DNA fragmentation dynamics allows the assessment of cryptic sperm damage in human: Evaluation of exposure to ionizing radiation, hyperthermia, acidic pH and nitric oxide

Sperm DNA fragmentation (SDF) is not a static seminal parameter, since the longevity of sperm DNA decreases progressively with time following ejaculation or thawing. While the dynamics of SDF is a species-specific characteristic, in the case of humans, there is still significant variation within patients. To evaluate the suitability of the dynamic SDF assay to assess the adverse effects of agents that cause genetic damage, fresh semen samples from different donors were exposed in vitro to (1) increasing acute doses of ionizing radiation, (2) elevated temperature (41°C and 45°C), (3) acidic pH (pH 4) and (4) the nitric oxide (NO) donor sodium nitroprusside (SNP). Sperm DNA fragmentation was analyzed after an incubation period of chronic (24h), or acute (1h) exposure to each treatment followed by incubation at 37°C over a period of 24h. SDF was assessed using the sperm chromatin dispersion (SCD) test. Dynamic SDF for each treatment was analyzed using Kaplan-Meier survival curves. All agents, except for ionizing radiation, accelerated SDF kinetics following chronic exposure over a 24h period. Transient exposure to NO and heat but not acidic pH increased the basal (T0) level of SDF. Despite the removal of the three toxicants, the remaining sperm following acute exposure showed a decrease in their expected DNA longevity. It is concluded that the assessment of sperm DNA fragmentation dynamics is an effective methodological approach for revealing latent damage associated with toxicants that is not initially expressed following a single initial observation of SDF.     

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.     

The effect of two cryopreservation methods on human sperm DNA damage

Several methods are currently available for selection when conducting sperm cryopreservation, however, these methods might cause different degrees of damage on sperm DNA. The aim of the this study is to compare the effects of storage at −80 °C (in ultra-low temperature refrigerator) and at −196 °C (in liquid nitrogen) on sperm DNA damage, thus to provide a reference for choosing the right method according to different aims. We randomly collected 28 semen samples from college students of Chongqing city. The samples stored at −80 °C were neat semen samples and the samples stored at −196 °C were mixed with additional cryoprotectants. Each sample was subjected to two freezing-thawing cycles, and the sperm DNA damage levels of fresh and thawed samples were measured by single cell gel electrophoresis (SCGE) and sperm chromatin structure assay (SCSA). Both SCGE and SCSA assays showed cryopreservation induced significant damage to sperm DNA. However, storage at −196 °C lead to more severe damage to sperm DNA than storage at −80 °C measured by SCSA. Sperm DNA damage increased simultaneously with the higher frequency of freezing-thawing cycles. We concluded that storage of neat semen samples at −80 °C had milder damage to sperm DNA than storage at −196 °C mixed with cryoprotectants. To avoid additional sperm DNA damage, repeated freezing and thawing should be prevented.     

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.     

Effect of Sperm DNA Fragmentation on Clinical Outcome of Frozen-Thawed Embryo Transfer and on Blastocyst Formation

During the last decades, many studies have shown the possible influence of sperm DNA fragmentation on assisted reproductive technique outcomes. However, little is known about the impact of sperm DNA fragmentation on the clinical outcome of frozen-thawed embryo transfer (FET) from cycles of conventional in vitro fertilization (IVF) and intra-cytoplasmic sperm injection (ICSI). In the present study, the relationship between sperm DNA fragmentation (SDF) and FET clinical outcomes in IVF and ICSI cycles was analyzed. A total of 1082 FET cycles with cleavage stage embryos (C-FET) (855 from IVF and 227 from ICSI) and 653 frozen-thawed blastocyst transfer cycles (B-FET) (525 from IVF and 128 from ICSI) were included. There was no significant change in clinical pregnancy, biochemical pregnancy and miscarriage rates in the group with a SDF >30% compared with the group with a SDF ≤30% in IVF and ICSI cycles with C-FET or B-FET. Also, there was no significant impact on the FET clinic outcome in IVF and ICSI when different values of SDF (such as 10%, 20%, 25%, 35%, and 40%) were taken as proposed threshold levels. However, the blastulation rates were significantly higher in the SDF ≤30% group in ICSI cycle. Taken together, our data show that sperm DNA fragmentation measured by Sperm Chromatin Dispersion (SCD) test is not associated with clinical outcome of FET in IVF and ICSI. Nonetheless, SDF is related to the blastocyst formation in ICSI cycles.     

The influence of the storage temperature and cryopreservation conditions on the extent of human sperm DNA fragmentation

We explored the influence of different storage temperature conditions, including different methods of cryopreservation, on the structure of DNA organization in human sperm using a direct labeling procedure for detecting DNA fragmentation. Nineteen sperm samples that were obtained from healthy men with normozoospermia (according to the criteria of the World Health Organization) were used for the investigation. A significant increase in human sperm DNA fragmentation was observed 8 h after the incubation at +39°C (by 76.7%) and at +37°C (by 68.9%). It was found that cooling the sperm with a cryoprotectant immediately after thawing did not produce a significant difference in the extent of DNA fragmentation; however, the samples that contained cryoprotectants showed a sharp increase in the DNA fragmentation 24 h after the incubation, which could suggest cryoprotectant cytotoxicity.     

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 degrees 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 degrees C and stored for 1h before commencing the analysis; cryopreserved semen was thawed and immediately analyzed. Following chilling or cryopreservation, the semen samples were incubated at 37 degrees 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 1h of incubation at 37 degrees C, both chilled and frozen-thawed spermatozoa showed a significant increase in the proportion of sDFI; after 6h 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 6h 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.     

Bacteria in bovine semen can increase sperm DNA fragmentation rates: a kinetic experimental approach

Cryopreserved straws of semen (n = 228) from Holstein bulls (n = 47) were examined for bacterial presence and sperm DNA fragmentation (SDF) dynamics. Commercial semen doses (representing six ejaculates per individual) were randomly selected from a bull stud in Spain. The dynamics of SDF were assessed after thawing (T0) and at 4, 24, 48, 72 and 96 h of incubation at 37 °C, using the commercial variant of the sperm chromatin dispersion test for Bovine (Halomax^®). One group of bulls showed a bacterial presence in semen samples between 0 and 96 h of incubation (n = 23, group A) while the other did not (n = 24, group B). Immediate post-thaw differences in SDF were not observed when both groups were compared. However, the rate of increase in SDF (rSDF) over time, considered as an estimate of the kinetic behaviour of sperm DNA survival, was significantly higher (P < 0.05) in semen samples from group A (0.7% per hour) versus group B (0.05% per hour). Polymerase Chain Reaction (PCR) assay was used for DNA amplification using primers designed for specific regions of the bacterial gene that codifies for 16S rRNA. Different species within the phyla Bacteroidetes, Firmicutes, Proteobacteria, Cyanobacteria, Fusobacteria and Actinobacteria were identified. The results show that (1) SDF at baseline (T0) may not be affected by the presence of bacteria but the rSDF can increase due to bacterial growth during incubation, (2) the increase in the rSDF is characteristic of some bulls but not for others, and (3) certain bacterial strains are repeatedly found in separate ejaculates from the same bull.     

Heat induced infertility of boars: The inter-relationship between depressed sperm output and fertility and an estimation of the critical air temperature above which sperm output is impaired

Eight groups of Large-White gilts were each inseminated with different numbers of normal motile sperm, in the range 0.28–7.0 × 10⁹. A significant (P < 0.05) relationship between conception rate and the number of motile sperm inseminated was shown. This relationship can be used to equate output of motile sperm with levels of fertility of boars. The optimal number of motile sperm for conception following intra-cervical insemination was near 5 × 10⁹ and the threshold number, below which animals did not conceive, was c. 4 × 10⁸.In a second experiment, three Large-White boars were subjected to graded thermal treatment (air temperature was increased by 1°C per day for 20 days, from a basal level of 20°C to a maximal level of 40°C) and responses of ejaculate and other physiological characteristics were monitored. Scrotal surface temperature, respiration rate and rectal temperature increased (P < 0.05) beyond basal levels at air temperatures of 30°C, 33°C and 35°C, respectively. Motility of sperm in ejaculates decreased when air temperature reached 30°C and this response was presumed to reflect hyperthermia in epididymal tissues, consistent with increasing scrotal surface temperature at this same air temperature. Motility fell below a pre-treatment level of about 93%, to 19% (P < 0.05), 3 weeks after heating. Volumes of seminal plasma and gel in ejaculates were also lower (P < 0.05) following heating. Changes in daily sperm production were minor and, as a result, daily motile sperm production levels paralleled changes in motility. Proportions of abnormal types of sperm increased (P < 0.05) to maximal levels in the last week of heating and all returned to pretreatment values 5 weeks later. High proportions of sperm with kinoplasmic droplets appeared in ejaculates collected after heating (P < 0.05), evidence that epididymal cell types in the boar are sensitive to heat.As a result of heat treatment, normal motile sperm production decreased from control levels (1.28 × 10¹⁰·day^?1) to 0.15 × 10¹⁰·day^?1, 3 weeks after heating ceased. However, the results suggest that normal sperm output by Large-White boars can be maintained at air temperatures as high as 29°C.By relating the results of both experiments, it is concluded that fertility of the boars in the second experiment (if mated once daily) would be depressed for about 5 weeks after heat treatment ceased. The findings support many field reports which indicate a contribution of boars to lower conception rates of sows during and immediately following summer and the results can be used in formulating strategies to circumvent this widespread problem.     

Double Stranded Sperm DNA Breaks,Measured by Comet Assay,Are Associated with Unexplained Recurrent Miscarriage in Couples without a Female Factor

It is known that sperm samples from recurrent pregnancy loss (RPL) couples have an increase in their sperm DNA fragmentation (SDF), but no studies have been performed in order to identify differences between single stranded SDF (ssSDF) and double stranded SDF (dsSDF) in these patients. This could be relevant because the type of DNA damage could have different effects. Semen samples were classified attending their clinical status: 25 fertile donors and 20 RPL patients with at least two unexplained first trimester miscarriages. SDF was analysed using alkaline and neutral Comet assay, SCD test and pulsed-field gel electrophoresis (PFGE), and ROC analysis including data from 105 more infertile patients (n = 150) was performed to establish predictive threshold values. SDF for alkaline and neutral Comet, and the SCD test was analysed in these categories of individuals. Data revealed the presence of two subgroups within fertile donors. The values obtained were 21.10±9.13, 23.35±10.45 and 12.31±4.31, respectively, for fertile donors with low values for both ssSDF and dsSDF; 27.86±12.64, 80.69±12.67 and 12.43±5.22, for fertile donors with low ssSDF and high dsSDF; and 33.61±15.50, 84.64±11.28 and 19.28±6.05, for unexplained RPL patients, also showing a low ssSDF and high dsSDF profile. This latter profile was seen in 85% of unexplained RPL and 33% of fertile donors, suggesting that it may be associated to a male risk factor for undergoing RPL. ROC analysis regarding recurrent miscarriage set the cut-off value at 77.50% of dsDNA SDF. PFGE for low ssSDF and high dsSDF profile samples and positive controls treated with DNase, to induce dsDNA breaks, showed a more intense band of about 48 kb, which fits the toroid model of DNA compaction in sperm, pointing out that some nuclease activity may be affecting their sperm DNA in RPL patients. This work identifies a very specific SDF profile related to the paternal risk of having RPL.     

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.     

Diurnal variation in sperm DNA fragmentation: analysis of 11,382 semen samples from two populations and in vivo animal experiments

ABSTRACT

Circadian rhythms have been found in some reproductive functions phenotypes but remain unclear for sperm DNA fragmentation index (DFI). The present study aims to investigate the diurnal variation of DFI in mice model and men sperm. Adult male mice were sacrificed for sperm DFI with Sperm Chromatin Structure Assay (SCSA) in 24 hours at 6 evenly distributed time points. A cosinor pattern of DFI was observed with a nadir at zeitgeber time 10 AM. In a community population with 630 semen samples collected between 8 AM and 20 PM, the temporal variation of DFI also fit a cosinor pattern with a ? 343° acrophase and a nadir at 11 AM (P = .031). In a reproductive-medical-center dataset of 10752 semen samples collected between 7 AM and 11 AM, the decreasing trend of DFI was also confirmed. For the males with multiple samples, intra-individual comparison between different timepoints was performed, and each consecutive hour after 7 AM was also associated with 2.5 (95% CI: ?1.0, 5.9)% lower DFI by SCSA or 4.9 (1.9, 7.8)% lower DFI by SCD. Our study reveals a daily diurnal variance in sperm DFI which may suggest a practical approach to get more qualified sperms for natural or assisted reproduction.

Abbreviations: BMI, Body mass index; DFI, DNA fragmentation index; MARHCS, Male Reproductive Health in the Chongqing College Students; RMC, Reproductive Medical Center; SCD, Sperm Chromatin Dispersion; SCSA, Sperm Chromatin Structure Assay.     

Temperature dependence of capacitation in bat sperm monitored by zona-free hamster ova

The temperature dependence of capacitation in bat sperm (Myotis lucifugus lucifugus) was studied by monitoring fertilizations rates of zona-free hamster ova at different temperatures. Spermatozoa were cultured in BWW medium at temperatures 4°C, 24°C, 32°C, 42°C, and 55°C from 0–24 hr. Activation of sperm could be determined visually due to the change in movement seen through light microscopy. Activation was later confirmed by higher rates of fertilization. Preincubation of the bat sperm was found to have a direct effect on the success of penetration of the zona-free hamster ova. Holding bat spermatozoa at low temperature for long intervals allowed them to remain motile but unable to fertilize. Sperm are not irreversibly damaged, however, and activation, when the temperature is increased to 32°C, is faster than when sperm are intitially put at 32°C, resulting in good fertilization rates.     

Incubation of sperm heads impairs fertilization and early embryo development following intracytoplasmic sperm injection (ICSI) by decreasing oocyte activation in mice

When intracytoplasmic sperm injection (ICSI) is performed in mice, isolation of sperm heads is usually performed prior to injections in order to increase the efficiency of the procedure. Consequently, the isolated sperm heads undergo an inevitable incubation in vitro. However, little is known about the effects of this incubation step on fertilization and embryo development following ICSI. When we incubated sperm heads at 37 °C, there was a significant time-dependent decrease in fertilization and blastocyst formation. Moreover, the DNA integrity of the sperm heads was maintained over 12 h incubation. Using assisted oocyte activation, these defects in fertilization and embryo development were rescued. Taken together, incubation of sperm heads following isolation can affect the oocyte-activating capacity of sperm thereby compromising fertilization and embryo development associated with ICSI.     

Cryoprotectants synergy improve zebrafish sperm cryopreservation and offspring skeletogenesis

The synergy obtained by the combination of cryoprotectants is a successful strategy that can be beneficial on the optimization of zebrafish sperm cryopreservation. Recently, a protocol was established for this species using an electric ultrafreezer (−150 °C) performing cooling rate (−66 °C/min) and storage within one step. The ultimate objective of sperm cryopreservation is to generate healthy offspring. Therefore, the objective of this study was to select the most adequate cryoprotectant combination, for the previously established protocol, that generate high quality offspring with normal skeletogenesis. Among the permeating cryoprotectant concentrations studied 12.5% and 15% of N,N-dimethylformamide (DMF) yielded high post-thaw sperm quality and hatching rates. For these two concentrations, the presence of bovine serum albumin (10 mg/mL), egg yolk (10%), glycine (30 mM) and bicine (50 mM) was evaluated for post-thaw sperm motility, viability, in vitro fertilization success and offspring skeletal development (30 days post fertilization). Higher concentration of permeating cryoprotectant (15%) decreased the incidence of deformed arches and severe skeletal malformations, which suggests higher capacity to protect the cell against cold stress and DNA damage. Extender containing 15% DMF with Ctrl, Bicine and egg yolk were the non-permeating cryoprotectants with higher post-thaw quality. The use of these compounds results in a reduction in vertebral fusions, compressions and severity of skeletal malformations in the offspring. Therefore, these extender compositions are beneficial for the quality of zebrafish offspring sired by cryopreserved sperm with −66 °C/min freezing rate. To the best of our knowledge, this is the first report on skeletal development of the offspring sired by cryopreserved sperm performed with different freezing media compositions in zebrafish.     

Rapid evolution of sperm length in response to increased temperature in an ectothermic fish

The Intergovernmental Panel on Climate Change predicts an average global temperature increase of 1.8–4.0 °C by 2100. Tropical ectotherms are expected to be particularly sensitive to this temperature increase because they live close to their thermal limits. We investigated the phenotypic plasticity and evolutionary response of sperm traits in guppies (Poecilia reticulata) to increased temperatures after 6, 18, and 24 months. Guppies with evolution temperatures of 25 °C (control) or 28 °C were reared in either 25 or 28 °C in a 2 × 2 common garden design. The plastic response to increased temperature was a decreased sperm length, velocity, and path linearity. The evolutionary response was a subsequent increase in sperm length, resulting in complete compensation after just 6 months (at most four generations) in 28 °C water. Sperm velocity and linearity showed no sign of evolution even after 24 months. This study provides evidence that some reproductive traits can respond via rapid evolution to the temperature increase associated with climate change.     

A new method to analyze boar sperm DNA fragmentation under bright-field or fluorescence microscopy

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.     

Effect of cholesterol-loaded cyclodextrins on bull and goat sperm processed with fast or slow cryopreservation protocols

Cholesterol-loaded cyclodextrins (CLC) added to the sperm before cryopreservation enhance sperm quality after freeze-thawing in several cold shock-sensitive species, including cattle and goats. However, all studies conducted to date have used conventional protocols, in which sperm are cooled slowly to 5°C before freezing. As cholesterol plays a significant role in sperm cold shock resistance, it is possible that CLC-treated sperm can withstand cooling damage when the sperm are not cooled slowly to 5°C before freezing. In this study, we determined whether CLC-treated goat (1 mg CLC/120×10⁶ sperm) and bull (2 mg CLC/120×10⁶ sperm) sperm quality, after thawing, was different for sperm frozen using conventional protocols (including a slow cooling phase to 5ºC) and protocols in which the sperm were frozen from room temperature, without cooling the sperm slowly to 5°C before freezing. CLC-treated sperm exhibited higher percentages of plasma membrane-intact sperm than control sperm when cryopreserved using conventional protocols. In addition, CLC treatment enhanced both sperm motility and plasma membrane integrity when sperm were frozen directly from room temperature. However, this treatment did not fully prevent the damage of the sperm after cooling rapidly and subsequent freezing, as the sperm quality was lower than that presented by the samples frozen using the conventional protocol. The results are promising, but studies to optimize the protocols for freezing sperm directly from room temperature need to be conducted, as well as studies to determine how cryopreserving sperm in this manner affects other sperm functions.     

Induction of meiotic gynogenesis in ship sturgeon Acipenser nudiventris using UV-irradiated heterologous sperm

Diploid gynogenesis was induced in ship sturgeon Acipenser nudiventris using UV-irradiated sperm from Siberian sturgeon Acipenser baerii. The optimal condition for the retention of the second polar body in ship sturgeon was determined to be 10 min after activation/fertilization in experiments. The temperature of cold shock and its duration were 2.5 °C and 30 min, respectively. A total of 30 gynogens of known parentage from experimental treatments were screened using microsatellite DNA analysis, and uniparental transmission in meiogens was confirmed. The results show that heterologous Siberian sturgeon sperm is applicable as UV-irradiated sperm for the induction of gynogenesis in ship sturgeon. This technique may recover the critically endangered sturgeon species that are becoming extinct.     

Cryopreservation of Plagiognathops microlepis sperm

Sperm was collected from cultured male fish and cryopreserved in 0.25 ml straws for the study of sperm cryopreservation. Different parameters were evaluated, including extender, dilution ratio, cryoprotectant type and concentration, equilibrium time, cooling height (in a two-step cooling protocol), and thawing temperature. The optimum result was obtained when the sperm was diluted at a 1:7 ratio in D-16 with 5% DMSO as a cryoprotectant, equilibrated for 20 min, held at 3 cm above liquid nitrogen for 10 min, and then stored in liquid nitrogen. After thawing in a water bath at 40 °C, the percentage of motile cells and fertilization rates of frozen-thawed sperm were 35.33 ± 2.52% and 39.00 ± 4.58%, respectively, while the corresponding rates for fresh sperm were 87.67 ± 3.06% and 88.67 ± 4.62%. We also used a programmed cooling protocol in which temperature was decreased from 4 °C to −80 °C by a rate of 30 °C/min, and then straws (0.25 ml) were placed above the surface of liquid nitrogen for 2 min before being stored in liquid nitrogen. This protocol provided a post-thaw activation rate of 36.67 ± 4.77%. Further parametric optimization is required to improve the quality of frozen-thawed sperm.