在体外成熟培养基中用卵泡液替代血清和激素添加剂：对水牛卵母细胞体外成熟、受精及后续发育的影响

Replacement of serum and hormone additives with follicular fluid in the IVM medium: effects on maturation, fertilization and subsequent development of buffalo oocytes in vitro.

作者信息

Chauhan M S, Palta P, Das S K, Katiyar P K, Madan M L

机构信息

Embryo Biotechnology Centre National Dairy Research Institute, Karnal-132001, India.

出版信息

Theriogenology. 1997 Aug;48(3):461-9. doi: 10.1016/s0093-691x(97)00255-0.

DOI:10.1016/s0093-691x(97)00255-0

PMID:16728142

Abstract

Buffalo follicular fluid was used in the IVM medium in place of serum and hormone additives for stimulating nuclear and cytoplasmic maturation of buffalo oocytes in vitro. Follicular fluid (buFF) was aspirated from visible surface follicles from buffalo ovaries. Cumulus oocyte complexes (COCs) were matured for 24 to 26 h at 38.5 degrees C, 5% CO(2) in air in the maturation medium (TCM-199). When used, the concentration of fetal bovine serum (FBS) was 10% and that of FSH-P was 5 mug/ml. In Experiment 1 TCM-199 was supplemented with 1) FBS, 2) FBS + FSH-P, 3) 20% buFF and 4) 40% buFF. The matured oocytes were denuded and stained with Giemsa stain to study nuclear maturation. The proportion of oocytes which completed nuclear maturation was similar in medium containing FSH (74%) and 20 or 40% buFF (67%), which was higher (P < 0.05) than in medium with FBS but without FSH or buFF (47%). In Experiment 2, which was aimed at examining the effects of buFF on cumulus expansion and rates of fertilization and subsequent development to the blastocyst stage after IVF, the maturation medium was supplemented with 1) FBS + FSH-P, 2) 20% buFF and 3) 40% buFF. The COCs matured in medium containing 20 or 40% buFF had significantly higher (P < 0.01) cumulus expansion than those matured in medium with FBS + FSH-P. Of the COCs matured in medium with FBS + FSH-P and 20 or 40% buFF, the fertilization rates indicated by the incidence of cleavage (56, 51 and 52%, respectively) and the proportion of cleaved COCs developing to morula (58, 54 and 57%, respectively) and blastocyst stage (30, 31 and 35%, respectively) were not significantly different. In Experiment 3, supplementation of the maturation medium with 1) FBS + FSH-P and 2) FBS + FSH-P + 20% buFF resulted in similar rates of morulae (41 and 38%, respectively) and blastocysts (31 and 25%, respectively), indicating that simultaneous presence of FBS, FSH-P and buFF did not have an additive effect on embryo yield. The results show that the gonadotropin and serum source in the IVM medium can be replaced by buFF at the 20% level to achieve comparable morula and blastocyst yields.

摘要

在体外成熟（IVM）培养基中，使用水牛卵泡液替代血清和激素添加剂，以刺激水牛卵母细胞的核成熟和胞质成熟。从水牛卵巢可见表面卵泡中抽吸卵泡液（buFF）。卵丘-卵母细胞复合体（COCs）在成熟培养基（TCM-199）中于38.5℃、5%二氧化碳（CO₂）的空气中培养24至26小时。使用时，胎牛血清（FBS）的浓度为10%，促卵泡素-P（FSH-P）的浓度为5微克/毫升。在实验1中，向TCM-199中添加：1）FBS；2）FBS + FSH-P；3）20% buFF；4）40% buFF。对成熟的卵母细胞进行去卵丘处理，并用吉姆萨染色以研究核成熟情况。在含有FSH的培养基（74%）和20%或40% buFF的培养基（67%）中，完成核成熟的卵母细胞比例相似，这一比例高于（P < 0.05）不含FSH或buFF的FBS培养基（47%）。在实验2中，旨在研究buFF对卵丘扩展、体外受精（IVF）后的受精率以及随后发育至囊胚阶段的影响，向成熟培养基中添加：1）FBS + FSH-P；2）20% buFF；3）40% buFF。在含有20%或40% buFF的培养基中成熟的COCs，其卵丘扩展显著高于（P < 0.01）在含有FBS + FSH-P的培养基中成熟的COCs。在含有FBS + FSH-P以及20%或40% buFF的培养基中成熟的COCs，由卵裂发生率表示的受精率（分别为56%、51%和52%）以及卵裂后的COCs发育至桑葚胚阶段（分别为58%、54%和57%）和囊胚阶段（分别为30%、31%和35%）的比例并无显著差异。在实验3中，向成熟培养基中添加：1）FBS + FSH-P；2）FBS + FSH-P + 20% buFF，得到的桑葚胚率（分别为41%和38%）和囊胚率（分别为31%和25%）相似，表明FBS、FSH-P和buFF同时存在对胚胎产量没有累加效应。结果表明，IVM培养基中的促性腺激素和血清来源可以用20%水平的buFF替代，以获得相当的桑葚胚和囊胚产量。