生產青殼蛋與(或)白殼蛋鴨隻蛋殼色素沉積作用之機制

Abstract

構成禽類蛋殼顏色之重要色素成員，已知包括：原吡咯紫質(protoporphyrin)與膽綠質(biliverdin)及其衍生物，例如鋅-膽綠質螯合物(Zn-biliverdin chelate)；其中膽綠質為血色質(heme)裂解後之一產物，係構成青色蛋殼之主要色素，而原吡咯紫質環(protoporphyrin ring)則為血色質形成過程之一中間產物，係棕色蛋殼之主要構成色素。褐色菜鴨為台灣本土主要之產蛋品系，其蛋殼顏色可由白色、淡青色至深青色不等。鴨隻之產出青殼蛋者，其殼腺可能較產出白殼蛋者，累積有更高量之膽綠質，然釐清此等假設之有力試驗證據，迄今尚且付之闕如。 本論文旨在探討鴨隻蛋殼色素沉積之作用模式，並嘗試藉由殼腺中相關mRNA表現量及其活性之論證，以釐清鴨隻殼腺中具有較高活性之血基質氧化酶，對於青殼蛋之形成是否扮演關鍵性角色？ 應用紫外光分光光度計與高壓液相層析儀偵測產青、白殼蛋鴨產蛋後6、12、18、20及23.5小時之殼腺、子宮液及蛋殼中膽綠質的濃度。結果顯示，就蛋殼及子宮液中含有之膽綠質濃度而言，鴨隻品種間確實呈現有顯著性之差異(P < 0.05)，惟就殼腺中之膽綠質濃度而言，則無顯著性之差異。相對地，就殼腺中血基質氧化酶活性而言，兩品種鴨隻分別均於全程排卵周期(Ovulatory cycle)中維持穩定；此外，免疫組織化學染色結果亦顯示，血基質氧化酶多被表現於殼腺複層立體上皮細胞之細胞質，且二品種鴨隻彼此間亦無顯著性之差異。同時，殼腺中胺基酮戊酸合成酶、第一型及第二型血基質氧化酶 mRNA之表現量，在產出青殼與白殼蛋之鴨隻間，彼此亦無顯著性之差異。鴨隻之生產白殼蛋者，其殼腺腔中經外源注入膽綠質後，確能有效增加沉積於蛋殼上之膽綠質濃度，導致蛋殼由白色轉為青色。應用蛋殼逐層溶解法，針對各層蛋殼中含有之膽綠質濃度進行分析結果發現，青殼蛋之蛋殼自第一層至第六層中含有膽綠質之濃度，分別皆顯著高於白殼蛋者；惟青殼蛋者之最外層，並未有大量膽綠質之累積，顯示鴨隻蛋殼色素之累積，確係以逐層持續不斷進行沉積模式而為之。 綜合上述試驗結果顯示，鴨隻蛋殼之為青色或白色，其顏色之差異性深受子宮液中膽綠質濃度高低之影響，而非殼腺中擁有高量膽綠質所使然，蓋殼腺內之膽綠質若未能有效釋出於子宮液中，則各層蛋殼仍然無從累積足量之膽綠質，令白殼蛋轉變成為青殼蛋。此等結果暗示，控制膽綠質自殼腺運輸至子宮液之相關機制，對於調節鴨隻蛋殼顏色，扮演重要之角色。

The pigmentation of avian eggshell is a complex process controling by two types of pigments, biliverdin and protoporphyrines. Biliverdin, a green pigment, is a byproduct of hemoglobin breakdown and will eventually display a blue and/or green color on the eggshell. In contrast to biliverdin, protoporphyrines is an immediate precursor of the heme molecule, and will give eggshells a reddish and/or brown color. The native Brown Tsaiya duck (Anas platyrhynchos) is a major laying duck breed in Taiwan and their eggshell color can vary from white to dark blue. It is possible that the shell gland of blue-shelled ducks (BSD) can accumulate higher concentration of biliverdin than white-shelled ducks (WSD) do. However there is no strong evidence to prove this assumption. In the present study, attempts were made to identify whether the higher bioactivity of heme oxygenase found in the BSD shell gland was responsible for the higher concentration of biliverdin observed. The processes of pigment deposition of eggshell in ducks were also investigated. Ultraviolet spectrophotometery and HPLC were applied to determine the concentrations of biliverdin within tissues of shell glands, uterine fluid as well as eggshells from both of BSD and WSD, at 6, 12, 18, 20 and 23.5 h post oviposition, respectively. Significant differences in biliverdin concentrations were found in the eggshell and uterine fluid of BSD and WSD, but not in the shell gland. Comparatively, the activity of heme oxygenase in the shell gland remained constant throughout the ovulatory cycle in both breed. HO1 immunoreactivity was observed in all the shell glands studied and present in the cytoplasma of stratified cuboid epithelial cells in the crypts of shell glands. The mRNA levels of aminolevulenic acid synthase one, heme oxygenase one, and heme oxygenase two in the shell gland were not significantly different between WSD and BSD. The exogenous injection of biliverdin into the shell gland antrum in the WSD resulted in increase of biliverdin deposition in the eggshell. Using the layer-by-layer dissolution assay, it was found that biliverdin concentration in the first to sixth layers of the eggshell in the BSD was significantly higher than that in the white-shelled counterpart. However, the BSD eggshells did not accumulate a large quantity of biliverdin in the most external layer, the deposition tended to increase layer by layer. Taken together, the data indicated that the color differences between egg shell of BSD and WSD were influenced by the amount of biliverdin in the uterine fluid rather than by the amount of biliverdin in the shell gland. The results imply that a mechanism controlling the biliverdin transportation from the shell gland into the uterine fluid may play a key role in regulating the duck eggshell colors.