Stage-specific requirement of phosphate for development of rat 1-cell embryos in a chemically defined medium

Zygote ◽  
1997 ◽  
Vol 5 (1) ◽  
pp. 67-73 ◽  
Author(s):  
Kazuchika Miyoshi ◽  
Koji Niwa
Keyword(s):  
Embryo Development ◽  
Defined Medium ◽  
Blastocyst Stage ◽  
Chemically Defined Medium ◽  
Specific Requirement ◽  
Blastocyst Formation ◽  
The Mean ◽  
Almost All

SummaryRat 1-cell embryos, recovered from naturally mated females, were cultured in a chemically defined medium (mR1ECM). When examined after 24, 56, 64 and 80h of culture, embryos developed to the 2-cell (100%), 4-cell (93%), 4-cell to 8-cell (97%) and ≥8-cell (95%) stages, respectively. When 0.4 M phosphate (NaH2PO4) was added to the medium after 0, 24, 56 and 64 h of culture, percentages (0–67%) of embryos that developed to the blastocyst stage after 115h of culture were lower than that (86%) in the medium without phosphate. However, addition of phosphate after 80h of culture accelerated blastocyst formation; a significantly higher percentage (94%) of blastocysts was obtained after 110 h of culture in this medium compared with when phosphate was not added (67%). When phosphate was added to the medium after 64h, almost all (97%) 8-cell embryos developed to the blastocyst stage by 100h of culture but development of 4-cell to 7-cell embryos was inhibited (22–63%). Acceleration of blastocyst formation was caused by addition of phosphate rather than by the exchange of the medium. The stimulatory effect of phosphate on embryo development was observed at concentrations of 0.1–1.2mM. The mean numbers of cells (54.5–60.9 cells) in blastocysts examined after 115 h of culture were increased by the addition of 0.4–1.6mM phosphate at 80 h as compared with blastocysts from cultures without phosphate (46.6 cells).

On the origin of variants of the marine bacteriumDeleya aesta134 able to grow at low Na+concentration

1997 ◽  
Vol 43 (9) ◽  
pp. 868-878 ◽  
Author(s):  
Robert A. MacLeod ◽  
Patricia R. MacLeod ◽  
Marc Berthelet
Keyword(s):  
Liquid Medium ◽  
Marine Bacteria ◽  
Lag Time ◽  
Defined Medium ◽  
Adaptive Mutation ◽  
Chemically Defined Medium ◽  
Colony Type ◽  
Lag Period ◽  
The Mean ◽  
Concentration Cells

Deleya aesta 134 grows optimally at 200 mM Na+in a chemically defined medium but at 10 mM Na+only after an extended lag period which was reduced if the cells that grew were reinoculated into medium of the same low Na+concentration. Cells that eventually grew at low Na+formed colonies on agar containing 17 mM Na+in the agar supernatant (the liquid released when the agar was compacted). Cells of the parent failed to form colonies at this Na+concentration when 102cells were plated. Colonies that formed on low Na+agar differed in appearance from colonies of the parent and three colony types were distinguished. When 106cells of D. aesta grown in liquid medium containing optimum Na+were spread on plates containing 17 mM Na+, a few variant colonies first appeared on day 4 and then increased in numbers over a 20-day period. In nine similar cultures the yield of colonies varied over a 3-log range. Fluctuation tests applied to the numbers arising from the similar cultures after different periods of incubation of the plates showed that the ratio of the variance to the mean was much greater than one initially and then increased with time. A total of seven different variants were isolated. These could be distinguished by the colony type formed, the length of the lag time preceding the first appearance of colonies, and the rate of colony accumulation on low (and in one case, high) Na+plates. The variants retained their distinctive characteristics when replated at low Na+after growth at optimum Na+. Differences in lag time and rate of colony accumulation were related to differences in Na+requirement of the variants and to the presence of other colonies on the plates. The variants appear to arise as the result of random mutations in the growing culture. There was no evidence of adaptive mutation.Key words: Deleya aesta, marine bacteria, variants, Na+response, colony accumulation, adaptive mutation.

scholarly journals In vitro production of cattle blastocysts in chemically defined medium with or without insulin supplementation

1996 ◽  
Vol 5 (5) ◽  
pp. 509-514 ◽  
Author(s):  
Kristiina Bredbacka ◽  
Peter Bredbacka
Keyword(s):  
Bovine Serum Albumin ◽  
Culture Medium ◽  
Defined Medium ◽  
Blastocyst Stage ◽  
Chemically Defined Medium ◽  
In Vitro Production ◽  
Cell Numbers ◽  
Vitro Production ◽  
Bovine Oocytes

In this study we evaluated the use of a chemically defined medium in the production of blastocysts from bovine oocytes fertilized in vitro. As culture medium we used CRI-PVP, a modification of CRlaa medium with bovine serum albumin replaced by polyvinylpyrrolidone. After 168 h of culture (192 h after insemination) 8.7%, 10.5 and 12.8% of the cleaved embryos developed to the blastocyst stage in the presence of 0, 2 or 200 nM insulin, respectively. The supplementation of 200 nM insulin tended to increase cell numbers in morulae and blastocysts (P=0.10). It is concluded that CRI-PVP can be used as a chemically defined medium in the production of blastocysts from bovine 1-cell embryos. However, further modifications are needed, and the insulin concentrations used may be below the optimum for blastocyst production.

scholarly journals Morphologic changes in boar sperm nuclei with reduced disulfide bonds in electrostimulated porcine oocytes

Reproduction ◽  
2006 ◽  
Vol 131 (3) ◽  
pp. 603-611 ◽  
Author(s):  
Michiko Nakai ◽  
Naomi Kashiwazaki ◽  
Akiko Takizawa ◽  
Naoki Maedomari ◽  
Manabu Ozawa ◽  
...  
Keyword(s):  
Electrical Stimulation ◽  
Blastocyst Stage ◽  
Blastocyst Formation ◽  
The Mean ◽  
Sperm Nuclei ◽  
Morphologic Changes ◽  
Number Of Cells ◽  
Stimulation Of ◽  
Nuclear Decondensation

In pigs, failure of sperm nuclear decondensation has been reported after injection into oocytes. We examined the effects of pretreating sperm heads with Triton X-100 (TX-100) and dithiothreitol (DTT) and of electrical stimulation of oocytes after sperm head injection on time-dependent morphologic changes in sperm nuclei andin vitrodevelopment to the blastocyst stage. In experiment 1, spermatozoa were pretreated with 1% TX-100 and 5 mM DTT (T + D) or not treated, and then injected intoin vitromatured oocytes. Electrical stimulation (1.5 kV/cm, 20 μs DC pulse) was applied to the oocytes 1 h after injection (stimulated group) or was not applied (unstimulated group). Some of the oocytes in each group were evaluated at hourly intervals until 10 h after injection for morphologic changes in the sperm nuclei. Unstimulated oocytes injected with untreated spermatozoa showed a delayed peak in the rate of nuclear decondensation (39.4–44.1%, 3–6 h after injection) compared with oocytes injected with T + D-treated spermatozoa (57.0% and 52.6%, 1 and 2 h, respectively). The rate of male pronucleus formation peaked 6 h after stimulation (by 40–60%) after injected oocytes had been stimulated with an electrical pulse, irrespective of whether or not the spermatozoa had been pretreated. In unstimulated oocytes, the rate of male pronucleus formation did not increase and stayed at the basal level (less than 20%) throughout the culture period, regardless of the sperm treatment. Thus, T + D treatment of spermatozoa did not affect completion of fertilization. In experiment 2, we evaluated the effects of electrical stimulation and sperm treatment with T + D on the rate of blastocyst formation and the mean number of cells per blastocyst. Oocytes stimulated after injection with either T + D-treated or untreated spermatozoa showed significantly higher percentages of blastocyst formation (24.8% and 27.1% respectively) than did unstimulated oocytes (1.1% and 4.1% for T + D-treated and untreated respectively;P< 0.01 by Duncan’s multiple-range test). The rate of blastocyst formation did not differ between the T + D-treated and untreated groups. The mean number of cells per blastocyst did not differ among any of the groups (14.0–29.4 cells). These results suggest that pretreatment of sperm with TX-100 and DTT shifted the timing of sperm nuclear decondensation forward. However, pronucleus formation and development to the blastocyst stagein vitrowere not improved by sperm treatment. Thus, electrical stimulation of injected oocytes enhancesin vitrodevelopment to the blastocyst stage in pigs.

scholarly journals 141 BENEFICIAL EFFECT OF ETHYLENEDIAMINETETRAACETIC ACID COMBINED WITH HEMOGLOBIN ON PRE-IMPLANTATION DEVELOPMENT OF PORCINE IN VITRO PRODUCED EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 221
Author(s):  
J.H. Kim ◽  
G.S. Lee ◽  
H.S. Kim ◽  
S.H. Lee ◽  
D.H. Nam ◽  
...  
Keyword(s):  
Beneficial Effect ◽  
Embryo Development ◽  
Ethylenediaminetetraacetic Acid ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Blastocyst Formation ◽  
Total Cell Number ◽  
Porcine Embryo

Developing a porcine embryo culture system is important for increasing the rates of implantation and pregnancy of somatic cell nuclear transfer (SCNT) embryos. Ethylenediaminetetraacetic acid (EDTA) was shown to inhibit glycolytic activity of cleavage stage embryos, thereby preventing the premature stimulation of glycolysis and enhancing development. However, EDTA should not be used for later-stage embryos as the inhibition of glycolysis reduces energy production at the blastocyst stage and significantly inhibits inner cell mass development. On the other hand, addition of a nitric oxide (NO) scavenger, hemoglobin (Hb), to the culture medium is known to promote embryo development to the blastocyst stage. This study was conducted to evaluate the beneficial effect of EDTA combined with Hb on pre-implantation development of porcine embryos in vitro. Porcine embryos produced by in vitro maturation and fertilization were cultured for 6 days in North Carolina State University (NCSU)-23 medium supplemented with EDTA or/and Hb. All data were subjected to one-way ANOVA and protected least significant difference (LSD) test using the general linear models (GLM) procedure of the statistical analysis system (SAS Institute, Inc., Cary, NC, USA) program to determine differences among experimental groups. Statistical significance was determined when the P value was less than 0.05. In Exp. 1, culturing porcine zygotes with 100 mM EDTA (n = 537) significantly increased cleavage rates (85.3%) at 48 h post-insemination compared to supplementing with 0, 1, or 10 mM EDTA (78.9, 79.7, or 78.2%, respectively). However, EDTA at these concentrations did not promote blastocyst formation compared to the control. In addition, no difference was observed in total cell numbers in blastocysts among the experimental groups (41.8, 42.6, 45.8, 44.5, respectively). In Exp. 2, in vitro-fertilized oocytes were cultured with 0, 1, or 10 mg/mL Hb. Culturing with Hb did not promote porcine embryo development, but significantly increased the total cell number of blastocysts obtained from 1 mg/mL Hb supplementation (n = 566) compared to that of the control (56.8 vs. 41.6). In Exp. 3, culturing embryos (n = 548) with 100 mM EDTA + 1 mg/mL Hb significantly improved rates of cleavage (84.0% vs. 75.2%) and blastocyst formation (19.2% vs. 12.7%), and the total number of cells in blastocysts compared to those of the control (58.4 vs. 42.3). In conclusion, our results demonstrated that EDTA or Hb have different roles in supporting in vitro pre-implantation development of porcine embryos; EDTA mainly stimulated early cleavage up to the 2- to 4-cell stage, and Hb promoted the total cell number of blastocysts. However, combined supplementation with these two chemicals improved cleavage, blastocyst formation, and total cell number in blastocysts. This study was supported by a grant from Korea Ministry of Science and Technology (Biodiscovery).

241 DIBUTYRYL CYCLIC AMP AND EGF-LIKE FACTORS SUPPORT IN VITRO MATURATION OF PORCINE OOCYTES IN A CHEMICALLY DEFINED MEDIUM WITHOUT GONADOTROPINS

2009 ◽  
Vol 21 (1) ◽  
pp. 218
Author(s):  
Y. Akaki ◽  
K. Yoshioka ◽  
H. Funahashi
Keyword(s):  
Cyclic Amp ◽  
In Vitro Maturation ◽  
Defined Medium ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Chemically Defined Medium ◽  
Dibutyryl Cyclic Amp ◽  
Vitro Fertilization ◽  
Porcine Oocyte

Exposure of porcine oocyte–cumulus complexes (OCC) to gonadotropins induces meiotic resumption, but the details of this mechanism are still unknown. The present study was undertaken to examine combinational effects of EGF-like factors and dibutyryl cyclic AMP (dbcAMP) in a chemically defined medium on in vitro maturation (IVM) of porcine oocytes. The OCC were aspirated from 3- to 6-mm-diameter follicles of prepuberal ovaries and used in the current study. The basic culture medium was a chemically defined medium, Porcine Oocyte Medium (POM; Research Institute for the Functional Peptides, Yamagata, Japan). In the first experiment, various concentrations (0, 10, and 1000 ng mL–1) of EGF-like factors (EGF, amphiregulin, and betacellulin) were added to POM during an entire IVM period (44 h). In the second experiment, to determine the additive effect of EGF-like factors, each EGF-like factor with an effective concentration was combined with the others. In the last experiment, to examine the combined effect with dbcAMP, OCC were exposed to EGF (10 ng mL–1), amphiregulin (1000 ng mL–1), and dbcAMP (1 mm) during the first 20 h of IVM and then the culture was continued in the absence of EGF-like factors and dbcAMP. After culture, in all experiments, meiotic resumption and the progress of oocytes were examined after denuding, fixing, and staining. Statistical analyses was performed by ANOVA with a Bonferroni-Dunn post hoc test (significance, P < 0.05). In the first experiment, all treatments without supplementation with 10 ng mL–1 amphiregulin increased the incidence of oocytes maturing to the MII phase, as compared with controls (29.1 to 39.3% v. 11.1%, P < 0.05). In the second experiment, combinations with 2 kinds of EGF-like factor slightly (but not significantly) improved the percentage of oocytes at the MII stage (37.7 to 47.4%). In the last experiment, supplementation with 1 mm dbcAMP during the first 20 h of IVM, regardless of the presence of EGF-like factors, significantly increased the incidence of MII oocytes as compared with controls, whereas the incidence was the highest when 1 mm dbcAMP, 10 ng mL–1 EGF, and 1000 ng mL–1 amphiregulin were supplemented (75.5%). When those oocytes were cultured in a chemically defined medium after in vitro fertilization, the developmental competence of oocytes to the blastocyst stage (25.0%) was not different from oocytes matured in the presence of gonadotropins and dbcAMP during the first 20 h of IVM (17.3%). These observations indicate that supplementation of a chemically defined maturation medium with EGF-like factors and dbcAMP during the first 20 h of IVM can support the meiotic progress and developmental competence of porcine oocytes well. Currently, we are examining the developmental competence of those oocytes after embryo transfer. The results will be presented at the meeting. This study was supported by MAFF AgriBio1605.

65 IMPACT OF AIRPORT RADIATION ON BOVINE SPERM DNA INTEGRITY, FERTILIZING ABILITY, AND EMBRYO DEVELOPMENT

2009 ◽  
Vol 21 (1) ◽  
pp. 132
Author(s):  
K. E. M. Hendricks ◽  
D. Evenson ◽  
P. J. Hansen ◽  
M. Kaproth ◽  
L. M. Penfold
Keyword(s):  
Embryo Development ◽  
Blastocyst Stage ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Bovine Sperm ◽  
Sperm Dna Integrity ◽  
Sperm Dna ◽  
Fertilizing Ability ◽  
The Mean ◽  
The Impact

Biological samples, including cryopreserved sperm, are routinely shipped using air transportation, in dry shippers that are x-rayed along with routine baggage. Accordingly, it is important to demonstrate that there are no potential risks associated with semen transport. The goal of this study was to investigate the impact of airport radiation used for a) checked luggage and b) carry-on luggage on bovine sperm DNA integrity, fertilizing ability, and embryo development. Frozen domestic bull sperm collected from known fertile bulls (n = 9) and stored in a dry shipper (–196°C) were x-rayed 0, 1, 2, and 3 times as a) checked luggage and b) carry-on luggage. Duplicate straws were thawed and assessed for DNA damage using the sperm chromatin structure assay (SCSA®, SCSA Diagnostics, Brookings, SD) and fertilization and embryo development by in vitro fertilization. The SCSA® parameters are the mean and SD of the DNA fragmentation index (mean DFI and SD DFI). Multiple x-rays did not significantly (P > 0.05) affect sperm chromatin heterogeneity assessed by SCSA® and no differences were observed in the mean, SD, and DFI for any of the sperm treatments. No differences (P > 0.05) were seen in embryo cleavage or blastocyst development rates (expressed as percentage of oocytes becoming blastocysts or percentage of cleaved embryos becoming blastocysts) for sperm x-rayed 0, 1, 2, or 3 times using either checked or carry-on luggage doses. The percentage of oocytes developing to the blastocyst stage was 13.8, 11.5, 12.8, and 9.0% (SEM = 2.3%) for sperm exposed to the checked luggage dose 0, 1, 2, and 3 times. The percentage of oocytes developing to the blastocyst stage was 13.0, 12.8, 14.0, and 13.5% (SEM = 3.5%) for sperm exposed to the carry-on luggage dose 0, 1, 2, and 3 times. As future x-ray machines are planned that deliver greater doses of radiation to scan large quantities of baggage with a single scan, it is important that continued monitoring of shipped sperm is performed. The authors are grateful to Lara Metrione, Brian Delauter, and the TSA staff at Jacksonville Airport for assistance with this study.

Additional effect of epidermal growth factor during in vitro maturation for individual bovine oocytes using a chemically defined medium

Zygote ◽  
2004 ◽  
Vol 12 (2) ◽  
pp. 143-150 ◽  
Author(s):  
Takahiro Oyamada ◽  
Hiroshi Iwayama ◽  
Yutaka Fukui
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Defined Medium ◽  
Control Medium ◽  
Chemically Defined Medium ◽  
Blastocyst Formation ◽  
Epidermal Growth ◽  
The Individual ◽  
Bovine Oocytes

This study was performed to establish an individual bovine oocyte-IVP system using a chemically defined simple medium (mSOFaa containing 1 mg/ml polyvinyl alcohol: PVA) and to investigate the effects of epidermal growth factor (EGF) during oocyte maturation on in vitro maturation, fertilization and embryonic development. Cumulus–oocyte complexes were collected from bovine ovaries and were matured in mSOFaa containing PVA (control medium) supplemented with 0, 1, 10 or 50 ng/ml of EGF. Two further groups (TCM199 and mSOFaa, supplemented with 10% fetal calf serum were also included. In this study, mSOFaa containing PVA were used as a basic medium for fertilization and embryo development in vitro. Experiments were conducted in both group- and individual-IVP systems. In the group-IVP system, the proportion of matured oocytes (MII) in the control medium (62.7%±5.0%) was significantly (p<0.05) lower than in all other treatments, and in the individual-IVP system, the addition of 1 ng/ml EGF significantly (p<0.05) increased the maturation rate (1 ng/ml EGF vs control: 76.2%±5.4% vs 57.1%±14.4%). The addition of EGF did not affect the proportions of penetrated and normally fertilized oocytes in either individual- or group-culture systems. In the group-IVP system, no significant difference among treatments was found in the rate of blastocyst formation, whereas in the individual-IVP system the control medium supplemented with 10 ng/ml EGF resulted in a significantly (p<0.05) higher the rate of blastocyst formation (20.0±5.2%) than that in the control medium (6.2%±3.5%). These results indicate that bovine oocytes can successfully develop to blastocysts in an individual-IVP system using a single chemically defined medium, and that the group-IVP system also resulted in a similar level of blastocyst formation to that in a standard multiple-media system in our laboratory. The effect of EGF during oocyte maturation medium differed depending on whether embryos were cultured individually or in groups.

scholarly journals 290EFFECTS OF HYALURONAN ON IN VITRO DEVELOPMENT OF PORCINE EMBRYOS CULTURED IN A CHEMICALLY DEFINED MEDIUM

2004 ◽  
Vol 16 (2) ◽  
pp. 264
Author(s):  
K. Yoshioka ◽  
H. Ekwall ◽  
H. Rodriguez-Martinez
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Defined Medium ◽  
Blastocyst Stage ◽  
Cell Counting ◽  
Transmission Electron Microscopy Data ◽  
Chemically Defined Medium ◽  
In Vitro Production ◽  
Microscopy Data

Hyaluronan (HA), a glycosaminoglycan present in follicular and oviductal fluids, has been related to sperm capacitation, fertilization and embryo development. We have previously developed an in vitro-production (IVP) system of porcine embryos, where porcine blastocysts can be produced by IVF and IVC in chemically defined media and can develop to full-term by transfer to recipients. The application of a chemically defined medium to IVP in pigs allows the analysis of the physical action of substances on the development of pre-implantation embryos. In the present study, the effects of HA on the development of porcine embryos in a chemically defined medium were investigated. Porcine presumptive zygotes were produced by IVM and IVF of COC from pre-pubertal gilts and frozen-thawed ejaculated boar semen. The zygotes were cultured in Porcine Zygote Medium (PZM)-5 containing different concentrations of HA (0 [control], 1, 2, 5, 10, 20 and 50μgmL−1) until 6 days after IVF, and representative specimens were fixed for cell counting and transmission electron microscopy. Data of percentages and cell numbers were statistically analyzed by one-way ANOVA and Fisher’s PLSD test. The percentage of embryos that developed to the blastocyst stage (15.8% [23/144] to 19.5% [27/139]) did not differ among treatments. However, addition of 5 or 10μgmL−1 HA increased (P&lt;0.05) the total number of cells in blastocysts (56.1 and 58.3 cells [n=22 and 23], respectively) compared to control (no HA, 42.0 cells [n=23]). To evaluate proliferation rates of inner cell mass (ICM) and trophectoderm (TE), embryos were cultured in PZM-5 for various periods of exposure to 10μgmL−1 HA. The numbers of ICM and TE cells in Day-6 blastocysts cultured in the presence of exogenous HA from Day 0 to Day 3 (18.3 and 34.4 cells, respectively [n=38]) or Day 6 (17.9 and 35.9 cells, respectively [n=36]) were significantly (P&lt;0.05) higher than those cultured without HA through the culture period (13.5 and 24.2 cells, respectively [n=26]). In the presence of HA from Day 3 to 6, only the number of TE cells (37.1 cells [n=33]) increased (P&lt;0.05), compared to PZM-5 alone. Differences in ultrastructure were noticed among blastocysts cultured with or without 10mgmL−1 HA. Blastocysts cultured with HA had mainly mature mitochondria while many mitochondria appeared morphologically immature in the blastocysts cultured without HA. Lipid droplets in the blastocysts cultured with HA seemed to be more homogeneous in comparison with those in the blastocysts cultured in PZM-5 alone. Further differences were seen in the numbers of lysosome-like structures, which were greater in blastocysts cultured with HA. This study demonstrates that exogenous HA improves cell proliferation and normality of ICM and TE in porcine embryos cultured in a chemically defined medium, depending on the exposure periods to HA. (Supported by MAFF, Japan and STINT, Sweden.)

Variations in the Metabolism of the Daughter Sporocysts of Microphallus pygmaeus in a Chemically Defined Medium

1972 ◽  
Vol 46 (1) ◽  
pp. 107-116 ◽  
Author(s):  
R. J. Richards ◽  
D. Pascoe ◽  
B. L. James
Keyword(s):  
Oxygen Uptake ◽  
Metabolic Rate ◽  
Metabolic Activity ◽  
Nutrient Medium ◽  
Sea Water ◽  
Defined Medium ◽  
Chemically Defined Medium ◽  
Nutrient Media ◽  
Mean Size ◽  
The Mean

A comparison is made of the variations in the mean size (length), reduced weight, oxygen uptake, metabolic rate and the number of contained fully formed metacercariae undergoing autolysis, in mature daughter sporocysts of Microphallus pygmaeus, in sea water, artificial sea water and in a chemically defined nutrient medium (modified medium 199). The work indicates that the sporocysts begin to degenerate almost immediately in the non-nutrient media but have a higher metabolic activity and remain healthy for up to 36 days in the nutrient medium.

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