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.

26 Baobab oil supplemented extender preserves post-thaw bull sperm quality parameters

2020 ◽  
Vol 32 (2) ◽  
pp. 139
Author(s):  
Z. Raphalalani ◽  
F. Ramukhithi ◽  
R. Ndhlala ◽  
K. Nephawe ◽  
T. Nedambale
Keyword(s):  
Sperm Quality ◽  
Membrane Integrity ◽  
Quality Parameters ◽  
Control Group ◽  
Dna Integrity ◽  
Sperm Dna ◽  
Frozen Semen ◽  
Morphological Defects ◽  
Bull Sperm ◽  
Semen Extender

The processes of semen cryopreservation and thawing affect sperm membrane integrity and motility and increases morphological defects as well as DNA damage. The most influential cause of this is oxidative stress. When endogenous antioxidant capacity of seminal plasma is reduced during the freeze-thawing process, plant extracts exhibiting strong antioxidant activity can be used as supplements for compensation. Baobab oil has gained interest because it is rich in powerful antioxidants, which could protect sperm cells from oxidative damage during cryopreservation. Our study aimed to assess the effects of baobab oil on post-thaw sperm quality parameters in an egg-yolk-based extender. Thirty semen ejaculates were collected from 15 Nguni bulls using an electro ejaculator. Semen samples were randomly allocated to control (no baobab oil), 20μL (1%), 50μL (2.5%), and 100μL (5%) baobab oil per millilitre extender. Following dilution, semen samples were loaded into 0.25-mL semen straws, equilibrated for 4h at 5°C, and transferred into a controlled rate programmable freezer. The frozen semen straws were stored in a liquid nitrogen tank (−196°C) until thawing. Semen straws were thawed (37°C/60 minutes) after 1 week of cryopreservation and analysed for (1) sperm motility using a computer-aided sperm analyser, (2) morphological defects and viability using eosin-nigrosin stain, (3) membrane integrity by hypo-osmotic swelling test, and (4) DNA integrity by terminal deoxynucleotidyl transferase dUTP nick end labelling assay. Data was analysed using analysis of variance. Treatment means were compared in relation to the control group by Dunnett's test. We found that supplementing semen extender with baobab oil at 1% significantly (P&lt;0.05) preserved sperm DNA integrity (88.3±3.7) and membrane integrity (74.0±4.2) when compared with the control group (71.7±3.7 and 55.8±4.4, respectively). Baobab oil supplementation either at 1% (5.9±0.5), 2.5% (7.2±0.5), or 5% (6.0±0.5) significantly reduced sperm morphological defects compared with control (9.5±0.5). Total motility (1% (72.7%), 2.5% (72.7%), 5% (71.9%), control (59.3%)) and viability (1% (79.1%), 2.5% (79.8%), 5% (77.8%), control (67.6%)) were also improved by supplementation; however, the difference was not significant. In conclusion, it was demonstrated that supplementing bull semen extender with 1% baobab oil protects sperm from morphological defects, maintains membrane integrity, as well as preserves sperm DNA. All the baobab oil supplementation levels preserved post-thaw bull-sperm quality parameters.

scholarly journals Seminal Plasma: Effect on Motility, Membrane Functionality, and Spermatic Chromatin Dispersion of Equine Sperm Treated with N-acetyl-L-cysteine at 5°C

2018 ◽  
Vol 44 (1) ◽  
pp. 6
Author(s):  
Murilo Farias Rodrigues ◽  
Janislene Mach Trentin ◽  
Laurence Boligon de Araujo ◽  
Luiz Augusto Machado Centeno ◽  
Ricardo Olimpio Schenatto ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Skim Milk ◽  
Dna Lesions ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Membrane Function ◽  
Motile Cells ◽  
Semen Preservation

Background: N-acetyl-L-cysteine (NAC) is a low molecular weight thiol studied as an antioxidant for stallion semen preservation without changes on sperm viability. Equine seminal plasma is rich in sulfur proteins (cysteine residues) named CRISPS, which, when combined with sulfur-containing antioxidants, can enhance the appearance of DNA lesions. The aim of this study was to assess and compare the effect of different concentrations of NAC by evaluating motility, membrane function and sperm chromatin integrity of equine semen cooled at 5°C in 50% of seminal plasma.Materials, Methods & Results: Nine ejaculates from 9 stallions were divided into 4 aliquots, diluted and divided in nonsupplemented skim milk group (0.0 mM), or supplemented with 5.0, 2.5 and 0.5 mM NAC. Evaluations were made at 0 h, 24 h and 48 h of cooling, except for motility which was evaluated only up to 24 h. The 0.5 (59.7 μM2) and 5.0 mM NAC (55.5 μM2) groups showed similar areas of sperm chromatin dispersion among all groups. However, the area of chromatin dispersion between the non-supplemented group was higher = 65.3 μM2 than the group supplemented with 2.5 mM. The percentage of cells with a functional plasma membrane was similar between supplemented and non-supplemented (0.0 mM) groups, but higher (P < 0.05) in the 0.5 mM NAC (39.7 and 39.8%, respectively) than that of 2.5 mM (34.5%) and 5.0 mM (34.2%) concentrations. Progressive motility was similar among all groups supplemented with NAC. The 0.5 mM NAC group showed 35.2% motile cells while the non-supplemented group exhibited 36.2%. Although 50% seminal plasma was used, NAC did not affect sperm chromatin integrity.Discussion: Seminal plasma interfered more in the results of different concentrations of NAC. This statement is proven by the motility analysis where all NAC concentrations showed similar results. Plasma percentage higher than 20% in diluted semen causes deleterious effects on sperm, such as decreased motility and fertilizing capacity. The membrane analysis in our study was compromised because NAC (2.5 to 5.0 mM) showed high osmolarity. As this was not adjusted, it affected the result. The 2.5 mM NAC group showed a lower area of sperm chromatin dispersion than none-treated sperm, although showing similar results to the other treatments. In a study with semen of Mangalarga Marchador stallions, the 2.5 mM of NAC was able to protect sperm membrane integrity. However, in another study, where semen was kept cooled between 5 and 15°C, no change was observed on sperm quality over different concentrations of NAC. This reinforces that 2.5 mM of NAC provides adequate protection to semen exposed to harmful conditions.The high percentage of plasma associated with this sulfur antioxidant did not compromise DNA integrity, as NAC concentration used was 100 times less than the concentration needed to induce DNA lesions.

scholarly journals Effects of freezing and activation on membrane quality and DNA damage in Xenopus tropicalis and Xenopus laevis spermatozoa

2017 ◽  
Vol 29 (8) ◽  
pp. 1556 ◽  
Author(s):  
S. Morrow ◽  
J. Gosálvez ◽  
C. López-Fernández ◽  
F. Arroyo ◽  
W. V. Holt ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Membrane Damage ◽  
Membrane Integrity ◽  
Subsequent Development ◽  
Xenopus Tropicalis ◽  
Model Organisms ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Sperm Cryopreservation ◽  
Sperm Plasma Membrane

There is growing concern over the effect of sperm cryopreservation on DNA integrity and the subsequent development of offspring generated from this cryopreserved material. In the present study, membrane integrity and DNA stability of Xenopus laevis and Xenopus tropicalis spermatozoa were evaluated in response to cryopreservation with or without activation, a process that happens upon exposure to water to spermatozoa of some aquatic species. A dye exclusion assay revealed that sperm plasma membrane integrity in both species decreased after freezing, more so for X. laevis than X. tropicalis spermatozoa. The sperm chromatin dispersion (SCD) test showed that for both X. tropicalis and X. laevis, activated frozen spermatozoa produced the highest levels of DNA fragmentation compared with all fresh samples and frozen non-activated samples (P < 0.05). Understanding the nature of DNA and membrane damage that occurs in cryopreserved spermatozoa from Xenopus species represents the first step in exploiting these powerful model organisms to understand the developmental consequences of fertilising with cryopreservation-damaged spermatozoa.

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 Finding an Effective Freezing Protocol for Turkey Semen: Benefits of Ficoll as Non-Permeant Cryoprotectant and 1:4 as Dilution Rate

Animals ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 421 ◽  
Author(s):  
Michele Di Iorio ◽  
Giusy Rusco ◽  
Roberta Iampietro ◽  
Maria Antonietta Colonna ◽  
Luisa Zaniboni ◽  
...  
Keyword(s):  
Dilution Rate ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Quality Parameters ◽  
Dna Integrity ◽  
Osmotic Resistance ◽  
Progressive Motility ◽  
Nitrogen Vapor ◽  
Combined Use ◽  
Cryopreservation Protocol

The present study aimed to find an effective cryopreservation protocol for turkey semen through the combined use of dimethylsulfoxide (DMSO) and three non-permeant cryoprotectants (NP-CPAs), sucrose, trehalose, and Ficoll 70. In addition, the action of two dilution rates (1:2 and 1:4) were also investigated. Semen was processed according to two final dilution rates and the following treatments: Tselutin extender (TE)/DMSO (control), TE/DMSO + sucrose or trehalose 50, 100, 200, or 400 mM, and TE/DMSO + Ficoll 0.5, 0.75, 1, or 1.5 mM. In total 26 different combinations treatments were achieved. The diluted semen was filled up into straws and frozen on liquid nitrogen vapor. The post-thawing sperm quality was assessed by analyzing motility, membrane integrity, osmotic resistance, and DNA integrity. The results obtained revealed a significant effect of NP-CPA concentration on total and progressive motility, on most of the kinetic parameters, on membrane integrity and DNA integrity, while the post-thaw quality was less affected by dilution rate. The highest post-thaw quality for all sperm quality parameters assessed except curvilinear velocity (VCL) and DNA integrity were found in semen frozen with 1 mM Ficoll/1:4 (p < 0.05). Our findings provide an important contribution for the identification of a reference procedure for turkey semen cryopreservation, in order to create the first national avian semen cryobank.

scholarly journals Antibiotics Versus Natural Biomolecules: The Case of In Vitro Induced Bacteriospermia by Enterococcus Faecalis in Rabbit Semen

Molecules ◽  
2019 ◽  
Vol 24 (23) ◽  
pp. 4329 ◽  
Author(s):  
Michal Duracka ◽  
Norbert Lukac ◽  
Miroslava Kacaniova ◽  
Attila Kantor ◽  
Lukas Hleba ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Enterococcus Faecalis ◽  
Antioxidant Properties ◽  
Membrane Integrity ◽  
Bioactive Molecules ◽  
Human Reproduction ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Computer Assisted ◽  
Microbiological Analysis

Male subfertility is a global issue in human reproduction as well as in animal reproduction. Bacterial infection and semen contamination are still widely overlooked. As the collection of ejaculates is not a sterile process, it is necessary to add antimicrobial agents to avoid a possible depreciation of semen samples. As traditionally used antibiotics have been questioned because of an ever-increasing bacterial resistance, natural bioactive molecules could offer an alternative because of their antibacterial and antioxidant properties. As such, we decided to compare the effects of selected natural biomolecules (resveratrol-RES, quercetin-QUE and curcumin-CUR) with routinely used antibiotics in animal biotechnologies (penicillin-PEN, gentamicin-GEN and kanamycin-KAN) on the rabbit sperm vitality in the presence of Enterococcus faecalis. Changes in the sperm structural integrity and functional activity were monitored at 0, 2, 4 and 6 h. Computer-assisted sperm analysis (CASA) was used for the assessment of spermatozoa motility. Production of reactive oxygen species (ROS) was evaluated using chemiluminiscence, while the mitochondrial membrane potential (ΔΨm) was examined using the JC-1 dye. Finally, the sperm chromatin dispersion (SCD) test was used to assess DNA fragmentation, and changes to the membrane integrity were evaluated with the help of annexin V/propidium iodide. The motility assessment revealed a significant sperm motility preservation following treatment with GEN (p < 0.001), followed by PEN and CUR (p < 0.01). QUE was the most capable substance to scavenge excessive ROS (p < 0.001) and to maintain ΔΨm (p < 0.01). The SCD assay revealed that the presence of bacteria and antibiotics significantly (p < 0.05) increased the DNA fragmentation. On the other hand, all bioactive compounds readily preserved the DNA integrity (p < 0.05). In contrast to the antibiotics, the natural biomolecules significantly maintained the sperm membrane integrity (p < 0.05). The microbiological analysis showed that GEN (p < 0.001), KAN (p < 0.001), PEN (p < 0.01) and CUR (p < 0.01) exhibited the strongest antibacterial activity against E. faecalis. In conclusion, all selected biomolecules provided protection to rabbit spermatozoa against deleterious changes to their structure and function as a result of Enterococcus faecalis contamination. Therefore, administration of RES, QUE and/or CUR to rabbit semen extenders in combination with a carefully selected antibacterial substance may be desirable.

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>

scholarly journals Initial Characterization of Male Southern Stingray (Hypanus americanus) Reproductive Parameters and Preliminary Investigation of Sperm Cryopreservation

Animals ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 2716
Author(s):  
James D. Gillis ◽  
Linda M. Penfold ◽  
Natalie D. Mylniczenko
Keyword(s):  
Sperm Count ◽  
Preliminary Investigation ◽  
Membrane Integrity ◽  
Ringer Solution ◽  
Ex Situ ◽  
Total Testosterone ◽  
Sperm Cryopreservation ◽  
Slow Cooling ◽  
Freezing Method ◽  
Elasmobranch Species

This study investigated the reproductive biology and sperm cryopreservation of ex situ southern stingrays (Hypanus americanus) by semen collection and characterization and the development and validation of an enzyme-linked immunoassay for plasma total testosterone. Semen was collected in March and June using a manual massage technique, and the ejaculates were assessed for volume, pH, osmolarity, motility, status (0–5 scale: 0 = no forward progression, 5 = rapid linear progression) and total sperm count. Semen was extended in Hank’s elasmobranch ringer solution containing 10% DMSO, 10% glycerol or 5% glycerol with 5% N-methylformamide and cryopreserved using a conventional freezing method (~−50 °C/min) or a modified slow freezing method (~−3 °C/min). Body condition was scored from 1–5 and was noted to be low in March (1.93 ± 0.07) due to feeding practices and increased by June (2.93 ± 0.05) after dietary corrections were made. A concomitant increase (p < 0.05) in plasma total testosterone concentration and sperm motility was noted between March (8.0 ± 7.2 ng/mL, 5.71 ± 2.77%) and June (97.3 ± 11.3 ng/mL, 51.4 ± 14.3%). Samples cryopreserved using a modified slow freeze method (~−3 °C/min) had higher post-thaw motility and plasma membrane integrity than conventionally cryopreserved samples. Data indicate that southern stingray sperm morphometrics adheres to those of other elasmobranch species and that a slow cooling rate may be an avenue of research to improve southern stingray sperm survival during cryopreservation.

scholarly journals Development of Sperm Cryopreservation Protocols for Sharks and Rays: New Tools for Elasmobranch Conservation

2021 ◽  
Vol 8 ◽  
Author(s):  
Pablo García-Salinas ◽  
Victor Gallego ◽  
Juan F. Asturiano
Keyword(s):  
Sperm Quality ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Ex Situ ◽  
Conservation Strategies ◽  
Sperm Cryopreservation ◽  
In Captivity ◽  
Reproductive Techniques ◽  
Elasmobranch Species ◽  
Reproductive Cycles

Elasmobranchs are one of the most endangered vertebrate groups on the planet, but despite this situation the use of reproductive techniques in elasmobranch conservation strategies has been scarce. Among these techniques, sperm preservation is a potential tool for ex situ conservation and aquaria sustainability. However, there are no widespread preservation protocols for elasmobranch sperm, and shark sperm cryopreservation has never been achieved before. Here we present the establishment of successful cryopreservation protocols for elasmobranch sperm, tested in several species. We have formulated a sperm extender that can be used for different elasmobranch species, capable of maintaining sperm motility for several weeks. Additionally, we achieved the cryopreservation of sperm by previously diluting it in our extender and supplementing it with different combinations of cryoprotectants. The effects of methanol and dimethyl sulfoxide as permeating cryoprotectants were evaluated, as well egg yolk as a non-permeating cryoprotectant. Sperm quality was assessed by studying the motility and membrane integrity post-thawing, demonstrating its effectiveness in the 10 species tested, including two which are considered Critically Endangered. This is the first time that shark sperm cryopreservation has been reported, broadening our knowledge of the reproductive techniques that can be applied to elasmobranchs and laying the foundations for the first cryobanks for shark and ray sperm. Outcomes from this study will be useful for ex situ conservation efforts developed by public aquaria. A regular supply of frozen sperm will reduce the problems that result from the transport of specimens, inbreeding or lack of synchronized reproductive cycles in captivity.

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