scholarly journals Transcriptional Profiling of Porcine Blastocysts Produced In Vitro in a Chemically Defined Culture Medium

Animals ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 1414
Author(s):  
Josep M. Cambra ◽  
Emilio A. Martinez ◽  
Heriberto Rodriguez-Martinez ◽  
Maria A. Gil ◽  
Cristina Cuello
Keyword(s):  
Culture Medium ◽  
Embryo Quality ◽  
Transcriptional Profiling ◽  
Defined Medium ◽  
Platelet Factor ◽  
Defined Media ◽  
Defined Culture ◽  
The Impact

The development of chemically defined media is a growing trend in in vitro embryo production (IVP). Recently, traditional undefined culture medium with bovine serum albumin (BSA) has been successfully replaced by a chemically defined medium using substances with embryotrophic properties such as platelet factor 4 (PF4). Although the use of this medium sustains IVP, the impact of defined media on the embryonic transcriptome has not been fully elucidated. This study analyzed the transcriptome of porcine IVP blastocysts, cultured in defined (PF4 group) and undefined media (BSA group) by microarrays. In vivo-derived blastocysts (IVV group) were used as a standard of maximum embryo quality. The results showed no differentially expressed genes (DEG) between the PF4 and BSA groups. However, a total of 2780 and 2577 DEGs were detected when comparing the PF4 or the BSA group with the IVV group, respectively. Most of these genes were common in both in vitro groups (2132) and present in some enriched pathways, such as cell cycle, lysosome and/or metabolic pathways. These results show that IVP conditions strongly affect embryo transcriptome and that the defined culture medium with PF4 is a guaranteed replacement for traditional culture with BSA.

Partially desulfated heparin modulates the interaction between anti-protamine/heparin antibodies and platelets

2016 ◽  
Vol 115 (02) ◽  
pp. 324-332 ◽  
Author(s):  
Rabie Jouni ◽  
Heike Zöllner ◽  
Ahmad Khadour ◽  
Jan Wesche ◽  
Anne Grotevendt ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Platelet Factor ◽  
Standard Drug ◽  
Platelet Surface ◽  
Minimal Impact ◽  
Platelet Survival ◽  
Heparin Complexes ◽  
The Impact

SummaryProtamine (PRT) is the standard drug to neutralise heparin. PRT/heparin complexes induce an immune response similar to that observed in heparin-induced thrombocytopenia (HIT). Partially desulfated heparin (ODSH) was shown to interfere with anti-platelet factor 4/heparin antibodies (Abs), which are responsible for HIT. In this study, we analyse the impact of ODSH on the interaction between anti-PRT/heparin Abs and platelets. The ability of ODSH to prevent anti-PRT/heparin Ab-induced platelet destruction in vivo was investigated using the NOD/ SCID mouse model. ODSH improved platelet survival in the presence of PRT, heparin and anti-PRT/heparin Abs (median platelet survival after 300 minutes (min) with 20 μg/ml ODSH: 75 %, range 70–81 % vs without ODSH: 49%, range 44–59%, p=0.006). Furthermore, when ODSH was applied 60 min after Ab injection platelet survival was improved (median platelet survival after 300 min with ODSH: 83 %, range 77–93 % vs without ODSH: 59 %, range 29–61 %, p=0.02). In in vitro experiments ODSH inhibited platelet activation at concentrations > 16 μg/mL (p< 0.001), as well as PRT/heparin complex binding to platelets (mean fluorescence intensity [MFI] without ODSH: 85 ± 14 vs with ODSH: 15 ± 0.6, p=0.013). ODSH also displaced pre-bound complexes from the platelet surface (MFI without ODSH: 324 ± 43 vs with 32 μg/ml ODSH: 53 ± 9, p< 0.001). While interfering with platelet activation by anti-PRT/heparin Abs, up to a concentration of 16 μg/ml, ODSH had only minimal impact on neutralisation of heparin by PRT. In conclusion, our study shows that ODSH is able to inhibit platelet activation and destruction suggesting a potential clinical use to reduce anti-PRT/heparin Ab-mediated adverse effects.

97 SUPPLEMENTATION WITH FOLATE IN VITRO INCREASES TROPHECTODERM AND TOTAL CELL NUMBER IN IN VITRO-DERIVED PORCINE BLASTOCYSTS

2012 ◽  
Vol 24 (1) ◽  
pp. 161 ◽  
Author(s):  
B. K. Redel ◽  
L. D. Spate ◽  
A. N. Brown ◽  
R. S. Prather
Keyword(s):  
Embryo Culture ◽  
Culture Medium ◽  
Transcriptional Profiling ◽  
Cell Number ◽  
Total Cell ◽  
Differential Staining ◽  
Total Cell Number ◽  
Culture Systems

It is vital that improvements are made to current culture environments because in vitro culture systems are suboptimal compared with in vivo. A previous transcriptional profiling endeavour conducted by Bauer et al. (2010 Biol. Reprod. 83, 791–798) identified hundreds of mRNA transcripts that were mis-expressed in porcine embryos fertilized in vivo and then cultured in vitro to Day 6 compared with in vivo Day-6 embryos. Enriched in the downregulated transcripts were 4 genes involved with the one carbon pool by folate KEGG pathway. This downregulation of genes involved with folate metabolism may illustrate an impaired folate homeostasis in embryos cultured in the current culture environment. The objective of this study was to determine the effects folate had on embryo development of in vitro fertilized embryos. Porcine cumulus–oocyte complexes were matured for 44 h in M199 supplemented with epidermal growth factor (EGF), FSH and LH. Oocytes with a visible polar body were selected and fertilized in modified tris buffered medium for 5 h and then placed into porcine zygote medium 3 with 0 mM, 0.2 mM, 0.4 mM and 0.8 mM folate to find the optimal concentration of folate. Twenty-eight hours post-fertilization, cleaved embryos were selected and moved into 25-μL drops of respective culture medium and cultured to Day 6 in a water-saturated atmosphere of 5% CO2, 5% O2, 90% N2, at 38.5°C. To determine the effect folate had on development, the blastocyst rate for each treatment group was measured. Results were log-transformed and analysed by using PROC GLM in SAS (SAS Institute Inc., Cary, NC). A least-significant difference post-test comparison was completed to determine if significant differences existed between treatment groups. The percentage of cleaved embryos on Day 6 that developed to blastocyst was 56.2%, 55.9%, 66.9% and 61.8% (n = 133, 149, 135 and 135) in 0 mM, 0.2 mM folate, 0.4 mM folate and 0.8 mM, respectively. The 0.4 mM folate group tended (P = 0.07) to have a higher number of cleaved embryos that developed to the blastocyst stage. Consequently, this concentration was used for all further embryo culture experiments. Differential staining was completed to compare the number of trophectoderm and inner cell mass nuclei for embryos cultured in 0 mM or 0.4 mM folate concentrations. Staining revealed that embryos cultured with folate had an increase in number of trophectoderm (29.7 ± 1.5 vs 24.4 ± 1.4 cells; P = 0.0058) and total cell (36.9 ± 1.0 vs 31.7 ± 1.0; P = 0.0007) numbers compared with embryos cultured without folate. These results illustrate that the addition of folate to current culture medium doesn't hinder development to blastocyst and by increasing trophectoderm and total cell number may give rise to better-quality in vitro-derived embryos. It is evident that using transcriptional profiling can be a great method of identifying ways to improve embryo culture systems and, in this case, supplementing with folate. Funded by Food for the 21st Century.

Preliminary Experiments with a Defined Culture Medium for Larval Fasciola hepatica

1973 ◽  
Vol 47 (2) ◽  
pp. 181-189 ◽  
Author(s):  
R. S. V. Pullin
Keyword(s):  
Amino Acids ◽  
Culture Medium ◽  
Fasciola Hepatica ◽  
Dry Weight ◽  
Defined Medium ◽  
Inorganic Salts ◽  
Final Maturation ◽  
Defined Culture ◽  
Stock Solutions

A defined medium is described as a basis for in vitro culture work with larval Fasciola hepatica. This medium, termed BCM, can be quickly made up by using a system of stock solutions. BCM contains inorganic salts, glucose, amino acids, vitamins and antibiotics, but no lipid or proteins. Rediae can be dissected from infected snails for culture, but many appear to be contaminated with bacteria. Large rediae cannot survive in BCM but free immature cercariae can complete their final maturation in vitro. This final maturation, from the 30th to the 35th day after miracidial penetration of donor snails, includes tail growth and appearance of body pigmentation. Cercariae matured in vitro encyst successfully when transferred from BCM to water. Small rediae survive in BCM for 5 days, but show no growth or development measured as dry weight and total nitrogen.

Glucose and glycine synergistically enhance the in vitro development of porcine blastocysts in a chemically defined medium

2012 ◽  
Vol 24 (3) ◽  
pp. 443 ◽  
Author(s):  
Tomomi Mito ◽  
Koji Yoshioka ◽  
Shoko Yamashita ◽  
Chie Suzuki ◽  
Michiko Noguchi ◽  
...  
Keyword(s):  
Culture Medium ◽  
Survival Rates ◽  
Defined Medium ◽  
Blastocyst Stage ◽  
Atp Content ◽  
Blastocyst Development ◽  
In Vitro Development ◽  
Defined Culture ◽  
Vitro Development

In the present study, the effects of glucose and/or glycine on the in vitro development of Day 5 (Day 0 = IVF) porcine blastocysts were determined. The addition of 2.5–10 mM glucose to the chemically defined culture medium porcine zygote medium (PZM)-5 significantly increased blastocyst survival rates compared with those of blastocysts cultured in the absence of glucose. The addition of 5 and 10 mM glycine to PZM-5 containing 5 mM glucose significantly enhanced the development to hatching and the number of hatched blastocysts compared with no addition of glycine. However, the addition of glycine to PZM-5 with no glucose did not improve blastocyst development. The ATP content of Day 6 blastocysts cultured with glucose was significantly higher than that of blastocysts cultured in the absence of glucose, regardless of glycine supplementation. The diameter and total cell numbers were significantly greater, and the apoptotic index was significantly lower, in Day 6 blastocysts cultured with both glucose and glycine. These results indicate that glucose is an important energy source for the porcine blastocyst and that glucose and glycine act synergistically to enhance development to the hatching and hatched blastocyst stage in vitro.

scholarly journals The Two-Component System 09 of Streptococcus pneumoniae Is Important for Metabolic Fitness and Resistance during Dissemination in the Host

2021 ◽  
Vol 9 (7) ◽  
pp. 1365
Author(s):  
Stephanie Hirschmann ◽  
Alejandro Gómez-Mejia ◽  
Thomas P. Kohler ◽  
Franziska Voß ◽  
Manfred Rohde ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Regulatory System ◽  
Immunoblot Analysis ◽  
Defined Medium ◽  
Host Cells ◽  
Two Component ◽  
Regulatory Effects ◽  
The Impact

The two-component regulatory system 09 of Streptococcus pneumoniae has been shown to modulate resistance against oxidative stress as well as capsule expression. These data and the implication of TCS09 in cell wall integrity have been shown for serotype 2 strain D39. Other data have suggested strain-specific regulatory effects of TCS09. Contradictory data are known on the impact of TCS09 on virulence, but all have been explored using only the rr09-mutant. In this study, we have therefore deleted one or both components of the TCS09 (SP_0661 and SP_0662) in serotype 4 S. pneumoniae TIGR4. In vitro growth assays in chemically defined medium (CDM) using sucrose or lactose as a carbon source indicated a delayed growth of nonencapsulated tcs09-mutants, while encapsulated wild-type TIGR4 and tcs09-mutants have reduced growth in CDM with glucose. Using a set of antigen-specific antibodies, immunoblot analysis showed that only the pilus 1 backbone protein RrgB is significantly reduced in TIGR4ΔcpsΔhk09. Electron microscopy, adherence and phagocytosis assays showed no impact of TCS09 on the TIGR4 cell morphology and interaction with host cells. In contrast, in vivo infections and in particular competitive co-infection experiments demonstrated that TCS09 enhances robustness during dissemination in the host by maintaining bacterial fitness.

scholarly journals Propagation of edible honeysuckle (Lonicera edulis Turcz) in in vitro conditions

2018 ◽  
Vol 5 (2) ◽  
pp. 18-26
Author(s):  
Ya. Zapolsky ◽  
T. Medvedeva ◽  
T. Natalchuk ◽  
M. Bublyk
Keyword(s):  
Culture Medium ◽  
Positive Impact ◽  
Mercury Chloride ◽  
Free Medium ◽  
Aseptic Culture ◽  
Effi Ciency ◽  
Comparison Results ◽  
The Impact

Aim. To propagate edible honeysuckle (Lonicera edulis Turcz) in in vitro conditions; to study the impact of sterilization agents on honeysuckle explants; to investigate the impact of the culture medium composition on the coeffi cient of propagation and rooting; to study the capability to adapt to in vivo conditions. Methods. Laboratory, mathematical, estimation and comparison. Results. The impact of sterilizing substances on obtain- ing the aseptic culture of edible honeysuckle in in vivo conditions was studied. The experiments were con- ducted on the following species: Alicia, Spokusa, Chaika, Nimfa, Doch Velikana, Karina. Lisoformin 3000 and mercury chloride were used as sterilizing agents. In the variant with Lisoformin 3000 it was studied in three exposures – 5, 7, and 10 minutes. In terms of explant regeneration effi ciency after sterilization with Lisofor- min 3000, three groups of edible honeysuckle species were isolated: 1 – with high regeneration capacity (94– 96 %) – Alicia, Karina and Spokusa; 2 – medium capacity (86–87 %) – Chaika and Doch Velikana, 3 – low capacity (80 %) – Nimfa. The experiments aimed at studying the impact of culture medium components on the propagation effi ciency determined the increase in the latter in case of rotating media with different quantitative and qualitative composition. Permanent application of uniform media leads to a sharp decrease in the prolif- eration coeffi cient in all the investigated species. Both hormone-free medium and the medium with growth regulators are effi cient for rooting. High indices of rooting were achieved in both variants. The use of auxins promoted the formation of a larger amount of plant roots (from 3.09 in Spokusa to 4.21 in Alicia) which in its turn impacted the survivability of plants in in vivo conditions. Conclusions. It was established that Lisoformin 3000 in the concentration of 3 % and at the exposure duration of 5 min ensured optimal effi ciency of steriliza- tion and regeneration of edible honeysuckle explants and did not decrease their propagation coeffi cients. With corresponding concentrations and sterilization duration, this preparation may be recommended for obtaining the aseptic culture of honeysuckle. It was demonstrated that the rotation of media, rich and poor in macro- and microsalts was effi cient for obtaining high indices of proliferation: the plants had a larger amount of tillering even in case of using not high concentrations of cytokinin. The introduction of rhizogenesis inducer, IBA, (1 mg/l) into the culture medium did not increase the percentage of rooted plants compared to hormone-free medium, but stimulated the formation of a larger amount of roots, which had further positive impact on the adaptation properties.

scholarly journals The change of behavior of two strains of Leishmania after cultivation in a defined medium

1987 ◽  
Vol 82 (4) ◽  
pp. 557-561 ◽  
Author(s):  
M. N. Melo ◽  
Paul Williams ◽  
N. M. Magalhães Rocha ◽  
E. H. Babá ◽  
W. Mayrink ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Electrophoretic Mobility ◽  
Growth Rates ◽  
Culture Medium ◽  
Defined Medium ◽  
Mobility Patterns ◽  
Golden Hamsters ◽  
Defined Culture

Attempts have been made to characterize two strains of Leishmania that became infective to golden hamsters only after they had been maintained for several years in a chemically defined culture medium. Observations were made on the growth rates of promastigotes in vitro, course of infection in hamsters, morphology of amastigotes, and electrophoretic mobility patterns of eight isoenzymes. Information was obtained about the buoyant densities of n-DNA and k-DNA, and one strain was tested against monoclonal antibodies. The identity of both strains remains obscure.

scholarly journals 123VITRIFICATION OF IN VITRO-PRODUCED BOVINE BLASTOCYSTS BY ADDITION OF CRYOPROTECTANT IN ONE STEP

2004 ◽  
Vol 16 (2) ◽  
pp. 184
Author(s):  
D.J. Walker ◽  
L.F. Campos-Chillon ◽  
G.E. Seidel
Keyword(s):  
Ethylene Glycol ◽  
Embryo Quality ◽  
Survival Rates ◽  
Defined Medium ◽  
Embryo Cryopreservation ◽  
Equilibration Time ◽  
One Step ◽  
Hatching Rates

Vitrification combined with in-straw dilution may replace conventional cryopreservation of bovine embryos, but this requires further study for practicality. Our objectives were to compare three ethylene glycol concentrations (6, 7, and 8M) and two equilibration times (2.5 and 3.5min) for one-step addition of cryoprotectant. In vitro-matured oocytes from slaughterhouse ovaries, fertilized using sperm of 3 bulls, were cultured in chemically defined medium (CDM-1/CDM-2) plus FAF-BSA to produce 420 blastocysts. Day 7.5 embryos were placed into HCDM-2 (HEPES-buffered medium) and then transferred to a 6μL drop of vitrification solution (V) (6, 7, or 8M ethylene glycol, 0.5M galactose, and 18% w/v Ficoll 70 in HCDM-2). Immediately thereafter, 1cm column of DHCDM (0.5M galactose in HCDM-2) was drawn into a 0.25mL straw, followed by a 0.5cm column of air and another 7cm of DHCDM. Another 0.5cm column of air was aspirated before the 6μL of V (0.5cm) containing the embryos were aspirated; then 0.5cm of air followed. Finally, DHCDM was drawn until the first column came into contact with the cotton plug. Straws were then heat-sealed and plunged into liquid nitrogen slightly above the embryos after 2.5 or 3.5min equilibration. The rest of the straw was then submerged slowly. Straws were thawed in air for 10s and then in 37°C water for 20s. Straws were held at room temperature (24°C) for 4min before being expelled into HCDM-2. They were then placed into CDM-2+5% FCS for culture. Quality score (1=excellent, 2=fair, 3=poor), survival (S) as determined by expansion of blastocysts, and hatching (H) were assessed at 24 and 48h post-thaw. Data from 6 replicates (2/bull) were analyzed by ANOVA after arc sin transformation of percentage data. S and H responses were calculated as a percentage of non-frozen controls in the same replicate. Control survival and hatching rates were: 24S: 90%, 24H: 50%, 48S: 90%, 48H: 72%. Quality scores at both 24 and 48h were higher (P&lt;0.05) for 8M than 6M ethylene glycol (2.68 and 3.24 for 24h; 2.55 and 3.17 for 48h); values for 7M ethylene glycol were intermediate. Equilibration time had no effect on embryo quality (P&gt;0.1). Neither ethylene glycol concentration nor exposure time affected survival or hatching at 24 or 48h (P&gt;0.1). Survival rates (as a % of control) at 48h were: 8M: 57%, 7M: 55%, 6M: 36% and hatching: 8M: 39%, 7M: 30%, and 6M: 21%; 2.5min tended to be better than 3.5min for survival at 24h, hatching at 24h, survival at 48h, but not hatching at 48h (56% and 43%, 30% and 26%, 55% and 44%, 28% and 32% respectively). Higher concentrations of ethylene glycol proved beneficial in terms of embryo quality, with the same trend for survival and hatching rates. One-step addition of cryoprotectant for vitrification shows potential for simplifying embryo cryopreservation. However, further research is needed to produce more acceptable survival rates and to study vitrification of in vivo-produced embryos.

Half-Life of Platelet Factor 4 (PF-4) in Plasma and Platelets from Macaca Mulatta

1977 ◽  
Vol 37 (01) ◽  
pp. 073-080 ◽  
Author(s):  
Knut Gjesdal ◽  
Duncan S. Pepper
Keyword(s):  
Macaca Mulatta ◽  
Half Life ◽  
Human Platelet ◽  
Gel Filtration ◽  
Platelet Factor 4 ◽  
Active Protein ◽  
Platelet Factor ◽  
Membrane Bound

SummaryHuman platelet factor 4 (PF-4) showed a reaction of complete identity with PF-4 from Macaca mulatta when tested against rabbit anti-human-PF-4. Such immunoglobulin was used for quantitative precipitation of in vivo labelled PF-4 in monkey serum. The results suggest that the active protein had an intra-platelet half-life of about 21 hours. In vitro 125I-labelled human PF-4 was injected intravenously into two monkeys and isolated by immuno-precipita-tion from platelet-poor plasma and from platelets disrupted after gel-filtration. Plasma PF-4 was found to have a half-life of 7 to 11 hours. Some of the labelled PF-4 was associated with platelets and this fraction had a rapid initial disappearance rate and a subsequent half-life close to that of plasma PF-4. The results are compatible with the hypothesis that granular PF-4 belongs to a separate compartment, whereas membrane-bound PF-4 and plasma PF-4 may interchange.

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