In vitro developmental competence of in vitro-matured bovine oocytes fertilized and cultured in completely defined media
In vitro developmental competence of in vitro-matured bovine oocytes fertilized and cultured in completely defined media
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DOI：
10.1095/biolreprod55.2.333
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发表时间：
1996-08-01
影响因子：
3.6
通讯作者：
Brackett, BG
中科院分区：
文献类型：
--
作者：
Keskintepe, L;Brackett, BG
目的是建立一个体外系统，使牛卵母细胞可以在没有血清、牛血清蛋白或体细胞支持的情况下成熟、受精并培养到囊胚期。培养基由添加羊LH （oLH）的改良组织培养基199 （mTCM 199）组成，用于成熟（IVM），实验性改变改良的定义培养基（mDM）用于精子选择和授精（IVF），柠檬酸盐+合成输卵管液+非必需氨基酸（c-SOF+NEA）用于受精卵/胚胎培养（IVC）。研究了肝素、BSA、聚乙烯醇（PVA）、青霉胺(P)、Hepes和碳酸氢钠（NaHCO3）的作用。结果包括卵母细胞在受精后48 h分裂，120 h达到胚乳，168 h形成囊胚，216 h形成囊胚的比例（pi）。当体外受精培养基中含有0.5 mg P + 1.0 mg PVA/ml， Hepes不超过10 mM时，当IVC中存在3.0 mg PVA/ml和10 mM Hepes时，获得最佳结果。不同浓度的NaHCO3，从25 mM到50 mM，在试管婴儿期间没有改变结果。在特定条件下产生的胚胎在玻璃化后仍能存活，解冻后96小时的体外发育证明了这一点。在添加LH的培养基中成熟的牛卵母细胞在化学条件下受精并培养到囊胚期，其结果与之前报道的包含血清、牛血清蛋白和/或体细胞后的结果相当。
The objective was to establish an in vitro system in which bovine oocytes can be matured, fertilized, and cultured up to the blastocyst stage without support of serum, BSA, or somatic cells. Media consisted of modified tissue culture medium 199 (mTCM 199) with ovine LH (oLH) for maturation (IVM), experimental alterations of modified defined medium (mDM) for sperm selection and insemination (IVF), and citrate + synthetic oviductal fluid + nonessential amino acids (c-SOF+NEA) for zygote/embryo culture (IVC). Effects of heparin, BSA, polyvinyl alcohol (PVA), penicillamine (P), Hepes, and sodium bicarbonate (NaHCO3) were studied. Results included proportions of oocytes that cleaved by 48 h and that reached morulae by 120 h, blastocysts by 168 h, and expanded blastocysts by 216 h postinsemination (pi). Best results were obtained when the IVF medium included 0.5 mg P + 1.0 mg PVA per milliliter with no more than 10 mM Hepes, and when 3.0 mg PVA/ml and 10 mM Hepes were present for IVC. Different concentrations of NaHCO3, up to 50 mM from 25 mM, during IVF did not alter results. Embryos produced in defined conditions yielding the best results remained viable after vitrification as evidenced by continued development in vitro for 96 h postthawing. Bovine oocytes matured in defined medium supplemented with LH were fertilized and cultured up to the blastocyst stage in chemically defined conditions that afforded results comparable to those reported earlier after inclusion of serum, BSA, and/or somatic cells.