VALIDATION OF QUANTITATIVE FLUORESCENCE FOR DETERMINING LIPID CONTENT OF BOVINE BLASTOCYSTS STAINED WITH NILE RED DYE

2007 ◽  
Vol 77 (Suppl_1) ◽  
pp. 87-88 ◽  
Author(s):  
Moises Barcelo-Fimbres ◽  
George Seidel
Keyword(s):  
Lipid Content ◽  
Nile Red ◽  
Red Dye ◽  
Quantitative Fluorescence

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

scholarly journals Generation of microplastics from the opening and closing of disposable plastic water bottles

2021 ◽  
Author(s):  
Tvisha Singh
Keyword(s):  
Particle Density ◽  
Nile Red ◽  
Bottled Water ◽  
Particle Generation ◽  
Large Spread ◽  
Total Particle ◽  
Contamination Levels ◽  
Opening And Closing ◽  
Red Dye ◽  
Disposable Plastic

Abstract There has recently been a significant increase in interest regarding the prevalence of microplastics in bottled water. Previous studies have shown that the composition of many of the microplastics in bottled water is consistent with the materials of the bottle and bottle cap. The focus of this study is to quantify microplastic particle generation from the cap and bottle interaction during open and close cycles. Nile Red dye was used for the detection of microplastics &gt;4.7 μm in size. Microplastic contamination levels in the water were found to increase as the bottle cap is opened and closed repeatedly. The rate of generation of particles with bottle opening and closing cycles (553 ± 202 microplastics/L/cycle) is adequate to account for the total particle density in the water. This clearly demonstrates that the abrasion between the bottle cap and bottleneck is the dominant mechanism for the generation of microplastic contamination detected in bottled water. A large spread between the maximum and minimum levels of microplastic contamination for bottles from the same lot, regardless of the number of times the cap is opened and closed, suggests that mechanical tolerances in the manufacturing of bottles and caps might play an important role in microplastic generation.

scholarly journals Silicon Nanomembrane Filtration and Imaging for the Evaluation of Microplastic Entrainment along a Municipal Water Delivery Route

2020 ◽  
Vol 12 (24) ◽  
pp. 10655
Author(s):  
Gregory R. Madejski ◽  
S. Danial Ahmad ◽  
Jonathan Musgrave ◽  
Jonathan Flax ◽  
Joseph G. Madejski ◽  
...  
Keyword(s):  
Drinking Water ◽  
Water Transport ◽  
Transport Process ◽  
Nile Red ◽  
Machine Learning Algorithms ◽  
Hydrophobic Polymers ◽  
Silicon Nanomembrane ◽  
Debris Transport ◽  
End Stage ◽  
Red Dye

To better understand the origin of microplastics in municipal drinking water, we evaluated 50 mL water samples from different stages of the City of Rochester’s drinking water production and transport route, from Hemlock Lake to the University of Rochester. We directly filtered samples using silicon nitride nanomembrane filters with precisely patterned slit-shaped pores, capturing many of the smallest particulates (<20 µm) that could be absorbed by the human body. We employed machine learning algorithms to quantify the shapes and quantity of debris at different stages of the water transport process, while automatically segregating out fibrous structures from particulate. Particulate concentrations ranged from 13 to 720 particles/mL at different stages of the water transport process and fibrous pollution ranged from 0.4 to 8.3 fibers/mL. A subset of the debris (0.2–8.6%) stained positively with Nile red dye which identifies them as hydrophobic polymers. Further spectroscopic analysis also indicated the presence of many non-plastic particulates, including rust, silicates, and calcium scale. While water leaving the Hemlock Lake facility is mostly devoid of debris, transport through many miles of piping results in the entrainment of a significant amount of debris, including plastics, although in-route reservoirs and end-stage filtration serve to reduce these concentrations.

106 USING A BUOYANT DENSITY GRADIENT AND NILE RED STAINING TO EVALUATE THE LIPID CONTENT OF BOS TAURUS AND BOS INDICUS OOCYTES

2007 ◽  
Vol 19 (1) ◽  
pp. 170 ◽  
Author(s):  
C. B. Ballard ◽  
C. R. Looney ◽  
B. R. Lindsey ◽  
J. H. Pryor ◽  
J. W. Lynn ◽  
...  
Keyword(s):  
Survival Rate ◽  
Density Gradient ◽  
Lipid Content ◽  
Bos Taurus ◽  
Buoyant Density ◽  
Bos Indicus ◽  
Nile Red ◽  
Intracellular Lipid ◽  
Lower Survival Rate ◽  
Nile Red Staining

Bos indicus embryos have a lower survival rate compared with Bos taurus after cryopreservation. It has been hypothesized that the lower survival rate is due to higher intracellular lipid content. The objective of this study was to determine if there is a difference in intracellular lipid content of oocytes from mature purebred Brahman and Angus cows. Donor females used in the study were maintained on pasture prior to the onset of the experiment and on a grain-supplemented hay ration during the study. Oocytes were collected from cows at 30-day intervals (3 aspirations/donor) by transvaginal ultrasound-guided oocyte aspirations (TUGA). Mature oocytes were evaluated using a sucrose step gradient procedure and Nile Red staining. FSH (Folltropin-V®; Bioniche Animal Health, Beltsville, Ontario, Canada) administration began on Day 4 of the estrous cycle (estrus = Day 0) twice daily for 3 days in decreasing doses (Brahman 232 mg and Angus 280 mg total), and on Day 8 oocytes were recovered. The mean number of follicles aspirated/donor and oocytes recovered/donor were 20 and 16.61 oocytes for the Brahman donors (n = 6) and 12 follicles and 7.06 oocytes/donor for the Angus donors (n = 10). Oocytes (individual donor basis) were then incubated in TCM-199 supplemented with 10% fetal bovine serum + bLH and bFSH (0.01 U mL−1) at 38.5°C. After 20 h, mature oocytes were denuded by vortexing for 3 min in HEPS + BSA (4 mg mL−1). Buoyancy was tested for individual mature oocytes using a sucrose step density gradient column prepared with sucrose and Dulbecco's PBS. Results from the sucrose gradients ranged from 23% sucrose (indicating high lipids) to 35% sucrose (indicating lower lipids). Oocytes recovered from the sucrose were fixed for 24 h in paraformaldehyde for evaluation with Nile Red stain. Oocytes were stained for 24 h, and then placed in Prolong® Gold (Invitrogen, Carlsbad, CA, USA) and evaluated under fluorescence. Oocytes images were evaluated using a Scion Image camera (Scion Corp., Frederick, MD, USA) to calculate mean (± SE) Nile Red units (NRU) (higher NRU = higher lipid content). Treatment groups were analyzed using one-way ANOVA. In summary, Brahman M-II oocytes had significantly lower (P ≤ 0.05) buoyant density, with a significantly higher mean NRU score, when compared with oocytes harvested from Angus donors (Table 1). Based on these results, Brahman oocytes have a higher intracellular lipid content then Angus oocytes. Table 1.Percent sucrose levels and Nile Red units for bovine oocytes from three replicates per donor

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

109 EFFECTS OF LIPOLYTIC AGENTS FORSKOLIN, EPINEPHRINE AND CAFFEINE ON EMBRYONIC DEVELOPMENT AND LIPID CONTENT OF BOVINE EMBRYOS PRODUCED IN VITRO

2009 ◽  
Vol 21 (1) ◽  
pp. 154 ◽  
Author(s):  
M. Barcelo-Fimbres ◽  
G. E. Seidel
Keyword(s):  
Amino Acids ◽  
Fatty Acid ◽  
Embryonic Development ◽  
Lipid Content ◽  
Lipid Accumulation ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Nile Red ◽  
Essential Amino Acids

The objective of this experiment was to evaluate lipid accumulation and embryonic development of bovine morulae treated with different chemicals. A total of 2619 slaughterhouse oocytes from heifers and mature cows were matured in CDM medium (similar to SOF) plus 0.5% fatty acid-free BSA and hormones (M-CDM) for 23 h at 38.5°C in 5% CO2 in air. Frozen–thawed sperm were centrifuged through a Percoll gradient and co-cultured with matured oocytes for 18 h in F-CDM (CDM+heparin). Zygotes were cultured at 38.5°C in 5% CO2/5% O2/90% N2 in CDM-1 with nonessential amino acids, 10 μm EDTA, 0.5% fatty acid free BSA, and 0.5 mm fructose. After 60 h, resulting 8-cell embryos were cultured 120 h in CDM-2 (CDM-1+essential amino acids and 2 mm fructose). A factorial design was used with 7 treatments, 2 ovary sources (cows v. heifers), and 3 bulls (A, B and C) replicated twice for each bull (6 replicates). At Day 2.5 embryo cleavage and 8-cell rates were evaluated, and on Day 6 a total of 755 morulae were randomly assigned to the 7 treatments (control, 2 and 8 mm caffeine, 1 and 4 μm epinephrine, and 10 and 40 μm forskolin). To quantify lipid accumulation, Day 7 blastocysts were fixed and stained with 1 μg mL–1 Nile red dye, after which a digital photograph of the equatorial part of the embryo (including the inner cell mass) was taken at 200×, and fluorescence intensity was measured with Image Pro software from 0 to 255 shades for each pixel (0 = no lipids; 255 = greatest lipid accumulation), as previously reported (Biol. Reprod. 2007 (Suppl. 1), 87–88). Data were analyzed by ANOVA. No differences in cleavage rates (75 v. 68 ± 3.6%) or eight cell rates (61 ± v. 57 ± 2.8%) were found for heifer v. cow oocytes (P > 0.1); however, blastocyst rates per oocyte and per 8-cell embryo were greater for cows than heifers (20 v. 10 ± 2.1%, and 68 v. 35 ± 3.8%, respectively; P < 0.05). Treatments: 2 and 8 mm caffeine produced fewer blastocysts per morula than 1 and 4 μm epinephrine, 10 and 40 μm forskolin and the control (39, 5 v. 54, 49, 48, 54 and 52 ± 5.8%; respectively) (P < 0.01). More lipid content was found in whole embryos and trophoblast of heifer-derived than cow blastocysts (P < 0.05), and forskolin resulted in less lipid content than control, caffeine- and epinephrine-treated morulae in whole embryos, embryonic mass and trophoblasts (P < 0.05; Table 1). In conclusion, mature cows were a better source of oocytes than feedlot heifers for embryonic development. High doses of caffeine were detrimental to embryos, and the addition of the lypolitic agent forskolin reduced lipid content relative to control, caffeine and epinephrine-treated embryos. Table 1.Main effect treatment means of lipid content (arbitrary fluorescence units)

45 SURVIVAL OF HOLSTEIN IN VITRO-PRODUCED EMBRYOS CULTURED IN NOVEL SYNTHETIC OVIDUCTAL FLUID MEDIA (SCF1) AND DEHYDRATED PRIOR TO CRYOPRESERVATION

2017 ◽  
Vol 29 (1) ◽  
pp. 129 ◽  
Author(s):  
C. M. Owen ◽  
M. Barceló-Fimbres ◽  
J. L. Altermatt ◽  
L. F. Campos-Chillon
Keyword(s):  
Lipid Content ◽  
Nile Red ◽  
Equilibration Time ◽  
Step Size ◽  
A Cell ◽  
Blastocyst Rate ◽  
Mitotracker Red ◽  
Main Effects ◽  
Conventional Freezing

In vitro-produced (IVP) embryos experience poor cryotolerance due to metabolic changes during in vitro culture causing increased lipid accumulation and apoptosis post-thaw. We hypothesised that embryos cultured in a novel SOF for conventional freezing media (SCF1), dehydrated, and allowed longer equilibration before conventional slow freezing would increase post-thaw survival and decrease apoptosis. IVP embryos were produced in 9 replicates by oocytes (n = 3172) aspirated from abattoir ovaries, matured for 23 h, fertilized with semen from 1 of 4 bulls, and cultured in conventional SOF media or SCF1 in 38.5°C in 5% O2, 5% CO2, and 90% N2. Stage 7 blastocysts were stained with 1 µg mL−1 Nile Red for lipid content and 300 nM Mitotracker Red CMX-Rosamine for mitochondrial polarity. Remaining blastocysts were slow-frozen by 1 of 4 protocols: 2-min dehydration in 0 or 0.6 M sucrose in holding media before equilibration (10 or 20 min) in conventional freezing media (1.5 M ethylene glycol and 0.5 M sucrose in holding media). Embryos were thawed and assessed for re-expansion at 48 h and surviving embryos were stained with 4′6-diamidino-2-phenylindole (DAPI) and a TUNEL assay to determine apoptosis. Ten images per embryo were acquired by confocal microscopy using a 5-µM step size at 40× magnification. Fluorescence of Nile Red and Mitotracker was measured by IMAGE PRO software, and cells stained for TUNEL were analysed by a cell counter plug-in. Blastocyst rate, Nile Red, and Mitotracker data (Table 1) were analysed by one-way ANOVA and means separated by Tukey’s HSD. Post-thaw survival and apoptotic levels (Table 1) were analysed as a factorial 2 (SOF and SCF1) × 2 (0 and 0.6 M sucrose) × 2 (10 and 20 min), and means separated by Tukey’s HSD. No interactions occurred between factors so they were dropped from the model and only main effects are shown. Results indicate that SCF1 increased blastocyst rate, mitochondrial polarity, and post-thaw survival and decreased lipid content and post-thaw apoptosis (P < 0.01). A 20-min equilibration time decreased apoptosis (P < 0.01) and tended to increase post-thaw survival (P < 0.1), suggesting that cryotolerance is improved in embryos cultured in SCF1 and equilibrated for 20 min. Table 1.Effect of media on development, lipid content and mitochondrial polarity (top) and of media, equilibration and dehydration on post-thaw survival and apoptosis (bottom)

Evaluation of the Lipid Content in Bovine Oocytes and Embryos with Nile Red: a Practical Approach

2005 ◽  
Vol 40 (1) ◽  
pp. 76-78 ◽  
Author(s):  
JLMR Leroy ◽  
G Genicot ◽  
I Donnay ◽  
A Van Soom
Keyword(s):  
Lipid Content ◽  
Nile Red ◽  
Practical Approach ◽  
Bovine Oocytes

Influence of the composition and structure of epoxy siloxane matrix on the spectral behavior of the nile red dye: I. Sol-gel system based on tetraethoxysilane and a mixture of epoxy resins

2009 ◽  
Vol 35 (1) ◽  
pp. 87-93 ◽  
Author(s):  
T. G. Movchan ◽  
T. V. Khamova ◽  
O. A. Shilova ◽  
Yu. A. Plachev ◽  
N. P. Sokolova ◽  
...  
Keyword(s):  
Epoxy Resins ◽  
Sol Gel ◽  
Nile Red ◽  
Spectral Behavior ◽  
Composition And Structure ◽  
Red Dye ◽  
Gel System
Sign in / Sign up

Export Citation Format

Share Document