102 PROTEOMIC ANALYSIS OF CONDITIONED MEDIUM SUPPLEMENTED WITH PORCINE FOLLICULAR FLUID

2011 ◽  
Vol 23 (1) ◽  
pp. 156
Author(s):  
S. Hwang ◽  
K. B. Oh ◽  
H.-C. Lee ◽  
B.-C. Yang ◽  
D. Lim ◽  
...  
Keyword(s):  
Conditioned Medium ◽  
Follicular Fluid ◽  
Culture Medium ◽  
Cell Metabolism ◽  
Eukaryotic Cell ◽  
Amino Acid Sequences ◽  
Homology Search ◽  
Surface Binding ◽  
Culture Methods

Follicular fluid (FF) contains growth factors, electrolytes, hormones, amino acids, and unknown factors. Supplementation of porcine FF (pFF) to in vitro maturation (IVM) medium was reported to improve the oocyte maturation, monospermic fertilization and embryonic development. This study aimed at investigating whether pFF supplementation affects the characteristics of donor cells for somatic cell nuclear transfer and the proteomic composition of the culture medium. Ear fibroblast cells from an NIH major histocompatibility complex (MHC) inbred miniature pig were cultured with different culture methods: 1) DMEM + 10% FBS (FBS); 2) DMEM + 10% FBS + 10% pFF (pFF). The conditioned medium was collected at 72 h. After isoelectric focusing (IEF), the equilibrated strips were submitted to SDS-PAGE. Normalized protein spots were considered significantly different between the two groups if expression levels varied by two standard deviations. To identify the protein spots, an Ettan MALDI-TOF method was used. Upon submission of the amino acid sequences, proteins were identified by a homology search using ProteinInfo or BLAST search using the ExPASy Molecular Biology Server. The proportion of G0/G1 stage cells in the pFF group was significantly higher than the proportions in the other groups (P < 0.05). Among 42 differentially expressed spots, 36 proteins were identified in the pFF group. Some molecular functions of the spots were: catalytic or methytransferase activity, eukaryotic cell surface binding, or ferric iron binding. It can be concluded that pFF supplementation of culture medium positively affects cell-cycle synchronization and cell metabolism. Further studies are needed to analyse the function of these important cellular proteins. This work received grant support from the Agenda Program (No. PJ006688) and (No. PJ007189), Rural Development Administration, Republic of Korea.

P–218 Analysis of the occurrence of microbial contamination in IVF culture system and the effect of microorganisms on embryo development and clinical outcomes

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
F Du ◽  
R Li ◽  
Q Zhang ◽  
W Wang
Keyword(s):  
Escherichia Coli ◽  
In Vitro Culture ◽  
Clinical Outcomes ◽  
Embryo Transfer ◽  
Follicular Fluid ◽  
Culture System ◽  
Microbial Contamination ◽  
Culture Medium ◽  
Ivf Et

Abstract Study question what is the source, prevalence, and influence of microbial contamination on in vitro fertilization (IVF) and embryo transfer (ET) cycles? Summary answer Microbial contamination mainly occurs on Day 2, most caused by Escherichia coli carried with semen. ICSI could prevent contamination effectively and get good clinical outcomes. What is known already Microbial contamination occurs in IVF-ET system occasionally, which is hard to stop happening. The IVF culture system and laboratory environment, the patients’ follicular fluid and semen are not absolutely sterile, while the antibiotics in culture medium isn’t effective for all microbe types, and the artificial operations may bring in microbes. Generally, microbial contamination leads to degradation of embryos, reduction the number of embryos available, and infection of female reproductive tract, which would increase the cost of patients’ time, money, and bring psychological damages. A better understanding of embryo contamination in IVF culture system is of added value. Study design, size, duration A total of 29583 IVF-ET cycles were enrolled in this prospective observational study, from January 2010 to December 2020, included 70 microbial contamination cycles discovered in Day1-Day3 (D1-D3) of in vitro culture. Follicular fluid and semen saved on oocyte retrieval day, and culture medium contaminated were examined and identified for microorganisms at each contamination cycle. Participants/materials, setting, methods Compared the contamination rate of different insemination methods (IVF/ICSI/IVF+ICSI), different in vitro culture days (D1-D3), and different samples examination (follicular fluid, semen, culture medium) respectively, identified the source of microorganism types, compared the IVF culture outcomes and clinical outcomes between total contamination group (TC group, 42 cases) and partial contamination group (PC group, 28 cases). Main results and the role of chance A total of 70 microbial contamination cases occurred in 29583 oocyte retrieving cycles (0.24%), and it was observed only in IVF embryos but never in ICSI (Intracytoplasmic sperm injection) embryos. 38 contamination cases occurred on D2 with a highest ratio (54.3%) compared to D1 (32.9%) and D3(12.9%); Compared with follicular fluid, semen was the main cause inducing contamination from D1 to D3, and Escherichia coli in semen and culture medium, Enterococcus faecalis in follicular fluid proved to be the most common sources. Compared with TC group, the PC group showed a lower rate of No-available embryos (21.4% vs 81.0%) and a higher rate of blastocyst formation (41.2% vs 28.6%), In addition, the clinical pregnancy rate of PC group was higher than that of TC group in both fresh and frozen-thawed embryo transfer cycles (31.3% vs 16.7%, 38.5% vs 0.0%). Limitations, reasons for caution Further study is still necessary to better understand the sources that induce microbial contamination embryos, and more efficient methods are required to remove the microbes on these contaminated embryos so as better develop and manage a sterile micro-environment for successful embryo growth. Wider implications of the findings: The differential embryonic microbe types associated to different IVF culture and clinical outcomes in patients undergoing IVF-ET might have profound implications for understanding the microbial sources and making a better management of IVF culture system. Trial registration number Not applicable

scholarly journals Efficiency of using different types induction culture medium for hexaploid triticale anther cultivation

2019 ◽  
Vol 25 ◽  
pp. 260-265
Author(s):  
E. V. Lagunovskaya ◽  
O. I. Zaitseva ◽  
V. A. Lemesh
Keyword(s):  
Culture Medium ◽  
Medium Composition ◽  
Induction Medium ◽  
Hexaploid Triticale ◽  
Culture Methods ◽  
Study Results ◽  
Short Period ◽  
Androgenesis In Vitro ◽  
The Republic

Aim. Triticale is one of the main grain crops of the Republic of Belarus. Further progress in the selection of this culture involves the accelerated creation of highly productive early ripening varieties resistant to abiotic and biotic factors. The method of induced androgenesis in vitro makes it possible to obtain stable homozygous lines in a short period of time and to eliminate the lengthy process of inbreeding used in classical breeding to fix the desired traits. Methods. The tissue and cell culture methods for plants was used in the study. Results. The influence of the induction medium composition on the efficiency of in vitro induced androgenesis in varieties and lines of hexaploid triticale is assessed. The influence of three types of induction culture medium, the type of phytohormones and the presence or absence of cefotaxime in the medium are analyzed. Results. It has been shown that using the C-17 culture medium supplemented with 2.0 mg/l 2,4-D and 0.5 mg/l kinetin without adding cefotaxime is most effective for the anther triticale cultivation. Keywords: triticale, anther culture, induction nutrient medium, embryoids, calli, regenerant plants, cefotaxime.

Studies on growth of the mink blastocyst

Development ◽  
1967 ◽  
Vol 17 (2) ◽  
pp. 293-302
Author(s):  
Joseph C. Daniel
Keyword(s):  
Culture Medium ◽  
Present Report ◽  
Blastocyst Stage ◽  
Pregnant Female ◽  
Culture Methods ◽  
Direct Treatment ◽  
Breeding Seasons

In those mammals in which implantation is delayed, the embryo enters a diapause at the blastocyst stage. The present report describes experiments with mink over the last three breeding seasons, attempting to define the factors that limit development at this stage. Four approaches to the problem were used: (1) determination of growth of mink blastocysts in vitro with specific modifications of media; (2) transplantation of mink embryos to rabbit uteri; (3) direct treatment of pregnant female mink with ergosterol; (4) growth of rabbit blastocysts in vitro in medium containing mink serum. (1) The culture methods used for mink embryos were those developed for the rabbit and described in earlier publications (Daniel, 1963, 1965). Mink blastocysts (Plate 1) were isolated in culture medium after being flushed from the uteri of mothers bred 9-20 days earlier. Various components were added to the medium, F10 (Ham, 1963), in concentrations that were previously tested against rabbit blastocysts and found to be non-toxic, and, in some cases, beneficial to growth.

338 EFFECTS OF CUMULUS CELLS AND FOLLICULAR FLUID ON PLASMINOGEN ACTIVATOR ACTIVITY AND OOCYTE MATURATION IN VITRO IN THE PIG

2006 ◽  
Vol 18 (2) ◽  
pp. 276
Author(s):  
C.-K. Park ◽  
J.-Y. An ◽  
S.-J. Sa ◽  
H.-T. Cheong ◽  
B.-K. Yang ◽  
...  
Keyword(s):  
Conditioned Medium ◽  
Oocyte Maturation ◽  
Follicular Fluid ◽  
Sodium Dodecyl ◽  
Cumulus Cells ◽  
The Other ◽  
Tissue Type ◽  
Bromophenol Blue ◽  
Maturation In Vitro

Plasminogen activators (PAs) are serine proteases, known to be secreted by a large number of cell type. PAs are reported to play a role in variety of physiologic processes, including fibrinolysis, ovulation, mammary involution, implantation, and fertilization. The present study investigated the effects of cumulus cells and porcine follicular fluid (pFF) on PA activity and oocyte maturation in vitro in the pig. Porcine oocytes were harvested from slaughterhouse ovaries, selected, and matured in modified North Carolina State University-23 (NCSU-23) media. After culture, cumulus-oocyte complexes (COCs) and denuded oocytes (DOs) were separately put into microtubes containing 20 �L of sample buffer [5.0% (w:v) sodium dodecyl sulfate, 20% (v:v) glycerol, and 0.0025% (w:v) bromophenol blue in 0.125 M Tris-HCl buffer] and frozen at -80�C until used for zymographic analysis. Differences in data were evaluated by Duncan's multiple-range test using the General Linear Models procedure in the Statistical Analysis System (SAS Institue, Inc., Cary, NC, USA). To determine the effect of porcine follicular fluid (pFF) on PA activity in porcine oocytes during maturation, the COCs and DOs were incubated in NCSU-23 medium with or without 10% (v/v) pFF for 0, 24, or 48 h. In the presence of cumulus cells, the proportions of oocytes matured to metaphase-II stage were significantly (P < 0.05) higher in medium with pFF than without pFF (69.8% vs. 37.7%, respectively). When COCs and DOs were cultured in the presence of pFF, tissue-type PA (tPA), urokinase-type PA (uPA), and tPA-PA inhibitor (tPA-PAI) were observed in COCs, and PA activities were higher at 48 h than 24 h. However, no PA activity was detected in DOs. Under the same conditions, when COCs and DOs were cultured in the absence of pFF, tPA and tPA-PAI were observed in COCs, and PA activities were increased as duration of culture increased. However, no PA activity was detected in DOs. When porcine oocytes were cultured in the presence of pFF, the activities of tPA-PAI, tPA, and uPA were observed in conditioned medium with COCs and DOs cultured for 24 h and 48 h. In the absence of pFF, PA activities were observed only in conditioned medium with COCs, and no PA activities were detected in conditioned medium with DOs. On the other hand, three plasminogen-dependent lytic bands (tPA-PAI, tPA, and uPA) were observed in pFF cultures. Particularly uPA activity was higher than the other kinds of PA activity. When oocytes and cumulus cells were separated from porcine COCs at 0 h of cultrue, tPA-PAI, tPA, and uPA were detected in cumulus cells at 48-h culture, but no PA activities were in DOs. The presence of pFF and cumulus cells in maturation medium stimulated not only nuclear and cytoplasmic maturation in porcine COCs, but also PA production by cumulus cells and COCs. It is possible that PAs produced by cumulus cells migrated through the gap junction between oocyte and cumulus cells. These results suggest that porcine oocytes have no ability to produce PA themselves.

Influence of oviductal cells and conditioned medium on porcine gametes

Zygote ◽  
2000 ◽  
Vol 8 (2) ◽  
pp. 139-144 ◽  
Author(s):  
Mariève Bureau ◽  
Janice L. Bailey ◽  
Marc-André Sirard
Keyword(s):  
Epithelial Cells ◽  
Conditioned Medium ◽  
Embryo Development ◽  
Follicular Fluid ◽  
Maturation Medium

The aim of this study was to optimise porcine in vitro fertilisation (IVF) with cryopreserved semen with the exploitation of the oviduct secretion. The oocytes were cultured in NCSU37 supplemented with db-cAMP (1 mM), porcine follicular fluid (pFF; 10%), cysteine (0.1 mg/ml) and β-mercaptoethanol (25 μM) for 44 h (the first 20 h with 10 IU/ml hCG and PMSG). The oviductal epithelial cells (OEC) were cultured in TCM-199 medium (with 10% FCS, 0.2 mM pyruvate and 50 μg/ml gentamicin) for 48 h. To determine the effects of OEC and conditioned medium, oocytes were separated into five groups for the last 3 h of maturation and placed in: fresh maturation medium (controls), OEC-cNCSU with OEC in the maturation medium for 24 h; OEC-fNUSU with fresh OEC in maturation medium; cTCM with TCM-199 conditioned with OEC for 48 h; or fTCM with fresh TCM-199. Results indicate that OEC-cNCSU and OEC-fNCSU increase the number of oocytes reaching the two pronucleus (2PN) stage (p < 0.01) and decrease the polyspermy rate (p < 0.01) compared with controls. The rates are significantly lower than controls when cTCM and fTCM were used (p < 0.01). As regards blastocyst rates, an increase was observed in the OEC-cNCSU and cTCM groups (p < 0.05). For the second experiment, spermatozoa were incubated with OEC in IVF medium (mTBM medium supplemented with 0.1% BSA) without caffeine for 4 h prior to IVF. Results indicate that sperm treatment with OEC increases the 2PN rate (p < 0.01) compared with controls and reduces the polyspermy rate (p < 0.01). In conclusion, our study shows that co-incubation of OEC with both oocytes and sperm before IVF reduces polyspermy rates and improves embryo development.

scholarly journals Effect of conditioned medium of umbilical cord-derived mesenchymal stem cells as a culture medium for human granulosa cells: An experimental study

2022 ◽  
pp. 1037-1044
Author(s):  
Kanadi Sumapraja ◽  
Andon Hestiantoro ◽  
Isabella Kurnia Liem ◽  
Arief Boediono ◽  
Teuku Z Jacoeb
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Conditioned Medium ◽  
Umbilical Cord ◽  
Granulosa Cells ◽  
Culture Medium ◽  
Culture Media ◽  
Controlled Ovarian Hyperstimulation ◽  
Vitro Fertilization

Background: The umbilical cord-derived mesenchymal stem cells conditioned medium (UC-MSCs-CM) produces secretomes with anti-apoptotic properties, and has the potential to prevent apoptosis of granulosa cells (GC) during controlled ovarian hyperstimulation. Objective: To observe the effect of UC-MSCs-CM on the interaction between pro- and anti-apoptotic proteins and the influence of growth differentiation factor 9 (GDF9) production in GC. Materials and Methods: UC-MSCs-CM was collected from umbilical cord stem cell culture on passage 4. GC from 23 women who underwent in vitro fertilization were cultured and exposed to UC-MSCs-CM for 24 hr. Then RNA of the GC was extracted and the mRNA expression of BCL-2 associated X (BAX), survivin and GDF9 were analysed using quantitative real-time PCR. The spent culture media of the GC were collected for measurement of insulin growth factor 1 using ELISA. Results: The expression of BAX was significantly different after UC-MSCs-CM exposure (4.09E-7 vs. 3.74E-7, p = 0.02). No significant changes occurred in survivin, BAX/survivin ratio, and GDF9 expression after UC-MSCs-CM exposure (p > 0.05). The IGF-1 level of the CM was significantly higher after the CM was used as a culture medium for GC (2.28 vs. 3.07 ± 1.72, p ≤ 0.001). A significant positive correlation was found between survivin and GDF9 (r = 0.966, p ≤ 0.001). Conclusion: IGF-1 produced by UC-MSCs-CM can work in paracrine fashion through the IGF receptor, which can inhibit BAX and maintain GDF9 production. Moreover, under the influence of UC-MSCs-CM, GC are also capable of producing IGF-1, which can impact GC through autocrine processes. Key words: Conditioned medium, BAX, Survivin, GDF9, IGF-1.

scholarly journals Effects of Various Levels of Luteinizing Hormone and Caprine Follicular Fluid on In Vitro Embryo Production of Shami Goat

2021 ◽  
Vol 5 (3) ◽  
pp. 575-584
Author(s):  
Omar Mardenli ◽  
Hadi Awad Hassooni ◽  
Mahdi Saleh Mohammad Al-Kerwi
Keyword(s):  
Luteinizing Hormone ◽  
Follicular Fluid ◽  
Culture Medium ◽  
Goat Breed ◽  
Embryo Production ◽  
Follicle Size ◽  
In Vitro Embryo Production ◽  
Type 3

In the current study, the hypothesis of the effects of luteinizing hormone (LH) and follicular fluid (FF) derived from follicles of varying size on in vitro embryo production of the Shami goat breed were tested. The caprine follicular fluid (cFF) was obtained from healthy female’s ovaries by aspiration method and classified into two main classes (follicles with a diameter of ≤ 2mm and ≥3mm). The resulting cFF was added to the culture medium TCM-199 through six Treatments (A, B and C with a source of follicle size of ≤ 2mm; D, E and F with a source of size of ≥3mm). LH was added only to four of the previous Treatments with the levels of 50 µg ml-1 (B and E) and100 µg ml-1 (C and F). Results of the study showed that the oocytes incubated in Treatment F achieved a clear superiority (p=0.001) in the rates of maturation (87.0%), fertilization (80.0%) and cleavage (82.3%). The oocytes incubated in the same Treatment (F) continued to outperform (p= 0.006) by achieving the best rates across cleavage stages at 2-16 cell (16%; the lower value of arrest) and blastocyst (42%). Significant differences (P=0.03) were observed among the rates of Type 1embryos (the highest rate: 45.3%; Treatment F) and Type 3 embryos (the highest rate: 45.1%; Treatment A). No significant differences were observed in the rates of morula and Type 2 embryos. It is advised to add 15% of the cFF derived from follicles with a diameter of ≥3mm and 100 µg of LH ml-1 in the maturation media to obtain higher rates of maturation and cleavage of goat oocytes.  

scholarly journals Selection of Boar Sperm by Reproductive Biofluids as Chemoattractants

Animals ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 53
Author(s):  
Luis Alberto Vieira ◽  
Alessia Diana ◽  
Cristina Soriano-Úbeda ◽  
Carmen Matás
Keyword(s):  
Calcium Channel ◽  
Conditioned Medium ◽  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Mammalian Species ◽  
Boar Sperm ◽  
Oviductal Fluid ◽  
Chemotactic Effect ◽  
Selection Of

Chemotaxis is a spermatozoa guidance mechanism demonstrated in vitro in several mammalian species including porcine. This work focused on follicular fluid (FF), periovulatory oviductal fluid (pOF), the medium surrounding oocytes during in vitro maturation (conditioned medium; CM), progesterone (P4), and the combination of those biofluids (Σ) as chemotactic agents and modulators of spermatozoa fertility in vitro. A chemotaxis chamber was designed consisting of two independent wells, A and B, connected by a tube. The spermatozoa are deposited in well A, and the chemoattractants in well B. The concentrations of biofluids that attracted a higher proportion of spermatozoa to well B were 0.25% FF, 0.25% OF, 0.06% CM, 10 pM P4 and 0.25% of a combination of biofluids (Σ2), which attracted between 3.3 and 12.3% of spermatozoa (p < 0.05). The motility of spermatozoa recovered in well B was determined and the chemotactic potential when the sperm calcium channel CatSper was inhibited, which significantly reduced the % of spermatozoa attracted (p < 0.05). Regarding the in vitro fertility, the spermatozoa attracted by FF produced higher rates of penetration of oocytes and development of expanded blastocysts. In conclusion, porcine reproductive biofluids show an in vitro chemotactic effect on spermatozoa and modulate their fertilizing potential.

scholarly journals Metabolomics Guides Rational Development of a Simplified Cell Culture Medium for Drug Screening against Trypanosoma brucei

2013 ◽  
Vol 57 (6) ◽  
pp. 2768-2779 ◽  
Author(s):  
Darren J. Creek ◽  
Brunda Nijagal ◽  
Dong-Hyun Kim ◽  
Federico Rojas ◽  
Keith R. Matthews ◽  
...  
Keyword(s):  
Cell Culture ◽  
Trypanosoma Brucei ◽  
Culture Medium ◽  
Culture Media ◽  
Culture Methods ◽  
Content Type ◽  
Drug Activity ◽  
Rational Development ◽  
High Concentrations

ABSTRACTIn vitroculture methods underpin many experimental approaches to biology and drug discovery. The modification of established cell culture methods to make them more biologically relevant or to optimize growth is traditionally a laborious task. Emerging metabolomic technology enables the rapid evaluation of intra- and extracellular metabolites and can be applied to the rational development of cell culture media. In this study, untargeted semiquantitative and targeted quantitative metabolomic analyses of fresh and spent media revealed the major nutritional requirements for the growth of bloodstream formTrypanosoma brucei. The standard culture medium (HMI11) contained unnecessarily high concentrations of 32 nutrients that were subsequently removed to make the concentrations more closely resemble those normally found in blood. Our new medium, Creek's minimal medium (CMM), supportsin vitrogrowth equivalent to that in HMI11 and causes no significant perturbation of metabolite levels for 94% of the detected metabolome (<3-fold change; α = 0.05). Importantly, improved sensitivity was observed for drug activity studies in whole-cell phenotypic screenings and in the metabolomic mode of action assays. Four-hundred-fold 50% inhibitory concentration decreases were observed for pentamidine and methotrexate, suggesting inhibition of activity by nutrients present in HMI11. CMM is suitable for routine cell culture and offers important advantages for metabolomic studies and drug activity screening.

Bovine dominant follicular fluid promotes the in vitro development of goat preantral follicles

2012 ◽  
Vol 24 (3) ◽  
pp. 490 ◽  
Author(s):  
A. B. G. Duarte ◽  
V. R. Araújo ◽  
R. N. Chaves ◽  
G. M. Silva ◽  
D. M. Magalhães-Padilha ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
Culture Medium ◽  
Follicular Development ◽  
Ultrastructural Analysis ◽  
In Vitro Development ◽  
Preantral Follicles ◽  
Nuclear Membranes ◽  
Vitro Development ◽  
First Time

The aim of this study was to evaluate the effect of follicular fluid collected from bovine dominant follicles (bFF) on the in vitro development of goat preantral follicles and determine the best time to add this supplement to the culture medium. The preantral follicles were isolated and randomly distributed into four treatments in absence (control) or presence of 10% of bFF added on Days 0 (FF0–18), 6 (FF6–18) or 12 (FF12–18) of culture onwards. After 18 days, follicular development was assessed based on follicular survival, antral cavity formation, increased follicular diameter as well as fully grown oocyte (>110 μm) viability and meiosis resumption. The oocytes from the cultured follicles were in vitro-matured and processed for fluorescence or ultrastructural analysis. The results showed that on Day 18 the treatment FF0–18 had a significantly higher (P < 0.05) survival than control and FF12–18, but not FF6–18. The addition of bFF at the beginning of culture (FF0–18 and FF6–18) promoted a high percentage of follicular growth, meiosis resumption and early antrum formation. Moreover, this study described for the first time the ultrastructural analysis of caprine oocytes grown in vitro. This evaluation revealed that in the presence of bFF on (FF0–18) the in vitro-grown oocytes presented normal organelle distribution and well-defined, intact plasma and nuclear membranes. In conclusion, bFF originating from dominant follicles maintain the survival and promote the in vitro growth of goat preantral follicles when added at the beginning of culture.

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