L-精氨酸对猪孤雌激活胚胎及体细胞克隆胚胎发育的影响

发布时间：2018-03-20 02:07

本文选题：猪　切入点：L-Arg　出处：《安徽农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：猪体细胞克隆技术在畜牧业遗传育种和生物医学疾病模型中起着重要的作用。而猪体细胞克隆还存在较多问题,如克隆效率较低、胎盘发育异常和克隆胎儿存在生理缺陷等。因此要实现体细胞克隆技术走出实验室,服务于动物生产以及在医学领域广泛应用,必须改进克隆胚胎的生产体系,充分发挥其发育潜力。L-精氨酸(L-Arginine,L-Arg)作为条件必需氨基酸,在日粮中补饲可提高自然受孕妊娠母猪生产性能,在体外培养液中添加也有利于猪体外受精胚胎发育。然而,关于L-Arg对猪体细胞克隆胚发育是否也有积极影响报道很少。本文探讨了在培养基中添加L-Arg对猪孤雌激活胚胎(PAEs)、体细胞克隆胚胎(NTEs)体外发育的影响;研究了在日粮中添加L-Arg对移入NTEs的代孕母猪妊娠率、产仔率的影响,具体试验及结果如下:试验一研究了在卵母细胞成熟液中添加L-Arg对猪卵母细胞体外成熟及早期PAEs发育的影响。试验组为在卵母细胞化学限定性成熟培养基中补充0.12m M、0.36 m M、0.72 m M、1.08 m M的L-Arg,对照组则采用相同培养基但不补充L-Arg。结果是相对于对照组,添加L-Arg的各试验组卵母细胞成熟率,及其来源的PAEs卵裂率、囊胚率和囊胚总细胞数均有略微改善,但差异不显著(P0.05)。结果表明,仅在卵母细胞成熟阶段添加L-Arg,不能有效提高卵母细胞成熟及着床前胚胎发育质量。试验二探讨了在胚胎培养液中添加L-Arg对PAEs发育的影响。在胚胎培养基中补充0.12 m M、0.36 m M、0.72 m M、1.08 m M的L-Arg作为试验组,采用相同培养基不补充L-Arg的为对照组。我们发现,与对照组相比,添加0.12 m M的L-Arg显著提高了PAEs的囊胚率和囊胚总细胞数(P0.05);其它三个试验组也均改善了囊胚率与囊胚总细胞数,但是差异不显著(P0.05)。结果显示,在胚胎培养阶段添加L-Arg,有助于提高着床前胚胎发育的效率和质量。试验三进一步探索了在卵母细胞成熟和胚胎培养阶段全程添加L-Arg对猪卵母细胞体外成熟及其PAEs发育的影响。以在成熟培养和胚胎培养阶段全程补充0.12 m M、0.36 m M和0.72 m M的L-Arg作为试验组,采用相同培养基不补充L-Arg的为对照组。结果显示,试验组的囊胚形成率和对照组相比有改善的趋势,不过差异并不显著(P0.05);只有添加0.72 m M的L-Arg处理组明显提高了着床前胚胎的囊胚率(P0.05)。结果提示,在卵母细胞成熟和胚胎培养阶段全程添加L-Arg,能提高着床前胚胎的发育潜力;相对于只在胚胎培养阶段添加L-Arg,可能需要更高浓度才能发挥作用。试验四探索了在胚胎培养不同的时间段添加L-Arg对PAEs发育的影响。试验组选择在胚胎培养的0-168 h、0-72 h和72-168 h补充0.12 m M的L-Arg,对照组采用相同的培养基但不补充L-Arg。试验组的卵裂率、囊胚率和总细胞数相对于对照组均有改善,但差异不显著(P0.05)。其中0-72 h组改善的趋势最不明显,而72-168 h组改善的趋势最明显。结果暗示,L-Arg对猪胚胎早期发育的影响可能主要发生在后期。试验五研究了胚胎培养阶段添加L-Arg对NTEs早期发育的影响。试验组在胚胎培养液中补充0.12 m M和0.36 m M L-Arg,对照组不补充L-Arg。相对于对照组,试验组的NTEs的卵裂率、囊胚率和囊胚总细胞数均有改善。其中0.12 m M组的囊胚率和囊胚总细胞数显著高于对照组(P0.05)。结果表明,补充L-Arg利于NTEs的体外培养,NTEs的发育效率及质量都可以得到显著提高。试验六揭示了在代孕妊娠母猪日粮中添加L-Arg对克隆胎儿发育的影响。在严格执行日粮饲喂标准的情况下,分别在试验组22头移植NTEs的代孕母猪日粮中补充0.8%的L-Arg,在对照组25头NTEs代孕母猪日粮中补充1.64%的L-丙氨酸(等氮对照)。我们发现,试验组的窝活仔胎盘平均重、窝活仔平均重、窝断奶前活仔平均重均有提高,虽然差异不显著(P0.05)。结果暗示,在NTEs代孕母猪日粮中补充L-Arg对NTEs的体内发育存在积极影响,且有利于断奶前克隆仔猪健康。综上所述,在胚胎体外培养时补充0.12 m M L-Arg,无论是PAEs或是NTEs都可以明显促进囊胚形成,并且显著增加囊胚的总细胞数。当我们在卵母细胞体外成熟与胚胎体外培养时补充0.72 m M的L-Arg,促进PAEs的囊胚形成,但囊胚质量并没有得到显著的改善。在胚胎培养不同时间段补充0.12 m M的L-Arg都能略微改善PAEs的卵裂率、囊胚率,其中72-168 h即后期补充对胚胎发育能力改善趋势较更明显(P0.05)。首次报道了在代孕母猪日粮中补饲0.8%的L-Arg,可改善胎盘的质量和NTEs的发育能力,更利于出生后克隆猪的发育。这些结果共同揭示了适量补充L-Arg可以改善猪PAEs和NTEs的发育质量和命运。
[Abstract]:Porcine somatic cell cloning technology plays an important role in the model of animal husbandry breeding and biomedical diseases. While porcine somatic cloning has many problems, such as low efficiency of cloning, cloning and abnormal development of placenta fetal physiological defects. So to realize the somatic cell cloning technology out of the laboratory service in animal production as well as in the widely used in medical field, must improve the cloned embryo production system, give full play to the developmental potential of.L- arginine (L-Arginine, L-Arg) as a condition of essential amino acids in the diet supplement can improve the production performance of pregnant sows natural conception, in vitro in culture medium is also beneficial to in vitro fertilization of porcine embryonic development. However, about L-Arg of porcine somatic cell cloned embryos have positive influence is rarely reported. This paper discusses the L-Arg is added in the medium of parthenogenetic activation embryos, somatic cell culture (PAEs) Cloned embryo development in vitro (NTEs) effect; the effect of L-Arg on pregnancy rate in NTEs surrogate sows in the diets, affect the birth rate, the specific test and the results are as follows: one was conducted to study the effect on oocyte maturation in liquid L-Arg on in vitro maturation of porcine oocytes and early development of PAEs in the test group. The oocytes chemically defined culture medium supplemented with 0.12M M of 0.36 m M, 0.72, m M 1.08, m M L-Arg, the control group with the same medium but not L-Arg. results are compared with the control group, the experimental group added L-Arg oocyte maturation rate and its origin the PAEs cleavage rate, blastocyst rate and total cell number of blastocyst was slightly improved, but the difference was not significant (P0.05). The results show that only adding L-Arg during oocyte maturation, can improve oocyte maturation and preimplantation embryo development test of two quality effectively. In the embryo culture effect of L-Arg solution on PAEs development. In embryo culture medium supplemented with 0.12 m M, 0.36 m M, 0.72 m M, 1.08 m M L-Arg as experimental group, using the same medium supplemented with L-Arg as the control group. We found that, compared with the control group, adding 0.12 m M L-Arg PAEs was significantly increased blastocyst rate and total cell number of blastocyst (P0.05); the other three experimental groups were also improved the blastocyst rate and total cell number of blastocyst, but the difference was not significant (P0.05). The results showed that in the stage of embryo culture add L-Arg, helps to improve the efficiency and quality of preimplantation development embryo. Experiment three further explored the cultivation period overall effect of L-Arg on in vitro maturation of porcine oocytes and the development of PAEs in oocyte maturation and embryo stage. In order to cultivate the whole supplement of 0.12 m M in vitro maturation and embryo, 0.36 m M and 0.72 m M L-Arg as the test Group, using the same medium supplemented with L-Arg as the control group. The results showed that the test group and the blastocyst formation rate compared with the control group improved trend, but the difference was not significant (P0.05); only 0.72 m M add L-Arg treatment group significantly improved the preimplantation blastocyst rate (P0.05). The results suggest that the addition of L-Arg in the whole culture stage, oocyte maturation and embryo, can improve the developmental potential of preimplantation embryos; compared to only in the embryo culture stage added L-Arg may need higher concentration can play a role. Experiment four explored the embryos cultured for different periods of time in the effect of L-Arg on PAEs development. In the test group embryo culture of 0-168 h, 0-72 h and 72-168 h m M L-Arg 0.12, the control group used the same cleavage medium but not complement the L-Arg. test group rate, blastocyst rate and total cell number compared with the control group were improved, but The difference was not significant (P0.05). The 0-72 group H trend of improvement is not obvious, and the 72-168 H group improved the most obvious trend. The results suggest that L-Arg may mainly occur in the late effects on the development of porcine embryos. Experiment five studied the effects of L-Arg on embryo culture stage of early development of the NTEs test group. In embryo culture supplemented with 0.12 m M and 0.36 m M L-Arg solution, the control group did not add L-Arg. to the control group, the test group NTEs cleavage rate, blastocyst rate and total cell number of blastocyst were improved. The number of blastocyst rate and blastocyst total cell m 0.12 M group is significantly higher than the control group (P0.05). The results show that the supplement of L-Arg for NTEs in vitro, the development efficiency and quality of NTEs can be significantly improved. Six test revealed in the surrogate pregnancy sow diets add the effect of L-Arg on cloned fetal development. In the strict implementation of the standard diet Under the condition of L-Arg 0.8% NTEs in group 22 were transplanted surrogate sows diets added 1.64% L- alanine in the control group 25 NTEs surrogate sow diets (such as nitrogen control). We found that the experimental group nest alive placenta average weight, average weight of live litter Wo, wo the average weight of live piglets before weaning were increased, although the difference was not significant (P0.05). The results suggested that L-Arg supplementation on the development of NTEs in vivo has positive effect on NTEs surrogate sow diets, and conducive to the health of cloned piglets before weaning. To sum up, m M added 0.12 L-Arg in embryos cultured in vitro, either PAEs or NTEs can significantly promote the blastocyst formation, and significantly increase the total number of cells in blastocysts. When we added 0.72 m M in the cultured in vitro maturation of oocytes and embryos in vitro L-Arg, promote PAEs blastocyst formation, but blastocyst quality has not been significantly improved. In the embryo culture in different periods of time of 0.12 m M L-Arg can slightly improve PAEs cleavage rate, blastocyst rate, of which 72-168 h to late embryonic development trend is more obvious improvement (P0.05). For the first time reported feeding in 0.8% surrogate sows diets L-Arg, can improve the development ability of placenta the quality and NTEs, more conducive to the development of cloned pigs after birth. These results reveal the amount of L-Arg can improve the growth quality of porcine PAEs and NTEs and destiny.

【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S828

【相似文献】