R. Simões

Ovulation rate and its relationship with follicle diameter and gene expression of the LH receptor (LHR) in Nelore cows

The objective was to determine the relationship among the diameter of ovarian follicles, ovulation rate, and gene expression of the LH receptor (LHR) in Nelore cattle. In Experiment 1, ovulation was synchronized in 53 Nelore cows. Three days after ovulation, ovaries were assessed with ultrasonography, all cows were given 6.25 mg LH im, and they were allocated into three groups, according to diameter of their largest ovarian follicle: G1 (7.0-8.0 mm); G2 (8.1-9.0 mm); and G3 (9.1-10.0 mm). For these three groups, ovulation rates were 9, 36, and 90%, respectively, (P<0.03; each rate differed significantly from the other two). In Experiment 2, granulosa and theca cells were subjected to total RNA extraction, and gene expression of the LHR was determined by RT-PCR. Follicles were allocated in three groups based on their diameter (similar to the Experiment 1), which were denoted Groups A, B, and C. Expression of the LHR gene in granulosa cells was lower in Group A than Group C (P<0.05). However, there were no significant differences among groups in expression of the LHR gene in theca cells. We concluded that ovulatory capacity in Nelore cattle was related to increased follicular diameter and expression of the LHR gene in granulosa cells.

Exogenous DNA uptake by bovine spermatozoa does not induce DNA fragmentation

Sperm-mediated gene transfer (SMGT) is a fast and low-cost method used to produce transgenic animals. The objective of this study was to evaluate the effects of the concentration of exogenous DNA and the duration of incubation on DNA uptake by bovine spermatozoa and subsequently the integrity of sperm DNA and sperm apoptosis. Spermatozoa (5 x 10(6) cells/mL) were incubated with 100, 300, or 500 ng of exogenous DNA (pEYFP-Nuc plasmid) for 60 or 120 min at 39 degrees C. The amount of exogenous DNA associated with spermatozoa was quantified by real-time PCR, and the percentages of DNA fragmentation in spermatozoa were evaluated using SCSA and a TUNEL assay, coupled with flow cytometry. Uptake of exogenous DNA increased significantly as incubation increased from 60 to 120 min (0.0091 and 0.028 ng, respectively), but only when the highest exogenous DNA concentration (500 ng) was used (P < 0.05). Based on SCSA and TUNEL assays, there was no effect of exogenous DNA uptake or incubation period on sperm DNA integrity. In conclusion, exogenous DNA uptake by bovine spermatozoa was increased with the highest exogenous DNA concentration and longest incubation period, but fragmentation of endogenous DNA was apparently not induced.

Vitrification with Glutamine Improves Maturation Rate of Vitrified / Warmed Immature Bovine Oocytes

The current study examined the protective effects of l-glutamine and cytochalasin B during vitrification of immature bovine oocytes. Oocyte vitrification solution (PBS supplemented with 10% FCS, 25% EG, 25% DMSO and 0.5 m trehalose) was the vitrification control. Treatments were the addition of 7 μg/ml cytochalasin B, 80 mm glutamine or both cytochalasin and glutaminine for 30 s. After warming, oocytes were matured in vitro for 24 h, fixed and stained with Hoechst (33342) for nuclear maturation evaluation. L-glutamine improved the vitrified/warmed immature bovine oocytes viability (32.8%), increasing the nuclear maturation rates compared to other treatments and the no treatment vitrified control (17.4%). There was, however, no effect of cytochalasin B on in vitro maturation (14.4%).

410 USE OF SPERMATOZOA AS VECTORS OF EXOGENOUS DNA FOR IN VITRO PRODUCTION OF BOVINE TRANSGENIC EMBRYOS

There are many methods to produce transgenic animals, although when considering bovine species, those methods offer low repeatability, high costs, and low transgene integration efficiency. To overcome these difficulties, sperm mediated gene transfer (SMGT) could be used as an alternative to produce transgenic embryos. This technology allows carrying exogenous DNA into the oocyte during the fertilization period, once spontaneous binding exists between spermatozoa and exogenous DNA. The aim of this study was to compare 4 methods for incorporating DNA into sperm cells—sperm incubation, capacitation, electroporation, and lipofection—to verify the efficiency of embryo production with exogenous DNA, employing spermatozoa as a vector. Cumulus–oocyte complexes (COCs) from abattoir-derived bovine ovaries were randomly divided into 5 groups (4 experimental groups: sperm incubation, sperm capacitation with calcium ionophore, electroporation, and lipofection; and 1 control group). In vitro maturation was performed in TCM-199 medium supplemented with 10% FCS, sodium pyruvate, gentamycin, FSH, hCG, and estradiol in an incubator at 39°C, in an atmosphere of 5% CO2 in air and high humidity for 24 h. After Percoll gradient (45/90%) separation at 600g for 30 min, spermatozoa were washed in Talp Semen medium (200g for 5 min). For in vitro fertilization (IVF), 5 × 106 spermatozoa were used to inseminate microdroplets with 20 matured oocytes from all groups: incubation with exogenous DNA for 1 h, sperm capacitation (250 nM of calcium ionophore) for 5 min followed by sperm incubation for 1 h, electroporation (500V), and lipofection (Effectene®; Qiagen, Mississauga, Ontario, Canada). The EYFP-Nuc plasmid (500 ng mL-1; Clontech, Mountain View, CA, USA) was used as exogenous DNA. Oocytes inseminated with non-treated sperm were considered as the control group. The presumptive zygotes were co-cultured with a granulosa cell monolayer in SOFaa medium in an incubator at 39°C, with an atmosphere of 5% CO2 in air and high humidity. The blastocyst rate was analyzed by ANOVA. Embryos from all experimental and control groups were subjected to PCR to detect internalized EYFP, using primers specific to this exogenous DNA, and considering positive embryos those that showed a fragment of 440 bp after electrophoresis. The fragment of 440 bp from positive embryos was sequenced to check correspondence to the EYFP. Electroporation (17.95%) and sperm capacitation (15.12%) were more efficient for EYFP-positive embryo production when compared to the lipofection protocol (6.25%). Sperm incubation (12.5%) did not show a significant difference from the other groups. Sequenced PCR-positive products for EYFP showed 100% homology with nucleotides of EYFP at GenBank. In conclusion, it is possible to use SMGT to deliver exogenous DNA into an oocyte at the time of IVF. This work was supported financially FAPESP 03/08542-5 and 03/07456-8.

270 APOPTOSIS AND ONCOSIS ASSESSMENT IN CRYOPRESERVED MOUSE EMBRYOS

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 -7°C until it reached -31°C and then plunged and stored in liquid nitrogen. The embryos were thawed in air for 10 s and in a 25°C 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 25°C 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; Wilcoxons 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.

315 EFFICIENCY OF CHEMICAL OR ELECTRICAL ACTIVATION OF BOVINE OOCYTES

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 donors 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 38.5°C 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 µs, 2 pulses). After treatments, oocytes were kept in 6-dimethylaminopurine for 3 h and cultured in SOFaa medium for 7 days at 38.5°C 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) 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.