体细胞克隆牛生产及新生犊牛护理
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摘要
细胞核移植包括胚胎细胞核移植(Embryo cell nuclear transplantation,简称ECNT)和体细胞核移植( Somatic cell nuclear transfer,简称SCNT),是哺乳动物无性繁殖的主要手段。虽然已经获得了多种存活的克隆动物,但克隆效率的低下仍然是制约该项技术走向产业化的主要瓶颈。除了受体胞质、供体细胞、及克隆操作过程可能影响克隆效率外,围产期受体动物的护理与新生犊牛的护理也极大的影响出生后动物的存活率。到目前为止,尚未有专门对新生克隆犊牛进行系统观察、管理、护理并且提高克隆牛存活率的报道。本研究主要是对牛克隆胚胎的生产,受体牛围产期饲养管理以及新生犊牛出生后的护理等进行了较为系统而详尽的观察与研究。在实验室,主要开展了牛体细胞培养、牛卵母细胞的成熟培养、核移植操作及克隆胚胎的培养等工作。在围产期、犊牛出生过程及出生后进行了详细的观察,主要针对新生犊牛体弱、易感疾病等特点,不同阶段给与不同的护理与治疗措施,尤其是新生克隆犊牛的异物性肺炎、脐带炎及犊牛腹泻等疾病进行了治疗研究。使得体细胞克隆胚妊娠率、出生率以及犊牛出生后存活率大大提高。体细胞克隆胚60d妊娠率为50%,6个月的妊娠率达44%,出生率为33.3%,新生克隆牛犊存活率80%以上。通过我们的系统观察与研究,获得了一套管理、护理克隆犊牛的技术体系,对克隆牛技术产业化,尤其是对转基因克隆牛的生产具有重要的实际意义。
Nuclear transfer including embryonic cell nuclear transfer (ECNT) and somatic cell nuclear transfer (SCNT), is a alternative approach in addition to the dominately sexual reproduction. A variety of animal species have been cloned through SCNT technology, the cloning efficiency, however, is extremely low which become the the major bottle-neck obstacle for the economical application of this technology. Although the exact mechanisms affecting the cloning efficiency are not clear, the oocyte cytoplasmic status, donor cells and detailed cloning protocols probably contribute to the extremely low offspring rate. More important, the high death rate of the newborn animals, usually over 50%, makes the cloners and breeders somewhat depression. The recipient cow’s periparturient management and newborn calves’care should have a great impact on the survival rate of the neonatal cloning animals. To our knowledge, this is the first study focused on specific and systematic observations of the cloned calves. During perinatal period, perinatal and the birth of the caves, the recipient cows and the calves were kept an eye on almost 24 hr per day. After transfer of the cloned embryos to the synchronized recipients the pregnancy rate was 50% after 60 days transfer. Sixth month later 44% pregnancy were still maintained, and 33.3% of the recipients delivered healthy calves. Different treatment and management were conducted according to the newborn calf’s appearances and physiological state. A series of physiological indexes such as body temperature, heart rate, respiratory frequency, nose wetness, etc, were measued and careful theropy were taken for the susceptible diseases. Foreign body pneumonia, omphalitis and calf diarrhea are the most susceptible diseases of the newborn cloned calves. With great careful careful treatments and nursings of the recipient cows and the calves the survival rate reached over 80%.
Through our careful observations and treatments, a practical protocols for the the managements and nursing of cloned calves were established, which would be beneficial for the industrialization of the cattle cloning and transgenic cloning.
Nuclear transfer including embryonic cell nuclear transfer (ECNT) and somatic cell nuclear transfer (SCNT), is a alternative approach in addition to the dominately sexual reproduction. A variety of animal species have been cloned through SCNT technology, the cloning efficiency, however, is extremely low which become the the major bottle-neck obstacle for the economical application of this technology. Although the exact mechanisms affecting the cloning efficiency are not clear, the oocyte cytoplasmic status, donor cells and detailed cloning protocols probably contribute to the extremely low offspring rate. More important, the high death rate of the newborn animals, usually over 50%, makes the cloners and breeders somewhat depression. The recipient cow’s periparturient management and newborn calves’care should have a great impact on the survival rate of the neonatal cloning animals. To our knowledge, this is the first study focused on specific and systematic observations of the cloned calves. During perinatal period, perinatal and the birth of the caves, the recipient cows and the calves were kept an eye on almost 24 hr per day. After transfer of the cloned embryos to the synchronized recipients the pregnancy rate was 50% after 60 days transfer. Sixth month later 44% pregnancy were still maintained, and 33.3% of the recipients delivered healthy calves. Different treatment and management were conducted according to the newborn calf’s appearances and physiological state. A series of physiological indexes such as body temperature, heart rate, respiratory frequency, nose wetness, etc, were measued and careful theropy were taken for the susceptible diseases. Foreign body pneumonia, omphalitis and calf diarrhea are the most susceptible diseases of the newborn cloned calves. With great careful careful treatments and nursings of the recipient cows and the calves the survival rate reached over 80%.
Through our careful observations and treatments, a practical protocols for the the managements and nursing of cloned calves were established, which would be beneficial for the industrialization of the cattle cloning and transgenic cloning.
引文
1张忠诚.家畜繁殖学(第三版) [ M] .北京:中国农业出版社,2000 ,255.
2 Wilmut, I., Schnieke, A. E., Mc Whir, J. , et al. Viable offspring derived from fetal and adult mammalian cells [ J] . Nature, 1997, 385:8102813.
3 Wilmut I, Schnieke AE, McWhir J, et al. Viable offspring derived form fetal and mammalian cells. Nature, 1997, 385: 810-813.
4 Kato Y, Tetsuya T, Sotomaru Y, et al. Eight calves cloned from somatic cells of a single adult. Science, 1998, 282: 2095-2098.
4 Baguisi A, Behboodi E, Melican DT, et al. Production of goats by somatic cell nuclear transfer. Nat Biotechnol. 1999, 17(5): 456-461.
5 Polejaeva I A, Chen S H, Vaught T D, et al. Cloned pigs produced by nuclear transfer from adult somatic cells. Nature, 2000, 407: 86-90.
7 Galli C, Lagutina I, Crotti G, et al. Pregnancy: a cloned horse born to its dam twin. Nature, 2003, 424 (6949): 635.
8 Woods GL, White KL, Vanderwall DK, et al. A mule cloned from fetal cells by nuclear transfer. Science, 2003, 301(5636): 1063.
9李光华,叶绍辉.动物体细胞克隆技术的研究进展.家畜生态学报第,2006,第27卷.
10陈大元,李劲松,韩之明等.体细胞克隆牛:供体细胞和受体的影响.科学通报, 2003, 48(8): 768-773.
11龚国春,戴蕴平,朱化彬等.供体细胞类型对体细胞克隆牛生产效率的影响.中国科学, C辑, 2004, 34(3): 257-262.
12戴蕴平,连正兴,朱化彬等.中国红系冀南黄牛的克隆及其繁殖性能.科学通报, 2005, 50(24):2763-2768.
13 Yang P, Wang J, Gong G, et al. Cattle mammary bioreactor generated by a novel procedure of transgenic cloning for large-scale production of functional human lactoferrin. PLoS One, 2008, 3(10): e3453.
14 Wang J, Yang P, Tang B, et al. Expression and characterization of bioactive recombinant human alpha-lactalbumin in the milk of transgenic cloned cows. J Dairy Sci, 2008, 91(12): 4466-4476.
15 Wang S, Sun X, Ding F, et al. Removal of selectable marker gene from fibroblast cells in transgenic cloned cattle by transient expression of Cre recombinase and subsequent effects on recloned embryo development. Theriogenology, 2009, 72(4): 535-541.
16 Wilmut I, Beaujean N, de Sousa P A, et al. Somatic cell nuclear transfer. Nature, 2002, 419 (10): 583-587.
17程金华,朱化彬,戴蕴平等.我国体细胞克隆牛的研究进展.中国生物工程杂志, 2006, 26(10): 77-82.
18 Bordignon V, Clarke HJ, Smith LC. Factors controlling the loss of immunoreactive somatic histone H1 from blastomere nuclei in oocyte cytoplasm: a potential marker of nuclear reprogramming. Dev Biol 2001, 233 (1): 192-203.
19 Dean W, Santos F, Stojkovic M, et al. Conservation of methylation reprogramming in mammalian development: aberrant reprogramming in cloned embryos. Proc Natl Acad Sci USA, 2001, 98(24): 13734-13738.
20 Kang YK, Park JS, Koo DB,et al. Limited demethylation leaves mosaic-type methylation states in cloned bovine pre-implantation embryos. Embo J, 2002, 21(5): 1092-1100.
21 Santos F, Zakhartchenko V, Stojkovic M, et al. Epigenetic marking correlates with developmental potential in cloned bovine preimplantation embryos. Curr Biol, 2003,13(13): 1116-1121.
22 Cezar GG, Bartolomei MS, Forsberg EJ, et al. Genome-wide epigenetic alterations in cloned bovine fetuses. Biol Reprod, 2003, 68(3): 1009-1014.
23 Wrenzycki C, Wells D, Herrmann D,et al. Nuclear transfer protocol affects messenger RNA expression patterns in cloned bovine blastocysts. Biol Reprod, 2001, 65(1): 309-317.
24 Nakao M. Epigenetics: interaction of DNA methylation and chromatin. Gene, 2001, 278(1/2): 25-31.
25 Kang YK, Yeo S, Kim SH, et al. Precise recapitulation of methylation change in early cloned embryos. Mol Reprod Dev, 2003, 66(1): 32-37.
26 Shi W, Zakhartchenko V, Wolf E. Epigenetic reprogramming in mammalian nuclear transfer. Differentiation, 2003, 71(2): 91-113. Review.
27 Enright BP, Jeong BS, Yang X, et al. Epigenetic characteristics of bovine donor cells for nuclear transfer: levels of histone acetylation. Biol. Reprod, 2003, 69 (5): 1525-1530.
28 Li GP, Bunch TD, White KL, et al. Development, chromosomal composition, and cell allocation of bovine cloned blastocyst derived from chemically assisted enucleation and cultured in conditioned media. Mol Reprod Dev, 2004a, 68(2): 189-197.
29 Li GP, White KL, Aston KI, et al. Conditioned medium increases the polyploid cell composition of bovine somatic cell nuclear-transferred blastocysts. Reproduction, 2004b, 127(2): 221-228.
30 Li GP, Liu Y, White KL. Cytogenetic analysis of diploidy in cloned bovine embryos using an improved air dry caryotyping method. Theriogenology, 2005, 63(9): 2434-2444.
31 Aston KI, Li GP, Hicks BA, et al. Genetic reprogramming of transcription factor ap-2gamma in bovine somatic cell nuclear transfer preimplantation embryos and placentomes. Cloning Stem Cells, 2009, 11(1): 177-86.
32 Buczinski S, Fecteau G, Comeau G, et al. Ultrasonographic fetal well-being assessment, neonatal and postpartum findings of cloned pregnancies in cattle: A preliminary study on 10 fetusesand calves. Can Vet J, 2009, 50: 261-269.
33 Meirelles FV, Birgel EH Jr, Perecin F, et al. Delivery of cloned offspring: experience in Zebu cattle (Bos indicus). Reproduction, Fertility and Development, 2010, 22: 88-97.
34陈秀兰谭丽玲荣瑞章等编著《家畜胚胎移植》上海科学出版社1983
35 K.J.贝特里吉编《家畜胚胎移植》科学技术文献出版社重庆分社1980年8月
36〔美」E.S.E.哈弗士主编许怀让等译《农学繁殖学》科学技术文献出版社重庆分社1982年10月
37陈启飞,王振兴,周元军,等.犊牛腹泻的诊断与治疗[J].上海畜牧兽医通讯,2006,(04):75
38.张会子.如何防治犊牛腹泻.畜牧兽医杂志[J],2007,(01):14
39.董改琳.犊牛腹泻综合情况报告.今日畜牧兽医[J],2006,(06):15～[6].
40.施睎.口服补液盐及其应用[J].当代畜禽养殖业, 2004,(04):20
41梁鑫平.犊牛腹泻的症状和防治[J].江西畜牧兽医杂志, 2006,(05):24
42.张军,王振军,张在华,等.中西结合治疗犊牛传染性腹泻[J].中兽医杂志,2007,(02):49～50
43.刘长松.犊牛腹泻[M].奶牛疾病诊疗大全,2005,(第一版),144～150
44.郭定宗.幼畜消化不良[M].家畜内科学, 2005,(第一版),81～83
45.兰磊.要防犊牛腹泻就须精心饲养.陕西科技报,2007,(01):1
46.赵刚,哀丽娜,等.犊牛真胃炎-真胃溃疡的治疗方法与体会[J].北方牧业,2007,(03):21
47刘万平.犊牛腹泻肛诊的临床观察[J].中兽医学杂志, 2002,(04):20～21
48谭丽玲等.1987-1990年奶牛胚胎分割研究(J).国外畜牧学——草食家蓄,1990(增刊):44-46.
49荒木武纪。牛胚胎移植技术的实践与普及.J.Reprod.Dev.J.1994,40(6):131-136
50旭日干,张锁链,薛晓先等.牛体外受精的移植及妊娠产犊.科学通报, 1990, 21: 1666-1667.
2 Wilmut, I., Schnieke, A. E., Mc Whir, J. , et al. Viable offspring derived from fetal and adult mammalian cells [ J] . Nature, 1997, 385:8102813.
3 Wilmut I, Schnieke AE, McWhir J, et al. Viable offspring derived form fetal and mammalian cells. Nature, 1997, 385: 810-813.
4 Kato Y, Tetsuya T, Sotomaru Y, et al. Eight calves cloned from somatic cells of a single adult. Science, 1998, 282: 2095-2098.
4 Baguisi A, Behboodi E, Melican DT, et al. Production of goats by somatic cell nuclear transfer. Nat Biotechnol. 1999, 17(5): 456-461.
5 Polejaeva I A, Chen S H, Vaught T D, et al. Cloned pigs produced by nuclear transfer from adult somatic cells. Nature, 2000, 407: 86-90.
7 Galli C, Lagutina I, Crotti G, et al. Pregnancy: a cloned horse born to its dam twin. Nature, 2003, 424 (6949): 635.
8 Woods GL, White KL, Vanderwall DK, et al. A mule cloned from fetal cells by nuclear transfer. Science, 2003, 301(5636): 1063.
9李光华,叶绍辉.动物体细胞克隆技术的研究进展.家畜生态学报第,2006,第27卷.
10陈大元,李劲松,韩之明等.体细胞克隆牛:供体细胞和受体的影响.科学通报, 2003, 48(8): 768-773.
11龚国春,戴蕴平,朱化彬等.供体细胞类型对体细胞克隆牛生产效率的影响.中国科学, C辑, 2004, 34(3): 257-262.
12戴蕴平,连正兴,朱化彬等.中国红系冀南黄牛的克隆及其繁殖性能.科学通报, 2005, 50(24):2763-2768.
13 Yang P, Wang J, Gong G, et al. Cattle mammary bioreactor generated by a novel procedure of transgenic cloning for large-scale production of functional human lactoferrin. PLoS One, 2008, 3(10): e3453.
14 Wang J, Yang P, Tang B, et al. Expression and characterization of bioactive recombinant human alpha-lactalbumin in the milk of transgenic cloned cows. J Dairy Sci, 2008, 91(12): 4466-4476.
15 Wang S, Sun X, Ding F, et al. Removal of selectable marker gene from fibroblast cells in transgenic cloned cattle by transient expression of Cre recombinase and subsequent effects on recloned embryo development. Theriogenology, 2009, 72(4): 535-541.
16 Wilmut I, Beaujean N, de Sousa P A, et al. Somatic cell nuclear transfer. Nature, 2002, 419 (10): 583-587.
17程金华,朱化彬,戴蕴平等.我国体细胞克隆牛的研究进展.中国生物工程杂志, 2006, 26(10): 77-82.
18 Bordignon V, Clarke HJ, Smith LC. Factors controlling the loss of immunoreactive somatic histone H1 from blastomere nuclei in oocyte cytoplasm: a potential marker of nuclear reprogramming. Dev Biol 2001, 233 (1): 192-203.
19 Dean W, Santos F, Stojkovic M, et al. Conservation of methylation reprogramming in mammalian development: aberrant reprogramming in cloned embryos. Proc Natl Acad Sci USA, 2001, 98(24): 13734-13738.
20 Kang YK, Park JS, Koo DB,et al. Limited demethylation leaves mosaic-type methylation states in cloned bovine pre-implantation embryos. Embo J, 2002, 21(5): 1092-1100.
21 Santos F, Zakhartchenko V, Stojkovic M, et al. Epigenetic marking correlates with developmental potential in cloned bovine preimplantation embryos. Curr Biol, 2003,13(13): 1116-1121.
22 Cezar GG, Bartolomei MS, Forsberg EJ, et al. Genome-wide epigenetic alterations in cloned bovine fetuses. Biol Reprod, 2003, 68(3): 1009-1014.
23 Wrenzycki C, Wells D, Herrmann D,et al. Nuclear transfer protocol affects messenger RNA expression patterns in cloned bovine blastocysts. Biol Reprod, 2001, 65(1): 309-317.
24 Nakao M. Epigenetics: interaction of DNA methylation and chromatin. Gene, 2001, 278(1/2): 25-31.
25 Kang YK, Yeo S, Kim SH, et al. Precise recapitulation of methylation change in early cloned embryos. Mol Reprod Dev, 2003, 66(1): 32-37.
26 Shi W, Zakhartchenko V, Wolf E. Epigenetic reprogramming in mammalian nuclear transfer. Differentiation, 2003, 71(2): 91-113. Review.
27 Enright BP, Jeong BS, Yang X, et al. Epigenetic characteristics of bovine donor cells for nuclear transfer: levels of histone acetylation. Biol. Reprod, 2003, 69 (5): 1525-1530.
28 Li GP, Bunch TD, White KL, et al. Development, chromosomal composition, and cell allocation of bovine cloned blastocyst derived from chemically assisted enucleation and cultured in conditioned media. Mol Reprod Dev, 2004a, 68(2): 189-197.
29 Li GP, White KL, Aston KI, et al. Conditioned medium increases the polyploid cell composition of bovine somatic cell nuclear-transferred blastocysts. Reproduction, 2004b, 127(2): 221-228.
30 Li GP, Liu Y, White KL. Cytogenetic analysis of diploidy in cloned bovine embryos using an improved air dry caryotyping method. Theriogenology, 2005, 63(9): 2434-2444.
31 Aston KI, Li GP, Hicks BA, et al. Genetic reprogramming of transcription factor ap-2gamma in bovine somatic cell nuclear transfer preimplantation embryos and placentomes. Cloning Stem Cells, 2009, 11(1): 177-86.
32 Buczinski S, Fecteau G, Comeau G, et al. Ultrasonographic fetal well-being assessment, neonatal and postpartum findings of cloned pregnancies in cattle: A preliminary study on 10 fetusesand calves. Can Vet J, 2009, 50: 261-269.
33 Meirelles FV, Birgel EH Jr, Perecin F, et al. Delivery of cloned offspring: experience in Zebu cattle (Bos indicus). Reproduction, Fertility and Development, 2010, 22: 88-97.
34陈秀兰谭丽玲荣瑞章等编著《家畜胚胎移植》上海科学出版社1983
35 K.J.贝特里吉编《家畜胚胎移植》科学技术文献出版社重庆分社1980年8月
36〔美」E.S.E.哈弗士主编许怀让等译《农学繁殖学》科学技术文献出版社重庆分社1982年10月
37陈启飞,王振兴,周元军,等.犊牛腹泻的诊断与治疗[J].上海畜牧兽医通讯,2006,(04):75
38.张会子.如何防治犊牛腹泻.畜牧兽医杂志[J],2007,(01):14
39.董改琳.犊牛腹泻综合情况报告.今日畜牧兽医[J],2006,(06):15～[6].
40.施睎.口服补液盐及其应用[J].当代畜禽养殖业, 2004,(04):20
41梁鑫平.犊牛腹泻的症状和防治[J].江西畜牧兽医杂志, 2006,(05):24
42.张军,王振军,张在华,等.中西结合治疗犊牛传染性腹泻[J].中兽医杂志,2007,(02):49～50
43.刘长松.犊牛腹泻[M].奶牛疾病诊疗大全,2005,(第一版),144～150
44.郭定宗.幼畜消化不良[M].家畜内科学, 2005,(第一版),81～83
45.兰磊.要防犊牛腹泻就须精心饲养.陕西科技报,2007,(01):1
46.赵刚,哀丽娜,等.犊牛真胃炎-真胃溃疡的治疗方法与体会[J].北方牧业,2007,(03):21
47刘万平.犊牛腹泻肛诊的临床观察[J].中兽医学杂志, 2002,(04):20～21
48谭丽玲等.1987-1990年奶牛胚胎分割研究(J).国外畜牧学——草食家蓄,1990(增刊):44-46.
49荒木武纪。牛胚胎移植技术的实践与普及.J.Reprod.Dev.J.1994,40(6):131-136
50旭日干,张锁链,薛晓先等.牛体外受精的移植及妊娠产犊.科学通报, 1990, 21: 1666-1667.