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The pig is an important animal model for the study of human diseases. An important step for better use of this species in biomedical research is to obtain genetically identical individuals by procedures such as somatic cell nuclear transfer (SCNT). As the in vitroculture environment is usually sub-optimal for embryo development, the oviductal transfer of cloned embryos at the 1-cell stage may be more efficient for the establishment of pregnancies. However, the transfer at such an early stage usually requires the presence of zona pellucida or agar embedding to protect embryos from the recipient''s immune system (Loi et al.1999 Livest. Prod. Sci. 60, 281-294). This study aimed to evaluate the developmental viability of 1-cell-stage porcine handmade cloned embryos directly transferred to the oviduct of female recipients without the zona pellucida or agar embedding. After 40 h of IVM in TCM-199 +10% follicular fluid, COCs obtained from sows were denuded, selected for the presence of a polar body (459/689), and submitted to a 0.2% pronase solution in 25% fetal bovine serum (FBS) for partial zona removal, followed by rinses in manipulation medium and pure FBS. Subsequent to oocyte splitting by manual bisection in a 5 μg mL-1cytochalasin B solution (CCB), hemi-oocytes (87.1%) were screened under fluorescent microscopy using Hoechst 33 342 stain, resulting in 57.6% enucleated halves (461/800). A somatic cell culture established from a fetal clone pig biopsy (Adam et al.2007 Oncogene 26, 1038-1045) at passage 4 was used for embryo reconstruction, which was done in a 0.05% phytohemagglutinin (PHA) solution, by sticking 2 cytoplasts and a somatic cell in a linear orientation. Reconstructed couplets, rinsed in calcium- and magnesium-free fusion medium, were electrofused in a fusion chamber after exposure to a 30-V AC pulse for 20 s for alignment, followed by two 24-μs-long DC fusion pulses of 1.3 kV cm-1. Fused couplets (154/214) were exposed for 10 min to a solution containing 5 μg mL-1CCB and 10 μg mL-1cycloheximide, followed by electrical activation (two 24-μs-long DC pulses of 0.9 kV cm-1) in fusion medium containing calcium and magnesium. Activated embryos were cultured in vitrofor 12 h in 500 μL of PZM-3 medium in the well of the well (WOW) system, in a plastic bag filled with gas mixture (90% N2, 5% O2, 5% CO2), at 38.5°C. Then, a total of 70 and 80 non-agar-embedded, zona-free 1-cell-stage cloned porcine embryos were transferred directly to the oviducts of a sow and a gilt, respectively, both synchronous at approximately 12 h before ovulation. The recipient sow was diagnosed pregnant by ultrasonography on Day 66 of gestation. Although the sow was diagnosed open on Day 72, this study demonstrates that the transfer of 1-cell-stage zona-free embryos directly to the oviduct of a synchronous sow can result in pregnancy. [ABSTRACT FROM AUTHOR]

Despite the apparent success of cloning by somatic cell nuclear transfer (SCNT), the efficiency in development to term remains low, with a high rate of losses occurring throughout pregnancy due to faulty reprogramming and conceptus abnormalities. As the ideal fusion-activation interval for optimal nuclear reprogramming after cloning is still ill-defined, the aim of this study was to determine the effect of 2 distinct fusion-activation intervals and embryo aggregation on in vitrodevelopment of cloned bovine embryos. Bovine COCs from slaughterhouse ovaries were used after IVM for the production of cloned embryos by handmade cloning, according to our established procedures (Ribeiro et al.2009 Cloning Stem Cells, in press). Following cumulus and zona removal, oocytes were manually bisected, with hemi-cytoplasts selected by DNA staining. Two hemi-cytoplasts and an adult skin somatic cell were attached and fused with a 15V AC pre-pulse for 5 s, followed by a double 1.2 kV cm-1DC pulse for 20 μs. Reconstructed embryos were activated in ionomycin exactly at 2 or 4 h post-fusion (2 hpf or 4 hpf), followed by an incubation in 6-DMAP for 4 h. Cloned embryos from both fusion-activation intervals were in vitro-cultured in the well of the well (WOW) system for 7 days, allocating one (1 × 100%) or two (2 × 100%) cloned embryos per WOW. Grade 1 Day-7 blastocysts were transferred to synchronous recipients. Cleavage (Day 2) and blastocyst (Day 7) rates, on a per WOW basis, and pregnancy (Days 30 and 150) rates were compared using the chi-square or the Fisher test, with results from 9 replications summarized in Table 1. Increasing the fusion-activation interval to 4 h decreased cleavage but not blastocyst rates in 1 × 100% embryos. Also, blastocyst rates were lower in 1 × 100% embryos activated 2 h post-fusion. In general, cleavage and blastocysts rates for 2 × 100% embryos (91.5 and 46.0%) were higher than for 1 × 100% embryo counterparts (74.4 and 31.3%), respectively, regardless of the activation time. In addition, blastocyst rates for 4 hpf-activated embryos (50.3%), based on cleavage, were higher than for 2 hpf-activated embryos (38.3%), irrespective of the aggregation scheme. Nonetheless, despite differences in in vitrodevelopment, pregnancy rates and conceptus development in the first half of pregnancy were similar between groups. A longer fusion-activation interval (4 hpf) or embryo aggregation (2 × 100%) increased blastocyst yield but did not improve in vivodevelopment and pregnancy maintenance following the transfer to female recipients in cattle. Table 1.In vitroand in vivodevelopment of cloned bovine embryosThis study was supported by FAPESP and CAPES, Brazil. [ABSTRACT FROM AUTHOR]

Despite the rather successful and widespread use of cloning in various species, distinct cell types from the same species and even the same genotype display differences in blastocyst yield. Moreover, variations in the protocol for embryo production can influence development to the blastocyst stage and subsequent fetal development. The aim of this study was to evaluate the effect of 2 cell types and 2 embryo pre-activation protocols with or without the presence of FCS in the in vitroculture medium on development of handmade pig cloned embryos to the blastocyst stage. Cumulus-oocyte complexes recovered from sow ovaries were in vitro-matured for 38 to 40 h. Denuded matured oocytes selected by the presence of a polar body had the zona pellucida removed in a 0.2% protease HEPES-buffered solution +25% FCS, followed by manual bisection and UV screening of enucleated halves using Hoechst stain. Clone embryo reconstruction was performed using a phytohemoagglutinin solution to adhere 2 cytoplasts and a somatic cell. Adipocyte-derived mesenchymal stem cells (ADMSC) from a Yorkshire pig or granulosa cells (GC) from an Ossabaw pig were used as nuclear donors. Following electrical fusion, couplets were pretreated with a brief exposure to cytochalasin B (CB) or cytochalasin B + cycloheximide (CB+CX) in the presence of serum before the electrical activation (Naruse et al.2007 Theriogenology 68, 709-716; Du et al.2009 Reprod. Fertil. Dev. 21, 114). Activated embryos were in vitro-cultured in the well of the well (WOW) system, with 2 embryos per microwell, for 7 days in PZM-3 medium +0.3% BSA in the presence (FBS+) or absence (FBS-) of 10% FCS. Cleavage (Day 2, chi-square test) and blastocyst (Day 7, Fisher test) rates, on a per WOW basis, were compared for a level of significance of 5%. Our preliminary data indicate that the presence of serum in the IVC affected cleavage and blastocyst yield in a cell-type-dependent manner. The presence of serum enhanced the blastocyst yield for ADMSC, whereas for GC, only the absence of serum allowed any blastocyst development. The cell type and the pre-activation protocol did not appear to affect cleavage and embryo development to the blastocyst stage. Despite the low number of replications, our results reinforce the importance of optimizing the embryo production system taking into consideration the individual requirements for distinct cell types, procedures, and culture conditions. Table 1.Effects of cell type, pre-activation process and in vitroculture (IVC) medium on development of handmade pig cloned embryos [ABSTRACT FROM AUTHOR]

The coordination of the cell cycle of the donor nucleus and the recipient cytoplasm is thought to be one of the major essential factors needed for successful development of cloned embryos and offspring from somatic cell populations. Cell cycle synchronization protocols used for somatic cell nuclear transfer (SCNT) vary in preference among groups, with the confluence inhibition by contact appearing to be one of the most widely used methods today. However, the relationship between the level of cell confluence in a culture dish at or near the plateau phase of growth and blastocyst yield following cloning by SCNT still needs to be better characterized. The aim of this study was to examine the effect of 3 distinct cell culture confluences before nuclear transfer and embryo aggregation on in vitrodevelopment of clone bovine embryos. In vitro-matured bovine COC were used for the production of clone embryos by handmade cloning, according to our established procedures (Ribeiro et al.2009 Cloning Stem Cells 11). Oocytes were manually bisected following cumulus and zona removal. After selection of hemi-cytoplasts by DNA staining, 1 (50%) or 2 (100%) enucleated hemi-cytoplasts were paired with an adult Nelore skin somatic cell and then electrofused (15 V AC pre-pulse for 5 s, followed by a double 1.2 kV cm-1DC pulse for 20 μs). Cells were selected from 1 out of 3 distinct culture confluences: (1) 70 to 80%; (2) 80 to 90%; and (3) >90%; assessed by morphological evaluation before the SCNT procedure. Reconstructed clone embryos and groups of zona-intact oocytes (parthenote controls) were activated in ionomycin and 6-DMAP. Clone embryos (100%) and hemi-embryos (50%) reconstructed from the 3 groups underwent IVC in the well of the well (WOW) system for 7 days, allocating 1 embryo (1 × 100%) or aggregating two hemi-embryos (2 × 50%) per WOW. After 11 replications, cleavage (Day 2) and blastocyst (Day 7) rates, on a per WOW basis, were compared using the chi-square test. Results are summarized in Table 1. Cleavage rates were similar for all groups. The aggregation scheme did not appear to have influenced, either positively or negatively, the developmental outcome to the blastocyst stage. However, blastocyst rates increased nonlinearly (7.0, 17.5, and 29.4%) with the increase in cell confluence. A highly confluent cell culture has already been shown to have a greater proportion of cells in G0/G1than cycling cells at the log phase (>91% v.59%; Sun et al.2008 Zygote 16, 111-116). However, blastocyst development in this study was lower than anticipated for cells in the early plateau phase (70 to 80%), when predicting such development based on the expected G0/G1proportion in that cell population. Table 1.In vitrodevelopment of bovine cloned embryos from distinct cell culture confluences and aggregation schemeThis study was supported by FAPESP and CAPES/Brazil. [ABSTRACT FROM AUTHOR]