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不同胚體外培養系統及卵母細胞亮甲酚藍染色分級對體外生產山羊胚後續發育能力之影響

Effect of culture methods of embryos and brilliant cresyl blue staining of oocytes on developmental capacity of in vitro produced caprine embryos

摘要


本研究目的是探討不同胚體外培養系統及卵母細胞成熟前利用亮甲酚藍(brilliant cresyl blue, BCB)染色分級,對體外生產山羊胚後續發育能力之影響。自活體摘除所得之卵巢表面濾泡中收集卵丘卵母細胞複合體(cumulus-oocyte complexes, COCs),隨之進行體外成熟與體外受精作用後,將其分別培養在TCM199輸卵管上皮細胞共培養系統(TCM199-goat oviduct epithelial cell, TCM199-GOEC)與合成輸卵管液(synthetic oviduct fluid plus amino acid, SOFaa)培養系統。結果顯示在卵裂率部份,培養於SOFaa顯著(p<0.05)高於培養於TCM199-GOEC者,然而在囊胚率與囊胚平均細胞數方面,培養於TCM199-GOEC者則顯著(p<0.05)高於培養於SOFaa者。於卵母細胞染色分級試驗中,COCs於體外成熟前經亮甲酚藍(BCB)染色篩選後,依卵胞質呈色狀態區分為BCB+與BCB-,將之分別進行體外成熟、體外受精作用後,培養在TCM199-GOEC系統。試驗結果顯示無論於卵裂率及囊胚率,BCB+組(77.7±15.9% and 27.5±8.1%)皆顯著(p<0.05)高於BCB-組(42.5±14.3% and 3.9±7.8%, p<0.05)。源自體外生產於TCM199-GOEC所得之6顆新鮮囊胚及9顆冷凍-解凍後囊胚,分別移置於2頭及3頭代孕母羊後,懷孕率分別為50.0%(1/2)及33.3%(1/3),胚存活率則分別為16.7%(1/6)及11.1%(1/9)。試驗結果顯示,TCM199-GOEC之體外培養系統為較佳之胚體外培養系統,且可成功產下健康的仔畜。卵母細胞成熟前利用BCB染色分級,可提升體外山羊胚生產效率。

關鍵字

山羊 體外生產 共培養 亮甲酚藍

並列摘要


The aim of this study was to evaluate the effect of different culture systems of embryos and brilliant cresyl blue (BCB) staining of oocytes prior to in vitro maturation (IVM) on subsequent developmental capacity of caprine embryos. Cumulus-oocyte-complexes (COCs) collected from the follicles in the ovariectomized ovaries were subjected to maturation, fertilization and culture in vitro. The presumed zygotes were cultured either in TCM199 with monolayer of goat oviduct epithelial cell (TCM-GOEC) or in synthetic oviduct fluid plus amino acid (SOFaa). The mean cleavage rate of presumed zygotes cultured in SOFaa was significant higher(p < 0.05) than that in TCM199-GOEC. However, the blastocyst rate and the cell number of blastocyst in TCM199-GOEC were significant higher (p < 0.05) than those in SOFaa. Thereafter, the developmental capacity of embryos derived from BCB staining selected oocytes was evaluated with TCM199-GOEC culture system. The cleavage rate and blastocyst rate were significant higher in BCB+ group (77.7 ± 15.9% and 27.5 ± 8.1%) than those in BCB- group (42.5 ± 14.3% and 3.9 ± 7.8%, p < 0.05). Six fresh blastocysts and 9 vitrified/thawed blastocysts derived from TCM199-GOEC culture system were transferred to two and three recipients, respectively, which resulted in 50% (1/2) and 33.3% (1/3) pregnancy rate. One kid was born in each of group. The results show that co-culture with oviduct epithelial cells is a superior culture method for in vitro produced caprine embryos in terms of subsequent developmental capacity of embryos. Selection of oocytes with BCB staining prior to IVM increases the efficiency of embryo production in vitro.

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