猪卵母细胞玻璃化冷冻及体外成熟的研究
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摘要
猪卵母细胞的冷冻和体外成熟技术已经成为现代养猪业与生物医学领域中的一项重要技术。然而目前这些技术存在较多的问题,特别是猪卵母细胞的冷冻一直没有得到有效的解决。为进一步提高猪卵母细胞的冷冻效果和体外成熟率,本试验研究了渗透性冷冻保护剂、非渗透性冷冻保护剂、冷冻解冻步骤、不同处理方法对猪未成熟卵母细胞玻璃化冷冻的影响,并从成熟液体积、生长因子、卵泡液和条件化培养体系等方面对猪未成熟卵母体外成熟进行了研究,获得如下结果。
1.乙二醇(EG)组卵母细胞所取得的成熟率最高达到12.9%,显著好于丙二醇(PROH, 3.7%)、甘油(GLY, 1.9%)和对照组(0.0%, P<0.05),但与二甲基亚砜(DMSO)组(8.6%)比较差异不显著(P >0.05);采用两两组合后, EG+DMSO组获得的成熟率最高(9.6%),显著优于PROH+GLY组(0.0%)和对照组(0.0%,P<0.05),但与其它组相比较差异不显著。
2.在添加葡萄糖组,使用1.5M冻后获得最高成熟率(8.7%),但与使用其它浓度的葡萄糖组比较无显著差异。在蔗糖组,添加0.75M冻后获得最高成熟率(19.9%),显著高于0.25M组(8.1%, P<0.05),但与1.0M(16.9%)和0.5M(18.6%)组相比较差异不显著(P>0.05);当添加3.0 g/ml聚蔗糖时冻后成熟率最高(13.4%),但和其他各浓度聚蔗糖组比较无显著差异(P>0.05)。在枸杞多糖组,添加0.10 g/ml组冻后成熟率达21.9%,显著高于0.05 g/ml(10.5%)和0.025 g/ml(6.9%, P<0.05),但是与添加0.075 g/ml组比较差异不显著(18.7%, P>0.05)。
3.六步法(12.3%)和三步法(11.1%)所取得的冻后成熟率显著高于一步法(1.3%, P<0.05),六步法稍优于三步法,差异不显著;四步解冻法(12.7%)和三步解冻法(10.5%)所取得的冻后成熟率显著高于一步法(6.7%, P<0.05),但与二步法相比较差异不显著(7.3%, P>0.05)。卵母细胞在用CB处理的情况下较长时间暴露于冷冻液的冻后成熟率(17.3%)要显著高于短时间暴露组(4.8%, P<0.05),而无CB处理时,结果相反,较长时间暴露于冷冻液组的冻后成熟率(6.9%)显著低于短时间暴露组(16.4%, P<0.05).
4.培养猪卵母细胞时,使用500μl(73.6%)、750μl(73.3%)和1000μl(69.1%)成熟液卵母细胞成熟率要显著好于微滴培养(52.4%, P<0.05),但与使用250μl成熟液培养无显著差异(66.0%, P>0.05)
5.使用10ng/mlEGF获得的成熟率(75.7%)要显著高于使用10ng/ml FGF(60.5%)和空白对照组(57.9%, P<0.05),但与使用10ng/ml IGF-I组取得的成熟率无显著差异(68.0%, P>0.05)。
6.使用卵泡期、排卵期和黄体期卵泡液组取得的成熟率(69.8%, 65.5%, 68.4%)与对照组(67.0%)相比较无显著差异(P>0.05),但使用白体期卵泡液组取得的成熟率(54.4%)显著低于对照组(P<0.05)。
7.猪卵母细胞与几种体细胞共培养,颗粒细胞对卵母细胞体外成熟有显著的促进作用(75.8%),明显优于对照组(66.7%, P<0.05)。输卵管上皮细胞和胰岛细胞对成熟也有促进作用,但与对照组比较差异不显著(P>0.05)。
The technology of immature porcine oocyte’s vitrification and maturation has become the basic technology of modern agriculture as well as biomedical study, however, the many problems have not be resolved, especially vitrification of immature porcine oocyte. For resolving these difficulties, in present study, we studied the effect of some factors on maturation rates after vitrified-thawed immature porcine oocytes, and these factors as follow: membrane permeating, membrane non-permeating, the processes of vitrified-thawed, different pretreatments. It is the first time for Lycium barbarum polysaccharide (LBP) to be studied as membrane non-permeating cryoprotectant. In addition, the effect of the volume of culture medium, growth factor, porcine follicular fluid and co-culture medium on the maturation in vitro have been studied. The results showed:
1. The maturation rate of the group adding ethylene glycol (EG, 12.9%) was significantly higher these adding 1, 2-propanediol (PROH, 3.7%), glycerol (GLY, 1.9%) and control (0.0%, P<0.05), but was not significantly different from that adding dimethyl sulfoxide (DMSO, 8.6%). When using mixture of two cryoprotectants, the highest maturation rates was obtained in EG +DMSO group (9.6%), was significantly higher than PROH+GLY (0.0%) group and control (0.0%, P<0.05).
2. In glucose group, adding 1.5 M obtained the highest maturation rate (8.7%), but was not significantly different from other concentrations (P>0.05). In sucrose group, the highest maturation rate (19.9%) when adding 0.75 M was significantly higher than that adding 0.25 M (8.1%, P<0.05), but was not significantly different from those adding 1.0 M (16.9%) and 0.5 M (18.6%, P>0.05). The highest maturation rate (13.4%) was obtained in the group adding 3.0 g/ml ficoll, was not significantly different from those adding other concentrations ficoll. In addition, the highest maturation rate was obtained in 0.10 g/ml LBP (21.9%), and it was significantly higher than those in 0.05 M (10.5%, P<0.05) and 0.025 M (6.9%, P<0.05), but it was not significantly different from that vitrified in 0.075 g/ml LBP (18.7%, P>0.05).
3. The maturation rates of six-step (12.3%) and three-step treatment (11.1%) were significantly higher than that using one-step treatment (1.3%, P<0.05). The maturation rates of four-step (12.7%) and three-step thaw (10.5%) was significantly higher than that using one-step thaw (6.7%, P<0.05), but was not significantly different from that using two-step thaw (7.3%, P>0.05). When using cytochalasin B, the maturation rate of oocyte exposed to vitrification solution long time (17.3%) were significantly higher than that short time(4.8%,P<0.05), and without cytochalasin B, the maturation rate of oocyte exposed to vitrification solution long time (6.9%)were significantly lower than that short time(16.4%, P<0.05).
4. In vitro maturation, the maturation rates using 500 ul (73.6%)、750 ul (73.3%) and 1000 ul (69.1%) culture medium were significantly higher than that culture in 50 ul drop (52.4%, P<0.05), and were not significantly different from that using 250 ul medium(66.0%, P>0.05).
5. In vitro maturation, the maturation rate using 10 ng/ml EGF (75.7%) was significantly higher than that using 10 ng/ml FGF (60.5%) and control (57.9%, P<0.05), but was not significantly different from that using 10 ng/ml IGF-1(68.0%, P>0.05).
6. In vitro maturation, the maturation rates using porcine follicular fluid (PFF) in follicular term (69.8%) , in ovulation(65.5%) and luteal term (68.4%) were not significantly different from that control (69.0%, P>0.05). But the maturation rate using PFF in lean type term (54.4%, P<0.05) was significantly lower than that control (69.0%, P<0.05).
7. In vitro co-culture medium, the maturation rate co-cultured with granulose cells (75.8%) was significantly higher than that control group (66.7%, P<0.05). Co-culturing with oviduct epithelial or pancreas cells could improved the maturation rates, but it was not significantly different from that control (P>0.05).
1.乙二醇(EG)组卵母细胞所取得的成熟率最高达到12.9%,显著好于丙二醇(PROH, 3.7%)、甘油(GLY, 1.9%)和对照组(0.0%, P<0.05),但与二甲基亚砜(DMSO)组(8.6%)比较差异不显著(P >0.05);采用两两组合后, EG+DMSO组获得的成熟率最高(9.6%),显著优于PROH+GLY组(0.0%)和对照组(0.0%,P<0.05),但与其它组相比较差异不显著。
2.在添加葡萄糖组,使用1.5M冻后获得最高成熟率(8.7%),但与使用其它浓度的葡萄糖组比较无显著差异。在蔗糖组,添加0.75M冻后获得最高成熟率(19.9%),显著高于0.25M组(8.1%, P<0.05),但与1.0M(16.9%)和0.5M(18.6%)组相比较差异不显著(P>0.05);当添加3.0 g/ml聚蔗糖时冻后成熟率最高(13.4%),但和其他各浓度聚蔗糖组比较无显著差异(P>0.05)。在枸杞多糖组,添加0.10 g/ml组冻后成熟率达21.9%,显著高于0.05 g/ml(10.5%)和0.025 g/ml(6.9%, P<0.05),但是与添加0.075 g/ml组比较差异不显著(18.7%, P>0.05)。
3.六步法(12.3%)和三步法(11.1%)所取得的冻后成熟率显著高于一步法(1.3%, P<0.05),六步法稍优于三步法,差异不显著;四步解冻法(12.7%)和三步解冻法(10.5%)所取得的冻后成熟率显著高于一步法(6.7%, P<0.05),但与二步法相比较差异不显著(7.3%, P>0.05)。卵母细胞在用CB处理的情况下较长时间暴露于冷冻液的冻后成熟率(17.3%)要显著高于短时间暴露组(4.8%, P<0.05),而无CB处理时,结果相反,较长时间暴露于冷冻液组的冻后成熟率(6.9%)显著低于短时间暴露组(16.4%, P<0.05).
4.培养猪卵母细胞时,使用500μl(73.6%)、750μl(73.3%)和1000μl(69.1%)成熟液卵母细胞成熟率要显著好于微滴培养(52.4%, P<0.05),但与使用250μl成熟液培养无显著差异(66.0%, P>0.05)
5.使用10ng/mlEGF获得的成熟率(75.7%)要显著高于使用10ng/ml FGF(60.5%)和空白对照组(57.9%, P<0.05),但与使用10ng/ml IGF-I组取得的成熟率无显著差异(68.0%, P>0.05)。
6.使用卵泡期、排卵期和黄体期卵泡液组取得的成熟率(69.8%, 65.5%, 68.4%)与对照组(67.0%)相比较无显著差异(P>0.05),但使用白体期卵泡液组取得的成熟率(54.4%)显著低于对照组(P<0.05)。
7.猪卵母细胞与几种体细胞共培养,颗粒细胞对卵母细胞体外成熟有显著的促进作用(75.8%),明显优于对照组(66.7%, P<0.05)。输卵管上皮细胞和胰岛细胞对成熟也有促进作用,但与对照组比较差异不显著(P>0.05)。
The technology of immature porcine oocyte’s vitrification and maturation has become the basic technology of modern agriculture as well as biomedical study, however, the many problems have not be resolved, especially vitrification of immature porcine oocyte. For resolving these difficulties, in present study, we studied the effect of some factors on maturation rates after vitrified-thawed immature porcine oocytes, and these factors as follow: membrane permeating, membrane non-permeating, the processes of vitrified-thawed, different pretreatments. It is the first time for Lycium barbarum polysaccharide (LBP) to be studied as membrane non-permeating cryoprotectant. In addition, the effect of the volume of culture medium, growth factor, porcine follicular fluid and co-culture medium on the maturation in vitro have been studied. The results showed:
1. The maturation rate of the group adding ethylene glycol (EG, 12.9%) was significantly higher these adding 1, 2-propanediol (PROH, 3.7%), glycerol (GLY, 1.9%) and control (0.0%, P<0.05), but was not significantly different from that adding dimethyl sulfoxide (DMSO, 8.6%). When using mixture of two cryoprotectants, the highest maturation rates was obtained in EG +DMSO group (9.6%), was significantly higher than PROH+GLY (0.0%) group and control (0.0%, P<0.05).
2. In glucose group, adding 1.5 M obtained the highest maturation rate (8.7%), but was not significantly different from other concentrations (P>0.05). In sucrose group, the highest maturation rate (19.9%) when adding 0.75 M was significantly higher than that adding 0.25 M (8.1%, P<0.05), but was not significantly different from those adding 1.0 M (16.9%) and 0.5 M (18.6%, P>0.05). The highest maturation rate (13.4%) was obtained in the group adding 3.0 g/ml ficoll, was not significantly different from those adding other concentrations ficoll. In addition, the highest maturation rate was obtained in 0.10 g/ml LBP (21.9%), and it was significantly higher than those in 0.05 M (10.5%, P<0.05) and 0.025 M (6.9%, P<0.05), but it was not significantly different from that vitrified in 0.075 g/ml LBP (18.7%, P>0.05).
3. The maturation rates of six-step (12.3%) and three-step treatment (11.1%) were significantly higher than that using one-step treatment (1.3%, P<0.05). The maturation rates of four-step (12.7%) and three-step thaw (10.5%) was significantly higher than that using one-step thaw (6.7%, P<0.05), but was not significantly different from that using two-step thaw (7.3%, P>0.05). When using cytochalasin B, the maturation rate of oocyte exposed to vitrification solution long time (17.3%) were significantly higher than that short time(4.8%,P<0.05), and without cytochalasin B, the maturation rate of oocyte exposed to vitrification solution long time (6.9%)were significantly lower than that short time(16.4%, P<0.05).
4. In vitro maturation, the maturation rates using 500 ul (73.6%)、750 ul (73.3%) and 1000 ul (69.1%) culture medium were significantly higher than that culture in 50 ul drop (52.4%, P<0.05), and were not significantly different from that using 250 ul medium(66.0%, P>0.05).
5. In vitro maturation, the maturation rate using 10 ng/ml EGF (75.7%) was significantly higher than that using 10 ng/ml FGF (60.5%) and control (57.9%, P<0.05), but was not significantly different from that using 10 ng/ml IGF-1(68.0%, P>0.05).
6. In vitro maturation, the maturation rates using porcine follicular fluid (PFF) in follicular term (69.8%) , in ovulation(65.5%) and luteal term (68.4%) were not significantly different from that control (69.0%, P>0.05). But the maturation rate using PFF in lean type term (54.4%, P<0.05) was significantly lower than that control (69.0%, P<0.05).
7. In vitro co-culture medium, the maturation rate co-cultured with granulose cells (75.8%) was significantly higher than that control group (66.7%, P<0.05). Co-culturing with oviduct epithelial or pancreas cells could improved the maturation rates, but it was not significantly different from that control (P>0.05).
引文
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