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Production of transgenic mouse embryos by microinjection is a well established and successful technique. However, when microinjection protocols were used for bovine, the amount of the oocyte lipid content did not allow the production of bovine transgenic embryos. Sperm-mediated gene transfer (SMGT) is an alternative for this species because it has lower cost and does not require microinjection handling. One of the procedures to introduce exogen DNA into oocytes is by means of sperm capacitated with calcium ionophore (CaI). The aim of this work was to evaluate different CaI concentrations ([CaI]), sperm incubation times with CaI (tCa), and incubation times of sperm capacitated with DNA (tDNA) (EYFP; Clontech, Palo Alta, CA, USA) to establish a satisfactory method for IVP of bovine transgenic embryos. Slaughterhouse oocytes with compact cumulus and uniform ooplasm were in vitro maturated in TCM-199 medium + 10% FCS + FSH + hCG + estradiol (E2) + piruvate + gentamicin under 5% CO2 in air, at 39C and high humidified atmosphere for 24 h. Semen was thawed in a water bath at 37C for 30 s and separated by Percoll gradient (45/90%) at 600g for 30 min. After this procedure, sperm cells were washed in TALP-semen medium by centrifugation at 200g for 5 min at room temperature. Supernatant was removed and capacitation (5 106 spermatozoa/group) was induced with CaI (250 nM or 500 nM for 1 or 5 min). Capacitated sperm cells were incubated with 500 ng/mL DNA for 1 or 2 h. Nontreated spermatozoa were used as control group. Sperm cells (1 105) were used to inseminate 20 oocytes/90 mL microdroplets for 18 h. The presumptive zygotes were co-cultured in SOFaa medium with a granulosa cell monolayer under high humidified atmosphere, at 39C and 5% CO2 in air. Blastocyst rates were analyzed by ANOVA. Independent variables were replicate, [CaI], tCa, tDNA, and the double and triple interactions among the last three variables; when appropriate, means were compared by orthogonal contrasts. There was [CaI] tCa tDNA interaction for blastocyst rate (P < 0.02). Treatments with 250 nM ([CaI]), 5 min (tCaI), and 1 h (tDNA) or 500 nM ([CaI]), 1 min (tCaI), and 1 h (tDNA) resulted in 36.1% and 37.4% blastocyst rates, respectively, similar to the control group (30.5%; P > 0.4). These results demonstrated that it is possible to capacitate spermatozoa with CaI to produce transgenic embryos, without alteration of blastocyst rate.This work was supported by FAPESP 03/08542-5 and 03/07456-8.

Cryopreservation of mammalian embryos is an important tool for the application of reproductive biotechnology. Recent evidence indicates that apoptosis of cryopreserved embryos may be a negative factor for their viability. The aim of this study was to detect apoptosis and to characterize and quantify the embryonic cell death caused by cryopreservation. Mouse morulae were separated to be subjected to two cryopreservation protocols (slow freezing and vitrification) and a control group (fresh). In the slow-freezing procedure, embryos were exposed to 10% ethylene glycol (EG) for 10 min. Straws were placed in a methanol bath at -7C until it reached -31C and then plunged and stored in liquid nitrogen. The embryos were thawed in air for 10 s and in a 25C water bath for 20 s. In the vitrification method, embryos were exposed to 10% and 20% EG for 5 min, followed by 40% EG + 18% Ficoll + 10% sucrose (EFS) for 30 s and then plunged and stored in liquid nitrogen. These embryos were thawed in a 25C water bath for 20 s. For the cell death evaluation, cell membrane integrity from the fresh and cryopreserved embryos was assessed by Hoechst and propidium iodide (H/PI staining). Morphology and apoptosis were assessed by means of the haematoxylin-eosin staining (HE) and by electron microscopy (MET). To confirm apoptosis, 64 cryopreserved mouse morulae (34 submitted to slow freezing and 30 to vitrification) were used to evaluate Caspase-3 activity. The cryopreserved embryos were divided into experimental and control groups and incubated with Caspase-3 and buffer solution, respectively. Afterward, the embryos were incubated with rhodamine and the Caspase activity was determined under a fluorescence microscope. H/PI staining detected more membrane permeability in the vitrification (69.7%) than in the slow-freezing (48.4%) or fresh (13.8%) groups (P < 0.05; Wilcoxon's test). Nuclear evaluation by HE revealed that vitrification and slow freezing induced pyknosis and chromatin condensation. HE staining revealed weakly staining cytoplasm and degenerated cells in the vitrification group (indicating oncosis), whereas in the slow-freezing the presence of cytoplasmic condensation and eosinophilic structures indicating apoptosis were observed. MET examination of the ultrastructure confirmed the HE results. The Caspase-3 activity showed a fluorescence increase in both experimental groups compared with the control group. In conclusion, staining with HE allows detection of oncosis and apoptosis in cryopreserved embryos. Regarding the cryopreservation techniques, both slow freezing and vitrification showed oncosis and apoptosis injuries. However, in this experiment vitrification caused more cellular injuries, with less embryo viability, than slow freezing.This work was supported by FAPESP 04/01252-4 and CAPES.

Activation of in vitro matured oocytes is essential for the success of nuclear transfer embryo production. Oocyte activation is promoted by the release of intracellular calcium and influx of extracellular ions, and can be chemically induced by calcium ionophores such as A23187 (CA) or ionomycin (IO). Electrical stimulation (EL) is an essential stage in nuclear transfer protocols for the fusion of enucleated oocytes with the donor's cell nucleus. Moreover, EL can be used as an alternative method to induce calcium influx through the formation of pores in the plasma membrane. This work aimed to evaluate the effect of electrical pulse vs the use of different calcium ionophores (A23187 or ionomycin) as primary agents of bovine oocyte activation, with or without the addition of BSA, on the rate of blastocyst formation and blastocyst quality. BSA was used to quench the activation process after a 5-min exposure to CA or IO. Cumulus-oocyte complexes were matured in TCM-199 medium with FCS and hormones for 18 h at 38.5C and 5% CO2 in air. After removal of cumulus cells, oocytes presenting the first polar body were selected and maintained in SOFaa medium to complete 24 h of maturation. They were then divided into five treatments groups 1-CA (CA 5 mM, 5 min); 2-CAB (CA 5 mM, 5 min; BSA, 5 min); 3-IO (IO 5 mM, 5 min); 4-IOB (IO 5 mM, 5 min; BSA, 5 min); and 5-EL (EL 1.5 kV/cm, 20 s, 2 pulses). After treatments, oocytes were kept in 6-dimethylaminopurine for 3 h and cultured in SOFaa medium for 7 days at 38.5C and 5% CO2 in air. Rates of cleavage and blastocyst were evaluated respectively on Days 2 and 7 of culture. To evaluate embryo quality, Hoechst 33342/propidium iodide staining was used. Data were evaluated by ANOVA and submitted to LSD test for embryo rates and t-test for embryo quality. Four replicates were carried out with a total of 89 oocytes per treatment. There was a difference (P < 0.05) in rate of development to blastocyst between treatments 1-CA (54.4%a), 3-IO (51.4%a), and 5-EL (54.5%a) compared with 4-IOB (18.3%b). Treatment 2-CAB (39.8%ab) did not show any difference from the others. There was no difference (P > 0.05) among treatments in total number of cells: 1-CA (63.1a), 2-CAB (57.2a), 3-IO (60.9a), 4-IOB (72.4a), and 5-EL (58.4a). However, there was a difference (P < 0.01) in the percentage of viable cells between treatments 1-CA (49.9%a), 2-CAB (45.8%a), 3-IO (64.9%a), and 4-IOB (50.9%a) in comparison to 5-EL (82.7%b). In conclusion, BSA, when associated with IO, had a negative effect on embryonic developmental rates. The different calcium ionophores used and the BSA did not improve embryo quality. Although there were no significant differences between electrical and chemical activation on the rate of blastocyst formation, it is important to point out that higher quality embryos were achieved by using electrical activation.This work was supported by FAPESP 03/00156-9.

The aim of this study was to evaluate the viability of in vitro-produced bovine embryos after exposure to different cryoprotectant solutions and cryopreservation. Bovine ovaries were collected at slaughterhouse and oocytes were matured, fertilized, and cultured in vitro. The embryos were co-cultured on a granulosa cell monolayer in SOF + 5% FCS and nonessential amino acids. In Experiment 1, expanded blastocysts were exposed to 10% ethylene glycol (EG) solution for 10 min (Group EG) or to 10% EG solution for 10 min and to 20% EG + 20% glycerol (Gly) solution for 30 s (Group EG/Gly). Cryoprotectants were diluted with PBS + 0.2% BSA + 0.3 M sucrose and PBS + 0.2% BSA solutions, both for 3 min, and the hatching rate was evaluated after culture. In Experiment 2, after exposure, EG Group was cryopreserved by slow freezing procedure (1.2C/min) and EG/Gly Group was vitrified on nitrogen vapor for 2 min. After thawing, cryoprotectants were diluted using PBS + 0.2% BSA + 0.3 M sucrose and PBS + 0.2% BSA solutions, both for 3 min; hatching rate was evaluated after culture. As a control group for both experiments, non exposed embryos were cultured and evaluated for hatching rate. In Experiment 1, the hatching rates were 59.72% (43/72) for control, 62.38% (63/101) for EG, and 69.00% (69/100) for EG/Gly groups. In Experiment 2, hatching rates were 59.72% (43/72) for control, 15.22% (7/46) for EG, and 0.00% (0/46) for EG/Gly groups. Results were analyzed by chi-square test. In Experiment 1, no differences were observed among groups (P > 0.05) and in Experiment 2, differences were observed among control, EG, and EG/Gly groups (P < 0.05). In conclusion, the cryoprotectants were not deleterious to the development of in vitro bovine embryos until hatching, but the cryopreservation procedures decreased embryo viability.This work was supported by FAPESP 04/05335-1.