Figure 2

Technical improvement. (A) Animal reproductive cloning process; (a) a common process for bovine cloning; from the ovaries derived from the slaughterhouse, the immature oocytes are aspirated from the microneedles. These eggs become mature oocytes when cultured in in vitro maturation medium containing various hormones and growth factors for 22 h. The matured oocytes undergo a sequential process of somatic cell nuclear transfer. Afterward, the cell and cytoplasm couplets are subjected to a fusion process using electrical stimulation and then the activation of the reconstructed embryos is performed. Successfully fused cloned embryos are cultured in IVC medium for 7 days to develop into blastocysts and then transplanted into estrus synchronized surrogate mothers. (b) The method employed to produce Snuppy is still in use by other groups. Serum progesterone is measured from oocyte donors that have not undergone any hormonal treatment to determine the time of mature oocytes. The activation process is performed for 4 h after the fusion is performed. (c) Currently, we are using the method of dog cloning as an electrical stimulation to give simultaneously fusion and activation, the reconstructed embryos is directly implanted to the surrogate. (B) The simultaneous activation method was 1.5 times higher in cloning efficiency and 1.3 times higher in pregnancy rate although there was no significant difference due to variation. (C) (a) A catheter inserted into the fallopian tube, and a mature oocyte is collected by flowing a perfusion liquid through the catheter. Left 16G Right 18G. (b) Mature oocytes K = canine, B = bovine, P = porcine, the canine mature oocytes have irregular and inhomogeneous zona pelucidas compared to other species of oocytes. The cytoplasm is dark, and the nucleus and the polar body cannot be observed without Hoechst staining. (c) The nucleus of the oocyte located in the cytoplasm under the polar body is moved to the periphery of the cytoplasm when the polar body is protruded, but in canine, it is often very separated from the polar body as shown in the second and third pictures. (d) The squeezing method that we used previously is favorable for reducing damage to the cytoplasmic membrane, but cytoplasmic loss is greater than in the aspiration method.