Non-centrifuged Samples Research Articles

Comparison of DNA extraction procedures for detection of Mycoplasma bovis directly from extended bovine semen straw samples using a commercial M. bovis PCR

BackgroundMycoplasma bovis is a global pathogen of cattle but was detected for the first time in New Zealand in 2017, triggering a response under their Biosecurity Act as an “unwanted organism”. Following a lengthy eradication campaign, the Ministry of Primary Industries (MPI) now requires all bovine semen destined for export to New Zealand to be screened with an M. bovis-specific real-time PCR (rtPCR) compliant with amended import health standard (IHS) test requirements aimed at preventing the accidental importation of M. bovis. The standard stipulates that semen samples cannot be centrifuged prior to DNA extraction. To comply with these strict requirements, one of the listed tests was validated together with different DNA preparation steps and compared with existing in-house procedures. DNA was extracted from semen straws using the current in-house semi-automated platform procedures for processing culture, tissue and body fluid sample submissions and was compared with the stipulated test requirements. DNA from centrifuged and unspun semen samples spiked with M. bovis was also compared.ResultsThe rtPCR had a sensitivity and specificity of 100% (95% confidence interval = 79–100% and 74–100%, respectively) when testing DNA from other Mycoplasma species or bovine semen spiked with the latter, with a high level of repeatability for within- and between- run replicates. The consistent limit of detection was 0.001 pg/µl M. bovis DNA and between 5.3 × 102 and 7.5 × 102 CFU/ml M. bovis when artificially spiked in semen. DNA extracted using the KingFisher Flex was detected with lower Cq values than the Maxwell 16, but the comparable improvements in sensitivity were mainly associated with non-centrifuged samples (p < 0.001). None of the procedures tested impeded the detection sensitivity of M. bovis in the presence of competitor organisms Acholeplasma laidlawii, Mycoplasma bovigenitalium and Ureaplasma diversum, confirming M. bovis specificity of the polC target.ConclusionsUnder the experimental conditions applied, this rtPCR test efficiently detected M. bovis in extended bovine semen straw samples from DNA extracted using both semi-automated extraction platforms, irrespective of prior centrifugation of extended semen.

Histological Properties of Adipose Tissue as an Autologous Tissue Filler Harvested from Different Donor Areas and Impact of Centrifugation.

As a burgeoning technique in reconstructive and aesthetic surgery, lipofilling's success is hindered by the unpredictability of graft integrity and quality. This study addresses the critical need to enhance consistency and reproducibility by exploring the clinical utility of adipose tissue from specific body areas, considering the influence of patient-specific factors and mechanical processing on fat graft integrity and morphology. In a prospective, randomized, single-blind study, 52 patients undergoing surgical reconstruction due to significant deformities were enrolled. Lipoaspiration from four areas was performed. Adipose tissue was compared using five parameters of tissue damage and 10 parameters of graft integrity, assessed immediately postcollection and after centrifugation. The study aimed to evaluate the structural integrity and clinical applicability of adipocytes. Morphological assessment revealed no significant differences in adipose tissue quality across donor sites, suggesting consistent graft quality regardless of the harvesting location. Centrifugation induced more morphological damage than noncentrifuged samples, but the overall graft integrity was maintained due to increased cell density. Higher graft acceptance parameters were noted in noncentrifuged samples compared with centrifuged ones. Despite centrifugation-induced morphological changes, adipose tissue integrity remains relatively unaffected, supporting a flexible approach to donor site selection. The consistent quality of adipose tissue underscores the potential for autologous fat transplantation across various clinical scenarios. Optimizing graft processing techniques is crucial for enhancing the predictability and efficacy of lipofilling.

Effects of certain pre-analytical factors on the performance of plasma phospho-tau217

IntroductionPre-analytical factors can cause substantial variability in the measurements of cerebrospinal fluid (CSF) and plasma biomarkers of Alzheimer’s disease (AD). However, their effects on the performance of one of the most promising plasma AD biomarkers, phosphorylated tau (p-tau)217, are not known.MethodsWe included 50 amyloid-β positive (Aβ+) and 50 Aβ− participants from the Swedish BioFINDER-1 study. Plasma and CSF p-tau217 were measured using an immunoassay developed by Lilly Research Laboratories. We examined the effect of four plasma handling conditions, i.e., (1) thawing at room temperature (RT) with no centrifugation, (2) thawing at RT followed by centrifugation, (3) thawing on ice with no centrifugation, and (4) thawing on ice followed by centrifugation. In addition, we also tested the effects of up to 3 freeze–thaw cycles on the associations of plasma p-tau217 with AD-related pathologies measured with CSF p-tau217 and CSF Aβ42/Aβ40.ResultsIn the whole cohort (combining Aβ+ and Aβ− participants), we found significant correlations between plasma p-tau217 and both CSF p-tau217 (Rrange, 0.614–0.717, p < 0.001) and CSF Aβ42/Aβ40 (Spearman Rrange, − 0.515 to − 0.652, p < 0.001) for each of the four tested conditions. Correlations between plasma and CSF p-tau217 were also significant for all conditions in the Aβ+ group (Rrange, 0.506–0.579, p < 0.001). However, in this Aβ+ subgroup, correlations with CSF Aβ42/Aβ40 were only significant for centrifuged samples (thawed at RT, R = − 0.394, p = 0.010; thawed on ice, R = − 0.406; p = 0.007). In Aβ− participants, correlations between plasma and CSF p-tau217 were again significant only for centrifuged samples (thawed at RT, R = 0.394, p = 0.007; thawed on ice, R = 0.334; p = 0.022), with no correlations seen between plasma p-tau217 and CSF Aβ42/Aβ40 for any of the conditions. While the accuracy of plasma p-tau217 to identify individuals with abnormal CSF Aβ42/Aβ40 or CSF p-tau217 status was high, the AUCs for samples thawed at RT and analyzed without centrifugation were numerically lower than the AUCs of other conditions (CSF Aβ42/Aβ40 = 0.845 vs 0.872–0.884; CSF p-tau217 = 0.866 vs 0.908–0.924, pdiff > 0.11). P-tau217 concentration was consistently higher in non-centrifuged samples than in centrifuged samples (p ≤ 0.021). There were no differences between samples freeze-thawed once, twice, or three times.ConclusionCentrifugation improved the performance of plasma p-tau217, but thawing temperatures and up to three freeze–thaw cycles did not have a significant impact. These results may inform the future development of standardized sample-handling protocols for AD biomarkers.

Synovial fluid crystal analysis with compensated polarization using a gout analyser inclinical practice.

To validate the gout analyzer as a clinical method of synovial fluid crystal analysis. Thirty knee synovial fluid samples with suspected calcium pyrophosphate (CPP) crystals were analyzed. Within 48 hours after collection, each non-centrifuged sample was examined blindly and independently by one or more rheumatologists in the following order: 1) with an optical microscope under ordinary light, 2) with the same microscope under compensated polarization provided by a gout analyzer, and 3) with a fully equipped compensated polarized microscope with a rotating stage as the gold standard. As a reference, laboratory technicians analyzed fresh, centrifuged synovial fluid using a gout analyzer. Of the 30 samples analyzed, CPP and monosodium urate (MSU) crystals were detected in 11 and four, non-centrifuged samples, respectively, using a fully equipped compensated polarized microscope. The rheumatologists' detection rate of crystals in the non-centrifuged synovial fluid under ordinary light and with a gout analyzer was 73.3% and 80%, respectively. The laboratory technicians' detection rate in fresh centrifuged synovial fluid using a gout analyzer was 100%. A gout analyzer may be used to diagnose gout and calcium pyrophosphate deposition disease definitively if a fully equipped compensated polarized microscope is unavailable.

Application of zincon analysis to investigate the zinc speciation in aqueous media for further understanding of ZnO solubility

The environmental accumulation and toxicity potential of metal oxide nanoparticles because of their increasing use in various commercial products have increased the interest in understanding the behavior of these materials in the aquatic environment. For example, zinc oxide (ZnO) is used in various industrial applications but exhibits partial solubility in aqueous media, therefore it has potential aquatoxic action. ZnO dissolves in water by releasing Zn2+ ions; thus, understanding the complexation behavior of these ions with water is crucial to be able to give a complete description of the fate of ZnO in aquatic environment. In this study, we propose application of a reliable and facile route based on the zincon analysis to further understand the Zn2+ complexation when ZnO is placed in aqueous media. To evaluate this, eight Zn2+ standards at a certain concentration were first prepared in the pH range between 7.0 and 10.0. Samples were analyzed via Inductive Coupled Plasma-Mass spectrometry (ICP-MS/MS) and the zincon analysis by monitoring their UV absorbance maximum at 620 nm. Analysis before and after centrifugation aided the investigation of the pH dependent complexation of Zn2+. In the centrifuged samples, the measured zinc ion fraction decreased, suggesting precipitation of zinc as zinc hydroxide (Zn(OH)2(s)) in the solid form from the solution when the pH was increased from 7.0 to 10.0. However, there was no significant decrease in the non-centrifuged samples in the same pH range. These results show that the zincon analysis can be used to quantify both Zn2+(aq) ions and Zn(OH)2. Moreover, the zincon results were consistent with the results of ICP-MS/MS analyses. The centrifugation helps to sediment solid hydroxide particles enabling one to predict fraction of the ions and the hydroxides. Solid hydroxide precipitate was characterized by FTIR, XRD, SEM and EDX; and formation of orthorhombic Zn(OH)2 phase was confirmed.

Transcriptional Response in Human Jurkat T Lymphocytes to a near Physiological Hypergravity Environment and to One Common in Routine Cell Culture Protocols.

Cellular effects of hypergravity have been described in many studies. We investigated the transcriptional dynamics in Jurkat T cells between 20 s and 60 min of 9 g hypergravity and characterized a highly dynamic biphasic time course of gene expression response with a transition point between rapid adaptation and long-term response at approximately 7 min. Upregulated genes were shifted towards the center of the nuclei, whereby downregulated genes were shifted towards the periphery. Upregulated gene expression was mostly located on chromosomes 16-22. Protein-coding transcripts formed the majority with more than 90% of all differentially expressed genes and followed a continuous trend of downregulation, whereas retained introns demonstrated a biphasic time-course. The gene expression pattern of hypergravity response was not comparable with other stress factors such as oxidative stress, heat shock or inflammation. Furthermore, we tested a routine centrifugation protocol that is widely used to harvest cells for subsequent RNA analysis and detected a huge impact on the transcriptome compared to non-centrifuged samples, which did not return to baseline within 15 min. Thus, we recommend carefully studying the response of any cell types used for any experiments regarding the hypergravity time and levels applied during cell culture procedures and analysis.

Replacing centrifugation with mixing in urine analysis enriches protein pool in the urine samples

Urine is the basic diagnostic material, easy to collect, not requiring invasive approach. During standard procedure the urine samples are centrifuged and the supernatant analysed physically, biochemically, and microscopically. The centrifugation step removes proteins including those forming aggregates especially in the state of illness and after transplantation. Here, we analysed the effect of urine centrifuging on specific protein content in urine samples obtained from cardiovascular patients (CVD) and after kidney or liver transplantation.We tested homogeneous whole urine samples, standardly centrifuge one, and the pellet after centrifuging. Protein content was examined using Western blot analysis and mass spectrometry (MS) of samples from CVD patients or the one after transplantation.The average of 21% proteins from non-centrifuged samples were found in the pellet removed after standard centrifugation. MS analysis confirmed that diagnostically important proteins were located there in. In 90% of cases whole urine samples contained more proteins than standard supernatant, among them e.g. proteins involved in immunological response like immunoglobulins and complement compounds secreted by leucocytes.Replacing centrifuging with intensive mixing of urine samples provides a method of enriching the samples with proteins removed during standard procedure, thus increasing possibility of finding new biomarkers for diseases undiagnosable with classic urine analysis.

Adsorption of aqueous copper chlorophyllin mixtures on model surfaces

Measured were the colloidal and adsorption properties of copper chlorophyllin (CuChl), a complex mixture derived from chlorophyll that is used as a food additive and in other applications. CuChl has an “apparent solubility” (it is a mixture) of 0.1 g/L in PBS buffer. Dynamic light scattering of dilute solutions showed the presence of particles with diameters in the range 10−800 nm. Nanoparticle tracking analysis (NanoSight LM10) showed the majority of nanoparticles had hydrodynamic diameters 100−200 nm. Dialysis experiments revealed that 58 % of a 0.1 g/L CuChl was present as dispersed particles. QCM-D measurements showed that aqueous CuChl adsorbs on surfaces giving a maximum adsorbed coverage, Γmax, that is sensitive to the type of surface; cellulose > polystyrene>> pullulan > silica = 0. No adsorption was observed on silica. Centrifuged CuCe6, a pure chlorin, showed essentially the same adsorption behaviors as centrifuged CuChl mixtures on polystyrene, suggesting that chlorins are the dominant adsorbed species from CuChl mixtures on hydrophobic surfaces. Centrifuged CuChl and CuCe6 usually gave lower Γmax values compared to non-centrifuged samples. In all cases, rinsing with buffer after CuChl or CuCe6 adsorption resulted in the immediate removal of some of the adsorbed chlorins. With cellulose there was no further dissolution, whereas with polystyrene, the adsorbed layer eventually was entirely removed. AFM measurements showed the adsorbed materials included nanoparticles. The surfactants SDS or DTAB could displace CuChl adsorbed on cellulosic filter paper only if the surfactant concentrations were above the critical micelle concentration.

Establishment of a reference procedure to measure urine-formed elements and evaluation of an automated urine analyzer

A standardized reference method is needed to accurately and precisely measure urine-formed elements (UFEs; red blood cells [RBCs], white blood cells [WBCs], and squamous epithelial cells [sECs]). We compared the results from a standard method with those from an automated analyzer. Trained technicians used standardized bright-field microscopy of fresh non-centrifuged urine samples, and disposable 1 µl chambers. Fifteen experienced technicians from 5 hospitals (3 per hospital) each performed 6 manual counts of 10 different native urine samples using a manual chamber and standard methods. The sEC counts were at least 50/µL, and the coefficient of variation (CV) was less than 14%; the RBC and WBC counts were at least 200/µL and the CVs were less than 7%. The same samples were also analyzed 6 times using automated analyzers. The means, CVs, and biases were determined. The median CVs for the manual measurements were 6.4% (WBCs), 6.6% (RBCs), and 12.7% (sECs). The CVs of the automated analyzer were 4.7% (WBCs), 5.6% (RBCs), and 9.2% (sECs). Biases between the automated and manual methods were −2.9% to 5.0%(WBCs), −0.8% to 8.8% (RBCs) and −2.8% to 9.4% (sECs). The count mean values and expanded uncertainties of these counts were (224.5 ± 15.0) cells/µL, (234.2 ± 16.2) cells/µL, and (61.5 ± 7.9) cells/µL, respectively. The standardized manual method for measuring UFEs had high precision and accuracy, making it a suitable reference method. Use of this reference method to calibrate an automated analyzer improved the accuracy of automated analysis.

Fouling behaviours of two stages microalgae/membrane filtration system applied to palm oil mill effluent treatment

Fouling by solids and microorganisms is the major obstacle limiting the efficient use of membrane wastewater treatment. In our previous study, two stages microalgae/membrane filtration system was proposed to treat anaerobic digested palm oil mill effluent (AnPOME). This two stages microalgae/membrane filtration system had showed great potential for the treatment of AnPOME with high removal of COD, NH3-N, PO43-, TSS, turbidity, and colour. However, fouling behavior of the membrane in this two stages microalgae/membrane filtration system was still unknown. In this study, empirical models that describe permeate flux decline for dead-end filtration (pore blocking - complete, intermediate, and standard; and cake layer formation) presented by Hermia were used to fit the experimental results in identifying the fouling mechanism under different experimental conditions. Both centrifuged and non-centrifuged samples were taken from the medium with 3 days RT intervals, from day 0 to day 12 to study their influence on fouling mechanisms described by Hermia for ultrafiltration (UF), nanofiltration (NF), and reverse osmosis (RO) filtration mode. Besides, a more detailed study on the use of resistance-in-series model for dead-end filtration was done to investigate the fouling mechanisms involved in membrane filtration of AnPOME collected after microalgae treatment. The results showed that fouling of UF and NF membrane was mainly caused by cake layer formation and it was also supported by the analysis for resistance-in-series model. Whereas, fouling of RO membrane was dominated by concentration polarization.

The effects of transport by car on coagulation tests.

This research investigated the effects of the transport of blood samples between centers/laboratories by car on coagulation tests. Five tubes of blood samples were taken from 20 healthy volunteers. The samples consisted of a baseline (control) group, centrifuged and noncentrifuged transported samples; centrifuged and noncentrifuged untransported samples. The groups of centrifuged and noncentrifuged samples were transported by car for 2 h. The centrifuged and noncentrifuged untransported samples were incubated in the laboratory until the transported samples arrived. Prothrombin time (PT) and activated partial thromboplastin time (APTT) tests were conducted for all samples. Significant differences between the baseline group and the centrifuged and noncentrifuged transported samples and the noncentrifuged untransported samples were found for APTT levels (p<0.05, for all). In addition, significant mean percentage differences in PT values were found between the baseline group and the noncentrifuged transported samples (p<0.001) and the noncentrifuged untransported samples (p=0.005). The mean level of PT in the noncentrifuged transported samples was outside the upper limit of the clinical decision level. Noncentrifuged transported samples showed clinically significant differences in PT test results that may have stemmed from mechanical agitation during transportation. Therefore, we recommend not transporting noncentrifuged specimens for PT testing by car.

Factors that alter the biochemical biomarkers of environmental contamination in Chironomus sancticaroli (Diptera, Chironomidae)

Changes in physiology of the nervous system and metabolism can be detected through the activity of acetylcholinesterase (AChE), alpha esterase (EST-α) and beta esterase (EST-β) in chironomids exposed to pollutants. However, to understand the real effect of xenobiotics on organisms, it is important to investigate how certain factors can interfere with enzyme activity. We investigated the effects of different temperatures, food stress and two steps of the enzymatic protocol on the activity of AChE, EST-α and EST-β in Chironomus sancticaroli. In experiment of thermal stress individuals from the egg stage to the fourth larval instar were exposed to different temperatures (20, 25 and 30°C). In food stress experiment, larvae were reared until IV instar in a standard setting (25°C and 0.9g weekly ration), but from fourth instar on they were divided into groups and offered different feeding regimes (24, 48 and 72h with/without food). In sample freezing experiment, a group of samples was processed immediately after homogenization and another after freezing for 30 days. To test the effect of centrifugation on samples, enzyme activity was quantified from centrifuged and non-centrifuged samples. The activity of each enzyme reached an optimum at a different temperature. The absence of food triggered different changes in enzyme activity depending on the period of starvation. Freezing and centrifugation of the samples significantly reduced the activity of three enzymes. Based on these results we conclude that the four factors studied had an influence on AChE, EST-α and EST-β and this influence should be considered in ecotoxicological approaches.

Effect of Tween-20 on Core Biomarkers Measured in Cerebrospinal Fluid from Patients with Alzheimer's Disease, Mild Cognitive Impairment, or Healthy Control Individuals.

There is substantial variation caused by preanalytical procedures in the measurement of cerebrospinal fluid (CSF) biomarkers for Alzheimer's disease (AD) reported in the literature. Determine whether the detergent Tween-20 improves diagnostic accuracy. CSF proteins (Aβ42, Aβ40, total tau, and phosphorylated tau) were measured by standard ELISA, in uncentrifuged CSF with or without 0.05% Tween-20 from patients with AD or amnestic mild cognitive impairment, and healthy elderly controls. In the main study, collection tubes containing Tween-20 (Sarstedt 15 mL) were filled with 5 mL CSF to ensure consistent detergent concentration across subsequent aliquots into Corning 2 mL tubes. These latter were also the primary collection vessel for samples without Tween-20. The effect of centrifugation, and extra tube transfer of samples with Tween-20 were also examined. 0.05% Tween-20 significantly increased mean measured CSF concentration of Aβ42 (30% ), Aβ40 (23% ), and total tau (4% ), but not phosphorylated tau. Generally, these increases were similar in all groups, although for Aβ42, the mean percentage increase with Tween-20 was slightly larger for AD. Areas under receiver-operator characteristic curves were similar whether Tween-20 was present or not. Centrifuged CSF without Tween-20 significantly reduced the measured concentration of Aβ42 versus non-centrifuged samples, a difference not seen when detergent was added. Similar CSF Aβ42 levels were found whether Tween-20 was added at collection in an extra tube or directly to the main collection tube. Addition of Tween-20 to CSF did not improve differentiation of patients from controls.

Importance and impact of preanalytical variables on Alzheimer disease biomarker concentrations in cerebrospinal fluid.

Analyses of cerebrospinal fluid (CSF) biomarkers (β-amyloid protein, total tau protein, and hyperphosphorylated tau protein) are part of the diagnostic criteria of Alzheimer disease. Different preanalytical sample procedures contribute to variability of CSF biomarker concentrations, hampering between-laboratory comparisons. The aim of this study was to explore the influence of fractionated sampling, centrifugation, freezing temperature, freezing delay, and freeze-thaw cycles on CSF biomarker analyses. We studied fractionated sampling in sequential aliquots of lumbar CSF. Centrifuged and noncentrifuged samples from the same fraction were compared. CSF samples were subjected to different protocols (liquid nitrogen, -80 °C, and -20 °C; 24 h at 2-8 °C; and 24 and 48 h at room temperature). To study the influence of freeze-thaw cycles, samples were thawed up to 4 times and refrozen at -80 °C. CSF was collected in polypropylene tubes. We measured CSF biomarker concentrations with commercially available single-analyte Innotest assays. CSF biomarker concentrations from non-blood-contaminated samples are not influenced by centrifugation or fractionated sampling. Freezing temperature and delayed storage can affect biomarker concentrations; freezing of CSF samples at -80 °C as soon as possible after collection is recommended. Consecutive freezing and thawing of CSF samples up to 3 times had little effect. Temperature of freezing, delay until freezing, and freeze-thaw cycles significantly influence CSF biomarker concentrations, stressing the need for standard operating procedures for preanalytical sample handling. The differences observed in this study are, however, relatively small, and the impact on the clinical value of these CSF biomarkers needs to be determined.

Targeted Metabolomics Identifies Reliable and Stable Metabolites in Human Serum and Plasma Samples

BackgroundInformation regarding the variability of metabolite levels over time in an individual is required to estimate the reproducibility of metabolite measurements. In intervention studies, it is critical to appropriately judge changes that are elicited by any kind of intervention. The pre-analytic phase (collection, transport and sample processing) is a particularly important component of data quality in multi-center studies.MethodsReliability of metabolites (within-and between-person variance, intraclass correlation coefficient) and stability (shipment simulation at different temperatures, use of gel-barrier collection tubes, freeze-thaw cycles) were analyzed in fasting serum and plasma samples of 22 healthy human subjects using a targeted LC-MS approach.ResultsReliability of metabolite measurements was higher in serum compared to plasma samples and was good in most saturated short-and medium-chain acylcarnitines, amino acids, biogenic amines, glycerophospholipids, sphingolipids and hexose. The majority of metabolites were stable for 24 h on cool packs and at room temperature in non-centrifuged tubes. Plasma and serum metabolite stability showed good coherence. Serum metabolite concentrations were mostly unaffected by tube type and one or two freeze-thaw cycles.ConclusionA single time point measurement is assumed to be sufficient for a targeted metabolomics analysis of most metabolites. For shipment, samples should ideally be separated and frozen immediately after collection, as some amino acids and biogenic amines become unstable within 3 h on cool packs. Serum gel-barrier tubes can be used safely for this process as they have no effect on concentration in most metabolites. Shipment of non-centrifuged samples on cool packs is a cost-efficient alternative for most metabolites.

Developing a dynamic test to identify stallions with reduced sperm DNA stability

Sperm DNA integrity is usually evaluated using a ‘static’ test. However, recent studies suggest that a ‘dynamic’ test to examine how rapidly spermDNA degenerates at 37 C is a more sensitive index of (sub)-fertility. This study validated a ‘DNA stress’ test by examining whether sperm DNA from different stallions degraded repeatedly at different rates during incubation at 37 C. After daily semen collection for 1 week to reach daily sperm output, semen was collected on three consecutive days from each of eight 3-year old Warmblood stallions, i.e. three ejaculates per stallion. All ejaculates had >60% morphologically normal and >60% motile sperm. The semen was diluted to 50x106 sperm/mL in an egg yolk-skimmed milk extender (Spervital EVD ; Spervital) and divided into two portions; one portion was centrifuged (800xg, 20min) and re-suspended. Centrifuged and non-centrifuged samples were split again and incubated at 37 C and 5 C. Semen was assessed during 37 C incubation after 4, 8, 24 and 48h; during 5 C incubation after 48, 96, 144 and 192h. At each time-point, sperm motility was evaluated using a computerized system (SpermVision ; Minitub), viability by SYBR14/PI staining assessed by fluorescence microscopy, and DNA integrity by SCSA . Differences in percentage of motile, viable or DNAintact sperm were compared between incubation temperatures, times and stallions using analysis of variance. Percentages of motile and viable sperm at 5 C were higher (p<0.05) for centrifuged than non-centrifuged semen, and there were no between-stallion differences in the rate of loss of motility or viability. Centrifugation did not reduce the rate at which the percentage of motile or viable sperm decreased during 37 C incubation. The rate of change of sperm DNA degradation, as reflected by the alpha t value, did not differ between these 8 stallions when semen was

CHARACTERIZATION AND CRYOPRESERVATION OF SEMEN FROM ENDANGERED MARKHOR GOATS (CAPRA FALCONERI HEPTNERI) WITH EVALUATION OF REPRODUCTIVE SEASONALITY

The aims of this study were to determine the reproductive seasonality of four captive markhor goats (Capra falconeri heptneri), to characterize semen collected by electroejaculation, and to compare extenders and processing techniques for semen cryopreservation. Over the course of 1 yr, mean monthly scrotal circumference, serum testosterone, and fecal testosterone were measured and found to be inversely associated with day length. Maximum scrotal circumference (25.2 +/- 0.9 cm), serum testosterone (521.0 +/- 103.4 ng/dl), and fecal testosterone (382.5 +/- 90.3 ng/g) occurred in November, when day length was short (9.7 +/- 0.1 hr). Once a month for 3 mo (December, January, and February), bucks were anesthetized for electroejaculation and semen evaluation. Semen samples were divided into six aliquots for extension and cryopreservation in soy-based Bioxcell or Tris-based extender with 5 or 15% egg yolk, with and without centrifugation. Samples were then thawed for repeat evaluation 1-3 mo later. Postthaw evaluation revealed no significant differences between centrifuged and noncentrifuged samples. Sperm in Tris 5% and 15% egg yolk displayed higher total motility at 0, 3, and 6 hr postthaw and higher progressive motility postthaw compared with sperm in Bioxcell (P < 0.05). Sperm in Bioxcell displayed higher viability than sperm in both Tris-egg yolk extenders (P < 0.01), more intact acrosomes than sperm in Tris-15% egg yolk (P < 0.05), and a tendency for more intact acrosomes than sperm in Tris-5% egg yolk (P < 0.10). Sperm in Tris-5% egg yolk tended to have a higher percentage of morphologically normal sperm compared with Bioxcell (P < 0.10). This study provides evidence that markhor goats exhibit seasonality in scrotal circumference and testosterone levels and that centrifugation may be eliminated from the processing of markhor semen.

Quantitative and Qualitative Aspects of Bacterial Communities Associated with Cultures of Chlorella minutissima

Microbiological conditions in 200 L cultures of microalgae Chlorella minutissima in polyethylene sleeves were examined. The influence of addition of antibiotic (nitrofurantoin) was studied. Samples were taken 2, 5, 9, and 14 d after inoculation and were spread on solid Zobell medium and on thiosulfate citrate bile salts sucrose agar for the enumeration of total numbers of bacteria and numbers of presumptive Vibrio bacteria, respectively. Microalgae grew well in the cultures and high numbers of culturable bacteria (about 107 bacteria/mL) were present in all microalgae cultures. Addition of antibiotic in 200‐L cultures of C. minutissima did not result in a significant decrease of total counts of bacteria per unit volume (P &gt; 0.05), while the numbers of presumptive Vibrio were higher in cultures added antibiotic compared with the control treatment (P &lt; 0.05). The numbers of presumptive Vibrio per unit volume and the percentage of fast‐growing bacteria were higher in microalgae supernatant than in noncentrifuged samples. Representative colonies were taken from all samples and in total 649 bacterial strains were isolated during this study. A range of phylotypes was identified by amplification and sequencing of the 16S rDNA gene and phylogenetic trees were constructed. Several members of the Roseobacter clade were dominant among the culturable isolates.

Effect of Pneumatic Tube Delivery System Rate and Distance on Hemolysis of Blood Specimens

We evaluated the effects of pneumatic tube system (PTS) transport rates and distances on routine hematology and coagulation analysis. PTS effects on centrifuged blood samples were also examined. The study was completed at Dicle University Hospital, which has the longest pneumatic tube system in Turkey. Blood samples were collected at three different locations within the hospital and an emergency department, and delivered to the central laboratory by the PTS or a human carrier. Samples were transported at different rates and over varying distances. Each specimen's potassium (K) and lactic dehydrogenase (LDH) levels, in both the serum and plasma, were tracked to monitor hemolysis. Measurements of LDH and K were obtained using heparin or citrate. A positive correlation was observed between distance and hemolysis in serum samples transported at 4.2 m/sec, and at 3.1 m/sec for more than 2200 m (r = 0.774 and r = 0.766, respectively). Distance and hemolysis were also correlated in non-centrifuged samples (r = 0.871). The alterations in plasma LDH and K levels at different rates and PTS lengths were not statistically significant. The rate of hemolysis in PTS transported samples, dependent on PTS length and rate, may seriously affect routine tests of non-centrifuged samples.

The influence of cryopreservation and seminal plasma on the chromatin structure of dog spermatozoa

It was the aim of the current study to investigate effects of seminal plasma on the chromatin structure of frozen-thawed canine ( Canis lupus familiaris) spermatozoa. A total of 20 ejaculates were collected. Ejaculates were divided, and one half was centrifuged for removal of seminal plasma (c) while the other was left uncentrifuged (nc) before cryopreservation. This was performed according to the Uppsala system in a computerized freezing machine. Before freezing (bf) and after thawing (at), samples were investigated for motility (M), viability (CASA), and chromatin status (sperm chromatin structure assay; SCSA). Before freezing, the average DFI% and the SD-DFI from 20 nc ejaculates were 1.7 ± 4.0% and 18.6 ± 1.2, respectively. After thawing, all motility parameters decreased and were significantly lower in centrifuged than in noncentrifuged samples, whereas the percentage of morphologically abnormal spermatozoa (Morph) was significantly higher (nc: M bf, 84.1 ± 20.6%; M at, 51.9 ± 15%; c: M bf, 84.1 ± 20.6%; M at, 43.3 ± 22.2%; Morph nc: 28.3 ± 7.8% vs. c: 31.0 ± 9.8%). Furthermore, only in c samples did the DFI increase within 6 h after thawing (DFI c: bf, 41.8 ± 1.5%; 6 h at, 45.4 ± 6.6%; P < 0.01). The SD-DFI as well as the DFI% increased within 3 h of storage in both groups (SD-DFI nc: bf, 18.6 ± 1.2%; 3 h at, 25.8 ± 5.4%; DFI% nc: bf, 1.1 ± 4.0%; 3 h at, 6.1 ± 12.9%; P < 0.05). For both parameters, there was no significant difference between c and nc samples at any time investigated. In conclusion, centrifugation of semen samples before freezing decreased postthaw motility and increased the percentage of morphologically abnormal spermatozoa as well as the degree of sperm chromatin denaturation over time. Centrifugation of canine ejaculates before cryopreservation can therefore no longer be recommended.