Zhang M, Lu K H, Seidel G E
XY, Inc., ARBL Building, CSU Foothills Campus, Fort Collins, CO 80523, USA.
Theriogenology. 2003 Dec;60(9):1657-63. doi: 10.1016/s0093-691x(03)00177-8.
The objective of this study was to determine the effect of staining bovine sperm, with or without flow cytometry, on in vitro fertilization of bovine oocytes and blastocyst development. Bovine oocytes (n=4273) were fertilized with frozen-thawed sperm from three bulls that was: stained with Hoechst 33342 and sorted (into X- or Y-chromosome-bearing sperm) with flow cytometry; stained but not sorted; and not stained or sorted (Control). Oocytes, aspirated from slaughterhouse ovaries, were matured in TCM199 (supplemented with 10% fetal calf serum and 15 ng FSH, 1.0 microg LH, 1.0 microg E2/ml) for 22-24h at 39 degrees C in 5% CO(2) in air with maximum humidity. Presumptive zygotes were removed from culture and placed in chemically defined medium (CDM-1) 6-7h after insemination and cultured for 65-66h. Embryos that had cleaved by 72h post-insemination were cultured an additional 96h in CDM-2 containing 0.12 IU insulin/ml. Cleavage and blastocyst rates per oocyte inseminated were recorded on Day 3 and Days 7-8 after insemination, respectively. There was no significant difference in blastocyst rate among the three types of sperm; however, cleavage rates with stained and sorted sperm (53.1%) and unsorted, stained sperm (59.9%) were lower (P<0.05) than Control sperm (69.7%). Furthermore, there were significant differences due to semen from different bulls in cleavage and blastocyst rates.
本研究的目的是确定对牛精子进行染色（无论是否使用流式细胞仪）对牛卵母细胞体外受精和囊胚发育的影响。用来自三头公牛的冻融精子使牛卵母细胞（n = 4273）受精，这些精子分别是：用Hoechst 33342染色并用流式细胞仪分选（分为携带X或Y染色体的精子）；染色但未分选；未染色也未分选（对照）。从屠宰场卵巢吸出的卵母细胞在含有10%胎牛血清、15 ng促卵泡素、1.0 μg促黄体素、1.0 μg雌二醇/毫升的TCM199中，于39℃、5%二氧化碳、空气饱和湿度条件下培养22 - 24小时。授精后6 - 7小时，将推定的受精卵从培养物中取出，置于化学成分明确的培养基（CDM - 1）中，并培养65 - 66小时。在授精后72小时发生卵裂的胚胎，在含有0.12 IU胰岛素/毫升的CDM - 2中再培养96小时。分别在授精后第3天和第7 - 8天记录每个授精卵母细胞的卵裂率和囊胚率。三种类型精子的囊胚率没有显著差异；然而，染色并分选的精子（53.1%）和未分选、染色的精子（59.9%）的卵裂率低于对照精子（69.7%）（P<0.05）。此外，不同公牛的精液在卵裂率和囊胚率方面存在显著差异。
