Technical Note: Effect of density gradient centrifugation with trypsin on the in vivo fertilising capability of bovine spermatozoa

2008 ◽  
Vol 20 (7) ◽  
pp. 784 ◽  
Author(s):  
Brock A. Blevins ◽  
Morne de la Rey ◽  
Naida M. Loskutoff
Keyword(s):  
Density Gradient ◽  
Standard Method ◽  
Silica Particle ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Bos Indicus ◽  
Egg Yolk ◽  
Technical Note ◽  
Bovine Spermatozoa

The present study investigated the effect of a novel density gradient centrifugation (DGC) treatment using recombinant trypsin on the in vivo fertilising capability of bovine spermatozoa compared with a standard method. In Trial 1, semen collected from Boran and Ankole (Bos indicus) bulls was treated either with a silane-coated silica particle colloid formulated for humans with a recombinant trypsin or processed using a standard method (dilution in an egg yolk-based diluent). Semen processed by the two methods was used to artificially inseminate (AI) superovulated cattle. Day 7 embryos were flushed and assessed for fertilisation rates and embryo quality. Trial 2 used a trypsinised silane-coated silica particle colloid formulated specifically for bovine semen. Trial 1 resulted in significantly higher fertilisation rates using the trypsinised human DGC treatment than cows inseminated using the standard method (75.2% v. 67%, respectively; P < 0.01), but the numbers of transferable-quality Day 7 embryos did not differ between the two groups (P > 0.05). Results for Trial 2 indicated that cows inseminated with the trypsinised bovine DGC treatment had significantly increased fertilisation rates compared with the standard method (88.4% v. 63.1%, respectively; P < 0.01) and had significantly higher numbers of transferable-quality embryos (70.3% v. 51.8%, respectively; P < 0.01). In summary, bovine sperm treatment before AI by DGC and recombinant trypsin increases fertilisation rates and can result in more transferable-quality embryos compared with standard methods.

7 EFFECT OF DENSITY GRADIENT CENTRIFUGATION WITH TRYPSIN ON THE FERTILIZING CAPABILITY OF BOVINE SPERM

2008 ◽  
Vol 20 (1) ◽  
pp. 84 ◽  
Author(s):  
B. A. Blevins ◽  
M. de la Rey ◽  
N. M. Loskutoff
Keyword(s):  
Protease Inhibitor ◽  
Standard Method ◽  
Silica Particle ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
The Novel ◽  
Fertilization Rates ◽  
Bovine Sperm ◽  
Fertilizing Capability

The goal of this research was to investigate the effect of a novel density gradient centrifugation (DGC) treatment on the fertilizing capability of bovine sperm as compared to a standard method. Domestic bull (Bos taurus) semen was used for AI and the production of embryos from in vivo-matured bovine oocytes. In 2004, a preliminary study compared the novel semen treatment using a trypsinized PVP-coated silica particle suspension (Percoll; Sigma, St. Louis, MO, USA) to a standard method (4� dilution with an egg yolk diluent) on the fertilizing capacity in vivo of bovine sperm (de la Rey et al. 2005 J. Reprod. Fertil. 17, 242 abst). Although not statistically significant (P = 0.69), there were more transferable quality embryos recovered from cows inseminated using the treated sperm method v. control (58.9 v. 43%, respectively). In this report we provide the results of two additional trials utilizing the novel semen treatment and substituting Percoll with a silane-coated silica particle medium containing a recombinant trypsin (r-protease). In the second trial (2005), semen samples collected from three bulls were processed by DGC: 2 mL of 40% PureSperm (NidaCon International AB, M�lndal, Sweden) containing recombinant trypsin (TrypLE Select, Gibco/Invitrogen, Carlsbad, CA, USA), which overlaid 2 mL of 80% PureSperm containing 10 µg mL–1 soy-based protease inhibitor (Sigma), was overlaid with the semen sample using a novel centrifuge tube insert (ProInsert, Nidacon) and then centrifuged at 300g for 20 min. The sperm pellets were recovered and washed (500g for 10 min) in 10 mL pre-warmed TL-HEPES medium (Cambrex Corp., East Rutherford, NJ, USA). The washed sperm pellets were then resuspended in the same total volume of pre-warmed Biladyl� A (Minit�b, Tiefenbach, Germany) as the standard method and used to AI a total of 42 (control) and 47 (treatment) superovulated cows three times at 12 h intervals. Day 7 embryos were recovered and assessed for stage and morphological quality. In Trial 3 (2006), semen samples collected from three bulls were processed by DGC containing r-protease and a soybean protease inhibitor (BoviPure Pro, NidaCon), media specifically formulated for domestic bull semen. Sperm pellets were washed in 10 mL BoviWash medium (NidaCon). The washed sperm pellet was resuspended in the same total volume of pre-warmed Biladyl A as the standard method and used to AI a total of 23 (control) and 25 (treatment) superovulated cows and embryos evaluated as in Trial 2. The results between control and treated groups were compared using the Mann-Whitney (Wixcoxon rank sum) test. Trial 2 using PureSperm tended to result in higher fertilization rates than for cows inseminated using the standard method (75.2% v. 67%, respectively) but the results were not statistically significant (P = 0.63). Results for Trial 3 indicated that cows inseminated with BoviPure Pro-treated sperm had significantly increased fertilization rates as compared to the standard method (88.4% v. 63.1%, respectively; P = 0.02) and had higher numbers of transferable quality embryos (70.3% v. 51.8%, respectively; P = 0.38). In summary, BoviPure Pro sperm treatment before AI significantly increases fertilization rates and can result in as much as an 18.5% increase in transferable quality embryos as compared to standard methods.

scholarly journals Separation of Degranulated Platelets from Normal Platelets

1977 ◽  
Author(s):  
P. Cieslar ◽  
J.P. Greenberg ◽  
M.A. Packham ◽  
R.L. Kinlough-Rathbone ◽  
J.F. Mustard
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Adenine Nucleotide ◽  
Thrombus Formation ◽  
Gradient Centrifugation ◽  
Rabbit Platelets ◽  
Fraction I

Platelets degranulated by thrombin (TDP) can be recovered, are effective in hemostasis and survive normally upon infusion into rabbits. Two approaches to determine whether platelets have been degranulated in vivo are: (1) measurement of circulating released materials; (2) detection of circulating degranulated platelets. We have used arabino-galactan (Stractan II) density gradient centrifugation to separate normal and degranulated platelets. The following distribution was obtained with washed rabbit platelets.The serotonin, PF4 and adenine nucleotide contents of the TDP were less than those of normal platelets and the TDP in fraction I had the lowest amounts. When TDP were labeled with 51cr and mixed with equal numbers of normal platelets, 85% of the platelets in fraction I were found to be TDP. 51Cr-TDP were injected into normal rabbits and reharvested after 18 hours. The greatest proportion of TDP was isolated in fraction I. Thus this method may make it possible to separate platelets that have lost their granule contents during participation in reversible thrombus formation in vivo.(* Visiting Fellow from the Faculty of Medicine, Charles University, Prague, Czechoslovakia.)

Analysis of selected sperm by density gradient centrifugation might aid in the estimation of in vivo fertility of thawed ram spermatozoa

2010 ◽  
Vol 74 (6) ◽  
pp. 979-988 ◽  
Author(s):  
O. García-Álvarez ◽  
A. Maroto-Morales ◽  
M. Ramón ◽  
E. del Olmo ◽  
V. Montoro ◽  
...  
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation

Microgravity Separation of Alginate Empty Capsules from Encapsulated Pancreatic Islets Using a Microfluidic System

2015 ◽  
Vol 15 (10) ◽  
pp. 7876-7880 ◽  
Author(s):  
Soojeong Shin ◽  
Young Je Yoo ◽  
Jong Wook Hong
Keyword(s):  
Pancreatic Islets ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Biomedical Application ◽  
Gradient Centrifugation ◽  
Previous Method ◽  
High Yield ◽  
One Pot ◽  
Optimal Separation

Although microencapsulated pancreatic islets have merits, such as ease of transplantation, viability and functionality improvement, and immune protection in vivo, the co-production of alginate empty capsules during the encapsulation of islets with alginate makes them unusable for biomedical application. In previous research, the removal of empty alginate capsules with high yield was achieved using density-gradient centrifugation. Here, we report advanced microgravity-based separation techniques in a microfluidic format for alginate empty capsules. The optimal separation conditions were mathematically evaluated using Stokes’ law and the separation of the encapsulation product was accomplished. A microfluidic chip was designed with two inlets and two outlets at different elevations to mimic the vertical percoll gradient in density-gradient centrifugation. The separation of alginate empty capsules using microgravitational force resulted in effective separation of encapsulated islets from alginate empty capsules with more than 70% efficiency. Moreover, no loss of encapsulated islets was expected because the process is a one-pot separation, unlike the previous method. This type of microgravitational particle separation could be used both for the fractionization of heterogeneous encapsulated cells and to remove empty capsules.

The polypeptide components of scintillons, the bioluminescence organelles of the dinoflagellate Gonyaulax polyedra

1993 ◽  
Vol 71 (3-4) ◽  
pp. 176-182 ◽  
Author(s):  
Michel Desjardins ◽  
David Morse
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Binding Protein ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Close Packing ◽  
Gonyaulax Polyedra

Scintillons, the bioluminescence organelles of Gonyaulax polyedra, were purified by isopycnic density gradient centrifugation until only low levels of contaminating chloroplasts and mitochondria were detected by fluorescence and electron microscopy. Purified scintillons catalyzed the luminescent reaction with kinetics identical to those observed during the bioluminescence flash in vivo. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the organelles appeared to contain only two proteins. These proteins were identified as luciferase (135 kilodaltons) and luciferin-binding protein (75 kilodaltons) based on their size and their known functions in the bioluminescence reaction in vitro. The staining of luciferin-binding protein by Coomassie blue was 2.4 ± 0.3 (n = 19) times greater than the luciferase, suggesting that there are four binding protein monomers for every luciferase monomer. A model is proposed for the close packing of the two proteins inside the scintillons.Key words: luciferase, luciferin-binding protein, density gradient centrifugation, dinoflagellate.

scholarly journals Delays during PBMC isolation have a moderate effect on yield, but severly compromise cell viability

2022 ◽  
Author(s):  
Tanja Golke ◽  
Patrick Mucher ◽  
Patricia Schmidt ◽  
Astrid Radakovics ◽  
Manuela Repl ◽  
...  
Keyword(s):  
Cell Viability ◽  
Density Gradient ◽  
Standard Method ◽  
Mononuclear Cells ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Initial Density ◽  
Minor Effect ◽  
Peripheral Blood Mononuclear ◽  
The Impact

Background: Peripheral blood mononuclear cells (PMBCs) are a versatile material for clinical routine as well as for research projects. However, their isolation via density gradient centrifugation is still time-consuming. When samples are taken beyond usual laboratory handling times, it may sometimes be necessary to pause the isolation process. Our aim was to evaluate the impact of delays up to 48 hours after the density gradient centrifugation on PBMC yield, purity and viability. Methods: PBMCs were isolated from samples of 20 donors, either with BD Vacutainer CPT tubes (CPT) or with the standard Ficoll method. Isolation was paused after initial density gradient centrifugation for 0, 24, or 48 hours. PBMC yield, purity and viability were compared. Results: The yield did not change significantly over time when CPT were used (55%/52%/47%), but did after isolation with the standard method (62%/40%[p<0.0001]/53%[p<0.01]). Purity was only affected if CPT were used (95%/93%[p=n.s./92%[p<0.05] vs. 97% for all time points with standard method). Whereas viable PBMCs decreased steadily for CPT isolates (62%/51%[p<0.001]/36%[p<0.0001]), after standard Ficoll gradient isolation, cell apoptosis was more pronounced already after 24h delay, and viability did not further decrease after 48h (64%/44%[p<0.0001]/40%[p<0.0001]). Conclusions: In conclusion, our data suggests that post-centrifugation delays of up to 48h might have only a minor effect on cell yield and purity. However, at the same time, a relevant decrease in cell viability was observed, which could be partially compensated by the use of CPT if the isolation was resumed latest the day after blood withdrawal.

Inhibition of RNA Synthesis by α-Amanitin in Vivo

1970 ◽  
Vol 25 (10) ◽  
pp. 1119-1125 ◽  
Author(s):  
J. Niessing ◽  
B. Schnieders ◽  
W. Kunz ◽  
K. H. Seifart ◽  
C. E. Sekeris
Keyword(s):  
Density Gradient ◽  
Column Chromatography ◽  
Orotic Acid ◽  
Rna Synthesis ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation

Injection of α-amanitin inhibits both the synthesis of ribosomal and DNA-like RNA as measured by the incorporation of radioactively labelled orotic acid and ortho-phosphate into RNA extracted by phenol and subsequently fractionated by density gradient centrifugation and MAK column chromatography.

Investigation of In Vivo Factors that May Influence Platelet Survival and Density

1979 ◽  
Author(s):  
M.A. Packham ◽  
J.F. Mustard ◽  
M.A. Guccione ◽  
P. D. Winocour ◽  
H.M. Groves ◽  
...  
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Platelet Membrane ◽  
Membrane Glycoproteins ◽  
Aortic Endothelium ◽  
Platelet Survival ◽  
Rabbit Platelets

Platelet survival CPS) is shortened in a number of conditions but the mechanisms responsible are unclear. In rabbits, removal of the aortic endothelium or injury of the neointima does not shorten PS. However, induction of thrombi in rabbit aortae with Indwelling cannulae (IDC) shortens FS (IDC 37.0 hr, control 79.6 hr), and Increases the proportion of platelets in the lightest fraction upon straetan density gradient centrifugation. Therefore we examined the effect of agents to which platelets may be exposed during thromboembolism (ADP, thrombin, plasmin) on PS and platelet density. ADP treatnent of washed rabbit platelets did not alter their survival but did increase the proportion in the lightest fraction. Treatment of platelets with thrombin did not shorten PS but increased the proportion in the lightest fraction. Treatment with plasmin in vitro shortened PS (plasmin, 57.6 ± 6.0 hr, control 80.2 ± 4,2 hr) and increased the proportion in the lightest fraction. Thus changes in platelet density are not necessarily associated with changes in PS. Of the factors investigated that are known to be involved in thromboembolism, only plasmin shortened PS. This may be due to its ability to alter major platelet membrane glycoproteins(principally glycoproteins I and II of rabbit platelets).

Studies on Human Platelets

1971 ◽  
Vol 26 (01) ◽  
pp. 167-176 ◽  
Author(s):  
F. M Booyse ◽  
Dorothea Zschocke ◽  
T. P Hoveke ◽  
M. E Rafelson
Keyword(s):  
Amino Acids ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Protein A ◽  
Human Platelets ◽  
Lower Content ◽  
Gradual Loss

SummaryHuman platelets have been separated into 4 more homogeneous populations (A, B, C, and D; D population of highest density) by isopycnic density gradient centrifugation. Newly formed platelets have been identified in band C. These newly formed platelets age in vivo and shift into band D containing mature and old platelets. Newly formed platelets are more active in their ability to incorporate labeled amino acids into protein than mature and/or older platelets. The maturation of newly formed platelets (shift from band C to band D) is concurrent with a gradual loss in their ability to synthesize protein, a increase in density and decrease in size. In addition, newly formed platelets have a lower content of thrombosthenin than do mature (old) platelets.

Complement Activation and Cytotoxicity In CLL Cells Treated with Alemtuzumab and Ofatumumab: Evidence for Multiple Mechanisms of Resistance to Complement Dependent Cytotoxicity

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3575-3575 ◽  
Author(s):  
Nisar A Baig ◽  
Amy K Church ◽  
Grzegorz S. Nowakowski ◽  
Ronald P Taylor ◽  
Margaret A Lindorfer ◽  
...  
Keyword(s):  
Density Gradient ◽  
Complement Activation ◽  
Research Funding ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Mechanisms Of Resistance ◽  
Intrinsic Resistance ◽  
Cd20 Expression ◽  
Small Subpopulation

Abstract Abstract 3575 Unconjugated monoclonal antibodies (MoAb) have proven value in the therapy of CLL, and alemtuzumab (ALEM) has efficacy in patients with purine analog refractory/p53 defective disease. Although the mechanisms of action of ALEM in vivo are not fully defined, complement dependant cytotoxicity (CDC) appears to play an important role. ALEM CDC resistance in a subpopulation of CLL cells in vivo could thus contribute to treatment failure. We hypothesized that the CD20 targeted complement activating human MoAb ofatumumab (OFA) would increase complement activation (as manifested by C3b deposition) as well as CDC in CLL cells treated with ALEM in vitro. Methods: We have completed experiments on 4 of 15 specimens collected from patients with progressive untreated CLL. CLL cells were isolated from blood by density gradient centrifugation and negative selection with magnetic beads. CD52 and CD20 expression were measured with fluorescent beads (Quantum MESF, Bangs Laboratories, IN). CLL cells were treated at a concentration of 2 × 106/ml with 10 mcg/ml of ALEM (Genzyme) and/or OFA (G.S.K.) in AIM V medium (Invitrogen CA) and 10% standard normal human serum (NHS) (Sigma, MO) as a source of complement for 1 hour at 37C. Absolute viable cell counts were assayed with counting beads (TruCOUNT, BD, CA) and propidium iodide (PI) staining (Sigma) using a FACSCalibur (BD) and CellQuest Pro software (BD). CDC was determined by comparing counts to CLL cells treated with NHS alone. Membrane levels of CD59, bound MoAb, and deposited C3b on the CLL cells membranes were measured by flow cytometry using using the anti-CD59 antibody FITC-CD59 (BD), mouse anti-human Fc antibody FITC-HB43, and the anti-C3b antibody FITC-7C12. To study ALEM CDC resistant cells, CLL cells were first treated with ALEM and NHS as detailed above. Surviving viable cells were isolated using density gradient centrifugation and then retreated. C5 deficient serum (Sigma) was used in experiments to determine MoAb binding and C3b deposition in the entire CLL cell population. Results: Binding of either ALEM or OFA to the CLL cells in NHS resulted in activation of complement and deposition of large amounts of membrane C3b on the cells. However, ALEM induced higher levels of C3b deposition (delta MFI 220) and caused more CDC (73% vs. 18%) compared to OFA. This correlated well with the higher levels of expression of CD52 vs. CD20 expression in CLL cells (143 ×103, range 131 ×103 – 159 ×103 vs. mean 20 ×103 molecules/cell, range 10 ×103 – 28 ×103). Studies with the anti-Fc antibody showed that only a small subpopulation of CLL cells had weak MoAb binding. Addition of OFA to ALEM increased complement-mediated C3b deposition. In three patients with high initial ALEM CDC, addition of OFA did not appreciably increase CDC (81% to 87%). However, in one patient with cells that were less sensitive to ALEM CDC, addition of OFA did appreciably increase CDC (51% to 86%). There was no relationship between levels of CD59 expression and CDC. All four CLL specimens studied contained a subpopulation of cells that were resistant to CDC (mean 13%, range 9–18%) despite robust C3b deposition. Conclusions: These data suggests that there are multiple mechanisms of resistance to MoAb-mediated CDC including: 1. Low MoAb binding because of low target antigen expression; 2. Insufficient C3b deposition after MoAb ligation and; 3. Intrinsic resistance in some cells in cells in which high levels of C3b deposition were clearly demonstrable. OFA can increase MoAb binding, C3b deposition, and CDC in CLL cells treated with ALEM. However, addition of OFA does not overcome the intrinsic resistance of a small subpopulation of CLL cells to CDC. Future studies will be needed to examine these resistance mechanisms and to develop interventions to disrupt them. In addition, membrane C3b could be a target for complement dependent cellular cytotoxity in these CDC resistant cells. Disclosures: Taylor: G.S.K.: Research Funding; Genmab: Research Funding. Zent:Genzyme: Research Funding; Genentech: Research Funding; Novartis: Research Funding; G.S.K.: Research Funding.

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