76 Extracellular vesicles from oviduct and uterus in sequential culture improve the quality of bovine embryos produced invitro

2020 ◽  
Vol 32 (2) ◽  
pp. 164
Author(s):  
C. Leal ◽  
K. Cañon-Beltrán ◽  
Y. Cajas ◽  
P. Gallego ◽  
P. Beltrán-Breña ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Lipid Content ◽  
Luteal Phase ◽  
Cell Number ◽  
Total Cell ◽  
Bovine Embryos ◽  
Total Cell Number ◽  
Transmission Electron

Extracellular vesicles (EVs) are released by cells and transport cargo that affect functions of other cells. Oviductal fluid (OF) and uterine fluid (UF) have been shown to improve quality of embryos during invitro culture (Hamdi et al. 2017 Reprod. Fertil. Dev. 30, 935-945) which may be due to their content of EV (Lopera-Vásquez et al. 2017 Reproduction 153, 461-470). Thus, the aim of this study was to evaluate the effect of EVs from OF and UF on a sequential invitro culture system on the development and quality of bovine embryos. Zygotes were cultured in synthetic oviduct fluid (SOF) supplemented with 3mgmL−1 bovine serum albumin (BSA; n=1228) or 5% EV-depleted fetal calf serum (dFCS, n=1261) in the presence (BSAEV, n=1265 and dFCSEV, n=1253) or absence of 3×105 EVmL−1 from OF (Day 1 to Day 4) and UF (Day 4 to Day 9), mimicking invivo conditions. The EVs pooled from 5 oviducts (early luteal phase) and 5 uterine horns (middle luteal phase) from slaughtered heifers were isolated by a Size Exclusion Chromatography kit (Hansa BioMed). The EV size and concentration were assessed by the nanotracking analysis system and morphology by transmission electron microscopy. Embryo development was recorded on Days 7/9. Day 7/8 blastocysts were assessed for quality by staining with (a) Hoechst 33342 (10 µgmL−1, 30min) for total cell number, (b) Bodipy 493/503 (20 µgmL−1, 1h) for lipid content (lipid droplet area in µm2), and (c) for survival rate after vitrification/warming. Data were analysed by one-way ANOVA and Tukey test. The EV concentration was 2.97 and 7.98×1010 particlesmL−1, and mode size 137.2 and 151.2nm for OF and UF, respectively. Transmission electron microscopy confirmed EV presence and size, showing typical cup-shaped morphology. Blastocyst yield was lower (P<0.05) on Day 7 in the BSA groups (BSA: 15.7±1.9 and BSAEV: 15.2.4%) compared with serum groups (dFCS: 28.1±2.6 and dFCSEV: 30.1±2.9%) irrespective of EV supplementation; however, these differences were compensated at Days 8 and 9 (range: 30.0±3.2-40.8±3.9%). The EVs increased (P<0.05) blastocyst total cell number in dFCSEV (152.6±2.9) and BSAEV (140.5±1.5) compared with dFCS (117.9±2.0) and BSA groups (122.4±1.1). However, lipid content was decreased (P<0.05) in the presence of EVs only in dFCSEV (0.231±0.05µm2) compared with BSA (0.393±0.03µm2) and BSAEV (0.379±0.03µm2) groups. The dFCS did not differ from any group (0.371±0.05µm2; P>0.05). Blastocyst survival after vitrification/warming was high in all groups up to 72h (range: 80.0±3.8-100%; P>0.05). In conclusion, mimicking physiological conditions using EV from OF and UF during invitro culture does not affect development but improves embryo quality by increasing blastocyst total cell numbers and decreasing lipid contents. These results provide evidence of the association of the reproductive tract environment and developing embryo, confirming embryo-maternal communication. Funding was provided by MINECO-Spain AGL2015-70140-R; Y. N. Cajas, SENESCYT-Ecuador; C. L. V. Leal, FAPESP-Brazil 2017/20339-3.

Application and Study of Reactive Polyacrylate Microgel in Pigment Paint of Printed Fabric

2011 ◽  
Vol 311-313 ◽  
pp. 1044-1048
Author(s):  
Hong Long Xing ◽  
Shui Lin Chen
Keyword(s):  
Electron Microscopy ◽  
Mechanical Properties ◽  
Transmission Electron Microscopy ◽  
Sodium Dodecyl ◽  
Ammonium Persulfate ◽  
Color Fastness ◽  
Adhesive Film ◽  
Transmission Electron ◽  
Divinyl Benzene

Polyacrylate microgel emulsion was prepared by emulsion polymerization using styrene, α-n-butyl acrylate and methyl methacrylate as monomer, polyoxyethylene octylphenol ether (TX-30) and sodium dodecyl sulfate(SDS) as combine emulsifier, divinyl benzene and ammonium persulfate (APS) as initiator，respectively. The prepared microgel was analyzed by a variety of measurment methods, such as Fourier transform infrared spectroscopy and transmission electron microscopy. The effect of microgel on the rheological properties of adhesives, leveling, mechanical properties and pigment printing performance was studied. The rhelogy and the color fastness of the pigment printing binder of printed fabrics were measured by rheometer and friction color fastness test instruments, respectively. At the same time, the mechanical properties of the adhesive film was measured by strength tester. The results show that the thixotropy, leveling and mechanical properties of adhesive printing binder and pringting quality of coating fabrics were improved when the microgel was added.

Cleaning Treatment to Advance the Quality of FIB Samples for Transmission Electron Microscopy

2004 ◽  
Vol 10 (S02) ◽  
pp. 1166-1167
Author(s):  
Wolfgang Gruenewald ◽  
Hans-Juergen Engelmann ◽  
Beate Volkmann
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Transmission Electron ◽  
Cleaning Treatment

Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.

A method of precise misorientation determination

2003 ◽  
Vol 36 (6) ◽  
pp. 1319-1323 ◽  
Author(s):  
A. Morawiec
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Spatial Resolution ◽  
Standard Procedure ◽  
Good Spatial Resolution ◽  
Transmission Electron ◽  
Order Of Magnitude ◽  
Achievable Accuracy ◽  
Better Than

A method that improves the accuracy of misorientations determined from Kikuchi patterns is described. It is based on the fact that some parameters of a misorientation calculated from two orientations are more accurate than other parameters. A procedure which eliminates inaccurate elements is devised. It requires at least two foil inclinations. The quality of the approach relies on the possibility to set large sample-to-detector distances and the availability of good spatial resolution of transmission electron microscopy. Achievable accuracy is one order of magnitude better than the accuracy of the standard procedure.

Use of chemically defined system for the direct comparison of inner cell mass and trophectoderm distribution in murine, porcine and bovine embryos

Zygote ◽  
1997 ◽  
Vol 5 (4) ◽  
pp. 309-320 ◽  
Author(s):  
Rabindranath de la Fuente ◽  
W. Allan King
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Total Cell ◽  
Differential Staining ◽  
Similar Proportion ◽  
Bovine Embryos ◽  
Total Cell Number ◽  
Cell Numbers

SummaryThe mammalian blastocyst comprises an inner cell mass (ICM) and a trophectoderm cell layer. In this study the allocation of blastomeres to either cell lineage was compared between murine, porcine and bovine blastocysts. Chemical permeation of trophectoderm cells by the Ca2+ ionophore A23187 in combination with DNA-specific fluorochromes resulted in the differential staining of trophectoderm and ICM. Confocal microscopy confirmed the exclusive permeation of trophectoderm and the internal localisation of intact ICM cells in bovine blastocysts. Overall, differential cell counts were obtained in approximately 85% of the embryos assessed. Mean (±SEM) total cell numbers were 72.2 ± 3.1 and 93.1±5 for in vivo derived murine (n = 41) and porcine (n = 21) expanded blastocysts, respectively. Corresponding ICM cell number counts revealed ICM/total cell number ratios of 0.27 and 0.21, respectively. Comparison of in vivo (n = 20) and in vitro derived bovine embryos on day 8 (n = 29) or day 9 (n = 29) revealed a total cell number of 195.25±9.9, 166.14±9.9 and 105±6.7 at the expanded blastocyst stage with corresponding ICM/total cell ratios of 0.27, 0.23 and 0.23, respectively. While total cell numbers differed significantly among the three groups of bovine embryos (p<0.05), the ICM/total cell ratio did not. These results indicate that a similar proportion of cells is allocated to the ICM among blastocysts of genetically divergent species.

155 QUALITY OF PORCINE EMBRYO PRODUCED IN TWO EMBRYO CULTURE MEDIA AS ASSESSED BY TOTAL CELL NUMBER AND TIME COURSE OF BLASTOCYST HATCHING

2006 ◽  
Vol 18 (2) ◽  
pp. 185 ◽  
Author(s):  
Y. Agca ◽  
H. Men ◽  
S. F. Mullen ◽  
L. K. Riley ◽  
R. S. Prather ◽  
...  
Keyword(s):  
Embryo Culture ◽  
Time Course ◽  
Culture Media ◽  
Cell Number ◽  
Total Cell ◽  
Total Cell Number ◽  
Blastocyst Hatching ◽  
Hatching Rates

The ability to produce porcine embryos of good quality will have a significant impact on a number of porcine assisted reproductive technologies, such as cloning, intracytoplasmic sperm injection, and embryo cryopreservation. However, porcine embryos resulting from current serum-free embryo culture systems differ significantly both structurally and functionally from those derived in vivo (Wang et al. 1999 Mol. Reprod. Dev. 53, 99-107). In this experiment, the quality of porcine embryos produced by North Carolina State University (NCSU)-23 medium (Petters and Wells 1993 J. Reprod. Fertil. Suppl. 1993, 48, 61-73) and porcine zygote medium (PZM)-1 (Yoshioka et al. 2002 Biol. Reprod. 66, 112-119) were compared by assessing the total cell number and the time course of in vitro blastocyst hatching. Porcine embryos were produced by in vitro maturation and fertilization using serum-free systems. After fertilization, presumptive zygotes were randomly allocated to either PZM-1 or NCSU-23 for subsequent development. On Day 4 of culture, the embryo culture media were supplemented with 10% fetal bovine serum (FBS). Day 6 blastocysts from each group were counted and the blastocysts were subsequently fixed in 4% formalin for counting the total cell number. The cell number in each embryo was determined by counting the nuclei after staining with bisbenzimide (Hoechst 33342). To assess the hatching ability of blastocysts, Day 6 blastocysts were cultured until Day 9 and hatched blastocysts were counted daily. Day 6 blastocyst rates (ratio of blastocysts to oocytes) and total cell number count were replicated three times. The time course of blastocyst hatching experiment was repeated four times. The data were analyzed using a chi-square test, Fisher's exact test, or Student's t-test. The blastocyst rate from culture in PZM-3 was 19.4 � 0.96% (mean � SEM), which was similar to that (16.7 � 3.2%) resulting from culture in NCSU-23 (P > 0.05). However, the total cell number in Day 6 blastocysts cultured in PZM-3 was significantly higher than for blastocysts cultured in NCSU-23 (57 � 3.1 vs. 46 � 1.7; P < 0.01). The total hatching rates (ratio of hatched blastocysts to total blastocysts) by Day 9 were similar between the two culture systems (50.1 � 9.1% vs. 50.7 � 4.1%; P > 0.05). However, on Day 6, 2.1% of blastocysts from PZM-3 culture hatched whereas no blastocysts from NCSU-23 culture hatched. The cumulative hatching rates from PZM-3 culture on Day 7 were significantly higher than those from NCSU-23 culture (15.1 � 3.8% vs. 2.6 � 1.1%; P < 0.01). In conclusion, these data suggest that blastocysts produced in PZM-3 medium have better quality than blastocysts produced in the NCSU-23 culture system as assessed by the total cell number and the time course of blastocyst hatching. This project was supported by a grant from the National Institutes of Health (U42 RR 018877).

scholarly journals High revivability of vitrified–warmed bovine mature oocytes after recovery culture with α-tocopherol

Reproduction ◽  
2015 ◽  
Vol 149 (4) ◽  
pp. 347-355 ◽  
Author(s):  
Ikuko Yashiro ◽  
Miho Tagiri ◽  
Hayato Ogawa ◽  
Kazuya Tashima ◽  
Seiji Takashima ◽  
...  
Keyword(s):  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Control Group ◽  
Mitochondrial Activity ◽  
Cortical Granules ◽  
Total Cell Number ◽  
Recovery Culture ◽  
Bovine Oocytes

The objective of this study was to investigate whether developmental competence of vitrified–warmed bovine oocytes can be improved by antioxidant treatment during recovery culture. In experiment 1, one of the two antioxidants (either l-ascorbic acid or α-tocopherol) was added as a supplement to the recovery culture medium to which postwarming oocytes were exposed for 2 h before IVF. The exposure to α-tocopherol had a positive effect on rescuing the oocytes as assessed by the blastocyst yield 8 days after the IVF (35.1–36.3% vs 19.2–25.8% in untreated postwarming oocytes). Quality of expanding blastocysts harvested on Day 8 was comparable between α-tocopherol-treated vitrification group and fresh control group in terms of total cell number and chromosomal ploidy. In experiment 2, level of reactive oxygen species, mitochondrial activity, and distribution of cortical granules in α-tocopherol-treated postwarming oocytes were assessed. No obvious differences from the control data were found in these parameters. However, the treatment with α-tocopherol increased the percentage of zygotes exhibiting normal single aster formation (90.3% vs 48.0% in untreated postwarming oocytes; 10 h post-IVF). It was concluded that α-tocopherol treatment of vitrified–warmed bovine mature oocytes during recovery culture can improve their revivability, as shown by the high blastocyst yield and the higher mean total cell number in the blastocysts.

Characterization and Growth Mechanism of B12As2 Epitaxial Layers Grown on (1-100) 15R-SiC

2008 ◽  
Vol 1069 ◽  
Author(s):  
Hui Chen ◽  
Guan Wang ◽  
Michael Dudley ◽  
Zhou Xu ◽  
James. H. Edgar ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
High Resolution ◽  
Growth Mechanism ◽  
Heteroepitaxial Growth ◽  
High Quality ◽  
X Ray ◽  
Transmission Electron ◽  
Substrate Choice

ABSTRACTA systematic study is presented of the heteroepitaxial growth of B12As2 on m-plane 15R-SiC. In contrast to previous studies of B12As2 on other substrates, including (100) Si, (110) Si, (111) Si and (0001) 6H-SiC, single crystalline and untwinned B12As2 was achieved on m-plane 15R-SiC. Observations of IBA on m-plane (1100)15R-SiC by synchrotron white beam x-ray topography (SWBXT) and high resolution transmission electron microscopy (HRTEM) confirm the good quality of the films on the 15R-SiC substrates. The growth mechanism of IBA on m-plane 15R-SiC is discussed. This work demonstrates that m-plane 15R-SiC is potentially a good substrate choice to grow high quality B12As2 epilayers.

The Influence of the Temperature Gradient on the Defect Structure of 3C-SiC Grown Heteroepitaxially on 6H-SiC by Sublimation Epitaxy

2010 ◽  
Vol 645-648 ◽  
pp. 367-370 ◽  
Author(s):  
Maya Marinova ◽  
Alkyoni Mantzari ◽  
Milena Beshkova ◽  
Mikael Syväjärvi ◽  
Rositza Yakimova ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
High Resolution ◽  
Temperature Gradient ◽  
Defect Structure ◽  
Temperature Gradients ◽  
Structural Quality ◽  
Fault Density ◽  
Transmission Electron

In the present work the structural quality of 3C-SiC layers grown by sublimation epitaxy is studied by means of conventional and high resolution transmission electron microscopy. The layers were grown on Si-face 6H-SiC nominally on-axis substrates at a temperature of 2000°C and different temperature gradients, ranging from 5 to 8 °C /mm. The influence of the temperature gradient on the structural quality of the layers is discussed. The formation of specific twin complexes and conditions for lower stacking fault density are investigated.

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