scholarly journals Elimination of apoptotic boar spermatozoa using magnetic activated cell sorting

2014 ◽  
Vol 83 (1) ◽  
pp. 13-18 ◽  
Author(s):  
Janko Mrkun ◽  
Tamara Dolenšek ◽  
Tanja Knific ◽  
Anja Pišlar ◽  
Marjan Kosec ◽  
...  
Keyword(s):  
Cell Sorting ◽  
Semen Quality ◽  
Annexin V ◽  
Alexa Fluor ◽  
Liquid Storage ◽  
Boar Semen ◽  
And Control ◽  
Boar Spermatozoa ◽  
First Time ◽  
Magnetic Activated Cell Sorting

One of the features of apoptosis is the externalization of phosphatidylserine which could be used to remove apoptotic cells from semen preparations. Magnetic-activated cell sorting using annexin V-conjugated microbeads which bind to phosphatidylserine could be used to enhance semen quality. Twelve boar semen samples after 3 days of liquid storage at 16­­–17 °C were subjected to magnetic-activated cell sorting. Bound and unbound fractions and control samples were subjected to flow cytometry following the staining of spermatozoa with Annexin V conjugated with Alexa Fluor 488 and propidium iodide. Four subpopulations were obtained: live, early apoptotic live, late apoptotic, early necrotic dead and late necrotic dead. The frequency of early apoptotic and late necrotic spermatozoa was significantly higher (P< 0.05) in bound (14.1 ± 10.6% and 24.1 ± 10.2%, respectively) than in unbound fractions (3.4 ± 2.1% and 12.7 ± 3.1%) and control (3.5 ± 1.6% and 12.0 ± 5.0%). The lowest concentration of live spermatozoa was found in the bound fraction (10.6 ± 8.0 %), which differed significantly (P< 0.05) from the control. In unbound fractions there was a significantly higher concentration (P< 0.05) of morphologically normal spermatozoa (31.8 ± 12.6%) compared to bound ones (5.9 ± 7.3%). A significantly (P< 0.05) lower proportion of morphologically normal spermatozoa was observed in both fractions compared to control (67.2 ± 17.0%). Boar spermatozoa were separated by the above method for the first time, however, the results showed this method to be inappropriate for boar semen separation under the tested conditions.

8 THE USE OF ANNEXIN V MAGNETIC-ACTIVATED CELL SORTING TO SEPARATE APOPTOTIC SPERM FROM THE EJACULATE OF STALLIONS

2011 ◽  
Vol 23 (1) ◽  
pp. 110
Author(s):  
M. A. Coutinho da Silva ◽  
C. R. F. Pinto ◽  
J. M. Young ◽  
K. Cole
Keyword(s):  
Cell Sorting ◽  
Semen Quality ◽  
Sperm Quality ◽  
Cleveland Clinic ◽  
Membrane Integrity ◽  
Annexin V ◽  
Sperm Parameters ◽  
Control Group ◽  
Frozen Semen ◽  
Magnetic Activated Cell Sorting

Magnetic-activated cell sorting (MACS) has been used successfully in humans to remove apoptotic sperm from the ejaculate. Annexin V-conjugated microbeads recognise sperm with externalized phosphatidylserine, which is considered one of the features of apoptosis, and the labelled sperm is separated by MACS. The goals of the study were to determine if MACS can be used to separate apoptotic sperm from the ejaculate of stallions; and to determine if removal of apoptotic sperm improves the quality of stallion sperm. Our hypothesis was that MACS would improve semen quality by removing apoptotic sperm, resulting in samples with higher motility and viability. Two ejaculates from three different stallions of good fertility were used. Sperm were diluted with Tyrode’s albumin lactate pyruvate (TALP) and incubated with annexin V-conjugated microbeads for 15 min at 37°C. Control samples were incubated in the absence of annexin V microbeads. The suspension was then loaded into the separation column containing iron globes, which were fitted in a magnet (MiniMACS; Miltenyi Biotec Inc., Auburn, CA, USA). The effluent sample containing annexin-negative sperm was collected and then, the column was removed from the magnetic field and rinsed with TALP to collect the annexin-positive cells. Sperm viability, motility, morphology and caspase activation were determined in all three samples: control, annexin-negative, and annexin-positive. Data were evaluated by ANOVA and individual comparisons were performed by Tukey’s hsd test. Significance was set at P < 0.05 and data is presented as means ± SEM (Table 1). The main effect of stallion was significant only for sperm motility parameters. Sperm recovery rate following MACS was 46 ± 3%. In conclusion, the use of MACS was effective in removing apoptotic sperm from the ejaculate. The annexin-positive population displayed a higher proportion of sperm with activated caspases and lower membrane integrity and motility. However, removal of apoptotic sperm from the ejaculate did not improve sperm parameters in the annexin-negative group compared to control group. In addition, sperm morphology was not affected by MACS. Further studies are necessary to determine if MACS could be used successfully to improve sperm quality from subfertile stallions and frozen semen. Table 1.Sperm parameters following annexin V MACS (mean ± SEM) The authors are thankful to Mark Williams at Miltenyi Biotec Inc. for providing supplies; and Dr Ashok Agarwal at The Center for Reproductive Medicine, Cleveland Clinic, for scientific input.

scholarly journals Analysis of proapoptotic changes in boar spermatozoa stored at 16 °C in different short-term semen extenders

2013 ◽  
Vol 57 (3) ◽  
pp. 425-428
Author(s):  
Paweł Wysocki ◽  
Aleksandra Łyjak ◽  
Władysław Kordan
Keyword(s):  
Annexin V ◽  
Point Of View ◽  
Sperm Cells ◽  
Hoechst 33258 ◽  
Short Term ◽  
Semen Storage ◽  
Boar Semen ◽  
Boar Spermatozoa ◽  
First Time ◽  
Cell Analyzer

Abstract The aim of this study was to evaluate the effect of boar semen storage in different short-term extenders (BTS, Kortowo-3, and M III) on the percentage of spermatozoa showing proapoptotic and necrotic changes. For the first time in this study, Annexin V isolated from swine placenta has been used to determine proapoptotic changes in stored boar spermatozoa. The changes were determined using the IN Cell Analyzer 2000. A gradual decrease in motility was observed on successive days of storage. Spermatozoa incubated in the BTS extender were characterised by the highest average motility, which reached 75% on the 1st d and 39% on day 5. Motility of spermatozoa stored in BTS was significantly higher than those stored in Kortowo-3 and M III extenders after 5 d of storage. Diluted semen contained 1.5% to 2.8% spermatozoa with proapoptotic changes. The discussed process was intensified on the 3rd d of storage when the percentage of apoptotic spermatozoa was determined at 8.3% to 14.6%, and the content of dead spermatozoa exceeded 25%. The analysed extenders differed insignificantly in their ability to protect semen against proapoptotic changes during storage. From the methodological point of view, Hoechst 33258 could be used additionally to stain sperm cells regardless of their status.

scholarly journals Binding of boar spermatozoa to oviductal epithelium in vitro in relation to sperm morphology and storage time

Reproduction ◽  
2006 ◽  
Vol 131 (2) ◽  
pp. 311-318 ◽  
Author(s):  
D Waberski ◽  
F Magnus ◽  
F Ardón ◽  
A M Petrunkina ◽  
K F Weitze ◽  
...  
Keyword(s):  
Semen Quality ◽  
Binding Capacity ◽  
Storage Period ◽  
Sperm Function ◽  
Sperm Binding ◽  
Oviductal Epithelium ◽  
Semen Storage ◽  
Boar Semen ◽  
Boar Spermatozoa

In vitro short-term storage of boar semen for up to 72 h before insemination negatively affects fertility, but this often remains undetected during semen quality assessment. One important sperm function is the ability to form the functional sperm reservoir in the oviduct. In the present study, we used the modified oviductal explant assay to study sperm binding to oviductal epithelium in vitro in diluted boar semen stored for 24 or 72 h. First, we determined the kinetics of in vitro sperm binding to oviductal epithelium in relation to co-incubation time of sperm and oviductal tissue pieces. Then, we studied how the binding of sperm to oviductal epithelium was affected by in vitro semen storage and by differences among individual boars. Sperm binding after different incubation times was significantly higher when semen was stored 24 h than after 72-h storage (P < 0.05), and peaked at 30–90 min of incubation. Sperm binding differed between boars (n = 44), and was negatively correlated to the percentage of sperm with cytoplasmic droplets (R = −0.51, P < 0.001). There were no significant changes in motility, acrosome integrity and propidium iodide stainability during the 72-h storage period. However, sperm-binding indices were significantly lower after 72 h in vitro storage than after 24-h storage in sperm from boars with normal semen quality (P < 0.05); in contrast, the binding capacity of sperm from boars with higher percentages of morphologically altered sperm remained at a low level. The sperm-binding capacity of sperm from four of the five boars with known subfertility was lower than the mean binding index minus one standard deviation of the boar population studied here. It is concluded that changes in the plasma membrane associated with in vitro ageing reduce the ability of stored boar sperm to bind to the oviductal epithelium. This study shows the potential of sperm–oviduct binding as a tool to assess both male fertility and changes in sperm function associated with in vitro ageing.

scholarly journals Cryopreservation of boar semen

2020 ◽  
Vol 65 (No. 4) ◽  
pp. 115-123
Author(s):  
Marija Jovičić ◽  
Eva Chmelíková ◽  
Markéta Sedmíková
Keyword(s):  
Animal Species ◽  
Semen Quality ◽  
Egg Yolk ◽  
High Rate ◽  
Freezing And Thawing ◽  
Long Term Storage ◽  
Boar Semen ◽  
Boar Spermatozoa ◽  
Term Storage

Sperm cryopreservation is the best technology for long-term storage of the semen. However, the damage of boar spermatozoa by cryopreservation is more severe than in other animal species and a standardized freezing protocol for efficient cryopreservation has not been established yet. Semen quality and freezability vary greatly between breeds as well as between individual boars and even the season. Boar spermatozoa are sensitive to low temperatures; they sustain damage and a high rate of mortality and freezing/thawing the boar semen may strongly impair the sperm function and decrease the semen quality. The freezability of boar semen can be influenced by a cryopreservation procedure, and also by using various additives to freezing and thawing extenders such as antioxidants. In order to obtain acceptable results after thawing the boar semen, it is necessary to combine an optimal amount of additives (glycerol, egg yolk, sugars, antioxidants), cooling and warming velocities.

The use of butylated hydroxytoluene in cryopreservation of boar semen

2016 ◽  
Vol 12 (2) ◽  
pp. 21-28
Author(s):  
Monika Trzcińska ◽  
Magdalena Baryła
Keyword(s):  
Dna Fragmentation ◽  
Sperm Quality ◽  
Annexin V ◽  
Egg Yolk ◽  
Butylated Hydroxytoluene ◽  
Preimplantation Embryos ◽  
Progressive Motility ◽  
Boar Semen ◽  
Boar Spermatozoa

The objective of the study was to determine the effect of butylated hydroxytoluene (BHT) on the quality and fertilizing capacity of frozen-thawed (FT) boar semen. Semen from five boars (36 ejaculates) was resuspended in lactose-egg yolk-glycerol extender supplemented with 0 (control), 1.0 (R1), 1.5 (R2) or 2.0 mM BHT (R3). Sperm quality was assessed based on motility (CASA; TM: total motility; PM: progressive motility), phosphatidylserine (PS) translocation across the plasma membrane (Annexin-V-FLuos Staining Kit) and DNA fragmentation (TUNEL Assay). The FT semen was also used for intrauterine artificial insemination (AI) of synchronized gilts. The fertilizing capacity of the FT semen was assessed on the basis of the gilt insemination rate and the number of morphologically normal embryos. The quality of the preimplantation embryos was determined by observing a TUNEL-positive reaction. The highest percentage of progressive motile and viable spermatozoa was noted in extender R3 (74.8 ±4.4% and 63.7 ±5.8%), as compared with the control (38.3 ±2.8% and 36.1 ±2.6%). The addition of BHT to the extender did not increase early apoptotic changes in the frozen-thawed spermatozoa with respect to the control. Irrespective of the variant of the extender, cryopreservation and thawing did not induce fragmentation in the boar spermatozoa. The highest number of morphologically normal embryos from inseminated gilts was observed in the case of semen cryopreserved in extender supplemented with 1.5 mM BHT. No significant differences were observed in DNA fragmentation in the expanded blastocysts from gilts inseminated with FT semen cryopreserved in the extenders analysed.

Effects of negative pressure applied before storage (liquid storage, 17°C) on boar semen quality and fertilization ability

2019 ◽  
Author(s):  
LI. Jingchun ◽  
LI. Qi ◽  
LI. Yanbug ◽  
WEI Guosheng ◽  
SUN Dongbo
Keyword(s):  
Sperm Motility ◽  
Negative Pressure ◽  
Semen Quality ◽  
The Other ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Liquid Storage ◽  
Fertilizing Ability ◽  
Boar Semen ◽  
Acrosome Integrity

The present study was aimed to investigate the effects of negative pressure applied before storage on the quality and fertilization ability of boar semen. Boar semen samples were collected and pooled, and diluted with Modena solution containing 0.4% (w/v) of bovine serum albumin. Negative pressure was applied for 2–5 min using a vacuum pump with a barometer. The pressure applied were 0 (Control), -0.02 MPa (P200), -0.04 MPa (P400), and -0.08 MPa (P800). The sperm motility, acrosome integrity and sperm fertilizing ability were evaluated. Application of –0.04 MPa improved the sperm motility, acrosome integrity and fertilizing ability, compared with the other groups. The sperm motility and acrosome integrity decreased with increasing storage time in vitro. After 5 days, the sperm motility and acrosome integrity of the P400 group were all higher than those of the other groups (P less than 0.05). The cleavage rate (64.5% ± 2.4%) and blastocyst development rate (33.9% ± 2.8%) for semen stored for 7 days were similar to those of fresh semen. In conclusion, application of –0.04 MPa before liquid storage at 17°C can improve the quality and fertilization ability of boar semen.

Effect of commercial long-term extenders on metabolic activity and membrane integrity of boar spermatozoa stored at 17°C

2013 ◽  
Vol 16 (3) ◽  
pp. 517-525 ◽  
Author(s):  
A. Dziekońska ◽  
L. Fraser ◽  
A. Majewska ◽  
M. Lecewicz ◽  
Ł. Zasiadczyk ◽  
...  
Keyword(s):  
Metabolic Activity ◽  
Storage Time ◽  
Membrane Integrity ◽  
Prolonged Storage ◽  
Atp Content ◽  
Liquid Storage ◽  
Semen Storage ◽  
Boar Semen ◽  
Boar Spermatozoa

Abstract This study was aimed to analyze the metabolic activity and membrane integrity of boar spermatozoa following storage in long-term semen extenders. Boar semen was diluted with AndrohepR EnduraGuardTM (AeG), DILU-Cell (DC), SafeCell PlusTM (SCP) and Vitasem LD (VLD) extenders and stored for 10 days at 17oC. Parameters of the analyzed sperm metabolic activity included total motility (TMOT), progressive motility (PMOT), high mitochondrial membrane potential (MMP) and ATP content, whereas those of the membrane integrity included plasma membrane integrity (PMI) and normal apical ridge (NAR) acrosome. Extender type was a significant (P < 0.05) source of variation in all the analyzed sperm parameters, except for ATP content. Furthermore, the storage time had a significant effect (P < 0.05) on the sperm metabolic activity and membrane integrity during semen storage. In all extenders the metabolic activity and membrane integrity of the stored spermatozoa decreased continuously over time. Among the four analyzed extenders, AeG and SCP showed the best performance in terms of TMOT and PMI on Days 5, 7 and 10 of storage. Marked differences in the proportions of spermatozoa with high MMP were observed between the extenders, particularly on Day 10 of storage. There were not any marked differences in sperm ATP content between the extenders, regardless of the storage time. Furthermore, the percentage of spermatozoa with NAR acrosomes decreased during prolonged storage, being markedly lower in DC-diluted semen compared with semen diluted with either AeG or SCP extender. The results of this study indicated that components of the long-term extenders have different effects on the sperm functionality and prolonged semen longevity by delaying the processes associated with sperm ageing during liquid storage.

18 EFFECTS OF SKIM MILK ON THE QUALITY AND FERTILITY OF BOAR SEMEN FOLLOWING LIQUID PRESERVATION AT 5°C AND 15°C

2011 ◽  
Vol 23 (1) ◽  
pp. 115 ◽  
Author(s):  
Z. Namula ◽  
R. Kodama ◽  
Y. Kaedei ◽  
F. Tanihara ◽  
V. L. Vien ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Storage Temperature ◽  
Semen Quality ◽  
Sperm Concentration ◽  
Membrane Integrity ◽  
Skim Milk ◽  
Control Group ◽  
Boar Semen ◽  
Boar Spermatozoa

Liquid preservation of semen can be an alternative to frozen–thawed semen for artificial insemination. The success of a selection of boar semen extenders has been studied over storage periods of 5 to 7 days. The objective of this study was to evaluate the effects of skim milk on the viability and in vitro fertility of boar spermatozoa preserved in Modena-based extenders at 5°C and 15°C for 2 weeks. A total of 7 ejaculates were collected from one boar. The sperm-rich fraction of each ejaculate was centrifuged and diluted in Modena extenders supplemented with 0 (control), 7.5, and 15 mg mL–1 of dry skim milk. The final sperm concentration was adjusted to 1 × 108 cells mL–1, and then the semen was stored at 5°C and 15°C for 2 weeks. In the first experiment, the motility, viability (live/dead fluorescence viability assay), plasma membrane integrity (hypoosmotic swelling test; HOST), and acrosome integrity (FITC-labelled peanut agglutinin staining) of semen stored for 2 weeks were assessed. In the second experiment, the fertilization of stored semen after 20 h of co-incubation with in vitro matured oocytes and their development were examined. Data were analysed using ANOVA. When the semen was stored at 5°C for 2 weeks, the mean total sperm motility of semen stored with 7.5 and 15 mg mL–1 of dry skim milk was significantly higher than that of semen in the control group (41.4% and 41.5% v. 17.4%; P < 0.05). However, the beneficial effects of skim milk on the sperm motility were not observed in the semen stored at 15°C. Moreover, there were no significant differences in the other parameters of semen quality among the groups in each storage temperature. Significantly higher penetration rates of semen stored with 7.5 and 15 mg mL–1 of dry skim milk were observed in the storage at 5°C (41.1% and 34.8% v. 19.8%; P < 0.05) but not at 15°C (38.9% and 26.0% v. 30.0%; P > 0.05) when compared with the control group. When the semen was stored at 5°C, the development rate to the blastocyst stage of oocytes fertilized with semen stored with 7.5 mg mL–1 of dry skim milk was significantly higher than that with control and 15 mg mL–1 of dry skim milk (15.4% v. 1.1% and 7.8%; P < 0.01). However, there were no significant differences in the development rates of oocytes fertilized with semen stored at 15°C among the groups (9.6–11.9%). In conclusion, our results indicate that the effect of skim milk on the viability and in vitro fertility of liquid-stored boar spermatozoa is dependent on the storage temperature. The addition of 7.5 mg mL–1 of dry skim milk may be effective for the improvement of viability and fertility of semen stored at 5°C but not at 15°C.

Art outcome of non-apoptotic spermatozoa selected by magnetic-activated cell sorting with annexin V-conjugated microbeads

2013 ◽  
Vol 100 (3) ◽  
pp. S433
Author(s):  
Y. Fujino ◽  
Y. Nakamura ◽  
E. Wakimoto ◽  
K. Koike ◽  
L. Yamamoto ◽  
...  
Keyword(s):  
Cell Sorting ◽  
Annexin V ◽  
Art Outcome ◽  
Magnetic Activated Cell Sorting

Effect of the MACS technique on rabbit sperm motility

2011 ◽  
Vol 6 (6) ◽  
pp. 958-962
Author(s):  
Jaromir Vasicek ◽  
Alexander Makarevich ◽  
Peter Chrenek
Keyword(s):  
Harmful Effect ◽  
Sperm Concentration ◽  
Annexin V ◽  
Outer Leaflet ◽  
Significant Difference ◽  
Sperm Plasma Membrane ◽  
And Control ◽  
Casa System ◽  
Control Samples ◽  
Magnetic Activated Cell Sorting

AbstractMagnetic-activated cell sorting (MACS) separates apoptotic spermatozoa by the use of annexin V-conjugated nanoparticles which bind to phosphatidylserine that is externalized on the outer leaflet of the sperm plasma membrane. This technique yields two fractions: annexin V-negative (AnV−) and annexin V-positive (AnV+). The aim of the study was to evaluate the effect of MACS application on the motility parameters of rabbit spermatozoa. Rabbit semen samples collected separately from 4 bucks (I, II, III, and IV) were filtered and separated in a MACS system. The semen samples from a control (untreated) group, AnV− and AnV+ fraction were evaluated using CASA system. The experiment was replicated 4 times for each buck. The AnV+ sperm had significantly lower concentration than the AnV− fractions and the control samples (P<0.05 for bucks I, II, III, but not IV). We observed that the proportion of apoptotic spermatozoa in the semen of NZW bucks is about 20%. There was no significant difference in the percentage of motile and progressively motile spermatozoa between the AnVfractions and control samples. In conclusion, the MACS technique has no harmful effect on the rabbit sperm concentration and motility.

Sign in / Sign up

Export Citation Format

Share Document