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

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

This study was conducted to investigate the expression of three genes related to early embryonic development in bovine transgenic cloned embryos. To accomplish this, development of bovine transgenic somatic cell nuclear transfer (SCNT) embryos was compared with non-transgenic embryos. Next, mRNA transcription of three specific genes (DNMT1, Hsp 70.1, and Mash2) related to early embryo development in transgenic SCNT embryos was compared between transgenic and non-transgenic SCNTs, parthenogenetic embryos, and in vitro fertilization (IVF) embryos. Transgenic SCNT embryos showed significantly lower rates of development to the blastocyst stage than non-transgenic ones. To investigate normal gene expression, RNA was extracted from ten blastocysts derived from parthenogenesis, IVF, non-transgenic, and transgenic SCNT embryos and reverse-transcribed to synthesize cDNA. The cDNA was then subjected to PCR amplification and semi-quantified. More DNMT1 mRNA was detected in the transgenic SCNT group than the other three groups. Hsp 70.1 mRNA was detected in the IVF embryos, while lower levels were found in SCNT and parthenogenetic embryos. Mash2 mRNA was present at the highest levels in transgenic SCNT embryos. In conclusion, the higher levels of methylation and lower protein synthesis after heat shock in the transgenic SCNT embryos expected based on our results may cause lower embryonic development.
Animals ; Animals, Genetically Modified/genetics ; Basic Helix-Loop-Helix Transcription Factors/*genetics/metabolism ; Cattle/embryology/*genetics ; DNA (Cytosine-5-)-Methyltransferase/*genetics/metabolism ; Embryo, Mammalian/embryology/metabolism ; Female ; Fertilization in Vitro ; *Gene Expression Regulation, Developmental ; HSP70 Heat-Shock Proteins/*genetics/metabolism ; Nuclear Transfer Techniques/veterinary ; Parthenogenesis ; Pregnancy ; RNA, Messenger/genetics/metabolism ; Transcription, Genetic

The objective of this study was to determine the effects of superovulation (SOV) on serum and uterine biochemical parameters, uterine bacteriology and cytology and number of transferable embryos (TE). Dairy cows were placed on a Presynch/CIDR Synch protocol. The SOV group was superovulated, induced in estrus, and inseminated, whereas the control group was induced in estrus and inseminated without SOV. Uterine bacteriology and cytology and uterine and serum biochemical parameters were measured at day 7 of the estrous cycle to start the SOV protocol, as well as on the day of embryo recovery (DER). The SOV group produced 7.5 +/- 6.7 oocytes/embryos, of which 3.4 +/- 4.7 were TE. Serum urea and E2 and uterine Glu, CK, LDH, TP, P4 and PGFM in the control group and serum P4 and PGFM and uterine LDH and PGFM in the SOV group were significantly higher (p < 0.01) at DER than day 7. At DER, uterine urea, LDH, PGFM and TP and serum urea, LDH, PGFM, and P4 concentrations were higher (p < 0.01) in the SOV group than the control. There was no significant variation in uterine bacteriology or cytology. Overall, these results infer that SOV affects both serum profile and uterine secretions, and that these changes may influence the number of TE.
Animals ; Blood Chemical Analysis/veterinary ; Cattle/blood/*embryology/*physiology ; Embryo Transfer/veterinary ; *Embryonic Development ; *Estrous Cycle ; Female ; Superovulation ; Uterus/*chemistry/cytology/*microbiology

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

Various somatic cell nuclear transfer (SCNT) techniques for mammalian species have been developed to adjust species-specific procedures to oocyte-associated differences among species. Species-specific SCNT protocols may result in different expression levels of developmentally important genes that may affect embryonic development and pregnancy. In the present study, porcine oocytes were treated with demecolcine that facilitated enucleation with protruding genetic material. Enucleation and donor cell injection were performed either simultaneously with a single pipette (simplified one-step SCNT; SONT) or separately with different pipettes (conventional two-step SCNT; CTNT) as the control procedure. After blastocysts from both groups were cultured in vitro, the expression levels of developmentally important genes (OCT4, NANOG, EOMES, CDX2, GLUT-1, PolyA, and HSP70) were analyzed by real-time quantitative polymerase chain reaction. Both the developmental rate according to blastocyst stage as well as the expression levels CDX2, EOMES, and HSP70 were elevated with SONT compared to CTNT. The genes with elevated expression are known to influence trophectoderm formation and heat stress-induced arrest. These results showed that our SONT technique improved the development of SCNT porcine embryos, and increased the expression of genes that are important for placental formation and stress-induced arrest.
Animals ; Biological Markers/metabolism ; Cloning, Organism ; Embryo, Mammalian/metabolism ; Female ; *Gene Expression Regulation, Developmental ; Nuclear Transfer Techniques/instrumentation/*veterinary ; Oocytes/metabolism ; Pregnancy ; Real-Time Polymerase Chain Reaction ; Swine/*embryology/*genetics

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

Genetically modified (GM) animals are unique mutants with an enormous scientific potential. Cryopreservation of pre-implantation embryos or spermatozoa is a common approach for protecting these lines from being lost or to store them in a repository. A mutant line can be taken out of a breeding nucleus only if sufficient numbers of samples with an appropriate level of quality are cryopreserved. The quality of different donors within the same mouse line might be heterogeneous and the cryopreservation procedure might also be error-prone. However, only limited amounts of material are available for analysis. To improve the monitoring of frozen/thawed spermatozoa, commonly used in vitro fertilization (IVF) followed by embryo transfer were replaced with animal-free techniques. Major factors for assessing spermatozoa quality (i.e., density, viability, motility, and morphology) were evaluated by fluorescence microscopy. For this, a live/dead cell staining protocol requiring only small amounts of material was created. Membrane integrity was then examined as major parameter closely correlated with successful IVF. These complex analyses allow us to monitor frozen/thawed spermatozoa from GM mice using a relatively simple staining procedure. This approach leads to a reduction of animal experiments and contributes to the 3R principles (replacement, reduction and refinement of animal experiments).
Animals ; Benzimidazoles/chemistry ; Cryopreservation/veterinary ; Embryo Transfer/veterinary ; Female ; Fertilization in Vitro/veterinary ; Fluorescent Dyes/chemistry ; Male ; Mice ; Mice, Transgenic ; Microscopy, Fluorescence/*methods/veterinary ; Propidium/chemistry ; Semen Analysis/*methods/veterinary ; Semen Preservation/veterinary ; Spermatozoa/*physiology

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

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