本實驗做了形態上的觀察，發現莪朮及三稜在胚胎受精後 24 小時加藥的組別會有 個體出現心包膜膨大的情形。心包膜膨大的情況如圖 4-3，紅色虛線處為心包膜的位置，
在 a 圖對照組中可看到從卵黃囊的曲線向頭部延伸時，在心包膜的位置會略為凹陷，但 在 b,c 二圖莪朮及三稜組從卵黃囊向頭部的曲線到心包膜時，曲線有向體外突出，藉此
40
判斷莪朮及三稜在胚胎受精後 24 小時加藥對心包膜有影響。
41
a
b
c
圖 4-3 斑馬魚胚胎受藥物影響後的形態變化
(a)對照組(b)三稜胚胎受精後 24 小時加藥(3.0g/L) (c)莪朮胚胎受精後 24 小時加藥 (1.950g/L)。
相較對照組，三稜及莪朮組心包膜膨大。
*紅色虛線處為心包膜位置，藍色虛線為卵黃囊
三棱 24hr 3.0g/L
莪朮 24hr 1.950g/L
0ppm
42
莪朮組在高劑量(1.950g/L 以上)時，孵化率在胚胎受精後 0、24&48 小時有明顯的 影響，而在死亡率的部分則在胚胎受精後 0&24 小時有影響。三稜組在高劑量組(3.0g/L 以上)，孵化率在胚胎受精後 12&24 小時有較明顯影響，死亡率與莪朮影響的時間相同 都在胚胎受精後 0&24 小時有較明顯影響。由此可看到三稜跟莪朮影響其死亡的時期在 胚胎發育初期(0 小時)及中期(24 小時)；而孵化率的部分，莪朮影響的範圍為胚胎發育 的初期及中後期，三稜則為發育中期。而水蛭在本研究中只有在胚胎受精後 12 加藥對 其孵化率有影響；死亡率方面則差異不大。另在形態學的觀察上發現莪朮及三稜在胚胎 受精後 24 小時加藥對心包膜發育有影響。
43 響胚胎孵化率及死亡率。水蛭在黃超培等人(2011)對妊娠大鼠灌餵 15g/kg 的水蛭凍乾 粉發現無畸形等結果與本研究結果相同，但在以水蛭煎液灌餵妊娠 7~11 天的小鼠及 細胞生長因子(Fibrolast growth factor 1, FGF-1)降低等症狀，以及用 3~12g/kg 三稜水煎 液餵食餵食母鼠的實驗中發現幼鼠流產率上升，與前兩者相似，本研究也發現三稜濃 度大於 4.5g/L 會造成胚胎孵化率大幅下降，由此可推斷三稜導致胚胎發育不良為妨礙
44 22-25 小時為胚胎的造血期(Jagadeewaran, Liu, & Eddy, 1997)，故本研究認為莪朮及三 稜對胚胎受精後 0 及 24 小時加藥造成胚胎死亡的原因可能為其抗凝血成分的影響。
而在莪朮與三稜對胚胎受精後 24 小時加藥的組別中，本研究發現胚胎有心包膜膨大 的狀況出現，此現象在前述水蛭素的研究也有發現(Jagadeewaran, Liu, & Eddy, 1997)。
引發心包膜腫大的藥物有抑制心血管生成藥物 Sutent (Hung, Zhang, Li, Lei, Yuan, Cui, Hoi, Chan& Lee, 2012)、致斑馬魚胚胎心包膜膨大藥物 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) 、 凝 血 酶 原 抑 制 劑 (prothrombin inhabit)
45 法。取自 http://www.niea.gov.tw/analysis/method/m_n_1.asp?m_niea=B902.13B 黃超培、何為濤、趙鵬、李彬、王彥武、傅傳忠、何勵、蘇愛榮（2011）。水蛭素凍乾
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Defective soil for a fertile seed? Altered endometrial development is detrimental to pregnancy success. PloS one 7, e53098.
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Stages of Embryonic Development of the Zebrafish. Developmental Dynamics, 203(3), 253-310
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.
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附錄一
豐年蝦軟化卵殼方法
1. 豐年蝦卵 36 g 加入自來水(4-5 L)於 5 L 塑膠燒杯中打氣 1 h 2. 加入 5.4 g NaOH 於 600 ml 自來水以打氣幫助溶解
3. 以 100 μ m 直徑 20 cm 的大濾網撈起豐年蝦卵
4. 加入 300 ml 漂白水(6% 次绿酸鈉)於含 NaOH 水溶液中,並放入濾網上的豐年蝦卵, 置於攪拌器攪拌 3- 6 分鐘 (大陸金魚牌—6 分鐘; 美國紅/黃標- 3 分鐘;要嚴格遵守時間) 5. 以 100 μ m 濾網撈起豐年蝦卵,並在自來水下沖洗至少 3 分鐘
6. 在養殖桶中放入 12 L 養殖水及 300 g 海鹽並放入處理過的豐年蝦卵,打氣並照光 24 h 就可收獲 (Note: 天冷時必需加入加溫棒並調到 31℃)
Note:
1. 若不馬上孵處理過的豐年蝦卵,可以 300 ml 自來水將豐年蝦卵由濾網移在小塑膠袋中, 加入 50 g 海鹽, 攪拌溶解海鹽,放入 4 ℃冰箱 3-4 天後仍可使用。
節自臺灣斑馬魚中心中研院分支(2012)。
50
附錄二
Maintenance, breeding and typical conditions for zebrafish embryo toxicity tests Zebrafish (Danio rerio)
Origin of species India, Burma, Malakka, Sumatra
Sexual dimorphism Females: protruding belly, when carrying eggs
Males: more slender, orange tint between blue longitudinal stripes (particularly evident at the anal fin)
Feeding regime Dry flake food (max. 3% fish weight per day) 3 - 5 times daily; from three days before spawning, plus frozen adult brine shrimp (Artemia spec.) nauplii or small daphnids of appropriate size obtained from an uncontaminated source twice daily (ad libitum). To guarantee for optimal water quality, excess feces should be removed approx. one hour after feeding.
Approximate weight of adult fish
Maintenance of parenthal fish
Illumination Fluorescent bulbs (wide spectrum); 10 - 20 μmol/m2/s, 540 - 1080 lux, or 50 - 100 ft-c (ambient laboratory levels); 12 - 16 hrs photoperiod
Water temperature 26±1 °C
Water quality O2＞80% saturation, hardness: e.g., ~ 30 - 300 mg/L CaCO3, NO3-:<48mg/L, NH4+ and NO2-: <0.001 mg/L, residual
chlorine <10 μg/L, total organic chlorine <2 ng/L, pH = 6. - 8.5
Further water quality criteria
Particulate matter <20 mg/L, total organic carbon <2 mg/L, total organophosphorus pesticides <50 ng/L, total
organochlorine pesticides plus polychlorinated biphenyls <50 ng/L
Tank size for maintenance
e.g., 180 L , 1 fish/L
Water purification Permanent (charcoal filtered); possible are combinations with semi-static renewal maintenance or flow-through system with continuous water renewal
Recommended male to female ratio for breeding
2:1 (or mass spanwing)
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Breeding tanks e.g., 4 L tanks equipped with steel grid bottom and plant dummy as spawning stimulant; external heating mats, or mass spawning within the maintenance tanks
Egg structure and appearance
Stable chorion, highly transparent, non-sticky, diameter ~ 0.8 – 1.2 mm
Spawning rate A single mature female spawns at least 50 - 80 eggs per day.
Depending on the strain, spawning rates may be considerably higher. The fertilisation rate should be ≧70%. For first time spawning fish, fertilisation rates of the eggs may be lower in the first few spawns.
Test type Static, semi-static renewal, flow-through, 26±1 °C, 24-well plates (2 ml per cavity)
節自 OECD(2013)test No.236
52
附錄三
96 小時 LC50 之計算流程圖
節自中華民國行政院環境保護署環境檢驗所(2013)