F. Arroyo

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.

Frozen-thawed rhinoceros sperm exhibit DNA damage shortly after thawing when assessed by the sperm chromatin dispersion assay

This study reports on the successful validation (via in situ nick translation and neutral comet assay) of the equine Sperm-Halomax kit as an appropriate methodology for the assessment of sperm DNA fragmentation in three species of rhinoceros. Rhinoceros sperm nuclei with fragmented DNA (validated using in situ nick translation) were evident as large halos with dispersed DNA fragments, whereas those with nonfragmented DNA displayed small halos of nondispersed DNA within the microgel. There was a high correlation (r) of 0.974 (R(2) value=0.949; P<0.01; n=16) between the respective assessments of the Sperm Chromatin Dispersion test (SCDt) and the neutral comet assay. Application of the SCDt to determine the DNA fragmentation dynamics of rhinoceros (n=6) sperm frozen in liquid nitrogen vapor and incubated postthaw at 37 degrees C for up to 48 h to mimic in vitro conditions in the female reproductive tract, revealed an increase (P=0.001) in DNA damage, as soon as 4h after the start of incubation. Linear regression equations were calculated for all six rhinoceroses over the first 6h of incubation and revealed individual animal variation. Freshly collected and incubated (37 degrees C) rhinoceros (n=3) sperm had no increase in the basal level of DNA fragmentation for up to 48 h, indicating that the cryopreservation of rhinoceros sperm in liquid nitrogen vapor, as used in this study, appeared to result in freeze-thaw DNA damage.

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.

Characterization of sperm DNA damage in Kartagener's syndrome with recurrent fertilization failure: Case revisited

Single- and double-strand sperm DNA breaks was assessed in a Kartageners syndrome with four failures of fertilization after ISCI in TESA samples. It is concluded that in addition to failure of sperm motility, this patient was infertile because a high level of non-reparable sperm DNA damage was present. Although four cycles of insemination were performed at patients request, if the argument of an exacerbated level of sperm DNA damage could be used at the time of the medical advice, repeated failed cycles of insemination using ICSI could be avoided. Pregnancy was achieved with a semen donor.

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

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 1h (T1) and 24h (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.

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.

Effects of freezing and activation on membrane quality and DNA damage in Xenopus tropicalis and Xenopus laevis spermatozoa

There is growing concern over the effect of sperm cryopreservation on DNA integrity and the subsequent development of offspring generated from this cryopreserved material. In the present study, membrane integrity and DNA stability of Xenopus laevis and Xenopus tropicalis spermatozoa were evaluated in response to cryopreservation with or without activation, a process that happens upon exposure to water to spermatozoa of some aquatic species. A dye exclusion assay revealed that sperm plasma membrane integrity in both species decreased after freezing, more so for X. laevis than X. tropicalis spermatozoa. The sperm chromatin dispersion (SCD) test showed that for both X. tropicalis and X. laevis, activated frozen spermatozoa produced the highest levels of DNA fragmentation compared with all fresh samples and frozen non-activated samples (P<0.05). Understanding the nature of DNA and membrane damage that occurs in cryopreserved spermatozoa from Xenopus species represents the first step in exploiting these powerful model organisms to understand the developmental consequences of fertilising with cryopreservation-damaged spermatozoa.

The assessment of sperm DNA fragmentation in the saltwater crocodile (Crocodylus porosus)

Herein we report a method of assessing DNA fragmentation in the saltwater crocodile using the sperm chromatin dispersion test (SCDt) after including frozen-thawed spermatozoa in a microgel (Halomax; Halotech DNA, Madrid, Spain). Following controlled protein depletion, which included a reducing agent, sperm nuclei with fragmented DNA showed a homogeneous and larger halo of chromatin dispersion with a corresponding reduced nucleoid core compared with sperm with non-fragmented DNA. The presence of DNA damage was confirmed directly by incorporation of modified nucleotides using in situ nick translation (ISNT) and indirectly by studying the correlation of the SCDt with the results of DNA damage visualisation using a two-tailed comet assay (r=0.90; P=0.037). Results of the SCDt immediately following thawing and after 5h incubation at 37°C in order to induce a range of DNA damage revealed individual crocodile differences in both the baseline level of DNA damage and DNA longevity.