Animal cloning has been associated with developmental abnormalities, with the level of heteroplasmy caused by the procedure being one of its potential limiting factors. The aim of this study was to determine the effect of the fusion of hemicytoplasts or aggregation of hemiembryos, varying the final cytoplasmic volume, on development and cell density of embryos produced by hand-made cloning (HMC), parthenogenesis or by in vitro fertilization (IVF). One or two enucleated hemicytoplasts were paired and fused with one skin somatic cell. Activated clone and zona-free parthenote embryos and hemiembryos were in vitro cultured in the well-of-the-well (WOW) system, being allocated to one of six experimental groups, on a per WOW basis: single clone or parthenote hemiembryos (1 x 50%); aggregation of two (2 x 50%), three (3 x 50%), or four (4 x 50%) clone or parthenote hemiembryos; single clone or parthenote embryos (1 x 100%); or aggregation of two clone or parthenote embryos (2 x 100%). Control zona-intact parthenote or IVF embryos were in vitro cultured in four-well dishes. Results indicated that the increase in the number of aggregated structures within each WOW was followed by a linear increase in cleavage, blastocyst rate, and cell density. The increase in cytoplasmic volume, either by fusion or by aggregation, had a positive effect on embryo development, supporting the establishment of pregnancies and the birth of a viable clone calf after transfer to recipients. However, embryo aggregation did not improve development on a hemicytoplast basis, except for the aggregation of two clone embryos.

No abstract

The aim of this study was to evaluate the effect of the cytoplast type and activation process on development of cloned embryos. Bovine oocytes (MII) or zygotes at the one-cell stage (IVF) were manually bisected and segregated in MII or IVF hemi-cytoplasts or hemi-karyoplasts. Adult skin cells from a bovine female were used as nucleus donors (SC). Experimental groups were composed of IVF embryos; parthenogenetic embryos; hand-made cloned (HMC) embryos; and reconstructed HMC embryos using IVF hemi-cytoplast + MII hemi-cytoplast + SC (G-I); IVF hemi-cytoplast + IVF hemi-cytoplast + SC (G-II); MII hemi-cytoplast + IVF hemi-karyoplast (G-III); and IVF hemi-cytoplast + IVF hemi-karyoplast (G-IV). Embryos from G-I to G-IV were allocated to subgroups as sperm-activated (SA) or were further chemically activated (SA + CA). Embryos from all groups and subgroups were in vitro cultured in the WOW system. Blastocyst development in subgroup G-I SA (28.2%) was similar to IVF (27.0%) and HMC (31.4%) controls, perhaps due to a to a more suitable activation process and/or better complementation of cytoplasmic reprogramming factors, with the other groups and subgroups having lower levels of development. No blastocyst development was observed when using IVF hemi-karyoplasts (G-III and G-IV), possibly due to the manipulation process during a sensitive biological period. In summary, the presence of cytoplasmic factors from MII hemi-oocytes and the sperm activation process from hemi-zygotes appear to be necessary for adequate in vitro development, as only the zygote-oocyte hemi-complementation was as efficient as controls for the generation of bovine cloned blastocysts.

The main cause of low efficiency of in vitro produced porcine embryos is the high polyspermic penetration rates at fertilization, which is aggravated in low quality oocytes. Experiment 1 evaluated the embryo development in high and

Exposing bovine embryos and porcine oocytes to hydrostatic pressure has been shown to increase cryosurvival, possibly by a resulting expression of stress tolerance proteins. This study aimed to evaluate the effect of the negative pressure stress condition (a 5-min-long embryo exposure to a negative pressure) and the interval between vacuum exposure and vitrification (40 min or 2 h) on survival of bovine in vitro-produced (IVP) embryos. The negative pressure was achieved with the same apparatus used previously for the cryopreservation of embryos (Nitrocooler; Mezzalira et al. 2009 Reprod. Fert. Dev. (1) 134), in which a negative pressure (vacuum) is applied to liquid nitrogen to increase the cooling rate through the slush phenomenon, except that in this study, the vacuum was applied to the chamber without liquid nitrogen, at room temperature. Grades 1 and 2 bovine IVP expanded blastocysts were allocated to 1 of 5 experimental groups: embryos in vitro-cultured as fresh (control) or after vitrification (Vitri); and embryos subjected to the negative pressure for 5 min and then in vitro-cultured as fresh (NP-fresh) or after vitrification performed 40 min (NP-Vitri-40 min) or 2 h (NP-Vitri-2h) following the vacuum exposure. Embryos were vitrified in pulled glass micropipettes in a solution with 20% ethylene glycol + 20% dimethylsulfoxide + 20% fetal bovine serum and rewarmed in decreasing sucrose concentrations (Mezzalira et al. 1999 Acta Scientiae Veterinariae 27 262-262). In vitro culture was carried out in all treatments for 72 h for the assessment of re-expansion and hatching rates (Table 1), which were analyzed by the chi-square test, for P < 0.05. No differences in re-expansion rates were observed between groups. However, the vitrification of embryos after 2 h of exposure to a 5-min-long negative pressure (NP-Vitri-2h) improved embryo survival expressed by a higher hatching rate than for embryos vitrified without vacuum exposure (Vitri) or after 40 min following the 5-min-long exposure to vacuum. In addition, hatching rates in group NP-Vitri-2h were similar to those for fresh embryos (control and NP-fresh). Our results indicated that a short exposure of embryos to a negative pressure can improve cryotolerance following vitrification, which is dependent on the time interval between NP exposure and cryopreservation. Bovine IVP embryos should be allowed to recover for at least 2 h after NP exposure before the increase in cryotolerance is achieved. Table 1.Effect of negative pressure on re-expansion and hatching rates of fresh and vitrified

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 vitro culture 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-1 cytochalasin 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-1 CCB and 10 μg mL-1 cycloheximide, 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 vitro for 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.

This study aimed to evaluate the effect of three different concentrations of discontinuous gradients of percoll (90/45, 80/40, and 70/35) in the outcome of porcine in vitro fertilization (IVF) and its influence on further embryo development and quality. Embryo viability was assessed by the expression of estrogen receptors (E 2 R) and cleaved caspase-3 (CC3). The highest percoll concentration (90/45) resulted in the lowest embryo production (24.9%) in comparison with 80/40 (37.5%) and 70/35 (40.0%), with the production being similar between the two lowest concentrations. The hatching rate for 90/45 (26.2%) was lower than for 80/40 (45.5%), and both were similar to the Group 70/35 (32.9%). The hatched embryos from the concentration 90/45 showed the lowest proportion of E 2 R expression (3.6%), while the Groups 80/40 (22.6%) and 70/35 (39.3%) had a similar proportion of expression. The live embryos that did not hatch until Day 8 of culture presented a higher CC3 proportion for Group 90/45 (18.3%), in comparison with 80/40 (12.7%) and 70/35 (10.7%), with the latter two being similar. In conclusion, adjustments in percoll concentration used for sperm selection before porcine IVF can improve embryo production and competence for pregnancy recognition and establishment. K E Y W O R D S apoptosis, estrogen receptors, porcine IVF, sperm selection

Effect of the cryopreservation method used, the embryonic stage and the use of conjugated linoleic acid isomers on the cryotolerance of in vitro-produced bovine embryos Efeito do método de criopreservação utilizado, estágio de desenvolvimento embrionário e uso de isômeros do ácido linoleico conjugado na criotolerância de embriões bovinos produzidos in vitro AbstractConjugated linoleic acid (CLA) might be able to improve the cryotolerance of in vitro-produced (IVP) embryos. The effect of two CLA isomers on the cryotolerance of bovine IVP embryos, as well as that of the stage of embryonic development and the method used for cryopreservation was evaluated by three experiments. In Experiment 1, oocytes (n = 3,917) were fertilized in vitro and cultured with 0, 50, 100, or 200 µM trans-10, cis-12 (t10, c12 CLA). In Experiment 2, fertilized oocytes (n = 2,131) were cultured with 100 µM t10, c12 or cis-9, trans-11 (c9, t11 CLA), or a combination of both isomers. The embryos were vitrified at the blastocyst (BL) or the expanded blastocyst (EB) stage. In Experiment 3, oocytes (n = 1,720) were fertilized and cultured with or without 100 µM t10, c12 CLA, and the blastocysts were vitrified or frozen. Blastocyst development rate as well as the rates of re-expansion and hatching after thawing was recorded. Moreover, the mean cell number and mRNA expression of acetyl-CoA carboxylase (ACC1) and stearoyl-CoA desaturase (SCD1) as well as fatty acid synthase (FASN) multienzyme complex were determined. In Experiment 1, the highest concentration of t10, c12 CLA that did not reduce blastocyst development rate was 100 µM. In Experiment 2, the rates of re-expansion and hatching among the EBs obtained through IVP after supplementation with t10, c12 CLA (73.1% and 57.7%), with c9, t11 CLA (80.0% and 68.6%), with the combination (78.3% and 52.2%), and with the control group (85.4% and 58.3%) were similar. At the BL stage, the rates of re-expansion and hatching were lower than those at the EB stage, and CLA combination allowed a hatching rate (8.0%) lower than that observed in the control group (40.0%). In Experiment 3, the hatching rates for vitrified EBs (vitrified control; 67.4%) and vitrified CLA EBs (65.8%) were higher than those obtained for frozen EBs, exposed (13.3%) or not exposed (28.6%) to CLA. In addition, in Experiment 3, the hatching rate was higher at the EB stage in vitrified groups, while the rates of BL and EB were similar in frozen groups, thus proving that vitrification was more efficient than freezing for IVP bovine embryos. In Experiment 3, CLA isomer t10, C12 did not influence the embryonic cell number or mRNA expression of ACC1 and SCD1 enzymes, but decreased the mRNA expression of FASN. In conclusion, 100 µM CLA did not affect subsequent embryonic development. However, neither CLA isomer improved the cryotolerance of IVP bovine embryos. c12 CLA). No Experimento 2, oócitos fecundados (n = 2.131) foram cultivados com 100 µM de t10, c12 ou cis-9, trans-11 (c9, t11 CLA), ou ainda com uma associação de ambos. Os embri...