血清孕酮水平检测在克隆胚胎移植受体牛的筛选及妊娠诊断中的应用 <sup>*</sup>

doi:10.13523/j.cb.2006011

中国生物工程杂志

2020, Vol. 40

Issue (7): 1-8 DOI: 10.13523/j.cb.2006011

研究报告

血清孕酮水平检测在克隆胚胎移植受体牛的筛选及妊娠诊断中的应用 ^*

贾晓^1,³,邱瑾^1,³,舒娟^1,^2,³,李华^1,³,习书斌^1,³,曾溢滔^1,³,曾凡一^1,^2,^3,^4,^***(

)

1 上海交通大学附属儿童医院上海市儿童医院上海医学遗传研究所上海 200040
2 上海滔滔转基因工程有限公司上海 201604
3 卫生部医学分子生物学重点实验室上海市胚胎与生殖工程重点实验室上海 200040
4 上海交通大学基础医学院组织胚胎学与遗传发育学系上海 200025

Serum Progesterone Level Detection for the Screening of Recipient Cattle for Cloned Embryo Transfer and Their Pregnancy Diagnosis

JIA Xiao^1,³,QIU Jin^1,³,SHU Juan^1,^2,³,LI Hua^1,³,XI Shu-bin^1,³,ZENG Yi-tao^1,³,ZENG Fan-yi^1,^2,^3,^4,^***(

)

1 Shanghai Institute of Medical Genetics,Children’s Hospital of Shanghai,Children’s Hospital Affiliated to Shanghai Jiaotong University,Shanghai 20040,China
2 Shanghai Tao Tao Transgenic Engineering Co., Ltd., Shanghai 201604,China
3 Key Laboratory of Embryo Molecular Biology,Ministry of Health,Shanghai Key Laboratory of Embryo and Reproduction Engineering,Shanghai 20040,China
4 Department of Histoembryology, Genetics & Development, Shanghai Jiaotong University College of Basic Medical Sciences, Shanghai 200025, China

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摘要：

目的:孕酮(progesterone, P4)作为一种生殖激素,在牛的发情期和妊娠期呈规律性变化,且在妊娠的建立和维持过程中发挥着重要作用。拟应用孕酮浓度测定辅助人工观察筛选克隆胚胎移植受体牛并监测其整个妊娠过程。方法:通过对自然配种牛不同生殖阶段血液中孕酮水平的分析,建立其在发情初期、妊娠期孕酮浓度的变化规律,以此为依据辅助人工观察筛选适合胚胎移植的受体牛。同时将在体外培养7天后的体细胞核移植重构囊胚移植到所筛选出的同期发情的受体牛子宫内,并应用孕酮测定监测其妊娠状态。实验结果: (1)运用孕酮检测筛选克隆胚胎移植受体牛时,当孕酮浓度在发情第0天和第5天分别为≤0.64nmol/L和2~8nmol/L时,适宜作为胚胎移植受体牛,根据此筛选指标能够排除50%左右假发情牛。(2)运用孕酮检测较传统的人工观察方法,胚胎移植的克隆牛出生率提高了7.1倍。结论:运用牛血清孕酮检测方法能有效提高母牛生理周期判断的准确性,有利于选择合适的胚胎移植受体牛,既能实时、有效地监测怀孕受体牛的妊娠状态,又避免了靠人工观察受体牛返情、流产时的人为疏漏和误判,有效地提高受体牛的利用率,提高克隆牛的生产效率,并能推广应用于畜牧行业牛的生产繁育中。

关键词： 孕酮水平; 受体牛; 胚胎移植; 体细胞核移植/克隆牛

Abstract:

Objective: Progesterone (P4), as a reproductive hormone, changes regularly in the estrus cycle of mammals, and plays an essential role in the establishment and maintenance of pregnancy in cattle. This study through monitoring serum progesterone concentration in cattle, in addition to the routine manual estrus observation, screens for embryo transfer (ET) recipient cattle, as well as for the monitoring of reproductive status during the whole pregnancy of cattle upon Somatic Cell Nuclear Transfer (SCNT) experiments. Methods: Through the study of serum progesterone levels of natural-breeding cattle at different reproductive stages, the reference indexes of progesterone concentration at early estrus and during pregnancy were established. Based on these indexes, the recipient cattle suitable for embryo transfer were screened and selected by complementing manual estrus observation. At the same time, reconstructed blastocyst from SCNT cultured in vitro for seven days was transferred into the uterus of selected estrus-synchronized recipient cattle. The pregnancy state was monitored with serum progesterone level. Experimental results: (1) When using serum progesterone level to screen for SCNT embryo transfer recipient cattle, i.e. progesterone concentration at 0d (≤0.64nmol/L) and 5d (2-8nmol/L), more than 50% of pseudo-estrus cows can be excluded from the embryo transfer recipient list. (2) Birth rate from embryo transfer of cloned embryos through the progesterone monitoring is 7.1 fold higher than using the traditional monitoring method alone. Conclusion: The application of bovine serum progesterone monitoring can effectively improve the accuracy of cow reproductive cycle evaluation. It increases the effectiveness of suitable embryo transfer recipient cattle selection, as well as the accurate judgment of pregnancy status after SCNT embryo transfer in the recipient cattle. This practice can improve the utilization efficacy of recipient cattle selection, and the production efficiency of cloned cattle. It can be extended to other applications in the embryo engineering field as well as in the livestock production industry.

Key words: Progesterone Embryo transfer Recipient cattle SCNT/Cloned cattle

收稿日期: 2020-06-08 出版日期: 2020-08-13

ZTFLH:

Q819

基金资助: * 国家重点科学研究计划(2014CB964701);国家重点科学研究计划(2014CB964703);国家自然科学基金(31871484);上海市重中之重重点学科项目(2017ZZ02019);上海市自然科学基金资助项目(16ZR1428600)

通讯作者: 曾凡一 E-mail: fzeng@vip.163.com

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引用本文:

贾晓,邱瑾,舒娟,李华,习书斌,曾溢滔,曾凡一. 血清孕酮水平检测在克隆胚胎移植受体牛的筛选及妊娠诊断中的应用 ^*[J]. 中国生物工程杂志, 2020, 40(7): 1-8.

JIA Xiao,QIU Jin,SHU Juan,LI Hua,XI Shu-bin,ZENG Yi-tao,ZENG Fan-yi. Serum Progesterone Level Detection for the Screening of Recipient Cattle for Cloned Embryo Transfer and Their Pregnancy Diagnosis. China Biotechnology, 2020, 40(7): 1-8.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2006011 或 https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I7/1

图1 空怀母牛发情周期中血清孕酮浓度的变化(n=16)

图2 自然配种牛妊娠期血清孕酮浓度的变化(n= 13)

图3 妊娠终止/不同时期返情母牛孕酮浓度变化(怀孕牛n=10)

表1 受体牛发情人工观察与孕酮检测结果对比

图4 受体牛妊娠期血清孕酮浓度变化(n= 22 )

表2 体细胞核移植受体牛妊娠率情况总结

[1]	李权武. 家畜外周血浆孕酮水平及其在兽医产科上的应用(续). 国外兽医学:畜禽疾病, 1980,3:5-8.
	Li Q W. The level of progesterone in peripheral plasma of domestic animals and its application in veterinary obstetrics (continuation). Journal of Foreign Veterinary Medicine:Livestock and Poultry Disease, 1980,3:5-8.
[2]	Rosenberg M, Herz Z, Davidson M, et al. Seasonal variations in post-partum plasma progesterone levels and conception in primiparous and multiparous dairy cows. J Reprod Fertil, 1977,51(2):363-367. doi: 10.1530/jrf.0.0510363 pmid: 563452
[3]	张庆坤, 杨会平. 提高牛胚胎移植受胎率的关键环节. 中国草食动物, 2007,27(2):64-65.
	Zhang Q K, Yang H P. Critical steps to improve the conception rate of bovine embryo transfer. China Herbivores, 2007,27(2):64-65.
[4]	Polejaeva I A, Broek D M, Walker S C, et al. Longitudinal study of reproductive performance of female cattle produced by somatic cell nuclear transfer. PLoS One, 2013,8(12):e84283. doi: 10.1371/journal.pone.0084283 pmid: 24391930
[5]	Abreu F M, Coutinho da Silva M A, Cruppe L H, et al. Role of progesterone concentrations during early follicular development in beef cattle: I. characteristics of LH secretion and oocyte quality. Anim Reprod Sci, 2018,9(196):59-68.
[6]	Abreu F M, Geary T W, Coutinho da Silva M A, et al. Role of progesterone concentrations during early follicular development in beef cattle: II. ovulatory follicle growth and pregnancy rates. Anim Reprod Sci, 2018,9(196):69-76.
[7]	Forde N, McGettigan PA, Mehta JP, et al. Proteomic analysis of uterine fluid during the pre-implantation period of pregnancy in cattle. Reproduction, 2014,147(5):575-587. doi: 10.1530/REP-13-0010 pmid: 24478148
[8]	Pat L, Niamh F, Thomas S. Role of progesterone in embryo development in cattle. Reprod Fertil Dev, 2016,28(1-2):66-74. doi: 10.1071/RD15326 pmid: 27062875
[9]	Ayad A, Sousa N M, Sulon J, et al. Influence of progesterone concentrations on secretory functions of trophoblast and pituitary during the first trimester of pregnancy in the dairy cattle. Theriogenology, 2007,67(9):1503-1514. doi: 10.1016/j.theriogenology.2007.03.010 pmid: 17459464
[10]	Forde N, Beltman M E, Duffy G B, et al. Changes in the endometrial transcriptome during the bovine estrous cycle: effect of low circulating progesterone and consequences for conceptus elongation. Biol Reprod, 2011,84(2):266-278. doi: 10.1095/biolreprod.110.085910 pmid: 20881316
[11]	Blavy P, Friggens N C, Nielsen K R, et al. Estimating probability of insemination success using milk progesterone measurements. J Dairy Sci, 2018,101(2):1648-1660. doi: 10.3168/jds.2016-12453 pmid: 29174142
[12]	Parr M H, Mullen M P, Crowe M A, et al. Relationship between pregnancy per artificial insemination and early luteal concentrations of progesterone and establishment of repeatability estimates for these traits in Holstein-Friesian heifers. Dairy Sci, 2012,95(5):2390-2396. doi: 10.3168/jds.2011-4498
[13]	Forde N, Mehta J P, Minten M, et al. Effects of low progesterone on the endometrial transcriptome in cattle. Biol Reprod, 2012. 87(5):124. pmid: 23018184
[14]	Clemente M, Fuente J L, Fair T, et al. Progesterone and conceptus elongation in cattle: a direct effect on the embryo or an indirect effect via the endometrium. Reproduction, 2009,138(3):507-517. doi: 10.1530/REP-09-0152 pmid: 19556439
[15]	Henricks D M, Dickey J F, Niswender G D. Serum luteinizing hormone and plasma progesterone levels during the estrous cycle and early pregnancy in cows. Biology of Reproduction, 1970,2(3):346-351. doi: 10.1095/biolreprod2.3.346 pmid: 5527833
[16]	Holtz W, Niggemeyer H. Reliable identification of pregnant dairy cows by double milk progesterone analysis. Livestock Science, 2019,7(228):38-41.
[17]	Zhang C, Murphy B D. Progesterone is critical for the development of mouse embryos. Endocrine, 2014,46(3):615-623. doi: 10.1007/s12020-013-0140-7 pmid: 24366645
[18]	Ferguson C E, Kesler D J, Godke R A. Progesterone enhances in vitro development of bovine embryos. Theriogenology, 2012,77(1):108-114. pmid: 21872313
[19]	Muhammad S, Zaeem S, Muhammad S, et al. Effect of plasma progesterone on oocyte recovery, oocyte quality, and early in vitro developmental competence of embryos in Bos indicus dairy cows. Anim Reprod Sci, 2019,3(202):80-86.
[20]	宗哲英, 王帅, 苏力德, 等. 奶牛发情行为的监测研究现状及进展. 畜牧与兽医, 2018,50(2):147-150.
	Zong Z Y, Wang S, Su L D, et al. A review of research on monitoring the oestrus behavior of dairy cows. Animal Husbandry & Veterinary Medicine, 2018,50(2):147-150.
[21]	Wilmut I, Schnieke A E, McWhir J, et al. Viable offspring derived from fetal and adult mammalian cells. Nature, 1997,385(6619):810-813. pmid: 9039911
[22]	Farin P W, Piedrahita J A, Farin C E. Errors in development of fetuses and placentas from in vitro produced bovine embryos. Theriogenology, 2006,65(1):178-191. pmid: 16266745
[23]	Yang X Y, Li H, Ma Q W, et al. Improved efficiency of bovine cloning by autologous somatic cell nuclear transfer. Reproduction, 2006,132(5):733-739. doi: 10.1530/rep.1.01118 pmid: 17071774
[24]	Akagi S, Geshi M, Nagai T. Recent progress in bovine somatic cell nuclear transfer. Anim Sci J, 2013,84(3):191-199. doi: 10.1111/asj.12035 pmid: 23480698
[25]	Ha A,N, Fakruzzaman M, Lee K,L, et al. Effects of co-culture of cumulus oocyte complexes with denuded oocytes during in vitro maturation on the developmental competence of cloned bovine embryos. Reprod Domest Anim. 2015,50(2):292-298. doi: 10.1111/rda.12487 pmid: 25605137
[26]	Zhang Y, Xiong X R, Wang L J, et al. Different preferences of IVF and SCNT bovine embryos for culture media. Zygote, 2014,22(1):1-9. doi: 10.1017/S0967199412000184
[27]	张鹏. 体细胞核移植技术的研究进展. 中国医药科学, 2016,6(5):40-43.
	Zhang P. Research progress of somatic cell nuclear transfer technique. China Medicine and Pharmacy, 2016,6(5):40-43.
[28]	Biase F H, Rabel C, Guillomotet M, et al. Massive dysregulation of genes involved in cell signaling and placental development in cloned cattle conceptus and maternal endometrium. PNAS, 2016,113(51):14492-14501. doi: 10.1073/pnas.1520945114 pmid: 27940919
[29]	Pope W F. Uterine asynchrony: a cause of embryonic loss. Biol Reprod, 1988,39(5):999-1003. doi: 10.1095/biolreprod39.5.999 pmid: 3064819
[30]	Knott J G, Poothapillai K, Wu H, et al. Porcine sperm factor supports activation and development of bovine nuclear transfer embryos. Biol Reprod, 2002,66(4):1095-1103. doi: 10.1095/biolreprod66.4.1095 pmid: 11906930
[31]	赵磊文, 伏静, 管鹏飞, 等. 胚胎移植时机对牛体细胞克隆胚胎妊娠率的影响. 上海交通大学学报(农业科学版), 2008,26(2):96-100.
	Zhao L W, Fu J, Guan P F, et al. Effect of implantation time on the pregnancy of bovine somatic nuclear transfer embryos. Journal of Shanghai Jiaotong University (Agricultural Science), 2008,26(2):96-100.
[32]	伏静, 赵磊文, 管鹏飞, 等. 共培养体系中滋养层细胞对牛核移植胚胎早期发育的影响. 上海交通大学学报(农业科学版), 2007,25(6):519-524.
	Fu J, Zhao L W, Guan P F, et al. Effect of feeder cells in co-culture system on in vitro development of bovine nuclear transfer embryos. Journal of Shanghai Jiaotong University(Agricultural Science), 2007,25(6):519-524.
[33]	陈海燕, 傅衍. 中国荷斯坦牛发情周期中孕酮、雌二醇含量的变化规律. 上海交通大学学报(农业科学版), 2005,23(2):122-124.
	Chen H Y, Fu Y. Change of contents of progesterone and oestradiol in the milk of Chinese Holstein Cow during oestrous cycle. Journal of Shanghai Jiaotong University (Agricultural Science), 2005,23(2):122-124.
[34]	丁伟良. 试论牛繁殖技术性不育的原因及改进策略. 黑龙江动物繁殖, 2017,25(6):47-48.
	Ding W L. Causes and improvement strategies of technical sterility in cattle breeding. Heilongjiang Journal of Animal Reproduction. 2017,25(6):47-48.
[35]	黄承俊, 张文军, 李宁. 牛羊胚胎移植研究进展. 现代畜牧兽医, 2019,5:51-58.
	Huang C J, Zhang W J, Li N. Studies of embryo transfer technology inlivestock. Modern journal of Animal Husbandry and Veterinary Medicine. 2019,5:51-58.

[1]	徐庆毅. 我国生物技术发展的回顾与展望[J]. 中国生物工程杂志, 1995, 15(1): 3-7.
[2]	ReurenJMaplejort;白琴华. 用来提高动物繁殖力的胚胎移植技术[J]. 中国生物工程杂志, 1984, 4(4): 8-14.

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