Evaluation of DNA fragmentation of freeze-dried mouse sperm using a modified sperm chromatin structure assay

Freeze-dried sperm is applicable to the storage and transport of genetic material. We recently reported that freeze-dried mouse sperm required temperatures lower than −80 °C for long-term preservation and concluded that it was necessary to explore freeze-drying conditions before long-term preservation of sperm becomes viable. In the current study, we determined the percentage of sperm with elevated levels of DNA fragmentation using a sperm chromatin structure assay (SCSA), a technique not previously reported for the evaluation of freeze-dried mouse sperm. We applied SCSA to mouse sperm freeze-dried under four conditions (various combinations of primary drying pressure of 0.04 and 0.37 hPa and storage temperatures of 4 and −80 °C) and compared the results with the embryonic developmental rates of freeze-dried sperm after intracytoplasmic sperm injection (ICSI) and with comet assay results. The DNA fragmentation index values under the four conditions determined by SCSA had good correlation with the developmental rate to the blastocyst stage of embryos from ICSI with freeze-dried mouse sperm. We concluded that the SCSA method applied to freeze-dried mouse sperm after storage will lead to not only clarification of the developmental rate derived from ICSI using freeze-dried sperm but also to improvements in the freeze-drying and storage processes.     

Fragmentation dynamics of frozen-thawed ram sperm DNA is modulated by sperm concentration

This study investigated the hypothesis that post-thaw incubation of ram sperm at high concentrations results in a faster rate of DNA fragmentation than when sperm are incubated at a lower concentration. Ejaculates from 10 rams were frozen-thawed, prepared in sperm concentrations of 100, 50, 25, 12, and 6 × 10⁶ sperm/mL, and incubated for 6 h at 37 °C. Sperm DNA fragmentation was assessed using the sperm chromatin dispersion test (Sperm-Halomax®) at 1, 3, 4, and 6 h of incubation at 37 °C. On fitting a binary logistic regression with a cubic over time and treating ram and dilution levels as factors, there were significant effects with respect to the ram, dilution and time (all P-values were very much smaller than 0.001). Therefore, DNA fragmentation dynamics of incubated frozen-thawed ram sperm were not only dependent on the inherent sperm DNA fragmentation expressed immediately after thawing, but also on the concentration of sperm incubated in the sample. Although there was evidence of individual ram variation in SDF during the incubation period, the general finding of the current study was that lower sperm concentrations resulted in a slower rate of DNA fragmentation These findings have important implications for the post-thaw manipulation of ram sperm used for AI and advanced reproductive procedures that use sperm at low concentrations. Our data also emphasised the highly dynamic nature of sperm DNA fragmentation and the importance of conducting the procedure in a standardised manner.     

Intra- and interboar variability in flow cytometric sperm sex sorting

To improve the efficiency of porcine sperm sex sorting using flow cytometry, the aims of the present study were to determine the relevance of inter- and intraboar variability in sperm sortability and to evaluate the significance of ejaculate semen characteristics in such variability. In addition, the variability among boars in the ability of sex-sorted spermatozoa to survive liquid storage at 15 °C to 17 °C was also evaluated. In total, 132 ejaculates collected from 67 boars of different breeds that were housed at an artificial insemination center were used in three experiments. X- and Y-chromosome–bearing sperm were simultaneously separated according to the Beltsville sperm-sorting technology using a high-speed flow cytometer. In the first experiment, interboar variability in the ability of the ejaculated spermatozoa to undergo the flow-based sex-sorting procedure was observed; the ejaculates of nearly 15% of the boars (n = 67) did not exhibit well-defined X- and Y-chromosome–bearing spermatozoa peaks in the histogram, and the ejaculate sperm concentration demonstrated good predictive value for explaining this variation, as indicated by the area under the receiver operating characteristics curve (0.88, P < 0.001). In the second experiment, a certain degree of intraboar variability was observed only in the boars that showed poor sperm sortability (measured according to the presence or not a well-defined split together with sperm sortability parameters) in the first ejaculate (n = 3). In contrast, boars classified as having good sperm sortability in the first ejaculate (n = 5) maintained this condition in five ejaculates collected over the subsequent 5 months. In the third experiment, sex-sorted spermatozoa from boars with good sperm sortability (n = 5) remained viable and motile (above 70% in all boars) after 48 hours of storage at 15 °C to 17 °C, which may facilitate the commercial application of sex-sorted spermatozoa in swine artificial insemination programs.     

The relationship between sperm quality in cool-shipped semen and embryo recovery rate in horses

The relationship between the quality of cool-shipped stallion semen and fertility has not been adequately described. This study evaluated sperm quality of cool-shipped semen from 459 ejaculates (N = 130 stallions) that were used for insemination of 196 embryo donor mares (n = 496 estrous cycles). Embryo recovery rate (ERR; %) increased, as all sperm measures (e.g., motility, viability, DNA quality, morphology, concentration, and total number) increased. Threshold values are reported for each sperm quality measure (e.g., total sperm motility ≥ 65%) that separate two ERR groups (e.g., average: ∼50% ERR; high: ∼65% ERR).     

Evaluation of DNA fragmentation in llama (Lama glama) sperm using the sperm chromatin dispersion test

The integrity of sperm chromatin is now viewed as an important factor in male fertility and in early embryonic development. The objectives of this study were: (1) adapt the simple and inexpensive sperm chromatin dispersion (SCD) test to evaluate DNA fragmentation in llama sperm and establish the halo patterns observed in this species, (2) determine an effective and reliable positive control for this technique and (3) evaluate correlation between the SCD test and the toluidine blue (TB) stain. To adapt the SCD test, three different mercaptoethanol (ME) concentrations were assayed (2.5%, 5% and 10% ME). To determine an effective positive control, three treatments (incubation at 100 °C for 30 min, incubation with 0.3 M NaOH for 30 min at room temperature and exposure to UV light for 2h) were assayed. The concentration selected to use in the SCD test was 5% ME, because it produced the largest halo while still conserving the structure of the core. Four DNA dispersion patterns were clearly observed: (I) nuclei with large DNA dispersion halos; (II) nuclei with medium halos; (III) nuclei with very small halos and (IV) nuclei with no halo. All treatments used as positive controls were effective in producing DNA fragmentation. A high correlation (r=0.84, P=0.03) was observed between spermatozoa without halos and TB positive cells. To conclude, SCD patterns in llama sperm have been established as well as a repeatable positive control for the assay. The SCD test and TB stain are simple and inexpensive techniques that can be used to evaluate DNA damage in llama sperm.     

Effects of dilution and centrifugation on the survival of spermatozoa and the structure of motile sperm cell subpopulations in refrigerated Catalonian donkey semen

The aim of this work was to study the effects of dilution and centrifugation (i.e., two methods of reducing the influence of the seminal plasma) on the survival of spermatozoa and the structure of motile sperm cell subpopulations in refrigerated Catalonian donkey (Equus asinus) semen. Fifty ejaculates from nine Catalonian jackasses were collected. Gel-free semen was diluted 1:1, 1:5 or 1:10 with Kenney extender. Another sample of semen was diluted 1:5, centrifuged, and then resuspended with Kenney extender until a final dilution of 25 × 10⁶ sperm/ml was achieved (C). After 24 h, 48 h or 72 h of refrigerated storage at 5 °C, aliquots of these semen samples were incubated at 37 °C for 5 min. The percentage of viable sperm was determined by staining with eosin-nigrosin. The motility characteristics of the spermatozoa were examined using the CASA system (Microptic, Barcelona, Spain). At 24 h, more surviving spermatozoa were seen in the more diluted and in the centrifuged semen samples (1:1 48.71%; 1:5 56.58%, 1:10 62.65%; C 72.40%). These differences were maintained at 48 h (1:1 34.31%, 1:5 40.56%, 1:10 48.52%, C 66.30%). After 72 h, only the C samples showed a survival rate of above 25%. The four known donkey motile sperm subpopulations were maintained by refrigeration. However, the percentage of motile sperms in each subpopulation changed with dilution. Only the centrifuged samples, and only at 24 h, showed exactly the same motile sperm subpopulation proportions as recorded for fresh sperm. However, the 1:10 dilutions at 24 and 48 h, and the centrifuged semen at 48 h, showed few variations compared to fresh sperm. These results show that the elimination of seminal plasma increases the survival of spermatozoa and the maintenance of motility patterns.The initial sperm concentration had a significant (P < 0.05) influence on centrifugation efficacy, but did not influence the number of spermatozoa damaged by centrifugation. In contrast, the percentage of live spermatozoa in the fresh semen significantly influenced the number of spermatozoa damaged by centrifugation, but not centrifugation efficacy.     

In vitro characteristics of frozen-thawed, sex-sorted bull sperm after refreezing or incubation at 15 or 37 °C

The objective was to determine the in vitro characteristics of frozen-thawed dairy bull sperm after sex-sorting and refreezing and thawing (0, 2, and 4 h post-thaw; 37 °C) or post-sort incubation at 15 or 37 °C for 30 and 24 h, respectively. These sperm were compared with nonsorted frozen-thawed sperm (control) and with nonsorted sperm undergoing two cryopreservation procedures (FF; 0, 2, and 4 h). Frozen-thawed sex-sorted (FS) sperm maintained at 15 or 37 °C had higher (P < 0.001) progressive motility (PM), velocity, mitochondrial function, viability, and acrosome integrity than that of control sperm but similar total motility at 0 and 2 h of incubation. Frozen-thawed sex-sorted sperm incubated at 15 °C maintained high levels of motility (66.5 ± 1.6%) and viability/acrosome integrity (64.9 ± 1.2%) at 24 h incubation and, after rewarming and further 6 h incubation at 37 °C, acceptable levels of motility (35.8 ± 1.6%) and viability/acrosome integrity (51.2 ± 1.2%) were maintained. Frozen-thawed sex-sorted sperm maintained at 37 °C had lower levels of motility, integrity, mitochondrial respiration, and velocity from 4 h of incubation onward than that of those incubated at 15 °C. However, when frozen-thawed sex-sorted sperm were refrozen (FSF), motility and velocity were depressed at all hours compared with levels exhibited by control sperm, but membrane viability/acrosome integrity and mitochondrial respiration were similar at 0 and 2 h post-thaw. Acrosome integrity of sperm in all groups undergoing sorting was exceptionally high at 0 h (≥90%), even after re-cryopreservation and 4 h of incubation (77.5 ± 1.3%). Double frozen-thawed nonsorted sperm (FF) had similar motility to FSF sperm at 0 and 2 h post-thaw but at all time points had the lowest (P < 0.001) levels of acrosome intact/viable sperm and mitochondrial respiration and the lowest velocity at 0 h. In conclusion, whereas sex-sorting improved the functionality of frozen-thawed sperm, refreezing depressed motility, viability, and velocity but not acrosome integrity after extended incubation compared with that of control sperm. Furthermore, frozen-thawed, sex-sorted sperm may be stored for transport at 15 °C for up 24 h without detrimental effects on in vitro sperm characteristics.     

Major morphological sperm abnormalities in the bull are related to sperm DNA damage

The influence of sperm morphology and chromatin integrity on bull fertility suggests a strong but undefined biological relationship between these two parameters. In this study we explore this relationship, making use of the Sperm Chromatin Dispersion test, which allows simultaneous observation of sperm abnormalities and DNA fragmentation. Based on spermatozoa from 17 Holstein-Friesian bulls, we determined a relationship between DNA fragmentation and the presence of the “so called” major-type sperm defects. Values for DNA fragmentation index (mean ± SEM) calculated from cells with major-type abnormalities were significantly (P < 0.05) higher (85.05 ± 5.00%) than those from abnormal forms classified as minor-type (17.89 ± 5.55%). Some of the sperm abnormalities, such as double forms, narrow base heads, small heads, shortened tails and proximal cytoplasmic droplets, were only associated with sperm showing fragmented DNA. The simultaneous assessment of sperm morphology and DNA fragmentation has the potential to improve the efficacy of sperm quality assessment in this species.     

Evaluation of oxidative DNA damage promoted by storage in sperm from sex-reversed rainbow trout

Short-term storage and cryopreservation of sperm are two common procedures in aquaculture, used for routine practices in artificial insemination reproduction and gene banking, respectively. Nevertheless, both procedures cause injuries affecting sperm motility, viability, cell structure and DNA stability, which diminish reproductive success. DNA modification is considered extremely important, especially when sperm storage is carried out with gene banking purposes. DNA damage caused by sperm storage is not well characterized and previous studies have reported simple and double strand breaks that have been attributed to oxidative events promoted by the generation of free radicals during storage.The objective of this study was to reveal DNA fragmentation and to explore the presence of oxidized bases that could be produced by oxidative events during short-term storage and cryopreservation in sex-reversed rainbow trout (Oncorhynchus mykiss) spermatozoa. Sperm from six males was analyzed separately. Different aliquots of the samples were stored 2 h (fresh) or 5 days at 4 °C or were cryopreserved. Then spermatozoa were analyzed using the Comet assay, as well as combining this method with digestion with two endonucleases from Escherichia coli (Endonuclease III, that cut in oxidized cytosines, and FPG, cutting in oxidized guanosines). Both storage procedures yielded DNA fragmentation, but only short-term storage oxidative events were clearly detected, showing that oxidative processes affect guanosines rather than cytosines. Cryopreservation increases DNA fragmentation but the presence of oxidized bases was not noticed, suggesting that mechanisms other than oxidative stress could be involved in DNA fragmentation promoted by freezing.     

Relationship between pregnancy,embryo development,and sperm deoxyribonucleic acid fragmentation dynamics

The way the dynamics of DNA fragmentation affects the growth of embryos in real time, and effectiveness of infertility treatment using the ICSI procedure were determined in 148 couples treated with the ICSI technique. The percentage of sperm with fragmented DNA (known as the DNA fragmentation index [DFI]) in semen samples was determined at 3, 6 and 12 h. Embryo culture was assessed continuously during 12 h of observation monitoring.Statistically significant difference was found in DFI at 12 h and outcome of treatment. For the remaining time intervals, no statistically significant differences were noted. An analysis of relationship between the DFI dynamics over time at individual measurements and achievement of pregnancy, confirmed a statistically significant relationship between the rate measured at 6–12 h of observations of DFI changes (DFI 12 h%/h), and achieving pregnancy. Correlation was observed between DFI (during 0, 3, 6 and 12 h), the growth rate in DFI, and time of embryo development. A statistically significant relationship was found between the rate from the start to the end of observations of the DFI, and outcome of treatment.Intensity level regarding fragmentation of sperm DNA and its growth rate affected the time of embryo development in the ICSI procedure. The most significant prognostic factor for achieving pregnancy was intensification of sperm DNA fragmentation after 12 h.     

Seasonal changes in sperm DNA fragmentation of Murciano-Granadina goats: The compelling case for dynamic assessment

Evidence is provided of seasonal changes in the rate of DNA fragmentation dynamics of sperm collected from goats Murciano-Granadina breed), in a Spanish Breeding Centre at 41°N latitude. Results show that the initial assessment of sperm DNA fragmentation (T0) was not sufficient to differentiate sperm DNA fragmentation of bucks collected at different times of the year (Kruskal–Wallis 2.399; P = 0.493). However, when thawed semen was subsequently incubated at 37 °C for periods of between 2 and 48 h, differences were recorded in the rate of sperm DNA fragmentation (r-SDF), that are indicative of seasonal changes in reproduction (Log Rank: Mantel–Cox; χ² = 158.6; DF = 3; P < 0.001). Goat semen collected and cryopreserved in the Spanish winter and spring resulted in a poor post-thaw sperm DNA longevity and recorded a r-SDF of 0.8 and 1.3 per hour, after 48 h of incubation respectively. The r-SDF subsequently decreased to 0.16 and 0.36 per hour during summer and autumn. It is therefore, recommended that semen samples from Murciano-Granadina goats housed above the 40°N latitude be collected and cryopreserved in the summer and early autumn. This would result in a more stable DNA molecule, which is likely to improve the reproductive success.     

Novel and traditional traits of frozen-thawed porcine sperm related to in vitro fertilization success

Cryopreserved semen allows the use of single ejaculates for repeated analyses, potentially improving IVF consistency by eliminating interejaculate variability observed with fresh semen. However, the freezing and thawing processes result in compromised sperm function and IVF success. Semen samples are often screened for motility before use for IVF. Samples that are below a designated motility threshold may be discarded. Our objectives were to determine if post-thaw sperm motility, other traits that may be indicative of sperm function, or a novel assay of oviduct binding were related to IVF success. Semen from 16 boars was cooled to 15 °C for overnight shipment before cryopreservation. Semen was thawed and motility was recorded microscopically and confirmed using computer-automated sperm assessment. Each sample was tested by IVF in two to three independent replicates. Regression and correlation analyses were employed to determine the interrelationships between sperm traits and the relationships between post-thaw motility, sperm-oviduct binding and IVF outcomes. Among the sperm traits examined, sperm acrosome integrity was negatively correlated with post-thaw motility (r² = 0.64) but not with IVF results. The number of sperm bound to oviduct aggregates was correlated with IVF polyspermy rates (r² = 0.62, P < 0.05) but less with overall IVF rates (r² = 0.31, P > 0.10). There was some relationship of post-thaw motility with IVF monospermic fertilization (P = 0.06, r² = 0.08) but not to other IVF outcomes. Our results indicate that post-thaw motility of frozen-thawed boar sperm is strongly related to acrosome integrity but has limited use for predicting IVF success. The number of sperm bound to oviduct cells was related to IVF polyspermy rates and may be more indicative of in vitro sperm function than traditional sperm motility and acrosome status evaluation.     

Successful sperm cryopreservation of the brown-marbled grouper,Epinephelus fuscoguttatus using propylene glycol as cryoprotectant

This study developed the cryopreservation of brown-marbled grouper spermatozoa for practical application. We examined 32 cryodiluents, developed from four types of cryoprotectants [propylene glycol (PG), dimethyl-sulphoxide (Me₂SO), dimethyl-acetamide (DMA) and ethylene glycol (EG)] at four concentrations of 5, 10, 15 and 20% in combination with two extenders [Fetal bovine serum (FBS) and artificial seminal plasma (ASP). Cooling rates were examined by adjusting the height of straws (2.5–12.5 cm) from the liquid nitrogen (LN) vapor and cooled for 5 min before immersion into LN. DNA laddering was used to detect DNA damage in cryopreserved sperm. In fertilization trials, 0.5 g of eggs was mixed with cryopreserved sperm stored for 30 days in LN. The best motility of post-thaw sperm was achieved using 15% PG + 85% FBS (76.7 ± 8.8%); 10% PG + 90% FBS was also effective as cryodiluent. Generally, FBS gave better post-thaw motility compared to ASP. The optimum cooling rate was at 17.6 °C min⁻¹ obtained by freezing at the height of 7.5 cm surface of LN. The results obtained showed that cryopreserved sperm of brown-marbled grouper suffered slight DNA fragmentation, which resulted in significantly lower motility. However, the fertilization (90.9 ± 0.5%), hatching (64.5 ± 4.1%) and deformity rates (3.8 ± 0.2%) obtained from cryopreserved sperm showed no significant difference with fresh sperm. These findings show that the developed protocol for cryopreservation of brown-marbled grouper sperm was viable and will be useful for successful breeding and seed production of brown-marbled grouper.     

The synergistic effect of trehalose and low concentrations of cryoprotectants can improve post-thaw ram sperm parameters

The objective of this study was to compare the effects of different concentrations of two different cryoprotectants (glycerol, G and ethylene glycol, EG) and trehalose (T), added to the semen extender, on post-thaw ram sperm parameters. Ejaculates, collected from 6 Merino rams, were pooled and evaluated at 37 °C. The pooled samples were divided into six equal aliquots, and diluted in Tris-based extenders containing 5% G, 3% G + 60 Mm T, 1.5% G + 100 Mm T, 5% EG, 3% EG + 60 mM T, and 1.5% EG + 100 Mm T. Subsequently, the samples were cooled to 5 °C, frozen in 0.25-ml French straws, and stored in liquid nitrogen (LN₂). Frozen samples were thawed individually, at 37 °C for 25 s in a water bath, for evaluation. Sperm motility was assessed using a phase-contrast microscope with a warm stage maintained at 37 °C. Acrosome integrity (FITC/PNA-PI), sperm viability (SYBR-14/PI), mitochondrial activity (JC-1/PI), DNA damage (COMET assay) and DNA fragmentation (TUNEL test) were determined. The group of samples diluted in an extender containing 5% of glycerol (Group 5% G) displayed higher percentages of subjective motility, viability and mitochondrial activity of sperm, compared to the other groups (P < 0.05). On the other hand, Group 3% G + 60 mM T yielded the second-best results for subjective motility, viability and mitochondrial activity of sperm, when compared to the other groups. The post-thaw sperm parameters of Group 3% G + 60 Mm T did not show any statistically significant difference from those of Group 5% G. There were no statistically significant differences between the groups for acrosome integrity (P > 0.05).The results of the COMET assay showed that the use of low concentrations of cryoprotectants in combination with trehalose decreased sperm DNA damage. Accordingly, Group 1.5% G + 100 mM T and Group 3% EG + 60 mM T benefited from a significantly stronger cryoprotective effect on DNA integrity, in comparison to Group 5% G (P < 0.05). According to the results of the TUNEL test, the combined use of low concentrations of cryoprotectants with trehalose decreased sperm DNA damage, when compared to the use of 5% glycerol, but this difference was statistically insignificant (P > 0.05).In conclusion, G and EG concentrations can be reduced by adding various amounts of T (60 mM, 100 mM) to the semen extender. The addition of 5% of glycerol and 3% G + 60 mM T to the semen extender did not yield statistically different post-thaw sperm parameters, when compared for protection against cryoinjury. Post-thaw sperm parameters can be improved by the supplementation of the semen extender with 3% G + 60 mM T. Thus, we recommend the use of freezing extenders containing low cryoprotectant concentrations (3% G) combined with trehalose to avoid the high level of toxic and osmotic damage caused by 5% G.     

Dynamics of sperm DNA fragmentation in domestic animals: II. The stallion

The mixed success of equine artificial insemination programs using chilled and frozen-thawed semen is most likely associated with the variable response of the sperm cell to the preservation process and the fact that stallions are not selected on the basis of reproductive performance. We propose that the traditional indicators of sperm viability do not fully account for male factor infertility in the stallion and that knowledge of sperm DNA damage in the original semen sample and during semen processing may provide a more informed explanation of an individual stallion's reproductive potential. This study reports on the validation of a sperm DNA fragmentation test based on the sperm chromatin dispersion test (SCD) for stallion spermatozoa and on its application to semen that was chilled (4 °C; n = 10) or frozen-thawed (n = 13). Semen samples were collected by artificial vagina and the proportion of sperm with fragmented DNA determined. Seminal plasma was then removed by centrifugation and the sperm pellet re-suspended in commercial extenders prior to being chilled or cryopreserved using standard industry protocols. Chilled semen was cooled slowly to 4 °C and stored for 1 h before commencing the analysis; cryopreserved semen was thawed and immediately analyzed. Following chilling or cryopreservation, the semen samples were incubated at 37 °C and analyzed for SCD after 0, 4, 6, 24 and 48 h storage. The results of this investigation revealed that there was no significant difference in the sperm DNA fragmentation index (sDFI) of sperm evaluated initially after collection compared to those tested immediately after chilling or cryopreservation. However, within 1 h of incubation at 37 °C, both chilled and frozen-thawed spermatozoa showed a significant increase in the proportion of sDFI; after 6 h the sDFI had increased to over 50% and by 48 h, almost 100% of the sperm showed DNA damage. While the sDFI of individual stallions at equivalent times of incubation was variable, an analysis of the rate of change of sDFI revealed no difference between stallions or the way in which the semen was preserved. In terms of sperm DNA fragmentation dynamics, the highest intensity of sperm DNA damage occurred in the first 6 h of incubation. We suggest that the SCD test can be used as a routine assessment tool for the development and refinement of preservation protocols designed to reduce stallion sperm DNA damage.     

Single-layer centrifugation with Androcoll-E can be scaled up to allow large volumes of stallion ejaculate to be processed easily

The objective of the current study was to optimize the volumes of Androcoll-E and sperm sample used in various sizes of centrifuge tube to scale up single-layer centrifugation (SLC) for routine use in the field. Although sperm suspensions of equivalent quality were produced using Androcoll-E in small and large tubes, the sperm yield was much lower in the latter (P < 0.001). In contrast, in 200-mL tubes (XL), the yields were approximately 25% higher than those for the small tubes. An increased volume (4.5 mL) of extended ejaculate in small tubes (SLC-Inc) or 15 to 18 mL extended ejaculate on 15 mL of colloid of a reduced density, Androcoll-E-Large (SLC-Large), in 50-mL tubes were both found to give similar yields of motile spermatozoa as that of the SLC-Small method (SLC-Small, 49.7 ± 18.6%; SLC-Inc, 53.3 ± 17.1%; SLC-Large, 44.9 ± 18.3%) and were found to be equivalent in quality (motility: 88.0 ± 8.8%, 84.0 ± 3.5%, 90.0 ± 5.4%; normal morphology: 69.4 ± 17.0%, 69.4 ± 12.7%, 63.9 ± 15.6%; viability: 78 ± 16.7%, 83.8 ± 12.5%, 80.05 ± 14.6%; DNA fragmentation index: 14.7 ± 10.9%, 12.8 ± 8.1%, 11.6 ± 7.6%, respectively). The processing of an “average” stallion Equus caballus ejaculate in approximately twenty-seven 10-mL tubes (SLC-Inc) or eight 50-mL tubes (SLC-Large) is feasible, the latter being considered more practical for on-stud use.     

The effect of seminal plasma on alpaca sperm function

In order to advance the development of assisted reproductive technologies in alpacas and other Camelids, the objective of this study was to explore the role of seminal plasma concentration on motility and functional integrity of alpaca sperm. Sixteen male alpacas > 3 y of age were used. In Experiment 1, epididymal sperm were incubated for 0 to 6 h in 0, 10, 25, 50, or 100% seminal plasma and motility was assessed. In Experiment 2, epididymal sperm were incubated in 0, 10, or 100% seminal plasma for 3 h and motility, acrosome integrity and DNA integrity were assessed. In Experiment 3, ejaculated sperm were incubated in 10, 25, 50, or 100% seminal plasma for 0 to 6 h and motility assessed. In Experiment 4, ejaculated sperm were incubated in 10 or 100% seminal plasma for 3 h and motility, acrosome integrity, DNA integrity, and viability were assessed. Epididymal and ejaculated sperm maintained motility longer when incubated in the presence of 10% seminal plasma compared to 0, 25, 50, or 100% seminal plasma (P < 0.001). The mean ± SEM percentage of epididymal sperm with intact acrosomes was less (P < 0.001) in samples incubated in 0% seminal plasma (39.4 ± 3.73) compared to 10% (75.3 ± 1.20) or 100% (77.4 ± 0.90) within 1 h after incubation. However, DNA integrity of ejaculated and epididymal sperm was not significantly affected by seminal plasma concentration. The mean viability of ejaculated sperm was reduced in the presence of 100 (12.7 ± 2.33) compared to 10% (36.2 ± 4.68) seminal plasma (P < 0.001) within 1 h of incubation. We concluded that alpaca semen should be diluted to a final concentration of 10% seminal plasma to prolong motility, preserve acrosome integrity, and maintain viability of sperm.     

Chromatin structure analysis of spermatozoa from reciprocal chromosome translocation (RCT) carriers with known meiotic segregation patterns

The presence of reciprocal chromosome translocations (RCTs), as well as sperm chromatin disturbances, is known to exert negative influence on male fertility. The aim of this study was to identify an association between chromosome structural rearrangements in male RCT carriers and sperm seminological parameters (concentration, motility, morphology), chromatin status (fragmentation and maturity), meiotic segregation pattern and observed chromosomal hyperhaploidy. Sperm samples originated from ten male RCT carriers with reproductive failure/success. TUNEL assay (DNA fragmentation) and chromomycin A3 (CMA3)/aniline blue (AB) staining (chromatin maturity) were used to analyze sperm chromatin status while fluorescent in situ hybridization (FISH) was applied to observe meiotic segregation patterns and hyperhaploidy in spermatozoa. We found that the mean level of sperm DNA fragmentation in the RCT carrier group (18.0 ± 11.9%) was significantly higher (p = 0.0006) than the mean of the control group (7.5 ± 4.3%). There was no correlation observed between sperm DNA fragmentation levels (5.6–38.0%) and the frequency of genetically normal/balanced gametes (34.3–62.4%), sperm seminological quality or revealed reproductive failure. In contrast, a correlation between the frequencies of genetically normal/balanced spermatozoa and of gametes with mature chromatin was observed (CMA3: R = 0.4524, p = 0.2604; AB: R = 0.5238, p = 0.1827). A statistically significant increase in the hyperhaploidy level of selected chromosomes in all analyzed RCT carriers was documented but was not correlated to sperm seminology or fertility status. Further evaluation and additional assays toward sperm chromatin quality assessment in RCT carriers is suggested to explain the complexity of genomic structural rearrangements and its possible relevance to reproductive success or failure.     

Induction and removal of DNA damage in blood leukocytes of patients with type 2 diabetes mellitus undergoing hemodialysis

Type 2 diabetes mellitus (T2DM) is associated with a high production of reactive oxygen species, which may cause oxidative DNA damage. High levels of genomic damage have been associated with renal failure and hemodialysis. However, no information is available in the literature concerning the levels of DNA damage in T2DM individuals who are dependent on hemodialysis. This study used the comet assay to assess the levels of DNA damage before, immediately after and 48 h after the hemodialysis session in 25 patients with T2DM and in a group of 20 healthy individuals, selected according to mean age, sex and smoking habit. Our results showed increased levels of DNA damage in hemodialysis-dependent T2DM individuals (12.36 ± 8.04) when compared with healthy individuals (7.35 ± 7.41) (p = 0.014). Damage levels increased immediately after the hemodialysis session (19.76 ± 12.40) (p = 0.04), which suggests a possible action of pro-oxidative factors related to the therapy, with a genotoxic effect on cells. Results obtained 48 h after hemodialysis (6.44 ± 5.99) evidenced damage removal (p = 0.001), which may be suggestive of DNA repair.     

Effects of label-dose permethrin administration in yearling beef cattle: I. Bull reproductive function and testicular histopathology

Pyrethroid administration to a wide variety of laboratory animals has been shown to cause detrimental effects on male fertility, including sperm quality, by means of endocrine disruption. The objective of this experiment was to study the effects of a commercial, permethrin-containing pour-on product on reproductive variables and testicular histopathology of yearling beef bulls. Black Angus bulls (n = 60; aged 369 ± 17 days; 511 ± 33 kg; 6.2 ± 0.5 body condition scores) were assigned to either (1) saline control (CON) or (2) permethrin pour-on administered at label dose (PYR). Blood samples were collected, and industry standard breeding soundness examinations (BSE), via electroejaculation, were performed on all bulls at 5 days before and 14 days after treatment. Progressive sperm motility and eosin-nigrosin–stained sperm were analyzed using high-power phase-contrast microscopy. Plasma testosterone concentrations were analyzed via radioimmunoassay. Bulls were slaughtered at 34 days, and one testicle per bull was randomly collected for histologic examination. Change in sperm motility between BSEs was not different because of treatment; sperm morphology however improved across treatments, but PYR bulls had less improvement in percent of head (P < 0.001) sperm abnormalities compared to CON, resulting in less improvement of primary abnormalities (P = 0.04). Nonetheless, morphological differences did not change the overall outcome for satisfactory breeder status. Change in testosterone concentration did not differ because of treatment. Histopathologic examination identified that testicular degeneration and tubule diameter did not differ as a result of treatment. It should be noted, however, that degeneration score (higher score having more degeneration) was positively correlated with primary abnormalities (P < 0.01; r = 0.35) and negatively correlated with normal sperm cells (P < 0.001; r = −0.43). In summary, these data indicate that a single use of permethrin at label dose in yearling Angus bulls results in minimal detrimental effects on sperm morphology but not to a degree that impacts the ability of bulls to pass a standard BSE.