Comparative aspects of somatic cell nuclear transfer with conventional and zona-free method in cattle, horse, pig and sheep.
Abstract: Nuclear transfer (NT) is a complex procedure that requires considerable technical skills. Over the years attempts have been made to simplify the micromanipulations involved and to make the procedure more user-friendly. A significant step forwards has been the development of the zona-free NT methods. We have used zona-free NT with mechanical aspiration of the metaphase plate as a mean of enucleation, in a comparative approach with the conventional nuclear transfer zona-enclosed method in cattle, horse, sheep and pig. The absence of the zona considerably facilitates the enucleation step and significantly increases cell fusion success. On the other hand, the culture of zona-free NT embryos requires the embryos to be cultured individually or anyway separated from each other to avoid aggregation and also requires to prolong the in vitro culture up to the blastocyst stage before transfer. Blastocyst rate is equal or higher with zona-free method as compared to zona-enclosed method while survival after cryopreservation and development to term is comparable. In conclusion, our findings, together with published data, demonstrate that the zona-free system described in this paper can significantly increase the output of NT blastocysts over the conventional zona-enclosed system.
Publication Date: 2006-11-01 PubMed ID: 17081599DOI: 10.1016/j.theriogenology.2006.09.011Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
- Research Support
- Non-U.S. Gov't
- Review
Summary
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
This research investigates the benefits of using the zona-free method of nuclear transfer in various animals including cattle, horses, sheep, and pigs. The findings suggest that this method offers a more successful cell fusion rate compared to the conventional method and an equivalent or higher blastocyst rate, without a significant impact on the embryos’ survival or development.
Understanding the Context
- The research was conducted in the field of animal cloning, particularly focusing on a procedure known as nuclear transfer (NT). NT is a complex procedure that involves the transfer of a nucleus from a donor cell into an egg cell which has had its own nucleus removed. This forms an embryo, which can then develop into an animal that is genetically identical to the donor.
- Over time, scientists have been trying to simplify this NT process. The authors in this paper have focused on comparing two methods: the conventional NT method and the more recent zona-free NT method, which eliminates the outer covering of the egg cell, called the zona pellucida.
Experiments and Findings
- The researchers experimented with the conventional and zona-free NT methods in cattle, horse, sheep, and pig. They discovered that the zona-free method significantly facilitated the process of enucleation (removal of the egg cell’s nucleus) and increased the success rate of cell fusion.
- In addition, they discovered that zona-free NT embryos, while they needed to be separated to avoid clustering and required further in vitro culture before transfer, showed an equal or higher blastocyst rate (the stage of embryo development where a hollow sphere of cells is formed) than those with the conventional, zona-enclosed method.
- Important to note is that survival after cryopreservation (controlled freezing of biological material) and development to term was comparable between both methods. This means that the zona-free method didn’t negatively impact the embryos’ ability to survive and develop.
Conclusions
- Based on these findings and previously published data, the researchers conclude that the zona-free method can significantly increase the production of NT blastocysts compared to the conventional, zona-enclosed method. This potentially makes the zona-free NT method a better option for animal cloning, as it can provide a higher yield without compromising the survival and development of cloned embryos.
Cite This Article
APA
Lagutina I, Lazzari G, Duchi R, Turini P, Tessaro I, Brunetti D, Colleoni S, Crotti G, Galli C.
(2006).
Comparative aspects of somatic cell nuclear transfer with conventional and zona-free method in cattle, horse, pig and sheep.
Theriogenology, 67(1), 90-98.
https://doi.org/10.1016/j.theriogenology.2006.09.011 Publication
Researcher Affiliations
- Laboratorio di Tecnologie della Riproduzione, Istituto Sperimentale Italiano Lazzaro Spallanzani, CIZ srl, Via Porcellasco 7/f, 26100 Cremona, Italy.
MeSH Terms
- Animals
- Blastocyst / physiology
- Cattle / embryology
- Cell Fusion
- Cells, Cultured
- Cloning, Organism
- Horses / embryology
- Nuclear Transfer Techniques / veterinary
- Oocytes / cytology
- Oocytes / physiology
- Sheep / embryology
- Species Specificity
- Swine / embryology
- Zona Pellucida / physiology
Citations
This article has been cited 21 times.- Meinecke B, Meinecke-Tillmann S. Lab partners: oocytes, embryos and company. A personal view on aspects of oocyte maturation and the development of monozygotic twins. Anim Reprod 2023;20(2):e20230049.
- Ammari AA, ALGhadi MG, Amran RA, Al Malahi NM, Alhimaidi AR. Interspecific Nuclear Transfer Blastocysts Reconstructed from Arabian Oryx Somatic Cells and Domestic Cow Ooplasm. Vet Sci 2022 Dec 28;10(1).
- Lee EJ, Ji KB, Lee JH, Oh HJ, Kil TY, Kim MK. Application of the modified handmade cloning technique to pigs. J Anim Sci Technol 2021 Mar;63(2):281-294.
- Springer C, Wolf E, Simmet K. A New Toolbox in Experimental Embryology-Alternative Model Organisms for Studying Preimplantation Development. J Dev Biol 2021 Apr 2;9(2).
- Vajta G, Parmegiani L, Machaty Z, Chen WB, Yakovenko S. Back to the future: optimised microwell culture of individual human preimplantation stage embryos. J Assist Reprod Genet 2021 Oct;38(10):2563-2574.
- Hisey EA, Ross PJ, Meyers S. Genetic Manipulation of the Equine Oocyte and Embryo. J Equine Vet Sci 2021 Apr;99:103394.
- Haddad M, Stewart J, Xie P, Cheung S, Trout A, Keating D, Parrella A, Lawrence S, Rosenwaks Z, Palermo GD. Thoughts on the popularity of ICSI. J Assist Reprod Genet 2021 Jan;38(1):101-123.
- Perota A, Galli C. N-Glycolylneuraminic Acid (Neu5Gc) Null Large Animals by Targeting the CMP-Neu5Gc Hydroxylase (CMAH). Front Immunol 2019;10:2396.
- Kalds P, Zhou S, Cai B, Liu J, Wang Y, Petersen B, Sonstegard T, Wang X, Chen Y. Sheep and Goat Genome Engineering: From Random Transgenesis to the CRISPR Era. Front Genet 2019;10:750.
- Moulavi F, Hosseini SM. Development of a modified method of handmade cloning in dromedary camel. PLoS One 2019;14(4):e0213737.
- Min B, Park JS, Kang YK. Determination of Oocyte-Manipulation, Zygote-Manipulation, and Genome-Reprogramming Effects on the Transcriptomes of Bovine Blastocysts. Front Genet 2018;9:143.
- Olivera R, Moro LN, Jordan R, Pallarols N, Guglielminetti A, Luzzani C, Miriuka SG, Vichera G. Bone marrow mesenchymal stem cells as nuclear donors improve viability and health of cloned horses. Stem Cells Cloning 2018;11:13-22.
- Liu T, Dou H, Xiang X, Li L, Li Y, Lin L, Pang X, Zhang Y, Chen Y, Luan J, Xu Y, Yang Z, Yang W, Liu H, Li F, Wang H, Yang H, Bolund L, Vajta G, Du Y. Factors Determining the Efficiency of Porcine Somatic Cell Nuclear Transfer: Data Analysis with Over 200,000 Reconstructed Embryos. Cell Reprogram 2015 Dec;17(6):463-71.
- Verma G, Arora JS, Sethi RS, Mukhopadhyay CS, Verma R. Handmade cloning: recent advances, potential and pitfalls. J Anim Sci Biotechnol 2015;6:43.
- Gambini A, De Stefano A, Bevacqua RJ, Karlanian F, Salamone DF. The aggregation of four reconstructed zygotes is the limit to improve the developmental competence of cloned equine embryos. PLoS One 2014;9(11):e110998.
- Li Z, Shi J, Liu D, Zhou R, Zeng H, Zhou X, Mai R, Zeng S, Luo L, Yu W, Zhang S, Wu Z. Effects of donor fibroblast cell type and transferred cloned embryo number on the efficiency of pig cloning. Cell Reprogram 2013 Feb;15(1):35-42.
- Asseged BD, Habtemariam T, Tameru B, Nganwa D. The risk of introduction of equine infectious anemia virus into USA via cloned horse embryos imported from Canada. Theriogenology 2012 Jan 15;77(2):445-58.
- Sachs DH, Galli C. Genetic manipulation in pigs. Curr Opin Organ Transplant 2009 Apr;14(2):148-53.
- Rodríguez L, Navarrete FI, Tovar H, Cox JF, Castro FO. High developmental potential in vitro and in vivo of cattle embryos cloned without micromanipulators. J Assist Reprod Genet 2008 Jan;25(1):13-6.
- Su R, Ding F, Sorgog M, Li L, Bai S, Ritu X, Nasenochir N, Fan R, Herrid M. Optimization of handmade cloning enhances pregnancy rates and live calf production in cattle. Sci Rep 2025 Apr 29;15(1):15032.
- Vazquez-Avendaño JR, Cortez-Romero C, Bravo-Vinaja Á, Ambríz-García DA, Trejo-Córdova A, Navarro-Maldonado MDC. Reproduction of Sheep through Nuclear Transfer of Somatic Cells: A Bibliometric Approach. Animals (Basel) 2023 Jun 1;13(11).
Use Nutrition Calculator
Check if your horse's diet meets their nutrition requirements with our easy-to-use tool Check your horse's diet with our easy-to-use tool
Talk to a Nutritionist
Discuss your horse's feeding plan with our experts over a free phone consultation Discuss your horse's diet over a phone consultation
Submit Diet Evaluation
Get a customized feeding plan for your horse formulated by our equine nutritionists Get a custom feeding plan formulated by our nutritionists
