Growth and Differentiation of Rabbit Blastocysts in Defined Culture Media

1983 ◽  
pp. 371-386 ◽  
Author(s):  
H. M. Beier ◽  
U. Mootz ◽  
B. Fischer ◽  
R. Ströbele-Müller
Keyword(s):  
Culture Media ◽  
Defined Culture

136 ADDITION OF LOW DENSITY LIPOPROTEIN TO CHEMICALLY DEFINED PORCINE EMBRYO CULTURE MEDIUM CAN PARTIALLY REPLACE BOVINE SERUM ALBUMIN

2008 ◽  
Vol 20 (1) ◽  
pp. 148
Author(s):  
L. D. Spate ◽  
K. A. Walker ◽  
C. E. McHughes ◽  
R. S. Prather
Keyword(s):  
Embryo Culture ◽  
Culture Medium ◽  
Culture Media ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Blastocyst Stage ◽  
Differential Staining ◽  
Low Density ◽  
Cell Stage ◽  
Defined Culture

Embryo culture media typically contain undefined biologicals such as BSA. Our goal is to develop chemically defined culture media that are based on the biology and physiology of the embryo. To that end we evaluated the presence of message in embryos at various stages of development and determined that the message for the low density lipoprotein receptor (LDLR) increased from the germinal vesicle and 4-cell stage to the blastocyst stage of porcine embryogenesis. Thus, this study was conducted to determine if the addition of low density lipoprotein (LDL) would enhance the development and quality of in vitro produced porcine embryos in an already chemically defined culture medium. Slaughterhouse ovaries were aspirated, cumulous–oocyte complexes (COC) identified, and the COC were matured for 42 h in M199 base medium supplemented with EGF, FSH, and LH. Metaphase II oocytes were then selected. Fertilization was then preformed in modified Tris buffered medium and cocultured with 0.25 � 106/mL frozen thawed porcine semen for 5 h. The presumptive zygotes were then transferred to either porcine zygote medium with 0.3% BSA or 0.1% PVA (PZM3, PZM4). After 28 h, cleaved embryos were then sorted into six treatment groups (1. PZM3, 2. PZM3 + 20 µg mL–1 LDL, 3. PZM4, 4. PZM4 + 10 µg mL–1 LDL, 5. PZM4 + 20 µg mL–1 LDL, 6. PZM4 + 50 µg mL–1 LDL). The embryos were cultured in 5%O2 5%CO2 90%N until day 7. The percentage of development to the blastocyst stage was determined and analyzed with the SAS Proc GENMOD Procedure (a–cP ≤ 0.05). The percentage blastocyst was 51.3 � 0.09a, 51.6 � 0.09a, 33.1 � 0.99c, 35.8 � 0.09c, 36.9 � 0.09c, and 41.3 � 0.06b, for treatments 1–6, respectively. Culture in PZM4 (without BSA) significantly reduced development. However, addition of 50 µg mL–1 of LDL to PZM4 improved development above PZM4 alone. We interpret these data to indicate that a high concentration of LDL in the PZM4 media did improve embryo development and that LDL could partially substitute for BSA. Differential staining was performed on the blastocysts, and preliminary results suggest that the ICM to trophectoderm ratio in the high LDL treatment group is closer to the ratio found in in vivo produced embryos. This project was supported by USDA CSREES NRI (2006-35203-17282) and Food for the 21st Century.

Chemically defined culture media: rational recipes or witches' brew?

1996 ◽  
Vol 6 (0) ◽  
pp. 111
Author(s):  
Jeroen Van Bergen ◽  
Egbert A. J. F. Lakke
Keyword(s):  
Culture Media ◽  
Defined Culture

Induction of Tyloses in Eucalyptus Globulus 'chips'

IAWA Journal ◽  
1999 ◽  
Vol 20 (2) ◽  
pp. 193-201 ◽  
Author(s):  
Mathew A. Leitch ◽  
Rodney A. Savidge ◽  
Geoff M. Downes ◽  
Irene L. Hudson
Keyword(s):  
Acetic Acid ◽  
Eucalyptus Globulus ◽  
Culture Media ◽  
Inhibitory Effect ◽  
Naphthalene Acetic Acid ◽  
Distilled Water ◽  
Control Medium ◽  
Water Control ◽  
Significant Inhibitory Effect ◽  
Defined Culture

Cambial stem chips containing intact cambium between xylem and phloem, or with the phloem layer removed, were cut from the main stem axis of four-year-old Eucalyptus globulus during the winter and grown under controlled environmental conditions for seven weeks on fully defined culture media . Light microscopy revealed that tyloses were induced in sapwood vessels in the region adjacent to the cambium within these stem chips. When incubated in autoclaved double-distilled water (control medium) tyloses were produced in 3.7% and 4.7% of vessels in chips with the phloem layer intact and removed, respectively. When non-hormonal ingredients were included, tyloses developed in 69.5% and 76.1% of vessels in chips with the phloem layer intact and removed, respectively. Addition of 1.0 mg l-1 of l-naphthalene acetic acid (NAA, a synthetic auxin) to the medium had a slight, but significant, inhibitory effect on tylosis formation.

136 N-METHYL-D-ASPARTIC ACID AND HOMOCYSTEINE CAN BE USED TOGETHER AS A REPLACEMENT FOR BOVINE SERUM ALBUMIN IN EARLY PORCINE EMBRYO CULTURE

2011 ◽  
Vol 23 (1) ◽  
pp. 172
Author(s):  
L. D. Spate ◽  
B. K. Bauer ◽  
C. N. Murphy ◽  
R. S. Prather
Keyword(s):  
Dna Methylation ◽  
Bovine Serum Albumin ◽  
Surface Area ◽  
Embryo Culture ◽  
Bovine Serum ◽  
Culture Media ◽  
Cell Number ◽  
Global Dna Methylation ◽  
Treatment Groups ◽  
Defined Culture

Most mammalian embryo culture media contain some form of unidentifiable biological contaminant, usually associated with fetal bovine serum (FBS) or bovine serum albumin (BSA). Such factor(s) confound experiments attempting to evaluate culture media composition and decrease the repeatability of experiments when different lots or batches of FBS or BSA are used. The goal of this study was to formulate a completely chemically defined culture media for development of early porcine embryos based on adding ligands for which there is the presence of mRNA for the corresponding receptors in the blastocyst. Cumulus–cell oocyte complexes were matured for 42 h in M199 supplemented with EGF, FSH, and LH. Metaphase II oocytes were selected and fertilized in modified Tris-buffered media for 4 h. Presumptive zygotes were then placed into porcine zygote media with 0.3% BSA (PZM3) or 0.1% polyvinyl alcohol (PZM4). At 28 h post-fertilization, 2- to 4-cell stage embryos were selected and placed into treatment groups for 5 days. Fifteen embryos were put into 25 μL of media and cultured in 5% CO2, 5% O2, and 90% N at 38.5°C. The treatment groups were as follows: 1. PZM3, 2. PZM4, 3. PZM4 + 0.5 mM N-methyl D-aspartic acid (NMDA), 4. PZM4 + 0.5 mM NMDA + 10 μM homocysteine (HC), and 5. PZM4 + 10 μM HC. There were 120, 135, 120, 135, and 120 embryos for each treatment, respectively. The percentages of embryos that developed to the blastocyst stage were 63.3%a, 29.7%b, 46.1%c, 55.6%ac, and 49.2%ac, respectively [SAS; SAS Institute, Cary, NC, USA) Proc GLM (a,b,cP < 0.05)]. Total cell number was determined using bisbenzimide to stain the nuclei, and the data were analysed by SAS Proc GLM. There was no difference in cell number among treatments with a mean cell number of 31.4. To further investigate the equality of the chemically defined media, the surface area of the blastocysts was measured by using Nis Elements BR3.0 software under 20× magnification. There was less surface area in treatment 5 compared with 4 [296 180ab, 295 114ab, 303 451ab, 271 913b, and 316 773a arbitrary units (a,bP < 0.05), with n = 33, 26, 37, 31, and 32 embryos in each treatment, respectively]. Because HC has been shown to affect global DNA methylation of bovine embryos, we stained our embryos for 5-methylcytidine (Eurogentec anti 5-MECY-0100) and embryos were visualised by UV light with a Texas red filter, and intensity was measured by Nis Elements BR 3.0 software under 20X magnification. The mean intensities were lower for the NMDA treatment [26.7a, 19.4a, 13.6b, 17.0a, and 19.4a arbitrary units (a,bP < 0.05)] compared with the other treatments. When embryos were cultured without BSA development decreases, but adding NMDA and HC returns development to control levels as measured by percentage of blastocysts, surface area, and global DNA methylation. We conclude that PZM4 supplemented with 0.5 mM NMDA and 10 μM HC may replace PZM3 as a chemically defined culture media for early porcine embryos. Embryo transfer experiments will be necessary to confirm that these embryos have equal developmental competence. Funded by the NRI (2006-35203-17282) and Food for the 21st Century.

Effects of serum or fatty acid supplementation of synthetic oviduct fluid medium on development of bovine embryosin vitro

1999 ◽  
Vol 1999 ◽  
pp. 62-62
Author(s):  
N.C. Farrar ◽  
M.E. Staines ◽  
G.J. McCallum ◽  
P. Haggarty ◽  
J.J. Robinson ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Culture Medium ◽  
Culture Media ◽  
Bovine Embryo ◽  
Fluid Medium ◽  
Alternative Approaches ◽  
Defined Culture ◽  
Oviduct Fluid ◽  
Transmission Of Disease

Serum, which is routinely included in many embryo culture media, can decrease the viability of bovine and ovine embryos produced in cultures employing synthetic oviduct fluid (SOF; Kuran et al., 1999) and represents a possible route for transmission of disease. Alternative approaches include the use of chemically defined culture media but results from studies which avoid sera and its derivatives (e.g., albumin) are generally less favourable due to a lack of knowledge regarding the embryo's response to specific nutrients, most notably fatty acids. As a preliminary step towards investigating fatty acid influences on bovine embryo developmentin vitro, the present study examined the effect of adding palmitic acid (C16:0) to SOF plus bovine serum albumin (BSA) on the performance of this semi-defined culture medium and contrasted it with embryo production in SOF supplemented with serum.

Development of in vitro matured/in vitro fertilized bovine embryos into morulae and blastocysts in defined culture media

1992 ◽  
Vol 37 (1) ◽  
pp. 127-146 ◽  
Author(s):  
Barry D. Bavister ◽  
Teresa A. Rose-Hellekant ◽  
Tanu Pinyopummintr
Keyword(s):  
Culture Media ◽  
Bovine Embryos ◽  
Defined Culture

Life history and iodine nutrition of the marine brown alga, Petalonia fascia (O. F. Müll.) Kuntze

1969 ◽  
Vol 47 (10) ◽  
pp. 1611-1616 ◽  
Author(s):  
Stephen I. C. Hsiao
Keyword(s):  
Life History ◽  
Brown Alga ◽  
Culture Media ◽  
Essential Element ◽  
Iodine Nutrition ◽  
History Of ◽  
Defined Culture ◽  
Free Media ◽  
Marine Brown Alga ◽  
Petalonia Fascia

The morphological life history of the marine brown alga, Petalonia fascia (O. F. Müll.) Kuntze, has been investigated in unialgal culture, using defined culture media with different iodide concentrations. The zoospores produced from the plurilocular sporangia of P. fascia blades developed directly into protonemata, plethysmothalli, or Ralfsia-like thalli, depending upon the iodide concentration. Protonemata and plethysmothalli survived in iodide-free media. For development of Ralfsia-like thalli and blades the minimal iodide concentrations required were 50.76 × 102 μg/1 and 50.76 × 101 μg/1, respectively. Iodine appears to be an essential element for growth, morphogenesis, and reproduction of P. fascia.

Spore germination and the life cycle of Frankia in vitro

1989 ◽  
Vol 35 (8) ◽  
pp. 801-806 ◽  
Author(s):  
S. S. Tzean ◽  
John G. Torrey
Keyword(s):  
Life Cycle ◽  
Spore Germination ◽  
Culture Media ◽  
Spore Release ◽  
Optimum Ph ◽  
Defined Culture ◽  
Physical And Chemical ◽  
Strain Cci3 ◽  
Better Than

Bacterial spores of Frankia produced in defined culture media were collected by filtration after washing in amounts approximating 106 spores/mL. Frankia strains UFGCeI5 from Casuarina equisetifolia and UFGCgI1 from C. glauca showed spontaneous release of spores in culture; strains HFPCcI3 from C. cunninghamiana and HFPAllI1 from Allocasuarina lehmanniana showed low spore release in culture unless homogenized. Spore germination was tested on plates of agar nutrient media under different physical and chemical environments. Strain CeI5 showed about 15% germination within 2 days in a defined (BAP) medium with an optimum pH of 6.0–6.8 at 28–35 °C. Under these conditions, strain CcI3 germinated less than 0.5%. In a series of trials with increasingly complex media, strain CeI5 showed 75% spore germination in 3 days at 28 °C and pH 6.7 in the most complex medium tested. Additions of specific single organic compounds to BAP medium caused either strong inhibition or slight stimulation of spore germination. Frankia strains that showed spontaneous spore release germinated better than strains that did not release. Spore germination in Frankia strains is markedly influenced by their strain origin and by the physical and chemical environment in which they are placed.Key words: Casuarina, Frankia, life cycle, spore germination.

scholarly journals Sensitivity of bovine blastocyst gene expression patterns to culture environments assessed by differential display RT-PCR

Reproduction ◽  
2001 ◽  
pp. 687-693 ◽  
Author(s):  
DR Natale ◽  
PA De Sousa ◽  
ME Westhusin ◽  
AJ Watson
Keyword(s):  
Gene Expression ◽  
Differential Display ◽  
Culture Media ◽  
Expression Patterns ◽  
Molecular Techniques ◽  
Preimplantation Embryos ◽  
Gene Expression Patterns ◽  
Bovine Embryos ◽  
Rt Pcr ◽  
Defined Culture

The use of culture media to support the development of preimplantation embryos to the blastocyst stage is often associated with detrimental effects on normal development. These effects have been uncovered largely by investigating the phenotypic abnormalities displayed by fetuses and newborns derived from cultured preimplantation embryos. Research to understand the impact of culture on the embryonic developmental programme has focused on embryo metabolism, gene expression and genomic imprinting. We have used differential display RT-PCR to examine culture influences on global transcript pools in bovine embryos. Others have examined culture influences on candidate "marker genes" in cultured murine, ovine and bovine embryos. These studies have demonstrated that culture conditions influence the amount of marker gene transcripts and downregulate or induce the expression of novel genes during early development. Optimized defined culture media maintain embryonic gene expression patterns closely resembling those displayed by embryos derived in vivo. Preimplantation mammalian embryos display an impressive capacity to respond to the pressures that suboptimal culture environments place upon them. However, this plasticity operates within a defined range of tolerances. Continued research using molecular techniques will lead to increased understanding of developmental mechanisms causing culture-related phenotypic abnormalities in post-implantation embryos.

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