藍胸鶉胚生理零度現象之探討

Abstract

在現今的家禽產業中，將剛產下的蛋儲存在生理零度下，以益於運輸等農場管理作業為常見的做法。生理零度在部分的鳥類及爬蟲類中均可以觀察到，當溫度低於生理零度時，胚細胞活性會逐漸降低而導致胚發育將被抑制，再當溫度回升至生理零度以上時，胚則會繼續發育。雖然，早先就已發現持續儲存雞蛋超過7天，會逐漸降低胚的孵化率及雛雞的品質，並且增加總胚孵化的時間，但一直到近年來才有研究指出，長時間低溫儲存會誘導胚細胞走向細胞凋亡及細胞壞死，導致胚在發育的過程中缺乏足夠的細胞數，而促使胚的孵化率下降。至今，致使胚細胞能抵抗低溫或最終死亡之分子機制，吾人仍然一無所知。 本研究擬以藍胸鶉為研究模式，為瞭解藍胸鶉胚之早期發育過程，我們以中性紅就其早期胚發育進行染色，發現藍胸鶉胚於胚早期之發育速度較雞胚與日本鵪鶉胚快。為瞭解使藍胸鶉胚發育的最低溫度，我們將新鮮的藍胸鶉胚於28、25及21 °C孵化4天，發現藍胸鶉胚在21 °C的環境下就會停止發育。為了更進一步了解藍胸鶉胚最適生理零度之條件，我們將藍胸鶉胚以不同儲存溫度 (16及21 °C)、不同儲存天數 (3、7及14天) 以及有無預孵化6小時作為三種不同影響生理零度之因子，並且記錄其處理前蛋重、孵化率、孵化時間及胚死亡率，統計分析的結果顯示：隨著儲存時間的加長，孵化率隨之下降，且較低的儲存溫度 (16 °C) 相較於較高的儲存溫度 (21 °C) 具有較佳的孵化率；另一方面，經過長時間的儲存後，其總胚孵化時間相較於新鮮的藍胸鶉胚長。在胚的死亡情形中，隨著儲存時間的加長，早期胚的死亡率隨之上升，而晚期胚的死亡率，則不受儲存條件的影響。為瞭解藍胸鶉胚於生理零度下儲存對microRNA表現之影響，我們將新鮮剛產下的藍胸鶉胚及經過16 °C儲存3天的藍胸鶉胚進行小分子核糖核酸次世代定序及qPCR的分析，結果顯示藍胸鶉胚經過生理零度下的儲存後，會改變特定microRNA的表現量，包括：bmo-miR-2761-3p、mmu-miR-6995-3p,、tgu-miR-19b-2-5p及mmu-miR-7054-3p。 綜觀上述之結果，本研究指出藍胸鶉胚之生理零度為21 °C，且超過一週的長時間儲存需要比21 °C更低的溫度，對孵化率較佳。最後，本次試驗亦成功發現藍胸鶉胚於生理零度下儲存，會改變特定microRNA的表現量，因此，microRNA有可能在這生理零度的現象中扮演重要的角色。

The fertilized avian embryonic disc can remain dormant below the temperature of physiological zero. However, long-term storage of the eggs below physiological zero may negatively affects embryo performance. Previous studies indicate long-term storage may increase cell death and affect embryonic metabolism. However, the mechanisms that support the survival of the embryo after prolonged dormancy remain unclear. In this study, we used blue-breasted quail as our model animal due to its small size, high fecundity and short incubation period. To find out whether the phenomenon of physiological zero also exists in blue-breasted quail, we incubated freshly laid blue-breasted quail eggs at 28, 25 or 21 °C for 4 days. The embryonic development was slower as the temperature decreased and completely stopped at 21 °C. To test whether different storage conditions affect the performance of eggs, we incubated eggs with different temperatures (16 and 21 °C) and storage periods (3, 7 and 14 days) with or without a 6-hour pre-incubation at 37.5 °C, and the hatchability was calculated. The hatchability indicates that eggs stored at 16 °C was significantly better than at 21 °C and 3-day storage was significantly better than 14-day storage. As recent studies suggest that microRNA is a critical measure for cells to cope with cold-shock, the small RNA profiles were compared between freshly laid eggs and eggs stored at 16 °C for three days, and eleven significantly regulated miRNA candidates have been identified. In conclusion, we have found out that the early embryo of blue-breasted quail remains dormant below 21 °C and an even lower temperature should be used when the storage duration is longer than 7 days. In addition, microRNA profile is significantly changed during the storage under physiological zero and these microRNAs might play a role in this phenomenon.