Herrick Jason R, Bond Jennifer B, Magarey Genevieve M, Bateman Helen L, Krisher Rebecca L, Dunford Susan A, Swanson William F
Center for Conservation and Research of Endangered Wildlife, Cincinnati Zoo and Botanical Garden, Cincinnati, Ohio 45220, USA.
Biol Reprod. 2007 May;76(5):858-70. doi: 10.1095/biolreprod.106.058065. Epub 2007 Jan 31.
The objective of this study was to define the physiologic needs of domestic cat embryos to facilitate development of a feline-specific culture medium. In a series of factorial experiments, in vivo-matured oocytes (n = 2040) from gonadotropin-treated domestic cats were inseminated in vitro to generate embryos (n = 1464) for culture. In the initial study, concentrations of NaCl (100.0 vs. 120.0 mM), KCl (4.0 vs. 8.0 mM), KH(2)PO(4) (0.25 vs. 1.0 mM), and the ratio of CaCl(2) to MgSO(4)-7H(2)O (1.0:2.0 mM vs. 2.0:1.0 mM) in the medium were evaluated during Days 1-6 (Day 0: oocyte recovery and in vitro fertilization [IVF]) of culture. Subsequent experiments assessed the effects of varying concentrations of carbohydrate (glucose, 1.5, 3.0, or 6.0 mM; l-lactate, 3.0, 6.0, or 12.0 mM; and pyruvate, 0.1 or 1.0 mM) and essential amino acids (EAAs; 0, 0.5, or 1.0x) in the medium during Days 1-3 and Days 3-6 of culture. Inclusion of vitamins (0 vs. 1.0x) and fetal calf serum (FCS; 0 vs. 5% [v/v]) in the medium also was evaluated during Days 3-6. Development and metabolism of IVF embryos on Day 3 or Day 6 were compared to age-matched in vivo embryos recovered from naturally mated queens. A feline-optimized culture medium (FOCM) was formulated based on these results (100.0 mM NaCl, 8.0 mM KCl, 1.0 mM KH(2)PO(4), 2.0 mM CaCl(2), 1.0 mM MgSO(4), 1.5 mM glucose, 6.0 mM L-lactate, 0.1 mM pyruvate, and 0x EAAs with 25.0 mM NaHCO(3), 1.0 mM alanyl-glutamine, 0.1 mM taurine, and 1.0x nonessential amino acids) with 0.4% (w/v) BSA from Days 0-3 and 5% FCS from Days 3-6. Using this medium, ~70% of cleaved embryos developed into blastocysts with profiles of carbohydrate metabolism similar to in vivo embryos. Our results suggest that feline embryos have stage-specific responses to carbohydrates and are sensitive to EAAs but are still reliant on one or more unidentified components of FCS for optimal blastocyst development.
本研究的目的是确定家猫胚胎的生理需求，以促进猫科动物专用培养基的开发。在一系列析因实验中，将来自促性腺激素处理的家猫的体内成熟卵母细胞（n = 2040）进行体外受精，以产生用于培养的胚胎（n = 1464）。在初始研究中，评估了培养第1 - 6天（第0天：卵母细胞回收和体外受精[IVF]）培养基中NaCl（100.0对120.0 mM）、KCl（4.0对8.0 mM）、KH₂PO₄（0.25对1.0 mM）的浓度以及CaCl₂与MgSO₄·7H₂O的比例（1.0:2.0 mM对2.0:1.0 mM）。后续实验评估了培养第1 - 3天和第3 - 6天培养基中不同浓度的碳水化合物（葡萄糖，1.5、3.0或6.0 mM；L - 乳酸，3.0、6.0或12.0 mM；丙酮酸，0.1或1.0 mM）和必需氨基酸（EAA；0、0.5或1.0倍）的影响。在培养第3 - 6天还评估了培养基中维生素（0对1.0倍）和胎牛血清（FCS；0对5%[v/v]）的添加情况。将第3天或第6天的IVF胚胎的发育和代谢与从自然交配的母猫中回收的年龄匹配的体内胚胎进行比较。基于这些结果配制了一种猫科动物优化培养基（FOCM）（100.0 mM NaCl、8.0 mM KCl、1.0 mM KH₂PO₄、2.0 mM CaCl₂、1.0 mM MgSO₄、1.5 mM葡萄糖、6.0 mM L - 乳酸、0.1 mM丙酮酸和0倍EAA，含25.0 mM NaHCO₃、1.0 mM丙氨酰 - 谷氨酰胺、0.1 mM牛磺酸和1.0倍非必需氨基酸），第0 - 3天添加0.4%（w/v）牛血清白蛋白，第3 - 6天添加5% FCS。使用这种培养基，约70%的分裂胚胎发育成囊胚，其碳水化合物代谢特征与体内胚胎相似。我们的结果表明，猫科动物胚胎对碳水化合物有阶段特异性反应，对EAA敏感，但仍依赖FCS中的一种或多种未确定成分以实现最佳囊胚发育。
