Optimization and Application of Mouse Embryo Transfer Technology

doi:10.12300/j.issn.1674-5817.2026.069

Abstract

Abstract:

Objective To optimize the technical parameters of mouse embryo transfer (including oviduct opening method, pseudopregnant recipient body weight, number of embryos transferred per recipient, and unilateral vs bilateral oviduct transfer), improve the efficiency of embryo transfer (the pregnancy rate of recipient female mice after embryo transfer is used as the evaluation index), and improve the utilization rate of high-quality embryo germplasm resources (the birth rate of mice after embryo transfer is used as the evaluation index). Methods 560 SPF-grade ICR mice and 440 C57BL/6J mice were used as experimental animals. 2-cell embryos were obtained through in vitro fertilization. A single-factor controlled variable method was used to systematically investigate the effects of four factors, including oviduct opening method, pseudopregnant recipient body weight, number of embryos transferred per recipient, and unilateral vs bilateral oviduct transfer, on the pregnancy rate and birth rate of mouse embryo transfer. Results The pregnancy rate of recipients with an opening made in the oviduct using a 26G syringe needle reached 90%, which was significantly higher than the 43% in the group with oviduct openings cut by Vannas scissors (P<0.05). Pseudopregnant recipients weighing 29-35 g exhibited optimal embryo transfer outcomes, with a pregnancy rate of 90% and a birth rate of 44%, and no pups were crushed by the recipient mothers. The highest birth rate of 42% was observed in the group receiving 16-25 embryos per recipient. Transferring 26-35 embryos per recipient achieved a 90% pregnancy rate and maximized embryo utilization efficiency. However, transfer efficiency decreased significantly when more than 35 embryos were transferred. Transfer of 24-26 embryos per recipient balanced relatively high pregnancy and birth rates. Bilateral oviduct transfer of mouse embryos yielded slightly higher pregnancy and birth rates than unilateral transfer, but the difference was not statistically significant (P>0.05). Conclusion Selecting pseudopregnant recipient female mice weighing 29-35 g, using a 26G syringe needle to make an opening in the oviduct, and controlling the number of transferred embryos at 24-26 per recipient can effectively improve the success rate of embryo transfer and achieve efficient utilization of high-quality embryo germplasm resources.

Key words: Embryo transfer, In vitro fertilization, Technical optimization, Transfer efficiency, ICR mice, C57BL/6J mice

CLC Number:

ZHANG Jiaoling,LIN Aping,XU Shiying,et al. Optimization and Application of Mouse Embryo Transfer Technology[J]. Laboratory Animal and Comparative Medicine, 2026, 46(5): 750-758. DOI: 10.12300/j.issn.1674-5817.2026.069.

Figures/Tables 4

Figure 1 Effects of two oviduct opening methods （Vannas scissors cutting <i>vs</i> 26G needle puncture） on pregnancy rate and pup birth rate of pseudopregnant ICR mice during embryo transfer

Note： A， Schematic diagram of making an opening in the oviduct using Vannas scissors； B， Schematic diagram of making an opening in the oviduct using a 26G syringe needle； C， Statistical results of pregnancy rate in recipient mice with two oviduct opening methods； D， Statistical results of pup birth rate after embryo transfer with two oviduct opening methods. ^*** P<0.001.

Figure 2 Effects of different body weight ranges of pseudo-pregnant ICR recipients on pregnancy rate， pup birth rate， and 7-day pup survival rate following embryo transfer

Note： A， Comparison of pregnancy rate in recipient mice among different body weight groups； B， Comparison of pup birth rate among different body weight groups； C， Comparison of 7-day pup survival rate among different body weight groups. ^* P<0.05， ^** P<0.01， ^*** P<0.001， ^**** P<0.000 1.

Figure 3 Effects of the number of 2-cell embryos transferred per recipient in a single transfer on pregnancy rate and pup birth rate in pseudopregnant ICR mice

Note： A， Pregnancy rates in recipients transplanted with embryos at gradient doses of 5-15， 16-25， 26-35， and 36-45 embryos； B， Pup birth rates in the same four groups； C and D， Pregnancy and pup birth rates， respectively， in recipients grouped by finer embryo-number intervals with the range of 16-35 embryos. ^ns P>0.05， ^** P<0.01， ^*** P<0.001， ^**** P<0.000 1.

Figure 4 Effects of unilateral and bilateral oviduct embryo transfer on pregnancy rate and pup birth rate in pseudopregnant ICR mice

Note： A， Pregnancy rate of recipient mice subjected to unilateral or bilateral oviduct embryo transfer； B， Pup birth rate after unilateral or bilateral oviduct embryo transfer. ^ns P>0.05.

References 31

[1]	TARKOWSKI A K. Experiments on the development of isolated blastomeres of mouse eggs[J]. Nature, 1959, 184: 1286-1287. DOI: 10.1038/1841286a0.
[2]	刘伟, 陶芳标, 黄德武, 等. 应用胚胎移植技术培育SPF级小鼠的研究进展[J]. 河北联合大学学报(医学版), 2013, 15(1): 30-32. DOI: 10.3969/j.issn.1008-6633.2013.01.018.
	LIU W, TAO F B, HUANG D W, et al. Research progress in cultivating SPF mice by embryo transfer technology[J]. J Hebei United Univ (Health Sci), 2013, 15(1): 30-32. DOI: 10.3969/j.issn.1008-6633.2013.01.018.
[3]	王超. 基因修饰小鼠制备常用技术[M]. 北京: 中国农业大学出版社, 2013: 21.
	WANG C. Common techniques for the preparation of genetically modified mice[M]. Beijing: China Agricultural University Press, 2013: 21.
[4]	XUE Y M, LING C, ZHENG H X, et al. Supplementation with oviductal EVs from the estrus, metestrus, and diestrus stages improved developmental competence of IVF mouse embryos[J]. Sci Rep, 2025, 15: 23376. DOI: 10.1038/s41598-025-07195-z.
[5]	KORCHIVAIA E, IVANOVA A, VOLODYAEV I, et al. Comparison of preimplantation mouse embryos with different genetic backgrounds as models for evaluating human embryo culture media composition[J]. Reprod Sci, 2024, 31(12): 3688-3696. DOI: 10.1007/s43032-024-01726-1.
[6]	YOSHIMI K, KUNO A, YAMAUCHI Y, et al. Genome editing using type I-E CRISPR-Cas3 in mice and rat zygotes[J]. Cell Rep Methods, 2024, 4(8): 100833. DOI: 10.1016/j.crmeth.2024. 100833.
[7]	SAKURAI T, TAKEI N, WEI Y X, et al. Efficient genome editing of two-cell mouse embryos via modified CRISPR/Cas electroporation[J]. Sci Rep, 2024, 14: 30347. DOI: 10.1038/s41598-024-81198-0.
[8]	MONCAUT N. Streamlining mouse genome editing by integrating AAV repair template delivery and CRISPR-Cas electroporation[J]. Lab Anim, 2024, 53(5): 115-116. DOI: 10. 1038/s41684-024-01363-w.
[9]	BOUCHAREB A, BIGGS D, ALGHADBAN S, et al. Increasing knockin efficiency in mouse zygotes by transient hypothermia[J]. CRISPR J, 2024, 7(2): 111-119. DOI: 10.1089/crispr.2023.0077.
[10]	IWATA S, NAGAHARA M, IDO R, et al. A Recql5 mutant facilitates complex CRISPR/Cas9-mediated chromosomal engineering in mouse zygotes[J]. Genetics, 2024, 227(2): iyae054. DOI: 10.1093/genetics/iyae054.
[11]	聂永强, 王朝霞. 高校基因编辑小鼠一站式服务平台建设的探索与实践: 以上海交通大学为例[J]. 实验动物与比较医学, 2025, 45(5): 642-648. DOI: 10.12300/j.issn.1674-5817.2025.036.
	NIE Y Q, WANG Z X. Exploration and practice of building a one-stop service platform for gene-edited mice in university animal centers: a case study of Shanghai Jiao Tong University[J]. Lab Anim Comp Med, 2025, 45(5): 642-648. DOI: 10.12300/j.issn.1674-5817.2025.036.
[12]	王芊芊, 陶斯珏, 卫振, 等. 利用辅助生殖技术挽救基因修饰小鼠的实例分析[J]. 实验动物与比较医学, 2025, 45(1): 79-86. DOI: 10.12300/j.issn.1674-5817.2024.107.
	WANG Q Q, TAO S J, WEI Z, et al. A case study of using assisted reproductive technology to rescue genetically modified mice with reproductive disorder phenotypes[J]. Lab Anim Comp Med, 2025, 45(1): 79-86. DOI: 10.12300/j.issn.1674-5817.2024.107.
[13]	PELTIER M, RASPA M, PUTTI S, et al. Cryopreservation at -80 ℃ impacts sperm integrity and fertility in a mouse strain-dependent manner[J]. Theriogenology, 2025, 245: 117523. DOI: 10.1016/j.theriogenology.2025.117523.
[14]	KOÇAK G, YILDIZ C. The effects of ferulic acid, tryptophan, and L-glutamine on the cryopreservation of mouse spermatozoa[J]. Biopreserv Biobank, 2024, 22(3): 286-293. DOI: 10.1089/bio.2023.0067.
[15]	IGONINA T N, RAKHMANOVA T A, OMELCHENKO A N, et al. Effects of stearic acid on the embryo cryopreservation in mouse[J]. Cryo Letters, 2024, 45(1): 28-35.
[16]	CRISPO M, MEIKLE M N, RÜLICKE T. Cryopreservation of valuable mouse and rat lines[M]//Rodent Quality Control: Genes and Bugs: Monitoring Health and Genetics of Laboratory Animals. Cham: Springer International Publishing, 2024:97-112. DOI: 10.1007/978-3-031-59103-7_6.
[17]	HODGSON T, LANE H, HORSLER H, et al. An improved vitrification protocol for the fast and safe storage of mouse oocytes[J]. Biol Reprod, 2025, 113(6): 1355-1363. DOI: 10.1093/biolre/ioaf215.
[18]	XU S Y, LEI Y, WEI M L, et al. Transparent alumina ceramics-based microfluidic chip enables on-chip cryopreservation for mouse oocyte[J]. Adv Funct Mater, 2025, 35(33): 2503296. DOI: 10.1002/adfm.202503296.
[19]	YANG L D, YANG Z S, GUO X M, et al. Research progress on novel bionic cryoprotectants in gamete and embryo cryopreservation[J]. Front Vet Sci, 2026, 13: 1797831. DOI: 10.3389/fvets.2026.1797831.
[20]	CHEN S Q, ZHANG M Z, LI L, et al. Loss of methylation of H19-imprinted gene derived from assisted reproductive technologies can be mitigated by cleavage-stage embryo transfer in mice[J]. J Assist Reprod Genet, 2019, 36(11): 2259-2269. DOI: 10.1007/s10815-019-01575-x.
[21]	李劲松, 孙强, 巢琴美, 等. HBV-C转基因小鼠快速胚胎冷冻方法的建立[J]. 中国实验动物学杂志, 2001, 11(1): 44-46. DOI: 10.3969/j.issn.1671-7856.2001.01.011.
	LI J S, SUN Q, CHAO Q M, et al. A rapid method for HBV-C transgenic mouse embryo cryopreservation[J]. Chin J Lab Anim Sci, 2001, 11(1): 44-46. DOI: 10.3969/j.issn.1671-7856. 2001.01.011.
[22]	熊文静, 母丹丹, 任静, 等. C57BL/6J小鼠胚胎单侧和双侧移植的比较研究[J]. 实验动物科学, 2025, 42(6): 55-59. DOI: 10.3969/j.issn.1006-6179.2025.06.009.
	XIONG W J, MU D D, REN J, et al. Comparison of unilateral and bilateral embryo transfer in C57BL/6J mice[J]. Lab Anim Sci, 2025, 42(6): 55-59. DOI: 10.3969/j.issn.1006-6179.2025.06.009.
[23]	王倩, 刘亮亮. 小鼠体外受精代孕鼠体重和饲料对其生产的影响[J]. 中南农业科技, 2023, 44(8): 251-253. DOI: 10.3969/j.issn.1007-273X.2023.08.058.
	WANG Q, LIU L L. Effects of body weight and feed on the production of surrogate mice in vitro fertilization[J]. South Cent Agric Sci Technol, 2023, 44(8): 251-253. DOI: 10.3969/j.issn.1007-273X.2023.08.058.
[24]	桂飞, 杨伟伟, 孙筱品, 等. 小鼠胚胎移植影响因素的初步探索[J]. 实验动物与比较医学, 2020, 40(6): 483-488. DOI: 10.3969/j.issn.1674-5817.2020.06.004.
	GUI F, YANG W W, SUN X P, et al. Preliminary study on the influencing factors of mouse embryo transfer[J]. Lab Anim Comp Med, 2020, 40(6): 483-488. DOI: 10.3969/j.issn.1674-5817.2020.06.004.
[25]	陈通克, 陈锡文. 利用体外受精-胚胎移植技术对ALK3基因敲除小鼠进行保种鉴定[J]. 江西畜牧兽医杂志, 2011(1): 11-13. DOI: 10.3969/j.issn.1004-2342.2011.01.005.
	CHEN T K, CHEN X W. In vitro fertilization-embryo transfer technique was used to identify the species of ALK3 knockout mice[J]. Jiangxi J Anim Husb Vet Med, 2011(1): 11-13. DOI: 10.3969/j.issn.1004-2342.2011.01.005.
[26]	张景锋, 郝京京, 徐秋良, 等. 多种因素对小鼠卵母细胞体外受精效果影响的分析[J]. 家畜生态学报, 2021, 42(3): 38-42. DOI: 10.3969/j.issn.1673-1182.2021.03.007.
	ZHANG J F, HAO J J, XU Q L, et al. Effect of various factors on mouse oocytes in vitro fertilization[J]. J Domest Anim Ecol, 2021, 42(3): 38-42. DOI: 10.3969/j.issn.1673-1182.2021.03.007.
[27]	NAGY A, GERTSENSTEIN M, VINTERSTEN K, et al. Manipulating the mouse embryo[M]. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 2003.
[28]	于天飞, 徐兴军, 杨晓杰, 等. 动物胚胎工程研究进展[J]. 生物学教学, 2011, 36(8): 2-4. DOI: 10.3969/j.issn.1004-7549.2011.08.001.
	YU T F, XU X J, YANG X J, et al. Research progress of animal embryo engineering[J]. Biol Teach, 2011, 36(8): 2-4. DOI: 10.3969/j.issn.1004-7549.2011.08.001.
[29]	杜凯, 李文光, 吴德国, 等. 小鼠胚胎移植技术优化及对巨细胞病毒净化研究[J]. 实验动物科学, 2019, 36(5): 31-34. DOI: 10.3969/j.issn.1006-6179.2019.05.006.
	DU K, LI W G, WU D G, et al. Optimization of mouse embryo transfer technology and purification of cytomegalovirus[J]. Lab Anim Sci, 2019, 36(5): 31-34. DOI: 10.3969/j.issn.1006-6179.2019.05.006.
[30]	DORSCH M, WITTUR I, GARRELS W. Success of embryo transfer in mice with freshly collected and cryopreserved two-cell embryos with different genetic backgrounds correlated with the number of transferred embryos: a 5-year retrospective analysis[J]. Lab Anim, 2019, 53(6): 577-586. DOI: 10.1177/0023677219832922.
[31]	杜淼, 陈芳华, 战大伟. 不同遗传背景小鼠输卵管移植效率的研究[J]. 实验动物科学, 2022, 39(4): 44-46. DOI: 10.3969/j.issn.1006-6179.2022.04.009.
	DU M, CHEN F H, ZHAN D W. Study on the efficiency of tubal transplantation in mice with different genetic backgrounds[J]. Lab Anim Sci, 2022, 39(4): 44-46. DOI: 10.3969/j.issn.1006-6179.2022.04.009.