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Production of cloned horse foals using roscovitine-treated donor cells and activation with sperm extract and/or ionomycin.
Abstract: We evaluated the effect of different activation treatments on the production of blastocysts and foals by nuclear transfer. Donor cells were prepared using roscovitine treatment, which has previously been associated with increased production of viable offspring. All activation treatments were followed by culture in 6-dimethylaminopurine (6-DMAP) for 4 h. In experiment 1, blastocyst production after activation by injection of sperm extract followed by treatment with ionomycin was significantly higher than that for activation with a serial treatment of ionomycin, 6-DMAP, and ionomycin (12.5 vs 2.8%; P < 0.05) and tended to be higher than that for injection of sperm extract alone (3.4%; P = 0.07). In experiment 2, there were no significant differences in blastocyst development among treatments with ionomycin once or twice, sperm extract then ionomycin, or ionomycin then sperm extract (range 4.6-7.3%). Overall, transfer of 26 blastocysts resulted in 16 pregnancies (62%) and 9 live foals (35% of transferred embryos). Treatment with sperm extract followed by ionomycin produced a live foal rate per embryo transferred of 5/10 (50%). One foal died of pneumonia 48 h post partum and one foal died at 1 week of age after complications during induction of anesthesia; the remaining seven foals are currently 10-14 months of age.
Publication Date: 2007-07-31 PubMed ID: 17660241DOI: 10.1530/REP-07-0069Google 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
Summary
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The research article details an experiment on nuclear transfer in cloning horse foals using roscovitine-treated donor cells activated by sperm extract and/or ionomycin. It discusses how the injection of sperm extract succeeded in increasing blastocyst and live foal production.
Donor Cell Preparation
- The study used roscovitine treatment to prepare donor cells. Previous research has associated this treatment method with an increased production of viable offspring.
- After different activation treatments, the cells were exposed to a culture in 6-dimethylaminopurine (6-DMAP) for 4 hours.
Experiment Procedure & Results
- The first experiment showed that injectable sperm extract followed by ionomycin treatment significantly boosted blastocyst production compared to other activation methods. These methods involved a serial treatment of ionomycin, 6-DMAP, and ionomycin.
- A second experiment found that there were no notable differences in blastocyst development across various activation treatments. This included ionomycin used once or twice and either sperm extract followed by ionomycin or vice versa.
- In sum, the transfer of 26 blastocysts resulted in 16 pregnancies (representing 62% successful pregnancies) and yielded 9 live foals (representing 35% of transferred embryos).
Live Foal Production
- Sperm extract with subsequent ionomycin treatment produced a live foal rate per embryo transferred of 50%, which equals 5 out of 10 embryos.
- Though two foals unfortunately died due to post-birth complications, seven of the foals have survived and are currently 10-14 months old.
Conclusions
- The study successfully demonstrated the impact of different activation treatments on blastocyst and live foal production in horse cloning.
- The findings indicate that using roscovitine-treated donor cells and activation with an injection of sperm extract followed by ionomycin can improve the success rate of horse cloning.
Cite This Article
APA
Hinrichs K, Choi YH, Varner DD, Hartman DL.
(2007).
Production of cloned horse foals using roscovitine-treated donor cells and activation with sperm extract and/or ionomycin.
Reproduction, 134(2), 319-325.
https://doi.org/10.1530/REP-07-0069 Publication
Researcher Affiliations
- Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas 77843, USA. khinrichs@cvm.tamu.edu
MeSH Terms
- Adenine / analogs & derivatives
- Adenine / pharmacology
- Animals
- Blastocyst / physiology
- Calcium / metabolism
- Cell Culture Techniques
- Cleavage Stage, Ovum
- Cloning, Organism / methods
- Embryo Transfer
- Embryonic Development
- Female
- Horses
- Ionomycin / pharmacology
- Male
- Microinjections
- Nuclear Transfer Techniques
- Oocyte Donation / veterinary
- Oocytes / drug effects
- Oocytes / metabolism
- Pregnancy
- Pregnancy Outcome
- Protein Kinase Inhibitors / pharmacology
- Purines / pharmacology
- Roscovitine
- Sperm Injections, Intracytoplasmic
Citations
This article has been cited 11 times.- Samiec M. Molecular Mechanism and Application of Somatic Cell Cloning in Mammals-Past, Present and Future. Int J Mol Sci 2022 Nov 9;23(22).
- Samiec M, Skrzyszowska M. Extranuclear Inheritance of Mitochondrial Genome and Epigenetic Reprogrammability of Chromosomal Telomeres in Somatic Cell Cloning of Mammals. Int J Mol Sci 2021 Mar 18;22(6).
- Hisey EA, Ross PJ, Meyers S. Genetic Manipulation of the Equine Oocyte and Embryo. J Equine Vet Sci 2021 Apr;99:103394.
- Stein P, Savy V, Williams AM, Williams CJ. Modulators of calcium signalling at fertilization. Open Biol 2020 Jul;10(7):200118.
- 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.
- Meijer L, Nelson DJ, Riazanski V, Gabdoulkhakova AG, Hery-Arnaud G, Le Berre R, Loaëc N, Oumata N, Galons H, Nowak E, Gueganton L, Dorothée G, Prochazkova M, Hall B, Kulkarni AB, Gray RD, Rossi AG, Witko-Sarsat V, Norez C, Becq F, Ravel D, Mottier D, Rault G. Modulating Innate and Adaptive Immunity by (R)-Roscovitine: Potential Therapeutic Opportunity in Cystic Fibrosis. J Innate Immun 2016;8(4):330-49.
- 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.
- Keefer CL. Artificial cloning of domestic animals. Proc Natl Acad Sci U S A 2015 Jul 21;112(29):8874-8.
- Lee W, Song K, Lee I, Shin H, Lee BC, Yeon S, Jang G. Cloned foal derived from in vivo matured horse oocytes aspirated by the short disposable needle system. J Vet Sci 2015;16(4):509-16.
- 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.
- Brosnahan MM, Brooks SA, Antczak DF. Equine clinical genomics: A clinician's primer. Equine Vet J 2010 Oct;42(7):658-70.
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