A Case Study of Using Assisted Reproductive Technology to Rescue Genetically Modified Mice with Reproductive Disorder Phenotypes

doi:10.12300/j.issn.1674-5817.2024.107

Abstract

Abstract:

Objective The utilization of assisted reproductive technology to rescue genetically modified mouse strains with reproductive disorders provides a reference for improving techniques to preserve valuable experimental mouse strains. Methods In vitro fertilization-embryo transfer (IVF-ET) technology was performed on 28 strains of infertile male mice aged 9-18 months. Several indicators such as sperm density and sperm motility in infertile male mice were assessed to select the most viable sperm for IVF-ET experiments. Fertility rate, abnormal egg rate, and birth rate were recorded after the birth of the pups. An optimized ovarian transplantation procedure was applied to 12 strains of infertile female mice aged 8-18 months. 6-week-old female mice with the same genetic background were selected as recipients. One intact ovary was removed from each recipient mouse, and the contralateral oviduct was ligated. An ovary from a donor mouse was isolated and transplanted orthotopically into the side where the ovary had been removed in the recipient mouse. Twenty-one days post-surgery, recipient mice were co-housed with 8-week-old wild type male mice of the same genetic background for breeding. Data such as the pregnancy rate and live birth rate of the recipients were recorded after the birth of the pups. Results IVF-ET successfully rescued 28 mouse strains, with the oldest male mice being 18 months old. The success rate of the first round of IVF-ET experiments was 89.29% (25/28). The average fertility rate of IVF in infertile male mice was (51.01±14.97)%, the abnormal egg rate was (9.03±5.28)%, and the birth rate of offspring mice was (18.60±7.03)%. 39 out of 40 ovarian transplant recipient mice survived, with a pregnancy rate of 33.33% (13/39) for ovarian transplant recipients, and a live birth rate of 17.95% (7/39). Four mouse strains were successfully rescued using optimized ovarian transplantation technology, with the oldest female mice being 18 months old. 8 strains were not rescued as they failed to produce offspring that survived to sexual maturity. Conclusion IVF-ET is an effective approach for rescuing mice with reproductive disorders caused by different reasons, especially for those beyond the optimal breeding age. Ovarian transplantation technology can also be used as an alternative for aged female mice. But its success rate is relatively lower than that of IVF-ET, and carries a higher experimental risk.

Key words: Genetically modified mouse, Middle-aged and aged, Reproductive disorders, In vitro fertilization-embryo transfer, Ovarian transplantation

CLC Number:

Q95-33

WANG Qianqian,TAO Sijue,WEI Zhen,et al. A Case Study of Using Assisted Reproductive Technology to Rescue Genetically Modified Mice with Reproductive Disorder Phenotypes[J]. Laboratory Animal and Comparative Medicine, 2025, 45(1): 79-86. DOI: 10.12300/j.issn.1674-5817.2024.107.

Figures/Tables 5

Figure 1 Sperm masses collected from male mice and sperm status after 1 h of capacitationNote：A， The unilateral cauda epididymidis and a segment of the vas deferens from a male mouse； B， Sperm masses collected from the cauda epididymidis placed into droplets of sperm capacitation medium； C， The sperm status of two different strains of male mice after 1 h of capacitation： in the left culture droplet， the sperm were sparse， while in the right culture droplet， they were evenly dispersed.

Figure 2 Ovarian transplantation surgery in miceNote： A， Ligation was performed at two sites on the oviduct of one side of the recipient mouse； B， An intact ovary from one side of the donor mouse was harvested； C， The ovary and oviduct on the other side of the recipient were stabilized； D， The intact ovary from the donor was transplanted into the ovarian capsule of the recipient at the site where the ovary had been removed， followed by suturing of the capsule.

Table 1 Abnormal egg rate, fertility rate and birth rate of infertile genetically modified mice rescued by in vitro fertilization-embryo transfer

不育雄性小鼠月龄/月 Age of Infertile male mice/month	动物数量 n	异形卵率/% Abnormal egg rate/%	受精率/% Fertility rate/%	仔鼠出生率/% Birth rate/%
9	3	4.35±2.21	48.99±28.09	19.87±13.93
10	4	11.55±8.29	62.34±11.32	16.84±5.43
11	4	9.38±2.35	60.85±10.46	17.41±10.46
12	4	7.14±1.12	48.87±13.72	19.04±5.31
13	4	9.50±6.44	42.21±5.86	19.68±1.92
14	4	13.15±7.72	53.07±12.79	16.51±4.60
15	1	6.13	36.79	12.82
16	1	5.04	43.17	15.00
17	1	9.09	47.20	20.00
18	2	8.31±3.53	42.45±27.12	27.66±10.50

Figure 3 Comparison between the young group and the middle-aged and aged group in their fertility rates， abnormal egg rates， and birth ratesNote： A， Comparison of in vitro fertilization （IVF） fertilization rates between the young group and the middle-aged and aged group； B， Comparison of IVF abnormal egg rates between the young group and the middle-aged and aged group； C， Comparison of IVF-embryo transfer （ET） birth rate between the young group and the middle-aged and aged group. *P<0.05， ***P<0.001； ns， differences were not statistically significant； D， Correlation between male mice age and fertility rate.

Table 2 Age of donor mice, pregnancy rate and live birth rate of infertile genetically modified mice successfully rescued through ovarian transplantation

供体雌性小鼠月龄/月 Age of donor mice/month	左侧卵巢移植的受体小鼠数/只 The number of recipients for donor’s left ovarian transplantation	右侧卵巢移植的受体小鼠数/只 The number of recipients for donor’s right ovarian transplantation	妊娠率/% Pregnancy rate/%	活产率/% Live birth rate/%
10	1	1	100.00 （2/2）	100.00 （2/2）
15	1	1	50.00 （1/2）	100.00 （1/1）
15	1	2	66.67 （2/3）	100.00 （2/2）
18	1	1	100.00 （2/2）	100.00 （2/2）

References 27

1	KINTER L B, DEHAVEN R, JOHNSON D K, et al. A brief history of use of animals in biomedical research and perspective on non-animal alternatives[J]. ILAR J, 2021, 62(1-2):7-16. DOI: 10.1093/ilar/ilab020 .
2	高晗, 钟蓓. 转基因技术和转基因动物的发展与应用[J]. 现代畜牧科技, 2020(6):1-4, 18. DOI: 10.19369/j.cnki.2095-9737.2020.06.001 .
	GAO H, ZHONG B. The development and application of transgenic technology and transgenic animals[J]. Mod Anim Husb Sci Technol, 2020(6):1-4, 18. DOI: 10.19369/j.cnki.2095-9737.2020.06.001 .
3	LIU W, PAN H F, WANG Q, et al. The application of transgenic and gene knockout mice in the study of gastric precancerous lesions[J]. Pathol Res Pract, 2018, 214(12):1929-1939. DOI: 10.1016/j.prp.2018.10.022 .
4	CAMPBELL K M, XU Y D, PATEL C, et al. Loss of TDP-43 in male germ cells causes meiotic failure and impairs fertility in mice[J]. J Biol Chem, 2021, 297(5):101231. DOI: 10.1016/j.jbc.2021.101231 .
5	刘庆玲, 赵振军, 徐文秀, 等. AKR7A5基因敲除对雄性小鼠生殖的影响[J]. 烟台大学学报(自然科学与工程版), 2024, 37(1):102-106. DOI: 10.13951/j.cnki.37-1213/n.220634 .
	LIU Q L, ZHAO Z J, XU W X, et al. Effects of AKR7A5 gene knockout on reproduction of male mouse[J]. J Yantai Univ Nat Sci Eng Ed, 2024, 37(1):102-106. DOI: 10.13951/j.cnki.37-1213/n.220634 .
6	孙晓梅, 李名聪, 李涛, 等. LSS基因功能缺失对雄性小鼠生殖的影响[J]. 安徽医科大学学报, 2022, 57(2):208-212. DOI: 10.19405/j.cnki.issn1000-1492.2022.02.009 .
	SUN X M, LI M C, LI T, et al. The effects of knockout of LSS on female mice fertility[J]. Acta Univ Med Anhui, 2022, 57(2):208-212. DOI: 10.19405/j.cnki.issn1000-1492.2022.02.009 .
7	龚晓娟, 马盼, 孙敏, 等. DRD1基因敲除影响雄性生育的分子机理研究[J]. 实验动物科学, 2021, 38(2):53-56, 60. DOI: 10.3969/j.issn.1006-6179.2021.02.009 .
	GONG X J, MA P, SUN M, et al. Study on molecular mechanism of male mice reproduction in DRD1 knockout[J]. Lab Anim Sci, 2021, 38(2):53-56, 60. DOI: 10.3969/j.issn.1006-6179.2021.02.009 .
8	TAFT R. In vitro fertilization in mice[J]. Cold Spring Harb Protoc, 2017, 2017(11): pdb.prot094508. DOI: 10.1101/pdb.prot094508 .
9	MOREIRA P N, POZUETA J, PÉREZ-CRESPO M, et al. Improving the generation of genomic-type transgenic mice by ICSI[J]. Transgenic Res, 2007, 16(2):163-168. DOI: 10.1007/s11248-007-9075-1 .
10	LARSON M A. Embryo transfer surgery[J]. Methods Mol Biol, 2020, 2066:101-106. DOI: 10.1007/978-1-4939-9837-1_8 .
11	BEHRINGER R. Mouse ovary transplantation[J]. Cold Spring Harb Protoc, 2017, 2017(3):254-256. DOI: 10.1101/pdb.prot094458 .
12	黎桂玲, 刘科, 杨林, 等. 精子数量对小鼠体外受精与胚胎早期发育的影响[J]. 广东农业科学, 2020, 47(1):131-136. DOI: 10.16768/j.issn.1004-874X.2020.01.018 .
	LI G L, LIU K, YANG L, et al. Effects of sperm counts on in vitro fertilization and early embryonic development of mice[J]. Guangdong Agric Sci, 2020, 47(1):131-136. DOI: 10.16768/j.issn.1004-874X.2020.01.018 .
13	王芊芊, 刘迪文, 洪胜辉, 等. 小鼠生物净化技术平台的设计、运行及维护[J]. 中国比较医学杂志, 2023, 33(8):122-126. DOI: 10.3969/j.issn.1671-7856.2023.08.016 .
	WANG Q Q, LIU D W, HONG S H, et al. Design, operation, and maintenance of the technology platform of mouse rederivation[J]. Chin J Comp Med, 2023, 33(8):122-126. DOI: 10.3969/j.issn.1671-7856.2023.08.016 .
14	吴清洪, 顾为望, 张嘉宁, 等. SPF级C57BL/6J小鼠生长发育和繁殖性能指标的测定[J]. 中国比较医学杂志, 2006, 16(10):606-607, 614. DOI: 10.3969/j.issn.1671-7856.2006.10.009 .
	WU Q H, GU W W, ZHANG J N, et al. Characterization of growth and reproductive performance in SPF C57BL/6J mice[J]. Chin J Comp Med, 2006, 16(10):606-607, 614. DOI: 10.3969/j.issn.1671-7856.2006.10.009 .
15	孙侠, 刘科, 杨林, 等. 6种常用SPF级大小鼠繁殖性能测定与分析[J]. 中国比较医学杂志, 2016, 26(10):9-13. DOI: 10.3969.j.issn.1671-7856.2016.10.003 .
	SUN X, LIU K, YANG L, et al. Measurement and analysis of the reproductive performance in six commonly used SPF mice and rats[J]. Chin J Comp Med, 2016, 26(10):9-13. DOI: 10.3969.j.issn.1671-7856.2016.10.003 .
16	陆少君, 曾昭智, 张锦红, 等. 胎间隔和交配间隔时间对昆明小鼠繁殖性能的影响[J]. 动物医学进展, 2018, 39(8):74-77. DOI: 10.16437/j.cnki.1007-5038.2018.08.016 .
	LU S J, ZENG Z Z, ZHANG J H, et al. Effects of fetal interval and mating interval on reproductive performance in KM mice[J]. Prog Vet Med, 2018, 39(8):74-77. DOI: 10.16437/j.cnki.1007-5038.2018.08.016 .
17	聂永强, 王朝霞. 濒危基因编辑小鼠品系拯救技术及其应用探讨[J]. 实验动物与比较医学, 2023, 43(6):636-640. DOI: 10.12300/j.issn.1674-5817.2023.080 .
	NIE Y Q, WANG Z X. Rescue technology and its application of endangered gene-edited mice[J]. Lab Anim Comp Med, 2023, 43(6):636-640. DOI: 10.12300/j.issn.1674-5817.2023.080 .
18	RICHARDS J S, PANGAS S A. The ovary: basic biology and clinical implications[J]. J Clin Invest, 2010, 120(4):963-972. DOI: 10.1172/JCI41350 .
19	NELSON J F, BERGMAN M D, KARELUS K, et al. Aging of the hypothalamo-pituitary-ovarian axis: hormonal influences and cellular mechanisms[J]. J Steroid Biochem, 1987, 27(4-6):699-705. DOI: 10.1016/0022-4731(87)90139-7 .
20	郁丽丽, 刘丽均, 王俊风, 等. 同品系小鼠卵巢移植方法的建立[J]. 中国比较医学杂志, 2013, 23(7):41-43. DOI: 10.3969/j.issn.1671-7856.2013.07.010 .
	YU L L, LIU L J, WANG J F, et al. Established a method of ovarian heterotopically transplantation in same strain[J]. Chin J Comp Med, 2013, 23(7):41-43. DOI: 10.3969/j.issn.1671-7856.2013.07.010 .
21	左琴, 王劲松, 范涛, 等. 遗传工程小鼠冻存卵巢异体原位移植比较研究[J]. 实验动物科学, 2020, 37(3):65-68, 72. DOI: 10.3969/j.issn.1006-6179.2020.03.011 .
	ZUO Q, WANG J S, FAN T, et al. Study for cryopreservation and orthotopic transplantation of genetically engineering mice ovary[J]. Lab Anim Sci, 2020, 37(3):65-68, 72. DOI: 10.3969/j.issn.1006-6179.2020.03.011 .
22	NIIKURA Y, NIIKURA T, TILLY J L. Aged mouse ovaries possess rare premeiotic germ cells that can generate oocytes following transplantation into a young host environment[J]. Aging, 2009, 1(12):971-978. DOI: 10.18632/aging.100105 .
23	裴承斌, 俞晓丽, 马建军, 等. 小鼠卵巢异体不同部位无血管吻合移植后的血管重建及对促性腺激素的反应[J]. 宁夏医科大学学报, 2017, 39(6):616-620, 626, 739. DOI: 10.16050/j.cnki.issn1674-6309.2017.06.002 .
	PEI C B, YU X L, MA J J, et al. Vascular remodeling and response to gonadotropin in different parts of transplantation in mice without vascular anastomosis[J]. J Ningxia Med Univ, 2017, 39(6):616-620, 626, 739. DOI: 10.16050/j.cnki.issn1674-6309.2017.06.002 .
24	李宇彬, 麦庆云, 李涛, 等. 去势手术方式对小鼠卵巢组织自体皮下移植效果的影响[J]. 广东医学, 2014, 35(11):1647-1649. DOI: 10.13820/j.cnki.gdyx.2014.11.005 .
	LI Y B, MAI Q Y, LI T, et al. Influence of castration methods on the subcutaneous auto- transplantation of mouse ovaria[J]. Guangdong Med J, 2014, 35(11):1647-1649. DOI: 10.13820/j.cnki.gdyx.2014.11.005 .
25	GAVISH Z, SPECTOR I, PEER G, et al. Follicle activation is a significant and immediate cause of follicle loss after ovarian tissue transplantation[J]. J Assist Reprod Genet, 2018, 35(1):61-69. DOI: 10.1007/s10815-017-1079-z .
26	RONESS H, MEIROW D. FERTILITY PRESERVATION: Follicle reserve loss in ovarian tissue transplantation[J]. Reproduction, 2019, 158(5): F35-F44. DOI: 10.1530/REP-19-0097 .
27	李茗薇, 黄茹, 严青秀, 等. 小鼠卵巢组织冷冻保存和自体移植技术的研究[J]. 医学信息, 2024, 37(6):120-124.
	LI M W, HUANG R, YAN Q X, et al. Study on the cryopreservation and autologous transplantation technique of mouse ovarian tissues[J]. J Med Inf, 2024, 37(6):120-124.

[1]	JIAO Qingzhen, WU Guihua, TANG Wen, FAN Fan, FENG Kai, YANG Chunxiang, QIAO Jian, DENG Sufang. Dynamic Monitoring and Analysis of Ammonia Concentration in Laboratory Animal Facilities Under Suspension of Heating Ventilation and Air Conditioning System [J]. Laboratory Animal and Comparative Medicine, 2025, 45(4): 490-495.
[2]	LIU Wentao, LUO Yanhong, LONG Yongxia, LUO Qihui, CHEN Zhengli, LIU Lida. Common Environmental Problems and Testing Experiences in Laboratory Animal Facilities in Sichuan Province [J]. Laboratory Animal and Comparative Medicine, 2025, 45(4): 483-489.
[3]	ZHAO Xin, WANG Chenxi, SHI Wenqing, LOU Yuefen. Advances in the Application of Zebrafish in the Research of Inflammatory Bowel Disease Mechanisms and Drug Development [J]. Laboratory Animal and Comparative Medicine, 2025, 45(4): 422-431.
[4]	GONG Leilei, WANG Xiaoxia, FENG Xuewei, LI Xinlei, ZHAO Han, ZHANG Xueyan, FENG Xin. A Mouse Model and Mechanism Study of Premature Ovarian Insufficiency Induced by Different Concentrations of Cyclophosphamide [J]. Laboratory Animal and Comparative Medicine, 2025, 45(4): 403-410.
[5]	LIN Zhenhua, CHU Xiangyu, WEI Zhenxi, DONG Chuanjun, ZHAO Zenglin, SUN Xiaoxia, LI Qingyu, ZHANG Qi. Evaluation of the Safety and Efficacy of Bone Cement in Experimental Pigs Using Vertebroplasty [J]. Laboratory Animal and Comparative Medicine, 2025, 45(4): 466-472.
[6]	JIANG Juan, SONG Ning, LIAN Wenbo, SHAO Congcong, GU Wenwen, SHI Yan. Comparison of Histopathological and Molecular Pathological Phenotypes in Mouse Models of Intrauterine Adhesions Induced by Two Concentrations of Ethanol Perfusion [J]. Laboratory Animal and Comparative Medicine, 2025, 45(4): 393-402.
[7]	LIU Yueqin, XUE Weiguo, WANG Shuyou, SHEN Yaohua, JIA Shuyong, WANG Guangjun, SONG Xiaojing. Observation of Digestive Tract Tissue Morphology in Mice Using Probe-Based Confocal Laser Endomicroscopy [J]. Laboratory Animal and Comparative Medicine, 2025, 45(4): 457-465.
[8]	ZHENG Qingyong, YANG Donghua, MA Zhichao, ZHOU Ziyu, LU Yang, WANG Jingyu, XING Lina, KANG Yingying, DU Li, ZHAO Chunxiang, DI Baoshan, TIAN Jinhui. Recommendations for Standardized Reporting of Systematic Reviews and Meta-Analysis of Animal Experiments [J]. Laboratory Animal and Comparative Medicine, 2025, 45(4): 496-507.
[9]	WANG Tingjun, LUO Hao, CHEN Qi. Discussion on AI-Based Digital Upgrade and Application Practice of Laboratory Animal Centers [J]. Laboratory Animal and Comparative Medicine, 2025, 45(4): 473-482.
[10]	WANG Jiaoxiang, ZHANG Lu, CHEN Shuhan, JIAO Deling, ZHAO Heng, WEI Taiyun, GUO Jianxiong, XU Kaixiang, WEI Hongjiang. Construction and Functional Validation of GTKO/hCD55 Gene-Edited Xenotransplant Donor Pigs [J]. Laboratory Animal and Comparative Medicine, 2025, 45(4): 379-392.
[11]	QIN Chao, LI Shuangxing, ZHAO Tingting, JIANG Chenchen, ZHAO Jing, YANG Yanwei, LIN Zhi, WANG Sanlong, WEN Hairuo. Study on the 90-day Feeding Experimental Background Data of SD Rats for Drug Safety Evaluation [J]. Laboratory Animal and Comparative Medicine, 2025, 45(4): 439-448.
[12]	LIU Kun, LAN Qing, YI Bing, XIE Xiaojie. Key Challenges and Mitigation Strategies for Animal Pregnancy in Non-clinical Reproductive Toxicity Testing of Drugs [J]. Laboratory Animal and Comparative Medicine, 2025, 45(4): 449-456.
[13]	. [J]. Laboratory Animal and Comparative Medicine, 2025, 45(4): 508-514.
[14]	CHEN Ziyi, SUN Hongyan, KANG Pinfang, WU Wenjuan. Research Advances in Animal Experimental Models of Pulmonary Hypertension [J]. Laboratory Animal and Comparative Medicine, 2025, (): 1-12.
[15]	XU Yingtao, WANG Mengmeng, LIN Ping, CHI Haitao, WANG Yi, BAI Ying. Exosomes Improve Ischemic Stroke by Regulation of Ferroptosis Through the NRF2/SLC7A11/GPX4 Pathway in Mice [J]. Laboratory Animal and Comparative Medicine, 2025, (): 1-11.