F. Crespo

Sex-sorted bovine spermatozoa and DNA damage: I. Static features

This study examined the static response of Spermatozoa DNA Fragmentation (SDF) after sex selection in bulls using a MoFlo(®) SX (Beckman Coulter, Miami FL) spermatozoa sorter to produce three different subpopulations: 1) Spermatozoa bearing X- chromosomes with a purity of 95%, 2) Spermatozoa bearing Y-chromosomes with a purity of 95%, and 3) non-viable spermatozoa. The static response of SDF refers to the baseline values observed for DNA damage when analyzed pre- and post sex-sorting. Results showed that while the baseline level SDF in pre-sorted bull spermatozoa samples ranged from 5.3% to 11% with an average of 7.9% ± 2.1%, the level of SDF obtained in X- and Y-chromosome sorted samples was much lower (3.1% ± 1.9%) and statistical differences were obtained after comparing both groups (P < 0.01). Spermatozoa containing a fragmented DNA molecule tend to be accumulated in the non-viable subpopulation. The baseline SDF level in X- and Y-chromosome sorted subpopulations is reduced, by 63% on average when compared to the values obtained in the neat semen sample. Different bulls exhibit unique SDF reduction efficiencies via the X- and Y-chromosome sex selection process.

DNA fragmentation in frozen sperm of Equus asinus: Zamorano-Leonés, a breed at risk of extinction.

The dynamics of sperm DNA fragmentation (sDF) and sperm viability were analyzed in frozen-thawed sperm samples of Equus asinus (Zamorano-Leonés), a breed at risk of extinction. Sperm DNA fragmentation was assessed using an adaptation of the sperm chromatin dispersion test developed for stallions in five different frozen samples. Sperm were thawed and incubated at different temperatures (37 degrees C, 25 degrees C, and 4 degrees C) and sDF was assessed at different times and compared. The mean sDF after thawing at the beginning of the experiment was 18.20+/-14.77% and did not differ significantly from the results of a neutral comet assay (22.0+/-19.34%). The tendency in the sDF of all donkeys indicated that sperm DNA is more sensitive to breakage when incubated at 37 degrees C than when incubated at 25 degrees C or 4 degrees C. Interestingly, the tendency was not the same when different animals were compared, and differences in sDF dynamics were established among individuals. sDF correlated negatively with sperm viability in some individuals but not in others. From a conservation perspective, sDF analysis may offer a new way to assess sperm quality in endangered breeds in order to identify and select the best semen samples for artificial reproduction purposes. In particular, we recommend for artificial insemination the use of semen samples with a slow increase in sDF with time after thawing.

Simple and economic colloidal centrifugation protocols may be incorporated into the clinical equine sperm processing procedure

For many years in human assisted-reproduction procedures there have been special protocols to prepare and improve sperm quality. Colloidal centrifugation (CC) is a useful technique that has been proved to enhance semen quality by selection of the best spermatozoa for different species. Its use is recommended to improve fertility of subfertile stallions but current CC protocols are clinically complicated in the equine sperm processing technique due to economic and technical difficulties. The aim of this study was to determine the optimal processing procedures to adapt the use of a CC product (EquiPure™) in the equine reproduction industry. A total of nineteen ejaculates were collected from 10 Purebred Spanish Horses (P.R.E horses) using a Missouri artificial vagina. Gel-free semen aliquots were analyzed prior to treatment (control). Semen was subjected to one of six CC protocols with EquiPure™ and centrifuged samples were statistically evaluated by ANOVA and Duncan tests (p<0.05) for sperm quality and recovery rate. We obtained higher values by colloidal centrifugation in LIN, STR and BCF variables and DNA fragmentation index trended to be lower in most of the CC protocols. The studied protocols were shown to be as efficient in improving equine sperm quality as the current commercial EquiPure™, with the added advantage of being much more economical and simple to use. According to these results it seems to be possible to incorporate single layer and or high colloidal centrifugation volume protocols what would make them simple, economic and clinically viable for the equine sperm processing procedure.

Colloidal Centrifugation of Stallion Semen Results in a Reduced Rate of Sperm DNA Fragmentation

Stallion spermatozoa recovered and examined immediately after colloidal centrifugation resulted in a higher straight-line velocity (VSL) than sperm processed using direct conventional centrifugation (p = 0.000), but there was no differences in the progressive motility or sperm DNA fragmentation (SDF) as determined by the sperm chromatin dispersion assay. However, when centrifuged spermatozoa were incubated at 37 °C for 24 h to determine the rate of SDF (r-SDF), a lower r-SDF (p = 0.0011) was observed in those sperm recovered after colloidal separation (0.5 ± 0.1%/h) compared to direct (1.2 ± 0.4%/h) or no centrifugation (r-SDF = 1.2 ± 0.3%/h). These results confirm that colloidal separation of stallion spermatozoa results in prolonged sperm DNA longevity, but these differences were only apparent following a period of incubation and dynamic assessment. Consequently, we strongly recommend the use of the dynamic form of the SDF assay for evaluating centrifugation and/or other ex vivo procedures, as a single basal assessment of SDF may inadvertently result in a false-positive evaluation of DNA quality.

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.

Localization of alkali-labile sites in donkey (Equus asinus) and stallion (Equus caballus) spermatozoa

The presence of constitutive alkali-labile sites (ALS) has been investigated using a protocol of DNA breakage detection-fluorescence in situ hybridization and comet assay in spermatozoa of donkey (Equus asinus) and stallion (Equus caballus). These results were compared with those obtained using a similar experimental approach using somatic cells. The relative abundance of ALS was of the order of four times more in spermatozoa than in somatic cells. Alkali-labile sites showed a tendency to cluster localized at the equatorial-distal regions of the sperm. The amount of hybridized signal in the ALS in the sperm of donkey (Equus asinus) was 1.3 times greater than in stallion (Equus caballus), and the length of the comet tail obtained in donkey sperm was 1.6 times longer than that observed in stallion (P < 0.05); however, these differences were not appreciated in somatic cells. In conclusion, ALS localization in sperm is not a randomized event and a different pattern of ALS distribution occurs for each species. These results suggest that ALS represents a species-specific issue related to chromatin organization in sperm and somatic cells in mammalian species, and they might diverge even with very short phylogenetic distances.

The effect of two pre-cryopreservation single layer colloidal centrifugation protocols in combination with different freezing extenders on the fragmentation dynamics of thawed equine sperm DNA.

BackgroundVariability among stallions in terms of semen cryopreservation quality renders it difficult to arrive at a standardized cryopreservation method. Different extenders and processing techniques (such us colloidal centrifugation) are used in order to optimize post-thaw sperm quality. Sperm chromatin integrity analysis is an effective tool for assessing such quality. The aim of the present study was to compare the effect of two single layer colloidal centrifugation protocols (prior to cryopreservation) in combination with three commercial freezing extenders on the post-thaw chromatin integrity of equine sperm samples at different post-thaw incubation (37°C) times (i.e., their DNA fragmentation dynamics).ResultsPost-thaw DNA fragmentation levels in semen samples subjected to either of the colloidal centrifugation protocols were significantly lower (p<0.05) immediately after thawing and after 4 h of incubation at 37°C compared to samples that underwent standard (control) centrifugation. The use of InraFreeze® extender was associated with significantly less DNA fragmentation than the use of Botu-Crio® extender at 6 h of incubation, and than the use of either Botu-Crio® or Gent® extender at 24 h of incubation (p<0.05).ConclusionsThese results suggest that single layer colloidal centrifugation performed with extended or raw semen prior to cryopreservation reduces DNA fragmentation during the first four hours after thawing. Further studies are needed to determine the influence of freezing extenders on equine sperm DNA fragmentation dynamics.

Differences in preservation of canine chilled semen using simple sperm washing, single-layer centrifugation and modified swim-up preparation techniques

This study compared the efficacy of simple sperm washing (SW), single-layer centrifugation (SLC) and modified swim-up (SU) techniques in the preparation of dog spermatozoa for cooling. Eighteen ejaculates, collected from three dogs (six per dog), were pooled (three ejaculates per pool) and divided into three aliquots: (1) one aliquot was washed and cooled at 5°C for 72h, considered as control (SW-control), (2) the second aliquot was selected by SLC through Androcoll-C and subsequently cooled in the same way as the SW-control samples (SLC-AC) and (3) the last aliquot was selected by a modified SU method with Androcoll-C and cooled as mentioned above (SU-AC). Assessment of sperm motility, sperm morphology, sperm membrane integrity and acrosome integrity were performed on aliquots of fresh semen and chilled-rewarmed samples. Sperm membrane integrity and progressive motility were significantly (PPP>0.05). The recovery rates were not significantly (P>0.05) different between SW-control, SLC-AC and SU-AC samples. Our results confirm that SU-AC may be a successful method for the preparation of dog spermatozoa for cooling.

Stallion sperm selection prior to freezing using a modified colloid swim-up procedure without centrifugation

The aims of this study were to: 1) develop a new method for stallion sperm selection using a modified swim-up procedure through a colloid and 2) evaluate its impact in good quality ejaculates from bad freezers in comparison to methods involving centrifugation such as single layer centrifugation and sperm washing. Ejaculates were processed before freezing using three different procedures: sperm washing (SW), colloid single layer centrifugation (SLC) and a modified colloid swim-up (SU). After semen processing, sperm recovery rates were measured and sperm were frozen. Post-thaw sperm motility (assessed by computer-assisted sperm analysis), normal forms and plasma membrane integrity (evaluated under bright-field and fluorescence microscopy respectively), and DNA fragmentation (assessed by the Sperm-Halomax kit) were compared between treatments. Sperm recovery rates were similar between SU and SLC but lower than SW. Sperm motility after thawing was lower in SU in comparison to SLC and SW, maybe due to the incomplete removal of seminal plasma before freezing. Sperm DNA fragmentation was lower in SU and SLC selection methods, particularly in SLC selected samples during the first 6h of incubation. The remaining sperm parameters assessed were similar among treatments. In conclusion, SLC is more suitable than SW and SU to process stallion semen prior to freezing, in particular when sperm DNA damage is suspected. Further studies are needed in order to determine the potential benefits of SU in samples where centrifugation is not necessary, such as epididymal sperm, ejaculate fractioning or post-thaw semen samples.

Equivalent seminal characteristics in human and stallion at first and second ejaculated fractions.

Sperm quality was assessed in normozoospermic human (n = 10) and Spanish breed stallion (n = 10) after sperm fractionation during ejaculation. The first ejaculated fraction was separated from the second. A third sample was reconstituted using equivalent proportion of both fractions (RAW). Fraction 1, Fraction 2 and RAW semen were incubated for 30 min at 37°C to homogenise the impact of iatrogenic damage between both species. Sperm concentration, motility and sperm DNA damage were assessed in each fraction and RAW semen. The results showed two important facts: (i) spermatozoa confined at Fraction 1 exhibit superior parameters than those included at Fraction 2 in both species, and (ii) there is a certain level of concordance between species in the proportion of benefit observed when Fraction 1 is compared to RAW semen. Altogether, these results call into question whether the standard practice of whole ejaculate collection can be considered the best strategy when using male gametes for artificial insemination. In fact, the reconstituted RAW semen exhibits poorer semen characteristics than those found in Fraction 1.