牛体外受精早期胚胎培养体系优化的研究
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摘要
研究旨在筛选牛体外受精的最优培养体系,提高体外受精的囊胚孵化率。本研究将培养体系分为四组,CR1aa组,mCR组,CR1aa前+mCR后组,mCR前+CR1aa后,通过统计胚胎发育情况。结果显示CR1aa组卵裂率,八细胞率,囊胚率,孵化率分别为82.29±0.58,48.79±1.12,24.99±0.28,14.42±0.86;mCR组卵裂率,八细胞率,囊胚率,孵化率分别为57.18±1.61,26.48±0.53,22.15±1.20,18.12±0.57;CR1aa前+mCR后组卵裂率,八细胞率,囊胚率,孵化率分别为82.24±1.32,48.60±1.93,34.07±1.04,24.87±0.66;mCR前+CR1aa后组卵裂率,八细胞率,囊胚率,孵化率分别为58.80±2.26,26.77±1.21,24.15±0.73,13.10±1.09;从四组结果中可以看出CR1aa前培养液与mCR前相比可以显著提高牛体外受精的卵裂及八细胞率(P<0.05),但其后期发育不佳。mCR后与CR1aa后相比可以显著提高牛体外受精胚胎的囊胚率和孵化率(P<0.05)。由此得出mCR前+CR1aa后组可以显著的提高体外受精的囊胚和孵化率。为了进一步验证该组合的稳定性,利用mCR前+CR1aa后体系分别在不同季节进行验证,结果显示除夏季相对不佳外,其他季节均能维持较高的卵裂囊胚孵化率。进而认为CR1aa前+mCR后体系最有利于牛体外受精胚胎的生产且孵化率较高,有利于后期胚胎移植的进行。
In order to further find a optimal culture system of IVF and improve the blastocyst incubation rate in IVF.In this study,CR1aa group,mCR group,pre-CR1aa+ post-mCR group and pre-mCR + post-mCR group were programmed.The results showed that the cleavage rate,eight cell rate,blastocystrate and hatching rate of CR1aa group were 82.29±0.58,48.79±1.12,24.99±0.28,14.42±0.86;mCR group group were 57.18±1.61,26.48±0.53,22.15±1.20,18.12±0.57;and preCR1aa+ post-mCR group were 82.24±1.32,48.60±1.93,34.07±1.04,24.87±0.66;pre-mCR +post-mCR were 58.80±2.26,26.77±1.21,24.15±0.73,13.10±1.09;It can be seen from the results of four groups that compared with mCR,CR1aa anterior culture solution can significantly improve the cleavage and eight cell rate(P<0.05)of bovine in vitro fertilization,but its late development is poor.The blastocyst rate and hatching rate(P<0.05)of bovine in vitro fertilization embryos could be significantly increased post-mCR.It is concluded that the pre-CR1aa+post-mCR can significantly improve the cleavage blastocyst and hatching rate of in vitro fertilization.In order to further verify the stability of the combination,the results of the pre-CR1aa+ post-mCR applied in different seasons showed that the hatching rate of the cleavage blastocyst could be maintained in other seasons except for the relatively poor summer.Our study found that the pre-CR1aa+post-mCR system provided a stable culture system for the production of bovine IVF embryos and a high incubation rate,which was conducive to the subsequent embryo transfer.
In order to further find a optimal culture system of IVF and improve the blastocyst incubation rate in IVF.In this study,CR1aa group,mCR group,pre-CR1aa+ post-mCR group and pre-mCR + post-mCR group were programmed.The results showed that the cleavage rate,eight cell rate,blastocystrate and hatching rate of CR1aa group were 82.29±0.58,48.79±1.12,24.99±0.28,14.42±0.86;mCR group group were 57.18±1.61,26.48±0.53,22.15±1.20,18.12±0.57;and preCR1aa+ post-mCR group were 82.24±1.32,48.60±1.93,34.07±1.04,24.87±0.66;pre-mCR +post-mCR were 58.80±2.26,26.77±1.21,24.15±0.73,13.10±1.09;It can be seen from the results of four groups that compared with mCR,CR1aa anterior culture solution can significantly improve the cleavage and eight cell rate(P<0.05)of bovine in vitro fertilization,but its late development is poor.The blastocyst rate and hatching rate(P<0.05)of bovine in vitro fertilization embryos could be significantly increased post-mCR.It is concluded that the pre-CR1aa+post-mCR can significantly improve the cleavage blastocyst and hatching rate of in vitro fertilization.In order to further verify the stability of the combination,the results of the pre-CR1aa+ post-mCR applied in different seasons showed that the hatching rate of the cleavage blastocyst could be maintained in other seasons except for the relatively poor summer.Our study found that the pre-CR1aa+post-mCR system provided a stable culture system for the production of bovine IVF embryos and a high incubation rate,which was conducive to the subsequent embryo transfer.
引文
[1]Liang XW,Lu YQ,Chen MT,et al.In vitro embryo production in buffalo(Bubalus bubalis)using sexed sperm and oocytes from ovum pick up[J].Theriogenology.2008,69:822-826.
[2]Nedambale TL,Dinnys A,Groen W,et al.Comparision on in vitro fertilized bovine embryos cultured in KSOM or SOF and cryopreserved by slow freezing or vitrification[J].Theriogenoglogy.2003,62:437-449.
[3]成文敏,戴蕴平,朱士恩,等.培养体系对牛胚胎体外发育的影响[J].中国畜牧杂志.2005,41:9-11.
[4]Sagirkaya H,Misirlioglu M,Kaya A,et al.Developmental and molecular correlates of bovine preimplantation embryos[J].Reproduction.2006,1(31):895-904.
[5]Lonergan P,Rizos D,Gutierrez-Adan A,et al.Oocyte and embryo quality:effect of origin,culture conditions and gene expression patterns[J].Repro Dom Anim.2003,38:259-267.
[6]Dinnyés A,Carolan C,Lonergan P,et al.Survival of frozen or vitrified bovine blastocysts produced in vitro in synthetic oviduct fluid[J].Theriogenology.1996,46:1425-1439.
[7]Bavister BD.Culture of preimplantation embryos:facts and artifacts[J].Hum Reprod Update.1995,1(2):91-148.
[8]Gardner DK.Changes in requirements and utilization of nutrinets during mammalian prei-maturation embryo development and their signficance in embryo culture[J].Theriogenology.1998,49:83-102.
[9]Wright RWJ,Bonkioli KP.Aspects of in vitro fetilisation and embryo culture in domestic animals[J].JAnim Sci.1981,5:702-729.
[10]Eyestone WH,First NL.Characterisation of developmental arrest in early bovine embryos cultured in vitro[J].Theriogenoglogy.1991,35:613-623.
[11]Presicce GA,Senatore EM,Santis GD,et al.Hormonal stimulation and oocyte maturational competence in prepuberal Mediterranean Italian bufaloes(Bubalus bubalis)[J].Theriogenoglogy.2002,57:1877-1884.
[12]Brackett BG,Zuelke KA.Analysis of factors involved in the in vitro production of bovine embryos[J].Theriogenology.1993,39:43-64.
[13]Chatot CL,Ziomek CA,Bavister BD,et al.An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro[J].J Reprod Fert.1989,86:679-688.
[14]Gardner DK,Lane M.Culture and selection of viable blastocysts:a feasible proposition for human IVF[J].Hum Reprod Update.1997,3(4):367-382.
[15]Gardner DK,Leese HJ.Concentrations of nutrients in mouse oviduct fluid and their effects on embryo development and metabolism in vitro[J].J Reprod Fert.1990,88:361-368.
[16]Sagirkaya H,Misirlioglu M,Kaya A,et al.Developmental and molecular correlates of bovine preimplantation embryos[J].Reproduction.2006,131:895-904.
[17]Quinn P.Enhanced results in mouse and human embryo culture using a modified human tubal fluid medium lacking glucose and phosphate[J].J Assist Reprod Genet.1995,12:97-105.
[18]Sagirkaya H,Misirlioglu M,Kaya A,et al.Developmental potential of bovine oocytes cultured in different maturation and culture conditions[J].Anim Reprod Sci.2007,101:225-240.
[19]Gardner DK,Lane M.Alleviation of the'2-cell block'and development to the blastocyst of CF1mouse embryos;role of amino acids,EDTA and physical parameters[J].Hum Reprod.1996,11(12):1703-2712.
[20]Alomar M,Tasiaux H,Remacle S,et al.Kinetics offertilization and development,and sex ratio of bovine embryos produced using the semen of different bulls[J].Anim Reprod Sci.2008,107:48-61.
[21]刘东军,廛洪武,旭日干.不同种公牛精液对牛卵母细胞体外受精效果影响的研究[J].内蒙古大学学报(自然科学版).2000,31(3):307-310.
[22]Virro MR,Larson-Cook KL,Evenson DP.Sperm chromatin structure assay(SCSA)parameters are related to fertilization,blastocyst development,and ongoing pregnancy in vitro fertilization and intracytoplasmic sperm injection cycles[J].Fertil Steril.2004,81:1289-1295.
[23]朱淑文,吉泽绿,中泽昭人,等.不同季节牛体外受精胚的发育能力和细胞遗传学分析[J].草食家畜.2001,增刊:154-156.
[2]Nedambale TL,Dinnys A,Groen W,et al.Comparision on in vitro fertilized bovine embryos cultured in KSOM or SOF and cryopreserved by slow freezing or vitrification[J].Theriogenoglogy.2003,62:437-449.
[3]成文敏,戴蕴平,朱士恩,等.培养体系对牛胚胎体外发育的影响[J].中国畜牧杂志.2005,41:9-11.
[4]Sagirkaya H,Misirlioglu M,Kaya A,et al.Developmental and molecular correlates of bovine preimplantation embryos[J].Reproduction.2006,1(31):895-904.
[5]Lonergan P,Rizos D,Gutierrez-Adan A,et al.Oocyte and embryo quality:effect of origin,culture conditions and gene expression patterns[J].Repro Dom Anim.2003,38:259-267.
[6]Dinnyés A,Carolan C,Lonergan P,et al.Survival of frozen or vitrified bovine blastocysts produced in vitro in synthetic oviduct fluid[J].Theriogenology.1996,46:1425-1439.
[7]Bavister BD.Culture of preimplantation embryos:facts and artifacts[J].Hum Reprod Update.1995,1(2):91-148.
[8]Gardner DK.Changes in requirements and utilization of nutrinets during mammalian prei-maturation embryo development and their signficance in embryo culture[J].Theriogenology.1998,49:83-102.
[9]Wright RWJ,Bonkioli KP.Aspects of in vitro fetilisation and embryo culture in domestic animals[J].JAnim Sci.1981,5:702-729.
[10]Eyestone WH,First NL.Characterisation of developmental arrest in early bovine embryos cultured in vitro[J].Theriogenoglogy.1991,35:613-623.
[11]Presicce GA,Senatore EM,Santis GD,et al.Hormonal stimulation and oocyte maturational competence in prepuberal Mediterranean Italian bufaloes(Bubalus bubalis)[J].Theriogenoglogy.2002,57:1877-1884.
[12]Brackett BG,Zuelke KA.Analysis of factors involved in the in vitro production of bovine embryos[J].Theriogenology.1993,39:43-64.
[13]Chatot CL,Ziomek CA,Bavister BD,et al.An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro[J].J Reprod Fert.1989,86:679-688.
[14]Gardner DK,Lane M.Culture and selection of viable blastocysts:a feasible proposition for human IVF[J].Hum Reprod Update.1997,3(4):367-382.
[15]Gardner DK,Leese HJ.Concentrations of nutrients in mouse oviduct fluid and their effects on embryo development and metabolism in vitro[J].J Reprod Fert.1990,88:361-368.
[16]Sagirkaya H,Misirlioglu M,Kaya A,et al.Developmental and molecular correlates of bovine preimplantation embryos[J].Reproduction.2006,131:895-904.
[17]Quinn P.Enhanced results in mouse and human embryo culture using a modified human tubal fluid medium lacking glucose and phosphate[J].J Assist Reprod Genet.1995,12:97-105.
[18]Sagirkaya H,Misirlioglu M,Kaya A,et al.Developmental potential of bovine oocytes cultured in different maturation and culture conditions[J].Anim Reprod Sci.2007,101:225-240.
[19]Gardner DK,Lane M.Alleviation of the'2-cell block'and development to the blastocyst of CF1mouse embryos;role of amino acids,EDTA and physical parameters[J].Hum Reprod.1996,11(12):1703-2712.
[20]Alomar M,Tasiaux H,Remacle S,et al.Kinetics offertilization and development,and sex ratio of bovine embryos produced using the semen of different bulls[J].Anim Reprod Sci.2008,107:48-61.
[21]刘东军,廛洪武,旭日干.不同种公牛精液对牛卵母细胞体外受精效果影响的研究[J].内蒙古大学学报(自然科学版).2000,31(3):307-310.
[22]Virro MR,Larson-Cook KL,Evenson DP.Sperm chromatin structure assay(SCSA)parameters are related to fertilization,blastocyst development,and ongoing pregnancy in vitro fertilization and intracytoplasmic sperm injection cycles[J].Fertil Steril.2004,81:1289-1295.
[23]朱淑文,吉泽绿,中泽昭人,等.不同季节牛体外受精胚的发育能力和细胞遗传学分析[J].草食家畜.2001,增刊:154-156.