Altea Gosálbez

Dynamics of sperm DNA fragmentation in domestic animals III. Ram.

From a biological viewpoint spermatozoa are ejaculated by the male and received into the female while maintaining roughly constant temperature, which in most mammals is below the temperature of the soma. When ejaculated spermatozoa are used for artificial reproductive purposes a temperature excursion episode is produced, because the spermatozoa are often stored as frozen or chilled samples and the biological temperature is only recovered after insemination. In this study we have analyzed the effects of cooling (to 15 degrees C) and freezing ram spermatozoa on the subsequent sperm DNA fragmentation index (sDFI) during a varying period of storage at 37 degrees C. The aim was to emulate in vivo processes that cooled or frozen-thawed spermatozoa experience after insemination. The study was performed using commercial semen samples derived from rams regularly used for reproductive purposes. Semen samples were studied after a cooling or cryopreservation episode followed by biological temperature recovery and incubation up to 48 h. The results indicated that when spermatozoa experience a severe (frozen) or mild (cooled) temperature excursion episode, major effects on sperm viability and DNA fragmentation are induced and cause the subsequent rapid decline of ram sperm quality. This effect could be detected just at the onset of the biological temperature recovery. Sperm DNA damage in cooled samples was observed after 5 h of incubation at 37 degrees C, while this time was reduced to less than 60 min in frozen-thaw samples. The dynamics of sDFI in different animals, analyzed under the same experimental conditions, was different from one sample to another, regardless of the method used for storage. Sperm viability was better preserved in cooled rather than in frozen samples. While for the frozen-thawed samples sperm viability was almost abolished after 5 h of incubation, a stable proportion of viable spermatozoa (ranging from 20% to 60%) was observed in the cooled samples at the corresponding time points. Finally, with respect to the prevalence of sDFI in ram, the level commonly found was lower than 5% at the onset of the experiment. However, sDFI was higher than 5% in 25% of the samples and in 15% of rams this index exceeded 10%.

Dynamics of sperm DNA fragmentation in the swine: Ejaculate and temperature effects

The dynamics of sperm DNA fragmentation was examined in 16 boar ejaculates using the sperm chromatin dispersion (SCD) test and a two-tail comet assay. The net sperm rich fraction was preserved at two different temperatures (Trial 1: 15 degrees C, n=10; Trial 2: 37 degrees C, n=6) and sub-samples were taken every day until a sperm motility of zero. Significant differences in the dynamics of DNA fragmentation were observed among the different ejaculates and also according to the storage temperature. After analyzing the dynamic response of the sperm DNA damage, when the sperm samples are incubated at 15 or 37 degrees C, each ejaculate could be classified and a considerable variation among individuals for an increase in DNA damage was observed. Thus, while in some ejaculates no rise in DNA fragmentation was observed, in others, the sperm DNA fragmentation process was triggered during the initial days of the experiment. In general, sperm incubation at 37 degrees C diminished sperm DNA quality. The two-tail comet assay indicated that at time zero existing DNA damage mainly consisted of double stranded DNA breakage. During storage, DNA damage affected one of the DNA strands until a second wave of DNA damage, in which there was both single and double stranded DNA damage.

Rapid rates of sperm DNA damage after activation in tench (Tinca tinca: Teleostei, Cyprinidae) measured using a sperm chromatin dispersion test

Spermatozoal haplotypic DNA is prone to damage, leading to male fertility problems. So far, the assessment of sperm DNA breakage has been challenging because protamines render the nuclear chromatin highly compacted. Here, we report the application of a new test to quantify DNA fragmentation in spermatozoa of an externally fertilizing teleost fish. The sperm chromatin dispersion (SCD) test uses a species-specific lysing solution to generate controlled protein depletion that, followed by DNA-specific fluorescent labelling, allows an easy morphological discrimination between nuclei affected by DNA damage. Using tench (Tinca tinca) as our model, we first trialled the test against established, but more technically demanding, assays employing in situ nick translation (ISNT) and the comet assay. The SCD test showed high concordance with ISNT, comet assay measures and a chromatin-swelling test, confirming the application of this straightforward SCD technique to various aspects of reproductive biology. Second, we examined between-male variation in DNA damage, and measured changes through time following spermatozoal activation. Between-male variation in the basal levels of average DNA damage ranged from 0 to 20% of sperm showing damage, and all showed increases in DNA fragmentation through time (0-60 min). The rates of DNA damage increase are the fastest so far recorded in sperm for a living organism, and may relate to the external fertilization mode. Our findings have relevance for broodstock selection and optimizing IVF protocols routinely used in modern aquaculture.

New Application of the Comet Assay Chromosome–Comet Assay

The comet assay is a well-established, simple, versatile, visual, rapid, and sensitive tool used extensively to assess DNA damage and DNA repair quantitatively and qualitatively in single cells. The comet assay is most frequently used to analyze white blood cells or lymphocytes in human biomonitoring studies, although other cell types have been examined, including buccal, nasal, epithelial, and placental cells and even spermatozoa. This study was conducted to design a protocol that can be used to generate comets in subnuclear units, such as chromosomes. The new technique is based on the chromosome isolation protocols currently used for whole chromosome mounting in electron microscopy, coupled to the alkaline variant of the comet assay, to detect DNA damage. The results show that migrant DNA fragments can be visualized in whole nuclei and isolated chromosomes and that they exhibit patterns of DNA migration that depend on the level of DNA damage produced. This protocol has great potential for the highly reproducible study of DNA damage and repair in specific chromosomal domains.

Directional mapping of DNA nicking in ejaculated and cauda epididymidal spermatozoa of the short-beaked echidna (Tachyglossus aculeatus: Monotremata)

Prototherian spermatozoa are unique amongst the Mammalia in terms of their filiform morphology, tandem arrangement of chromosomes and formation of sperm bundles. In the present study, we provide observations of echidna spermatozoa and note that the superstructure of the bundle is engineered around the shape of the individual sperm head and that this in turn may be a consequence of the unusual circumferential and helicoidal condensation of the DNA during spermiogenesis. Frozen-thawed ejaculated echidna spermatozoa were incubated and examined for the presence of non-typical DNA conformation by means of in situ labelling of DNA breaks using Klenow polymerase and via alkaline single-cell comet assays for detection of fragmented DNA. Both techniques successfully revealed the presence of what appeared to be directional DNA nicking, co-localised with the presence of highly sensitive alkali sites along the length of the sperm nucleus. It was not possible to define whether these alternative DNA configurations were associated with a failure of the sperm nucleus to condense appropriately during spermiogenesis or were evidence of DNA fragmentation following post-thaw incubation or a sequential structural chromatin rearrangement necessary for fertilisation.

Assessing sperm DNA fragmentation in the field: an adaptation of sperm chromatin dispersion technology

The sperm chromatin dispersion (SCD) test is a new technique that allows assessment of sperm DNA fragmentation (SDF) in different species. The application of this technique, like other techniques, is restricted to the laboratory. Our investigation was aimed at exploring the possibilities of extending SCD methodology for use in the field, where electric powered facilities such as freezers, microscopes or heaters are not available. Our results showed that SCD methodology, with minor modifications to the standard protocol, can be performed readily in the field, offering reliable information about SDF. An Light Emitting Diode (LED)-equipped microscope attached to a laptop, a gas heater and a CO2 spray for cooling are sufficient to assess the quality of sperm DNA. The results obtained after assessing 10 different semen samples under different conditions (30° C in the laboratory and at 17° C and 4° C in the field) showed that except after processing the slides at 4° C, the results of SDF in different animals showed no significant differences. With the modifications suggested here, the SCD technique can be used to assess SDF in the wild. In particular, the DNA quality of spermatozoa obtained from animals post mortem can be assessed in the field.

Sperm fractions obtained following density gradient centrifugation in human ejaculates show differences in sperm DNA longevity

Abstract Objective To investigate the DNA longevity characteristics associated with each resultant fraction following density gradient centrifugation (DGC) in comparison to that of the original neat ejaculated sample. Methods An aliquot of neat semen (NSS) collected from 7 patients was processed using DGC resulting in 3 fractions; Fraction 1: seminal plasma/40% gradient interface (GI); Fraction 2: 40%GI/80%GI; Fraction 3: 80%GI/pellet. An aliquot of each fraction and NSS was cryopreserved, thawed and incubated at 37 ° for 24h; the increase of sperm DNA fragmentation was assessed using the Dyn-Halosperm assay following 0, 3, 6 and 24h of incubation. Results While there was a significant reduction in the incidence of baseline sperm DNA fragmentation following DGC in Fraction 3, sperm DNA longevity was shown to be higher in the NSS than in any other sub-population following incubation. The highest levels of baseline DNA damage were found in Fractions 1 and 2; these fractions also showed the highest rate DNA fragmentation following incubation, subsequently exhibiting the lowest DNA longevity. Conclusion 1) Unnecessary incubation of spermatozoa prior to artificial insemination or in vitro fertilization, should be avoided, since sperm DNA longevity is significantly reduced after ex vivo sperm handling and 2) Although sperm selection by DCG significantly reduces the baseline levels of SDF of sperm in Fraction 3, sperm DNA longevity in this fraction was ultimately lower following 24 h incubation when compared to sperm recovered from non-centrifuged NSS.

Improved Sensitivity for Cell Mapping of Hepatitis C Virus RNA Sequences and Cellular Surface Antigens in Blood Cells

Improved Sensitivity for Cell Mapping of Hepatitis C Virus RNA Sequences and Cellular Surface Antigens in Blood Cells