Choi Y H, Chung Y G, Walker S C, Westhusin M E, Hinrichs K
Department of Veterinary Physiology & Pharmacology, College of Veterinary Medicine, Texas A&M University, College Station 77843-4466, USA.
Zygote. 2003 Feb;11(1):77-86. doi: 10.1017/s0967199403001102.
This study was conducted to evaluate the effects of insulin-like growth factor I (IGF-I) and other media factors during oocyte maturation, and the presence of different compositions of amino acids in embryo culture medium, on the development of equine embryos. Oocytes recovered from slaughterhouse-derived ovaries were matured in vitro for 24 h and those with a polar body were subjected to intracytoplasmic sperm injection (ICSI) or nuclear transfer with adult fibroblasts (NT). For ICSI embryos, there were no significant differences in rates of morphological cleavage, cleavage with normal nuclei or average nucleus number at 96 h post-ICSI between the absence and presence of IGF-I in maturation medium, or between embryos cultured in G1.2 or a modified CZB medium (CZB-C). Embryos produced by interspecies NT (equine donor cells into bovine cytoplasts) also showed no difference in cleavage rate or average nucleus number whether cultured in G1.2 or in CZB-C. The rates of cleavage, cleavage with normal nuclei and average nucleus number of equine NT embryos were not significantly different among oocytes matured in M199 with FSH in the presence or absence of IGF-I, or in EMMI medium, which contains IGF-I, epidermal growth factor, steroid hormones, FSH and LH. There were no differences in development of equine NT embryos cultured in any of three amino acid treatments (with or without non-essential amino acids, or containing taurine, hypotaurine and cysteine only). The cleavage rate and average nucleus number of parthenogenetically activated oocytes (treated similarly to NT oocytes but not enucleated or subjected to donor cell injection) were significantly (p < 0.05) higher than those for NT embryos. These results indicate that the presence of IGF-I or of EMMI medium during in vitro maturation of equine oocytes does not have a beneficial effect on their developmental competence as assessed at 96 h. Presence or absence of non-essential amino acids in embryo culture medium does not affect development of NT embryos within the first 96 h of culture. Factors associated with enucleation or nuclear transfer decrease the developmental competence of equine NT embryos. CZB-C medium may be used for culture of equine embryos with results similar to those obtained with G1.2 medium, thus providing a base medium that may be modified for further study of culture requirements of equine embryos.
本研究旨在评估卵母细胞成熟过程中胰岛素样生长因子I（IGF-I）及其他培养基成分，以及胚胎培养基中不同氨基酸组成对马胚胎发育的影响。从屠宰场获取的卵巢中回收的卵母细胞进行体外成熟培养24小时，将排出极体的卵母细胞进行胞浆内单精子注射（ICSI）或与成年成纤维细胞进行核移植（NT）。对于ICSI胚胎，在成熟培养基中添加或不添加IGF-I，或在G1.2或改良的CZB培养基（CZB-C）中培养的胚胎，在ICSI后96小时的形态学分裂率、正常核分裂率或平均核数方面均无显著差异。种间核移植产生的胚胎（马供体细胞移入牛细胞质），无论在G1.2还是CZB-C中培养，其分裂率或平均核数也无差异。在添加或不添加IGF-I的含FSH的M199培养基中成熟的卵母细胞，或在含有IGF-I、表皮生长因子、类固醇激素、FSH和LH的EMMI培养基中成熟的卵母细胞，所产生的马核移植胚胎的分裂率、正常核分裂率和平均核数均无显著差异。在三种氨基酸处理（添加或不添加非必需氨基酸，或仅含牛磺酸、次牛磺酸和半胱氨酸）中的任何一种处理下培养的马核移植胚胎的发育情况均无差异。孤雌激活卵母细胞（处理方式与核移植卵母细胞相似，但未去核或进行供体细胞注射）的分裂率和平均核数显著高于核移植胚胎（p < 0.05）。这些结果表明，马卵母细胞体外成熟过程中IGF-I或EMMI培养基的存在，对其在96小时时评估的发育能力没有有益影响。胚胎培养基中添加或不添加非必需氨基酸在培养的前96小时内不影响核移植胚胎的发育。与去核或核移植相关的因素会降低马核移植胚胎的发育能力。CZB-C培养基可用于培养马胚胎，其结果与G1.2培养基相似，从而提供了一种可进行改良以进一步研究马胚胎培养需求的基础培养基。
