Evaluation of bovine embryos produced in high performance serum-free media.
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This review evaluates the quality of bovine embryos developed from in vitro-matured (IVM) and -fertilized (IVF) oocytes cultured in either serum-free or serum-containing media. Bovine embryos cultured in serum-supplemented medium contain numerous cytoplasmic lipid droplets and immature mitochondria compared to those cultured in serum-free medium. The accumulation of cytoplasmic lipids in embryos developed in serum-containing medium may be a result of incorporation of lipoproteins from the serum and may result in impaired function of mitochondria. The improved serum-free media (IVMD101 and IVD101) offer several advantages over culture in serum-containing medium, including increased rates of blastocyst formation and higher cell numbers. Additionally, the survival and hatching rates of embryos produced in serum-free media after post-thaw culture were superior to those of embryos produced in the serum-containing medium, suggesting that the abnormal accumulation of cytoplasmic lipids in embryos may have a negative effect on the sensitivity of embryos to chilling and freezing. These serum-free culture systems have proven to be beneficial for the production of good quality embryos from IVM-IVF bovine oocytes. Furthermore, recent studies have shown a correlation between mitochondrial function (oxygen consumption) and embryo quality. A new method using scanning electrochemical microscopy may be capable of assessing the viability and developmental potential of bovine embryos. (+info)
Effects of BSA and fetal bovine serum in culture medium on development of rat embryos.
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Rat 1-cell embryos, recovered from naturally mated females, were cultured in a chemically defined medium (mR1ECM) or in mR1ECM supplemented with BSA (4 mg/ml; mR1ECM-BSA) or fetal bovine serum (FBS; 10%, v:v; mR1ECM-FBS) instead of polyvinylalcohol. There was no difference in percentages of embryos that developed to the 2-cell to blastocyst stages between mR1ECM and mR1ECM-BSA, but in mR1ECM-FBS, no development beyond the 2-cell stage was observed. When embryos were transferred to mR1ECM-FBS from mR1ECM after 24 to 64 h of culture, development of embryos to and beyond the 4-cell stage was inhibited. However, when transferred after 80 h of culture, more embryos developed to blastocysts and hatching or hatched blastocysts than in embryos cultured in mR1ECM. When 8-cell embryos and early morulae obtained after 72 and 80 h of culture in mR1ECM, respectively, were cultured in mR1ECM-FBS, a higher proportion of early morulae developed to the blastocyst stage than did 8-cell embryos. When morulae obtained after culture in mR1ECM or mR1ECM-BSA were transferred to recipient females, there was no difference in proportions of fetuses obtained. However, a higher proportion of blastocysts cultured in mR1ECM-FBS developed to fetuses compared with those obtained in mR1ECM. These results indicate that BSA has neither deleterious nor beneficial effects on development of rat 1-cell embryos. In contrast, FBS has deleterious effects on early cleavage of embryos but it promotes more rapid development of morulae to blastocysts, resulting in better quality blastocysts. (+info)
A new alternative method for superovulation using passive immunization against inhibin in adult rats.
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The present study was undertaken to investigate the effects of passive immunoneutralization of endogenous inhibin on ovulation rate and embryo development in vivo and in vitro to establish a new alternative superovulation method in the adult rat. Female adult rats of Wistar strain were superovulated with a single injection of inhibin antiserum (inhibin-AS; 100 or 400 microl) or an injection of 20 IU eCG followed by an injection of 10 IU hCG. Untreated animals served as controls. Embryos were collected from oviducts or uteri on Days 1-5 of pregnancy, and the number of embryos and implantation sites were observed. On Day 1 of pregnancy, the two-cell-stage embryos were cultured and embryos from the 100-microl inhibin-AS group and the control group were transferred to recipient females to determine developmental competence. There were no significant differences between groups in fertilization rate. The numbers of normal embryos in the inhibin-AS-treated groups were significantly higher than the control and the eCG-hCG-treated groups throughout Days 1-4 of pregnancy. The number of implantation sites observed on Day 5 of pregnancy in the inhibin-AS-treated groups was significantly higher than both the control and the eCG-hCG-treated groups. Furthermore, the rate of blastocyst development in vitro in the inhibin-AS-treated groups and posttransfer viability in the 100-microl-inhibin-AS group were comparable with those of the control group. These results indicate that immunoneutralization of endogenous inhibin is a new practical alternative for induction of superovulation as a substitution for eCG-hCG method in the adult rat. (+info)
Use of assisted reproductive technologies in the propagation of rhesus macaque offspring.
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The assisted reproductive technologies (ARTs) as tailored to the production of rhesus monkeys at the Oregon National Primate Research Center (ONPRC) are described. Efficient fertilization of mature oocytes recovered by aspiration from females subjected to follicular stimulation was achieved with fresh or frozen sperm by intracytoplasmic sperm injection (ICSI). Embryo development to the early cleavage stage occurred at high frequency. Cryopreserved embryos showed high postthaw survival and were also transferred in efforts to establish pregnancies. Three methods of transfer were evaluated, two involving embryo placement into the oviduct, laparoscopy and minilaparotomy, and a nonsurgical, transcervical approach that resulted in uterine deposition. Early cleaving embryos (Days 1-4) were transferred into the oviducts of synchronized recipients with optimal results and pregnancy rates of up to 36%. Pregnancy rates were similar when two fresh or frozen embryos were transferred (28- 30%), although more than two embryos had to be thawed to compensate for embryo loss during freeze-thawing. Normal gestational lengths, birth weights, and growth curves were seen with ART-produced infants compared with infants produced by natural mating in the timed mated breeding (TMB) colony at the ONPRC. In 72 singleton pregnancies established following the transfer of ART-produced embryos, the live-birth rate, at 87.5%, was statistically identical to that for the TMB colony. Further development of the ARTs should result in increasing use of these techniques to augment conventional approaches to propagating monkeys, especially those of defined genotypes. (+info)
Morphometric analysis of embryonic rat trigeminal neurons treated with different neurotrophins.
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In whole-mount explant cultures of the trigeminal ganglion (TG) with intact peripheral and brainstem targets, exogenous application of nerve growth factor (NGF) and neurotrophin-3 (NT-3) leads to elongation and precocious arborization of embryonic trigeminal axons, respectively. In addition, neurotrophins play a major role in survival and differentiation of distinct classes of TG neurons. In the present study, we conducted morphometric analyses of trigeminal neurons exposed to exogenous NGF or NT-3 in whole-mount explant cultures. Explants dissected from embryonic day (E) 13 and E15 rats were cultured in the presence of serum-free medium (SFM) or in SFM supplemented with NGF or NT-3 for 3 days. TG neurons were then retrogradely labeled with lipophilic tracer DiI and their soma size distributions were compared following different treatments. The mean diameters of E13 and E15 trigeminal neurons grown in the presence of NT-3 were similar to those grown in SFM. On the other hand, in cultures supplemented with NGF, the mean diameters of neurons were larger at E13, but smaller at E15. Double immunolabeling with TrkA and TrkC antibodies confirmed the presence of large-diameter TrkA-positive neurons in E13 TG, but not in E15 TG. At both ages, other large-diameter neurons expressed only TrkC. These results show that exposure to NGF leads to phenotypic changes in TrkA-expressing trigeminal neurons at early embryonic development, but selective survival of small diameter neurons at later ages. (+info)
Rapid triple-labeling method combining in situ hybridization and double immunocytochemistry.
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A new, rapid method is described for combining in situ hybridization and immunocytochemistry to define cell populations and to map three-dimensional movements of groups of labeled cells within developing chick embryos. The method allows fluorescently labeled cells to be followed in living embryos and subsequently detected as a permanent reaction product for detailed three-dimensional analysis by immunocytochemistry in histological serial sections. Cell identity can be ascertained using a specific riboprobe and in situ hybridization. With this approach, the movements of two groups of cells can be mapped simultaneously (using two different fluorescent trackers and, subsequently, two different chromogens for immunocytochemistry) to analyze relative movements within an embryo, and when combined with in situ hybridization with a specific riboprobe for cell identity, allows fate mapping studies to be conducted using molecular criteria, rather than solely at morphological/positional criteria. The improved method enables the investigator to extract substantially more information from individual embryos, maximizing the results obtained from labor-intensive fate mapping studies. (+info)
Oxygen-regulated gene expression in bovine blastocysts.
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Oxygen concentrations used during in vitro embryo culture can influence embryo development, cell numbers, and gene expression. Here we propose that the preimplantation bovine embryo possesses a molecular mechanism for the detection of, and response to, oxygen, mediated by a family of basic helix-loop-helix transcription factors, the hypoxia-inducible factors (HIFs). Day 5 compacting bovine embryos were cultured under different oxygen tensions (2%, 7%, 20%) and the effect on the expression of oxygen-regulated genes, development, and cell number allocation and HIFalpha protein localization were examined. Bovine in vitro-produced embryos responded to variations in oxygen concentration by altering gene expression. GLUT1 expression was higher following 2% oxygen culture compared with 7% and 20% cultured blastocysts. HIF mRNA expression (HIF1alpha, HIF2alpha) was unaltered by oxygen concentration. HIF2alpha protein was predominantly localized to the nucleus of blastocysts. In contrast, HIF1alpha protein was undetectable at any oxygen concentration or in the presence of the HIF protein stabilizer desferrioxamine (DFO), despite being detectable in cumulus cells following normal maturation conditions, acute anoxic culture, or in the presence of DFO. Oxygen concentration also significantly altered inner cell mass cell proportions at the blastocyst stage. These results suggest that oxygen can influence gene expression in the bovine embryo during postcompaction development and that these effects may be mediated by HIF2alpha. (+info)
Effect of the post-fertilization culture environment on the incidence of chromosome aberrations in bovine blastocysts.
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We have previously shown that the postfertilization embryo culture environment has a significant influence on the quality of the resulting bovine blastocyst measured in terms of its cryotolerance and relative abundance for several developmentally important gene transcripts. Using three different culture conditions known to produce blastocysts of differing quality, the objective of this study was to examine whether the postfertilization culture environment had an effect on the incidence of mixoploidy in bovine blastocysts. Presumptive zygotes, produced by in vitro maturation and fertilization, were cultured in vitro in synthetic oviduct fluid (SOF) medium in the absence or presence of fetal calf serum (FCS), or in vivo in the ewe oviduct. Blastocysts were recovered from the three systems at Day 7 and the incidence of mixoploidy was assessed using fluorescence in situ hybridization with chromosome 6- and chromosome 7-specific probes. A total of 10 025 nuclei were scored in 122 blastocysts. The frequency of normal, diploid, blastocysts was 8.8%, 21.4%, and 34.8% in embryos derived from culture in SOF+FCS, SOF, and the ewe oviduct, respectively, the remainder showing some degree of mixoploidy. The incidence of mixoploidy was apparently not related to the presence of serum; omission of serum from SOF resulted in a reduction in the incidence of mixoploidy (91.2% vs. 78.6%), although this difference was not significant. Culture in vivo, however, resulted in a significant (P < 0.01) reduction in the incidence of mixoploidy compared with culture in vitro in the presence of serum (65.2% vs. 91.2%, respectively). Among the mixoploid blastocysts, the majority contained less than 10% polyploid cells, irrespective of culture group (SOF, 69.7%; SOF+FCS, 64.5%; ewe oviduct, 60.0%). More than one type of polyploidy was frequently observed in mixoploid blastocysts. Overall, diploidy-triploidy was the most frequent abnormality, but diploid-tetraploid and diploid-triploid-tetraploid mosaics were also observed. A significantly higher proportion (P < 0.05) of blastocysts derived from SOF+FCS had more than one type of abnormality (80.6%, 25/ 31) compared with those derived from SOF (45.4%, 15/33) or in vivo culture (53.3%, 16/30). In conclusion, the postfertilization culture environment of the developing embryo can affect the incidence and severity of mixoploidy in the resulting blastocyst. (+info)