Single Layer Centrifugation Samples Research Articles

Non-enzymatic extraction of spermatozoa from alpaca ejaculates by pipetting followed by colloid centrifugation

Viscous camelid ejaculates present problems for sperm handling and sperm preservation. In the present study, a technique that had been used for dromedary camel semen was tested with alpaca semen. Ejaculates (n=9) were collected by artificial vagina at San Marcos University, Lima, and were liquefied by gentle pipetting in tris-citrate-fructose. Half of the sample was prepared by Single Layer Centrifugation (SLC) through a colloid; the other half was centrifuged without colloid as a control. Each control and SLC sample was then split into two parts; one part was stored cooled for 24 h at 5 °C and the other part was frozen, resulting in 4 treatments for each ejaculate. All samples were evaluated for sperm motility, hypoosmotic swelling test (HOST), plasma membrane integrity, and morphology, immediately after centrifugation and again after storage Total motility and plasma membrane integrity were greater in samples prepared by SLC than controls (motility 72±13% vs. 57±7%; plasma membrane integrity 63±13% vs. 54±8%, for SLC and controls respectively). Normal morphology and HOST were not different between treatments (65±13 vs. 61±13% and 42±6 vs. 39±10%, for SLC and controls respectively). After 24 h cooled storage, motility and plasma membrane integrity were greater for SLC samples (motility: 51±16 vs. 34±15%; p<0.001; membrane integrity: 51±15 vs. 40±18%; P < 0.05 for SLC and controls, respectively); HOST (40±14 vs. 34±11%) and normal morphology (67±13 vs. 63±14%) were not different between treatments. Sperm quality decreased considerably after cryopreservation (P<0.001 for all parameters); however, motility (P<0.01), plasma membrane integrity (P<0.05) and morphology (P<0.05) were higher for SLC than for controls. These results indicate that alpaca spermatozoa can be extracted from semen using a combination of pipetting and SLC, potentially with a beneficial effect on sperm quality. Samples could be stored cooled for 24 h, retaining better motility than controls; motility and plasma membrane integrity were greater in SLC samples than controls after freezing and thawing but the freezing protocol requires improvement.

Variation among stallions in sperm quality after single layer centrifugation

Although single layer centrifugation (SLC) selects robust spermatozoa from stallion semen, the effect of individual variation has not been studied in detail. The objective of this study was to determine the variation among stallions in the effects of SLC on sperm quality during cooled storage for up to 48hr. Semen samples from seven stallions (18 ejaculates) were split, with one portion being used for SLC and the other serving as a control (CON). Sperm quality (kinematics, reactive oxygen species (ROS) production, membrane integrity (MI) and chromatin integrity) were analysed at 0, 24 and 48hr using computer-assisted sperm analysis and flow cytometry. Sperm quality was better in SLC than in CON at all timepoints, especially chromatin integrity and MI (p<.0001 for both), and some categories of ROS production (e.g. proportion of live hydrogen peroxide negative spermatozoa, p<.0001), but the degree of improvement varied among stallions and type of ROS (p<.05-p<.0001). Total and progressive motility were also better in SLC samples than in CON at 24 and 48hr (p<.0001), although the effect on sperm kinematics varied. The interaction of treatment, time and stallion was not significant. In conclusion, sperm quality was better in SLC samples than in CON, although there was considerable individual variation among stallions. The improvement in sperm quality, particularly in chromatin integrity, was clearly beneficial, and therefore the use of this technique would be warranted for all stallion semen samples.

Stored Stallion Sperm Quality Depends on Sperm Preparation Method in INRA82 or INRA96

Removal of seminal plasma facilitates stallion sperm survival during storage, but washing may damage sperm chromatin. Therefore, sperm quality was compared in samples following single-layer centrifugation (SLC) or sperm washing and controls (extension only) in two extenders, INRA82 and INRA96. Ejaculates from six stallions were split among six treatments: SLC, sperm washing, and controls, in INRA82 and INRA96. Sperm motility and acrosome status were evaluated at 0, 24, 48, 72 and 96 hours; morphology at 0, 24, 48, 72 hours and chromatin integrity at 0 and 96 hours, with storage at 6°C. Sperm samples in INRA96 had better motility, acrosome status, and normal morphology than samples in INRA82. The SLC samples had higher motility and fewer reacted acrosomes than controls, and lower fragmented chromatin than washed samples. Fewer spermatozoa with tail defects were observed after SLC than after sperm washing; spermatozoa washed in INRA82 had fewer tail defects than those washed in INRA96. In conclusion, sperm quality (except for morphology) was better in INRA96 than in INRA82 and was better in SLC samples than in washed samples or controls. The SLC method is a useful adjunct to stallion sperm preparation, especially for storage before artificial insemination.

14 Sperm membrane integrity in ejaculates from young bulls may be improved by single-layer centrifugation

Sperm quality in the first ejaculates of young bulls is often poor, leading to rejection of these samples for commercial freezing. It may be several weeks before sperm quality reaches the thresholds regarded as being acceptable for commercial production. Single-layer centrifugation (SLC) has been shown to separate robust bull spermatozoa from the rest of the ejaculate (Goodla et al. 2015 J. Dairy Sci. 97, 2204-2212; Nongbua et al. 2017 Reprod Domest. Anim. 52, 596-602). The aim of this study was to test whether SLC can be used to select spermatozoa from the ejaculates of young bulls and thus freeze samples earlier than is currently possible. Ejaculates (at least 3 per bull; n=33) were collected from 9 young Holstein or Swedish Red bulls, 255 to 415 days old. The sperm concentration was adjusted to 69×106 spermatozoa mL−1 with Andromed™ (Minitub, Tiefenbach, Germany) before layering part of the sample over 4mL of Bovicoll colloid. The remaining sample served as the unselected control. After centrifugation at 300×g for 20min, the sperm pellet was aspirated into fresh extender. Both control (uncentrifuged) and SLC samples were frozen and stored in LN pending analysis. Membrane integrity was evaluated by flow cytometry after staining for 10min at 37°C with SYBR14/propidium iodide (Live-Dead Sperm Viability Kit, L-7011; Invitrogen, Carlsbad, CA, USA). Paired t-test was used to compare results for control and SLC samples, and Pearson correlation was used for age and membrane integrity. The median age at which the samples were taken was 319 days (range: 255-415 days). Membrane integrity varied considerably among bulls (range: 11-72%). The mean proportion of membrane-intact spermatozoa in controls and SLC samples was 40±15 and 47±16%, respectively (P&amp;lt;0.01). In 21 of the 33 ejaculates (64%), the SLC sample had a higher membrane integrity than the controls (Table 1). Age in days was positively correlated with membrane integrity for SLC samples (r=0.40; P&amp;lt;0.05) but not for controls. These results suggest that SLC might be a useful technique for selecting membrane-intact spermatozoa from the ejaculates of young bulls. However, there is considerable variation among bulls, possibly due to age, which is currently being investigated. Other parameters of sperm quality, such as chromatin integrity, mitochondrial membrane potential reactive oxygen species production, and motility, are also being evaluated. Table 1.Age and membrane integrity (mean±standard deviation) in ejaculates from young bulls before and after SLC (n=33) This project was funded by the Royal Swedish Academy of Agriculture and Forestry, Viking Genetics, and the Faculty for Veterinary Medicine and Animal Health, SLU. We thank Viking Genetics for supplying the semen samples.

Improved cryosurvival of stallion spermatozoa after colloid centrifugation is independent of the addition of seminal plasma

Addition of seminal plasma (SP) prior to cryopreservation may influence stallion sperm cryosurvival. The objective of this study was to investigate the addition of pooled SP from “good” or “bad” freezer stallions to spermatozoa selected by single layer centrifugation (SLC) prior to cryopreservation on post-thaw sperm quality. Semen from 12 stallions was collected; 5 mL was frozen as control (C) and the remainder was processed by SLC to remove SP and was divided into three aliquots: i) SLC sample without SP (SLC); ii) SLC plus pooled SP from “good freezer” stallions (SLC-GF); iii) SLC plus pooled SP from “bad freezer” stallions (SLC-BF). After thawing, the following parameters were evaluated: chromatin integrity (DNA fragmentation index; %DFI), mitochondrial membrane potential (MMP), membrane integrity (MI), reactive oxygen species (ROS) and sperm kinematics. The %DFI was reduced (P < 0.0001) in SLC samples compared to controls. The SLC group showed a lower proportion of spermatozoa with low MMP and a higher proportion of spermatozoa with high MMP than other groups (P < 0.0001), and had lower hydrogen peroxide content than control. Sperm kinematics were not different. In conclusion, selection by SLC prior to cryopreservation improved post-thaw sperm quality; inclusion of SP from “good” and “bad” freezer stallions did not have an additional beneficial effect.

New approach to assess sperm DNA fragmentation dynamics: Fine-tuning mathematical models

BackgroundSperm DNA fragmentation (sDF) has been proved to be an important parameter in order to predict in vitro the potential fertility of a semen sample. Colloid centrifugation could be a suitable technique to select those donkey sperm more resistant to DNA fragmentation after thawing. Previous studies have shown that to elucidate the latent damage of the DNA molecule, sDF should be assessed dynamically, where the rate of fragmentation between treatments indicates how resistant the DNA is to iatrogenic damage. The rate of fragmentation is calculated using the slope of a linear regression equation. However, it has not been studied if sDF dynamics fit this model. The objectives of this study were to evaluate the effect of different after-thawing centrifugation protocols on sperm DNA fragmentation and elucidate the most accurate mathematical model (linear regression, exponential or polynomial) for DNA fragmentation over time in frozen-thawed donkey semen.ResultsAfter submitting post-thaw semen samples to no centrifugation (UDC), sperm washing (SW) or single layer centrifugation (SLC) protocols, sDF values after 6 h of incubation were significantly lower in SLC samples than in SW or UDC. Coefficient of determination (R2) values were significantly higher for a second order polynomial model than for linear or exponential. The highest values for acceleration of fragmentation (aSDF) were obtained for SW, followed by SLC and UDC.ConclusionSLC after thawing seems to preserve longer DNA longevity in comparison to UDC and SW. Moreover, the fine-tuning of models has shown that sDF dynamics in frozen-thawed donkey semen fit a second order polynomial model, which implies that fragmentation rate is not constant and fragmentation acceleration must be taken into account to elucidate hidden damage in the DNA molecule.

Effect of Single Layer Centrifugation on reactive oxygen species and sperm mitochondrial membrane potential in cooled stallion semen.

Additional means are needed for evaluating the quality of stallion spermatozoa in semen doses for AI. Mitochondrial membrane potential (ΔΨm) has been linked to fertility in some species, but is rarely used in the evaluation of cooled stallion semen; metabolic activity may be associated with reactive oxygen species production (ROS). In the present study, ΔΨm and ROS production were measured in doses of cooled stallion semen. The effect of colloid centrifugation on these parameters was also investigated. In this case, colloid centrifugation involves centrifuging a sperm sample through a silane-coated silica colloid formulation to retrieve the most robust spermatozoa. High and low ΔΨm in cooled stallion semen varied between stallions and between ejaculates, but was not affected by single-layer centrifugation (SLC). The SLC-selected spermatozoa produced significantly less hydrogen peroxide than controls (P < 0.001), which could explain the increased longevity and retention of fertilising capacity seen in previous studies. For SLC samples, ΔΨm was positively associated with viable spermatozoa that were not producing reactive oxygen species (r = 0.49; P < 0.001) and negatively associated with ROS production (for superoxide: r = -0.4, P < 0.01; for hydrogen peroxide: r = -0.39, P < 0.05). There was no clear association between ΔΨm and ROS production in control samples.

Effect of single-layer centrifugation or washing on frozen–thawed donkey semen quality: Do they have the same effect regardless of the quality of the sample?

The aims of this study were to determine the sperm quality of frozen–thawed donkey sperm samples after single-layer centrifugation (SLC) using Androcoll-E in comparison to sperm washing or no centrifugation and to determine if the effect on the sperm quality after SLC or sperm washing depends on the quality of the sample. Frozen–thawed sperm samples from Andalusian donkeys were divided into three aliquots, and they were processed using three different techniques after thawing: uncentrifuged diluted control (UDC), sperm washing (SW), and SLC. Afterward, sperm quality index was estimated by integrating all parameters (total and progressive sperm motility, membrane integrity, and DNA fragmentation) in a single value. The relationship between the sperm quality of thawed UDC samples and the effect on sperm parameters in SW and SLC-selected samples was assessed. Sperm quality index was significantly higher (P < 0.001) in SLC (0.8 ± 0.0) samples than that in UDC (0.6 ± 0.0) and SW (0.6 ± 0.0) samples, regardless of the sperm quality index after thawing of the sperm sample. In conclusion, SLC of frozen–thawed donkey spermatozoa using Androcoll-E-Small can be a suitable procedure for selecting frozen–thawed donkey sperm with better quality, in particular in those samples where an improvement in motility is needed.

Changes in Bull Sperm Kinematics after Single Layer Centrifugation

The objective of this study was to evaluate bull sperm kinematics after centrifugation through a single layer of a colloid [Single Layer Centrifugation (SLC)]. Ejaculates from 20 bulls were extended and stored at 4-6°C for 24h during transport to the laboratory for SLC through Androcoll-B, followed by measurement of sperm kinematics in all samples. Total motility (86% and 88% for uncentrifuged and SLC samples, respectively) and progressive motility (84% for both the groups) were similar (p>0.05). In contrast, straightness (STR) (0.65 vs 0.69), linearity (LIN) (0.32 vs 0.35) and beat cross frequency (BCF) (22.3 vs 23.6Hz) were significantly higher in the SLC-selected samples than in the uncentrifuged samples, whereas velocity of the average path (VAP) (95 vs 90μm/s), curvilinear velocity (VCL) (192 vs 180μm/s), amplitude of lateral head deviation (ALH) (7μm vs 6.5μm) and hypermotility (49% vs 38%) were significantly decreased. The kinematics of the samples with the poorest motility was improved most by SLC. In conclusion, even though SLC had no direct effect on total and progressive motility, it appeared to have a positive influence on several other kinematic parameters that may be important for fertilization after artificial insemination.

Comparison of the Effect of Heterologous and Homologous Seminal Plasma on Motility and Chromatin Integrity of Stallion Spermatozoa Selected by Single Layer Centrifugation.

The effect on sperm motility and chromatin integrity of adding homologous or heterologous equine seminal plasma (SP) to fresh stallion spermatozoa selected by single layer centrifugation (SLC) was studied. No statistical difference in mean progressive motility was seen after adding SP at time 0 h, although there were differences for individual stallions. The proportion of spermatozoa with high velocity was increased compared to untreated SLC-selected spermatozoa (P < 0.05), with significant differences between individuals (P < 0.01). When the SLC samples were stored for 24 h before adding SP, a significant increase in mean progressive motility was seen for SLC + homologous SP (P < 0.01) and for SLC + heterologous SP (P < 0.056). Whether homologous SP or heterologous SP had a greater effect on progressive motility depended on the individual. Adding either type of SP caused a significant increase in chromatin damage compared to SLC after storage for 24 h (homologous SP, P < 0.05; heterologous SP, P < 0.01). These preliminary data showed that storage of SLC-spermatozoa mixed with SP should be avoided because of the risk of increased chromatin damage. If SP is to be added to take advantage of a transient increase in progressive motility for a particular individual stallion, different combinations of SP and spermatozoa should be tested first to optimize the effect.

Quality of bull spermatozoa after preparation by single-layer centrifugation

The present study aimed to evaluate the effect of single-layer centrifugation (SLC) through a species-specific colloid (Androcoll-B; patent pending, J. M. Morrell) on bull sperm quality. Computer-assisted sperm analysis of motility and flow cytometric analysis of sperm viability (SYBR-14/propidium iodide staining), chromatin integrity (acridine orange staining), reactive oxygen species production [Hoechst 33258-hydroethidine-2′,7′-dichlorodihydrofluorescein diacetate (HO-HE-DCFDA) staining], mitochondrial membrane potential (staining with JC-1 probe), and protein tyrosine phosphorylation (specific antibody staining) were performed on unselected and SLC-selected sperm samples. Single-layer centrifugation of bull spermatozoa resulted in the selection of a sperm population that had high mitochondrial membrane potential, a higher content of phosphorylated protein, and more reactive oxygen species than control samples. Sperm chromatin damage was lower in the SLC samples although sperm viability and motility did not differ between SLC samples and controls. These observations suggest that SLC of bull semen in a soybean-containing extender improved some, but not all, parameters of sperm quality.

Reactive oxygen species in stallion semen can be affected by season and colloid centrifugation

There are anecdotal reports that equine fertility may decline towards the end of the breeding season. Previous studies have examined differences in sperm quality between the breeding season and non-breeding season but few studies have investigated the proportions of superoxide or peroxide containing spermatozoa at different times during the breeding season. The purpose of this study was to measure the content of these reactive oxygen species (ROS) at the beginning and end of the Swedish breeding season, using flow cytometric analysis of the fluorescence produced after staining with hydroethidium and dichlorodihydrofluorescein diacetate. In addition, the effects of a new method of selecting good quality spermatozoa by colloid centrifugation, known as Single Layer Centrifugation (SLC), on ROS-content were investigated. Superoxide production by stallion spermatozoa was found to be higher at the start than at the end of the breeding season in Sweden (22±16% versus 9±6%, P<0.05), whereas sperm motility was lower (total motility 80±9% versus 90±6%, P<0.01; progressive motility 55±12% versus 60±8%, P<0.05, at the beginning and end of the breeding season respectively). The mean values of the other parameters of sperm quality measured did not differ with time within the breeding season although differences did occur for individual stallions. SLC was found to select motile spermatozoa that contained less superoxide (16±14% versus 23±18%, P<0.01) and less peroxide (0.3±0.8 versus 1±2%, P<0.01) than uncentrifuged controls, although they were capable of producing ROS when stimulated with menadione. This reduced peroxide production may contribute to the enhanced sperm survival (retention of motility) seen in the SLC samples during storage.

Improvement of boar sperm cryosurvival by using single-layer colloid centrifugation prior freezing

The aim of this experimental study was to evaluate the effectiveness of sperm selection using single-layer centrifugation (SLC) prior to freezing on the sperm cryosurvival of boar ejaculates. Twenty-four sperm rich ejaculate fractions (SREF), collected from 24 boars (one per boar), were divided into two groups according to their initial semen traits: standard (n = 15) and substandard (n = 9). Semen samples from each SREF were split in two aliquots, one remained untreated (control samples) and the other was single-layer centrifuged (500g for 20 min) using 15 mL of Androcoll-P Large (SLC samples). The yield of total, motile (assessed by CASA) and viable (cytometrically evaluated after staining with H-42, propidium iodide (PI) and FITC-PNA) sperm after SLC was higher (P < 0.05) in standard than substandard semen samples. The semen samples were cryopreserved using a standard 0.5-mL straw freezing protocol. Post-thaw sperm motility and viability (assessed at 30 and 150 min post-thawing) were higher (P < 0.05) in SLC than in control samples, regardless of the initial semen traits of the ejaculates. Additionally, thawed spermatozoa from SLC samples were more resistant (P < 0.05) to lipid peroxidation (BIOXYTECH MDA-586 Assay Kit) than those from control samples, regardless of the initial semen traits of the ejaculates. The SLC-treatment also influenced the functionality of thawed spermatozoa undergoing an in vitro capacitation process. The percentage of viable sperm showing high membrane fluidity (assessed with merocyanine 540) was lower (P < 0.05) in the SLC than in the control samples, regardless of the initial semen traits of the ejaculates. Thawed viable spermatozoa of SLC samples generated less (P < 0.05) reactive oxygen species (assessed with CM-H2DCFDA) than those of control samples in the substandard ejaculates. These findings indicate that the sperm selection before freezing using SLC improves the freezability of boar sperm.

Restoration of seminal plasma to stallion spermatozoa selected by colloid centrifugation increases sperm progressive motility but is detrimental to chromatin integrity

There is controversy about whether the presence of some seminal plasma (SP) in an equine insemination dose is necessary for promoting fertility. A new technique for improving stallion sperm quality, single layer centrifugation (SLC) using a species-specific colloid, Androcoll-E, selects a sperm subpopulation that is highly motile with normal morphology, intact membranes and good chromatin integrity from the rest of the ejaculate and removes SP. The present study was designed to investigate the effect of restoring homologous SP (5% and 10%) on the progressive motility, velocity, and chromatin integrity of SLC-selected stallion spermatozoa in 44 semen samples over time. Sperm progressive motility (P < 0.01) and the proportion with class A velocity (>50 μm/sec) were increased in samples where SP was restored, whereas the proportion with class B velocity (10 to 50 μm/sec) was decreased compared with SLC samples. However, after 24 h cold storage of treated samples, progressive motility was not different for the SP-treated groups compared with SLC, whereas chromatin damage DNA fragmentation index (%DFI) was higher. In contrast, adding SP to untreated 24 h-stored SLC samples did not affect progressive motility although it did increase the proportion of spermatozoa with class A velocity. There was individual variation between stallions whether 5% or 10% SP produced a greater increase in progressive motility. In conclusion, 5% to 10% SP can be added back to SLC-selected samples if considered necessary to optimize fertility. However, it should be added immediately before insemination rather than before storage of the sperm dose, to benefit from the transient increase in sperm progressive motility and avoid increased chromatin damage.

Single layer centrifugation of stallion spermatozoa improves sperm quality compared with sperm washing

This study compared the effect on semen quality of different handling methods used in the preparation of stallion semen doses for artificial insemination. The three methods were (i) extending the ejaculate to 30-50 x 10(6)/ml, (ii) single layer centrifugation (SLC) and (iii) sperm washing (centrifugation without a colloid). An additional treatment was to add seminal plasma (SP) in various proportions to some SLC preparations. The resulting samples were evaluated for sperm motility by computer assisted sperm analysis, membrane integrity using the Nucleocounter SP-100 and chromatin integrity by the sperm chromatin structure assay. SLC samples consistently had better sperm quality than the extended samples. Sperm washing did not confer any beneficial effect compared with the extended samples and these samples had significantly worse sperm quality than the SLC samples (motility, P<0.01; viability, P<0.001). There was no evidence to suggest that adding SP to the SLC samples could enhance sperm motility for more than a few hours. Longer term cold storage of spermatozoa in the presence of small concentrations of SP resulted in a reduction in total motility and progressive motility compared with SLC alone. High concentrations of SP were detrimental to sperm survival.