Reply of the Authors: Acridine orange binding to RNA interferes DNA fragmentation index calculation in sperm chromatin structure assay

2010 ◽  
Vol 94 (1) ◽  
pp. e38-e38
Author(s):  
Marij Smit ◽  
Gert R. Dohle ◽  
Johannes C. Romijn
Keyword(s):  
Acridine Orange ◽  
Dna Fragmentation ◽  
Chromatin Structure ◽  
Sperm Chromatin ◽  
Sperm Chromatin Structure Assay ◽  
Fragmentation Index ◽  
Index Calculation ◽  
Dna Fragmentation Index

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 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.

313 ASSESSMENT OF SPERM DNA FRAGMENTATION IN CANINE EJACULATES USING THE Sperm-Halomax® KIT: PRELIMINARY RESULTS

2010 ◽  
Vol 22 (1) ◽  
pp. 312 ◽  
Author(s):  
M. Hidalgo ◽  
M. R. Murabito ◽  
M. J. Gálvez ◽  
S. Demyda ◽  
L. J. De Luca ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Sperm Concentration ◽  
Semen Parameters ◽  
Sperm Chromatin ◽  
Sperm Dna Fragmentation ◽  
Fragmentation Index ◽  
Sperm Dna ◽  
Commercial Kit ◽  
Sperm Dna Fragmentation Index ◽  
Dna Fragmentation Index

Recently, a new procedure for the analysis of sperm DNA fragmentation has been developed for humans and different mammalian species, using a commercial kit based on the sperm chromatin dispersion (SCD) test; however, a descriptive study in canine semen has not been performed. The aim of this work was to assess the sperm DNA fragmentation in canine ejaculates using the SCD test and 2 different staining techniques. For this purpose, ejaculates were collectedby digital manipulation from4 healthy dogs of different breeds (1 German Pointer, 2 Spanish Greyhounds, and 1 Crossbreed). After collection, the sperm-rich fraction of the ejaculates from 3 dogs were pooled each time (n = 4) and then extended in Dulbecco’s phosphate buffered saline. All the pooled semen samples presented physiological values concerning routine semen parameters (motility, morphology, and sperm concentration). The sperm DNA fragmentation was assessed using the Sperm-Halomax® commercial kit specifically developed for canine semen (Halotech DNA SL, Madrid, Spain). Two semen aliquots of the diluted pooled semen samples were processed on each pre-treated slide provided in the kit following the manufacturer’s instructions. The last step was the staining technique. We stained each slide with 2 different staining procedures. The first half of the slide was stained with propidium iodide (Sigma-Aldrich, St. Louis, MO, USA) mixed in a proportion 1 : 1 with an antifading solution. The second half of the slide was stained for 15 min in Wright solution (1.01383.0500, Merck, Whitehouse Station, NJ, USA) 1 :1 in Phosphate Buffer pH 6.88 (1.07294.1000, Merck). The stained slides were observed using fluorescence and light microscopy, respectively. Five hundred sperm per slide were counted. Spermatozoa with fragmented DNA showed a large and spotty halo of chromatin dispersion. Unfragmented sperm only showed a small and compact halo. Statistical analyses were performed using the Statistical Package for Social Science version 12.0 (SPSS Inc., Chicago, IL, USA). The sperm DNA fragmentation index was compared between Wright and fluorescence staining methods by ANOVA. Results were expressed as mean ± standard error of the mean. The first report of the sperm DNA fragmentation index in canine ejaculates was 2.26 ± 0.53% for Wright staining and 1.99 ± 0.10% for fluorescence technique. No differences were found between staining procedures. In conclusion, it was possible to assess the sperm DNA fragmentation of canine ejaculates using 2 different staining procedures, expecting that continuous research could be useful in defining the role of DNA fragmentation SCD test in canine semen evaluation and cryopreservation.

82 DNA FRAGMENTATION DYNAMICS OF KOALA SPERMATOZOA

2009 ◽  
Vol 21 (1) ◽  
pp. 141 ◽  
Author(s):  
Y. P. Zee ◽  
C. Lopez-Fernandez ◽  
J. Gosalvez ◽  
W. V. Holt ◽  
S. D. Johnston
Keyword(s):  
Dna Fragmentation ◽  
Dna Degradation ◽  
Sperm Chromatin ◽  
Sperm Dna Fragmentation ◽  
Fragmentation Index ◽  
Sperm Dna ◽  
Rate Of Increase ◽  
Fragmentation Dynamics ◽  
Sperm Dna Fragmentation Index ◽  
Dna Fragmentation Index

Koala sperm chromatin has a tendency to relax following incubation and thawing but the background incidence and dynamics of DNA fragmentation during semen processing at 35°C and following chilled and frozen preservation has not been investigated. This study (n = 10) was designed to establish the fragmentation dynamics of koala sperm DNA at body temperature (35°C), after chilling (4°C) for upward of 16 days, and following a standard freeze–thaw protocol (Johnston SD et al. 2006 Cryobiology 53, 218–228). Sperm DNA fragmentation index (sDFI) was determined using a Halomax kit (ChromaCell SL, Madrid, Spain), which had been customized and validated for koalas (Johnston SD et al. 2007 J. Androl. 28, 891–899). All semen was assessed for sDFI over a 48-h incubation period (T0, T2, T6, T24, and T48) at 35°C. After incubation at 35°C for 48 h, the sDFI and rate of DNA degradation of freshly diluted spermatozoa were highly variable between individuals; the sDFI for 2 koalas remained consistently low (≤2%) whereas the other 8 had sDFI of 8 to 12% after incubation. Chilled storage increased sDFI in all animals, but the rate of increase and the time at which the DNA started to fragment also varied between koalas; sDFI for 1 koala increased immediately upon rewarming after being chilled for 4 h, whereas that of another koala did not increase until after 8 days of chilling and 24 h of incubation at 35°C. Animals also responded to cryopreservation differently; sDFI increased after thawing for 2 of the koalas but did not increase in the others. Subsequent evaluation of frozen–thawed spermatozoa from a greater number of captive koalas (n = 22), and under extended conditions of post-thaw incubation (up to 17 days at 35°C) permitted categorization of the koalas into 3 distinctive groups based on their DNA fragmentation dynamics and rate of DNA degradation. For 7 of the animals, sDFI remained close to the basal level when incubated at 35°C over 7 days, whereas 2 of the koalas had sDFI ranging from 40 to 70% after 24 h of incubation. This study confirmed the occurrence of inter-animal variability in the dynamics of DNA fragmentation, a finding that was apparent whether or not the spermatozoa had been subjected to chilling or cryopreservation.

247 COMPARISON OF THE DNA FRAGMENTATION INDEX BETWEEN CRYOPRESERVED EJACULATED SPERM AND EPIDIDYMAL SPERM IN STALLIONS

2013 ◽  
Vol 25 (1) ◽  
pp. 271
Author(s):  
G. A. Monteiro ◽  
C. P. Freitas-DellAqua ◽  
P. N. Guasti ◽  
Y. F. R. Sancler-Silva ◽  
C. Ramires-Neto ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Sperm Motility ◽  
Epifluorescence Microscopy ◽  
Sperm Chromatin ◽  
Computer Assisted ◽  
Epididymal Sperm ◽  
Fragmentation Index ◽  
Motion Parameters ◽  
Motility Parameters ◽  
Dna Fragmentation Index

The development of a reliable technique for freezing epididymal semen would provide a unique opportunity to preserve valuable genetic material from unexpectedly lost stallions. The semen analysis method with the best ability to predict fertility is an examination of the sperm chromatin structure. This test evaluates the susceptibility of spermatozoa DNA to denaturation. The ability of spermatozoal DNA to maintain an intact double-stranded configuration is determined by exposure to an acid environment. The aim of this study was to compare the DNA fragmentation index of sperm obtained from ejaculate (G1) and sperm from the cauda epididymis (G2). For G1, two ejaculates from each of seven stallions were collected and then subjected to cryopreservation using BotuCrioTM extender. One week after the last semen collection, the stallions underwent bilateral orchiectomy. Sperm from the cauda epididymis was harvested immediately after castration (G2) by retrograde flushing of the caudal portion of the epididymis using a skim milk-based extender (BotuSemenTM). The recovered sperm was then cryopreserved using BotuCrioTM extender. The sperm motility parameters were analysed by computer-assisted sperm analysis (HTM IVOS 12, Hamilton Thorne Inc., Beverly, MA, USA), and the DNA fragmentation index was estimated using acridine orange test epifluorescence microscopy. The samples were evaluated immediately (0 h) and 8 h after thawing. The total motility, progressive motility, and percentage of rapid cells of the G1 v. G2 samples at 0 h were, respectively, 62.3 ± 12.9a v. 72.6 ± 8.4a, 31.6 ± 9.2a v. 35.3 ± 10.32a, and 49.3 ± 14.33a v. 59.7 ± 13.59a. At 8 h, the results were 26.0 ± 21.6b v. 54.7 ± 12.2a, 6.1 ± 6.4b v. 17.4 ± 8.54a, and 13.7 ± 14.85b v. 37.6 ± 14.15a. Evaluation of the DNA fragmentation in the G1 and G2 samples yielded 6.7 ± 1.41a v. 5.7 ± 1.60a at 0 h and 8.3 ± 1.78b v. 7.2 ± 1.19b at 8 h for percentage of DNA fragmentation after thawing. At 0 h, no differences in the sperm parameters were observed between groups, but statistical differences were observed in the sperm motility parameters between the treatment groups after 8 h. For the DNA fragmentation index, no difference was found at 0 and 8 h between the groups. However, after thawing, a higher percentage of DNA fragmentation was observed in the ejaculated sperm (8 h) as compared with the epididymal sperm (0 h). On the basis of these results, we can conclude that frozen–thawed cauda epididymal sperm had similar or higher motion parameters than ejaculated sperm after thawing. In addition, incubating the sperm at 20°C for 8 h after thawing resulted in higher motion parameters and less DNA fragmentation of the epididymal sperm. This finding suggests that epididymal sperm are more resistant to the cold shock caused by cryopreservation. FAPESP for financial support and Botupharma for donation of BotuSemenTM and BotuCrioTM extender.

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