scholarly journals 9 DOES STRAW CONCENTRATION AFFECT POST-THAW SPERM QUALITY IN AI BULL SIRES?

2005 ◽  
Vol 17 (2) ◽  
pp. 155
Author(s):  
J. Ballester ◽  
A. Johannisson ◽  
M. Håård ◽  
H. Gustafsson ◽  
H. Rodriguez-Martinez
Keyword(s):  
Plasma Membrane ◽  
Sperm Quality ◽  
Chromatin Condensation ◽  
Membrane Integrity ◽  
Annexin V ◽  
Membrane Stability ◽  
Semen Parameters ◽  
Sperm Chromatin ◽  
Sperm Viability ◽  
Low Concentrations

There is increasing interest in decreasing the number of spermatozoa per AI dose, owing to the discrimination of threshold concentration and fertility, economical revenues and the use of sexed semen. This study evaluated the quality (as sperm viability, acrosome integrity, membrane stability and chromatin stability) post-thaw of semen collected from four elite AI sires and frozen at 15 × 106 (control) or 2 × 106 spermatozoa (spz) per dose to disclose eventual deleterious extension effects. Semen collected via a.v. from 4 élite AI sires was split-processed and frozen in 0.25 mL plastic straws under commercial conditions, at either 2 × 106 or 15 × 106 spz/straw (the latter as control). The semen parameters were within acceptable limits of normality. The post-thaw samples showed acceptable motility (above 50%). Sperm viability and stability were assessed post-thaw using flow cytometry of cells loaded with SYBR-14 and propidium iodide (PI) for sperm viability (membrane integrity), and with carboxy-SNARF-1 (SNARF; Invitrogen AB, Frolunda, Sweden), PI, and FITC-Pisum sativum agglutinin (PSA, triple stain) for acrosome status. Membrane stability status was measured with Annexin-V/PI, while sperm chromatin condensation and stability were assessed following in situ acid-induced DNA denaturation and staining with acridine orange. No significant differences were seen between the two concentrations regarding sperm motility, plasma membrane integrity (SYBR/PI, SNARF), or chromatin stability. The highly extended semen (2 × 106 spz/straw), however, showed a higher (P < 0.05) frequency of spermatozoa with translocated phosphatydil serine (as detected with Annexin-V), indicating their plasma membranes had become unstable. Also, there were more spermatozoa showing acrosomal damage (PSA). In conclusion, low concentrations of spermatozoa do not properly sustain conventional cryopreservation, although damages appear restricted to the sperm membrane. These changes in the stability of the plasma membrane apparently did not affect the viability of the spermatozoa, although they may negatively affect their lifespan. These findings may have an impact on the care that must be taken when cryopreserving low concentrations of spermatozoa with conventional freezing protocols. This work was supported by the Swedish Farmers’ Foundation for Research in Agriculture (SLF) and FORMAS, Stockholm.

scholarly journals Effects of the platelet-activating factor (PAF) on selected quality parameters of cryopreserved bull semen (AI) with reduced sperm motility

2016 ◽  
Vol 19 (1) ◽  
pp. 147-158 ◽  
Author(s):  
M. Lecewicz ◽  
W. Kordan ◽  
A. Majewska ◽  
S. Kamiński ◽  
A. Dziekońska ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Sperm Motility ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Platelet Activating Factor ◽  
Quality Parameters ◽  
Sperm Viability ◽  
Plasma Membrane Integrity ◽  
Positive Effects ◽  
Bull Semen

Abstract The aim of the study was to determine the effects of platelet-activating factor (PAF) on selected quality parameters of cryopreserved bull semen with reduced sperm motility used for artificial insemination. The aim of experiment 1 was to identify the optimal concentration of the phospholipid able to preserve sperm viability. Cryopreserved semen was treated with different PAF concentrations: 1×10-5M, 1×10-6M, 1×10-7M, 1×10-8M and 1×10-9M. The experiment demonstrated that PAF at concentration 1×10-9M increased most the sperm viability parameters (motility parameters, plasma membrane integrity and mitochondrial function) after 120 min of incubation of thawed semen at 37°C. Cryopreserved bull semen with reduced sperm motility (below 70%) was supplemented with PAF in a concentration of 1×10-9M. A statistically significant increase in sperm motility, percentage of linear motile spermatozoa and VSL value was observed after 120 min incubation of sperm with 1×10-9M PAF. Sperm supplementation with PAF also had positive effects on plasma membrane integrity and percentage of spermatozoa with preserved mitochondrial transmembrane potential, but the differences were not statistically significant. The results indicated positive effects of PAF supplementation at a concentration of 1×10-9M on the selected sperm quality parameters in cryopreserved bull semen with reduced motility.

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.

54 SINGLE LAYER CENTRIFUGATION BEFORE CRYOPRESERVATION IMPROVES BULL SPERM QUALITY

2017 ◽  
Vol 29 (1) ◽  
pp. 134
Author(s):  
T. Nongbua ◽  
A. Utta ◽  
N. Am-In ◽  
J. Suwimonteerabutr ◽  
A. Johannisson ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Chromatin Structure ◽  
Mitochondrial Membrane ◽  
Linear Mixed Model ◽  
Sperm Quality ◽  
Single Layer ◽  
Sperm Chromatin ◽  
Sperm Viability ◽  
Bull Sperm

Single layer centrifugation (SLC) with Bovicoll is a technique to enhance sperm quality. The purpose of this study was to investigate the effect of SLC before cryopreservation on bull sperm quality after thawing. Semen was collected from 8 bulls (American Brahman, n = 5 and Sahiwal, n = 3) at the North Eastern Bull Centre (KhonKaen, Thailand). The ejaculate was split: one part was prepared following the standard procedure at the bull centre (n = 88) as control. The other part was used for SLC with Bovicoll-B (Johannisson et al. 2016 Theriogenology 86, 140). The SLC-selected sperm samples were frozen using the same protocol as control (n = 88). After thawing at 37°C for 12 s, motility analysis was performed using the CEROS II® (Hamilton Thorne, Beverly, MA, USA); sperm chromatin structure, mitochondrial membrane potential, and sperm viability were assessed using a FC500 flow cytometer (Beckman Coulter, Brea, CA, USA). Treatment means were compared using the linear mixed model (Proc MIXED, SAS®, 9.3, SAS Institute Inc., Cary, NC, USA). Results are reported as least-squares means ± standard error. The sperm kinematics for SLC samples were higher than controls for progressive motility (26.37 ± 1.59%, 19.56 ± 1.59%), Linearity (LIN) (52.80 ± 0.87%, 44.94 ± 0.87%), Straightness (STR) (83.06% ± 0.59, 76.20 ± 0.59%), beat cross frequency (BCF) (29.25 ± 0.50 Hz, 24.35 ± 0.50 Hz) and wobble (WOB) (61.78 ± 0.63%, 57.40 ± 0.63%) (all P < 0.0001) respectively, whereas SLC-selected samples were lower than controls for slow motility (13.61 ± 0.71%, 15.56 ± 0.71%; P < 0.05), Amplitude of lateral head displacement (ALH) (4.88 ± 0.18 μm, 6.67 ± 0.18 μm), velocity average path, (VAP) (61.17 ± 1.93μ/s, 67.88 ± 1.93μ/s), and curvilinear velocity (VCL) (99.78 ± 3.77 μ/s, 122.91 ± 3.77 μ/s) (all P < 0.0001), respectively. Other parameters of sperm quality were not different between treatments, although there was considerable variation among individual bulls in sperm chromatin structure assay, mitochondrial membrane potential, and sperm viability. These results suggest that SLC can be used before cryopreservation to improve the kinematics of thawed bull sperm samples without adversely affecting other parameters of sperm quality.

scholarly journals O papel do dimetilsulfóxido na criopreservação de sêmen de Curimba (Prochilodus lineatus)

2015 ◽  
Vol 36 (5) ◽  
pp. 3471
Author(s):  
Antonio Sergio Varela Junior ◽  
Estela Fernandes Silva ◽  
Tainã Figueiredo Cardoso ◽  
Érica Yokoyama Namba ◽  
Rodrigo Desessards Jardim ◽  
...  
Keyword(s):  
Sperm Quality ◽  
Membrane Integrity ◽  
Fertilization Rate ◽  
Dna Integrity ◽  
Hatching Rate ◽  
Sperm Viability ◽  
Prochilodus Lineatus ◽  
Dmso Concentration ◽  
Fresh Semen

<p class="Pa7">Cryopreservation of Curimba semen (Prochilodus lineatus) is ecological and commercial importance. The objective of this study was to evaluate the effect of different concentrations (2, 5, 8 and 11%) of dimethyl sulfoxide (DMSO) diluted in Betsville Thawing Solution (BTS) on the quality of post-thaw semen Curimba. We analyzed the rate and period motility, sperm viability, membrane integrity and DNA, mitochondrial functionality, and fertilization and hatching rate. The plasma membrane and DNA integrity of a DMSO concentration of 11% obtained better results than the concentration of 5% (p &lt;0.05). However, treatment of 5% DMSO resulted in a longer latency and a higher fertilization rate and hatching, in other sperm quality equal to that of fresh semen. The results of this study indicate that 5% DMSO is ideal for cryopreservation of semen Curimba.</p>

The effects of chronic administration of ghrelin on rat sperm quality and membrane integrity

2009 ◽  
Vol 59 (2) ◽  
pp. 159-168 ◽  
Author(s):  
Arash Kheradmand ◽  
Majid Taati ◽  
Homayoon Babaei
Keyword(s):  
Plasma Membrane ◽  
Treatment Group ◽  
Sperm Motility ◽  
Sperm Quality ◽  
Sperm Concentration ◽  
Membrane Integrity ◽  
Chronic Administration ◽  
Control Group ◽  
Plasma Membrane Integrity ◽  
Significant Difference

AbstractAlthough ghrelin acts as a modulator of feeding behavior and energy metabolism in the central nervous system, recent studies have implicated the peripheral actions of ghrelin in reproductive tissues. Here, we investigated the effects of chronic administration of ghrelin on the motility, plasma membrane integrity and concentration of rat spermatozoa. 45-d male Wistar rats were scheduled for the study and were divided into control and treatment groups. In the treatment group, 1 nmol of ghrelin was administered as sc injection for 10 consecutive days or vehicle (physiological saline) to the control rats. Sperm collection was achieved by killing of the rats on days 15, 25 and 50 after first injection. Total sperm motility and forward progressive movement did not exhibit significant difference during the experiment, although, there was a tendency for greater motion rate on d 15 and 25 in the treated rats compared to the control group. Plasma membrane integrity (HOS-reacted spermatozoa) was significantly higher in the treated animals, especially on day 15 as well as day 25, because of possible antioxidant properties of ghrelin. This value was statistically higher on day 15 than that of day 25 (P <0.05). Likewise, there was a significant correlation between the FPM (P <0.0001, r = 0.79) and TSM (P <0.01, r = 0.52) with the HOS test percentage in the treatment group. It was not observed statistically difference in the sperm concentration between groups during all of the experimental days. In conclusion, chronic administration of ghrelin (similar to induced by energy deficiency such as fasting) increased the integrity of sperm membrane, however, the sperm motility and concentration did not display any alterations.

scholarly journals Effects of cryopreservation on semen quality and the expression of sperm membrane hexose transporters in the spermatozoa of Iberian pigs

Reproduction ◽  
2007 ◽  
Vol 134 (1) ◽  
pp. 111-121 ◽  
Author(s):  
S Sancho ◽  
I Casas ◽  
H Ekwall ◽  
F Saravia ◽  
H Rodriguez-Martinez ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Glucose Transporter ◽  
Semen Quality ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Membrane Receptors ◽  
Plasma Membrane Integrity ◽  
Sugar Transporters ◽  
Hexose Transporters

This study evaluated the effects of cooling, freezing and thawing on the plasma membrane integrity, kinetics and expression of two sugar transporters glucose transporter-3 and -5 (GLUT-3 and GLUT-5) in spermatozoa from Iberian boars. Semen samples were collected twice weekly from eight young, fertile Iberian boars of the ‘Entrepelado’ and ‘Lampiño’ breeds. The samples were suspended in a commercial extender and refrigerated to 17 °C for transport to the laboratory (step A), where they were further extended with a lactose–egg yolk-based extender and chilled to 5 °C (step B) prior to freezing in the presence of glycerol (3%). Spermatozoa were assessed for plasma membrane integrity and sperm motility at each of the steps, including post-thaw (step C). Aliquots were also prepared for immunocytochemical localisation of the sugar transporters (fixed and thin smears for transmission and scanning electron microscopy levels respectively) and for SDS–PAGE electrophoresis and subsequent western blotting, using the same antibodies (rabbit anti-GLUT-3 and anti-GLUT-5 polyclonal antibodies). The results showed lower percentages of progressively motile spermatozoa at step C in both breeds, while the percentage of live spermatozoa was significantly lower only in the ‘Entrepelado’ breed. The results obtained from electron microscopy clearly showed that Iberian boar spermatozoa expressed the hexose transporters, GLUT-3 and GLUT-5. The pattern of expression, in terms of location and concentration, was characteristic in each case but, in the case of isoform GLUT-5, it remained constant during the different steps of freezing–thawing protocol. These results indicate that cryopreservation affects the status of sperm cells of Iberian boars by altering the distribution of some membrane receptors and decreasing the percentage values of parameters linked to sperm quality.

Effect of platelet activating factor (PAF) supplementation in semen extender on viability and ATP content of cryopreserved canine spermatozoa

2010 ◽  
Vol 13 (4) ◽  
pp. 571-579 ◽  
Author(s):  
W. Kordan ◽  
M. Lecewicz ◽  
R. Strzeżek ◽  
A. Dziekońska ◽  
L. Fraser
Keyword(s):  
Plasma Membrane ◽  
Sperm Motility ◽  
Mitochondrial Function ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Platelet Activating Factor ◽  
Computer Assisted ◽  
Atp Content ◽  
Plasma Membrane Integrity ◽  
Semen Extender

Effect of platelet activating factor (PAF) supplementation in semen extender on viability and ATP content of cryopreserved canine spermatozoa The aim of this study was to investigate the effect of platelet activating factor (PAF) on the quality characteristics of cryopreserved canine spermatozoa. Cryopreserved semen of 5 mixed-breed dogs was treated with different concentrations of exogenous PAF (1 × 10-3M, 1 × 10-4M, 1 × 10-5M and 1 × 10-6M) and examined at different time intervals (0, 30, 60 and 120 min). Cryopreserved semen treated without PAF was used as the control. Sperm quality was evaluated for motility (computer-assisted semen analysis, CASA), mitochondrial function (JC-1/PI assay) and plasma membrane integrity (SYBR-14/PI assay and Hoechst 33258). Also, ATP content of spermatozoa was determined using a bioluminescence assay. Treatment of cryopreserved semen with 1 × 10-3 M PAF at 120 min of incubation resulted in significantly higher total sperm motility compared with the control. It was observed that PAF-improved total sperm motility was concurrent with enhanced sperm motility patterns after treatment of cryopreserved semen. Treatment of cryopreserved semen with PAF did not improve either sperm mitochondrial function or plasma membrane integrity, as monitored by different fluorescent membrane markers. Furthermore, ATP content of cryopreserved spermatozoa was significantly higher when PAF was used at a concentration of 1 × 10-3 M compared with the control and other PAF treatments, regardless of the incubation time. The findings of this study indicated that treatment with 1 × 10-3 M PAF at 120 min of incubation rendered better quality of cryopreserved canine semen, which was associated with improved sperm motility parameters and ATP content. It can be suggested that exogenous PAF addition is beneficial as a supplement for canine semen extender used for.

scholarly journals Evaluation of motility, membrane status and DNA integrity of frozen–thawed bottlenose dolphin (Tursiops truncatus) spermatozoa after sex-sorting and recryopreservation

Reproduction ◽  
2012 ◽  
Vol 143 (6) ◽  
pp. 799-813 ◽  
Author(s):  
G A Montano ◽  
D C Kraemer ◽  
C C Love ◽  
T R Robeck ◽  
J K O'Brien
Keyword(s):  
Bottlenose Dolphin ◽  
Sperm Quality ◽  
Critical Evaluation ◽  
Membrane Integrity ◽  
Ex Situ ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Freezing Method ◽  
Frozen Semen ◽  
Motility Parameters

Artificial insemination (AI) with sex-sorted frozen–thawed spermatozoa has led to enhanced management of ex situ bottlenose dolphin populations. Extended distance of animals from the sorting facility can be overcome by the use of frozen–thawed, sorted and recryopreserved spermatozoa. Although one bottlenose dolphin calf had been born using sexed frozen–thawed spermatozoa derived from frozen semen, a critical evaluation of in vitro sperm quality is needed to justify the routine use of such samples in AI programs. Sperm motility parameters and plasma membrane integrity were influenced by stage of the sex-sorting process, sperm type (non-sorted and sorted) and freezing method (straw and directional) (P<0.05). After recryopreservation, sorted spermatozoa frozen with the directional freezing method maintained higher (P<0.05) motility parameters over a 24-h incubation period compared to spermatozoa frozen using straws. Quality of sperm DNA of non-sorted spermatozoa, as assessed by the sperm chromatin structure assay (SCSA), was high and remained unchanged throughout freeze–thawing and incubation processes. Though a possible interaction between Hoechst 33342 and the SCSA-derived acridine orange was observed in stained and sorted samples, the proportion of sex-sorted, recryopreserved spermatozoa exhibiting denatured DNA was low (6.6±4.1%) at 6 h after the second thawing step and remained unchanged (P>0.05) at 24 h. The viability of sorted spermatozoa was higher (P<0.05) than that of non-sorted spermatozoa across all time points after recryopreservation. Collective results indicate that bottlenose dolphin spermatozoa undergoing cryopreservation, sorting and recryopreservation are of adequate quality for use in AI.

scholarly journals Correlations Amongst Functional and Morphological Attributes and Oxidative Markers of Fresh and Cryopreserved Semen of Gir and Murrah Bulls

2020 ◽  
Vol 15 (03) ◽  
pp. 24-29
Author(s):  
A. J. Dhami ◽  
Tapasvi M Patel ◽  
DV Chaudhari
Keyword(s):  
Plasma Membrane ◽  
Sperm Motility ◽  
Semen Quality ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Winter Season ◽  
Correlation Coefficients ◽  
Plasma Membrane Integrity ◽  
Murrah Buffalo ◽  
Oxidative Markers

This study was undertaken during the winter season on healthy mature Gir cattle and Murrah buffalo bulls (n=3 each). The semen samples (6 ejaculates/bull, total 36 ejaculates) collected in the morning using artificial vagina were evaluated for routine seminal attributes, including acrosomal and plasma membrane integrity. The samples were then diluted @ 100 million sperm/ml with tris fructose yolk glycerol extender without and with sericin @ 0.1, 0.25, 0.5 and 1.0% (w/v), filled in French mini-straws, and frozen in LN2 using biofreezer as per standard freezing protocol. Straws were thawed in water bath at 37°C for 30 sec and evaluated for post-thaw quality, viz., motility, viability, morphology, acrosome integrity and plasma membrane integrity (HOST). Lipid peroxidation (malondialdehyde - MDA production) and activities of enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx) were assessed as oxidative markers in seminal plasma of freshly diluted and frozen-thawed semen samples. Sericn at 0.5% level significantly (p less than 0.01) improved the post-thaw sperm quality with reduced oxidative stress in both the species. The breed-wise correlation coefficients (r) among sperm quality attributes and oxidative markers were studied in fresh and frozen-thawed semen of each species, and also for fresh with frozen-thawed semen. The findings revealed significant interrelationships amongst most of the attributes of fresh as well as post-thawed semen and also of fresh semen attributes with those of cryopreserved semen including oxidative markers in both the species. Sperm motility estimation in fresh, pre-freeze and post-thawed semen was a legitimately good indicator of quality of spermatozoa at various steps of semen processing/freezing, and its fertilizing potential. Thus, the sperm motility, HOS test and either MDA or SOD/GPx activity alone may be used as valuable and practical tools for routine assessment of bovine semen quality considering significant correlations found between them.

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