Mingoti G Z, Caiado Castro V S D, Méo S C, Barretto L S S, Garcia J M
School of Veterinary Medicine, Department of Animal Health, UNESP, Rua Clóvis Pestana, 793, 16050-680, Araçatuba, São Paulo, Brazil.
Zygote. 2009 Nov;17(4):321-8. doi: 10.1017/S0967199409005450. Epub 2009 May 22.
SummaryAiming to improve in vitro production of bovine embryos and to obtain supplements to replace serum for in vitro maturation (IVM), this study evaluated the effects of macromolecular supplementation of IMV medium (bovine serum albumin - BSA, polyvinyl alcohol - PVA, polyvinyl pyrrolidone - PVP, Ficoll, KnockoutSR, or fetal calf serum - FCS) and oxygen tension [5% CO2 in air (20% O2) or 5% CO2, 5% O2 and 90% N2 (5% O2)] on oocyte maturation and embryo development. Nuclear progression to germinal vesicle breakdown, metaphase I and metaphase II stages were evaluated and overall results revealed that undefined (FCS) and semi-defined (BSA) media gave better results at 20% O2 and defined media (PVA, PVP and Ficoll) at 5% O2. Independent of macromolecule supplement, IVM at 20% O2 was considered optimal for nuclear maturation. To evaluate embryo development, oocytes matured in the previously described conditions were fertilized and cultured at the same oxygen tension used for IVM and assessed for cleavage (43.0 to 74.8%) and development to morulae (16.4 to 33.8%), blastocyst (7.7 to 52.9%) and hatched blastocyst (9.6 to 48.1%). Apart from oxygen tension, all treatments, except Knockout (22.7%), gave similar results for blastocyst development (26.5 to 38.7%). Independently of macromolecule supplement, higher development rates were obtained in an oxygen tension of 20% O2 (67.4% cleavage, 29.2% morulae, 40.8% blastocyst and 34.0% hatched blastocyst) when compared with 5% O2 (52.5, 21.8, 18.2 and 15.6%, respectively). This study indicates that BSA, PVA, PVP and Ficoll can replace serum during IVM and that the optimal atmospheric condition for in vitro production of bovine embryos is 5% CO2 and 20% O2.
摘要
为了提高牛胚胎的体外生产效率,并获得可替代血清用于体外成熟(IVM)的补充剂,本研究评估了IMV培养基的大分子补充剂(牛血清白蛋白 - BSA、聚乙烯醇 - PVA、聚乙烯吡咯烷酮 - PVP、聚蔗糖、KnockoutSR或胎牛血清 - FCS)以及氧张力[空气中5%二氧化碳(20%氧气)或5%二氧化碳、5%氧气和90%氮气(5%氧气)]对卵母细胞成熟和胚胎发育的影响。评估了核进展至生发泡破裂、中期I和中期II阶段的情况,总体结果显示,未定义培养基(FCS)和半定义培养基(BSA)在20%氧气条件下效果更好,而定义培养基(PVA、PVP和聚蔗糖)在5%氧气条件下效果更好。与大分子补充剂无关,20%氧气条件下的IVM被认为对核成熟最为适宜。为了评估胚胎发育情况,将在上述条件下成熟的卵母细胞进行受精,并在用于IVM的相同氧张力下进行培养,评估其卵裂率(43.0%至74.8%)以及发育至桑葚胚(16.4%至33.8%)、囊胚(7.7%至52.9%)和孵化囊胚(9.6%至48.1%)的情况。除了氧张力外,所有处理组(除Knockout组为22.7%外)的囊胚发育情况相似(26.5%至38.7%)。与大分子补充剂无关,与5%氧气条件(分别为52.5%、21.8%、18.2%和15.6%)相比,20%氧气条件下的发育率更高(67.4%卵裂、29.2%桑葚胚、40.8%囊胚和34.0%孵化囊胚)。本研究表明,BSA、PVA、PVP和聚蔗糖在IVM过程中可替代血清,并且牛胚胎体外生产的最佳气体条件是5%二氧化碳和20%氧气。