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研究生:蔡承良
研究生(外文):Stevhen Juniardi
論文名稱:優化斑馬魚之顏色偏好實驗條件
論文名稱(外文):Optimization of color preference test conditions in zebrafish
指導教授:蕭崇德賴昱衡
指導教授(外文):Chung-Der HsiaoYu-Heng lai
學位類別:碩士
校院名稱:中原大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2019
畢業學年度:107
語文別:英文
論文頁數:48
中文關鍵詞:顏色喜好斑馬魚行為學
外文關鍵詞:Zebrafish behavioridTrackerColor preferences
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斑馬魚有四色視覺,可使它們能區別紫外光和可見光,且辨別顏色能力比人類更好。近年來斑馬魚的顏色喜好已得到很多關注,乃因測試設備簡易且測試結果可用於篩選與行為相關的刺激物,但已發表的多篇斑馬魚顏色喜好相關文章有互相牴觸的結果,這更強調了顏色喜好測定方法標準化的重要性。不同實驗室發表不同結果,乃因它們使用不同的光源、不同強度的光或不同的其他因子如魚的年齡及性別等。在此研究中,我們考慮光源位置、光源強度、魚的性別和年齡因素,來標準化斑馬魚顏色喜好測試。拍攝的影像用idTracker軟體分析來決定顏色喜好指標,用紅外光相機攝影可減少因不同顏色強度造成idTracker軟體判定上所受到的干擾。我們的實驗結果顯示斑馬魚的顏色喜好受光源位置、受測魚隻的年齡和密度影響,但是不受魚的性別影響。使用標準化方法測量,我們發現pycr1與pycr2基因敲除魚展現相反的顏色喜好,從喜好紅色轉成喜好藍色(在紅藍組合測試)。我們也發現酒精可改變顏色喜好,這可能和酒精所誘發之憂鬱及抑鬱有關。我們的結論是斑馬魚顏色喜好篩檢是一種有力的工具,可應用在高通量神經藥物篩檢,且本研究所建立的方法可提供斑馬魚社群一個有用的參考方案。
Zebrafish have a tetrachromatic vision that enables them to distinguish UV from visible wavelength and discern color better than human. Recently zebrafish’s color preferences have gained a lot of attention because of the easy setup for the test and the usefulness to screen behavior-linked stimulus, but several published papers in zebrafish color preferences have contradicting results which underscore the importance of method standardization in this field. Different laboratories may report different results because of differences in light source, color intensity and several other parameters such as age and gender of fish. In this study, we aimed to standardize the color preferences test in zebrafish by considering the light source position, light intensity, gender, and age. The recorded videos were analyzed using idTracker software determine the color preferences indices. An infrared camera was used to diminish the color intensity differences which might interfere with the intensity threshold determination by idTracker software. Results showed that color preferences for zebrafish are affected by light position, age, and fish density, while not affected by fish gender. By applying optimized conditions, we found two genetic mutants carrying pycr1 or pycr2 gene deficiency display reversed color preference from red to blue color preference in a red-blue combination test. We also found ethanol has an effect on color preferences which may be related with anxiety and depression. In conclusion, zebrafish color preferences screening is a powerful tool for high-throughput neuropharmacological applications and the standard protocol established in this study provides a useful reference for the zebrafish community.
CONTENTS
摘要 I
ABSTRACT II
ACKNOWLEDGEMENT III
CONTENTS IV
LIST OF FIGURES V
LIST OF TABLES V

OPTIMIZATION OF COLOR PREFERENCES TEST IN ZEBRAFISH
1. INTRODUCTION 1
2. RESEARCH DESIGN AND MATERIALS 4
2.1 COLOR PREFERENCES ASSAY 4
2.2 DATA ANALYSIS 6
2.3 CREATION OF PYCR2 KO FISH 6
2.4 STATISTICS 7
3. RESULTS 8
3. 1 LIGHT SOURCE POSITION ON ZEBRAFISH COLOR PREFERENCES 8
3.2 TANK SIZE ON ZEBRAFISH COLOR PREFERENCES 10
3.3 SOCIAL INTERACTION ON ZEBRAFISH COLOR PREFERENCES 11
3.4 GENDER DIFFERENCE IN ZEBRAFISH COLOR PREFERENCES 13
3.5 AGE DIFFERENCE IN ZEBRAFISH COLOR PREFERENCES 13
3.6 PYCR2 KO FISH COLOR PREFERENCES 15
3.7 ETHANOL 1% 24H AND 96H TREATMENT COLOR PREFERENCES 17
4. DISCUSSION 19
5. REFERENCES 22
6. SUPPLEMENTARY DATA 25

LIST OF FIGURES
Figure 1 Experimental setup for color preferences assay in zebrafish 5
Figure 2 The effect of light intensity difference and light source position towards zebrafish swimming activity choice index 8
Figure 3 The effect of different color and light position on the zebrafish swimming activity choice index 9
Figure 4 The effect of different tank size on the zebrafish swimming activity choice index 10
Figure 5 The effect of social interaction on color preference results using the top light source 12
Figure 6 The effect of social interaction on the zebrafish swimming activity choice index 12
Figure 7 The effect of gender on color preference using the top light source 13
Figure 8 The effect of different age on color preference using top light source 14
Figure 9 The effect of light intensity difference on pycr2 KO color preference using top light source 16
Figure 10 The effect of single pycr2 KO fish in one tank on color preference using top light source 16
Figure 11 The effect of multiple pycr2 KO fish in one tank on color preference using top light source 17
Figure 12 The effect of ethanol 1% on treated wild-type using top light source 18

LIST OF TABLE
Table 1 Comparison of the conditions of the zebrafish color preferences tests used in various laboratories 8
Table 2 illumination condition for the top and bottom light source 9
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