scholarly journals Whole genome integrity and enhanced developmental potential in ram freeze-dried spermatozoa at mild sub-zero temperature

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Luca Palazzese ◽  
Debora Agata Anzalone ◽  
Federica Turri ◽  
Marco Faieta ◽  
Anna Donnadio ◽  
...  
Keyword(s):  
Dna Damage ◽  
Chromatin Structure ◽  
Mechanical Damage ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Residual Water ◽  
Gravimetric Analysis ◽  
Technological Advancement ◽  
Developmental Potential ◽  
Freeze Dried

Abstract Freeze-dried spermatozoa typically shows a reduction in fertility primarily due to the DNA damage resulting from the sublimation process. In order to minimize the physical/mechanical damage resulting from lyophilization, here we focused on the freezing phase, comparing two cooling protocols: (i) rapid-freezing, where ram sperm sample is directly plunged into liquid nitrogen (LN-group), as currently done; (ii) slow-freezing, where the sample is progressively cooled to − 50 °C (SF-group). The spermatozoa dried in both conditions were analysed to assess residual water content by Thermal Gravimetric Analysis (TGA) and DNA integrity using Sperm Chromatin Structure Assay (SCSA). TGA revealed more than 90% of water subtraction in both groups. A minor DNA damage, Double-Strand Break (DSB) in particular, characterized by a lower degree of abnormal chromatin structure (Alpha-T), was detected in the SF-group, comparing to the LN-one. In accordance with the structural and DNA integrity data, spermatozoa from SF-group had the best embryonic development rates, comparing to LN-group: cleaved embryos [42/100 (42%) versus 19/75 (25.3%), P < 0.05, SL and LN respectively] and blastocyst formation [7/100 (7%) versus 2/75 (2.7%), P < 0.05, SF and LN respectively]. This data represents a significant technological advancement for the development of lyophilization as a valuable and cheaper alternative to deep-freezing in LN for ram semen.

Increased DNA damage in sperm from leukocytospermic semen samples as determined by the sperm chromatin structure assay

2002 ◽  
Vol 78 (2) ◽  
pp. 319-329 ◽  
Author(s):  
Juan G Alvarez ◽  
Rakesh K Sharma ◽  
Mario Ollero ◽  
Ramadan A Saleh ◽  
Mari C Lopez ◽  
...  
Keyword(s):  
Dna Damage ◽  
Chromatin Structure ◽  
Sperm Chromatin ◽  
Sperm Chromatin Structure Assay

69 AN EASY-TO-PERFORM METHOD TO ASSESS VIABILITY OF FELINE FREEZE-DRIED SPERM

2013 ◽  
Vol 25 (1) ◽  
pp. 182 ◽  
Author(s):  
L. C. O. Magalhães ◽  
C. M. Melo-Oña ◽  
M. J. Sudano ◽  
D. M. Paschoal ◽  
L. F. Crocomo ◽  
...  
Keyword(s):  
Dna Damage ◽  
Acridine Orange ◽  
Freeze Drying ◽  
Membrane Damage ◽  
Daily Basis ◽  
Control Group ◽  
Dna Integrity ◽  
Sperm Cells ◽  
Significance Level ◽  
Freeze Dried

Freeze-drying sperm seems to be the ultimate alternative to preserve sperm cells from endangered species because of its easiness in transportation and storage of samples. This technique has already been used for equine, bovine, murine, rabbit, canine, and feline sperm cells. However, it is still important to verify the DNA integrity of such samples to produce viable offspring. The objective of this study was to verify the reliability of acridine orange (AO) staining to assess DNA damage of feline freeze-dried sperm samples. Three normospermic cats were used as sperm donors, and sperm samples were collected with the use of an artificial vagina without the presence of an oestrous queen. The control group (G1) used only fresh semen. To increase the range of sperm variability before freeze-drying, the sperm samples were supplemented with 2 extenders, namely SOF (G2) and human tubal fluid (G3). These extenders were selected because of their lack of cryoprotectant agents with a view to producing membrane damage and thus putting DNA integrity at risk. Samples were taken to a freeze-dryer (Edwards do Brasil, Brazil) to produce stable free-dried sperm. Over 50 samples were assessed for DNA damage by using AO and alkaline comet assay (CA). Acridine orange taints red/orange the sperm cells that present sperm damage, and CA shows long comet tails for cells with DNA damage. The CA was selected to countercheck the AO results owing to its higher accuracy. Nevertheless, both AO and CA count 100 cells per analysis, and a total of 3 repetitions per sample were sufficient. For the statistical analysis, SAS PROC GLM was used, and the significance level was set at 0.05. The AO staining showed that the G1 samples had a greater DNA damage (27.1%) if compared with G2 (15.2%) and G3 (23.1%; P < 0.0001). Nevertheless, G2 and G3 displayed sufficient DNA integrity throughout the whole lyophilization process. The CA, which is a more precise evaluation technique, proved that the groups had distinguished results but still were ordered in the same way: G1 was the one with more DNA damage (17.7%), G3 was second (14.4%), and G2 was third (3.2%) in DNA alterations. Just like AO, the CA showed difference among treatments (P < 0.0001). Since AO is a technique that requires very little sophistication and presented about the same results as the very elaborate CA method, it might be used on a daily basis to assess feline sperm DNA that underwent the freeze-drying process.

Levels of apoptosis in ejaculated spermatozoa are significantly correlated with sperm chromatin structure assay (SCSA)-defined DNA damage

2002 ◽  
Vol 78 ◽  
pp. S265-S266
Author(s):  
Mohamed H Moustafa ◽  
Ramadan A Saleh ◽  
Rakesh K Sharma ◽  
Mohammed A Abdel Hafez ◽  
Anthony J Thomas ◽  
...  
Keyword(s):  
Dna Damage ◽  
Chromatin Structure ◽  
Sperm Chromatin ◽  
Sperm Chromatin Structure Assay

scholarly journals Drying and temperature induced conformational changes of nucleic acids and stallion sperm chromatin in trehalose preservation formulations

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Raffaele Brogna ◽  
Juezhu Fan ◽  
Harald Sieme ◽  
Willem F. Wolkers ◽  
Harriëtte Oldenhof
Keyword(s):  
Reactive Oxygen Species ◽  
Dna Damage ◽  
Nucleic Acids ◽  
Conformational Changes ◽  
Chromatin Structure ◽  
Reactive Oxygen ◽  
Sperm Chromatin ◽  
Oxygen Species ◽  
Spectral Changes ◽  
And Storage

AbstractEven though dried sperm is not viable, it can be used for fertilization as long as its chromatin remains intact. In this study, we investigated drying- and temperature-induced conformational changes of nucleic acids and stallion sperm chromatin. Sperm was diluted in preservation formulations with and without sugar/albumin and subjected to convective drying at elevated temperatures on glass substrates. Accumulation of reactive oxygen species was studied during storage at different temperatures, and the sperm chromatin structure assay was used to assess DNA damage. Fourier transform infrared spectroscopy was used to identify dehydration and storage induced conformational changes in isolated DNA and sperm chromatin. Furthermore, hydrogen bonding in the preservation solutions associated with storage stability were investigated. Reactive oxygen species and DNA damage in dried sperm samples were found to accumulate with increasing storage temperature and storage duration. Non-reducing disaccharides (i.e., trehalose, sucrose) and albumin counteracted oxidative stress and preserved sperm chromatin during dried storage, whereas glucose increased DNA damage during storage. When sperm was dried in the presence of trehalose and albumin, no spectral changes were detected during storage at refrigeration temperatures, whereas under accelerated aging conditions, i.e., storage at 37 °C, spectral changes were detected indicating alterations in sperm chromatin structure.

scholarly journals 45 DNA Fragmentation of Epididymal Freeze-Dried Ram Spermatozoa Impairs Embryo Development

2018 ◽  
Vol 30 (1) ◽  
pp. 162
Author(s):  
L. Palazzese ◽  
D. A. Anzalone ◽  
J. Gosálvez´ ◽  
P. Loi ◽  
J. Saragusty
Keyword(s):  
Dna Damage ◽  
Embryonic Development ◽  
Dna Fragmentation ◽  
Embryo Development ◽  
Blastocyst Stage ◽  
Sperm Chromatin ◽  
Sperm Dna Fragmentation ◽  
Strand Breaks ◽  
Sperm Dna ◽  
Freeze Dried

Sperm freeze-drying is a revolutionary technique that resolves many of the drawback of long-term storage under liquid nitrogen. The first significant result of this method was provided by Wakayama and Yanagimachi (1998 Nat. Biotechnol. 16, 639-641, 10.1038/nbt0798-639), demonstrating for the first time the birth of healthy offspring from epididymal freeze-dried (mouse) spermatozoa. Besides models in the mouse and rat, which are the first small mammals born from epididymal lyophilized sperm by intracytoplasmic sperm injection (ICSI), most studies in this field have used ejaculated sperm. In this work, aiming to repeat the result of Wakayama and Yanagimachi, we tried to apply this technique to epididymal spermatozoa from a large mammal (ram). Moreover, we checked the correlation between freeze-dried spermatozoa DNA integrity and embryo development. To do this, epididymal sperm from 4 rams was lyophilized in a medium containing trehalose, glucose, KCl, HEPES, and Trolox. To evaluate DNA damage and fragmentation at rehydration, part of the sperm was processed for sperm chromatin dispersion test (SCD) and two-tailed comet assay and the rest was used for ICSI. Compared with rams 1 and 3, rams 2 and 4 had higher rate of spermatozoa with intact DNA (median: 3.3% v. 16.5%, respectively), lower rate of single strand breaks (SSB; median: 94.2% v. 81.5%, respectively) and lower rate of double-strand breaks (DSB; median: 2.5% v. 2%, respectively). Embryo development following ICSI showed that blastocyst stage was reached only from rams that had sperm with more intact DNA: ram 2 (4.8%, n = 83) and ram 4 (6.3%, n = 64). Spermatozoa from rams 1 and 3 produced no blastocysts. This can be explained by the fact that rams 2 and 4 had higher rate of spermatozoa with intact DNA than rams 1 and 3. Definitively, the implication of sperm DNA damage in embryonic development should depend on the balance between the extent of sperm DNA fragmentation, the type of fragmentation (SSB or DSB), and the oocyte’s repair capacity. Rams 2 and 4 were the only rams that produced blastocyst probably because they had considerably more sperm with normal DNA; thus, it is important to select spermatozoa of the best quality to perform a good ICSI. Fragmentation of DNA due to the lyophilization process impairs embryonic development. To conclude, oocytes injected with epididymal freeze-dried ram spermatozoa can reach the blastocyst stage. These are preliminary results; more conclusive outcomes will be given following embryo transfer experiments that are now in progress.

scholarly journals Species-Specific Differences in Sperm Chromatin Decondensation Between Eutherian Mammals Underlie Distinct Lysis Requirements

2021 ◽  
Vol 9 ◽  
Author(s):  
Jordi Ribas-Maynou ◽  
Estela Garcia-Bonavila ◽  
Carlos O. Hidalgo ◽  
Jaime Catalán ◽  
Jordi Miró ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Condensation ◽  
Proteinase K ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Dna Breaks ◽  
Chromatin Decondensation ◽  
The Third ◽  
Core Length ◽  
Sperm Dna

Sperm present a highly particular DNA condensation that is acquired during their differentiation. Protamines are key elements for DNA condensation. However, whereas the presence of protamine 1 (P1) is conserved across mammalian species, that of protamine 2 (P2) has evolved differentially, existing only few species that use both protamines for sperm DNA condensation. In addition, altered P1/P2 ratios and alterations in the expression of P1 have previously been associated to infertility and DNA damage disorders. On the other hand, different methods evaluating DNA integrity, such as Sperm Chromatin Dispersion (SCD) and Comet tests, need a previous complete DNA decondensation to properly assess DNA breaks. Related with this, the present study aims to analyze the resilience of sperm DNA to decodensation in different eutherian mammals. Sperm samples from humans, horses, cattle, pigs and donkeys were used. Samples were embedded in low melting point agarose and treated with lysis solutions to induce DNA decondensation and formation of sperm haloes. The treatment consisted of three steps: (1) incubation in SDS + DTT for 30 min; (2) incubation in DTT + NaCl for 30 min; and (3) incubation in DTT + NaCl with or without proteinase K for a variable time of 0, 30, or 180 min. How incubation with the third lysis solution (with or without proteinase K) for 0, 30, and 180 min affected DNA decondensation was tested through analyzing core and halo diameters in 50 sperm per sample. Halo/core length ratio was used as an indicator of complete chromatin decondensation. While incubation time with the third lysis solution had no impact on halo/core length ratios in species having P1 and P2 (human, equine and donkey), DNA decondensation of pig and cattle sperm, which only present P1, significantly (P &lt; 0.05) increased following incubation with the third lysis solution for 180 min. In addition, the inclusion of proteinase K was found to accelerate DNA decondensation. In conclusion, longer incubations in lysis solution including proteinase K lead to higher DNA decondensation in porcine and bovine sperm. This suggests that tests intended to analyze DNA damage, such as halo or Comet assays, require complete chromatin deprotamination to achieve high sensitivity in the detection of DNA breaks.

scholarly journals Are semen quality parameters sufficient for biomonitoring spermatozoa DNA integrity and oxidatively damaged DNA

Biomonitoring ◽  
2015 ◽  
Vol 2 (1) ◽  
Author(s):  
Hueiwang Anna Jeng ◽  
Ruei-Nian Li ◽  
Wen-Yi Lin
Keyword(s):  
Dna Damage ◽  
Dna Fragmentation ◽  
Semen Quality ◽  
Quality Parameters ◽  
Semen Parameters ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Phase System ◽  
Sperm Dna Fragmentation ◽  
Sperm Dna

Abstract:The present study aimed to investigate the relationship between semen quality parameters and DNA integrity, and determine whether semen quality parameters could serve as a reliable biomarker for monitoring sperm DNA damage. Conventional semen parameters from a total of 202 male human subjects were analyzed. DNA fragmentation and 8-oxo-7,8-dihydro-2′- deoxyguanosine (8-oxoGuo) were used to assess sperm DNA integrity. DNA fragmentation was analyzed by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and sperm chromatin structure assay (SCSA), while 8-oxodGuo was quantified by the liquid chromatography/tandem mass spectrometry (LC-MS/MS) coupled with an on-line solid phase system. The levels of 8-oxodGuo levels in sperm were related to the percentages of DNA fragmentation measured by both the TUNEL and SCSA (r = 0.22, p = 0.048; r = 0.12, p = 0.039). Sperm vitality, motility and morphology from all of the participants exhibited a weak correlation with the levels of 8-oxodGuo and the percentages of DNA fragmentation. Semen quality parameters may be independent of the formation of DNA fragmentation and oxidative adducts in sperm. Semen quality parameters may be insufficient to monitor sperm DNA fragmentation and oxidative damage. DNA damage in sperm is recommended to be included in routine measurements.

DNA Damage Measured by Sperm Chromatin Structure Assay (SCSA) and Birth Characteristics in Children Conceived by IVF or ICSI.

2008 ◽  
Vol 78 (Suppl_1) ◽  
pp. 182-182
Author(s):  
Leif Bungum ◽  
Mona Bungum ◽  
Peter Humaidan ◽  
Lars Rylander ◽  
Lena Wedlund ◽  
...  
Keyword(s):  
Dna Damage ◽  
Chromatin Structure ◽  
Sperm Chromatin ◽  
Sperm Chromatin Structure Assay ◽  
Birth Characteristics
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