影响水牛卵母细胞体外成熟、受精因素的研究
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摘要
本文主要利用屠宰场废弃的牛卵巢,比较了来自不同地区的水牛卵
    母细胞的回收数量及质量上的差异,并将其与同样方法获得的黄牛的卵
    母细胞进行比较;比较了水牛和黄牛的卵母细胞体外成熟、体外受精的
    不同;研究了影响水牛卵母细胞体外成熟、体外受精及早期胚胎发育的
    因素。研究发现:
    1.来自安徽滁洲地区的水牛的卵母细胞在数量与质量上显著高于河南
     信阳地区(p<0.01)。水牛卵母细胞在数量与质量上显著低于黄牛,
     (p<0.01)。
    2.采用发情牛血清(ECS)代替犊牛血清(FCS)进行体外成熟培养水
     牛卵母细胞,获得了更高的成熟率,(p<0.05)。
    3.在含发情牛血清的成熟培养液中添加一定量的促性腺激素,比单纯
     使用发情牛血清获得的水牛卵母细胞成熟率更高,(p<0.05)。
    4.在水牛的体外受精液中添加牛磺酸,能显著提高受精率和随后的胚
     胎发育率,(p<0.05)。
    5.将获能的精子和成熟的卵母细胞一起与输卵管上皮+颗粒细胞单层共
     同培养,结果水牛的体外受精率和囊胚率都得到显著提高,(p<0.01)。
    6.经过比较发现,输卵管上皮+颗粒细胞共培养与受精液中含牛磺酸的
     卵母细胞受精率无明显差异,但早期胚胎发育能力前者却显著高于后者,
     (p<0.01)。
    7.早期胚胎与输卵管上皮+颗粒细胞单层共同培养,比单独培养获得的
     囊胚率更高,(p<0.01)。
The buffalo has been identified as a domestic animal of great economic importance
     for Asia and Mediterranean. Although births of calves from embryos produced in vitro
     and transferred to recipient buffaloes have been recorded, only a few reports are
     available on the technology. Inherent problems of low follicular oocyte population
     coupled with fragility of buffalo semen result in low cleavage rate and
     morulalblastocyst production. Buffalo follicular oocytes fertilized and developed in
     vitro. The procedures for in vitro maturation, fertilization and culture of embryos in
     buffaloes must be improved and optimized.
    
     Nine experiments were conducted in this study to determine factors affecting the in
     vitro maturation (IVM) and in vitro fertilization (IVF) of buffalo oocytes.
    
     In experiment 1,2, the study was designed to evaluate the effects of area and types
     on ovarian follicle population and oocytes quality in buffalo and cattle. Results
     indicated that Henna’s buffalo had lower number population and poor quality compared
     to Anhui’s buffalo(p<0.05),the oocyte of buffalo was lower number population and poor
     quality than cattle(p<0.0 1).
    
     In experiment 3,the aim of this study was to elucidate the possibility of applying
     bovine IVF procedure on buffalo IVF. Aspiration of 379 buffalo ovaries has provided an
     average of 4.75oocytes per ovary, which is about 1/2 obtained from bovine ovary. The
     maturation rate (60%) and cleavage rate (25%) of buffalo ,oocytes were significantly
     lower (p    
     In experiment 4 and 5, the cumulus-oocyte complexes were cultured for 24h in
    
     TCM-199 with or without gonadotrophins and supplemented with either IO%ECS or
    
     I O%FCS. The maturation rates of oocytes cultured in TCM- 199 supplemented with
    
     lO%ECS,l0%FCS were 83.9% and 73.4%, (p.O5)respectively. Addition of lOug/ ml
    
     FSH and I OugIml LH significantly improved (p<0.05) the maturation rate in the TCM-
     199 medium supplemented with I 5%ECS (84%)
    
     In experiment 6,7 and 8, frozen-thawed spermatozoa prepared in Bracket and
     Oliphant (BO) medium and treated 1t)uglml heparin and 5mM coffeine for sperm
     capaciation. The cleavage rate of in vitro matured bovine oocytes was compared after
     fertlization in 1): Bornedium alone ,2): in BO+BOEC+CCs , 3): in BQ+taurin .The
    
    
     overall cleavage rate at 45h post insemination was greater (p     rates for treatments 2 (39.5%) and 3 (40.6%) than for treatment 1(28.6G/o3 1.8%) , the
     oocyte cleavage rates for treats 2 and 3 were similar. Addition of BOEC+CCs to the
     fertilization medium resulted in more embryos at blastocyst by 6d post insemination
    
     (p     In experiment 9,the cleaved embryos were co-cultured with buffalo oviductal
     epithelial cells for 7d post insemination. The cleavage rate and the proportion of cleaved
     embryos that developed to morula and blastocyst stages were significantly higher(p
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