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Rat Oocytes Fertilized in Modified Rat 1-Cell Embryo: DISCUSSION(3)

DISCUSSION(3)

In the present studies, we demonstrated successful development to term after transfer of morulae and early blastocysts that were fertilized in mR1ECM-BSA containing 110 mM NaCl and developed in a chemically defined medium, mR1ECM. However, the litter size was very small, and more than half the recipients had failed to maintain the pregnancy around Days 13-20. Similar results were obtained after transfer of morulae or early blastocysts that had been fertilized in vivo and in vitro and then developed in mR1ECM. Therefore, the small litter size and low efficiency of offspring production after transfer of the embryos appear not to be due to unsuitable conditions in mR1ECM-BSA containing 110 mM NaCl, but possibly in mR1ECM. In porcine embryos, both the number of cells in a blastocyst and the incidence of blastocysts hatched were dramatically improved by supplementation with fetal bovine serum by the morula stage. Those characteristics of the embryos were not examined in the present studies. Further investigation will be required to improve the development of rat embryos cultured in mR1ECM and then transferred. buy cipro

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Rat Oocytes Fertilized in Modified Rat 1-Cell Embryo: DISCUSSION(2)

Currently, more than 80% of 1-cell rat embryos can develop to the blastocyst stage in a chemically defined mR1ECM. However, the developmental ability of 1-cell rat embryos in mR1ECM is known to be very low when obtained soon after penetration in vitro. This phenomenon has also been described in hamster embryos in a chemically defined medium, HECM-3. The low early embryonic development was also observed when rat zygotes were collected before pronuclear formation and then cultured in mR1ECM. Low embryonic development appears to be due to the absence of essential factor(s) in mR1ECM during pronuclear formation. The low development of the embryos has been overcome by supplementation of HECM-3 with hypotaurine in the hamster and by preincubation in mKRB. flovent inhaler

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Rat Oocytes Fertilized in Modified Rat 1-Cell Embryo: DISCUSSION(1)

DISCUSSION(1)

In the present studies, we demonstrated that successful sperm penetration was achieved in mR1ECM with modifications in which PVA was replaced with BSA and the NaCl concentration was increased to 100-130 mM. In hamsters, mice, and cattle, sperm penetration of oocytes in vitro was achieved when cumulus-oocyte complexes were cocultured in media not containing BSA. In the rat, in contrast, previous findings showing that BSA is essential in order to result in sperm penetration have not yet been overturned. Miyamoto and Chang also reported that in the presence of glucose, BSA was the most important component for in vitro fertilization of rat oocytes and that addition of lactate and pyruvate facilitated the process. In the present studies, however, neither simply replacing PVA with BSA, or replacing PVA with BSA and increasing the concentration of sodium lactate, resulted in successful sperm penetration in mR1ECM. Since we confirmed the necessity of supplementing with BSA in mKRB, we basically used mR1ECM-BSA in the other experiments. buy birth control online

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Rat Oocytes Fertilized in Modified Rat 1-Cell Embryo: RESULTS(2)

Optimal Concentration of NaCl in mR1ECM-BSA for Sperm Penetration (Experiment 3)

Table 5 shows the proportion of oocytes penetrated during coculture with spermatozoa in mR1ECM-BSA containing various concentrations (90-140 mM) of NaCl for 10 h. The incidence of oocytes penetrated was high, and no differences were noted (p > 0.05) among mR1ECM-BSA medium containing 100-130 mM NaCl (88.8 ± 4.1% to 93.1 ± 5.1%). Rates of total sperm and monospermic penetration in mR1ECM-BSA containing 100-130 mM NaCl were not different (p > 0.05) compared to those in mKRB (81.6 ± 3.8% and 24.0 ± 7.7%, respectively; Table 5). However, the incidence of sperm penetration was reduced (at least p < 0.05) in mR1ECM-BSA containing 90 and 140 mM NaCl (21.0 ± 5.1% and 58.8 ± 13.2%, respectively). ventolin 100 mcg

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Rat Oocytes Fertilized in Modified Rat 1-Cell Embryo: RESULTS(1)

RESULTS(1)

Effect of BSA in mR1ECM (Experiment 1)

The incidence of oocytes penetrated during coculture of oocyte-cumulus complexes with spermatozoa in mKRB, mKRB-PVA, mR1ECM, and mR1ECM-BSA for 10 h is shown in Table 2. Sperm penetration was observed only in mKRB (88.2 ± 2.7%). Although no sperm penetration occurred in mR1ECM-BSA, this medium was used in the subsequent experiments because we found the presence of BSA to be of importance for sperm penetration in mKRB. proventil inhaler

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Rat Oocytes Fertilized in Modified Rat 1-Cell Embryo: MATERIALS AND METHODS(5)

After coculture of cumulus-oocyte complexes with spermatozoa in the above-mentioned modified mR1ECM-BSA for 10 h, the incidence of sperm penetration was compared to that in mKRB. Further, to examine the effect on sperm penetration of supplementation to adjust osmolarity, the osmolarity of mR1ECM-BSA was increased to the same level (310 mOsm) in mKRB by increasing NaCl concentration (106.7 mM) or by adding 60 mM sorbitol. After coculture of cu-mulus-oocyte complexes with spermatozoa in the modified mR1ECM-BSA for 10 h, the incidence of sperm penetration was compared to that in mR1ECM-BSA. Cheap Diskus Advair

Experiment 3

To determine the optimal concentration of NaCl in mR1ECM-BSA for sperm penetration, cumulus-oocyte complexes were cocultured with spermatozoa in mR1ECM-BSA containing various concentrations (90-140 mM) of NaCl for 10 h.

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Rat Oocytes Fertilized in Modified Rat 1-Cell Embryo: MATERIALS AND METHODS(4)

MATERIALS AND METHODS(4)

The embryos were picked up with a mouth-controlled pipette with a curved tip 150-200 ^m in diameter The tip of the pipette was inserted into an opening made previously by inserting a 26-gauge needle through the uterine wall at the oviductal side, and embryos were transferred into each uterine horn. After transfer, the vaginal smears of the recipients were examined daily. Cyclicity was considered to have been reestablished when the day of proestrus was identified in the recipients by examination of vaginal smears. These nonpregnant recipients were immediately killed and examined for implantation sites.

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