Effect of sperm extract injection volume, injection of PLCzeta cRNA, and tissue cell line on efficiency of equine nuclear transfer.
Abstract: We evaluated the effect of different activation methods on blastocyst development after equine nuclear transfer. All activation treatments were followed by incubation in 2 mM 6-dimethylaminopurine for 4 h. In Experiment 1, reconstructed oocytes were injected with sperm extract for 0.1, 0.2, 0.4, 0.8, or 1.6 sec using a FemtoJet injection device, then treated with ionomycin. The blastocyst rate (9.8%) for 0.1-sec injection was significantly higher than that for 0.2 sec (0%) or 0.8 sec (1.4%). In Experiment 2, injection of murine PLCzeta cRNA before or after ionomycin treatment did not increase blastocyst development (0 and 4.5%) over a control treatment (injection of sperm extract after ionomycin exposure; 5.6%). Transfer of 10 blastocysts produced in Experiments 1 and 2 resulted in five pregnancies, all lost before 70 days of gestation. In Experiment 3, cells from a second biopsy sample from the same horse produced significantly more blastocysts than did the original sample (4/44 vs. 0/58; p < 0.05). Transfer of these four blastocysts produced two viable foals. In Experiment 4, blastocyst development rates did not differ between oocytes in metaphase I or II at the time of nuclear transfer (16.7 and 3.0%, respectively). A healthy foal was produced from a blastocyst originating from a metaphase I oocyte.
Publication Date: 2009-06-11 PubMed ID: 19508114DOI: 10.1089/clo.2008.0077Google Scholar: Lookup
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- Evaluation Study
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This study analyzes how various methods of activation influence the development of horse embryos after nuclear transfer, an essential step in cloning. The article shows that the volume of sperm extract injected, the use of murine PLCzeta cRNA, and the selection of consumable cells from different tissue cell lines can all impact the efficiency of equine nuclear transfer.
Experiment 1: Evaluating Sperm Extract Injection Volume
- This part of the research evaluated the effect of varying volumes of sperm extract on the development of horse blastocysts after nuclear transfer. The volumes were measured in seconds (0.1 sec, 0.2 sec, 0.4 sec, 0.8 sec, or 1.6 sec) using a FemtoJet injection device, followed by ionomycin treatment.
- The study found that an injection duration of 0.1 seconds had the highest blastocyst rate (9.8%). This rate was significantly greater than the rates for 0.2 seconds (0%) and 0.8 seconds (1.4%).
Experiment 2: Injection of Murine PLCzeta cRNA
- The second experiment evaluated whether the injection of murine PLCzeta cRNA before or after ionomycin treatment could enhance the development of blastocysts.
- It was found that the injection did not significantly increase the rate of development (0% and 4.5%), compared to a control treatment that involved the injection of sperm extract after ionomycin exposure (5.6%).
Experiment 3: Impact of Tissue Cell Line
- Researchers then compared the results from two different biopsy samples from the same horse. The cell lines were then used to create clones via nuclear transfer.
- The study found that the second biopsy sample line produced significantly more blastocysts compared to the first one (4 out of 44 as opposed to 0 out of 58).
- When four of these blastocyst were transferred, two viable foals were created.
Experiment 4: Metaphase State of Oocytes at Time of Transfer
- The last experiment compared the development rates of oocytes in two phases of mitosis — metaphase I or II — at the time of nuclear transfer.
- Results showed that the development rates didn’t differ significantly between the two phases, with rates at 16.7% for metaphase I and 3.0% for metaphase II.
- A healthy foal was successfully produced from a blastocyst originated from a metaphase I oocyte.
Cite This Article
APA
Choi YH, Hartman DL, Fissore RA, Bedford-Guaus SJ, Hinrichs K.
(2009).
Effect of sperm extract injection volume, injection of PLCzeta cRNA, and tissue cell line on efficiency of equine nuclear transfer.
Cloning Stem Cells, 11(2), 301-308.
https://doi.org/10.1089/clo.2008.0077 Publication
Researcher Affiliations
- College of Veterinary Medicine and Biomedical Sciences, Texas A&M University , College Station, TX 77843-4466, USA.
MeSH Terms
- Animals
- Blastocyst / cytology
- Blastocyst / physiology
- Cell Line
- Embryo, Mammalian / physiology
- Horses
- Isoenzymes / genetics
- Isoenzymes / metabolism
- Male
- Mice
- Nuclear Transfer Techniques
- Oocytes / physiology
- Phosphoinositide Phospholipase C / genetics
- Phosphoinositide Phospholipase C / metabolism
- RNA, Complementary / genetics
- RNA, Complementary / metabolism
- Spermatozoa / chemistry
Citations
This article has been cited 5 times.- Hisey EA, Ross PJ, Meyers S. Genetic Manipulation of the Equine Oocyte and Embryo. J Equine Vet Sci 2021 Apr;99:103394.
- Olivera R, Moro LN, Jordan R, Luzzani C, Miriuka S, Radrizzani M, Donadeu FX, Vichera G. In Vitro and In Vivo Development of Horse Cloned Embryos Generated with iPSCs, Mesenchymal Stromal Cells and Fetal or Adult Fibroblasts as Nuclear Donors. PLoS One 2016;11(10):e0164049.
- Kang H, Park JI, Roh S. Murine somatic cell nuclear transfer using reprogrammed donor cells expressing male germ cell-specific genes. J Vet Med Sci 2016 Jan;78(1):149-52.
- 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.
- Martino NA, Dell'Aquila ME, Filioli Uranio M, Rutigliano L, Nicassio M, Lacalandra GM, Hinrichs K. Effect of holding equine oocytes in meiosis inhibitor-free medium before in vitro maturation and of holding temperature on meiotic suppression and mitochondrial energy/redox potential. Reprod Biol Endocrinol 2014 Oct 11;12:99.
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