Production of nuclear transfer horse embryos by Piezo-driven injection of somatic cell nuclei and activation with stallion sperm cytosolic extract.

The study explores the use of the Piezo drill and stallion sperm cytosolic extract to create cloned horse embryos through nuclear transfer. The experimental approach led to an 82% successful reconstruction of horse oocytes with horse somatic cell donor nuclei, and indicated Piezo drill can effectively be used for nuclear transfer in cattle and horse oocytes.

Research Methodology

• In this study, the researchers employed two main methods: the direct injection of nuclear material using a Piezo drill, and the activation of the process by injecting stallion sperm cytosolic extract.
• In a controlled environment, equine metaphase II oocytes were activated either with one or two dosages of sperm extract. Results from subsequent culture after 20 hours showed near identical activation (88% vs. 90%) and cleavage rates (49% vs. 46%).
• The injection of the donor nuclei (from horse somatic cells) using the Piezo drill resulted in an 82% successful reconstruction rate of the horse oocytes. This suggests a high level of efficiency in the application of the Piezo drill method.

Observations and Results

• The researchers tested four different dosages of the sperm extract (59, 176, 293, or 1375 microg/ml protein) and also observed two different activation times (1.5-2 hours vs. 8-10 hours after the nuclear transfer).
• They observed that the cleavage and activation rates of oocytes injected with a sperm extract containing 59 microg/ml protein were considerably lower than any other dosage used.
• A significantly higher percentage of embryos with normal nuclei were observed in oocytes injected with, 1.5-2 hours after donor injection, the 1375 microg/ml preparation, in contrast with the 293 microg/ml preparation which was injected 8-10 hours after donor injection (22% vs. 6%).
• In terms of cell development, embryos developed to a maximum of 10 nuclei.

Inter-species Nuclear Transfer

• Inter-species nuclear transfer was also performed where horse nuclei were injected into bovine oocytes that were enucleated and then chemically activated.
• This approach resulted in a successful injection of the donor cell rate of 81%, cleavage rate of 88%, and a 73% cleavage rate with normal nuclei. These findings imply that the methods employed have potential application in inter-species nuclear transfer.

Conclusion

• The study essentially concludes that direct nuclear injection using the Piezo drill is not only an effective way of carrying out nuclear transfer in horse and cattle oocytes, but also that the stallion sperm extract can successfully activate horse oocytes both parthenogenetically and after nuclear transfer.