97 DIFFERENCES IN SPERM CHROMATIN STRUCTURE AMONG GOOD AND BAD BOAR SPERM FREEZERS

2006 ◽  
Vol 18 (2) ◽  
pp. 157
Author(s):  
M. Hernández ◽  
J. Roca ◽  
J. Ballester ◽  
J. M. Vázquez ◽  
E. A. Martínez ◽  
...  
Keyword(s):  
Chromatin Structure ◽  
Sperm Quality ◽  
Egg Yolk ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Computer Assisted ◽  
Sperm Analysis ◽  
Fragmentation Index ◽  
Sperm Dna ◽  
Multivariate Pattern

Inter- and intra-boar differences in sperm freezability are observed independent of the sperm quality before freezing, the breed, or the genetic line. This study aimed to determine whether boars with different post-thaw sperm quality also show differences in sperm DNA integrity. Sperm-rich fractions (3 to 10 ejaculates per boar) from 19 fertile mature boars were extended in Beltsville thawing solution (BTS) and cooled to 17�C for 16 h. Then, samples were centrifuged at 2400g for 3 min, extended in freezing extender (lactose/egg yolk/glycerol/Equex STM; Nova Chemical Sales, Inc., Scituate, MA, USA) to a final concentration of 1 � 109 spermatozoa/mL, dispensed into 0.5 mL straws, and frozen in a programmable cell freezer at a rate of -20�C min. Thawing was carried out in a water bath at 37�C for 20 s. Frozen-thawed spermatozoa were evaluated for progressive sperm motility (PSM) using a computer-assisted sperm analysis (CASA) system, and sperm viability (PMI) using flow cytometry. All data generated were used for a multivariate pattern analysis (PATN; CSIRO, Canberra, Australia) which objectively classified all boars into two groups, categorized as good (n = 10; >60% PSM and PMI) or bad (n = 9; <40% PSM and PMI) based on their sperm freezability. Post-thaw sperm quality was consistent for different ejaculates within boars (P < 0.05). The DNA-integrity of frozen-thawed spermatozoa was evaluated according to the sperm chromatin structure assay (SCSA; Evenson et al. 1980 Science 210, 1131-1133). All SCSA variables (X mean, DNA fragmentation index (DFI), and the standard deviation of the DFI), were significantly higher for bad freezers (P < 0.001). The percentage of spermatozoa with abnormal chromatin structure ranged from 1.06 to 3.42% for good and 3.06 to 6.04% for bad freezers. Although these differences exist between good and bad sperm freezers, and can only to some extent be the product of cryopreservation, the levels of affected spermatozoa can not explain the differences on post-thaw sperm survival seen in the two categories of sires. This work was supported by CICYT, AGL05-0471 (Spain), SLF and Formas (Sweden).

scholarly journals Thawing boar semen in the presence of seminal plasma improves motility, modifies subpopulation patterns and reduces chromatin alterations

2017 ◽  
Vol 29 (8) ◽  
pp. 1576 ◽  
Author(s):  
Rocío Fernández-Gago ◽  
Manuel Álvarez-Rodríguez ◽  
Marta E. Alonso ◽  
J. Ramiro González ◽  
Beatriz Alegre ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Chromatin Structure ◽  
Sperm Chromatin ◽  
Chromatin Compaction ◽  
Sperm Analysis ◽  
Positive Effects ◽  
Fragmentation Index ◽  
Non Linear ◽  
Boar Semen ◽  
Dna Fragmentation Index

Seminal plasma could have positive effects on boar semen after thawing. In the present study we investigated changes in the motility and chromatin structure in spermatozoa over 4 h incubation (37°C) of boar semen thawed in the presence of 0%, 10% or 50% seminal plasma from good-fertility boars. Cryopreserved doses were used from seven males, three of which were identified as susceptible to post-thawing chromatin alterations. Motility was analysed by computer-aided sperm analysis every hour, and data were used in a two-step clustering, yielding three subpopulations of spermatozoa (slow non-linear, fast non-linear, fast linear). Chromatin structure was analysed using a sperm chromatin structure assay and flow cytometry to determine the DNA fragmentation index (%DFI) as a percentage, the standard deviation of the DFI (SD-DFI) and the percentage of high DNA stainability (%HDS), indicating chromatin compaction. Thawing without seminal plasma resulted in a rapid loss of motility, whereas seminal plasma helped maintain motility throughout the incubation period and preserved the subpopulation comprising fast and linear spermatozoa. The incidence of chromatin alterations was very low in samples from non-susceptible males, whereas samples from males susceptible to post-thawing chromatin alterations exhibited marked alterations in %DFI and %HDS. Seminal plasma partly prevented these alterations in samples from susceptible males. Overall, 50% seminal plasma was the most efficient concentration to protect motility and chromatin. Some changes were concomitant with physiological events reported previously (e.g., semen thawed with 50% seminal plasma increased the production of reactive oxygen species and yielded higher fertility after AI). Thawing in the presence of seminal plasma could be particularly useful in the case of samples susceptible to post-thawing chromatin damage.

Evaluating human sperm DNA integrity: relationship between 8-hydroxydeoxyguanosine quantification and the sperm chromatin structure assay

Zygote ◽  
2003 ◽  
Vol 11 (4) ◽  
pp. 367-371 ◽  
Author(s):  
Isabelle Oger ◽  
Christelle Da Cruz ◽  
Gilles Panteix ◽  
Yves Menezo
Keyword(s):  
Dna Fragmentation ◽  
Chromatin Structure ◽  
Inverse Relationship ◽  
Sperm Concentration ◽  
Sperm Chromatin ◽  
Sperm Chromatin Structure Assay ◽  
Fragmentation Index ◽  
Sperm Dna ◽  
Oxidative Products ◽  
Dna Fragmentation Index

In our work, we have used 8-hydroxy-deoxyguanosine (8-OH-dG), one of the major oxidative products of sperm DNA, in a population of patients consulting for infertility. We found an inverse relationship between sperm concentration and the log of the ratio of 8-OH-dG to dG (P<0.01). On the same patients' sperm samples, the sperm chromatin structure assay (SCSA) was performed. An inverse relationship was observed between the DNA fragmentation index and sperm concentration (P<0.001). There was also a positive relationship between SCSA and log 8-OH-dG/dG. This indicates that DNA fragmentation measured by the SCSA originates in part from oxidative products. In a few patients, antioxidant treatment decreased the DNA fragmentation index below the threshold of 30% that is crucial for subfertility.

scholarly journals Improvement of Post-Thaw Sperm Kinematics and DNA Integrity of Cross-Bred Bovine Sperm by Incorporating DGC as Selection Method Prior to Cryopreservation

2017 ◽  
Vol 9 (13) ◽  
pp. 24
Author(s):  
Nur Hilwani Ismail ◽  
Khairul Osman ◽  
Farida Zuraina Mohd Yusof ◽  
Syarifah Faezah Syed Mohamad ◽  
Farah Hanan Fatihah Jaafar ◽  
...  
Keyword(s):  
Treatment Group ◽  
Sperm Quality ◽  
Gradient Centrifugation ◽  
Control Group ◽  
Dna Integrity ◽  
Sperm Selection ◽  
Sperm Analysis ◽  
Fragmentation Index ◽  
Bovine Spermatozoa ◽  
Kinematics Parameters

The aim of this study was to assess post-thaw sperm quality following initial sperm selection using density gradient centrifugation (DGC) prior to cryopreservation. Ejaculates from four mature Charolais cross Kedah-Kelantan bulls were collected using artificial vagina at IBVK Pahang, Malaysia. The ejaculates were aliquoted into 3 groups: non-cryopreserved group (NC); control group of cryopreserved sperm without DGC (ND) and treatment group of sperm undergoing DGC sperm selection before cryopreservation (CDGC). Prior to analysis, samples from both cryopreserved groups were thawed at 37 °C for 30 sec. All samples were analysed for kinematics parameters, viability and compromise in DNA integrity (evaluated as DNA Fragmentation Index, DFI). All kinematics parameters were analysed using computer aided sperm analysis (CASA). Results indicated significant (p < 0.05) kinematics parameter changes for all parameters of velocity (VCL, VSL, VAP) and progression (WOB, LIN, ALH and BCF). Unfortunately, changes in spermatozoa straightness were insignificant (STR) F(2, 68) = 1.004, p = 0.371. Spermatozoa viability had increased by 26.2% (p < 0.01) following the treatment. DFI revealed the treatment group recorded a significant reduction in DFI value (0.17% fragmented DNA). In conclusion, DGC sperm selection prior to cryopreservation reduced the effects of cryodamage and showed an improvement in post-thaw sperm quality, thus reducing the occurrence of asthenozoospermia in populations of frozen-thawed cross-bred bovine spermatozoa.

scholarly journals Efficiency of Commercial Egg Yolk-Free and Egg Yolk-Supplemented Tris-Based Extenders for Dromedary Camel Semen Cryopreservation

Animals ◽  
2019 ◽  
Vol 9 (11) ◽  
pp. 999 ◽  
Author(s):  
Ayman Abdel-Aziz Swelum ◽  
Islam M. Saadeldin ◽  
Hani Ba-Awadh ◽  
Mohsen G. Al-Mutary ◽  
Abdullah F. Moumen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Sperm Motility ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Dna Integrity ◽  
Computer Assisted ◽  
Dromedary Camel ◽  
Plasma Membrane Integrity ◽  
Semen Cryopreservation

This study compared the efficiency of commercial egg yolk-free (AndroMed, OPTIXcell) and egg yolk-supplemented (Triladyl, Steridyl) Tris-based extenders for semen cryopreservation in seven adult dromedary camels. The camel-specific extender SHOTOR was used as control. The collected semen samples were evaluated and diluted with SHOTOR, Triladyl, Steridyl, AndroMed, or OPTIXcell. The diluted semen was gradually cooled and equilibrated for two hours before liquid nitrogen freezing. Semen was evaluated prior to freezing and after freeze-thawing cycles for motility, kinetics, vitality, abnormality, plasma membrane integrity, and DNA fragmentation using computer-assisted sperm analysis. In pre-freezing evaluation, progressive sperm motility was higher in SHOTOR-diluted samples (21.54 ± 1.83) than in samples diluted with Steridyl, OPTIXcell, or AndroMed (15.76 ± 1.80, 17.43 ± 1.10, and 13.27 ± 1.07, respectively). Moreover, Triladyl and SHOTOR resulted in significantly (p < 0.05) better sperm vitality and DNA integrity than all other diluents, but Triladyl resulted in a significantly (p < 0.05) better plasma membrane integrity (87.77 ± 0.31) than SHOTOR (85.48 ± 0.58). In the post-thawing evaluation, Triladyl led to significantly (p < 0.05) higher sperm motility (38.63 ± 0.81%; p < 0.05) when compared to SHOTOR, Steridyl or AndroMed (35.09 ± 1.341%, 34.4 ± 0.84%, and 31.99 ± 1.48%, respectively), with OPTIXcell being the least efficient (28.39 ± 0.86%). Progressive sperm motility was the highest when using Triladyl. Post-thawing curvilinear, straight line and average path sperm velocities were highest with Triladyl and lowest with AndroMed. Triladyl led to the highest linearity coefficient and straightness sperm coefficient, while SHOTOR to the highest DNA and plasma membrane integrity. OPTIXcell and AndroMed resulted in poor post-thawing sperm vitality, while Steridyl was less efficient than Triladyl. The highest rate of sperm abnormalities was recorded with OPTIXcell and the lowest with SHOTOR or Triladyl. In conclusion, SHOTOR, Triladyl, Steridyl, AndroMed, and OPTIXcell can all be used for camel semen cryopreservation; however, SHOTOR and Triladyl provided the best post-thawing sperm quality. Based on our findings, Triladyl is the best commercially available extender for dromedary camel semen cryopreservation to date.

73 EFFECT OF THE BUFFER SYSTEM, CRYOPROTECTANT, AND ANTIOXIDANT ON SPERM FROM ENDANGERED CATALONIAN SHEEP XISQUETA AND ARANESA AND GOAT BLANCA DE RASQUERA BREEDS DURING PRESERVATION

2013 ◽  
Vol 25 (1) ◽  
pp. 184
Author(s):  
M.-J. Palomo ◽  
A. Tabarez ◽  
W. Garcia ◽  
M. Terre ◽  
A. Ferrando ◽  
...  
Keyword(s):  
Soybean Lecithin ◽  
Sperm Quality ◽  
Egg Yolk ◽  
Reproductive Period ◽  
Tris Buffer ◽  
Butylated Hydroxytoluene ◽  
Computer Assisted ◽  
Buffer System ◽  
Sperm Viability ◽  
Sperm Analysis

In an attempt to increase the possibilities of a better sperm cryopreservation from these endangered small ruminant catalonian breeds, we are studying different strategies. One of them is to study the effect of the buffer system Tes-Tris (TEST) compared with the Tris and citric acid (TRIS) buffer system, testing simultaneously both systems in a 1% (w/v) soybean lecithin or in a 15% (v/v) powdered egg yolk–based media supplemented both with 5% glycerol and to assess also the inclusion of 5 mM of butylated hydroxytoluene (BHT) as an antioxidant on the cryopreservation media. However, the main objective of the present study was to test first the effect of the different media when the sperm were cultured 4 h at 5°C. Briefly, fresh ejaculates from 6 young bucks of Blanca de Rasquera breed (1 year old) were collected by an artificial vagina in favourable reproductive period and immediately mixed in equal quantities. Pooled ejaculates were split into 2 equal aliquots and washed by centrifugation (twice for 10 min at 600g) in TRIS- or TEST-based media without cryoprotectants and antioxidant. Afterward each pellet was split into 4 equal aliquots, re-suspended in TRIS or TEST, depending on the experimental group, soybean lecithin, or powdered egg yolk-based media, and supplemented or not with BHT and kept for 4 h at 5°C. Likewise, fresh ejaculates from 8 young rams (4 rams of Xisqueta and 4 rams of Aranesa breed, 1 year old) were collected and processed as buck semen samples. Sperm survival before cryopreservation was determined by eosine-nigrosine stain, and sperm motion parameters were analysed by a computer-assisted sperm analysis system (ISAS®). Six replications were performed in both species, and General Lineal Model (SAS®, Cary, NC, USA) was used for the statistical analysis. The highest sperm viability percentage (mean ± SE) on goat sperm cultured 4 h at 5°C was observed in the extender with TRIS buffer system in powdered egg yolk-based media supplemented with BHT (81.1 ± 2.8), not showing significant differences with the others extenders, except with the viability of the samples in the extenders with TEST buffer system in soybean lecithin-based media supplemented (56.5 ± 2.9; P < 0.001) or not with BHT (60.1 ± 5.1; P < 0.001). On the other hand, no significant differences on sperm viability were observed on ram sperm between treatments. Nevertheless, the sperm quality motion characteristics (data not shown) were quite different between all the treatments in both species. Considering that the present results are still preliminary, we suggest that more analysis should be made to explain how the different composition of the extenders affect sperm quality during the cryopreservation process. Supported by INIA (RZ2009-00008-00-00), Generalitat de Catalunya (2009SGR0621 and CUR-DIUE), and FSE and Fundacion Carolina.

Probiotic administration improves sperm quality in asthenozoospermic human donors

2017 ◽  
Vol 8 (2) ◽  
pp. 193-206 ◽  
Author(s):  
D.G. Valcarce ◽  
S. Genovés ◽  
M.F. Riesco ◽  
P. Martorell ◽  
M.P. Herráez ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Viability ◽  
Dna Fragmentation ◽  
Sperm Motility ◽  
Sperm Quality ◽  
Bifidobacterium Longum ◽  
Fold Change ◽  
Sperm Chromatin ◽  
Computer Assisted ◽  
Sperm Analysis

The objective of this study is to analyse the effect of the ingestion of two selected antioxidant probiotics strains (Lactobacillus rhamnosus CECT8361 and Bifidobacterium longum CECT7347) on sperm quality parameters in asthenozoospermic males after three and six weeks of administration. Nine asthenozoospermic men without any medical treatment under similar diet conditions participated in the study. The quality of individual sperm samples was evaluated before (previous to ingestion), during (after 3 and 6 weeks of ingestion) and after probiotic administration (3 and 6 weeks after finishing the treatment). Sperm motility was evaluated by computer-assisted sperm analysis system, DNA fragmentation by sperm chromatin structure assay, cell viability by flow cytometry and measurement of intracellular H2O2 (reactive oxygen species; ROS) by flow cytometry using dichloro-dihydrofluorescein diacetate. Sperm motility was drastically improved after the treatment (approximately 6 fold change), DNA fragmentation was statistically reduced after probiotic administration from (approximately 1.2 fold change) and intracellular H2O2 level was decreased (approximately 3.5 fold change). Cell viability was not affected by the treatment. The significant improvement in sperm motility and the decrease in DNA fragmentation reported in this study provide preliminary evidence that probiotics could be administrated to improve motility and decrease DNA fragmentation and ROS levels in asthenozoospermic human males.

scholarly journals Effect of Trehalose and Sucrose in Post-thaw Quality of Crassostrea angulata Sperm

2021 ◽  
Vol 12 ◽  
Author(s):  
Catarina Anjos ◽  
Ana Luísa Santos ◽  
Daniel Duarte ◽  
Domitília Matias ◽  
Elsa Cabrita
Keyword(s):  
Plasma Membrane ◽  
Sperm Quality ◽  
Artificial Seawater ◽  
Live Cells ◽  
Dna Integrity ◽  
Computer Assisted ◽  
Sperm Cryopreservation ◽  
Sperm Analysis ◽  
Crassostrea Angulata ◽  
And Function

Sperm cryopreservation can be a helpful tool in reproductive management and preservation of biodiversity. However, the freezing methodologies lead to some damage in structure and function of cells that may compromise post-thaw sperm activity. Cryoprotectant supplementation with sugars proved to be a successful strategy to reduce cryodamage in sperm of several species, once allowing to stabilize the plasma membrane constituents. Therefore, this study intends to understand the effects of sugars in the plasma membrane, DNA integrity, and oxidative response during Portuguese oyster sperm cryopreservation. Three cryoprotectants solutions with an initial concentration of 20% dimethyl sulfoxide (DMSO) and 20% DMSO complemented with 0.9 M trehalose or sucrose in artificial seawater were employed. Sperm samples of mature males were individually collected and diluted 1:10 (v/v) in artificial seawater followed by addition of cryoprotectants [1:1 (v/v)]. Thereafter, sperm was loaded into 0.5 ml straws, maintained at 4°C for 10 min, frozen in a programmable biofreezer at −6°C/min from 0 to −70°C, and stored in liquid nitrogen. Samples were thawed in a 37°C bath for 10 s. Several techniques were performed to evaluate post-thaw quality. Sperm motility and DNA integrity were analyzed by using computer-assisted sperm analysis (CASA) software and comet assay. Flow cytometry was employed to determine membrane and acrosome integrity and to detect intracellular reactive oxygen species (ROS) and apoptosis activity. Lipid peroxidation was determined by malondialdehyde (MDA) detection by using spectrophotometry. Sperm antioxidant capacity was evaluated through glutathione peroxidase, glutathione reductase, and superoxide dismutase. Motility was not affected by the extenders containing sugars; these compounds did not reduce the DNA damage. However, both the trehalose and sucrose protected plasma membrane of cells by increasing cell viability and significantly reducing MDA content. The same finding was observed for the ROS, where live cells registered significantly lower levels of ROS in samples cryopreserved with sugars. The activity of antioxidant enzymes was higher in treatments supplemented with sugars, although not significant. In conclusion, the addition of sugars seems to play an important role in protecting the Crassostrea angulata sperm membrane during cryopreservation, showing potential to improve the post-thaw sperm quality and protect the cells from cryoinjuries.

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