無法辨識銀鰻性別、誘導性腺成熟的技術不完善、受精卵的品質不穩定、幼魚的營養代謝與需求尚未釐清，以及沒有合適的幼魚餌飼料和培育技術。在本研究中，我們著重在探討銀鰻的性別分型和胚胎及幼魚之營養代謝。首先，我們採集雌、雄銀鰻以及雄黃鰻的胸鰭組織，並萃取其總RNA來進行轉錄體定序和定量PCR的分析，從而找出性二態基因來做為性別分型之分子標記。然後我們也收集了野生胚胎、前柳葉鰻、柳葉鰻及玻璃鰻，與人工繁殖的胚胎和前柳葉鰻來進行轉錄體定序並探討其營養代謝。 在本研究中，我們預測了一個包含27,350條轉錄本的日本鰻基因模型。然後也建構了一個線上資料庫（MOLAS），可整合所有轉錄本的註解、進行轉錄體的比較分析與探索日本鰻的生理功能。在比較分析不同性別的胸鰭轉錄體後，篩選出29個候選的性二態基因。其中有10個候選基因會進行RT-qPCR，來初步評估作為標記的可行性。結果表明，三個有潛力的標記muc19、kera和dcn可用於性別分型上。此外，進一步分析所有樣本中三個標記的ΔCT值以推算閾值，結果表明若銀鰻的胸鰭中，muc19的ΔCT < 11.3，或kera的ΔCT > 11.4，或dcn的ΔCT > 6.5，則其可被評估為雌性。相反地，若銀鰻或黃鰻的胸鰭中，muc19的ΔCT > 11.3，或kera的ΔCT < 11.4，或dcn的ΔCT < 6.5，則其可被評估為雄性。性激素是否調控這些基因標記在兩性之間的差異表達，尤其是雌激素，還需要進一步探討。 另一方面，相較於野生的胚胎和前柳葉鰻，人工繁殖的胚胎和前柳葉鰻似乎有較旺盛的嘌呤、脂質、磷酸肌醇和蛋白質之代謝，這可能會加速其發育與卵黃囊和油球的消耗，進而影響人工繁殖的胚胎孵化和導致開口進食前之人工繁殖的前柳葉鰻能量缺乏。在所有的早期生活史階段，蛋白類的消化酵素以及營養運輸蛋白之表現比例，都比醣類和脂類的消化酵素以及營養運輸蛋白之表現比例來的高，這意味著高蛋白飼料可能對鰻魚的幼苗至關重要。另外，以單醣作為飼料中的醣類的主要成分可能有益於鰻魚的幼苗對醣類的利用與吸收。而且最重要的是鰻魚幼苗的營養需求可能隨著其發育而變化。為了促進鰻魚幼苗的存活率和生長速度，我們建議目前鰻魚幼苗的人工膏狀飼料之營養成分應進行改變，且隨著幼苗的發育，飼料也應進行調整。希望我們的研究成果能為日本鰻的人工繁殖帶來突破性的進展。
並列摘要
Japanese eel is an important aquaculture species in East Asia, but the seedlings for eel culture still depend on nature resources due to the scarcity of artificially propagated glass eels. Some reasons leading to fail mass production of propagated glass eels are unrecognizable sex for silver eels before hormonal injections, imperfect techniques of inducing gonadal maturation, unstable quality of fertilized eggs, unintelligible larval nutrition metabolisms, and inappropriate larval feed and larval cultivation methods. In this study, we focused on the sex typing of silver eels and nutrition metabolism of embryos and larvae. First, we sampled pectoral fins of female and male silver eels and male yellow eels to extract total RNA for transcriptome sequencing and RT-qPCR analysis, thereby identifying sexually dimorphic expressed genes as molecular markers for sex typing. Then, we collected wild embryos, preleptocephali, leptocephali, and glass eel and propagated embryos and preleptocephali to conduct transcriptome sequencing for investigating their nutrition metabolisms. In this study, we predicted a Japanese eel gene model, containing 27,350 transcripts. Then, an online database (MOLAS) for integrating the annotations of transcripts, performing the comparative transcriptome analysis, and exploring the physiological functions of Japanese eels was also constructed. The comparative analysis between the libraries of pectoral fins of different sexes identified 29 candidates of sexually dimorphic expressed genes. Ten candidates of them were conducted RT-qPCR to preliminarily assess their feasibility as markers. The RT-qPCR results indicated that there are three potential markers, muc19, kera, and dcn used for sex typing. Additionally, the ΔCT values of three markers in all samples were further analyzed to infer the thresholds, suggesting that a silver eel whose pectoral fins have the ΔCT of muc19 < 11.3, or the ΔCT of kera > 11.4, or the ΔCT of dcn > 6.5 can be assessed as a female. Conversely, a silver or yellow eel whose pectoral fins have the ΔCT of muc19 > 11.3, or the ΔCT of kera < 11.4, or the ΔCT of dcn < 6.5 can be assessed as a male. Whether the sex hormones regulate the differential expression of these gene markers between sexes or not, especially estrogen will need further exploration. On the other hand, compared to the wild embryos and preleptocephali, the propagated ones seemed to have stronger purine, lipid, inositol phosphate, and protein metabolisms. These might accelerate the development and the consumption of yolk sacs and oil globules of the propagated embryos and preleptocephali that would affect the hatching of embryos and cause the energy deficits in preleptocephali before opening their mouths to eat. The expressional percentage of protein digestive enzymes and transporters was higher than that of carbohydrate and lipid digestive enzymes and transporters at all early developmental stages, implying that high-protein feed is essential for the eel larvae. In addition, using monosaccharides as the main component of carbohydrates in the feed for propagated eel larvae might be beneficial to their utilization and absorption of carbohydrates. Most importantly, the nutritional requirements of eel larvae would vary with development. To promote the survival and growth of eel larvae, we suggest that the nutritional composition of current slurry-typed feed shall be changed, and the feed shall need modifying with the larval development. We hope that our research will bring a breakthrough to the artificial propagation of Japanese eels.