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In this study annual fluctuations of DNA fragmentation and quality of cold-stored equine sperm were evaluated. Ejaculates were collected weekly during one year from 15 stallions. Ejaculate volume, sperm concentration and total sperm count were determined and semen was then extended and cold-stored for 48 h. Sperm motility was evaluated by CASA before and after 24 as well as 48 h of cold storage. In addition, the percentages of sperm with intact plasma membrane and acrosome (PMAI %) and with low intracellular Ca 2+ level were determined in cold-stored semen (24 h, 48 h). SCSA™ was performed to assess mean DFI, SD of DFI and % DFI in raw frozen-thawed as well as in extended sperm after 24 and 48 h of storage. The month of semen collection affected (P < 0.05) all parameters evaluated in raw semen and all criteria except progressive motility as well as rapid cells in semen stored for 24 and 48 h, respectively. Ejaculate volume was higher and sperm concentration lower in summer compared to winter and motility lower in July than in any other month of the year (P < 0.05). In semen processed in April and stored for 24 h the percentage of rapid cells was improved compared to January and after 48 h of storage progressive motility (%) was higher in January and October than in July (P < 0.05). After 24 h of cold storage PMAI % was higher in October than in January and after 48 h values were higher in September compared to January and February as well as from April to July (P < 0.05). Regarding sperm with low intracellular Ca +2 level (%) after storage for 24 and 48 h, higher values were measured in winter and in October compared to April, June and July (P < 0.01). Seasonal changes in DNA fragmentation were most evident with respect to mean DFI. In raw frozen-thawed semen mean DFI was lower from August to November than in June and July (P < 0.001). Values were lower during winter compared to spring and early summer (P < 0.05) and lower in December than from April to September (P < 0.001). After 24 h of cold storage mean DFI was lower in September and October when compared to January, February, May, July and November (P < 0.05) and after 48 h storage mean DFI was reduced in spring and autumn compared to February, June and July (P < 0.05). In conclusion, a seasonal effect was evident on semen characteristics of raw and cold-stored sperm. Semen quality was impaired in midsummer when low sperm motility and viability were combined with an elevated DNA fragmentation and Ca 2+ level of sperm. [ABSTRACT FROM AUTHOR]

Abstract: The objective of the present study was to explore the potential relationships of ovine sperm chromatin integrity, quantified using the sperm chromatin structure assay (SCSA), to the heat load of the scrotum and the discomfort felt by the animals because of fluctuations of microclimatic factors at different time periods before ejaculation. Ejaculates were collected once per week from five Chios rams and four East Friesian rams for 12 months and stored in liquid nitrogen. Frozen-thawed semen samples were analyzed using the SCSA, to determine the DNA fragmentation index (DFI) and the percentage of cells outside the main sperm population (%DFI) in each one of the samples. Scrotal surface temperature (SST) of each ram was measured using an infrared thermometer on a daily basis. Ambient air temperature and relative humidity were recorded at hourly intervals throughout the experimental period and temperature-humidity index (THI) was used to assess the discomfort felt by the rams. Mean values of SST (SSTmean) and THI (THImean) were computed for eight different time periods (up to 61 days) preceding each ejaculation day (Day 0). A linear mixed-effect model analysis was performed to describe the relation of SCSA parameters to collection month, SSTmean, and THImean of different time periods before ejaculation. The results of the statistical analysis revealed a relation of %DFI to the SSTmean of the last 12 days preceding ejaculation, namely the period that resembled the phase of epididymal maturation. On the contrary, the variation of DFI was most adequately described by the linear mixed-effect model applied for Days 54 to 48 before ejaculation, which resembled the phase of spermatogonial mitoses. The effect of collection month was significant for DFI and %DFI, with semen samples collected in September and February exhibiting the lowest DFI values; a less profound seasonal pattern was detected for %DFI. The effect of THImean on DFI and %DFI was proven nonsignificant in regard to all time periods. In conclusion, a relation of SCSA parameters to SSTmean of different periods before ejaculation was shown in the present study, implying an effect of scrotal microenvironment on intratesticular and epididymal sperm population. In contrast, we failed to detect any effect of microclimate-induced discomfort felt by the animals on the chromatin integrity of frozen-thawed ram spermatozoa. [Copyright &y& Elsevier]

Abstract: Sperm chromatin status was assessed in 565 Zebu and Zebu crossbred beef bulls in extensive tropical environments using the sperm chromatin structure assay (SCSA). The SCSA involved exposure of sperm to acid hydrolysis for 0.5 or 5.0 minutes, followed by flow cytometry to ascertain relative amounts of double-stranded (normal) and single-stranded (denatured) DNA, which was used to generate a DNA fragmentation index (%DFI). With conventional SCSA (0.5-minute SCSA), 513 bulls (91%) had <15 %DFI, 24 bulls (4%) had 15 to 27 %DFI, and 28 bulls (5%) had >27 %DFI. In 5.0-minute SCSA, 432 bulls (76%) had <15 %DFI, 68 bulls (12%) had 15 to 27 %DFI and 65 bulls (12%) had >27 %DFI. For most bulls, the SCSA was repeatable on two to four occasions; however, because most bulls had <15 %DFI, repeatability of the SCSA will need to be determined in a larger number of bulls in the 15 to 27 %DFI and >27 %DFI categories. The %DFI was negatively correlated with several bull semen parameters and the strongest negative correlation was with normal sperm. There was a strong positive correlation between %DFI and sperm head abnormalities. Based on these findings, most Zebu beef bulls in extensive tropical environments had relatively stable sperm chromatin. Based on the apparent negative correlations with conventional semen parameters, we inferred that the SCSA measured a unique feature of sperm quality, which has also been suggested for other species. Further studies on the relationships between sperm chromatin stability and fertility are required in beef bulls before chromatin status can be used as an additional predictor of the siring capacity of individual bulls in extensive multiple-sire herds. [Copyright &y& Elsevier]

Abstract: The semen quality of seven young adult boars was assessed for percentages of sperm motility, normal acrosomes, abnormal sperm, cells positive to sHOST (short Hipoosmotic Swelling Test), HPNA cells (sHOST Positive with Normal Acrosome cells) and the percentage of sperm heads, which exhibited DNA fragmentation using the Sperm Chromatin Dispersion test (SCD). These parameters were analysed in sperm samples both undiluted and diluted using a commercial extender and stored at 15°C for 21 days. Results showed that semen quality decreases faster in the undiluted semen samples from day 0 to day 7 compared to diluted semen samples that remained with a high quality up to day 11. The undiluted semen exhibited a low DNA fragmentation index (DFI) during the first days and then a significant increase from day 7 up to day 21. This increase in the DFI coincided with the lowest levels of the other semen quality parameters. On the contrary, the samples diluted in the commercial extender showed very low levels of DNA fragmentation in all boars during the preservation period. When the evolution of DNA fragmentation was analysed in the undiluted samples, differences were found among boars. These differences were not shown in the samples diluted in the extender where the basal DFI remained stable during the 21 days. The main conclusion of this study was that some sperm extenders delay or partially prevent sperm DNA fragmentation. [Copyright &y& Elsevier]

Abstract: We present a new, rapid and simple method to study DNA Fragmentation Index (DFI) in sperm samples from boar under bright-field and fluorescence microscopy. Discrimination of sperm cells containing fragmented DNA relies on the extreme peripheral diffusion of their chromatin fragments, whereas those sperm nuclei without DNA fragmentation do not disperse or show very restricted spreading of DNA loops close to the flagellum. The basic methodology provided in the commercial kit Sperm-Sus-Halomax® allows, in addition to a direct estimation of DFI in a sperm sample under bright field microscopy, a direct visualization of DNA breaks by incorporation of labelled nucleotides using the DNA polymerase I following the in situ nick translation assay (ISNT methodology not provided in the kit). An external control using DBD-FISH (DNA breakage detection-fluorescence in situ hybridization) on human and boar sperm samples was used in this experiment. The results obtained show (i) low levels of background DNA fragmentation (from 0.7 to 10%), (ii) no significant differences for DFI after the application of Sperm-Sus-Halomax and ISNT, with a tendency to be underestimated after using DBD-FISH and (iii) a characteristic chromatin organization in boar sperm nucleus, with a particular response to chromatin loop relaxation and preferential DNA labelling by ISNT at the proximal nuclear area, close to the flagellum. This methodology allows the routine assessment of boar sperm samples for DFI, as well as basic and clinical research on this relevant topic in any laboratory of semen analysis. [Copyright &y& Elsevier]

The effect of the straw volume employed for semen freezing was studied in 14 ejaculates from seven boars, by evaluating the viability, IVF capacity and chromatin state of spermatozoa. Frozen–thawed semen from 0.5 and 5 ml straws was compared to fresh semen. The chromatin condensation degree was determined by flow cytometry, using propidium iodide as fluorochrome, and the chromatin stability was evaluated by inducing its decondensation with SDS and EDTA. The results obtained for IVF, motility and normal apical ridge (NAR) were: 91.64, 78.14 and 81.47% sperm penetration, 80.78, 68.38 and 70.83% monospermy, 10.86, 9.76 and 10.64% polyspermy, 87.14, 50.71 and 47.86% motility, 79.14, 56.14 and 53.36% NAR, for fresh semen, thawed semen in 0.5 and 5 ml straws, respectively. Frozen–thawed spermatozoa showed significantly increased (P<0.05) chromatin compactness compared to fresh spermatozoa (55.42, 48.41 and 47.08 fluorescence units (MIFU), for fresh semen, thawed semen in 0.5 and 5 ml straws, respectively). Chromatin was significantly more unstable (P<0.05) in spermatozoa frozen in 0.5 ml straws (174.7 MIFU) compared to those frozen in 5 ml straws (155.53 MIFU) or to those in fresh semen (149.74 MIFU). [Copyright &y& Elsevier]

We used the sperm chromatin structure assay (SCSA) to study the change in stallion sperm DNA susceptibility to denaturation after exposure of extended semen to three different storage temperatures (5, 20, or 37 °C) at 7, 20, 31, and 46 h. In addition, we compared the rates of sperm DNA denaturation in fertile and subfertile stallions. Among fertile stallions, spermatozoa stored at 20 and 37 °C showed a significant (P<0.05) rise in the SCSA measures (Mean(αt), S.D.(αt), and percent cells outside the main population—COMP(αt)) over time, with the degree of rise being more dramatic at 37 °C. Over all stallions, samples stored at 5 °C showed no significant (P>0.05) changes in the SCSA values measured over time, indicating maintenance of chromatin quality for up to 46 h. The COMP(αt) from stallions classified as subfertile showed an increased susceptibility to denaturation or decline in chromatin quality between 20 and 31 h when stored at 5 °C; however, spermatozoa from fertile stallions did not change during the time intervals analyzed. These data suggest that sperm DNA from some subfertile stallions may decline at a greater rate than spermatozoa from fertile stallions when exposed to similar storage conditions. [Copyright &y& Elsevier]