Méo Simone Cristina, Leal Cláudia Lima Verde, Garcia Joaquim Mansano
Departamento de Medicina Veterinária Preventiva e Reprodução Animal, FCAV-UNESP, Via de Acesso Paulo Donato Castellane s/n, CEP 14884-900, Jaboticabal, SP, Brazil.
Anim Reprod Sci. 2004 Mar;81(1-2):35-46. doi: 10.1016/j.anireprosci.2003.09.004.
Efficient artificial activation is indispensable for the success of cloning programs. Strontium has been shown to effectively activate mouse oocytes for nuclear transfer procedures, however, there is limited information on its use for bovine oocytes. The present study had as objectives: (1). to assess the ability of strontium to induce activation and parthenogenetic development in bovine oocytes of different maturational ages in comparison with ethanol; and (2). to verify whether the combination of both treatments improves activation and parthenogenetic development rates. Bovine oocytes were in vitro matured for 24, 26, 28, and 30 h, and treated with ethanol (E, 7% for 5 min) or strontium chloride (S, 10mM SrCl(2) for 5h) alone or in combination: ethanol+strontium (ES) and strontium+ethanol (SE). Activated oocytes were cultured in vitro in synthetic oviductal fluid (SOF) medium and assessed for pronuclear formation (15-16 h), cleavage (46-48 h) and development to the blastocyst stage (D7). Treatment with ethanol and strontium promoted similar results regarding pronuclear formation (E, 20-66.7%; S, 26.7-53.3%; P>0.05) and cleavage (E, 12.8-40.6%; S, 16.1-41.9%; P>0.05), regardless of oocyte age. The actions of both strontium and ethanol were influenced by oocyte age: ethanol induced greater activation rates after 28 and 30 h of maturation (48.4 and 66.7% versus 20.0 and 23.3% for 24 and 26 h, respectively; P<0.05) and strontium after 30 h (53.3%) was superior to 24 and 26 h (26.7% for both). Blastocyst development rates were minimal in all treatments (0.0-6.3%; P>0.05), however, when the mean (+/-S.D.) cell number in blastocysts at the same maturational period was compared, strontium treatment was superior to ethanol for activation rates (82+/-5.7 and 89.5+/-7.8 versus 54 and 61, at 28 and 30 h, respectively). Improved results were obtained by combined treatments. The combination of ethanol and strontium resulted in similar pronuclear formation (ES, 36.7-83.9%; SE, 53.1-90.3%) and cleavage rates (ES, 31.3-81.3%; SE, 65.6-80.7%). Regarding embryo development, there was no difference (P>0.05) between treatments, and blastocysts were only obtained in treatment SE at 24 and 26 h (6.5% for both). It is concluded that, SrCl(2) induces activation and parthenogenetic development in bovine oocytes.
高效的人工激活对于克隆程序的成功至关重要。已证明锶能有效激活小鼠卵母细胞用于核移植程序，然而，关于其用于牛卵母细胞的信息有限。本研究的目的是：（1）. 评估与乙醇相比，锶诱导不同成熟年龄牛卵母细胞激活和孤雌发育的能力；（2）. 验证两种处理方法的组合是否能提高激活率和孤雌发育率。牛卵母细胞在体外成熟24、26、28和30小时，分别用乙醇（E，7%处理5分钟）或氯化锶（S，10mM SrCl₂处理5小时）单独处理或联合处理：乙醇 + 锶（ES）和锶 + 乙醇（SE）。激活后的卵母细胞在合成输卵管液（SOF）培养基中进行体外培养，并评估原核形成（15 - 16小时）、卵裂（46 - 48小时）和发育至囊胚阶段（第7天）的情况。无论卵母细胞年龄如何，乙醇和锶处理在原核形成（E，20 - 66.7%；S，26.7 - 53.3%；P>0.05）和卵裂（E，12.8 - 40.6%；S，16.1 - 41.9%；P>0.05）方面产生了相似的结果。锶和乙醇的作用均受卵母细胞年龄影响：乙醇在成熟28和30小时后诱导的激活率更高（分别为48.4%和66.7%，而24和26小时分别为20.0%和23.3%；P<0.05），锶在30小时后（53.3%）优于24和26小时（均为26.7%）。所有处理的囊胚发育率都很低（0.0 - 6.3%；P>0.05），然而，当比较同一成熟时期囊胚的平均（±标准差）细胞数量时，锶处理在激活率方面优于乙醇（28小时和30小时分别为82±5.7和89.5±7.8，而乙醇分别为54和61）。联合处理获得了更好的结果。乙醇和锶的组合导致相似的原核形成率（ES，36.7 - 83.9%；SE，53.1 - 90.3%）和卵裂率（ES，31.3 -
