161 LIPID CONTENT, RESISTANCE TO CRYOPRESERVATION, AND SEX RATIO OF IN VITRO BOVINE BLASTOCYSTS PRODUCED IN A SERUM-FREE SYSTEM

2006 ◽  
Vol 18 (2) ◽  
pp. 188
Author(s):  
F. George ◽  
C. Daniaux ◽  
G. Genicot ◽  
F. Focant ◽  
B. Verhaeghe ◽  
...  
Keyword(s):  
Sex Ratio ◽  
Lipid Content ◽  
Culture Medium ◽  
Nile Red ◽  
Embryo Cryopreservation ◽  
Hatching Rate ◽  
Free System ◽  
Serum Free

In vitro-produced (IVP) bovine blastocysts are known to be more sensitive to cryopreservation than their in vivo counterparts. Removing serum from the culture medium decreases sanitary risk and could improve embryo resistance to cryopreservation by preventing the accumulation of intracellular lipids. Our objectives were to evaluate the lipid content, resistance to cryopreservation, and sex ratio of IVP embryos cultured in a serum-free system. Oocytes from slaughterhouse ovaries were matured in a serum-free enriched medium (Donnay et al. 2004 Reprod. Fertil. Dev. 16, 274) and cultured in 5% O2 in modified SOF supplemented with 5% FCS (FCS) or with insulin-transferrin-selenium (ITS) and 0.1 mg/mL polyvinylpyrrolidone (PVP) (ITS-PVP) or 4 mg/mL BSA (ITS-BSA) (Daniaux et al. 2005 Reprod. Fertil. Dev. 17, 217). Day 5 morulae were stained with the fluorescent dye Nile Red in order to evaluate their lipid content (Genicot et al. 2005 Theriogenology 63, 1181). Day 7 blastocysts (diameter ≥160 µm) were selected, classified according to their size, and frozen in HEPES-SOF containing 1.5 M ethylene glycol, 0.1 M sucrose, and 1.8 mg/mL wheat peptones (George et al. 2002 Reproduction 29, 51). The lipid content was significantly lower in morulae cultured in ITS-BSA compared with the two other media (320 ± 10 arbitrary fluorescence units vs. 383 ± 12 in FCS and 406 ± 10 in ITS-PVP; n = 271; ANOVA2: P < 0.01). After cryopreservation, a higher total hatching rate was found 24 h post-thawing in blastocysts cultured in ITS-BSA and for both serum-free conditions at 48 h (Table 1). In particular, embryos ≤180 µm cultured in FCS were less resistant to cryopreservation than embryos of the same size produced without serum. Expanded blastocysts cultured in ITS-BSA were sexed by PCR (Grisart et al. 1995 Theriogenology 43, 1097) and a higher proportion of male embryos was found (62.7%; n = 51). In conclusion, a complete serum-free system was set up from oocyte maturation to embryo cryopreservation that gave high quality embryos resistant to cryo-preservation. Embryos produced in ITS-BSA presented a lower lipid content, but a shift of the expanded blastocyst sex ratio toward males was observed. Table 1. Hatching rates post-thawing as a function of the blastocyst size and the culture medium

scholarly journals 133 INSULIN, TRANSFERRIN AND SELENIUM WITH OR WITHOUT BSA IN A SERUM-FREE CULTURE SYSTEM FOR BOVINE EMBRYO, AND ITS SUITABILITY FOR EMBRYOS CULTURED IN SMALL GROUPS

2005 ◽  
Vol 17 (2) ◽  
pp. 217 ◽  
Author(s):  
C. Daniaux ◽  
B. Verhaeghe ◽  
I. Donnay
Keyword(s):  
Small Groups ◽  
Culture Medium ◽  
Culture Media ◽  
Quality Parameters ◽  
Hatching Rate ◽  
Bovine Embryo ◽  
Serum Free ◽  
Abnormal Accumulation ◽  
Development In Vitro

Serum in embryo culture medium may be a potential cause of abnormal accumulation of lipid droplets, which is correlated to a higher sensitivity to cryopreservation. Moreover, serum may introduce pathogens. With the aim of developing a serum-free culture medium, we first (Experiment 1) investigated the effect of adding ITS (5 μg/mL insulin, 5 μg/mL transferrin, 5 ng/mL selenium) as a serum substitute in SOF medium on embryos cultured in large groups (20 embryos per culture drop of 20 μL) and we then (Experiment 2) analyzed the effect of adding BSA. In this second experiment, our serum-free culture media were also tested on embryos cultured in small numbers (5 embryos per drop of 20 μL) in order to mimic ovum pickup (OPU) conditions. Embryos were obtained from slaughterhouse oocytes, matured in vitro for 24 h in a serum-free enriched 199 medium (Donnay et al. 2004 Reprod. Fertil. Dev. 16, 274) containing ITS, and fertilized for 18 h. In experiment 1, embryos were cultured in SOF (Holm et al. 1999 Theriogenology 52, 683–700) supplemented with 0.1 mg/mL polyvinylpyrrolidane (PVP) without (SOF) or with ITS (SOF-ITS), or with 5% FCS (SOF-FCS). Cavitation occurred earlier in presence of serum (Table). Adding ITS to SOF increased blastocyst rates at Day 7 and Day 8 post-insemination (p.i.) and also the hatching rate. In experiment 2, embryos were cultured in SOF-FCS, SOF-ITS, or SOF-ITS supplemented with 4 mg/mL fatty acid free BSA (SOF-ITS-BSA). Within each condition, no differences were observed for blastocyst and hatching rates between embryos cultured in large or in small groups. Adding BSA to SOF-ITS increased blastocyst rate at Day 6 p.i. and also the hatching rate. At Days 7 and 8 p.i., blastocyst rates were higher in SOF-FCS than in SOF-ITS and tended to be higher than in SOF-ITS-BSA, especially for embryos cultured in small groups. Cell numbers of the resulting embryos were unaffected. These results indicate that: (1) ITS as supplement to SOF medium promotes embryo development in vitro. (2) BSA as protein supplement to SOF-ITS medium accelerates blastulation and improves hatching rate. (3) SOF-ITS and SOF-ITS-BSA are two serum-free culture media that can sustain development of embryos, also when cultured in small number, even though SOF-FCS tended to afford better rates of development. Further studies will include evaluation of other quality parameters including resistance to cryopreservation. This work was supported by the Ministery of Agriculture of the Region wallonne de Belgique.

scholarly journals Genetic influence on the reduction in bovine embryo lipid content by L-carnitine

2016 ◽  
Vol 28 (8) ◽  
pp. 1172 ◽  
Author(s):  
Luis Baldoceda ◽  
Dominic Gagné ◽  
Christina Ramires Ferreira ◽  
Claude Robert
Keyword(s):  
Lipid Content ◽  
Culture Medium ◽  
Cell Damage ◽  
Mitochondrial Activity ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Intracellular Lipid ◽  
Embryo Culture Medium

The decreased rate of pregnancy obtained in cattle using frozen in vitro embryos compared with in vivo embryos has been associated with over-accumulation of intracellular lipid, which causes cell damage during cryopreservation. It is believed that the higher lipid content of blastomeres of bovine embryos produced in vitro results in darker-coloured cytoplasm, which could be a consequence of impaired mitochondrial function. In this study, l-carnitine was used as a treatment to reduce embryonic lipid content by increasing metabolism in cultured bovine embryos. We have observed previously that in vivo embryos of different dairy breeds collected from cows housed and fed under the same conditions differed in lipid content and metabolism. As such, breed effects between Holstein and Jersey were also examined in terms of general appearance, lipid composition, mitochondrial activity and gene expression. Adding l-carnitine to the embryo culture medium reduced the lipid content in both breeds due to increased mitochondrial activity. The response to l-carnitine was weaker in Jersey than in Holstein embryos. Our results thus show that genetics influence the response of bovine embryos to stimulation of mitochondrial metabolism.

190 EFFICIENT GENERATION OF INDUCED PLURIPOTENT STEM CELLS FROM PORCINE ADIPOSE-DERIVED STEM CELLS WITH A FEEDER-INDEPENDENT AND SERUM-FREE SYSTEM

2014 ◽  
Vol 26 (1) ◽  
pp. 209
Author(s):  
Y. Zhang ◽  
C. Wei ◽  
P.-F. Zhang ◽  
X. Li ◽  
Y.-S. Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Adipose Derived Stem Cells ◽  
Free System ◽  
Serum Free ◽  
Induced Pluripotent ◽  
Serum Free Conditions

Somatic cells could be directly reprogrammed into stem state by ectopic expression of transcription factors, which share similar features of embryonic stem cells (ESC). Induced pluripotent stem cells (iPSC) possess promising application in producing genetically modified animals, whereas the generation of porcine offspring from iPSC is still difficult and controversial, and new materials are needed. In this study, we report the generation of iPSC from porcine adipose-derived stem cells (pADSC) using drug-inducible expression of defined human factors (Oct4, Sox2, Klf4, and c-Myc) and ‘2i’ plus leukemia inhibitory factor (LIF) culture system. pADSC were isolated from subcutaneous adipose tissue of a 28-day-old Danish Landrace, and subsequently characterised by high proliferation rate at low passages, long period passaging without significant replication senescence, mesenchymal stem cell-specific surface markers expression, including CD29 (0.995 ± 0.0577), CD44 (0.999 ± 0.0333), and CD90 (0.994 ± 0.0333), together with successful adipogenic and osteogenic differentiation ability in vitro. The reprogramming of iPSC from pADSC was evidently more efficient than the process from adult fibroblasts (P < 0.01), both of which were carried out under feeder-independent and serum-free conditions, and this may be due to the higher demethylation level of genomic DNA in pADSC. Two lines of porcine iPSC with naïve-like state were finally obtained through feeder-independent and serum-free conditions. The successful reprogramming of iPSC was demonstrated by short cell cycle interval, alkaline phosphatase (AP) staining positive, expression of stemness-related proteins including OCT-4, SOX2, NANOG, SSEA3, and SSEA4. Full reprogramming of iPSC was evaluated by the significant up-regulation of LIN28, ESRRB, UTF1, and DPPA5. Naïve-like state of porcine iPSC was further confirmed by the striking resemblance to naïve mESC, single-cell dissociation, LIF-dependency, up-regulation of STELLA and ERAS, and little translation of TRA-1-60 and TRA-1-81. In addition, porcine naïve-like iPSC possessed normal karyotypes, and could differentiate into cell types of all three germ layers in vitro and in vivo. Furthermore, in vivo studies to determine the capacity of these cells to integrate into the inner cell mass of blastocysts are still being undertaken for validation. Together, our study provided an efficient method to derive porcine naïve-like iPSC from pADSC, which may be useful for the production of living offspring. Y. Zhang and C. Wei contributed equally to this work. Y.-H. Zhang is the corresponding author. This work was supported by the National Natural Science Foundation Program 31272442.

113 EVALUATION OF DIFFERENT CRYOPROTECTANT AND FORSKOLIN IN THE CULTURE MEDIUM FOR IMPROVING THE EFFICACY OF VITRIFICATION OF BOS INDICUS IN VITRO-DERIVED EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 215 ◽  
Author(s):  
B. V. Sanches ◽  
B. D. O. Filho ◽  
J. H. F. Pontes ◽  
A. C. Basso ◽  
M. L. G. Meirinhos ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Lipid Droplets ◽  
Culture Medium ◽  
Bos Taurus ◽  
Bos Indicus ◽  
Calf Serum ◽  
Genetic Material ◽  
Embryo Cryopreservation ◽  
Hatching Rate

Embryo cryopreservation is an essential method for the biotechnology of reproduction. This is the safest option for interchange of genetic material for research and commercial purposes. For cattle, Brazil has become the leading country in the world for the number of in vitro-produced embryos, using mostly Bos indicus animals. However, considering the in vitro method of embryo production, field results have shown a lower resistance to cryopreservation for B. indicus when compared with Bos taurus embryos. A possible explanation for this is a great concentration of lipid droplets in the cytoplasm of cells fromB. indicus embryos. The objective of this study was to compare 2 cryoprotectants (Propanediol and DMSO) to vitrification and evaluate the effect of adding 10 μM forskolin to the SOF medium for embryo culture before cryopreservation. For all the experiments, ovaries from slaughtered Nelore Bos indicus donors were recovered and maintained at 30 to 35°C in NaCl solution until recovery of the COC. Embryos submmited to vitrification were expanded blastocysts at Day 7 of in vitro culture. In the first experiment embryos were first incubated in 10% ethylene glycol (EG) plus 10% DMSO dissolved in holding medium (TCM-HEPES with 20% calf serum) for 1 min and then transferred to droplet of 20% EG plus 20% DMSO in holding medium and 0.5 M sucrose for 20 s before immersing in liquid nitrogen (n = 107; group EG + DMSO). For the group EG + Propanediol (EG + PRO; n = 96), blastocysts were placed in 10% EG plus 10% PRO in holding medium for 1 min and then transferred to a droplet of 20% EG plus 20% PRO in holding medium and 0.5 M sucrose for 20 s before immersing in liquid nitrogen. Both treatments were performed using the Cryotop system. Results were compared with embryos (n = 118) not submitted to cryopreservation. The evaluation was done by the hatching rate of blastocysts at Day 9, being higher (86.4%) for embryos not cryopreserved, when comparing with 77.1% for group EG + PRO and 72.9% for group EG + DMSO (P < 0.05). In the second experiment, Day 5 embryos obtained in vitro from Nelore donors were cultured using SOF medium with 10 μM forskolin (n = 112) or not (control; n = 101), being all submitted to cryopreservation using Cryotop and the same vitrification method for group EG + DMSO. Results were compared with embryos cultured with SOF medium and not submitted to cryopreservation (n = 96). The evaluation was performed by considering hatching rate at Day 9, being higher (85.4%) for not cryopreserved, when compared with 63.3% for control and 70.5% for forskolin group (P < 0.05). Considering embryos submitted to cryopreservation, the hatching rate was higher (P < 0.05) for the forskolin group.

scholarly journals 49 EFFECTS OF DIETARY CONJUGATED LINOLEIC ACID SUPPLEMENTATION ON BOVINE OOCYTE LIPID METABOLISM, LIPID COMPOSITION, AND EMBRYO CRYOTOLERANCE

2015 ◽  
Vol 27 (1) ◽  
pp. 117
Author(s):  
C. L. Bailey ◽  
J. A. Sarmiento-Guzmán ◽  
S. E. Farmer ◽  
G. T. Gentry ◽  
J. W. Lynn ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Lipid Content ◽  
Milk Fat ◽  
Culture Medium ◽  
Nile Red ◽  
Dietary Supplementation ◽  
Milk Composition ◽  
Lipid Supplement ◽  
Relative Gene

Reduced tolerance to chilling and cryotolerance of oocytes and embryos has been associated with greater cytoplasmic lipids. Previous studies in the cow have demonstrated nutrition-induced modification of follicular components. trans-10, cis-12 conjugated linoleic acid (CLA) was identified as a potent inhibitor of milk fat synthesis in lactating cows, and inclusion of CLA in bovine embryo culture medium reduced embryo lipid content and improved post-thaw embryo survival. Dietary supplementation of cows with CLA could alter oocyte fatty acid metabolism, oocyte lipid composition, and embryo cryotolerance, and responses may be different between Bos indicus and Bos taurus cattle. Therefore, a series of experiments were conducted to evaluate effects of dietary CLA on bovine oocyte lipid content and lipid metabolism and cryosurvival of in vitro-produced embryos from CLA-supplemented oocyte donor cows. Lactating Holstein cows (n = 39) were supplemented 100 g per head/d CLA or Ca salts of palm oil to determine effects of dietary CLA on milk production and milk composition. Nonlactating Holstein (n = 8) and Brahman (n = 17) cows were individually supplemented with 150 g per head/d CLA or no lipid supplement. Cumulus-oocyte complexes (COC) were collected after 41 ± 1 day of CLA supplementation, and mRNA was isolated for qPCR. Relative gene expression in COC from CLA- and control-fed cows was evaluated for genes involved in lipid metabolism (CPT1, FADS2, and PPARα). Crossbred (Angus × Red Angus × Brangus) cows (n = 28) were randomly allotted to a 2 × 2 factorial experiment and fed 150 g per head/d CLA or no lipid supplement. Oocytes were collected (Day 129 and 143 of CLA supplementation), matured, fertilized, and cultured in vitro for 7 days in serum-free culture medium. Embryos were cryopreserved in individual 0.25-mL plastic straws containing 1.5 M ethylene glycol using a slow-cooled method. Post-thaw survival and hatching were evaluated using a 24-h in vitro culture (mSOF with 5% FBS) assay. Milk yield, milk composition, and Nile Red intensity were analysed using the GLM procedure of SAS (SAS Institute Inc., Cary, NC, USA). Follicle and oocyte responses were analysed with the Mixed procedure of SAS. Relative gene expression of COC was evaluated using the REST 2009 Software. In vitro embryo production, post-thaw survival, and hatching rates were analysed using Chi Square. Milk fat was depressed (P < 0.001) by 10.1% in lactating Holstein cows fed CLA. Dietary supplementation of Holstien and Brahman cows with CLA did not alter expression of genes (CPT1, FADS2, and PPARα) in COC. Dietary supplementation of crossbred cows with CLA before oocyte collection did not influence cryotolerance of in vitro-produced embryos. Lipid content of oocytes (measured by Nile Red florescence) and follicle, oocyte, and embryo production was not influenced by CLA supplementation of Holstein and Brahman cows. The highly regulated mechanisms involved in fatty acid uptake by ovarian components may help explain the lack of ovarian response to dietary CLA in the current study.

81 Effects of Day 5 or 6 HEPES-Buffered Transportation Culture Medium on Developmental Competence of Bovine In Vitro-Produced Embryos

2018 ◽  
Vol 30 (1) ◽  
pp. 179
Author(s):  
W. Choi ◽  
C. M. Owen ◽  
M. Barcelo-Fimbres ◽  
J. L. Altermatt ◽  
L. F. Campos-Chillon
Keyword(s):  
Lipid Content ◽  
Embryo Culture ◽  
Culture Medium ◽  
Nile Red ◽  
Developmental Competence ◽  
Fluorescent Intensity ◽  
Embryo Culture Medium ◽  
Thermo Fisher Scientific ◽  
And Control

Most in vitro-produced (IVP) bovine embryos are transferred fresh. Use of a HEPES/bicarbonate embryo culture medium for transportation would offer flexibility for embryo shipment and transfer. We hypothesized that embryos cultured for 36 (Day 6 embryos) or 60 h (Day 5 embryos) in a novel SCF1T medium (SOF for Conventional Freezing 1 supplemented with HEPES) would maintain developmental competence compared with bicarbonate-buffered medium SCF1 (control). In 5 replicates, IVP embryos were produced by aspirating cumulus–oocyte complexes (COC) from 2-to 8-mm follicles of abattoir ovaries. The COC (n = 1036) were matured for 23 h, fertilized with semen from 1 of 3 bulls, and cultured in SCF1 at 38.5°C, 5% CO2, 5% O2, and 90% N2 (Owen et al. 2017 Reprod. Fertil. Dev. 29, 129-130). Randomly, on Day 5 and 6 after fertilization, a subset of presumptive embryos were moved into 500-µL polystyrene vials containing 100 µL of SCF1T medium, covered with 300 µL of sterile mineral oil and cultured in a portable incubator (MicroQ iQ2, Scottsdale, AZ, USA) at 38.5°C for 60 and 36 h, respectively. On Day 7.5 post-fertilization, blastocyst rates were evaluated and embryos (n = 8) from each group were stained with 1 µg mL−1 Nile Red for lipid quantification, and 300 nM Mitotracker Red CMX-Rosamine (Thermo Fisher Scientific, Waltham, MA, USA) for mitochondrial polarity. Images were obtained with confocal microscopy and fluorescent intensity (AFU) was measured by Image J software (National Institutes of Health, Bethesda, MD, USA). Data were analysed by one-way ANOVA and means separated by Tukey’s HSD. Results (Table 1) indicate similar blastocyst rates and lipid content between embryos cultured for 36 to 60 h in SCF1T and control media (P > 0.05). However, mitochondrial polarity was lower in the Day 5 group (P < 0.05) compared with Day 6 and control groups. Results suggest that culturing embryos in SCF1T medium for 36 h maintains developmental competence compared with bicarbonate-buffered media and offers an alternative for shipment and transfer of IVP embryos. Studies involving evaluation of pregnancy rates of the present study are ongoing. Table 1.Effects of Day 5 or 6 SCF1T embryo culture medium on development, lipid content, and mitochondrial polarity

90 LIPID CONTENT OF IN VIVO- AND IN VITRO-PRODUCED JERSEY AND HOLSTEIN CATTLE EMBRYOS AND THE EFFECT OF FORSKOLIN ON EMBRYO LIPID REDUCTION

2016 ◽  
Vol 28 (2) ◽  
pp. 174
Author(s):  
K. Rhodes-Long ◽  
L. F. Campos-Chillon ◽  
M. Barceló-Fimbres ◽  
J. L. Altermatt
Keyword(s):  
Fluorescence Intensity ◽  
Lipid Content ◽  
Lipid Accumulation ◽  
Culture Media ◽  
Nile Red ◽  
Holstein Cattle ◽  
Embryo Viability ◽  
Red Dye

Jersey embryos have been suggested to have higher lipid content and lower tolerance to cryopreservation. In addition, in vitro-produced (IVP) bovine embryos have darker cytoplasm as a consequence of higher lipid accumulation than in vivo-derived embryos, associated with impaired embryo quality and reduced cryotolerance. Forskolin is an adenylate cyclase activator that regulates cAMP levels in cells and has been shown to induce lipolysis in IVP embryos. We hypothesised that the lipid content of in vivo-produced and IVP Jersey embryos is higher than respective Holstein embryos and that forskolin would reduce lipid content of IVP embryos. The objectives of this experiment were (1) to analyse lipid content of in vivo and IVP Jersey and Holstein cattle embryos and (2) to evaluate the effect of forskolin added to IVP culture media. The factorial experimental design used two breeds (Holstein and Jersey) and three embryo production methods (in vivo, IVP, and IVP + forskolin). IVP embryos (n = 27 blastocysts) were collected from super-stimulated donors by routine procedures 7.5 days after AI. IVP embryos (n = 259 blastocysts) were produced by standard procedures; briefly, oocytes were aspirated from 2- to 8-mm follicles from slaughterhouse ovaries and matured for 24 h in SMM medium (BoviPro, MOFA Global, Verona, WI, USA). Matured oocytes were fertilized using semen from two different bulls for each breed, and embryos were cultured in BBH7 medium (BoviPro, MOFA Global) alone or with the addition of forskolin (10 µM) at Day 5 of culture at 38.5°C in 5% O2, 5% CO2, and 90% N2. The lipid content of embryos was quantified by staining Day 7 blastocysts with 1 μg mL–1 Nile red dye (580–596 nm), after which a digital photograph of the equatorial part of the embryo was taken at 40×, and fluorescence intensity (FI) was measured with Image Pro software. Data (Table 1) were analysed by ANOVA, and means were compared using Tukey’s HSD. Jersey and Holstein IVP embryos had higher lipid content than Holstein in vivo-produced embryos (P < 0.05), but were not different than Jersey in vivo-derived embryos (P > 0.1). Forskolin lowered the lipid content (P < 0.05) of both IVP Jersey and Holstein embryos and was not different (P > 0.1) than in vivo-produced embryos. Addition of forskolin to embryo culture media has the potential to lower embryo lipid accumulation and possibly improve embryo viability and cryotolerance of IVP embryos. Further studies including cryopreservation and transfer of IVP + forskolin embryos to recipients are necessary to corroborate the findings of the present study. Table 1.Fluorescence intensity of in vivo-produced and IVP Jersey and Holstein embryos

Reduction in cytoplasmic lipid content in bovine embryos cultured in vitro with linoleic acid in semi-defined medium is correlated with increases in cryotolerance

Zygote ◽  
2015 ◽  
Vol 24 (4) ◽  
pp. 485-494 ◽  
Author(s):  
Mônica F. Accorsi ◽  
Beatriz Caetano da Silva Leão ◽  
Nathália Alves de Souza Rocha-Frigoni ◽  
Silvia Helena Venturoli Perri ◽  
Gisele Zoccal Mingoti
Keyword(s):  
Linoleic Acid ◽  
Lipid Content ◽  
Lipid Accumulation ◽  
Culture Medium ◽  
Calf Serum ◽  
Nile Red ◽  
Defined Medium ◽  
Blastocyst Stage ◽  
Embryo Survival

SummaryWe examined whether culturing embryos with linoleic acid (LA) in semi-defined medium reduces lipid accumulation and improves cryosurvival after vitrification. Embryos were cultured with LA (100 μM) and a semi-defined medium was used during in vitro culture (IVC), in which the fetal calf serum was substituted by bovine serum albumin (BSA). There was a reduction (P < 0.05) in the embryonic development rate (Control: 25.8% versus LA: 18.5%), but the proposed system was effective in promoting the decrease (P = 0.0130) in the intracellular lipid content (Control: 27.3 ± 0.7 versus LA: 24.6 ± 0.7 arbitrary fluorescence units of embryos stained with the fluorescent dye Nile Red), consequently increasing (P = 0.0490) the embryo survival after 24h of culture post-warming (Control: 50.0% versus LA: 71.7%). The results question the criteria used to evaluate the efficiency of an in vitro production system specifically with relation to the maximum number of blastocysts produced and suggest that might be more appropriate to improve the desired characteristics of embryos generated in accordance with the specific purpose of in vitro embryo production, commercial or scientific. In conclusion, supplying LA to serum-free culture medium was found to adversely affect the rates of embryo development to the blastocyst stage, but significantly reduced embryo lipid accumulation and improved cryopreservation survival.

Reprogrammed M1 macrophages with inhibited STAT3, STAT6 and/or SMAD3 extends lifespan of mice with experimental carcinoma

2017 ◽  
pp. 4-9
Author(s):  
С.В. Калиш ◽  
С.В. Лямина ◽  
А.А. Раецкая ◽  
И.Ю. Малышев
Keyword(s):  
Tumor Growth ◽  
Culture Medium ◽  
Ehrlich Carcinoma ◽  
Macrophage Phenotype ◽  
M1 Macrophages ◽  
M1 Macrophage ◽  
Ec Cells ◽  
Experimental Carcinoma

Цель исследования. Репрограммирование М1 фенотипа макрофагов с ингибированными факторами транскрипции М2 фенотипа STAT3, STAТ6 и SMAD и оценка их влияния на развитие карциномы Эрлиха (КЭ) in vitro и in vivo. Методика. Рост опухоли иницировали in vitro путем добавления клеток КЭ в среду культивирования RPMI-1640 и in vivo путем внутрибрюшинной инъекции клеток КЭ мышам. Результаты. Установлено, что M1макрофаги и in vitro, и in vivo оказывают выраженный противоопухолевый эффект, который превосходит антиопухолевые эффекты М1, M1, M1 макрофагов и цисплатина. Заключение. М1 макрофаги с ингибированными STAT3, STAT6 и/или SMAD3 эффективно ограничивают рост опухоли. Полученные данные обосновывают разработку новой технологии противоопухолевой клеточной терапии. Objective. Reprogramming of M1 macrophage phenotype with inhibited M2 phenotype transcription factors, such as STAT3, STAT6 and SMAD and assess their impact on the development of Ehrlich carcinoma (EC) in vitro and in vivo . Methods. Tumor growth in vitro was initiated by addition of EC cells in RPMI-1640 culture medium and in vivo by intraperitoneal of EC cell injection into mice. Results. It was found that M1 macrophages have a pronounced anti-tumor effect in vitro , and in vivo , which was greater than anti-tumor effects of M1, M1, M1 macrophages and cisplatin. Conclusion. M1 macrophages with inhibited STAT3, STAT6 and/or SMAD3 effectively restrict tumor growth. The findings justify the development of new anti-tumor cell therapy technology.

Differentiation of mesenchymal stem cells from humans and animals into insulin-producing cells: An overview in vitro induction forms

2020 ◽  
Vol 16 ◽  
Author(s):  
Bruna O. S. Câmara ◽  
Bruno M. Bertassoli ◽  
Natália M. Ocarino ◽  
Rogéria Serakides
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Culture Medium ◽  
Adipogenic Differentiation ◽  
Medium Composition ◽  
Cell Therapies ◽  
Insulin Producing Cells ◽  
Msc Differentiation

The use of stem cells in cell therapies has shown promising results in the treatment of several diseases, including diabetes mellitus, in both humans and animals. Mesenchymal stem cells (MSCs) can be isolated from various locations, including bone marrow, adipose tissues, synovia, muscles, dental pulp, umbilical cords, and the placenta. In vitro, by manipulating the composition of the culture medium or transfection, MSCs can differentiate into several cell lineages, including insulin-producing cells (IPCs). Unlike osteogenic, chondrogenic, and adipogenic differentiation, for which the culture medium and time are similar between studies, studies involving the induction of MSC differentiation in IPCs differ greatly. This divergence is usually evident in relation to the differentiation technique used, the composition of the culture medium, the cultivation time, which can vary from a few hours to several months, and the number of steps to complete differentiation. However, although there is no “gold standard” differentiation medium composition, most prominent studies mention the use of nicotinamide, exedin-4, ß-mercaptoethanol, fibroblast growth factor b (FGFb), and glucose in the culture medium to promote the differentiation of MSCs into IPCs. Therefore, the purpose of this review is to investigate the stages of MSC differentiation into IPCs both in vivo and in vitro, as well as address differentiation techniques and molecular actions and mechanisms by which some substances, such as nicotinamide, exedin-4, ßmercaptoethanol, FGFb, and glucose, participate in the differentiation process.

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