Production of Nuclear Transfer Horse Embryos by Piezo-Driven Injection of Somatic Cell Nuclei and Activation with Stallion Sperm Cytosolic Extract1

Abstract

We investigated the use of direct nuclear injection using the Piezo drill and activation by injection of stallion sperm cytosolic extract for production of cloned equine embryos. When metaphase II horse oocytes were injected with either of two dosages of sperm extract and cultured 20 h, similar activation rates (88% vs. 90%) and cleavage rates (49% vs. 46%) were obtained. The successful reconstruction rate of horse oocytes with horse somatic cell donor nuclei after direct injection using the Piezo drill was 82%. Four dosages of sperm extract (containing 59, 176, 293, or 1375 microg/ml protein) and two activation times (1.5-2 vs. 8-10 h after nuclear transfer) were examined. Cleavage and activation (pseudopronucleus formation) rates of oocytes injected with sperm extract containing 59 microg/ml protein were significantly (P < 0.05) lower than any other dosage. The percentage of embryos cleaving with normal nuclei in oocytes injected with the 1375 microg/ml preparation 1.5-2 h after donor injection was significantly (P < 0.05) higher than that of the 293 microg/ml preparation 8-10 h after donor injection (22 vs. 6%). Embryos developed to a maximum of 10 nuclei. Interspecies nuclear transfer was performed by direct injection of horse nuclei into enucleated bovine oocytes, followed by chemical activation. This resulted in 81% reconstruction (successful injection of the donor cell), 88% cleavage, and 73% cleavage with normal nuclei. These results indicate that direct nuclear injection using the Piezo drill is an efficient method for nuclear transfer in horse and cattle oocytes and that sperm extract can efficiently activate horse oocytes both parthenogenetically and after nuclear transfer

Piezo-actuated mouse intracytoplasmic sperm injection (ICSI)

“…In addition, and again in contrast to conventional micromanipulation, piezo permits multiple procedures to be performed with a single needle. Thus, although many oocytes (such as those of humans, cattle, pigs and rabbits) can be injected conventionally, the relative speed and ease of piezo means that it is advantageous even in these species 3,[15][16][17][18][19][20] . Ease of pipette fabrication (beveled tips are not required) and speed also make piezo suitable for the injection of blastocysts with ES cells 11 , and the method additionally allows partial ablation of the inner cell mass (i.e., the removal of potentially competing embryonic cells).…”

Factors inducing the activation of reconstructed oocytes of farm animals in somatic cloning procedures

“…However, the microinjection technique has not been in wide use as a method of activating reconstructed oocytes. Recently, an interesting proposition of chemical activation is injection of sperm cytosolic extract into cytoplasm of reconstructed oocytes (Choi et al, 2002).…”

Intracellular calcium oscillations and activation in horse oocytes injected with stallion sperm extracts or spermatozoa

“…Recently, crude sperm extracts obtained from the supernatant of frozen-thawed stallion sperm lysates have been used to activate equine oocytes subjected to nuclear transfer (Choi et al, 2002a …”