本文選題：豬　切入點(diǎn)：L-Arg　出處：《安徽農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：豬體細(xì)胞克隆技術(shù)在畜牧業(yè)遺傳育種和生物醫(yī)學(xué)疾病模型中起著重要的作用。而豬體細(xì)胞克隆還存在較多問題,如克隆效率較低、胎盤發(fā)育異常和克隆胎兒存在生理缺陷等。因此要實(shí)現(xiàn)體細(xì)胞克隆技術(shù)走出實(shí)驗(yàn)室,服務(wù)于動(dòng)物生產(chǎn)以及在醫(yī)學(xué)領(lǐng)域廣泛應(yīng)用,必須改進(jìn)克隆胚胎的生產(chǎn)體系,充分發(fā)揮其發(fā)育潛力。L-精氨酸(L-Arginine,L-Arg)作為條件必需氨基酸,在日糧中補(bǔ)飼可提高自然受孕妊娠母豬生產(chǎn)性能,在體外培養(yǎng)液中添加也有利于豬體外受精胚胎發(fā)育。然而,關(guān)于L-Arg對(duì)豬體細(xì)胞克隆胚發(fā)育是否也有積極影響報(bào)道很少。本文探討了在培養(yǎng)基中添加L-Arg對(duì)豬孤雌激活胚胎(PAEs)、體細(xì)胞克隆胚胎(NTEs)體外發(fā)育的影響;研究了在日糧中添加L-Arg對(duì)移入NTEs的代孕母豬妊娠率、產(chǎn)仔率的影響,具體試驗(yàn)及結(jié)果如下:試驗(yàn)一研究了在卵母細(xì)胞成熟液中添加L-Arg對(duì)豬卵母細(xì)胞體外成熟及早期PAEs發(fā)育的影響。試驗(yàn)組為在卵母細(xì)胞化學(xué)限定性成熟培養(yǎng)基中補(bǔ)充0.12m M、0.36 m M、0.72 m M、1.08 m M的L-Arg,對(duì)照組則采用相同培養(yǎng)基但不補(bǔ)充L-Arg。結(jié)果是相對(duì)于對(duì)照組,添加L-Arg的各試驗(yàn)組卵母細(xì)胞成熟率,及其來源的PAEs卵裂率、囊胚率和囊胚總細(xì)胞數(shù)均有略微改善,但差異不顯著(P0.05)。結(jié)果表明,僅在卵母細(xì)胞成熟階段添加L-Arg,不能有效提高卵母細(xì)胞成熟及著床前胚胎發(fā)育質(zhì)量。試驗(yàn)二探討了在胚胎培養(yǎng)液中添加L-Arg對(duì)PAEs發(fā)育的影響。在胚胎培養(yǎng)基中補(bǔ)充0.12 m M、0.36 m M、0.72 m M、1.08 m M的L-Arg作為試驗(yàn)組,采用相同培養(yǎng)基不補(bǔ)充L-Arg的為對(duì)照組。我們發(fā)現(xiàn),與對(duì)照組相比,添加0.12 m M的L-Arg顯著提高了PAEs的囊胚率和囊胚總細(xì)胞數(shù)(P0.05);其它三個(gè)試驗(yàn)組也均改善了囊胚率與囊胚總細(xì)胞數(shù),但是差異不顯著(P0.05)。結(jié)果顯示,在胚胎培養(yǎng)階段添加L-Arg,有助于提高著床前胚胎發(fā)育的效率和質(zhì)量。試驗(yàn)三進(jìn)一步探索了在卵母細(xì)胞成熟和胚胎培養(yǎng)階段全程添加L-Arg對(duì)豬卵母細(xì)胞體外成熟及其PAEs發(fā)育的影響。以在成熟培養(yǎng)和胚胎培養(yǎng)階段全程補(bǔ)充0.12 m M、0.36 m M和0.72 m M的L-Arg作為試驗(yàn)組,采用相同培養(yǎng)基不補(bǔ)充L-Arg的為對(duì)照組。結(jié)果顯示,試驗(yàn)組的囊胚形成率和對(duì)照組相比有改善的趨勢(shì),不過差異并不顯著(P0.05);只有添加0.72 m M的L-Arg處理組明顯提高了著床前胚胎的囊胚率(P0.05)。結(jié)果提示,在卵母細(xì)胞成熟和胚胎培養(yǎng)階段全程添加L-Arg,能提高著床前胚胎的發(fā)育潛力;相對(duì)于只在胚胎培養(yǎng)階段添加L-Arg,可能需要更高濃度才能發(fā)揮作用。試驗(yàn)四探索了在胚胎培養(yǎng)不同的時(shí)間段添加L-Arg對(duì)PAEs發(fā)育的影響。試驗(yàn)組選擇在胚胎培養(yǎng)的0-168 h、0-72 h和72-168 h補(bǔ)充0.12 m M的L-Arg,對(duì)照組采用相同的培養(yǎng)基但不補(bǔ)充L-Arg。試驗(yàn)組的卵裂率、囊胚率和總細(xì)胞數(shù)相對(duì)于對(duì)照組均有改善,但差異不顯著(P0.05)。其中0-72 h組改善的趨勢(shì)最不明顯,而72-168 h組改善的趨勢(shì)最明顯。結(jié)果暗示,L-Arg對(duì)豬胚胎早期發(fā)育的影響可能主要發(fā)生在后期。試驗(yàn)五研究了胚胎培養(yǎng)階段添加L-Arg對(duì)NTEs早期發(fā)育的影響。試驗(yàn)組在胚胎培養(yǎng)液中補(bǔ)充0.12 m M和0.36 m M L-Arg,對(duì)照組不補(bǔ)充L-Arg。相對(duì)于對(duì)照組,試驗(yàn)組的NTEs的卵裂率、囊胚率和囊胚總細(xì)胞數(shù)均有改善。其中0.12 m M組的囊胚率和囊胚總細(xì)胞數(shù)顯著高于對(duì)照組(P0.05)。結(jié)果表明,補(bǔ)充L-Arg利于NTEs的體外培養(yǎng),NTEs的發(fā)育效率及質(zhì)量都可以得到顯著提高。試驗(yàn)六揭示了在代孕妊娠母豬日糧中添加L-Arg對(duì)克隆胎兒發(fā)育的影響。在嚴(yán)格執(zhí)行日糧飼喂標(biāo)準(zhǔn)的情況下,分別在試驗(yàn)組22頭移植NTEs的代孕母豬日糧中補(bǔ)充0.8%的L-Arg,在對(duì)照組25頭NTEs代孕母豬日糧中補(bǔ)充1.64%的L-丙氨酸(等氮對(duì)照)。我們發(fā)現(xiàn),試驗(yàn)組的窩活仔胎盤平均重、窩活仔平均重、窩斷奶前活仔平均重均有提高,雖然差異不顯著(P0.05)。結(jié)果暗示,在NTEs代孕母豬日糧中補(bǔ)充L-Arg對(duì)NTEs的體內(nèi)發(fā)育存在積極影響,且有利于斷奶前克隆仔豬健康。綜上所述,在胚胎體外培養(yǎng)時(shí)補(bǔ)充0.12 m M L-Arg,無論是PAEs或是NTEs都可以明顯促進(jìn)囊胚形成,并且顯著增加囊胚的總細(xì)胞數(shù)。當(dāng)我們?cè)诼涯讣?xì)胞體外成熟與胚胎體外培養(yǎng)時(shí)補(bǔ)充0.72 m M的L-Arg,促進(jìn)PAEs的囊胚形成,但囊胚質(zhì)量并沒有得到顯著的改善。在胚胎培養(yǎng)不同時(shí)間段補(bǔ)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即后期補(bǔ)充對(duì)胚胎發(fā)育能力改善趨勢(shì)較更明顯(P0.05)。首次報(bào)道了在代孕母豬日糧中補(bǔ)飼0.8%的L-Arg,可改善胎盤的質(zhì)量和NTEs的發(fā)育能力,更利于出生后克隆豬的發(fā)育。這些結(jié)果共同揭示了適量補(bǔ)充L-Arg可以改善豬PAEs和NTEs的發(fā)育質(zhì)量和命運(yùn)。
[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.

【學(xué)位授予單位】：安徽農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S828

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