150 α6 Integrin for evaluating sperm quality in bulls with different capacities of invitro embryo production

2020 ◽  
Vol 32 (2) ◽  
pp. 201
Author(s):  
E. G. A. Perez ◽  
D. L. D'Ercole ◽  
M. H. Macedo ◽  
A. A. F. Rodrigues ◽  
R. F. Gonçalves
Keyword(s):  
Bovine Serum Albumin ◽  
Fluorescence Intensity ◽  
Bovine Serum ◽  
Sperm Quality ◽  
Flow Cytometric Analysis ◽  
Fluorescein Isothiocyanate ◽  
Embryo Production ◽  
Logarithmic Amplifier ◽  
Flow Cytometric ◽  
The Mean

The α6 integrin, an adhesion molecule, is expressed on bovine sperm, but major questions about the role of integrins in sperm-oocyte fusion remain unsolved. In this work, we show the results of characterisation of sperm α6 integrin from 4 bulls with different capacities of invitro embryo production using flow cytometric analysis. The bull capacities judged by the rate of blastocyst formation after invitro embryo production with semen from 5 ejaculates per animal were 44.3, 17.1, 13.2, and 15.0% for bulls 1, 2, 3, and 4, respectively (P<0.05). For flow cytometric analysis, surface expression of α6 integrin was evaluated using a fluorescence-activated cell sorter (FACScan, Coulter Electronics) using a 520-nm excitation from an argon laser at 150 mW for excitation. Frozen-thawed sperm were centrifuged at 700×g for 10min and washed once in warm phosphate buffered saline (PBS). Briefly, the spermatozoa (5×105 cells per sample) were resuspended in 100μL of PBS containing 1% bovine serum albumin. After washing 3 times, the live cells were incubated at room temperature for 1h with 100μL (1:500) of α6 monoclonal antibody (Chemicon) in PBS (0.1M, pH 7.4) with 1% bovine serum albumin. After washing three times, the cells were incubated at 4°C for 1h in the dark with the fluorescein isothiocyanate-conjugated F(ab)2 fragment of affinity isolated goat anti-mouse antibody (Invitrogen). The cells were washed three times, resuspended in 100μL of PBS, and analysed. For each sample, 10 000 cells were recorded at a flow rate of 200-300 cells s−1 using forward scatter (cell size) and side angle of light scatter (cell density), the first using a logarithmic amplifier and the second using a linear amplifier. The fluorescence data were collected using the logarithmic amplifier. The percentage of positive cells and the mean fluorescence channel on a 1023-channel scale were calculated using Epics Profile II software (Epics II Software). To define the forward and side-scatter regions corresponding to sperm, binding of fluorescein isothiocyanate-conjugated Pisum sativum agglutinin to the acrosome was used for setting the bitmap on the dot plot. Initially, the percentage of spermatozoa stained with α6 antibody was calculated on a per-individual basis. Subsequently, the mean±standard deviation was calculated for each group. The Mann-Whitney U test was used to compare the differences in the expression of α6 between groups. Expression of α6 integrin was higher in bull 1 (control) than in bulls with low capacities of invitro embryo production. The spermatozoa from bull 1 was distributed in a single broad peak with a mean fluorescence intensity of 36.16±4.17%. The increased distance of the fluorescence intensity in bull 1 compared with the weak fluorescence peaks in the others bulls reflects the increased width and distance of the population distribution. In conclusion, α6 integrin may be used as a biomarker to evaluate sperm quality. This study was supported by FAPESP grants 2010/01077-9, 2011/18085-7, and 2016/00976-6.

263. Substitution of skim milk with bovine serum albumin in a stallion semen diluent

2008 ◽  
Vol 20 (9) ◽  
pp. 63
Author(s):  
Z. Gibb ◽  
C. G. Grupen ◽  
L. H. A. Morris ◽  
G. Evans ◽  
W. M. C. Maxwell
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Bovine Serum ◽  
Sperm Quality ◽  
Skim Milk ◽  
Lower Percentage ◽  
Animal Science ◽  
Flow Cytometric ◽  
Alternative Protein ◽  
Protective Proteins

Skim milk has long been utilised as a source of protective proteins in stallion semen diluents. However, skim milk is also thought to contain components that are toxic to sperm and reduces the clarity of sperm suspensions, which impedes sperm assessments. This may also reduce the effectiveness of staining procedures used to process sperm for flow cytometric sex-sorting. The aim of this study was to ascertain the optimal concentration of bovine serum albumin (BSA) to replace skim milk in a traditional stallion semen diluent, Kenney's Modified Tyrode's (KMT) Medium1, for handling and processing stallion sperm before flow cytometric sex-sorting. Two ejaculates were collected from each of three pony stallions. Each ejaculate was divided into five aliquots and diluted in either KMT with skim milk or KMT supplemented with 0, 0.25, 0.5 or 1% BSA. Diluted samples were further divided into two aliquots and either stored at 15°C for 18 h before incubation and assessment, or incubated and assessed immediately upon arrival. Samples were incubated at 34°C and evaluated at 0, 45 and 90 min for objective motility and acrosome integrity. No interactions were observed between any treatments over time. There was a lower percentage of intact and a higher percentage of detached acrosomes for sperm incubated in KMT containing 0% BSA than all other treatments. A greater proportion of sperm incubated in KMT with skim milk had partial acrosome damage compared with other treatments. There was no difference in % total motility for sperm incubated in KMT with skim milk, and KMT containing 0.5 and 1% BSA (Table 1). These results indicate that BSA may be suitable as an alternative protein source in stallion semen diluents. Further studies are required to compare sex-sorting rates and sperm quality after sex-sorting, incubation and staining in skim milk compared with BSA-based media. (1) Padilla, A W and Foote, R H 1991, ‘Extender and centrifugation effects on the motility patterns of slow-cooled stallion spermatozoa’, Journal of Animal Science, vol. 69, no. 8, pp. 3308–331

Expression of the cell adhesion molecules on leukocytes that demarginate during acute maximal exercise

1999 ◽  
Vol 86 (3) ◽  
pp. 970-976 ◽  
Author(s):  
Stephan F. van Eeden ◽  
John Granton ◽  
Jennifer M. Hards ◽  
Barbara Moore ◽  
James C. Hogg
Keyword(s):  
Fluorescence Intensity ◽  
Mean Fluorescence Intensity ◽  
Maximal Exercise ◽  
Flow Cytometric Analysis ◽  
Cycle Ergometer ◽  
Phenotypic Characteristics ◽  
Flow Cytometric ◽  
The Mean ◽  
Low Levels

The pulmonary vascular bed is an important reservoir for the marginated pool of leukocytes that can be mobilized by exercise or catecholamines. This study was designed to determine the phenotypic characteristics of leukocytes that are mobilized into the circulation during exercise. Twenty healthy volunteers performed incremental exercise to exhaustion [maximal O2consumption (V˙o2 max)] on a cycle ergometer. Blood was collected at baseline, at 3-min intervals during exercise, atV˙o2 max, and 30 min after exercise. Total white cell, polymorphonuclear leukocyte (PMN), and lymphocyte counts increased with exercise toV˙o2 max( P < 0.05). Flow cytometric analysis showed that the mean fluorescence intensity of L-selectin on PMN (from 14.9 ± 1 at baseline to 9.5 ± 1.6 atV˙o2 max, P < 0.05) and lymphocytes (from 11.7 ± 1.2 at baseline to 8 ± 0.8 atV˙o2 max, P < 0.05) decreased with exercise. Mean fluorescence intensity of CD11b on PMN increased with exercise (from 10.2 ± 0.6 at baseline to 25 ± 2.5 atV˙o2 max, P < 0.002) but remained unchanged on lymphocytes. Myeloperoxidase levels in PMN did not change with exercise. In vitro studies showed that neither catecholamines nor plasma collected atV˙o2 maxduring exercise changed leukocyte L-selectin or CD11b levels. We conclude that PMN released from the marginated pool during exercise express low levels of L-selectin and high levels of CD11b.

RADIOIMMUNOASSAY OF OESTRONE AND OESTRADIOL IN THE PERIPHERAL PLASMA OF THE DOMESTIC FOWL IN VARIOUS PHYSIOLOGICAL STATES AND OF HYPOPHYSECTOMIZED AND OVARIECTOMIZED FOWLS

1974 ◽  
Vol 75 (1) ◽  
pp. 133-140 ◽  
Author(s):  
B. E. Senior
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Diethyl Ether ◽  
Bovine Serum ◽  
Domestic Fowl ◽  
Laying Hens ◽  
Peripheral Plasma ◽  
The Mean ◽  
Precision And Accuracy ◽  
Chicken Ovary

ABSTRACT A radioimmunoassay was developed to measure the levels of oestrone and oestradiol in 0.5–1.0 ml of domestic fowl peripheral plasma. The oestrogens were extracted with diethyl ether, chromatographed on columns of Sephadex LH-20 and assayed with an antiserum prepared against oestradiol-17β-succinyl-bovine serum albumin using a 17 h incubation at 4°C. The specificity, sensitivity, precision and accuracy of the assays were satisfactory. Oestrogen concentrations were determined in the plasma of birds in various reproductive states. In laying hens the ranges of oestrone and oestradiol were 12–190 pg/ml and 29–327 pg/ml respectively. Levels in immature birds, in adult cockerels and in an ovariectomized hen were barely detectable. The mean concentrations of oestrone and oestradiol in the plasma of four non-laying hens (55 pg/ml and 72 pg/ml respectively) and one partially ovariectomized hen (71 pg/ml and 134 pg/ml respectively) were well within the range for laying hens. It is evident that the large, yolk-filled follicles are not the only source of oestrogens in the chicken ovary.

scholarly journals Flow Cytometric Analysis of T, B, and NK Cells Antigens in Patients with Mycosis Fungoides

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Serkan Yazıcı ◽  
Emel Bülbül Başkan ◽  
Ferah Budak ◽  
Barbaros Oral ◽  
Şaduman Balaban Adim ◽  
...  
Keyword(s):  
Cell Surface ◽  
Mycosis Fungoides ◽  
Peripheral Blood ◽  
Prognostic Value ◽  
Flow Cytometric Analysis ◽  
Surface Antigens ◽  
Cell Surface Antigens ◽  
Flow Cytometric ◽  
Hla Dr ◽  
The Mean

We retrospectively analyzed the clinicopathological correlation and prognostic value of cell surface antigens expressed by peripheral blood mononuclear cells in patients with mycosis fungoides (MF). 121 consecutive MF patients were included in this study. All patients had peripheral blood flow cytometry as part of their first visit. TNMB and histopathological staging of the cases were retrospectively performed in accordance with International Society for Cutaneous Lymphomas/European Organization of Research and Treatment of Cancer (ISCL/EORTC) criteria at the time of flow cytometry sampling. To determine prognostic value of cell surface antigens, cases were divided into two groups as stable and progressive disease. 17 flow cytometric analyses of 17 parapsoriasis (PP) and 11 analyses of 11 benign erythrodermic patients were included as control groups. Fluorescent labeled monoclonal antibodies were used to detect cell surface antigens: T cells (CD3+, CD4+, CD8+, TCRαβ+, TCRγδ+, CD7+, CD4+CD7+, CD4+CD7−, and CD71+), B cells (HLA-DR+, CD19+, and HLA-DR+CD19+), NKT cells (CD3+CD16+CD56+), and NK cells (CD3−CD16+CD56+). The mean value of all cell surface antigens was not statistically significant between parapsoriasis and MF groups. Along with an increase in cases of MF stage statistically significant difference was found between the mean values of cell surface antigens. Flow cytometric analysis of peripheral blood cell surface antigens in patients with mycosis fungoides may contribute to predicting disease stage and progression.

scholarly journals Uptake of fluorescent-labeled BSA into root cells: endocytosis?

2014 ◽  
Vol 66 (3-4) ◽  
pp. 319-324 ◽  
Author(s):  
A. Kaźmierczak ◽  
J. Maszewski
Keyword(s):  
Zea Mays ◽  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Bovine Serum ◽  
Cytochalasin B ◽  
Fluorescein Isothiocyanate ◽  
Cell Type ◽  
Root Cells ◽  
Root Zones ◽  
Species Specific

Incorporation of rhodamine- and fluorescein-isothiocyanate labeled bovine serum albumin (BSA-TRITC, BSA-FITC) was examined in different root zones of the 3-day-old seedlings in <em>Melandrium noctiflorum</em>, <em>Allium cepa</em> and <em>Zea mays</em>. The uptake of fluorescent-labeled BSA was found: (1) species-specific, (2) cell-type dependent, and (3) cytochalasin B-sensitive. The characteristic punctute distribution of vesicles within the cytoplasm suggests the internalization of labeled proteins by endocytosis.

Platelet activation, upregulation of CD11b/CD18 expression on leukocytes and increase in circulating leukocyte-platelet aggregates in Indian women chronically exposed to biomass smoke

2006 ◽  
Vol 25 (11) ◽  
pp. 627-635 ◽  
Author(s):  
M R Ray ◽  
S Mukherjee ◽  
S Roychoudhury ◽  
P Bhattacharya ◽  
M Banerjee ◽  
...  
Keyword(s):  
Flow Cytometric Analysis ◽  
Surface Expression ◽  
Liquefied Petroleum Gas ◽  
Indian Women ◽  
Biomass Smoke ◽  
Flow Cytometric ◽  
Platelet Aggregates ◽  
The Mean ◽  
Biomass Smoke Exposure ◽  
Solid Biomass

The majority of households in rural India still rely on unprocessed solid biomass for domestic energy. The aim of this study was to investigate whether chronic exposure to biomass smoke causes activation of leukocytes and the formation of leukocyte-platelet aggregates. We conducted flow cytometric analysis of β2 Mac-1 integrin (CD11b/CD18) expression on polymorphonuclear leukocytes (PMN) and monocytes, and P-selectin (CD62P) expression on the platelets of 165 women from eastern India, who cook solely with wood, dung and agricultural wastes, and 155 age- and socio-economic condition-matched control subjects, who used relatively cleaner fuel, liquefied petroleum gas (LPG). Leukocyte-platelet aggregates were defined as CD11b-positive PMN and monocytes co-expressing platelet-specific markers CD41 or CD62P. A significant increase in leukocyte-platelet aggregates was found in women who used biomass as cooking fuel. In addition, they showed increased surface expression of CD11b/CD18 in circulating PMN and monocytes and CD62P expression on platelets. The mean fluorescence intensity (MFI) of CD11b on the surface of circulating monocytes and PMN of biomass users increased by 50 and 68%, respectively. Similarly, a 62 and 48% increase in MFI was observed in CD18 expression on the surface of these cells in biomass users. The results show that chronic biomass smoke exposure activates circulating platelets, PMN and monocytes, and increases the number of leukocyte-platelet aggregates, which are considered a risk factor for thrombosis.

Phosphotyrosine Levels by Flow Cytometric Assay for Therapeutic evaluation in Chronic Myelogenous Leukemias

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4246-4246
Author(s):  
Xuemei Sun ◽  
Junhao Chen ◽  
Pengjun Jiang ◽  
Xuejun Zhu
Keyword(s):  
Cell Lines ◽  
Fusion Gene ◽  
Flow Cytometric Analysis ◽  
Chronic Phase ◽  
Fluorescein Isothiocyanate ◽  
Myelogenous Leukemia ◽  
Clinical Monitoring ◽  
Flow Cytometric ◽  
Patient Values

Abstract Assay of phosphotyrosine levels using flow cytometry has been used to identify patients with chronic myelogenous leukemia (CML) positive for the Bcr-Abl fusion gene. We hypothesized that clinical monitoring could identify treatment response through reductions in intra-granulocyte phosphotyrosine. Initially, we studied cell lines FDC-P1 and HL60 (Bcr-Abl–negative) and FDrv210 and K562 (Bcr-Abl–positive) with our technique. A fluorescein isothiocyanate-conjugated monoclonal antibody was used along with fluorescence-conjugated microspheres for reference (ratio of sample fluorescence: bead fluorescence=relative fluorescence unit [RFU]). Samples from 20 controls and 32 patients undergoing treatment were analyzed using the same method. Bcr-Abl status for each patient was confirmed using fluorescent in situ hybridization (FISH) or polymerase chain reaction gene amplification (PCR). Testing of cell lines consistently produced expected results. Patient values were found to be significantly higher than control values (P&lt;0.001) and values for patients with advanced disease were significantly higher than for patients with chronic-phase disease (P&lt; 0.05). Results of clinical monitoring were consistent with results from PCR. Two patients who received allogeneic stem cell transplantation had molecular remission confirmed by PCR and had a marked decrease in RFU value (from 62 to 5 and from 131 to 23). No such fluorescence change was observed in patients who achieved clinical remission. Flow cytometric analysis of phosphotyrosine levels is a reliable and convenient adjuvant technique for diagnosis of Bcr-Abl–positive leukemias and shows promise for serial evaluation of patients undergoing treatment.

scholarly journals Selective exocytosis of zymogen granules induces non-parallel secretion in short-term cholecystokinin-stimulated rats

1999 ◽  
Vol 163 (2) ◽  
pp. 199-206 ◽  
Author(s):  
I de Dios ◽  
AC Garcia-Montero ◽  
A Orfao ◽  
MA Manso
Keyword(s):  
Pancreatic Enzyme ◽  
Flow Cytometric Analysis ◽  
Mean Value ◽  
Enzyme Secretion ◽  
Zymogen Granule ◽  
Zymogen Granules ◽  
Short Term ◽  
Flow Cytometric ◽  
Pancreatic Enzyme Secretion ◽  
The Mean

Parallel studies on pancreatic enzyme secretion and zymogen granule enzyme composition have been carried out in rats subjected to infusion of cholecystokinin (CCK) (1.25 microgram/kg per h) over 30 min. Flow cytometric analysis showed a significant decrease in the mean value of granule size after CCK stimulation. The amount of trypsinogen stored in each individual zymogen granule was significantly lower at 30 min of CCK infusion, but no variation in intragranular amylase content was observed. As a result, the amylase/trypsinogen ratio was significantly increased in the zymogen granules that remained in the pancreas of rats stimulated with CCK for 30 min. A significantly greater proportion of trypsin than amylase was secreted after 30 min CCK infusion. Our results support the existence of different types of granules loaded with different proportions of enzymes. We conclude that short-term CCK stimulation induces the selective release of large granules containing a high proportion of trypsinogen, which leads to a non-parallelism of enzyme secretion.

Oxygen concentration and protein source affect the development of preimplantation goat embryos in vitro

1991 ◽  
Vol 3 (5) ◽  
pp. 601 ◽  
Author(s):  
PA Batt ◽  
DK Gardner ◽  
AW Cameron
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Oxygen Concentration ◽  
Bovine Serum ◽  
Calf Serum ◽  
Cell Number ◽  
The Mean ◽  
Number Of Cells

The effect of oxygen concentration and the source of protein in culture medium on the development of 2- to 4-cell goat embryos in vitro was investigated. Embryos were collected from superovulated Angora-Cashmere-cross goats 48 h after ovulation and cultured for 6 days in synthetic oviduct fluid (SOF) medium under one of two oxygen concentrations (20% or 7%) and in the presence of one of five protein sources; Miles bovine serum albumin (Miles BSA), Commonwealth Serum Laboratory bovine serum albumin (CSL BSA), goat serum (GS), fetal calf serum (FCS) and human serum (HS). In the presence of 20% oxygen the percentage of embryos reaching the expanded and/or hatched blastocyst stage in SOF medium containing Miles BSA was 29%, with a mean cell number per embryo of 28.1 +/- 6.0 (+/- s.e.m.). Use of an oxygen concentration of 7% significantly increased the percentage of embryos reaching this stage (80%, P less than 0.01) and the mean number of cells per embryo (65.3 +/- 8.2, P less than 0.01). The mean number of cells of the early-cleavage-stage embryos was significantly lower when the medium contained CSL BSA, GS or FCS (42.7 +/- 5.6, 29.0 +/- 6.1 and 21.3 +/- 3.2, respectively) than with Miles BSA (92.8 +/- 6.4) or HS (104.8 +/- 17.2) (P less than 0.01). Under 7% oxygen and with Miles BSA or HS, embryos were morphologically comparable to those developed in vivo, but the mean cell numbers in vitro were only approximately half those obtained in vivo.

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