Recently, we reported the three wolves cloning with normal karyotype from somatic cells of endangered male gray wolves (Canis lupus), but one wolf had female external genitalia. In this study, we conducted further clinical, histological, and genetic analyses. This cloned wolf had a normal uterus but developed ovotestis. Through molecular analysis of the SRY gene, a mutation in the coding sequence of SRY gene could be excluded as a cause of intersexuality. This is the first report of a cloned wolf with a 78, XY ovotesticular disorder affecting sexual development characterized by bilateral ovotestes.
Animals ; Cloning, Organism/*veterinary ; Female ; Karyotyping ; Mutation ; Nuclear Transfer Techniques/*veterinary ; Ovotesticular Disorders of Sex Development/pathology/*veterinary ; *Wolves

In this study, we examined the feasibility of using subzonal cell injection with electrofusion for interspecies somatic cell nuclear transfer (iSCNT) to produce sei whale embryos and to improve their developmental capacity by investigating the effect of osmolarity and macromolecules in the culture medium on the in vitro developmental capacity. Hybrid embryos produced by the electrofusion of fetal whale fibroblasts with enucleated porcine oocytes were cultured in modified porcine zygote medium-3 to examine the effects of osmolarity and fetal serum on their in vitro developmental capacity. More than 66% of the whale somatic cells successfully fused with the porcine oocytes following electrofusion. A portion (60~81%) of the iSCNT whale embryos developed to the two- to four-cell stages, but no embryos were able to reach the blastocyst stage. This developmental arrest was not overcome by increasing the osmolarity of the medium to 360 mOsm or by the addition of fetal bovine or fetal whale serum. Our results demonstrate that sei whale-porcine hybrid embryos may be produced by SCNT using subzonal injection and electrofusion. The pig oocytes partly supported the remodeling and reprogramming of the sei whale somatic cell nuclei, but they were unable to support the development of iSCNT whale embryos to the blastocyst stage.
Animals ; Cloning, Organism/*veterinary ; Culture Media ; Embryo, Mammalian ; Karyotyping ; Nuclear Transfer Techniques/*veterinary ; *Oocytes ; Swine/*embryology ; Whales/*embryology

Somatic cell nuclear transfer (SCNT) is a cost-effective technique for producing transgenic pigs. However, abnormalities in the cloned pigs might prevent use these animals for clinical applications or disease modeling. In the present study, we generated several cloned pigs. One of the pigs was found to have intrapancreatic ectopic splenic tissue during histopathology analysis although this animal was grossly normal and genetically identical to the other cloned pigs. Ectopic splenic tissue in the pancreas is very rare, especially in animals. To the best of our knowledge, this is the first such report for cloned pigs.
Animals ; Animals, Genetically Modified ; Choristoma/pathology/*veterinary ; Cloning, Organism ; Nuclear Transfer Techniques/*veterinary ; *Pancreas ; Splenic Diseases/pathology/*veterinary ; Swine ; Swine Diseases/*pathology ; Swine, Miniature

The objective of the present study was to investigate the effects of three different culture media on the development of canine somatic cell nuclear transfer (SCNT) embryos. Canine cloned embryos were cultured in modified synthetic oviductal fluid (mSOF), porcine zygote medium-3 (PZM-3), or G1/G2 sequential media. Our results showed that the G1/G2 media yielded significantly higher morula and blastocyst development in canine SCNT embryos (26.1% and 7.8%, respectively) compared to PZM-3 (8.5% and 0%) or mSOF (2.3% and 0%) media. In conclusion, this study suggests that blastocysts can be produced more efficiently using G1/G2 media to culture canine SCNT embryos.
Animals ; Blastocyst/cytology ; Cloning, Organism/*veterinary ; Culture Media/metabolism ; Dogs/*embryology ; Embryo Culture Techniques/*veterinary ; *Embryonic Development ; Nuclear Transfer Techniques/*veterinary

Somatic cell nuclear transfer (SCNT) is considered to be a critical tool for propagating valuable animals. To determine the productivity calves resulting from embryos derived with different culture media, enucleated oocytes matured in vitro were reconstructed with fetal fibroblasts, fused, and activated. The cloned embryos were cultured in modified synthetic oviduct fluid (mSOF) or a chemically defined medium (CDM) and developmental competence was monitored. After 7 days of culturing, the blastocysts were transferred into the uterine horn of estrus-synchronized recipients. SCNT embryos that were cultured in mSOF or CDM developed to the blastocysts stages at similar rates (26.6% vs. 22.5%, respectively). A total of 67 preimplantational stage embryos were transferred into 34 recipients and six cloned calves were born by caesarean section, or assisted or natural delivery. Survival of transferred blastocysts to live cloned calves in the mSOF and the CDM was 18.5% (to recipients), 9.6% (to blastocysts) and 42.9% (to recipients), 20.0% (to blastocysts), respectively. DNA analysis showed that all cloned calves were genetically identical to the donor cells. These results demonstrate that SCNT embryos cultured in CDM showed higher viability as judged by survival of the calves that came to term compared to blastocysts derived from mSOF cultures.
Animals ; Blastocyst/physiology ; *Cattle ; Cloning, Organism/methods/*veterinary ; Culture Media/chemistry ; Embryo Culture Techniques ; Embryo Transfer ; Embryonic Development ; Female ; Fertilization in Vitro/*veterinary ; Nuclear Transfer Techniques/*veterinary ; Pregnancy

Inbred strains of pig become indispensable for a wide range of biological studies. In biomedical science, it is generally accepted that somatic cell nuclear transfer(SCNT)technology with inbreed strain of pig is essential for xenotransplantation. In this study, we observed the anal atresia in a cloned pig which was derived from fetal fibroblast of inbreed miniature pig. A presumptive anal site of the cloned pig was excised and the rectum was sutured to apposed skin for treatment. This cloned piglet seemed to be normal with healthy status after surgery. This report can be useful for the treatment of anal atresia of cloned piglets.
Animals ; Animals, Genetically Modified/*surgery ; Anus, Imperforate/genetics/surgery/*veterinary ; *Cloning, Organism ; Female ; Genetic Predisposition to Disease ; Swine/*abnormalities/genetics/*surgery

In order to enhance the efficiency of sheep somatic cell nuclear transfer, we used a chemically assisted enucleation with colchicine to study the effects of the concentration of colchicine, the incubation time of oocytes in colchicine and the maturation time of oocytes on the enucleation rates and the development of reconstructed embryos. The results showed that 1) there were no significant differences in the rates of cytoplast protrusion and enucleation between oocytes that were incubated in colchicine (0.4 microg/mL) for 0.5 h and oocytes that were incubated in colchicine (0.4 microg/mL) for 1 h, and the rate of cytoplast protrusion can be 85.4% while the rate of cytoplast enucleation is 100%. 2) There was no significant difference in oocyte enucleation between oocytes treated with medium containing 0.2 microg/mL colchicine for 0.5 h and oocytes treated with medium containing 0.4 microg/mL colchicine for 0.5 h. 3) A maturation time of 18-23 h did not affect the rates of cytoplast protrusion and enucleation by chemically assisted enucleation, whereas the rate of enucleation of oocytes by blind enucleation was found to decrease with a prolonged incubation time. 4) The development rates of reconstructed embryos could not be influenced by these two enucleation methods, increased from oocytes matured for 21-23 h. These results demonstrate that sheep oocytes can be enucleated fast and effectively by optimized colcholine chemically assisted enucleation, which can enhance the enucleation rate of sheep oocytes and the early development of reconstructed embryos in vitro.
Animals ; Cloning, Organism ; methods ; Colchicine ; pharmacology ; Embryo, Mammalian ; embryology ; Female ; Nuclear Transfer Techniques ; veterinary ; Oocytes ; cytology ; drug effects ; Sheep

To optimize program of bovine somatic nuclear transfer, we used two different enucleation procedures (by Spindle-view system & Hoechst 33342 staining), two different procedures to introduce donor nuclei (by ooplasm microinjection & electrofusion), and three different group electrofusion parameters (group 1: 1.9 kV/cm, 10 micros, two; group 2: 1.5 kV/cm, 25 micros, two; group 3: 0.6 kV/cm, 100 micros, one) to reconstruct bovine cloned embryos. The cleavation rates and blastocyst development rates of cloned embryos were used to assess the efficiency of different operational procedure. Finally, the best combination of operational procedure, that the spindle-viewer system was used for oocytes enucleating, and donor cell was electrofused into ooplasm by electrical pulse (1.9 kV/cm, 10 micros, two) to reconstruct bovine cloned embryos. Then the excellent blastocysts were transferred to fosters for producing cloned cattle 80 high-quality cloned blastocysts were transferred into 33 fosters, two cloned calves were produced. According to the results, the optimized program could be used to produce cloned cattle.
Animals ; Cattle ; Cell Nucleus ; physiology ; Cloning, Organism ; veterinary ; Embryo Transfer ; methods ; Embryo, Mammalian ; cytology ; physiology ; Female ; Microinjections ; Nuclear Transfer Techniques ; veterinary ; Oocytes ; cytology ; physiology

We compared the efficiency of cloning goat using human lactoferrin (hLF) with genetically modified donor cells marked by single (Neo(r)) or double (Neo(r)/GFP) markers. Single marker expression vector (pBLC14) or dual markers expression vector (pAPLM) was delivered to goat fetal fibroblasts (GFF), and then the transgenic GFF was used as donor cells to produce transgenic goats. Respectively, 58.8% (20/34) and 86.7% (26/30) resistant cell lines confirmed the transgenic integration by PCR. Moreover, pAPLM cells lines were subcultured with several passages, only 20% (6/30) cell lines was observed fluorescence from each cell during the cell passage. Somatic cell nuclear transfer using the donor cells harbouring pBLC14 or pAPLM construct, resulting in a total of 806 reconstructed embryos, a pregnancy rate at 35 d (53.8%, 39.1%) and 60 d (26.9%, 21.7%), and an offspring birth rate (1.9%, 1.4%) with 5 and 7 newborn cloned goats, respectively. Transgene was confirmed by PCR and southern-blot in all cloned offspring. There were no significant differences at the reconstructed embryo fusion rates, pregnancy rates and the birth rate (P > 0.05) between single and double markers groups. The Neo(r)/GFP double markers could improve the reliability for accurately and efficiently selecting the genetically modified donor cells. No adverse effect was observed on the efficiency of transgenic goat production by SCNT using somatic cells transfected with double (Neo(r)/GFP) markers vector.
Animals ; Animals, Genetically Modified ; genetics ; Cloning, Molecular ; Cloning, Organism ; methods ; veterinary ; Fetus ; Fibroblasts ; cytology ; Genetic Markers ; Goats ; embryology ; genetics ; Green Fluorescent Proteins ; genetics ; Humans ; Lactoferrin ; biosynthesis ; genetics ; Neomycin ; Nuclear Transfer Techniques ; veterinary ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection ; veterinary

The preimplantation development competences of somatic cell nuclear transfer (SCNT) embryos reconstructed with enuleated goat (Capra hircus) Metaphase II (MII) oocytes matured in vivo and whole cells derived from adult fibroblasts of several mammalian species (goat, boer goat, bovine, tahr, panda) and human patient were evaluated. Results obtained from our experiments revealed that these reconstructed SCNT embryos could complete preimplantation development to form blastocysts. The fusion rate and blastocyst rate of intra-species SCNT embryos (Capra hircus as control) was 78.67 (557/708); 56.29% (264/469), that of sub-species or inter-species SCNT embryos were: boer goat 78.18% (541/692); 33.90% (40/118), bovine 70.53% (146/207); 22.52% (25/111), tahr 53.51% (61/114); 5.26% (3/570), panda 79.82% (1159/1452); 8.35% (75/898) and human 68.76% (317/461); 5.41% (16/296), respectively. It is concluded that (1) there are no relationships between fusion rate and relativeness of the recipient cytoplasm to nucleus donor cells, (2) cytoplast of the goat MII oocyte can support the preimplantation development of SCNT embryos reconstructed with nucleus from other species, (3) the blastocyst rate of close relative inter-species SCNT embryos is higher than that of distant relative inter-species SCNT embryos.
Animals ; Cattle ; Cloning, Organism ; veterinary ; Embryo Culture Techniques ; methods ; veterinary ; Embryo, Mammalian ; physiology ; Embryonic Development ; physiology ; Female ; Fibroblasts ; cytology ; Goats ; embryology ; genetics ; Humans ; Nuclear Transfer Techniques ; veterinary ; Oocytes ; cytology ; physiology ; Pregnancy