170 ASSESSMENT OF FRESH AND FROZEN - THAWED BOTTLENOSE DOLPHIN (TURSIOPS TRUNCATUS) SPERM DNA FRAGMENTATION USING THE SPERM CHROMATIN DISPERSION TEST

There has been significant development over the last 20 years to improve genetic management of the captive bottlenose dolphin (Tursiops truncatus) by means of genome resource banking and assisted reproduction. Although standard semen parameters have been analysed in some detail, very little is known about sperm DNA fragmentation (SDF) in this species. The aim of this study was to develop a sperm chromatin dispersion test (SCDt) for the bottlenose dolphin to establish the baseline level of SDF immediately after ejaculation and cryopreservation and to determine the dynamic loss of sperm DNA quality after ex vivo handing and incubation in conditions that mimic the female reproductive tract. Semen from 8 bottlenose dolphins was collected by manual stimulation. Initial validation of the SCDt was conducted by means of in situ nick translation and neutral comet assay using a proven fertile male. To investigate the dynamic loss of sperm chromatin (rate of sDF loss), thawed sperm samples were incubated at 37.9°C for up to 48 h, and aliquots of spermatozoa were assessed after 1, 4, 8, 24, and 48 h. Dolphin sperm nuclei with fragmented DNA exhibited large halos of dispersed DNA fragments, whereas those with nonfragmented DNA displayed small halos of nondispersed DNA. A high correlation (r2 = 0.82; P ≤ 0.01) was found between the respective assessments of the SCDt and the neutral comet assay. All nucleoids resulting in a large halo of dispersed chromatin were intensely positive to in situ nick translation. The level of sDF fragmentation observed immediately after ejaculation in fresh and frozen samples was relatively low (1–5%). After comparing different ejaculates of the same individual, differences were found. Chromatin stability was high during the first 48 h of ejaculation or post-thawing and incubation. Evaluation of the sDF dynamics of fresh and frozen–thawed spermatozoa revealed no significant increase in the baseline level of sDF or in the relative increase of DNA damage after 48 h of incubation. Our data suggest that cryopreservation does not induce a dramatic increase in sperm chromatin damage. Interestingly, sperm samples derived from aged animals resulted in an increased rate of DNA loss, which was observed after 60 min post-incubation.

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Validation of the sperm chromatin dispersion (SCD) test in the amphibian Xenopus laevis using in situ nick translation and comet assay

Reproduction Fertility and Development ◽

10.1071/rd14070 ◽

2015 ◽

Vol 27 (8) ◽

pp. 1168 ◽

Cited By ~ 7

Author(s):

K. Pollock ◽

J. Gosálvez ◽

F. Arroyo ◽

C. López-Fernández ◽

M. Guille ◽

...

Keyword(s):

Dna Damage ◽

Comet Assay ◽

Xenopus Laevis ◽

Semen Quality ◽

Sperm Chromatin ◽

Sperm Dna Fragmentation ◽

Nick Translation ◽

In Situ Nick Translation ◽

Sperm Dna

The integrity of sperm DNA is becoming increasingly recognised as an important parameter of semen quality, but there are no published reports of this procedure for any amphibian. The primary aim of this study was to apply a modified sperm chromatin dispersion (SCD) test (Halomax) to an amphibian sperm model (African clawed frog; Xenopus laevis) and to validate the assay against in situ nick translation (ISNT) and the double-comet assay procedure. Inactivated spermatozoa were collected from fresh testes (n = 3). Sperm DNA fragmentation (SDF) for each sperm sample was conducted immediately following activation (T0) and again after 1 h (T1) and 24 h (T24) of incubation at room temperature in order to produce a range of spermatozoa with differing levels of DNA damage. The SCD procedure resulted in the production of three nuclear morphotypes; amphibian sperm morphotype 1 (ASM-1) and ASM-2 showed no evidence of DNA damage, whereas ASM-3 spermatozoa were highly fragmented with large halos of dispersed DNA fragments and a reduced nuclear core. ISNT confirmed that ASM-3 nuclei contained damaged DNA. There was a significant correlation (r = 0.9613) between the levels of ASM-3 detected by the SCD test and SDF revealed by the double-comet assay.

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Sperm chromatin dispersion test (SCDt) for the assessment of sperm DNA fragmentation in black tiger prawn, Penaeus monodon

Aquaculture ◽

10.1016/j.aquaculture.2018.03.041 ◽

2018 ◽

Vol 491 ◽

pp. 281-288 ◽

Cited By ~ 2

Author(s):

Tianyi Feng ◽

Jamie Gosálvez ◽

Carmen Lopez-Fernandez ◽

Francisca Arroyo ◽

Brian Paterson ◽

...

Keyword(s):

Dna Fragmentation ◽

Penaeus Monodon ◽

Sperm Chromatin ◽

Sperm Dna Fragmentation ◽

Sperm Dna ◽

Black Tiger Prawn ◽

Tiger Prawn ◽

Dispersion Test ◽

Sperm Chromatin Dispersion Test

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Assessment of Sperm DNA Fragmentation in Stallion (Equus caballus) and Donkey (Equus asinus) Using the Sperm Chromatin Dispersion Test

Reproduction in Domestic Animals ◽

10.1111/j.1439-0531.2008.01091.x ◽

2009 ◽

Vol 44 (5) ◽

pp. 823-828 ◽

Cited By ~ 10

Author(s):

EI Cortés-Gutiérrez ◽

F Crespo ◽

C Serres-Dalmau ◽

AL Gutiérrez de las Rozas ◽

MI Dávila-Rodríguez ◽

...

Keyword(s):

Dna Fragmentation ◽

Equus Caballus ◽

Sperm Chromatin ◽

Sperm Dna Fragmentation ◽

Equus Asinus ◽

Sperm Dna ◽

Dispersion Test ◽

Sperm Chromatin Dispersion Test

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P–009 A modified sperm chromatin dispersion test, LensHooke® R10, for quick and accurate determination of human sperm DNA fragmentation

Human Reproduction ◽

10.1093/humrep/deab130.008 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

L S Chang ◽

H C Lee ◽

C T Hsu ◽

H M Tsao ◽

C C Huang ◽

...

Keyword(s):

Male Infertility ◽

Dna Fragmentation ◽

Observer Agreement ◽

Sperm Chromatin ◽

Sperm Dna Fragmentation ◽

Test Results ◽

Sperm Dna ◽

Test Kit ◽

Dispersion Test ◽

Sperm Chromatin Dispersion Test

Abstract Study question The performance and efficiency of the LensHooke® R10 test kit were evaluated by the clinical examination for precision, accuracy, and time. Summary answer The LensHooke® R10 based on sperm chromatin dispersion test offers not only quick testing for sperm DNA fragmentation but also reliable and accurate test results. What is known already Sperm chromatin dispersion (SCD) test, one of the most commonly used testing for sperm DNA fragmentation (SDF), can be conducted promptly and without the need for expensive laboratory instruments. However, the main disadvantage of the SCD test is inter-observer variability in categorizing the size of characteristics halos surrounding the core of sperm. Moreover, it takes more than one hour to accomplish whole assay procedures making this testing an inefficient diagnostic tool. These may hinder its broad availability among andrology laboratories or prevent it from being routinely used for the evaluation of male infertility. Study design, size, duration A total of 108 participants was included in this prospective study. Data was collected from the reproductive medicine center between June and December 2020. Participants/materials, setting, methods This study included 108 consecutive male partners of couples attending for assisted reproductive treatment. SDF was simultaneously tested by using LensHooke® R10 (R10) and Halosperm® G2 (G2) respectively. We evaluated the correlation and agreement between two SCD-based test kits. The repeatability and reproducibility of the SCD kits were assessed by intra-and inter-observer agreement experiments. The sensitivity, specificity, positive predictive value, negative predictive value for the R10 was determined by receiver operator characteristics (ROC) curve analysis. Main results and the role of chance The R10 produced more clear sperm core and dispersed chromatin, therefore highly recognizable images can be easily and accurately categorized when scoring of SDF. It took 50% less time for SDF testing by the R10 compared to the G2 (38.26 ± 9.85 minutes vs. 76.52 ± 19.7 minutes, P < 0.0001). The SDF% results showed a strong correlation for the R10 and G2 with Spearman’s coefficients of rank correlation (rho) above 0.8 (P < 0.0001, N = 108). The R10 showed 89.8% accuracy with 87.9% sensitivity, 90.8% specificity, 82.9% PPV, and 93.7% NPV on the measurement of SDF% at the threshold value of 22%. Intraclass correlation coefficients (ICC) >0.9 showed a strong agreement between two observers on the testing of SDF using the R10. ICC >0.9 showed a high intra-observer agreement within 4 repeated testing on SDF using the R10. The R10 showed an intra-observer’s precision of coefficient variation, CV < 10% for SDF%. In addition, SDF% test results obtained by the R10 for asthenospermic (31.8% ± 16.7%), teratospermic (22.9% ± 14.4%), and oligoasthenoteratozoospermic samples (36.6% ± 14.4%) were significantly higher than that observed in normozoospermic samples (15.3% ± 10.2%, p < 0.05), was comparable with the G2. Limitations, reasons for caution The sample size of 4 semen specimens used to evaluate the intra-and inter-observer agreement was a limitation. Besides, evaluating the relationship between the SDF and clinical outcome of ART is necessary for further study. Wider implications of the findings: The new in vitro diagnostics reagent, LensHooke® R10, is a simple and quick test kit that offers reliable and accurate test results of sperm DNA fragmentation, can be routinely used in male infertility evaluation. Trial registration number CS2–20012

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Sperm DNA Fragmentation Among Smokers and Non-Smokers as Measured by Sperm Chromatin Dispersion Test

IIUM Medical Journal Malaysia ◽

10.31436/imjm.v17i1.917 ◽

2018 ◽

Vol 17 (1) ◽

Author(s):

Azha Syahril Azizan ◽

Kamarul Bahyah Mustafa ◽

Azantee Yazmie Abdul Wahab ◽

Roszaman Ramli ◽

Nurkhairulnisa Abu Ishak

Keyword(s):

Dna Fragmentation ◽

Seminal Fluid ◽

Sperm Concentration ◽

Sperm Chromatin ◽

Sperm Dna Fragmentation ◽

Cross Sectional ◽

Fluid Analysis ◽

Sperm Dna ◽

Dispersion Test ◽

Sperm Chromatin Dispersion Test

Introduction: Traditionally, seminal fluid analysis is done to assess male infertility particularly sperm concentration, morphology and motility. Sperm chromatin dispersion test explores the quality of the sperm DNA structures. High sperm DNA fragmentation is associated with unexplained infertility, failure of assisted reproduction and recurrent miscarriages. Smoking has been associated with high sperm DNA fragmentation in some studies but not in others. Materials and Methods: A comparative cross-sectional study, involving 32 smokers and 32 non-smokers to determine the association between cigarette smoking and sperm DNA fragmentation. Semen samples were collected from patients undergoing seminal fluid analysis (SFA) at the IIUM Fertility Centre and IIUM Medical Centre O&G Clinic from January 2017 to June 2018. Sperm chromatin dispersion test was done and level of 15% is considered upper limit of normal. Results: 53.1% in the smoker group have abnormal level of sperm DNA fragmentation compared to 34.4% of nonsmokers. The median and interquartile range for smokers were 15.30 and 19.0 (10.425 - 29.375) respectively while for non-smokers were 8.25 and 17.8 (4.075 - 21.850) which was statistically significant (p= 0.012). It was also noted that three participants in the smoker group (9.4%) has very high sperm DNA fragmentation index of more than 80% even though they have normal seminal fluid analysis. Conclusion: There is an association between smoking and high sperm DNA fragmentation. There is a trend of increased in DNA fragmentation in smokers even though their SFA results were normal.

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