優化黑猩猩精液冷凍保存方法

Abstract

黑猩猩與紅毛猩猩是世界上與人類親緣最接近的非人靈長類，然而，野外族群數量下降與原始棲地破壞等原因，都使這兩種物種都被國際自然保護聯盟瀕危物種紅色名錄列為瀕危物種。在域外保育部分，基於這兩種物種都有優勢個體才具較高交配權的原因，增加圈養族群數量成為域外保育中亟需解決之事。精液冷凍保存提供了一個高效率且能確保基因多樣性的解決方法。 然而，猩猩的精液在射精後即會形成凝膠，對精液冷凍保存造成了巨大影響。本篇論文旨在溶解精液形成的凝膠與隨之而來的精液冷凍保存方法之發展。根據我們實驗結果，在黑猩猩實驗中，使用0.1%第一型膠原蛋白酶，可以成功從凝膠化精液中更多溶解2.7倍之精子數，且並不影響活力、型態與受精能力相關參數。接下來，我們發展了一個使用卵黃當基礎冷凍液，並添加2.5%或7.8%甘油的慢速冷凍方式。結果顯示，在冷凍解凍後兩種方法皆可成功保存具良好品質之精液，2.5%甘油之方法，具有較高的頂體保護力；7.8%甘油之方法有較佳之活力與快速活動力。而之後，我們又改進了一個花費時間更短的快速冷凍方式且使用無添加卵黃的冷凍劑的冷凍方式。此方法與慢速冷凍法添加卵黃冷凍劑方法加相比，其具由相似的精子活力與保存更加的快速活動力、粒腺體功能性與受精能力相關參數。然而，在紅毛猩猩的實驗中，無論使用添加卵黃的慢速冷凍法搭配2.5%甘油、添加7.8%甘油，抑或是快速冷凍搭配無添加卵黃的方法，在冷凍解凍後皆不能成功保存良好的精子活力。 綜上所述，我們的研究顯示了第一型膠原蛋白酶可成功溶解黑猩猩凝膠化精液。慢速冷凍方法搭配添加卵黃冷凍劑，可成功保存黑猩猩精液。快速冷凍方法搭配無卵黃冷凍劑之方法可用於取代慢速冷凍方法搭配添加卵黃冷凍劑，此方法更容易操作，且保存粒線體保護能力更佳。

Chimpanzees (Pan troglodytes) and Orangutans (Pongo pygmaeus), have the most similar gene profile in non-human primates (NHP) to human. However, both species are endangered species on IUCN red list due to decreasing total populations and increasing destroy of their natural habitats. In both species, due to the dominant hierarchy male has the priority and exclusive mating right, which greatly affects the number of individuals within the population and subsequently decreases genes diversity of captive groups live in the extraterritorial conservation, alternative solution other than natural mating is urgently needed. To overcome these obstacles, sperm cryopreservation provides an effective way to preserve valuable genetic resources and to develop subsequent assisted reproductive technologies. However, semen ejaculates forming solid gel-like plug after ejaculation causes the difficulty for sperm cryopreservation. This thesis focuses on liquefaction of colloid semen plug and subsequently improve cryopreservation protocols for both chimpanzees and orangutans. According to our result, we showed 0.1% type I collagenase can successfully degelificated semen plug and released 2.7-fold more sperm cells without affected sperm motility, morphology, and fertilization relative parameter in chimpanzees. Subsequently, we demonstrated that the long process protocol with egg yolk -based cryoprotectant added with 2.5% and 7.8% glycerol can be used in chimpanzee and are favorable for different aspects of sperm physiology. The 2.5% protects sperm acrosome integrity with superior result, and 7.8% results in better sperm motility, progressive motility. Moreover, we also showed that improved shorter protocol with egg yolk-free cryoprotectant displayed similar motility and better progressive motility, mitochondrial functionality and fertilization relative parameter when compared with the traditional long and time-consuming protocol. However, no matter egg yolk containing, or egg yolk-free medium used in long process or short-term protocol cannot be applied to freeze orangutan semen as low sperm motility was observed after the cryopreservation procedure. In conclusions, our study demonstrated the type I collagenase could be used in semen liquefication in chimpanzees. The egg yolk medium with long process protocol can be used to preserved chimpanzee semen. The egg yolk-free medium with improve protocol can also be used to replaced egg yolk medium with beneficial effects in terms of shorter sperm handling time and better preservation of sperm mitochondria.