Finding a needle in a haystack: DNA Haemoproteus columbae enrichment using percoll density gradient and flow cytometry.

Isolation of genomic DNA of blood parasites in birds, reptiles, amphibians, and fishes is a challenging task, given that their red blood cells are nucleated; for that reason, parasite genomic DNA is only a fraction of the total extracted DNA, and it is challenging to obtain concentrated high-quality genetic material. Percoll Density Gradient (PDG) and flow cytometry are tools for separating and analyzing cell populations or even a single cell, and both represent potent approaches for isolating avian haemosporidians parasites. Our experimental design included several steps seeking to concentrate the parasite´s DNA. We used blood samples from a Rock pigeon infected with Haemoproteus columbae. After inducing parasite exflagellation and gametogenesis in vitro, we subjected the samples to a Percoll Density Gradient to separate the parasites from the rest of the blood cells. Following centrifugation, the layer containing extracellular parasites underwent a flow cytometry and cell sorting process, during which we selected two different subpopulations of cells for analysis. Based on qPCR analyses, we demonstrate parasite DNA enrichment in Percoll Density Gradient and flow cytometry samples; simultaneously, these samples showed the lowest concentration of Columba livia DNA. However, the concentration of parasite DNA was higher in the PDG than in the cell sorting sample. This study reports the concentration of the Haemoproteus parasite by flow cytometry without DNA-intercalating dyes, and this methodology can serve as a technique for DNA enrichment of blood parasites infecting nucleated red blood cells to improve techniques that allow obtaining complete genomes.

Equine Spermatozoa Selection by Magnetic Activation for Use in Assisted Reproduction.

This study aimed to select high-quality spermatozoa by sperm separation by magnetic activation of the fresh equine semen, compared to density gradient centrifugation and evaluating cell quality after selection. The semen of 10 stallions was collected by the artificial vagina technique. The samples analyzed were: (1) fresh semen; (2) density gradient centrifugation (DGC); (3) separation by magnetic activation (MASS) (nonapoptotic portion NAP); (4) separation by MASS (apoptotic portion-APT). Was analyzed: motility (light microscopy), concentration (Neubauer chamber), semen morphology (humid chamber in phase contrast), and supravital test (eosin/nigrosine). In DGC, 20 × 106 spermatozoa were used in the gradient of Percoll at 90% and 45% (400 µL each), centrifugation at 900 G/5 min, the pellet was diluted in HEPES. In MASS, 10 × 106 spermatozoa were diluted in 1.5 mL of HEPES, centrifugation at 300 G/10 min, pellet was resuspended in 150 µL of HEPES with 20 µL of nanoparticles bound to annexin V, incubation for 15 minutes and filtered in the magnetic separation column. The nonapoptotic fraction was collected directly and the apoptotic fraction after removal the column from the magnet and adding 300 µL of HEPES. The total abnormalities were 43.2% ± 2.78%, with the DGC and MASS being effective in reducing sperm abnormality by 15.6% ± 2.10% and 24.30% ± 1.63%, respectively, like the observed for the number of cells with intact membranes (50% lower in the APT portion). This nanotechnological method is efficient in producing high-quality semen samples for assisted reproduction procedures.

The influence of Percoll® density gradient centrifugation before cryopreservation on the quality of frozen wisent (Bison bonasus) epididymal spermatozoa.

BACKGROUND: The wisent (Bison bonasus) is a species that has undergone a population bottleneck. Homozygosity is prevalent within the population and may have a negative impact on semen quality in wisent bulls. Semen samples containing a large amount of functionally and morphologically impaired or dead spermatozoa have lower tolerance for cryopreservation process. Such samples are prone to involve damage acrosomes, to produce and release reactive oxygen which negatively affects proper function of spermatozoas. It is a good practice to select intact and viable gametes before subjecting the sample to cryopreservation to improve the efficiency of this process. The aim of this study was to assess the ability of Percoll® density gradient centrifugation in order to improve the quality of wisent spermatozoa after cryopreservation. Spermatozoa samples were analysed with computer-assisted semen analysis system and flow cytometry. RESULTS: Percoll® density gradient centrifugation resulted in increased percentage of motile spermatozoa, higher proportion of spermatozoa with normal morphology and proper functionality but also in a significant reduction of the total number of gametes. Nevertheless, the concentration of frozen spermatoza was still sufficient for obtaining a few complete insemination doses suggested for cattle from each epididymis. CONCLUSIONS: While creating a high-quality genetic reserve, for in vitro fertilisation purposes, eliminating detritus and improving the overall quality of samples is more important than total number of spermatozoa. For these reasons, the achievement of higher post thaw quality of spermatozoa justifies the purification of samples by centrifugation in a Percoll® density gradient prior to the cryopreservation process.

BoviPure® density-gradient centrifugation procedure enhances the quality of fresh and cryopreserved dog epididymal spermatozoa.

BoviPure® is a salt solution containing colloidal silica particles coated with silane used to select sperm (e.g., ruminants) by density-gradient centrifugation (DGC). This research assessed the suitability of the BoviPure-DGC and swim-up methods for selecting dog epididymal sperm in fresh, chilled and frozen-thawed samples on post-treatment sperm quality. Sperm samples (n = 60 epididymides) were recovered by retrograde flushing from thirty orchiectomized adult dogs. Thereafter, 20 sperm pools, containing sperm aliquots of three randomly selected animals, were used for chilling (at 5 ºC for 24 h) and freezing (in liquid nitrogen vapors). Sperm selection by BoviPure-DCG and swim-up was performed in both individual and pooled samples, including non-selected samples as controls. Overall, after BoviPure-DGC selection a higher sperm retrieval rate was obtained than the swim-up selection in both individual (P < 0.05) and pooled (P < 0.01) samples. BoviPure-DGC improved (P < 0.05) the total (TM) and progressive (PSM) sperm motilities, curvilinear (VCL) and straight-line (VSL) velocities, linearity (LIN), wobble (WOB), beat-cross frequency (BCF), and integrity of plasmatic (IPM) and acrosomal (IAM) membranes of individual samples in comparison with non-selected samples. In pooled samples, however, the BoviPure-DGC improved (P < 0.05) the PSM, VCL, WOB, and IPM of chilled and frozen-thawed samples. The swim-up method improved (P < 0.05) only some kinematic variables of the individual (VCL, WOB and BCF) and cryopreserved pooled samples (VCL and ALH) in comparison with non-selected samples. In conclusion, BoviPure-DGC was more effective for recovering and selecting both fresh and cryopreserved dog epididymal sperm than the swim-up procedure improving the kinematic variables, and membranes intactness.

Cushioned centrifugation during sperm selection increases the fertilization and cleavage rates of cattle embryos produced in vitro.

This study was conducted to evaluate the effect of utilization of an iodixanol-based solution as a cushioning method during the sperm selection utilizing discontinuous Percoll gradient centrifugation in in vitro production (IVP) of cattle embryos. In Experiment I, all aliquots of thawed semen were subjected to sperm selection using the same discontinuous Percoll® gradients, except for the following four conditions: presence of cushioning solution (Cushion Fluid, Minitube) during the first centrifugation process (C1), presence of cushioning solution during the second centrifugation process (C2), inclusion of cushioning solution in both centrifugation steps (C1-2), and no addiction of cushioning solution (C; control group). Recovery rates, sperm kinetics, and reactive oxygen species (ROS) production were evaluated. In Experiment II, sperm cells were processed using sperm selection conditions C and C1, and fertilization rates and embryonic development kinetics were compared between experimental groups. With use of condition C1, there was improvement in fertilization and cleavage rates when compared to use of condition C (56.4% compared with 45.5% and 80.0% compared 64.7%, respectively). In conclusion, results indicate the use of a cushioning solution during sperm selection positively affects the developmental potential of embryos.

Effects of selection by the Percoll density gradient method on motility, mitochondrial membrane potential and fertility in a subpopulation of Atlantic salmon (Salmo salar) testicular spermatozoa.

The aim of this study was to evaluate effects of selection using the Percoll density gradient method on motility, mitochondrial membrane potential (ΔΨMit) and fertility in a subpopulation of testicular spermatozoa obtained from Atlantic salmon (Salmo salar). Samples were divided into three groups: Control (C), T1 (45/90 % Percoll®) and T2 (45/60 % Percoll®). Sperm motility was evaluated using CASA (Computer-Assisted Sperm Analysis), ΔΨMit using flow cytometry, and fertility evaluating whether cleavage of fertilised eggs had occurred after 16 h of incubation at 10 °C. Results indicate that motility was greater in T1 (92 ± 2.91 %) and T2 (89 ± 2.88 %) than in the Control (83.2 ± 2.04 %). The percentage of ΔΨMit was 88.3 ± 0.58 % and 85 ± 2% for T1 and T2, respectively, compared to 35 ± 6.24 % for the control. The fertility rates were 76 ± 9.1 % and 70 ± 8.1 % for T1 and T2, respectively, compared with 66 ± 12 % for the control. The kinetic characteristics for T1 were curvilinear velocity (VCL): 92.44 ± 21.12 µm/s, average path velocity (VAP): 85.87 ± 21.83 µm/s; and for T2 VCL was 78.69 ± 17.63 µm/s and VAP was 73.62 ± 17.08 µm/s. The results indicate sperm motility and ΔΨMit were greater in T1 and T2 compared with the control (P < 0.05). Similarly, there was an increase in the fertilisation rate compared to the control. The results from this study are the first where sperm quality variables were evaluated for Salmo salar testicular sperm using the Percoll® density gradient method.

Prevalence of serum antibodies to Toxoplasma gondii in the small Indian mongoose (Herpestes auropunctatus) on Amami-Oshima Island, Japan.

Prevalence of antibodies to Toxoplasm gondii was studied using the latex agglutination (LA) method, followed by sucrose density gradient centrifugation (SDGC) method on the small Indian mongoose (Herpestes auropunctatus), which inhabits Amami-Oshima Island. Of the 362 samples, 38 (10.5%) revealed positive. Single or double peaks in the 7-8 and/or 12-14 fraction to LA titer by SDGC indicated the early stage of T. gondii infection. It is suggested that domestic/feral cats play an important role for spreading this zoonotic pathogen to the mongoose as well as other species that are endemic to this island. Future studies are warranted to prevent the transmission of T. gondii among cats and wild animals in order to maintain the ecosystem health.

Sperm sexing with density gradient centrifugation in dogs.

Sexed sperm in dogs is of interest because of being polytocous, and as a result, the greatest number of offspring of the same sex can improve the market, although few studies assessing sperm sexing have been performed in this species. The present study, therefore, was conducted to evaluate the effects on sperm quality and the effectiveness of three discontinuous density gradients to separate dog sperm containing X and Y chromosomes. Thirty ejaculates from ten adult dogs were collected by digital manipulation of the penis. Cells were separated using gradients of Percoll® and Percoll® associated with Nycodenz® or Ficoll. The cells were evaluated for motility by the CASA system (Computer-Aided Semen Analyzer) and for concentration and recovered sperm concentration (after centrifugation), sperm morphology, plasma and acrosomal membrane integrity, and mitochondrial function pre- and post-centrifugation. The percentage of sperm containing X and Y chromosomes was also evaluated pre- and post-centrifugation by quantitative real-time PCR (qPCR). The use of the Ficoll gradient resulted in the greatest sperm quality after centrifugation; however, no sperm enhancement containing X or Y chromosome occurred with use of any of the methods (Percoll® 54.8 ± 1.9 compared with 45.2 ± 1.9; Percoll® associated with Nycodenz® 53.2 ± 2.0 compared with 46.8 ± 2.0; and Percoll® associated with Ficoll 55.0 ± 1.5 compared with 45.0 ± 1.5 for the percentages of cells containing the X and Y chromosomes, respectively). Thus, it was concluded that the technique of sexing dog sperm using density gradients was not effective for commercial application.

Evaluation of density gradient ultracentrifugation serum lipoprotein profiles in healthy dogs and dogs with exocrine pancreatic insufficiency.

Changes in proportions of lipoprotein classes have been described in disease states in humans. In veterinary medicine, hyperlipidemia can cause complications, such as cutaneous xanthomas, liver disease, cholelithiasis, pancreatitis, glomerular disease, lipemia retinalis, or peripheral neuropathy, but there are few reports regarding lipoproteins in diseased animals. For canine serum, we partially validated continuous lipoprotein density profiling (CLPDP), a novel density gradient ultracentrifugation technique. We examined canine lipoproteins separated by CLPDP by transmission electron microscopy (TEM). We compared lipoprotein profiles between healthy control dogs ( n = 29) and dogs with exocrine pancreatic insufficiency (EPI; n = 28) using CLPDP. Dogs with EPI included those untreated (EPI-NT; n = 6) and those treated with enzyme supplementation (EPI-T; n = 22). Our preliminary assay validation showed that CLPDP was repeatable (CV = 11.2%) and reproducible (CV = 10.6%) in canine serum. The diameters of lipoproteins analyzed by TEM were similar to those reported previously. Dogs in the EPI-NT group had more severe dyslipidemia than dogs in the EPI-T group. Dogs in the EPI-T group had lipoprotein profiles similar to healthy control dogs. CLPDP might be a useful tool for evaluating dyslipidemia in dogs.

Reduction in Percoll volume increases recovery rate of sex-sorted semen of bulls without affecting sperm quality and early embryonic development.

The aim of the present study was to evaluate the effects of Percoll volume on recovery rate, sperm quality, and embryo development kinetics in in vitro production of cattle embryos. Straws of conventional and sex-sorted semen were allocated to three different volumes of Percoll: 300 µL of each Percoll gradient (90%, 60%, and 30%), Control; 100 µL of each Percoll gradient, P100; and 200 µL of each Percoll gradient, P200. Sperm quality, fertilization rate, and embryo morpho-kinetic development using time lapse cinematography up to 48 h post-insemination were evaluated. For conventionally processed semen, sperm motility, vigor, and recovery rate were greater in the P100 and P200 treatment groups compared to the Control (P < 0.05), whereas reactive oxygen species (ROS) concentrations, lipid peroxidation, and superoxide dismutase enzyme activity were not influenced by treatments. For sex-sorted semen, treatment with P100 increased sperm curvilinear velocity, average path velocity, and amplitude of lateral head displacement (P < 0.05). Recovery rate was greater in the P100 group than Control and P200 groups (P < 0.05), formation of ROS was less in the P100 than Control and P200 groups, and superoxide dismutase enzyme activity was less in the P100 than Control group. Fertilization and cleavage rates, time of first cleavage, and cell number were similar between the P100 and Control groups (P > 0.05). The inclusion of Percoll volumes of 100 µL resulted in an increased sperm recovery rate without damage to sperm quality or affecting early embryonic development.

Sephadex filtration as successful alternative to density-gradient centrifugation procedures for ram sperm selection with improved kinetics.

Density-gradients centrifugation (DGC) and filtration columns (FC) are used to separate deformed or dead sperm, debris, and other cells that may negatively affect the fertilizing capacity of sperm in fresh, chilled and frozen/thawed semen. The present study was conducted to evaluate the suitability of DGC (BoviPure®, Percoll® and Accudenz®) and FC (Sephadex G-15®) sperm selection procedures for fresh-extended and cold-stored ram semen by assessment of post-treatment sperm quality variables. Twenty normospermic ejaculates from ten adult Merino rams were used. Sperm concentration of recovered cells was greater (P < 0.001) after BoviPure treatment than other procedures in both fresh and cold semen. With the Sephadex method, there were more desirable values than with use of DGC procedures in several sperm motility variables measured by using the CASA system. In non-refrigerated semen samples, the percentage of progressive sperm motility (%PSM) after Sephadex filtration was greater (P < 0.05) than after BoviPure treatment; the straightline velocity (VSL) value after Sephadex filtration was greater (P < 0.01) than after Accudenz treatment; the amplitude of lateral head displacement (ALH) after Sephadex and Accudenz treatment was less than non-filtered semen (P < 0.001) and after Percoll (P < 0.01) and BoviPure (P < 0.05) treatments. In cold-stored semen samples, the %PSM after Sephadex filtration was greater than non-filtered (P < 0.05) semen and after BoviPure (P < 0.05), Percoll (P < 0.05) and Accudenz (P < 0.001) treatments. It is concluded that Sephadex column filtration can be used to select ram sperm in non-refrigerated and cooled semen, because percentage progressively motile sperm and some other sperm motility characteristics are greater with use of this techniques as compared with use of DGC methods.

Procedures for leukocytes isolation from lymphoid tissues and consequences on immune endpoints used to evaluate fish immune status: A case study on roach (Rutilus rutilus).

The effects of two protocols (density gradient versus hypotonic lysis) used for leukocyte isolation from three major lymphoid tissue of fish (head-kidney, spleen and blood) were examined on some cell functional activities (tissue leucocytes distributions, phagocytosis, basal and burst oxidative activities) classically used to estimate the fish immune status. Experiments were conducted on roach (Rutilus rutilus), a cyprinid fish model often studied in different eco-physiological contexts (aquaculture, ecotoxicology …). All of immune endpoints were assessed either immediately after cell isolation or after a 12 h of incubation in order to observe if a post-isolation incubation may influence the leukocytes activities. Compared to the density gradient, hypotonic lysis is associated with granulocytes enrichments of cell suspensions. This is particularly true for leukocyte suspensions isolated from head kidney where granulocytes are naturally abundant. However, important variabilities in leukocyte distributions were observed in head kidney and spleen cells samples obtained by the use of hypotonic lysis for two incubation conditions used (no incubation or 12 h of incubation at 4 °C). The density gradient protocol leads to a transitory increase in basal ROS production in spleen lymphocytes and macrophages The blood leukocytes isolated by this same method exhibit high basal oxidative activities after 12 h of incubation at 4 °C and for the three leukocyte types (lymphocytes, monocytes and granulocytes). The hypotonic lysis is associated with an increase in PMA-induced ROS production especially in head kidney leukocytes. The increases in cell oxidative activities are consistent with increases in granulocyte proportions observed in leukocyte suspensions obtained by hypotonic lysis. Finally, the two protocols have no effect on leukocyte mortality and phagocytic activity. Within limits of our experimental conditions, the spleen is the organ whose leukocyte oxidative activities (stimulated or not) are only slightly influenced by the methods used for leukocyte isolation. This is also the case for the anterior kidney, but for this tissue, it is necessary to incubate the isolated cells for 12 h at 4 °C before functional analyses. Each of the two methodologies used has advantages and disadvantages. The hypotonic lysis allows to isolate a greater variety of leukocytes types whereas the density gradient used ensures a better stability of cells distributions over time. However, for the same fish species and for the same tissue, the method used to isolate leukocytes influences results and must be taken into consideration during acquired data analysis for evaluation of fish immune status.

Seminal plasma removal by density-gradient centrifugation is superior for goat sperm preservation compared with classical sperm washing.

Seminal plasma removal is routine in goat sperm cryopreservation protocols. The classical washing procedure designed to accomplish this usually leaves the pellet resulting from use of this procedure contaminated with dead sperm, debris, and cells other than sperm. This contamination negatively affects viability of sperm after cryopreservation. The present research was conducted to compare the effect on chilled and frozen-thawed goat sperm of the classical washing method to that of a selective washing method involving density gradient centrifugation (DGC). In the first experiment, sperm variables were measured in freshly collected sperm, and again after its washing with both methods and chilling at 5°C for 0, 3, 24, 48, 72 or 96h. The DGC-washed sperm had greater (P<0.01) straight line velocity (VSL), average path velocity (VAP) and progression ratio values at all chilling times. The amplitude of lateral head displacement (ALH) was, however, less (P<0.001) in the DGC-washed sperm at all chilling times. There was a negative correlation (P<0.05) between ALH and VSL. In the second experiment involving the freezing-thawing of sperm washed by using either method, aliquots were post-wash diluted with a Tris-citric acid/glucose/egg yolk/glycerol-based medium and frozen in liquid nitrogen for 5days. After thawing, neither the VCL, VSL nor VAP of the DGC-washed samples were affected, whereas the traditionally washed samples had less motility. In conclusion, the use of DGC was associated with enhanced sperm motility variables after chilling and freezing-thawing. This procedure would, therefore, be a useful means of removing seminal plasma from goat semen and obtaining greater quality sperm for insemination purposes.

Lipid composition of the canine sperm plasma membrane as markers of sperm motility.

The fatty acid composition of the sperm membrane is an important factor involved in the overall sperm quality, including motility. However, in the canine species, the exact composition of the plasma membrane is still unknown. Therefore, the purpose of this study was to evaluate the plasma membrane lipid composition of motile sperm cells and to compare it with asthenospermic samples, as an attempt to determine possible involvements of membrane lipids in dog sperm cell motility. The sperm-rich fraction of ten mature dogs was collected, and samples were subjected to density gradient centrifugation by Percoll® , in order to separate motile and asthenospermic samples. Processed semen samples were evaluated for sperm motility, plasma and acrosome membrane integrity, mitochondrial activity and susceptibility to oxidative stress. Lipid plasma membrane composition was identified by mass spectrometry (MALDI-MS). The motile sperm samples presented the following phospholipids in a high frequency in the plasma membrane: phosphatidylcholine 38:4 (composed of stearic and arachidonic fatty acids), phosphatidylcholine 36:1 (stearic and oleic fatty acids), phosphatidylethanolamine 34:4 (myristic and arachidonic fatty acids), glycerophosphatidic acid 36:4 (palmitic and arachidonic fatty acids), phosphatidylcholine 40:4 plasmanyl and phosphatidylcholine 40:5 plasmenyl. Furthermore, no lipid markers were found in the asthenospermic samples. Results also indicate that differences on plasma membrane composition between motile and asthenospermic samples are crucial factors for determining sperm motility, sperm functionality and susceptibility to oxidative stress. In conclusion, plasma membrane lipid composition varies considerable between motile and asthenospermic samples. Therefore, lipid markers of sperm motility can be considered, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylcholine plasmanyl, phosphatidylcholine plasmenyl and phosphatidic acid.

Selection of red deer spermatozoa with different cryoresistance using density gradients.

The objective of sperm selection media is selecting the best spermatozoa and to remove seminal plasma and diluent for using them in assisted reproductive techniques. It is known that individuals show different cryoresistance in response to the same freezing procedure. Our hypothesis was that the efficacy of selection media could be dissimilar for samples with different sperm quality after thawing. Epididymal sperm samples from mature Iberian red deer were collected and frozen. Males were classified as with high post-thaw sperm quality when sperm motility (SM) ≥ 70%, or as with low post-thaw sperm quality when SM ≤ 69%. Samples were centrifuged using the following density gradients (DG): Percoll® , Puresperm® and Bovipure™ , and several functional sperm parameters were assessed after sperm selecting and washing. Males classified with high sperm quality had higher post-thawing values (p > .05) for all parameters evaluated, except for linearity index, than those categorized as low sperm quality. After selection, some sperm characteristics improved (viability, apoptosis and mitochondrial activity) for both groups, showing the males with high sperm quality higher values in all sperm parameters except for kinematic traits and DNA fragmentation index (%DFI), regardless of DG. Bovipure™ yield lower values of sperm motility, viability, apoptosis and mitochondrial activity in relation to Percoll® and Puresperm® considering both quality groups. There was an interaction between the type of DG and sperm quality group for sperm viability (p = .040) and apoptosis (p = .003). Thus, Percoll® selected less live and more apoptotic spermatozoa than Puresperm® and Bovipure™ for males with low sperm quality. In conclusion, the DG are more efficient selecting spermatozoa from samples with high sperm quality, acting differently depending on initial sperm quality.

Colloid centrifugation of fresh stallion semen before cryopreservation decreased microorganism load of frozen-thawed semen without affecting seminal kinetics.

Freezability of equine semen may be influenced by microorganism population of semen. The objective of this study was to verify the effect of single-layer density gradient centrifugation (SLC) of fresh semen before cryopreservation on semen's microbial load (ML) and sperm cells kinetics after freezing-thawing. For that, one ejaculate was collected from 20 healthy stallions and split into control (C) samples (cryopreserved without previous SLC) and SLC samples (subjected to SLC). Semen cryopreservation was performed according to the same protocol in both groups. Microbial load of each microorganism species and total microbial load (TML) expressed in colony-forming units (CFU/mL) as well as frozen-thawed sperm kinetics were assessed in both groups. Additional analysis of the TML was performed, subdividing the frozen-thawed samples in "suitable" (total motility ≥ 30%) and "unsuitable" (total motility < 30%) semen for freezing programs, and comparing the C and SLC groups within these subpopulations. After thawing, SLC samples had less (P < 0.05) TML (88.65 × 10(2) ± 83.8 × 10(2) CFU/mL) than C samples (155.69 × 10(2) ± 48.85 × 10(2) CFU/mL), mainly due to a reduction of Enterococcus spp. and Bacillus spp. A relationship between post-thaw motility and SLC effect on ML was noted, as only in samples with more than 30% total motility was ML reduced (P < 0.05) by SLC (from 51.33 × 10(2) ± 33.26 × 10(2) CFU/mL to 26.68 × 10(2) ± 12.39 × 10(2) CFU/mL in "suitable" frozen-thawed semen vs. 240.90 × 10(2) ± 498.20 × 10(2) to 139.30 × 10(2) ± 290.30 × 10(2) CFU/mL in "unsuitable" frozen-thawed semen). The effect of SLC on kinetics of frozen-thawed sperm cells was negligible.

Use of density centrifugation for delayed cryopreservation of stallion sperm: perform sperm selection directly after collection or after storage?

Equipment for cryopreservation of stallion sperm is not always available. In such cases, diluted semen can be shipped to a facility for later cryopreservation. The aim of this study was to evaluate if selection of sperm via density centrifugation yields higher survival rates when cryopreservation is to be delayed (i.e. carried out after 1 day of storage at 5°C). Two-layer iodixanol as well as single-layer Androcoll density centrifugation were tested and compared with samples prepared with standard centrifugation. Special emphasis was placed on comparing centrifugation on the day of semen collection with centrifugation after 1-day refrigerated storage. Sperm morphology and motility as well as membrane and chromatin integrity were evaluated before and after centrifugation. Sperm motility and membrane integrity were also assessed after cryopreservation. It was found that both two- and single-layer density centrifugation processing resulted in higher percentages of morphologically normal and motile sperm with higher membrane and chromatin integrity, as compared to standard centrifugation or diluted samples. Differences were only in the order of magnitude of 5%. Recovery rates after density centrifugation were only approximately 30-40%. When cryopreservation was carried out after 1-day refrigerated storage, centrifugation processing of sperm directly after semen collection resulted in higher percentages of plasma membrane intact sperm post-thaw as compared to performing centrifugation processing of stored sperm just prior to cryopreservation. No significant differences in progressively motile sperm post-thaw were seen. Taken together, for delayed cryopreservation, it is best to perform density centrifugation directly after collection rather than immediately prior to cryopreservation.

Salvaging urospermic ejaculates from brown bear (Ursus arctos).

The objective of this study was to reverse the osmotic stress of sperm in urine contaminated bear ejaculates that were obtained by electroejaculation using pre-freezing washing or density gradient centrifugation isolation. In Experiment 1, ejaculates were divided into six aliquots, five were diluted in each washing extender: 200, 300, 400, 500 and 700 mOsm/kg (prepared from a Tes-Tris-Fructose base, adding water or fructose as corresponds), at a 1:2 ratio (raw semen: washing solution, v/v); and the other aliquot was handled without washing (Control group). Samples were centrifuged at 600 × g for 6 min prior to freezing. In Experiment 2, ejaculates were divided into two aliquots: one was diluted 1:1 with TCG (Tris-Citric acid-Glucose) and centrifuged at 600 × g for 6 min (Centrifugation Control; C-Control); the other was treated with PureSperm density gradient column. After treatments, samples were cryopreserved. Sperm motility, viability (SYBR-14/propidium iodide (PI)) and acrosomal status (peanut agglutinin-fluorescein isothiocyanate (PNA-FITC)/PI) were analyzed before and after freezing. Ejaculates with an initial osmolality of less than 120 mOsm/kg treated with pre-freezing washing, and the Control sample had greater pre-freezing sperm motility than the raw ejaculate, but sperm viability was not different among these groups. The samples washed with 700 mOsm/kg solutions had the least pre-freezing viability. In the post-thawing evaluation, pre-freezing washing treatments did not provide any improvement in comparison with the Control sample, and treatment with 700 mOsm/kg extender had deleterious effects in all urospermic samples. PureSperm density gradient centrifugation applied to urospermic raw semen was suitable for improving sperm motility and viability of pre-freezing samples and the selected spermatozoa had greater freezing capacity.

Sperm selection by Capripure(®) density-gradient centrifugation versus the dextran swim-up procedure in wild mountain ruminants.

This study compares the effectiveness of two methods of sperm selection - Capripure(®) density-gradient centrifugation (DGC) and dextran swim-up (DSU) - in semen samples from Iberian ibex (Capra pyrenaica) and European mouflon (Ovis musimon). During the increasing photoperiod, Capripure(®) DGC improved the percentage of sperm with progressive motility (P<0.05) in ibexes, and selected 60.6% of the initial total number of spermatozoa contained in the ejaculate samples. In mouflon, Capripure(®) DGC selection was unaffected by photoperiod, had no influence on any sperm variable, and selected 47.8% of the initial total number of mouflon spermatozoa in ejaculate samples. Photoperiod had no influence on the effectiveness of DSU in either ibexes or mouflons. In the ibexes, DSU reduced (P<0.05) the percentage of sperm cells with morphological abnormalities, but only selected 11.3% of the initial total number of spermatozoa in ejaculate samples. In the mouflons, DSU had no significant influence on any sperm variable, and selected 27.8% of the initial total number. Capripure(®) DGC improved ibex and mouflon sperm motility (P<0.05) following 30min and 2h of post-centrifugation, stress-inducing incubation, respectively. In both ibexes and mouflon, sperm cells showing non-progressive motility were found after only 20 h of post-centrifugation incubation following Capripure(®) DGC selection. In conclusion, Capripure(®) DGC would seem a useful method for selecting the best spermatozoa from both ibex and mouflon ejaculates.

Dense spermatozoa in stallion ejaculates contain lower concentrations of mRNAs encoding the sperm specific calcium channel 1, ornithine decarboxylase antizyme 3, aromatase, and estrogen receptor alpha than less dense spermatozoa.

Stallions are unique among livestock in that, like men, they commonly receive medical treatment for subfertility. In both species, about 15% of individuals have normal semen parameters but are subfertile, indicating a need for novel analyses of spermatozoa function. One procedure for improving fertilizing capability of stallions and men is isolation of dense spermatozoa from an ejaculate for use in artificial insemination. In the current study, dense and less dense spermatozoa were purified by density gradient centrifugation from individual ejaculates from seven reproductively normal adult stallions. The RNA isolated from the spermatozoa seemed to be naturally fragmented to an average length of 250 bases, consistent with reports of spermatozoa RNA from other species. The DNAse treatment of RNA prepared from spermatozoa removed any genomic DNA contamination, as assessed by PCR with intron spanning primers for the protamine 1 (PRM1) gene. Concentrations of seven mRNAs in spermatozoa, correlated with the fertility of men and bulls, were quantified by reverse transcription polymerase chain reaction in dense and less dense spermatozoa. Concentrations of four mRNAs were two- to four-fold lower in dense spermatozoa compared with less dense spermatozoa: Encoding the spermatozoa-specific calcium channel (P < 0.03), ornithine decarboxylase antizyme 3 (P < 0.02), aromatase (P < 0.02), and estrogen receptor alpha (P < 0.08). In contrast, concentrations of three other mRNAs, encoding PRM1 and heat shock proteins HSPA8 and DNAJC4, were not different (P > 0.1). These results identify new differences in mRNA concentrations in populations of spermatozoa with dissimilar densities.