Although efficient nonsurgical transfer of embryos in mice would provide many advantages over a surgical method, the low success rate of nonsurgical transfer has hampered its acceptance and use. Here, a plastic catheter was used to mimic embryo transfer process and then the transfer efficiency was evaluated by intrauterine trypan blue dye dispersion. Also 3.5-day blastocysts from natural pregnant mice were transferred through cervix into uterine horns. The results show that 70.9% of CD-1 mouse 3.5-day blastocysts transferred into unilateral uterine horns of pseudopregnant 2.5-day recipients can be developed to live newborns, and an efficient mouse nonsurgical embryo transfer technique was established. The technique was simple, rapid, inexpensive, unlikely to get contaminated, ethical and do not need specialized apparatus, and can completely replace surgical embryo transfer techniques. Moreover, the mouse nonsurgical embryo transfer technique provides a research model for human and other large animal embryo transfer.
Animals ; Blastocyst ; Embryo Transfer ; methods ; veterinary ; Female ; Mice ; Pregnancy

This study was performed to produce transgenic Korean native goat (Capra hircus) by laparoscopic embryo transfer (ET) to overcome the limitations of ET performed by laparotomy. Transgenic embryos were produced by DNA pronuclear microinjection of in vivo zygotes. The recipient goats were synchronized for estrus by using an introvaginal progesterone devices as a controlled internal drugreleasing insert (CIDR) for 13 days and injection of 400 IU PMSG 48 h before removal of the insert. Embryos were transferred on day 3 and 4 after removal of the insert. Recipient goats were deprived of feed for 48 h, then suspended in a laparotomy cradle at an angle of 45degrees. After obtaining a sufficient pneumoperitoneum, the laparoscope and forceps were inserted abdominally through 5 mm trocar sleeves. Examination of the ovaries and uterus was performed and then 213 embryos were transferred into the oviducts via the infundibula of 76 recipient goats. To compare pregnancy rates, ET was also performed by laparotomy in 82 recipient goats. The pregnancies in the recipient goats were diagnosed by ultrasound on day 30 after embryo transfer. The pregnancy rate with laparoscopic ET was significantly higher than with ET performed by laparotomy (46.1% vs. 28.6%, p < 0.05). In addition, the pregnancy rates were compared between ovulated and non-ovulated ovaries of the recipient goats in the laparoscopic ET group. No significant difference was observed between the pregnancy rates of ovulated and non-ovulated ovaries (41.3% vs. 33.3%, p < 0.05) suggesting that ET may also be possible in non-ovulated recipients through artificial rupture of Graafian follicles. These results suggest that laparoscopic ET is a highly efficient method for the transfer of goat embryos.
Animals ; Animals, Genetically Modified/*embryology ; Embryo Transfer/methods/*veterinary ; Female ; Goats/*genetics/physiology ; Laparoscopy/*veterinary ; Laparotomy/*veterinary ; Microinjections/veterinary ; Oocytes

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

The purpose was to optimize the vitrification for porcine embryos cryopreservation. Blastocyst/Morula (5-6th day-embryos) were collected from superovulated Bama mini-pigs (sows/gilts). We compared different cryopreservation methods, cryopreservation tools, thining of zona pellucida (ZP) and recipient breeds on the efficiency of porcine embryo cryopreservation. The results showed that: in embryo survival rate and blastocyst cell number, there were no significant differences between cryopreservation method I [embryos were vitrified by two step method with open pulled straw (OPS) and glass micropipette (GMP) in solution 1 (TCM199 + 20% FBS + 10% EG + 10% DMSO) for 3 min, and solution 2 (TCM199 + 20% FBS + 20% EG + 20% DMSO + 0.4 mol/L SUC) for 1 min, stored in liquid nitrogen] and method II[Blastocysts were cultured for 25 min in NCSU23 + 7.5 microg/mL cytochalasin B, centrifuged at approximately 13 000 xg for 12-13 min, and recovered back into pNCSU23. They were then equilibrated for 5 min in 2 mol/L ethylene glycol in pNCSU23, washed quickly in the vitrification medium, 8 mol/L ethylene glycol, 7% polyvinylpyrrolidone (PVP) in pNCSU23, loaded into OPS/GMP, and plunged into liquid nitrogen]. GMP vitrification method was more suitable and efficient than OPS method (P < 0.05) in embryo survival rate (83.8% vs 77.6%) and blastocyst cell number (53.1 vs 47.5) after thawing. Thining of ZP did not increase the survival rate, but significantly improved blastocyst cell number in the survival blastcysts (60.1 and 46, P < 0.01). Local pig breeds (Fengjing sows) were more suitable as recipients for embryo transfer of vitrified/warmed blastcysts, which can improve pregnant rate and embryo efficiency.
Animals ; Blastomeres ; cytology ; Cryopreservation ; methods ; veterinary ; Embryo Transfer ; veterinary ; Embryo, Mammalian ; Swine ; Swine, Miniature ; Vitrification ; Zona Pellucida ; physiology

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

291 embryos (Blastocyst/Morula) from 20 donor sows were vitrified by two step method with OPS (open pulled straw) in solution I (TCM199 + 20% FBS + 10% EG + 10% DMSO) for 3min, and solution II (TCM199 + 20% FBS + 20% EG + 20% DMSO + 0.4mol/L SUC) for 1min, stored in liquid nitrogen for 3 months, and transferred into 8 recipient sows after warming, one recipient sow was pregnant and 8 alive piglets were born. This is the first paper to report getting alive piglets by vitrification in China.
Animals ; Blastocyst ; physiology ; Cryopreservation ; methods ; veterinary ; Embryo Transfer ; Female ; Pregnancy ; Swine ; embryology

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

This study examined effects on the developmental competence of pig oocytes after somatic cell nuclear transfer (SCNT) or parthenogenetic activation (PA) of : 1) co-culturing of oocytes with follicular shell pieces (FSP) during in vitro maturation (IVM); 2) different durations of maturation; and 3) defined maturation medium supplemented with polyvinyl alcohol (PVA; control), pig follicular fluid (pFF), cysteamine (CYS), or beta-mercaptoethanol (beta-ME). The proportion of metaphase II oocytes was increased (p < 0.05) by co-culturing with FSP compared to control oocytes (98% vs. 94%). However, blastocyst formation after SCNT was not improved by FSP coculture (9% vs. 12%). Nuclear maturation of oocytes matured for 39 or 42 h was higher (p < 0.05) than that of oocytes matured for 36 h (95-96% vs. 79%). Cleavage (83%) and blastocyst formation (26%) were significantly higher (p < 0.05) in oocytes matured for 42 h than in other groups. Supplementation of a defined maturation medium with 100 micrometer CYS or 100 micrometer beta-ME showed no stimulatory effect on oocyte maturation, embryo cleavage, or blastocyst formation after PA. beta-ME treatment during IVM decreased embryo cleavage after SCNT compared to pFF or PVA treatments, but no significant difference was found in blastocyst formation (7-16%) among the four treatment groups. The results indicated that maturation of oocytes for 42 h was beneficial for the development of SCNT embryos. Furthermore, the defined maturation system used in this study could support in vitro development of PA or SCNT embryos.
Animals ; Cysteamine ; Embryo Culture Techniques/*veterinary ; Embryo, Mammalian/*physiology ; Female ; Follicular Fluid ; Mercaptoethanol ; Nuclear Transfer Techniques/*veterinary ; Oocytes/*growth & development ; Sus scrofa/*physiology ; Time Factors

The level of P4 at the time of embryo transfer (ET) is important. P4 concentrations and numbers of corpora lutea for 126 recipients were evaluated. Nuclear transfer embryos were transferred into 126 surrogates. 11 maintained their pregnancy until full-term delivery, 17 miscarried, and implantation failed in 98 animals. P4 levels in the full-term group were significantly different from those of the pigs that aborted or in which implantation failed (p < 0.05). However, the numbers of corpora lutea were not significantly different. These findings indicate that the concentration of progesterone can be an important factor for successful ET in pigs.
Animals ; Corpus Luteum/*physiology ; Embryo Transfer/*veterinary ; Embryo, Mammalian/*physiology ; Female ; Nuclear Transfer Techniques ; Pregnancy ; *Pregnancy Rate ; Progesterone/*blood ; Retrospective Studies ; Sus scrofa/*physiology

The generation of reactive oxygen species (ROS) and subsequent mitochondrial and DNA damage in bovine somatic cell nuclear transfer (SCNT) embryos were examined. Bovine enucleated oocytes were electrofused with donor cells and then activated by a combination of Ca-ionophore and 6-dimethylaminopurine culture. The H2O2 and .OH radical levels, mitochondrial morphology and membrane potential (DeltaPsi), and DNA fragmentation of SCNT and in vitro fertilized (IVF) embryos at the zygote stage were analyzed. The H2O2 (35.6 +/- 1.1 pixels/embryo) and .OH radical levels (44.6 +/- 1.2 pixels/embryo) of SCNT embryos were significantly higher than those of IVF embryos (19.2 +/- 1.5 and 23.8 +/- 1.8 pixels/embryo, respectively, p < 0.05). The mitochondria morphology of SCNT embryos was diffused within the cytoplasm. The DeltaPsi of SCNT embryos was significantly lower (p < 0.05) than that of IVF embryos (0.95 +/- 0.04 vs. 1.21 +/- 0.06, red/green). Moreover, the comet tail length of SCNT embryos was longer than that of IVF embryos (515.5 +/- 26.4 microm vs. 425.6 +/- 25.0 microm, p < 0.05). These results indicate that mitochondrial and DNA damage increased in bovine SCNT embryos, which may have been induced by increased ROS levels.
Animals ; *Apoptosis ; Caspase 3/metabolism ; Cattle ; Colorimetry/veterinary ; Comet Assay/veterinary ; *DNA Damage ; DNA, Mitochondrial/*genetics/metabolism ; Embryo Transfer/veterinary ; Embryo, Mammalian/*cytology/embryology ; Fertilization in Vitro/veterinary ; In Situ Nick-End Labeling/veterinary ; Membrane Potential, Mitochondrial ; Microscopy, Confocal/veterinary ; Microscopy, Fluorescence/veterinary ; Mitochondria/*metabolism ; Nuclear Transfer Techniques/*veterinary ; Reactive Oxygen Species/*metabolism