120 Use of fixable dyes to analyze equine sperm membrane integrity and acrosome reaction after A23187 treatment

2020 ◽  
Vol 32 (2) ◽  
pp. 187
Author(s):  
I. Ortiz ◽  
M. Felix ◽  
H. Resende ◽  
C. Love ◽  
K. Hinrichs
Keyword(s):  
Flow Cytometry ◽  
Repeated Measures ◽  
Semen Analysis ◽  
Flow Cytometric Analysis ◽  
Membrane Integrity ◽  
Fluorescein Isothiocyanate ◽  
Computer Assisted ◽  
Cell Agglutination ◽  
Staining Protocol ◽  
Viable Sperm

Conventional IVF is not successful in the horse, and current work is focused on factors affecting sperm capacitation in this species. Challenges arise in assessing equine sperm incubated in media containing capacitation promotors, as some of these factors cause sperm head-to-head binding (aggregation). Our preliminary microscopic findings showed that sperm aggregates are largely of viable sperm, whereas nonviable sperm individualize. Thus, data obtained using technologies that analyse only individual cells and gate out aggregates, such as flow cytometry, may not accurately represent the study population. We developed a fixable live/dead/acrosome staining protocol (LD-PSA) that minimizes sperm aggregation. The aim of this study was to evaluate the percentage of viable and acrosome-reacted equine sperm after A23187 treatment, using either LD-PSA or a standard staining protocol (PI-PSA). Sperm from 9 ejaculates were suspended in Hanks’ balanced salt solution (HBSS) and exposed for 10min at 37°C to vehicle (V) or to 10 µM A23187 (C10). The sperm were washed, resuspended in HBSS medium with added lactate and pyruvate and containing 7mgmL−1 of bovine serum albumin (BSA), and assessed immediately (0h) or incubated at 37°C for 2h (the period needed for equine sperm to respond to A23187). Motility was analysed using computer-assisted semen analysis. Each treatment was stained by PI-PSA: propidium iodide and fluorescein isothiocyanate-Pisum sativum agglutinin (PSA) in DPBS; and by LD-PSA: Live/Dead Fixable Red, paraformaldehyde 2%, Triton×1%, and FITC-PSA in Dulbecco's phosphate-buffered saline with Accumax (Stem Cell Technologies), a commercial proprietary cell agglutination inhibitor, before flow cytometric analysis. Differences were analysed using repeated-measures two-way ANOVA. The% total motile sperm (TMOT) for V and C10 treatments were 76.3±3.0 and 71.2±4.7 at 0h (P>0.05), and 70.5±14.8 and 2.4±0.8 at 2h (P<0.05). On flow cytometry, the percentage of events outside the gate for V sperm (0h and 2h combined) was 31.9% in PI-PSA and 21.9% in LD-PSA samples (P<0.01). Measured viability in V samples was significantly lower when stained with PI-PSA than with LD-PSA at 0h (49.2±4.6 vs. 67.1±4.9) and tended to be lower (P=0.07) at 2h (44.0±4.9 vs. 55.1±2.8). Notably, the viability recorded in PI-PSA was 26 percentage points lower than was the TMOT at both 0h and 2h, indicating nonrepresentative results, as nonviable sperm should not be motile. By LD-PSA, this difference was 9 points at 0h and 15 points at 2h. Vehicle sperm showed significantly higher AR values in PI-PSA than in LD-PSA at 0h (30.4±4.0 vs. 17.7±2.4) and 2h (41.9±4.5 vs. 24.0±1.8), as did C10 sperm at 0h (28.9±2.7 vs. 18.0±2.5). The lower values for viability than total motility likely reflect agglutination of viable sperm and thus their exclusion from analysis on flow cytometry. The anti-clumping measures employed in the LD-PSA protocol were associated with increased correspondence of measured viability with TMOT. Thus, LD-PSA may offer a more accurate technique to assess viability and acrosome status of equine sperm incubated in capacitating conditions.

18 SEMENRATE: THE USE OF COMPUTER-ASSISTED SEMEN ANALYSIS AND FLOW CYTOMETRY FOR OBJECTIVE BOVINE SEMEN ANALYSIS IN THE UNITED KINGDOM

2017 ◽  
Vol 29 (1) ◽  
pp. 116
Author(s):  
M. W. Spilman ◽  
K. L. Burton ◽  
J. M. E. Statham
Keyword(s):  
Flow Cytometry ◽  
United Kingdom ◽  
Semen Quality ◽  
Semen Analysis ◽  
Computer Assisted ◽  
Motile Sperm ◽  
Bovine Semen ◽  
The United Kingdom ◽  
Frozen Semen ◽  
Viable Sperm

Routine assessment of bovine semen consists of a subjective assessment of morphology, motility and concentration. This subjective approach used during quality control at semen production centres (SPC) or investigations of poor reproductive performance in veterinary practice has been shown to be relatively inaccurate, imprecise, and operator dependent (Vincent, et al. 2012 Anim. Reprod. 9, 153–165). Assessment of frozen semen samples in a dedicated laboratory aimed to establish variations in multiple parameters associated with fertility using computer-assisted semen analysis and flow cytometry and evaluate their relationship to semen performance in the field. This has developed into a commercial service that is available to veterinarians and farmers across the United Kingdom. AI semen from 50 farms across Yorkshire, UK, that had been stored on farm was assessed for factors associated with fertility (motility, progressive motility, intact acrosome, viability, and polarised mitochondria). Data ranges and mean values for each parameter have been analysed. This analysis is ongoing as the dataset continues to expand and significance will be assessed. For frozen semen (n = 79), % viable sperm (max = 67.64, min = 0.00, mean = 43.44), % sperm with polarised mitochondria (max = 72.50, min = 0.26, mean = 38.56), % sperm with acrosome intact (max = 68.82, min = 0.06, mean = 35.29), % motile sperm (max = 66.90, min = 0.00, mean = 37.44) and % progressively motile sperm (max = 59.00, min = 0.00, mean = 26.11). 25% of the samples fell below the cut off for release of 30% motile sperm set by SPCs. For sexed AI semen (n = 9), % viable sperm (max = 66.31, min = 17.08, mean = 43.57), % polarised mitochondria (max = 26.74, min = 13.40, mean = 19.96), % intact acrosome (max = 52.62, min = 15.34, mean = 37.00), % motile (max = 38.00, min = 9.40, mean = 24.88) and % progressively motile (max = 22.80, min = 3.90, mean = 13.15). Objective semen analysis before beginning an embryo collection programme allows informed decisions to be made regarding semen choice and dosage depending on compensable v. non-compensable defects detected (Hudson et al. 2012 Dairy Herd Health 73–111; CABI Publishing). Use of semen that falls below the 30% cut off for SPCs is unlikely to perform as expected in the field (Phillips et al. 2004 Anim. Reprod. Sci. 80, 47–61). A European collaboration aims to establish correlations between semen quality parameters and fertility outcomes for UK cattle herds, providing unique data for the industry (Sellem et al. 2015 Theriogenology 84, 1447–1454.e5). These data should highlight to stakeholders in the industry how imperative optimal semen quality is and highlight the benefits to herd fertility and financial performance.

scholarly journals Flow-cytometric analysis of membrane integrity of stallion sperm in the face of agglutination: the “zombie sperm” dilemma

2021 ◽  
Author(s):  
Isabel Ortiz ◽  
Matheus Felix ◽  
Hélène Resende ◽  
Luisa Ramírez-Agámez ◽  
Charles C. Love ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Accurate Determination ◽  
Flow Cytometric Analysis ◽  
Membrane Integrity ◽  
Flow Cytometric ◽  
The Face ◽  
Viable Sperm ◽  
Sperm Membrane ◽  
Novel Method ◽  
Live Sperm

Abstract Purpose To define the effect of sperm agglutination, associated with incubation under capacitating conditions, on accuracy of membrane assessment via flow cytometry and to develop methods to mitigate that effect. Methods Sperm motility was measured by CASA. Sperm were stained with PI-PSA or a novel method, LD-PSA, using fixable live/dead stain and cell dissociation treatment, before flow-cytometric analysis. Using LD-PSA, acrosome reaction and plasma membrane status were determined in equine sperm treated with 10 μm A23187 for 10 min, followed by 0, 1, or 2 h incubation in capacitating conditions. Results Using PI-PSA, measured membrane integrity (MI; live sperm) was dramatically lower than was total motility (TMOT), indicating spurious results (“zombie sperm”). Sperm aggregates were largely of motile sperm. Loss of motility after A23187 treatment was associated with disaggregation and increased MI. On disaggregation using LD-PSA, MI rose, and MI then corresponded with TMOT. In equine sperm incubated after A23187 treatment, as the percentage of live acrosome-reacted sperm increased, TMOT decreased to near 0. Conclusion Flow cytometry assesses only individualized sperm; thus, agglutination of viable sperm alters recorded membrane integrity. As viable sperm become immotile, they individualize; therefore, factors that decrease motility, such as A23187, result in increased measured MI. Disaggregation before assessment allows more accurate determination of sperm membrane status; in this case we documented a mismatch between motility and live acrosome-reacted equine sperm that may relate to the poor repeatability of A23187 treatment for equine IVF. These findings are of profound value to future studies on sperm capacitation.

66 EFFECT OF ADDITION OF CHOLESTEROL-LOADED CYCLODEXTRIN BEFORE FREEZING ON QUALITY AND FERTILITY OF STALLION FROZEN SEMEN

2015 ◽  
Vol 27 (1) ◽  
pp. 126
Author(s):  
F. O. Papa ◽  
C. Ramires Neto ◽  
Y. F. R. Sancler-Silva ◽  
H. L. Resende ◽  
G. A. Monteiro ◽  
...  
Keyword(s):  
Kinetic Parameters ◽  
Semen Analysis ◽  
Membrane Integrity ◽  
Skim Milk ◽  
Fluorescein Isothiocyanate ◽  
Treated Group ◽  
Control Group ◽  
Computer Assisted ◽  
Exact Test ◽  
Frozen Semen

The aim of the present study was to evaluate the effect of cholesterol-loaded cyclodextrin on the quality and fertility of stallion frozen semen. Three ejaculates from each of 4 stallions were used. The semen was diluted (1 : 1) with a skim milk-based extender (Botu-SemenTM, Botupharma, Brazil). The samples were divided into 2 groups: control group (CG), composed of semen diluted only with extender, and treated group (TG), composed of semen diluted with extender plus 750 mg mL–1 of cholesterol-loaded cyclodextrin. Both groups were incubated for 15 min at 20°C. The semen was frozen with Botu-CryoTM (Botupharma, Brazil) extender according to the manufacturer's protocol in 0.5-mL straws containing 100 × 106 of total sperm. The sperm kinetic parameters were analysed by computer-assisted semen analysis, and plasma membrane integrity by flow cytometer (propidium iodide and fluorescein isothiocyanate -PSA) on post-thaw. The fertility trial was carried out inseminating 2 cycles of 20 mares (total of 40 cycles) immediately post-ovulation using 4 straws of CG or TG (400 × 106 total sperm), one from each stallion in a randomised design. Comparison of sperm parameters was performed by t-test and fertility by Fisher's exact test. No difference (P > 0.05) was observed in total motility (%, CG = 57.9 ± 6.5 v. TG = 60.2 ± 6.7), progressive motility (%, CG = 26.9 ± 4.8 v. TG = 28.5 ± 4.8), percentage of rapid sperm (%, CG = 43.5 ± 8.8 v. TG = 45.7 ± 7.6), membrane integrity (%, CG = 20.1 ± 5.1 v. TG = 20.3 ± 6.3), and fertility (CG = 60% v. TG = 70%) between the groups. The results of this study showed that the use of cholesterol-loaded cyclodextrin did not affect sperm kinetic parameters and fertility in stallion with good quality in post-thaw semen. Further studies must be performed with stallions sensitive to freeze-thawing process.

Cryopreservation of collared peccary (Pecari tajacu L., 1758) epididymal sperm using extenders based on Tris and powdered coconut water (ACP®-116c)

Zygote ◽  
2018 ◽  
Vol 26 (4) ◽  
pp. 301-307 ◽  
Author(s):  
José A. B. Bezerra ◽  
Andréia M. Silva ◽  
Patrícia C Sousa ◽  
Lívia B. Campos ◽  
Érica C. G. Praxedes ◽  
...  
Keyword(s):  
Sperm Quality ◽  
Semen Analysis ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Coconut Water ◽  
Computer Assisted ◽  
Epididymal Sperm ◽  
Sperm Plasma Membrane ◽  
Pecari Tajacu ◽  
Functional Membrane

SummaryThe aim of this study was to establish a functional freezing–thawing protocol for epididymal sperm of collared peccaries (Pecari tajacu L., 1758) by comparing different extenders. The epididymal sperm from 12 sexually mature males was recovered by retrograde flushing using Tris-based or coconut water-based (ACP®-116c) extenders. After initial evaluation, samples were diluted and frozen with the same extenders to which 20% egg yolk and 6% glycerol were added. After 2 weeks, thawing was performed at 37°C/60 s and sperm motility, vigour, morphology, functional membrane integrity, sperm viability, sperm plasma membrane integrity, and a computer-assisted semen analysis (CASA) were assessed. In addition, to evaluate the survival of frozen–thawed sperm, a thermal resistance test (TRT) was executed. Samples preserved using Tris were in better condition compared with those preserved using ACP®, showing higher values for most assessments performed, including CASA and the TRT (P<0.05). After determining Tris to be the better of the two extenders, additional samples were thawed using different thawing rates (37°C/60 s, 55°C/7 s, 70°C/8 s). Sperm thawed at 37°C/60 s had the greatest preservation (P<0.05) of viability (54.1 ± 5.9%) and functional membrane integrity (43.2 ± 5.4%), and had higher values for various CASA parameters. In conclusion, we suggest the use of a Tris-based extender added to egg yolk and glycerol for the cryopreservation of epididymal sperm obtained from collared peccaries. In order to achieve better post-thawing sperm quality, we suggest that samples should be thawed at 37°C/60 s.

90 EFFECT OF EGG YOLK ON THE KINEMATICS AND ACROSOME MEMBRANE INTEGRITY OF COOLED-REWARMED CANINE SPERMATOZOA

2010 ◽  
Vol 22 (1) ◽  
pp. 204
Author(s):  
J. Dorado ◽  
M. J. Galvez ◽  
M. R. Murabito ◽  
S. Demyda ◽  
L. J. De Luca ◽  
...  
Keyword(s):  
Semen Analysis ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Computer Assisted ◽  
Mean Values ◽  
Head Displacement ◽  
Healthy Dogs ◽  
Acrosome Integrity ◽  
Digital Manipulation ◽  
Lateral Head

Tris-egg yolk-based diluents provide adequate cryoprotection for the sperm of most species. This study was conducted to compare the ability of Tris-glucose extender containing 2 different concentrations of egg yolk to maintain sperm motility and acrosome integrity of canine spermatozoa during 72 h of preservation. For this purpose, a total of 20 ejaculates from 4 clinically healthy dogs (2 Spanish Greyhound, 1 German Pointer, and 1 Crossbreed) were collected by digital manipulation. The sperm-rich fraction of each ejaculate was divided into 2 aliquots. Then, they were diluted in Tris-based extender and centrifuged at 700g for 8 min. Sperm pellets were resuspended in either Tris buffer added to 20% (EY20) or 10% centrifuged egg yolk (EY10) and cooled to 5°C over 72 h. The effects of these extenders on motility and acrosome integrity were assessed objectively using a computer-aided semen analyzer (Sperm Class Analyzer, Microptic SL, Spain) and Spermac® staining, respectively. Each cooled-rewarmed semen sample was evaluated after 24, 48, and 72 h of preservation. Sperm motion parameters shown by computer-assisted semen analysis (CASA) are progressively motile (PMS) and motile spermatozoa (MS), curvilinear velocity (CLV), average path velocity (APV), progressive speed (SLV), and lateral head displacement (LHD). Data were statistically analysed by ANOVA. Dependent variables expressed as percentages were arsine-transformed before analysis. Differences between mean values were evaluated by the Duncan method. Data were presented as mean ± SEM. Differences were considered significant when P < 0.05. Analyses were performed using the statistical package SPSS 12.0. A total of 98 172 motile sperm trajectories were analyzed by CASA: 52 259 in EY20 and 45 913 in EY10. After 24, 48, and 72 h of preservation, MS and PMS were statistically higher (P < 0.01) in EY20. No significant differences were found for LHD using either extender over a 72-h period. No significant differences were observed for CLV using either extender during the first 2 days. At Day 3, CLV data were significantly higher (P < 0.01) in EY20. Similarly, from Day 2, APV was significantly higher (P < 0.001) in EY20. After 24 h of preservation, SLV was statistically higher (P < 0.001) in EY10, whereas the opposite tendency was found at Day 3. No significant differences were observed for SLV using either extender after 48 h of preservation. During the first 2 days, acrosome integrity was statistically higher (P < 0.001) in EY20. At hour 72, higher acrosome integrity (P < 0.001) was observed in EY10. In conclusion, we have observed that the EY20 extender provided higher motility after 72 h of chilled preservation; however, the acrosome membrane integrity was better preserved in EY10.

134 RELAXIN AND ITS RECEPTORS IN MATURE CANINE SPERMATOZOA

2017 ◽  
Vol 29 (1) ◽  
pp. 175 ◽  
Author(s):  
I. L. G. Almeida ◽  
C. L. Durfey ◽  
G. D. A. Gastal ◽  
D. Devos-Burnett ◽  
S. T. Willard ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Protein Extraction ◽  
Standard Procedure ◽  
Rna Extraction ◽  
Fluorescein Isothiocyanate ◽  
Housekeeping Gene ◽  
Computer Assisted ◽  
Solution Ph ◽  
Rna Transcripts ◽  
Reproductive Tissues

Relaxin and its receptors (RXPF1 and RXFP2) are found in reproductive and non-reproductive tissues of both males and females, across species. In dogs, both relaxin and receptors are found in reproductive tissues of females and play important roles during pregnancy; however, their presence in male reproductive tissues remains unclear. The goal of this study was to explore the presence of both relaxin and receptors in mature dog spermatozoa. Semen samples of 4 adult (≥4 years old) dogs were harvested from a local breeder and diluted (vol:vol) with a pre-warmed Tris extender solution (pH 6.9). Samples were centrifuged and sperm pellets were resuspended in the Tris extender for motility and velocity analyses using computer-assisted sperm analyzer. Semen with over 70% motility were retained for analyses. A subset of those samples (n = 4 dogs) was subjected to total RNA extraction, followed by RT-PCR to amplify relaxin and RXFP1/2 RNA transcripts, with β-actin used as the housekeeping gene. All PCR amplicons were run on a gel electrophoresis for imaging. Another subset of samples (n = 4 dogs) were used for total protein extraction, followed by immunoblotting using anti-human RXFP1 and RXFP2 antibodies. Finally, the remaining subset of samples (n = 4 dogs) was fixed in 4% paraformaldehyde and submitted to a standard procedure of immunofluorescence using an anti-human relaxin, and followed by fluorescein isothiocyanate-conjugated secondary antibody. Labelled spermatozoa were immediately analysed with flow cytometry and imaged with a confocal microscope. Gene expression data were collected after 40 cycles of PCR and all samples expressed β-actin. The detection of RXFP2 was observed at a low level in all samples, whereas RXFP1 and relaxin were absent. Protein analysis with western immunoblotting showed weak signal bands of both receptors in all samples; however, the band corresponding to RXFP1 (~82 kDa) appeared stronger than that of RXFP2 (~90 kDa). Flow cytometry revealed small proportions of relaxin immunopositive spermatozoa (less than 35%) that exhibited low fluorescence intensity. Confocal microcopy imaging confirmed the weak signals that were mostly located in the sperm head. Altogether, the findings showed that relaxin and its receptors RXFP1 and RXFP2 are present in mature spermatozoa of adult dogs. More studies are needed to better characterise the roles of the relaxin system on canine sperm fertility. Grant supports are from the USDA-ARS Biophotonics Initiative project #58–6402–3-018 and Brazil Scientific Mobility Program (BSMP) scholarship from CAPES-Brazil.

scholarly journals Exosomes derived from HEK293T cells interact in an efficient and noninvasive manner with mammalian sperm in vitro

Nanomedicine ◽  
2020 ◽  
Vol 15 (20) ◽  
pp. 1965-1980
Author(s):  
Teresa Vilanova-Perez ◽  
Celine Jones ◽  
Stefan Balint ◽  
Rebecca Dragovic ◽  
Michael L Dustin ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Mitochondrial Membrane ◽  
Membrane Integrity ◽  
Computer Assisted ◽  
Sperm Function ◽  
Sperm Analysis ◽  
Mammalian Sperm ◽  
Boar Sperm ◽  
Computer Assisted Sperm Analysis

Aim: To investigate exosomes as a noninvasive delivery tool for mammalian sperm. Materials & Methods: Exosomes were isolated from HEK293T cells and co-incubated with boar sperm in vitro. Results: Internalized exosomes were detected within 10 min of co-incubation. Computer-assisted sperm analysis and flow cytometry demonstrated that even after 5-h of exposure to exosomes, there were no significant deleterious effects with regard to sperm motility, viability, membrane integrity and mitochondrial membrane potential (p > 0.05), thus indicating that exosomes did not interfere with basic sperm function. Conclusion: HEK293T-derived exosomes interacted with boar sperm without affecting sperm function. Exosomes represent a versatile and promising research tool for studying sperm biology and provide new options for the diagnosis and treatment of male infertility.

180 ASSESSMENT OF BULL SEMEN QUALITY LOADED IN NEW SensiTemp STRAWS USING SEMEN AND IN VITRO PRODUCTION TECHNOLOGIES

2016 ◽  
Vol 28 (2) ◽  
pp. 221
Author(s):  
D. Le Bourhis ◽  
S. Camugli ◽  
P. Salvetti ◽  
L. Schibler ◽  
E. Schmitt
Keyword(s):  
Sperm Motility ◽  
Semen Quality ◽  
Sperm Quality ◽  
Semen Analysis ◽  
Membrane Integrity ◽  
Computer Assisted ◽  
Cleavage Rate ◽  
Fluid Medium ◽  
And Control

SensiTemp, a new in vitro maturation (IMV) bull straw concept, presents the advantage of colour changing while the straw is thawed. The colour of frozen straws is blue and straws start to become white when the temperature reaches 33°C, with a complete change of colour at 37°C. The objective of this study is to assess sperm quality after thawing of semen frozen in SensiTemp from 2 bulls, by analysing, in experiment 1, sperm motility and membrane integrity using computer-assisted semen analysis (CASA) and flow cytometry (FC), and, in experiment 2, the in vitro embryo production (IVP) using IVP technologies [IVM, IVF, and in vitro culture (IVC)]. The ejaculates of 2 bulls, selected during preliminary experiments on high in vitro fertility, were harvested at CIA L’Aigle, France, and split ejaculates were frozen in experimental (SensiTemp) and conventional (control) straws. In experiment 1 after thawing semen from the 2 types of straws (5 pooled straws each; 2 replicates), motility was assessed using the IVOS CASA system (Hamilton Thorne Inc., Beverly, MA, USA) and membrane integrity was evaluated through FC with Cytosoft software (Millipore-Guava Technologies Inc., Hayward, CA, USA). In experiment 2, IVF was used to evaluate the non-toxicity of SensiTemp and control straws. Cumulus-oocyte complexes (COC; n = 1178; 4 replicates) collected from slaughterhouse ovaries were matured in IVM medium (TCM-199 with bicarbonate, Sigma-Aldrich, Saint Quentin Fallavier, France; 10 µg mL–1 FSH-LH, Reprobiol, Liège, Belgium; and 10% FCS, Thermo Fisher, Illkirch, France) for 22 h. After fertilization, presumptive zygotes of each group (SensiTemp and control for each bull) were cultured in synthetic oviduct fluid medium (SOF, Minitube, Tiefenbach, Germany) with 1% estrous cow serum (ECS) and 0.6% BSA (Sigma-Aldrich, France) up to 8 days. All cultures were conducted at 38.5C in 5% CO2, and 5% O2. The cleavage and blastocysts rates were evaluated on Days 3 and 7, respectively, for each group. Embryo quality was recorded on Day 7 according to the IETS evaluation. Data from each bull were analysed separately using the chi-squared test (P < 0.05). In experiment 1, neither sperm motility from bull 1 (61.2 and 60.5%) and bull 2 (66.2 and 66.5%) nor membrane integrity from bull 1 (58.6 and 52.2%) and bull 2 (61.0 and 61.9%) were different between SensiTemp and control, respectively. Results from experiment 2 showed no difference (P > 0.05) in cleavage rate between SensiTemp and control for the 2 bulls: 92.1 and 91.7% for bull 1 and 94.2 and 94.6% for bull 2 respectively. The blastocysts rate on Day 7 did not differ (P > 0.05) among groups (47.5, 47.1 and 51.3, 50.4% for SensiTemp and control bull 1 and bull 2, respectively) nor the quality of embryos retrieved in the different groups: 25.4, 23.3, and 30.8, 29.6% in grade 1 embryo for SensiTemp and control bull 1 and bull 2, respectively. Those results demonstrate, in vitro, that the new SensiTemp straws were non-toxic and did not affect the semen quality after thawing nor did the SensiTemp straws affect the ability of sperm cells to fertilize oocytes and produce 8-day-old embryos.

HNA−1a, −1b, −2a, −3a and −4a Frequencies in Brazilian Persons.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3837-3837 ◽  
Author(s):  
Angela M. Norcia ◽  
Elisa Y. Kimura ◽  
Akemi K. Chiba ◽  
Elyse Moritz ◽  
Mihoko Yamamoto ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
African Americans ◽  
Blood Donors ◽  
Mononuclear Cells ◽  
Flow Cytometric Analysis ◽  
Fluorescein Isothiocyanate ◽  
Blue Staining ◽  
Drug Induced ◽  
Flow Cytometric ◽  
The Gift

Abstract Granulocyte reactive antibodies have been found to cause clinical disorders such as transfusion related acute lung injury (TRALI), febrile transfusion reactions, alloimmune neonatal neutropenia, immune neutropenia after bone marrow transplantation, refractoriness to granulocyte transfusion, drug-induced neutropenia and autoimmune neutropenia. Using the granulocyte immunofluorescence test (GIFT) by microscopic and flow cytometric analysis, the frequencies of the neutrophil antigens HNA−1a, −1b, −2a, −3a and −4a were determined among 100 random Brazilian blood donors from the Blood Center of Universidade Federal de Sao Paulo, SP, Brazil. Granulocytes were separated from mononuclear cells and red cells by sedimentation with 5% dextran, followed by centrifugation on Ficoll-paque (d = 1.077), and then incubated with anti-sera (anti-HNA−1a, −1b, −2a, −3a, and −4a obtained from American Red Cross, North Central Blood Services, St. Paul, MN) conjugated with fluorescein isothiocyanate (FITC) labeled F(ab’)2 fragments of anti-human IgG. Only cell suspensions containing ≥95% neutrophils with viability ≥90% according to the trypan-blue staining were analyzed. The frequencies of HNA−1a, −1b and −2a were 65%, 83% and 94%, respectively, and for such alloantigens exact same results were observed using either the GIFT performed by microscopy or by flow cytometry. The frequency of HNA-3a was 86% by the microscopic GIFT, and 95% by the flow cytometry analysis; while the frequency of HNA−4a was 93% by the microscopic GIFT, and 94% by the flow cytometric technique. These results indicate that: GIFT by flow cytometry is more sensitive than the GIFT by microscopy to detect HNA−3a; the phenotypic frequencies found for neutrophil antigens HNA−1a and −1b among Brazilian blood donors are quite similar to those reported among African Americans, but different from those reported for Japanese and Chinese individuals; the phenotype frequencies of the neutrophil antigens HNA−2a, −3a, and −4a in Brazilians are quite similar to those found among Caucasians (Table). (These studies were funded by FAPESP, SP, Brazil - 05/55237–9). Asians Brazilians Antigen African Americans Chineses Hindus Japaneses Caucasians M FC M, microscopy; FC, flow cytometry; nd, not done HNA-1a 46 – 68 90 44 88 52 – 54 65 65 HNA-1b 78 – 84 52 83 51 – 64 87 – 89 83 83 HNA-2a nd 99 nd 89 87 – 97 94 94 HNA-3a nd nd nd nd 99 86 95 HNA-4a nd nd nd nd 96 93 94

scholarly journals 216.The effects of Viagra on sperm function and early embryo development

2004 ◽  
Vol 16 (9) ◽  
pp. 216
Author(s):  
S. E. M. Lewis ◽  
D. R. J. Glenn ◽  
N. McClure
Keyword(s):  
Semen Analysis ◽  
Plasma Concentrations ◽  
Phosphodiesterase Inhibitor ◽  
Fluorescein Isothiocyanate ◽  
Human Sperm ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Computer Assisted ◽  
Sperm Function ◽  
Embryo Cleavage

In an audit of UK fertility units, we have demonstrated that 42% prescribe Viagra to aid patient semen production. Viagra is a phosphodiesterase inhibitor (PDE5) and as non-specific PDEs have been shown to affect fertility, safety concerns have been raised. The aims of this study are to investigate the effects of Viagra on sperm function and early embryo cleavage. Human semen was incubated with and without Viagra (450�ng/mL sildenafil citrate, equivalent to plasma concentrations after 100�mg oral dose; Pfizer, UK). Aliquots were also prepared by a 90/45% density centrifugation gradient to separate good and poor subpopulations. All samples were analysed by computer assisted semen analysis (HTM-IVOS) up to 60 and 120�min. Prepared samples were also labelled with fluorescein isothiocyanate–peanut agglutinin to determine acrosome status. Male mice were gavaged with Viagra (equivalent dose/body wt) and mated with superovulating females. Twenty females were sacrificed 12�h later, their oviducts flushed and viable fertilized oocytes counted. Another 20 females were sacrificed 4�days after mating and their embryo numbers and cleavage stages determined. Viagra increased % progressive motility in semen (n�=�22) by 38%, VAP by 21%, VSL by 21% and VCL by 16% at 60�min (all P values <0.001). These effects were sustained at 120�min. Sperm isolated from 90% (n�=�57) and 45% (n�=�15) fractions showed similar increases. Viagra also increased the proportion of acrosome reacted sperm in the 90% (+79%, P�<�0.001) and 45% (+77%, P�<�0.001) fractions. Further, Viagra caused a reduction in both the numbers of fertilised oocytes (–35%, P�<�0.001) and those reaching blastocyst stage (85%, P�<�0.001). This study demonstrates that Viagra increases human sperm motility. However, Viagra induces human premature acrosome reactions and impairs mouse fertilisation and embryo cleavage. This study raises significant concerns for its use in assisted reproduction.

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