水牛活体采卵和无透明带胚胎培养体系的优化

doi:10.16431/j.cnki.1671-7236.2020.12.024

摘要/Abstract

摘要： 本研究旨在探索哺乳动物体外受精（IVF）胚胎单独或少量培养时发育效率低、无透明带胚胎的体外发育潜能受阻的问题，以期建立提高水牛活体采卵（ovum-pick-up，OPU）和徒手克隆（handmade clone，HMC）胚胎发育潜能高效稳定的体外生产体系。研究首先比较了单个微滴内共培养的胚胎数量（1、3、5、10和20枚）对胚胎发育效果的影响；而后采用微穴体系（well-of-the-well，WOW）和辅助共培养体系（培养微滴中添加包埋IVF胚胎的琼脂糖小块）培养OPU-IVF胚胎，并用WOW体系培养无透明带的徒手克隆重构胚，与传统的微滴培养体系比较其体外发育效果。结果表明：单个微滴内培养的胚胎数量为1、3和5枚时，囊胚发育率极显著低于10枚和20枚组（P<0.01）；与微滴培养体系相比，辅助共培养和WOW体系均极显著提高OPU-IVF胚胎的囊胚率（P<0.01），且WOW培养体系极显著促进HMC重构胚的卵裂率和囊胚率（P<0.01）。综上所述，胚胎群体培养有助于胚胎发育，在保证系谱明确的前提下琼脂糖包埋辅助胚胎共培养体系和WOW体系提高了OPU-IVF体外胚胎发育效率，且WOW体系还可用于无透明带胚胎的高效培养。

关键词: 水牛; 活体采卵(OPU); 徒手克隆(HMC); 琼脂糖包埋; 微穴体系

Abstract: The purpose of this study was to explore the problems of low development efficiency of small amounts in vitro fertilization (IVF) embryos and limited development potential of zona pellucida-free embryos when cultured in vitro of mammals,to establish an efficient and stable in vitro production system for improving the developmental potential of living ovum-pick-up (OPU) and handmade clone (HMC) embryos in buffalos.The study first compared the effect of the number of fertilized eggs (1,3,5,10 and 20) co-cultured within a single microdroplet on the embryonic development effect.Then,OPU-IVF embryos were cultured using the well-of-the-well (WOW) system and the auxiliary co-culture system (adding agarosaccharide fragments of embedded in in vitro fertilization (IVF) fertilized egg in the cultivation of microdroplets).Furthermore,the embryos without zona pellucida were cloned and reconstructed by using the WOW system and compared with the traditional microdroplet system in vitro.The results showed that the blastocyst development rates of the 10 and 20 embryos groups were significantly higher than that of the 1,3 and 5 embryos groups.Compared with the microdroplet system,the assisted co-culture system and the WOW system significantly improved the blastocyst rate of OPU-IVF embryos(P<0.01).Moreover,the WOW culture system significantly promoted the cleavage rate and blastocyst rate of HMC reconstructed embryos(P<0.01).To sum up,embryo mass culture contributed to embryo development,and under the premise of ensuring a clear pedigree,the agar-sugar embedding assisted embryo co-culture system and the WOW system improved the in vitro development efficiency of OPU-IVF embryos,the WOW system would also be applied to the high-efficient culture of zona pellucida free embryos.

Key words: buffalos; ovum-pick-up(OPU); handmade clone (HMC); agarose-embedded helper; well-of-the-well system

中图分类号:

S813.7

杨春艳, 郑海英, 李玲玉, 黄加祥, 黄春丽, 尚江华. 水牛活体采卵和无透明带胚胎培养体系的优化[J]. 中国畜牧兽医, 2020, 47(12): 4008-4015.

YANG Chunyan, ZHENG Haiying, LI Lingyu, HUANG Jiaxiang, HUANG Chunli, SHANG Jianghua. Optimization of in vitro Culture System for Embryos Derived from Ovum-pick-up and Zona-free Embryos in Buffalos[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(12): 4008-4015.

参考文献

[1] LIANG X,ZHANG X,YANG B,et al.Pregnancy and calving rates following transfer of in-vitro-produced river and F1(river x swamp) buffalo (Bubalusbubalis) embryos in recipients on natural oestrus or synchronised for ovulation[J].Reproduction,Fertility and Development,2007,19(5):670-676.
[2] 杨春艳,庞春英,李日聪,等.水牛徒手克隆的初步研究[J].中国畜牧兽医,2011,38(4):147-150. YANG C Y,PANG C Y,LI R C,et al.Study on the hand-made cloning (HMC) in buffalo[J].China Animal Husbandry & Veterinary Medicine,2011,38(4):147-150.(in Chinese)
[3] CONTRAMAESTRE A P,SIFONTES F,MARIN R,et al.Secretion of stem cell factor and granulocyte-macrophage colony-stimulating factor by mouse embryos in culture:Influence of group culture[J].Zygote,2008,16(4):297-301.
[4] GARDBER D K,LANE M,SPITZER A,et al.Enhanced rates of cleavage and development for sheep zygotes cultured to the blastocyst stage in the absence of serum and somatic cells:Amino acids,vitamins,and culturing embryos in groups stimulate develop-ment[J].Biology Reproduction,1994,50:390-400.
[5] KHURANA N K,NIEMANN H.Effects of oocyte quality,oxygen tension,embryo density,cumulus cells and energy substrates on cleavage and morula/blastocyst formation of bovine embryos[J].Theriogenology,2000,54(5):741-756.
[6] BELLA A,MORENO J F,MANNINO M E,et al.Improved in vitro development of OPU-derived bovine (Bostaurus) embryos by group culture with agarose-embedded helper embryos[J].Theriogeno-logy,2010,74(9):1643-1651.
[7] DEB G K,JIN J,KWON T,et al.Improved blastocyst development of single cow OPU-derived presumptive zygotes by group culture with agarose-embedded helper embryos[J].Reproductive Biology and Endocrinology,2011,9:121.
[8] GARDNER D K,TRUONG T T.Culture of the mouse preimplantation embryo[J].Methods in Molecular Biology,2019,2006:13-32.
[9] TAN K,AN L,WANG S M,et al.Actin disorganization plays a vital role in impaired embryonic development of in vitro-produced mouse preimplantation embryos[J].PLoS One,2015,10(6):e0130382.
[10] FUKUI Y,LEE E S,ARAKI N.Effect of medium renewal during culture in two different culture systems on development to blastocysts from in vitro produced early bovine embryos[J].Journal of Animal Science,1996,74(11):2752-2758.
[11] FUKUI Y,KIKUCHI Y,KONDO H,et al.Fertilizability and developmental capacity of individually cultured bovine oocytes[J].Theriogenology,2000,53(8):1553-1565.
[12] VAJTA G,PEUTA T T,HOLM P,et al.New method for culture of zona-included or zona-free embryos:The well of the well (WOW) system[J].Molecular Reproduction and Development,2000,55(3):256-264.
[13] KANG S S,OFUJI S,IMAI K,et al.The efficacy of the well of the well (WOW) culture system on development of bovine embryos in a small group and the effect of number of adjacent embryos on their development[J].Zygote,2015,23(3):412-415.
[14] LEHNER A,KASZAS Z,MURBER A,et al.Embryo density may affect embryo quality during in vitro culture in a microwell group culture dish[J].Archives of Gynecology and Obstetrics,2017,296(2):345-353.
[15] VAJTA G,KOROSI T,DU Y,et al.The well-of-the-well system:An efficient approach to improve embryo development[J].Reproductive Biomedicine Online,2008,17(1):73-81.
[16] MINASI M G,FABZZI G,CASCIANI V,et al.Improved blastocyst formation with reduced culture volume:Comparison of three different culture conditions on 1128 sibling human zygotes[J].Journal of Assisted Reproduction and Genetics,2015,32(2):215-220.
[17] WYDOOGHE E,VANDAELE L,PIEPERS S,et al.Individual commitment to a group effect:Strengths and weaknesses of bovine embryo group culture[J].Reproduction,2014,148(5):519-529.
[18] DE M N,SANTOS R S,MATEIZEL I,et al.Reduced blastocyst formation in reduced culture volume[J].Journal of Assisted Reproduction and Genetics,2015,32(9):1365-1370.
[19] AGCA Y.In vitro culture of rat preimplantation embryos[J].Methods in Molecular Biology,2019,2006:33-43.
[20] HARDARSON T,BUNGUM M,CONAGHAN J,et al.Noninferiority,randomized,controlled trial comparing embryo development using media developed for sequential or undisturbed culture in a time-lapse setup[J].Fertility and Sterility,2015,104(6):1452-9.e1-4.
[21] IEDA S,AKAI T,SAKAGUCHI Y,et al.A microwell culture system that allows group culture and is compatible with human single media[J].Journal of Assisted Reproduction and Genetics,2018,35(10):1869-1880.
[22] MATSUURA K.Numerical calculations for diffusion effects in the well-of-the-well culture system for mammalian embryos[J].Reproduction,Fertility,and Development,2014,26(5):742-751.
[23] TAKA M,IWAYAMA H,FUKUI Y.Effect of the well of the well (WOW) system on in vitro culture for porcine embryos after intracytoplasmic sperm injection[J].The Journal of Reproduction and Development,2005,51(4):533-537.
[24] SUGIMURA S,AKAI T,IMAI K.Selection of viable in vitro-fertilized bovine embryos using time-lapse monitoring in microwell culture dishes[J]. The Journal of Reproduction and Development,2017,63(4):353-357.
[25] GAMBINI A,BEVACQUA R J,KARLANIAN F,et al.The aggregation of four reconstructed zygotes is the limit to improve the developmental competence of cloned equine embryos[J].PLoS One,2014,9(11):e110998.