【作者】 刘敏；

【导师】 刘长松；

【作者基本信息】 四川农业大学 ， 临床兽医学， 2009， 硕士

【摘要】 本试验以间情期母犬的卵母细胞为试验材料,研究了在含不同浓度促黄体素(LH)与促卵泡素(FSH)的培养液中体外培养效果的差异;不同浓度的发情犬血清对卵母细胞体外成熟效果的影响;培养时间的长短对卵母细胞核成熟效果的相关性,探讨了犬卵母细胞体外培养部分环节的影响因素。结果如下:1.LH对犬卵母细胞体外成熟的影响。在基础培养液中分别添加0、2、4、8IU/mL LH,将采集到的COGS随机移入培养液滴中,进行体外培养。结果表明:2IU/mL和4IU/mL LH组的成熟效果均明显高于对照组(P＜0.05),且2IU/mL LH组略高于4IU/mL LH组(P＞0.05)。2.FSH对犬卵母细胞体外成熟的影响。在基础培养液中分别添加0、2、4、8IU/mL FSH,将采集到的COCS随机移入培养液滴中,进行体外培养。结果表明:8IU/mL FSH组的成熟效果极显著高于对照组和2IU/mL FSH组(P＜0.01);略高于4IU/mL FSH组(P＞0.05)。3.FSH与LH协同对犬卵母细胞体外成熟的影响。将FSH和LH的各个实验浓度进行正交组合,基础液设为对照组(A组),成熟培养60h。在各协同添加组中,以FSH和LH浓度均为2IU/mL的成熟效果极显著高于对照组(P＜0.01),成熟率最高;在高浓度(8IU/mL)FSH下继续添加LH的三组培养后的成熟率与对照组比较差异不显著(P＞0.05),并极显著低于其他协同添加组(P＜0.01)。4.EDS(发情犬血清)对犬卵母细胞体外成熟的影响。筛选出的成熟培养效果最佳的FSH与LH浓度配比作为对照组(FSH与LH均为2IU/mL),试验组在对照组基础上分别添加10%和20%EDS,将采集到的COCS随机移入培养液滴中,进行体外培养。结果表明:添加10%EDS的成熟率最高,各组间差异不显著(P＞0.05)。5.培养时间对犬卵母细胞体外成熟的影响。将卵母细胞置于进一步筛选后成熟效果最好的培养液滴即基础液+2IU/mL LH+2IU/mL FSH+10%EDS中培养,分别于第0h、24h、36h、48h、60h和72h时检查卵母细胞成熟情况。结果表明:培养36h时处于GVBD期、60h处于MII期的卵母细胞比率极显著高于其它培养时段(P＜0.01)。本试验证明:间情期犬卵母细胞的体外成熟效果与FSH和LH浓度密切相关,单独添加时,FSH与LH的最佳剂量分别为8IU/mL和2IU/mL,协同添加时,当FSH和LH均为2IU/mL,成熟效果最佳;发情犬血清有助于进一步提高体外成熟效果;培养时间的长短对犬卵母细胞的核成熟效果密切相关,最佳培养时间建议36h～60h为宜。更多还原

【Abstract】 In this experiment, the dioestrus female dogs’ oocytes were chosed as test material ,the research was consisted of the difference of culturing result in vitro using different concentration of luteinizing Hormon (LH) and follicle Stimulating Hormon (FSH) nutrient fluid; the influence of different concentration of estrous dog serum in vitro mature; the raiseing time relevanced the oocytes in vitro mature effect, has discussed the dogs’ oocytes in vitro raise partial influencing factor. The result is as follows:1. Effect of LH on maturation in vitro of dogs’oocytes. 0, 2, 4, 8IU /mL LH were separately put in the basic nutrient fluid, then the random gathered COCs were moved into nutrient fluid drop and cultured in vitro. The result indicated: the maturation effect of 2IU/mL group and the 4IU/mL LH group were obviously better than the control group(P<0.05), and the 2IU/mL LH group was slightly better than the 4IU/mL LH group(P>0.05).2. Effect of FSH on maturation in vitro of dogs’oocytes..0, 2, 4, 8IU /mL FSH were separately put in the basic nutrient fluid, then the random gathered COCs were moved into nutrient fluid drop and cultured in vitro. The result indicated: the maturation effect of 8IU/mL FSH group was significantly better than the control group(P<0.01) and the 2IU/mL FSH group(P<0.01) ,and slightly better than the 4IU/mL FSH group (P>0.05).3. Effect of LH and FSH together on maturation of dogs’oocytes in vitro. Each concentration of FSH and LH was taken orthogonal combination, the basic fluid supposed for the control group (A group), raised 36h. The result indicated: in the synergetic groups , the maturing effect the FSH and LH concentration of 2IU/mL was extremely better than the control group(P<0.01 ),as well as maturing rate; the maturing rate of the three groups with highly concentration of (8IU/mL)FSH added LH were not significantly higher than the control group(P>0.05),and significantly lower than other synergetic groups(P<0.01). 4. Effect of EDS on maturation in vitro of dogs’oocytes. the best concentration of FSH and LH in vitro raise was choosed as the control group (the concentration of FSH and LH was 2IU/mL), the experimental groups were increased 10% and 20%EDS separately from the control group foundation, the randomly gathered COCs were moved into nutrient fluid drop and cultured in vitro. The result indicated: the maturing rate increased 10%EDS was highest, each group had no significant change (P>0.05).5. The raiseing time relevanced the oocytes in vitro mature effect, .the oocytes were cultured in further screening concentration of culture fluid (basic fluid +2IU/mL+ LH+2IU in /mL FSH +10%EDS), and the maturing result was checked at Oh, 24h, 36h, 48h, 60h and 72h. The result indicated: the proportion of oocyte cultured for 36h (GCBD time),60h (MII time), was extremely remarkable higher than other raiseing time (P<0.01).This experiment proved: The maturation of the dioestrus dogs’ oocytes in vitro were closely related to the concentration of FSH and LH, if add alone,the best concentration of FSH and LH was 8IU/mL, 2IU/mL; if add synergistically, FSH and LH were both 2IU/mL, the maturing result was best; the EDS was helpful to further maturing effect in vitro; the raiseing time was closely related to maturing effect of the dogs’oocyte nucleis, we suggested 36h-60h as the best of raiseing time.更多还原